Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
  • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
  • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D263/36—One oxygen atom
  • C07D263/42—One oxygen atom attached in position 5
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/022—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2
  • C07K5/0222—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -X-C(=O)-(C)n-N-C-C(=O)-Y-; X and Y being heteroatoms; n being 1 or 2 with the first amino acid being heterocyclic, e.g. Pro, Trp
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to the logical development of biochemical and biopharmaceutical agents and of new materials including fabricated materials such as fibers, beads, films, and gels.
• the invention relates to the development of molecular modules derived from oxazolone (azlactone) and related structures, and to the use of these modules in the assembly of simple and complex molecules, polymers and fabricated materials with tailored properties; where said properties can be planned and are determined by the contributions of the individual building modules.
• the molecular modules of the invention are preferably chiral, and can be used to synthesize new compounds and fabricated materials which are able to recognize biological receptors, enzymes, genetic materials, and other chiral molecules, and are thus of great interest in the fields of biopharmaceuticals, separation and materials science.
• the discovery of new peptide hormones has involved work with peptides; the discovery of new therapeutic steroids has involved work with the steroid nucleus; the discovery of new surfaces to be used in the construction of computer chips or sensors has involved work with inorganic materials, etc.
• the discovery of new functional molecules being ad hoc in nature and relying predominantly on serendipity, has been an extremely time-consuming, laborious, unpredictable, and costly enterprise.
• nucleotides can form complementary base pairs so that complementary single-stranded molecules hybridize resulting in double- or triple-helical structures that appear to be involved in
• a biologically active molecule binds with another molecule, usually a macromolecule referred to as ligand-acceptor (e.g. , a receptor or an enzyme), and this binding elicits a chain of molecular events which ultimately gives rise to a physiological state, e.g., normal cell growth and differentiation, abnormal cell growth leading to carcinogenesis, blood-pressure regulation, nerve-impulse-generation and -propagation, etc.
• ligand-acceptor e.g., a receptor or an enzyme
• the binding between ligand and ligand-acceptor is geometrically characteristic and extraordinarily specific, involving appropriate three-dimensional structural arrangements and chemical interactions.
• sequence of the native target DNA or RNA molecule is characterized and standard methods are used to synthesize oligonucleotides representing the
• disaccharides and four dissimilar monosaccharides can give rise to up to 35,560 unique tetramers, each capable of functioning as a fundamental discreet molecular messenger in a given physiological system.
• the gangliosides are examples of the versatility and effect with which organisms can use saccharide structures. These molecules are glycolipids (sugar-lipid composites) and as such are able to position themselves at strategic locations on the cell wall: their lipid component enables them to anchor in the hydrophobic interior of the cell wall, positioning their hydrophilic component in the aqueous extracellular milieu.
• the gangliosides (like many other saccharides) have been chosen to act as cellular sentries: they are involved in both the inactivation of bacterial toxins and in contact inhibition, the latter being the complex and poorly understood process by which normal cells inhibit the growth of adjacent cells, a property lost in most tumor cells.
• the structure of ganglioside GM a potent inhibitor of the toxin secreted by the cholera organism, featuring a branched complex pentameric structure is shown below.
• glycoproteins saliva-protein composites
• human blood-group antigens the A, B, and O blood classes
• glycoproteins on red blood cells belonging to incompatible blood classes cause formation of aggregates, or clusters and are the cause for failed transfusions of human blood.
• glycosylation i.e., the covalent linking with sugars.
• deglycosylation of erythropoetin causes loss of the hormone's biological activity
• deglycosylation of human gonadotropic hormone increases receptor binding but results in almost complete loss of biological activity (see Rademacher et al., Ann. Rev. Biochem 57, 785 (1988)
• TPA tissue plasminogen activating factor
• glycopolypeptide which is 30% more active than the
• polypeptide that has been glycosylated at two of the sites.
• a currently favored strategy for the development of agents which can be used to treat diseases involves the discovery of forms of ligands of biological receptors, enzymes, or related macromolecules, which mimic such ligands and either boost, i.e., agonize, or suppress, i.e., antagonize, the activity of the ligand.
• the discovery of such desirable ligand forms has traditionally been carried out either by random screening of molecules (produced through chemical synthesis or isolated from natural sources), or by using a so-called "rational" approach involving identification of a lead-structure, usually the structure of the native ligand, and optimization of its properties through numerous cycles of structural redesign and biological testing.
• peptide mimetics bind tightly, preferably in the nanomolar range, and can withstand the chemical and
• peptidomimetics however, in the majority of cases the results in one biochemical area, e.g., peptidase inhibitor design using the enzyme substrate as a lead, cannot be transferred for use in another area, e.g., tyrosine-kinase inhibitor design using the kinase substrate as a lead.
• the peptidomimetics that result from a peptide structural lead using the "rational" approach comprise unnatural alpha-amino acids. Many of these mimetics exhibit several of the troublesome features of native peptides (which also comprise alpha-amino acids) and are, thus, not favored for use as drugs.
• nonpeptidic scaffolds such as steroidal or sugar structures, to anchor specific receptor-binding groups in fixed geometric relationships have been described (see for example Hirschmann, R. et al., 1992 J. Am. Chem. Soc,
• V. D. Huebner and D.V. Santi utilized functionahzed polystyrene beads divided into portions each of which was acylated with a desired amino acid; the bead portions were mixed together, then divided into portions each of which was re-subjected to acylation with a second desirable amino acid producing dipeptides, using the techniques of solid phase peptide synthesis.
• antisense nucleotide mimetics Hoogstein-type binders or minor groove binding compounds such as those pioneered by Dervan and coworkers, have employed a variety of derivatives and variants of the naturally occuring sugar-phosphate backbone. Polyamide backbones have also been employed to support the base complements. While binding and desired functionality is observed in vitro withthese systems, they have inherent design drawbacks for in vivo use for hybridization against a rogue gene or its insidious RNA. The two main drawbacks of these polyamide systems are in (a) the persistent reliance upon an amide bond which is susceptible to proteolytic cleavage, and (b) the inability of the compound either as a class, or even singularly show efficient membrane
• a very useful source of information for the realization of the preferred "rational" drug discovery is the structure of the biological ligand acceptor which, often in conjunction with molecular modelling calculations, is used to simulate modes of binding of the ligand with its acceptor; information on the mode of binding is useful in optimizing the binding properties of the lead-structure.
• finding the structure of the ligand acceptor, or preferably the structure of a complex of the acceptor with a high affinity ligand requires the isolation of the acceptor or complex in the pure, crystalline state, followed by x-ray crystallographic analysis. The isolation and
• polypeptide substrates thereof are time-consuming, laborious, and expensive. Success in this important area of biological chemistry depends on the effective utilization of sophisticated separation technologies.
• Crystallization can be valuable as a separation technique but in the majority of cases, especially in cases involving isolation of a biomolecule from a complex biological milieu, successful separation is chromatographic.
• Chromatographic separations are the result of reversible differential binding of the components of a mixture as the mixture moves on an active natural, synthetic, or semisynthetic surface; tight-binding components in the moving mixture leave the surface last en masse resulting in separation.
• substrates or supports to be used in separations has involved either the polymerization-crosslinking of monomeric molecules under various conditions to produce fabricated materials such as beads, gels, or films, or the chemical modification of various commercially available fabricated materials e.g., sulfonation of polystyrene beads, to produce the desired new materials.
• fabricated materials such as beads, gels, or films
• chemical modification of various commercially available fabricated materials e.g., sulfonation of polystyrene beads
• prior art support materials have been developed to perform specific separations or types of separations and are thus of limited utility. Many of these materials are incompatible with biological macromolecules, e.g., reverse-phase silica frequently used to perform high pressure liquid chromatography can denature hydrophobic proteins and other polypeptides.
• chromatography and remains an extremely effective and widely used separation technique. It is certainly much more selective than traditional chromatographic techniques, e.g chromatography on silica, alumina, silica or alumina coated with long-chain hydrocarbons, polysaccharide and other types of beads or gels which in order to attain their maximum separating efficiency need to be used under conditions that are damaging to biomolecules, e.g., conditions involving high pressure, use of organic solvents and other denaturing agents, etc.
• Oxazolones, or azlactones are structures of the general formula:
• A is a functional group and n is 0-3.
• Oxazolones containing a five-membered ring and a single substituent at position 4 are typically encountered as transient
• An oxazolone can in principle contain one or two substituents at the 4-position. When these substituents are not equivalent, the carbon atom at the 4-position is asymmetric and two non-superimposable oxazolone structures (azlactones) result:
• Chiral oxazolones possessing a single 4 substituent also known as 5(4H)-oxazolones
• derived from (chiral) natural amino acid derivatives, including activated acylamino acyl structures have been prepared and isolated acyiamino acyl structures, have been prepared and isolated in the pure, crystalline state (Bodansky, M.; Klausner, Y. S.; Ondetti, M. A. in "Peptide Synthesis", Second Edition, John Wiley & Sons, New York, 1976, p. 14 and references cited therein).
• the facile, base-catalyzed racemization of several of these oxazolones has been studied in connection with investigations of the serious racemization problem
• the oxazolone-derivative building blocks of the invention can be used to synthesize novel molecules designed to mimic the three-dimensional structure and function of native ligands. and/or interact with the binding sites of a native receptor.
• This logical approach to molecular construction is applicable to the synthesis of all types of molecules, including but not limited to mimetics of peptides, proteins, oligonucleotides, carbohydrates, lipids, polymers and to fabricated materials useful in materials science. It is analogous to the modular construction of a mechanical apparatus that performs a specific operation wherein each module performs a specific task contributing to the overall operation of the apparatus.
• the invention is based, in part, on the following insights of the discoverer.
• All ligands share a single universal architectural feature: they consist of a scaffold structure, made e.g. of amide, carbon-carbon, or
• Binding modes between ligands and receptors share a single universal feature as well: they all involve attractive interactions between complementary structural elements, e.g., charge- and pi-type interactions, hydrophobic and van der Waals forces, hydrogen bonds.
• a continuum of fabricated materials exists spanning a dimensional range from about 100 Angstroms to 1 cm in diameter comprising various materials of construction, geometries, morphologies, and functions, all possessing the common feature of a functional surface which is presented to a biologically active molecule or a mixture of molecules to achieve recognition between the molecule (or the desired molecule in a mixture) and the surface.
• Oxazolone derivative structures heretofore regarded as unwanted intermediates which may form during the synthesis of peptides, would be ideal building blocks for constructing backbones or scaffolds bearing the appropriate functional groups that either mimic desired ligands. and/or interact with appropriate receptor binding sites, and for carrying out the synthesis of the various parts of the functionalized scaffold orthogonally, provided that racemization of the oxazolone structures is prevented or controlled.
• the invention is also based, in part, on the further recognition that such derivatives of ozaxolones, which do not racemize, can be used as universal building blocks for the synthesis of such novel molecules.
• oxazolone derivatives may be utilized in a variety of ways across the continuum of fabricated materials described above to produce new materials capable of specific molecular recognition. These oxazolone derivatives may be chirally pure and used to synthesize molecules that mimic a number of biologically active molecules, including but not limited to peptides, proteins, oligonucleotides, polynucleotides, carbohydrates and lipids, and a variety of other polymers as well as fabricated materials that are useful as new materials, including but not limited to solid supports useful in column chromatography, catalysts, solid phase immunoassays, drug delivery vehicles, films, and "intelligent" materials designed for use in selective separations of various components of complex mixtures.
• the molecular structures include functionalized silica surfaces useful in the optical resolution of racemic mixtures; peptide mimetics which inhibit human elastase, protein-kinase, and the HIV protease; carbohydrate, oligonucleotide and pharmacophore mimetics and polymers formed via free-radical or
• the oxazolone-derived molecules of interest possess the desired stereochemistry and, when required, are obtained
• oxazolone-derived molecules possess enhanced hydrolytic and enzymatic stabilities, and in the case of biologically active materials, are transported to target ligand-acceptor macromolecules in vivo, without causing any serious side-effects.
• chiral oxazolones in which the asymmetric center is a
• disubstituted carbon at the 4-position as well as synthetic nonchiral oxazolones may be synthesized readily and used as molecular modules capable of controlled reaction with a variety of other molecules to produce designed chiral recognition agents and conjugates.
• These chiral oxazolones may also be linked together, using polymerizing reactions carried out either in a stepwise or chain manner, to produce polymeric biological ligand mimics of defined sequence and stereochemistry.
• 4-disubstituted chiral oxazolones are extremely useful in the asymmetric functionalization of various solid supports and biological macromolecules and in the production of various chiral polymers with useful properties. The products of all of these reactions are surprisingly stable in diverse chemical and enzymological environments, and uniquely suitable for a variety of superior pharmaceutical and high-technological
• the 4 position of the oxazolone precursor does not need to be chiral, e.g., the construction of certain polymeric materials
• the use of oxazolones in the construction of linkers for the joining of two or more pharmaceutically useful or, simply, biologically active ligands, etc., symmetric or nonchiral oxazolones are used in chemical syntheses.
• the oxazolone-derived product does not need to incorporate the 4-position of the oxazolone precursor in the enantiomerically pure state, oxazolone precursors which are not enantiomerically pure may be used for syntheses.
• the invention is also directed to a method of making a polymer having a particular water solubility comprising the steps of; a) choosing a first monomer having the formula
• R and R' are the same or different and are chosen from those organic moieties exhibiting hydrophobicity; b) choosing a second monomer having the formula
• R and R' are the same or different and are chosen from those organic moieties exhibiting hydrophilicity; and c) reacting said monomers to provide an effective amount of each monomer in a developing polymer chain until a polymer having the desired water solubility is created.
• said hydrophobic organic moieties can include those which do not have carboxyl, amino or ester functionality. Also said hydrophilic moieties can include those which do have carboxyl, amino or ester functionality.
• This invention is further directed to using said method of preparing a synthetic compound to produce a compound that mimics or complements the structure of a biologically active compound of the formula.
• This method can be used to produce pharmacaphores, peptide mimetics, nucleotide mimetics, carbohydrate mimetics, and reporter compounds, for example.
• This invention is also further directed to a method of preparing a combinatorial library which comprises: a) preparing a compound having the formula;
• this invention is directed to a method of separating a desired compound from a plurality of compounds, which comprises; a) preparing a separator compound having the formula:
• the compounds of the present invention can be synthesized by many different routes. It is well known' in the art of organic synthesis that many different synthetic protocols can be used to prepare a given compound.
• Chiral 4,4'-disubstituted oxazolones may be prepared from the appropriate N-acyl amino acid using any of a number of standard acylation and cyclization
• the required chiral amino acid precursors for oxazolone synthesis may be produced using stereoselective reactions that employ chiral auxiliaries.
• An example of such a chiral auxiliary is (5)-(-)- 1 -dimethoxymethyl-2-methoxymethylpyrrolidine (SMPD) (Liebig's Ann. Chem. 1668 ( 1983)) as shown below, /
• R 2 CH 3 , i-Bu, or benzyl
• R 3 CH 3 , CHF 2 , C 2 H 5 , n-Bu, or benzyl.
• a second example involves 5H,10ß- Hoxazolo[3,2-c][1,3]benzoxazine-2(3H),5-diones (55 J. Org. Chem. 5437 (1990)),
• CH 2 CH-CH 2 .
• the desired chiral amino acid may be obtained using stereoselective biochemical
• Racemic mixtures of 4,4'-disubstituted oxazolones may be prepared from monosubstituted oxazolones by alkylation of the 4-position, as in the following transformation (Synthesis Commun., Sept. 1984, at 763; 23 Tetrahedron Lett. 4259 ( 1982)):
• Resolution of racemic mixtures of oxazolones may be effected using chromatography or chiral supports under suitable conditions which are well known in the art; using fractional crystallization of stable salts of oxazolones with chiral acids; or simply by hydrolyzing the racemic oxazolone to the amino acid derivative and resolving the racemic modification using standard analytical techniques.
• 4-monosubstituted azlactones may be readily prepared by reduction of the corresponding unsaturated derivatives obtained in high yield from the condensation reaction of aldehydes, ketones, or imines with the oxazolone formed from an N-acyl glycine (49 J. Org. Chem. 2502 (1984); 418 Synthesis Communications ( 1984))
• the hydrogenation may be carried out using a
• This product may
• Chiral recognition is a process whereby individual chiral enantiomers display differential binding energies with an
• This agent may be attached to a surface to produce a chiral stationary phase (CSP) for chromatographic use or may be used to form
• the interaction of the enantiomeric R and S species with the CSP can be envisioned as a "three point interaction". This does not mean that three actual points of attachment or association are necessary, but rather that any three kinds of attractive or repulsive interactions within the diastereomeric complexes can serve to differentiate (“recognize”) the enantiomers. Greater differentiation (“recognition”) betwen the complexes is promoted by multiple combinations of attractive and/or repulsive interactions, including hydrogen bonding, ionic interactions, dipole interactions,
• Chiral oxazolones may be subjected to ring opening reactions with a variety of nucleophiles producing chiral molecules as shown below:
• R 1 and R 2 differ from one another and taken alone each signifies one of the following: alkyl including carbocyclic and substituted forms thereof; aryl, aralkyl, alkaryl, and substituted or heterocyclic versions thereof; preferred forms of R1 and R2 are the side chain substituents occurring in native polypeptides,
• carbohydrates pharmacophores, variants or mimetics of these, or any other side chain substituent which can be attached to a scaffold or a backbone to produce a desired interaction with a target system.
• the above ring-opening reaction can be carried out either in an organic solvent such as methylene chloride, ethyl acetate, dimethyl formamide (DMF) or in water at room or higher temperatures, in the presence or absence of acids, such as carboxylic, other proton or Lewis-acids, or bases, such as tertiary amines or hydroxides, serving as catalysts. If structure BYH contains nucleophilic functional groups which may interfere with the ring-opening
• a and B shown may be of a variety of structures and may differ markedly in their physical or functional properties, or may be the same; they may also be chiral or symmetric.
• a and B are preferably selected from:
• Proteins including structural proteins such as collagen, functional proteins such as hemoglobin, regulatory proteins such as the dopamine and thrombin receptors.
• Nucleotide probes (N 2-25) and
• oligonucleotides including all of the various possible homo and heterosynthetic combinations and permutations of the naturally occuring nucleotides, derivatives and variants containing synthetic purine or pyrimidine species or mimics of these, various sugar ring mimetics, and a wide variety of alternate backbone analogues including but not limited to phosphodiester, phosphorothionate, phosphorodithionate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioformacetal, methylene(methylimino), 3-N-carbamate, morpholino carbamate and peptide nucleic acid analogues.
• natural physiologically active carbohydrates such as including related compounds such as glucose, galactose. sialii acids, beta-D-glucosylamine and nojorimycin which are both inhibitors of glucosidase, pseudo sugars, such as 5a-carba-2-D-galactopyranose, which is known to inhibit the growth of Klebs
• a naturally occurring or synthetic organic structural motif is defined as meaning an organic molecule having a specific structure that has biological activity, such as having a complementary structure to an enzyme, for instance.
• This term includes any of the well known base structures of pharmaceutical compounds including
• beta-lactams such as pennicillin, known to inhibit bacterial cell wall biosynthesis
• dibenzazepines known to bind to CNS receptors, used as anti depressants
• polyketide macrolides known to bind to bacterial ribosymes, etc.
• a reporter element such as a natural or synthetic dye or a residue capable of photographic
• reactive groups which may be synthetically incorporated into the oxazolone structure or reaction scheme and may be attached through the groups without adversely interfering with the reporting functionality of the group.
• Preferred reactive groups are amino, thio, hydroxy, carboxylic acid, carboxylic acid ester, particularly methyl ester, acid chloride, isocyanate alkyl halides, aryl halides and oxirane groups.
• a macromolecular component such as a macromolecular surface or structures which may be attached to the oxazolone modules via the various reactive groups outlined above in a manner where the binding of the attached species to a ligand-receptor molecule is not adversely affected and the interactive activity of the attached functionality is determined or limited by the macromolecule.
• porous and non-porous inorganic macromolecular components such as, for example, silica, alumina, zirconia, titania and the like, as commonly used for various applications, such as normal and reverse phase chromatographic separations, water purification, pigments for paints, etc.
• porous and non-porous organic macromolecular components including synthetic components such as styrene-divinyl benzene beads, various methacrylate beads, PVA beads, and the like, commonly used for protein purification, water softening and a variety of other applications, natural components such as native and
• celluloses such as, for example, agarose and chitin, sheet and hollow fiber membranes made from nylon, polyether sulfone or any of the materials mentioned above.
• a and/or B may be a chemical bond to a suitable organic moiety, a hydrogen atom, an organic moiety which contains a suitable electrophilic group, such as an aldehyde, ester, alkyl halide. ketone, nitrile, epoxide or the like, a suitable nucleophilic group, such as a hydroxyl, amino, carboxylate, amide, carbanion, urea or the like, or one of the R groups defined below.
• a and B may join to form a ring or structure which connects to the ends of the repeating unit of the compound defined by the preceding formula or may be separately connected to other moieties.
• composition of this invention is defined by the following formula:
• a and B are as defined above and A and B are optionally connected to each other or to other compounds;
• X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
• R and R' are the same or different and each represents B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
• linear chain or branched chained alkyl groups means any substituted or unsubstituted acyclic carbon-containing compounds, including alkanes, alkenes and alkynes. Alkyl groups having up to 30 carbon atoms are preferred.
• alkyl groups include lower alkyl, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl; upper alkyl, for example, cotyl, nonyl, decyl, and the like; lower alkylene, for example, ethylene, propylene, propyldiene, butylene, butyldiene; upper alkenyl such as 1-decene, 1 -nonene, 2,6-dimethyl-5-octenyl, 6-ethyl5-octenyl or heptenyl, and the like; alkynyl such as 1 -ethynyl, 2-butynyl, 1-pentynyl and the like.
• alkynyl such as 1 -ethynyl, 2-butynyl, 1-pentynyl and the like.
• the ordinary skilled artisan is
• alkyl group may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group.
• Functional groups include but are not limited to hydroxyl, amino, carboxyl, amide, ester, ether, and halogen (fluorine, chlorine, bromine and iodine), to mention but a few.
• Specific substituted alkyl groups can be, for example, alkoxy such as methoxy, ethoxy, butoxy, pentoxy and the like, polyhydroxy such as 1,2-dihydroxypropyl, 1 ,4-dihydroxy-1 -butyl , and the like; methylamino, ethylamino, dimethylamino, diethylamino, triethylamino, cyclopentylamino, benzylamino, dibenzylamino, and the like; propanoic, butanoic or pentanoic acid groups, and the like; formamido, acetamido, butanamido, and the like, methoxycarbonyl, ethoxycarbonyl or the like, chloroformyl, bromoformyl, 1 ,1 -chloroethyl, bromo ethyl ,and the like, or dimethyl or diethyl ether groups or the like.
• alkoxy
• substituted and unsubstituted carbocyclic groups of up to about 20 carbon atoms means cyclic carbon-containing compounds, including but not limited to cyclopentyl, cyclohexyl, cycloheptyl, admantyl, and the like, such cyclic groups may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group.
• Such functional groups include those described above, and lower alkyl groups as described above.
• the cyclic groups of the invention may further comprise a heteroatom.
• R2 is cycohexanol.
• substituted and unsubstituted aryl groups means a hydrocarbon ring bearing a system of conjugated double bonds, usually comprising an even number of 6 or more (pi) electrons.
• aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, toluyl, xylenyl and the like.
• aryl also includes aryloxy, aralkyl, aralkyloxy and heteroaryl groups, e.g., pyrimidine, morpholine, piperazine, piperidine, benzoic acid, toluene or thiophene and the like.
• These aryl groups may also be substituted with any number of a variety of functional groups.
• functional groups on the aryl groups can be nitro groups.
• R2 can also represent any combination of alkyl, carbocyclic or aryl groups, for example, 1-cyclohexylpropyl, benzylcyclohexylmethyl, 2-cyclohexylpropyl, 2,2-methylcyclohexylpropyl, 2,2methylphenylpropyl, 2,2-methylphenylbutyl, and the like.
• G is a chemical bond or a connecting group and G may be different in adjacent n units; and e. n is equal to or greater than 1.
• G is a chemical bond
• Y includes a terminal carbon atom for attachment to the quaternary nitrogen
• R and R' are the same, A and B are different and one is other than H or R
• a and/or B may be a chemical bond to a suitable organic moiety, a hydrogen atom, an organic moiety which contains a suitable
• electrophilic group such as an aldehyde, ester, alkyl halide, ketone, nitrile, epoxide or the like, a suitable nucleophilic group, such as a hydroxyl, amino, carboxylate, aminde, carbanion, urea or the like, or one of the R groups defined below.
• a and B may join to form a ring or structure which connects to the ends of the repeating unit of the compound defined by the preceding formula or may be separately connected to other moeities.
• composition of the invention is defined by the structure
• At least one of A and B are as defined above and A and B are optionally
• X and Y are the same or different and each represents a chemical bond or one or more atoms of carbon, nitrogen, sulfur, oxygen or combinations thereof;
• R and R' are the same or different and each is selected from the group consisting of A, B, cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic, aryl and substituted or heterocyclic derivatives thereof, wherein R and R' may be different in adjacent n units and have a selected stereochemical arrangement about the carbon atom to which they are attached;
• G is a connecting group or a chemical bond which may be different in adjacent n
• connection to the carbonyl group and G-B is other than an amino acid residue or a peptide; (3) if n is 1 and X, Y, and G each is a chemical bond, A and B each is other than a chemical bond, an amino acid residue or a peptide; and (4) if n is 1 , either X or A has to include a CO group for direct, connection to the NH group.
• compositions may be used to mimic various compounds such as peptides, nucleotides,
• At least one of A and B represents an organic or inorganic
• macromolecular surface functionalized with hydroxyl, sulfhydryl or amine groups.
• preferred macromolecular surfaces include ceramics such as silica and alumina, porous or nonporous beads, polymers such as a latex in the form of beads, membranes, gels, macroscopic surfaces, or coated versions or composites or hybrids thereof. A general structure of a chiral form of these materials is shown below:
• group A or B in the above structure is an aminimide moiety.
• This moiety may be introduced, for example by reacting the oxazolone with an asymmetrically substituted hydrazine and alkylating the resulting hydrazide, (e.g., by reaction with an alkyl halide, or epoxide).
• An example of such a surface is shown below.
• A, R and R' are as described above and q is zero or 1.
• Y is a chemical bond This ring is useful for preparing the desired oxazolone derivatives.
• a further embodiment of the invention exploits the capability of oxazolones with suitable substituents at the 2-position to act as reactive agents. Appropriate substituents include vinyl groups, which make the
• oxazolone a Michael acceptor, haloalkyl and alkyl sulfonate ester and epoxide groups.
• Michael addition to the double bond of a chiral 2-vinyloxazolone followed by a ring opening reaction results in a chiral conjugate structure.
• This general reaction scheme illustrated for the case of a 2-vinyl azlactone derivative, is as follows:
• X can represent a sulfur, oxygen or nitrogen atom
• Y can represent a sulfur, oxygen, or nitrogen atom
• substituents A and B, as described above, may adopt a variety of structures, differing markedly in their physical or functional properties or being the same, may be chiral or achiral, and may be preferably selected from amino acids, oligopeptides, polypeptides and proteins, nucleotides, oligonucleotides, ligand mimetics,
• the Michael reaction described above is usually carried out using stoichiometric amounts of the nucleophile, AXH, and the oxazolone in a suitable solvent, such as toluene, ethyl acetate, dimethyl formamide, an alcohol, or the like.
• a suitable solvent such as toluene, ethyl acetate, dimethyl formamide, an alcohol, or the like.
• the product of the Michael addition is preferably isolated by evaporating the reaction solvent in vacuo and purifying the material isolated using a technique such as recrystallization or chromatography. Gravity- or pressurechromatography, on one of a variety of supports, e.g., silica, alumina, under normal- or reversed-phase conditions, in the presence of a suitable solvent system, may be used for purification.
• the selectivities of the Michael and oxazolone ring-opening processes impose certain limitations on the choice of AXH and BYH nucleophiles shown above
• nucleophiles of the form ROH tend to add primarily via the ring-opening reaction, and usually require, acidic catalysts (e.g., BF3); thus, X should not normally be oxygen.
• acidic catalysts e.g., BF3
• X should therefore not be NH.
• Secondary amines readily add to the double bond under appropriate reaction conditions, but manv can also cause ring-opening; accordingly, X or Y can be N, provided A or B are not hydrogen.
• Nucleophiles of the form RSH will exclusively add via ring-opening if the sulfhydryl group is ionized, i.e. , in the presence of a (non-oxazolone-reactive) base strong enough to remove the SH proton; on the other hand, such sulfur containing nucleophiles will exclusively . add via Michael reaction under non-ionizing, i..e., neutral or mildly acidic conditions. During the Michael addition, it is important to limit the presence of hydroxylic species in the reaction mixture (e.g., moisture) to avoid ring-opening side-reactions.
• AXH can be a secondary amine or a thiol
• BYH can be a primary or secondary amine, a thiol, or an alcohol.
• A is a substituent selected from the foregoing list and BXH comprises an organic or inorganic macromolecular surface, e.g., a ceramic, a porous or nonporous bead, a polymer such as a latex in the form of a bead, a membrane, a gel or a composite, or hybrid of these; the macromolecular surface is functionalized with hydroxyl, sulfhydryl or amine groups which serve as the nucleophiles in the ring-opening reaction.
• a ceramic e.g., a ceramic, a porous or nonporous bead, a polymer such as a latex in the form of a bead, a membrane, a gel or a composite, or hybrid of these
• the macromolecular surface is functionalized with hydroxyl, sulfhydryl or amine groups which serve as the nucleophiles in the ring-opening reaction.
• reaction sequence is carried out under conditions similar to those given for the nonpolymeric cases; purification of the final product involves techniques used in the art to purify supports and other surfaces after derivatization, such as washing, dialysis, etc.
• the result of this reaction sequence is a structure such as the one shown below:
• oxazolones posessing reactive groups at the 2-position may be producedvia suitable acylation reactions, as shown for the specific example of a benzoyl chloride oxazolone derivative containing a reactive p-benzyl group:
• ring-opening addition with BYH may be carried out and followed by reaction with an appropriate AXH group, e.g. a primary amine, to give the product shown:
• electrophile competes with the oxazolone ring for the nucleophile BYH, a suitable protecting group, shown as B1 below, may be used to block the benzylic electrophile.
• the protected group is removed using standard techniques (e.g., if the protecting group is Boc, it is removed by using dilute TFA in CH2Cl2), and the resulting product is then reacted with an appropriate electrophile, e.g., A-CH 2 -Br, thus introducing substituent A into the molecule.
• an appropriate electrophile e.g., A-CH 2 -Br
• oxazolone-derived building blocks possessing functional groups capable of establishing predictable binding interactions with target molecules and using synthetic techniques such as those broadly described above to effect catenation (linking) of the building blocks, it is possible to construct sequences of oxazolone-derived subunits mimicking selected native oligomers or polymers, e.g. peptides and polypeptides, oligonucleotides, carbohydrates as well as any other biologically active species whose three dimensional binding geometry can be mimicked by various combinations of oxazolone derivitive containing scaffolds and side chains. This may be accomplished using a wide variety of side chain recognition group substituents, including, but not limited to the substituents found in the side chains of naturallv occuring amino acids; purine and pyrimidine groups as well as
• a generic "base” (purine or pyrimidine) group into an oxazolone-derived scaffold connected via a carbonyl- terminal spacer. While the example uses a base as the recognition group, it should be kept in mind that this group can be any group which will provide the desired end product, such as, for example, a carbohydrate, a pharmacophore moiety or a designed synthetic recognition element.
• the following specific sequence illustrates the construction of a ligand having bases attached to every other oxazolone-derived module.
• species may be constructed with bases attached to each sequential oxazolone module.
• the substituents on the recognition group-bearing modules may all posess the same chirality, may have regularly alternating chirality or may be racemic, depending on the desired structural relationships between the individual recognition groups and between each
• the assembled ligand may be subjected to
• oxazolone modules are catenated via ring-opening nucleophilic attack by the amino group of a (chiral) alpha,alpha-disubstituted amino acid derivative, usually a lithium salt; the resulting adduct is subsequently recyclized to form a terminal oxazolone (with retention of chirality).
• This oxazolone is then subjected to another nucleophilic ring-opening catenation reaction sequence, producing a growing chiral chain, as shown below. This procedure is repeated until the desired polymer is obtained.
• each member of the substituent pairs R 1 and R 2 , R 3 and R 4 , R 5 and R 6 and R n and R n- 1 differs from the other and, taken alone, each signifies alkyl, cycloalkyl, or substituted versions thereof, aryl, aralkyl or alkaryl, or substituted and heterocyclic versions thereof; these substituent pairs can also be joined into a carbocyclic or heterocyclic ring; preferred forms of R1 and R2 are the side chain substituents occuring in native polypeptides, oligonucleotides, carbohydrates, pharmacophores, variants or mimetics of these, or any other side chain substituent which can be attached to a scaffold or backbone to produce a desired interaction with a target system. ; X represents an oxygen, sulfur, or nitrogen atom; and A and B are the substituents described above.
• a chiral oxazolone derivative containing a blocked terminal amino group may be prepared from a blocked, disubstituted dipeptide, that was prepared by standard techniques known to those skilled in the art, as shown:
• B 1 is an appropriate protecting group, such as Boc
• acylation is followed by deblocking, using standard amine deprotection techniques compatible with the overall structure of the amide (i.e., the amine protecting group is reaactively orthogonal with respect to any other protecting or functional groups that may be present in the molecule), and the resulting amino group is used for reaction with a new bifunctional oxazolone, generating a growing chiral polymeric structure, as shown below:
• Y is a linker, such as, for example, a functionalized aryl group
• X is a nitrogen of suitable structure, an oxygen or a sulfur atom
• each member of the substituent pairs R 1 and R 2 , R 3 and R 4 , R n - 1 and R n differs from the other and, taken alone, each signifies alkyl, carbocyclic, or functionalized versions thereof, aryl, aralkyl or alkaryl or
• R1 and R2 are the side-chain
• substituents occuring in native polypeptides, oligonucleotides, carbohydrates, pharmacophores, variants or mimetics of these, or any other side-chain substituent which can be attached to a scaffold or a backbone to produce a desired interaction with a target system;
• substituent R can also be part of a carbocyclic or
• A is a substituent as described above;
• C. is a substituent selected from the set of structures for A; and
• B 1 is a blocking or protecting group.
• a preliminary step may be carried out with a suitable amino acid derivative as shown below, prepared via standard synthesis.
• individual module may carry a recognition group
• the recognition group-bearing modules can be separating from each other by one or more modules which do not carry recognition groups.
• These intervening modules may be achiral, alpha, alpha- disubstituted or, in cases where chirality is not important, they may be standard hydrogen-bearing alpha amino acid modules. These may serve as spacers, to regulate the periodicity of substitution or may serve various other co- functions, such as limiting the flexibility of the ligand.
• the substituents on the recognition group-bearing modules may be constructed to all posess the same chirality, may have regularly alternating chirality or may be racemic,
• modular "sub assemblies" capable of conferring higher order structural properties may be pre- constructed and assembled together using these same reaction sequences in a manner which allows control of the higher order, structure. This is illustrated for the case of a polymer formed witha repeating pattern of alternating modules of the type:
• This polymer will form 3-10 helices, driven by the conformational restrictions imposed by the repetitive viscinal disubstitution. This triadic periodicity results in the formation of a helical superstructure which has charged sulfonate groups lined up regularly along one side of the helix:
• alpha'disubstituted amino acid may be inserted in the polymer backbone as shown below;
• This process may be repeated, if desired, at each step in the synthesis where an oxazolone ring is produced.
• the bifunctional species used may be the same or different in each individual step of the synthesis.
• dipolar aprotic solvents e.g., dimethyl formamide, DMF, dimethyl sulfoxide, DMSO. N-methyl pyrolidone, etc.
• chaotropic agents e.g., urea
• HNu 1 -Z-Nu 2 H represents a structure containing two differentially reactive nucleophilic groups, such as methylamino-ethylamine, 1 - amino propane-3-thiol, and so on; groups Nu 1 , Nu 2 , Nu 3 and Nu 4 need not be identical and Z is a linker structure as described above.
• Structure HNu 1 -Z-Nu 2 H may contain two nucleophilic groups of differential reactivity, as stated above, or if Nu 1 and Nu 2 are of comparable reactivity one of the nucleophilic groups is protected to prevent it from competing with the other and is deprotected selectively following acylation; protecting groups commonly used in the art of peptide synthesis (e.g., for the nucleophilic groups such as amino, hydroxyl, thio, etc.) are useful in the protection of one of the Nu substituents of the structure HNu 1 -Z-Nu 2 H .
• oligomers are highly useful biochemically because of their structural similarity to biological scaffolds, particularly polypeptide scaffolds.
• the substituents R can be chosen to tailor the steric, charge or hydrophobicity characteristics of the oligomer such that a versatile mimetic results.
• nucleophilic ring-opening of asymmetrically disubstituted oxazolones may be utilized to introduce a chiral residue or sequence in selected positions in peptides or proteins to produce hybrid molecules with improved hydrolytic and enzymatic stability properties.
• the reaction of a chiral azlactone with the amino terminus of a synthetic tripeptide attached to a Merrifield support is shown below.
• the oxazolone used in the above aminolysis may contain a blocked amino terminus which, after the
• polypeptide synthesis may be continued, if desired, using standard peptidesynthesis techniques.
• the structure below illustrates a short polymer containing nine subunits prepared as above and detached from the solid phase synthesis support.
• each of the R groups signifies alkyl, carbocyclic, or substituted versions thereof; aryl, aralkyl, alkaryl, or substituted versions thereof, including heterocyclic versions; the R groups can also define a carbocyclic or heterocyclic ring; preferred structures for the R groups in this application are those mimicking the structures of the side-chains of naturally-occurring amino acids.
• disubstituted chiral azlactones may be utilized to introduce a variety of novel, unnatural residues into peptides or proteins using the following multistep procedure:
• each of the R groups signifies alkyl, cycloalkyl, aryl, aralkyl or alkaryl, or substituted or suitably heterocyclic versions thereof; the R groups may also define a carbocyclic or heterocyclic ring; preferably the R groups are structural mimetics of the side-chains of naturally-occurring amino acids.
• the reactions shown in steps a-d above are carried out using the conditions described above for related cases. Couplings of peptide segments on a support or in solution are carried out using the traditional
• the oxazolone peptide produced in step (b) above may be reacted with a variety of bifunctional nucleophilic
• the above acylation product may be coupled with a peptide to produce novel chiral hybrids; two coupling routes may be used.
• A is a group which can be condensed with an amino group, the condensation reaction is used for coupling. For example, if A is a carboxyl group,
• condensation with a peptide amine using DCC or a similar reagent produces the desired product.
• Reaction conditions and suitable (orthogonal) protecting groups well-known in the art, such as those described above, are expected to be useful.
• A is a suitable nucleophilic group (e.g., hydroxyl, amino, thio, etc.) it may be used to open a peptide oxazolone containing a protected amino terminus.
• residues may be attached to or inserted into peptide chains using oxazolones with reactive groups attached at the 2-position of the ring. This may be accomplished in either of two ways, as illustrated below for the case of 2-alkenyl azlactones.
• Oxazolone-derived mimetics can be produced, using the oxazolone-forming and catenation chemistries outlined above, so as to produce backbones having natural or synthetic recognition groups, such as purine or pyrimidine bases, carbohydrates, pharmacophores. etc., attached as side chain substituents via appropriate spacers, i.e. R or R' in the general structural formulas described above represents a recognition group-spacer sub assembly.
• guanidine, 5-fluorouricil(5FU)) onto oxazolone backbones to form an antisense strand, or nucleotide mimetic The resulting linkages and backbones are superior in their resistance to base, acid and proteolytic/phospholytic activity.
• the bases can be attached using appropriate spacers and the stereochemistry and periodiocity of substitution geometry and rigidity of the backbone scaffold can be designed such that the bases are arrayed and projected in space to provide the optimum arrangement and orientation of the bases to hybridize with their targeted counterparts. Specific examples of the synthesis of oxazolone-derived oligonucleotide mimetics are given below.
• carbohydrates increasingly are being viewed as the components of living systems with the enormously complex structures required for the encoding of the massive amounts of information needed to orchestrate the processes of life, e.g., cellular recognition, immunity, embryonic development, carcinogenesis and cell-death.
• This information is contained and utilized through highly specific binding interactions mediated by the detailed three dimensional-topological form of the specific carbohydrate. It is of great value to be able to arrange and to connect these moities in various arravs in a controlled manner. This may be done either by connecting carbohydrate recognition groups along an oligomeric backbone, as done by for random vinyl copolymers containing functionalized sialic acid groups, which were shown to inhibit hemagluttinin binding (J. Am. Chem.
• Oxazolone-derived carbohydrate mimetics may be synthesized from carbohydrate modules containing functional groups, such as carboxylic acid halides, carboxylic acids, alcohols, thiols, amines, aldehydes, ketones, together with any other groups which are compatible with the oxazolone-forming and catenating reactions outlined above, thus allowing the carbohydrates to be attached to a basic scaffold, or to be arrayed along a backbone in a precise controlled manner. Examples for the synthesis of such
• carbohydrate modules are outlined below.
• the objectives of any drug discovery program are:
• a biologically active compound for example a protein or polypeptide
• a solid support such as a resin or glass surface.
• linked compounds show diverse inhibitory activity, an indication that the ability of linked molecules to retain its binding properties despite the partial loss of mobility.
• oxazolone-derived molecular building blocks may be utilized to construct new macromolecular structures capable of recognizing specific molecules ("intelligent macromolecules").
• the "intelligent macromolecules” may be represented by the following general formula: P - C - L - R where , R is a structure capable of molecular
• L is a linker
• P is a macromolecular structure serving as a supporting platform
• C is a polymeric structure serving as a coating which surrounds P.
• Structure R may be a native ligand or a biological ligand-acceptor or a mimetic thereof, such as those described above.
• Linker L may be a chemical bond or one of the linker structures listed above, or a sequence of subunits such as amino acids, aminimide monomers, oxazolone-derived chains of atoms, etc.
• Polymeric coating C may be attached to the supporting platform either via covalent bonds or "shrink wrapping," i.e. the bonding that results when a surface is subjected to coating polymerization is well known to those skilled in the art.
• This coating element may be
• the controlled microporosity gel may be engineered to completely fill the porous structure of the support platform.
• the polymeric coatings may be constructed in a controlled way by carefully controlling a variety of reaction parameters such as the nature and degree of coating crosslinking, polymerization initiator, solvent, concentration of reactants, and other reaction conditions, such as temperature. agitation, etc., in a manner that is well known to those skilled in the art.
• the support platform P may be a pellicular material having a diameter (dp) from 100 Angstroms to 1000 microns, a latex particle (dp 0.1 - 0.2 microns), a microporous bead (dp 1 - 1000 microns), a porous membrane, a gel, a fiber, or a
• polymeric materials such as silica, polystyrene, polyacrylates, polysulfones, agarose, cellulose, etc. or synthetic oxazolone -containing polymers such as those described below.
• any of the elements P, C, L, or R containing an oxazolone-derived structure is derived from a form of the element containing a precursor to the oxazolone-derived structure.
• the multisubunit recognition agents above are expected to be very useful in the development of targeted therapeutics, drug delivery systems, adjuvants, diagnostics, chiral selectors, separation systems, and tailored catalysts.
• surface refers to either P, P linked to C or P linked to C and L as defined above.
• azlactone ring-opening addition reaction may be used to directly produce a wide variety of chiral vinyl monomers. These may be polymerized or
• copolymerized to produce chiral oligomers or polymers and may be further crosslinked to produce chiral beads,
• Other useful monomers which may be used to produce chiral crosslinkable polymers, may be produced by nucleophilic opening of a chiral 2-vinyl oxazolone with a suitable amino alkene or other unsaturated nucleophile.
• Vinyl polymerization and polymer-crosslinking techniques are well-known in the art (see, e.g., U.S. Patent No. 4,981,933) and are applicable to the above preferred processes .
• Solid phase libraries i. e. , libraries in which the ligand-candidates remain attached to the solid support particles used for their synthesis
• an enzyme e. g.
• combinatorial library is the encoded combinatorial library, which involves the synthesis of a unique chemical code (e.g. , an
• oligonucleotide or peptide that is readily decipherable (e.g. , by sequencing using traditional analytical methods), in parallel with the synthesis of the ligandcandidates of the library.
• the structure of the code is fully descriptive of the structure of the ligand and used to structurally characterize biologically active ligands whose structures are difficult or impossible to elucidate using traditional analytical methods. Coding schemes for construction of combinatorial libraries have been described recently (for example, see S. Brenner and R.A. Lerner, Proc. Natl. Acad. Sci. USA 89, 5381 ( 1992); J.M. Kerr, et al. J. Am. Chem. Soc. 115, 2529 (1993)). These and other related schemes are contemplated for use in constructing encoded combinatorial libraries of oligomers and other complex structures derived from oxazolones.
• each ligand-candidate is attached to one or more solid-phase synthesis support particles and each such particle contains a single ligand-canditate type.
• This library can be constructed and screened for biological activity in just a few days. Such is the power of combinatorial chemistry using oxazolone modules to construct new molecular candidates.
• a suitable solid phase synthesis support e.g. , the
• chloromethyl resin of Merrifield is split into three equal portions.
• Each amino acyl resin portion is treated with an acid solution such as neat trifluoroacetic acid (TFA), or preferably, a 1 : 1 mixture of TFA and CH 2 CI 2 , to remove the t-Bu blocking group.
• TFA trifluoroacetic acid
• the resulting acyl amino acid resin is treated with ethyl chloroformate as described above producing the oxazolone resin.
• each of the resin portions is coupled to a different glycine protected as t-butyl ester using the conditions described above; the amide product is deprotected as described above, for each of the resin portions and cyclized to the oxazolone using the reaction with ethyl chloroformate .
• beads each type containing a single oxazolonederived tripeptide analog linked to the support via a succinoyl linker; this linker may be severed using acidolysis to produce a "solution-phase" library of peptides whose N-terminus is succinoylated.
• polysaccharide structural motifs incorporating oxazolone-derived structures are contemplated including but not limited to the following.
• carbohydrate chemistry describes numerous sugars of variety of sizes with selectively blocked functional groups, which allows for selective reactions with oxazolone and related species producing the desired products (see
• ßglucosides can be reacted with unhindered alcohols to produce ßglucosides using well-known experimental conditions.
• the resulting sugar blocked at all positions except position 2.
• acid catalyst such as BF 3 in a suitable inert organic solvent to open a suitable oxazolone using the reaction conditions described above, e.g. , in the absence or presence of a Lewis (e.g., EtOAC, dioxane, etc.).
• a Lewis e.g., EtOAC, dioxane, etc.
• the sugar that results from reaction of D-glucose with benzaldehyde can be readily blocked at positions 1 and 6, by sequential reactions with an alcohol in the presence of acid, and tritylation using techniques well known in the art of carbohydrate chemistry.
• the resulting sugar, with position 3 unblocked can be used selectively as described above to derivatize a desired oxazolone structure.
• a suitable oxazolone can also be ring-opened by a sugar containing reactive amino substituents, i. e. , an aminosaccharide or polyaminosaccharide.
• reaction with muramic acid is expected to proceed as follows.
• tetrasaccharide scaffold supports peptidomimetic structures derived from oxazolones in a geometrically defined manner.
• oxazolonederived structures are contemplated including, but not limited to, the following.
• the (S)-isomer of p-hydroxyphenylglycine (oxfenacine) is an effective therapeutic agent for promoting the oxidation of carbohydrates when this process is depressed by high fatty acid utilization levels (such as occurs in ischemic heart disease), and is also an important chiral intermediate in the production of penicillin,
• Oxfenacine is prone to racemization, and the assay for chiral purity described in this example therefore represents a useful development and quality-control tool.
• a solution of the diastereomeric amides was prepared in methylene chloride at a concentration of 7 mg/ml. This solution was injected into a DuPont Model 830 liquid chromatograph equipped with a detector set at 254 nm using a 20 ul loop valve injection system. The sample was chromatographed on a 25 cm ⁇ 0.4 cm stainless steel HPLC column packed with 5_ Spherisorb S5W silica gel using a 98/1/1 cyclohexane/n-butanol/isopropanol mobile phase at a flow rate of 0.9 ml/min.
• the enantiomeric amide conjugates were then quantitated using a calibration curve generated with a series of synthetic mixtures containing varying ratios of the two pure enantiomers.
• the pure Lisomer was purchased from Schweizerhall Inc.
• the pure Disomer was prepared from the commercially available D,Lracemate obtained from MTM Research
• the resulting product was dried in a vacuum oven set for 30" and 60°C to yield 4.87 g functionalized silica.
• the bonded phase was packed into a 25 cm ⁇ 0.46 cm stainlesssteel HPLC column from methanol, and successfully used to separate a series of mandelic acid derivatives using standard conditions.
• triethylamine was added dropwise over a 10-min. period and the mixture was stirred at room temperature until gas evolution ceased ( 1.5 hours).
• the mixture was then cooled, the silica collected on a Buechner filter and washed with 100 ml benzene.
• the wet cake was reslurried in 100 ml methanol and refiltered a total of four times.
• the product was dried in a vacuum oven set for 30" and 60°C to give 9.72 g functionalized silica.
• the bonded phase was packed into a 25 cm ⁇ 0.46 cm stainless-steel HPLC column from methanol and successfully used to separate a series of pi-acceptor amine derivatives using standard conditions described in the Chromatography Catalog distributed by Regis Chemical, Morton Grove, III.
• This example teaches the synthesis of a competitive inhibitor for human elastase based on the structure of known N-trifluoroacetyl dipeptide analide inhibitors - see, e.g., 107 Eur. J. Biochem. 423 (1980); 162 L Mol. Biol. 645 (1982) and references cited therein.
• oxazolone The product was purified by recrystallization from acetone at -30-C.
• This mimetic is useful as a competitive inhibitor for proteases inhibited by pepstatin.
• the Boc-protected lithium salt prepared as described below simultaneously converted to the acid form and deprotected by treatment with acid under standard deprotection conditions.
• 5.17 g (0.01 mol) of N-isovaleryl(S)-2-methy derivative added to 100 ml dry acetonitrile, stirred at room temperature and 3.17 g (0.01 mol) of the valyl-(S)-4-methyl-4-isopropyl-5-oxazolone was added with cooling. Once addition was complete, the mixture was heated to reflux and held at reflux for 1 hour.
• N-isovaleryl-(S)-2-methylvalyl-(3S,4S)-statyl(S)-2-methylalanyl-(3S,4S)-statine useful as a pepstatinmimetic competitive inhibitor for aspartyl proteases which are inhibited by pepstatin (see, 23 J. Med. Chem. 27 (1980) and references cited therein).
• Boc-protected (3S,4S)-statine, [(3S,4S)-4-amino3-hydroxy-6- methylheptanoic acid] was produced from the commercially available amino acid, coupled with 2methylalanine using standard peptide synthesis methods and converted to the lithium salt using the method
• FTIR shows strong azlactone CO band in the 1820 cm - 1 region.
• 2-(S)-methylvaline was prepared from (S)-valine by the method described by Kolbe and Barth (Liebi gs Ann. Chem. at 1668 ( 1983)), and was acylated with isovaleryl chloride using standard acy ation methods to produce Nisovaleryl-(S)-methylvaline, this was
• This example teaches the synthesis of a competitive inhibitor for the HIV protease, based on the insertion of a chiral azlactone residue into a strategically important position in the scissile position of the known substrate, Ac-Ser-Leu-Asn-Phe-Pro-Ile-ValOMe. See, e.g., 33 J. Med. Chem. 1285 ( 1990) and references cited therein.
• the sidechain blocking groups are subsequently removed using standard peptide deprotection techniques to yield the product MeO-D-Ser-D-Leu-D-Asn-NH-CO-(S)-Phe-[Me]-NH-COCH2-CH2-L-N-Pro-L-Ile-L-Val-OMe, useful as a competitive inhibitor for the HIV protease.
• This example teaches the synthesis of another competitive inhibitor for the HIV protease.
• the phenyl substituent is replaced with a uracil derivative.
• 3-methyluracil-5-carboxylic acid was treated with HCL and CH 2 O using standard chloromethylation conditions to yield 3-methyl-5chloromethyluracil in 52% yield, following standard work-up and recrystallization from ethyl acetate.
• reaction mixture is stirred at 0°C for 1 hour and then allowed to come to room temperature.
• the mixture is then stirred at room temperature under a nitrogen blanket for 7 days.
• the solvent is removed under vacuum and the water is removed by freeze drying to give the product (V).
• (V) is useful as a probe for the study of receptor proteins that bind morphine and its derivatives.
• the mixture is heated to 70°C and stirred at this
• the dopamineconnected catechol functionality is a photographic developer, capable of photographic
• This material is dissolved in methanol (400 mL) to which water (25 mL) and p-toluenesulfonic acid (0.5 g) is added. The mixture is heated at reflux to exhaustion of the acetal. The reaction mixture is concentrated in vacuo and the residue partitioned between THF and an aqueous solution of sodium bicarbonate (10 % w/v, 300 mL). The aqueous phase is extracted with THF and the combined orgaincs are dried (sat'd aq NaCl, MgSO 4 ), filtered and concentrated to afford, after recrystallization, 6-benzoylamido-9-(4-oxobutyl)purine ( 16.38 g, 0.053 mole, 84%).
• Triethylamine (0.101 g, 1.0 mmol) is added to a solution of 2-phenyl-5-oxazolone ( 1.61 g, 10 mmol) and 6- benzoylamido-9-(4-oxobutyl)purine ((3.09 g, 10 mmol) in benzene (20 ml).
• the resultant mixture is heated to 50°C for 10 minutes and, after cooling to room temperature, the solvent is removed in vacuo.
• the residual pasty mass is triturated with ethanol to afford a solid which is subsequently
• a suspension of the adeninyl oxazolone (4.52 g, 10 mmol) and 10% palladium on carbon (106 mg, 1 mol%) in ethyl acetate ( 100 mL) is sparged with dry hydrogen gas until the exocyclic methylene is fully reduced (1 equivalent).
• the catalyst is removed by filtration through a pad of celite and the filtrate is concentrated.
• the residue is dissolved in tetrahydrofuran (100 mL) and aq NaOH (1.0 M, 100 mL), tetra-n-butylammonium hydroxide (0.26 g, 1.0 mmol) and quinine (0.324 g, 1.0 mmol) was added.
• the Erlenmeyer products also may be left unreduced to provide an alternative scaffolding from which to present the recognition groups. Whereas this provides a "flat" structure it will also provide a different spacing and presentation of those groups. Shown below is an experimental sequence to provide the seminal units for such a molecule.
• adeninyl oxazolone (2.84 g, 6.3 mmol) is dissolved in methanol (10 mL) and glycine lithium salt (0.77 g, 9.45 mmol)) is added. The mixture is warmed at 50°C for three hours.
• a three neck round-bottom flask is charged with 5 mL of a suitable solvent such as CH 2 CI 2 and 337 mL (3.9 mmol, 1.2 equiv) oxalyl chloride.
• a suitable solvent such as CH 2 CI 2 and 337 mL (3.9 mmol, 1.2 equiv) oxalyl chloride.
• the solution is stirred and cooled at -60 °C as 460 mL (505 mg, 6.5 mmol, 2 equiv) of DMSO in 5 mL dichloromethane is added dropwise at a rapid rate. After 5 min, compound 1 (1 g, 3.23 mmol, 1.0 equiv) is added dropwise over 10 min maintaining the temperature at -60 °C.
• a three neck round-bottom flask is charged with 10 mL of a suitable solvent such as CH 2 CI 2 and 540 mL (6.2 mmol, 1.2 equiv) oxalyl chloride.
• a suitable solvent such as CH 2 CI 2 and 540 mL (6.2 mmol, 1.2 equiv) oxalyl chloride.
• the solution is stirred and cooled at -60 °C as 740 mL (810 mg, 10.4 mmol, 2 equiv) of DMSO in 5 mL dichloromethane is added dropwise at a rapid rate.
• compound 6 (1 g, 51.8 mmol, 1.0 equiv
• triethylamine 7.2 mL, 51.8 mmol, 10 equiv
• TMSC1 (1.55 g, 14.2 mmol, 5.5 equiv) followed by triethylamine (2.9 mL, 20.7 mmol, 8 equiv).
• the reaction mixture is stirred at room temperature for 6 h. Water is added to quench the reaction. The organic layer is washed with water and saturared NaCl and dried over anhydrous magnesium sulfate. The filtered solution is concentrated by rotary evaporation to otain the silylated product 9 (1.3 g, 91 %).
• Da-aminobenzylpenicillin, methyl ester (36.3 g, 99.9 mmol, synthesized from the reaction of D(-)- a-aminobenzylpenicillin in as solution of anhydrous methanol in the presence of a resin-supported super acid, such as ⁇ afion) and 4-(diethoxymethyl)benzaldehyde (21.2 g, 101.8 mmol) in an anhydrous solvent such as THF or methanol (400 mL) under an inert atmosphere such as argon or nitrogen is stirred, typically overnight, until the imine intermediate is formed and the starting reagents consumed as shown by thin layer chromatography (TLC). The reaction mixture is cooled to 0°C .
• reaction mixture is cooled to room temperature, diluted in a suitable solvent such as methylene chloride or diethyl ether, extracted with saturated aqueous ⁇ aHCO 3 (2 ⁇ 100 mL) followed by brine (1 ⁇ 100 mL) and dried over Na 2 SO 4 .
• a suitable solvent such as methylene chloride or diethyl ether
• saturated aqueous ⁇ aHCO 3 (2 ⁇ 100 mL) followed by brine (1 ⁇ 100 mL) and dried over Na 2 SO 4 .
• the solvent is removed on a rotary evaporator to afford an off white solid (7.31 g, 89%). A portion is recrystallized to yield a sample for analysis.
• dibenzosuberenone (12.5 g, 60.6 mmol) in an appropriate anhydrous solvent such as THF (100 mL) is added dropwise with stirring over a period of 30 minutes. Stirring continues at 0 °C for another two hours. The reaction is quenched with the addition of water (50 mL). The solvent is partially evaporated by rotary evaporation. The residual is dissolved in a suitable solvent such as methylene chloride or diethyl ether, extracted with saturated aqueous NaHCO 3 (2 ⁇ 200 mL) followed by brine (1 ⁇ 100 mL), and dried over anhydrous Na 2 S O 4 . The organic solvent is concentrated by rotary evaporation to afford 39 g of a colored oil.
• THF 100 mL
• the crude material is purified with column chromatography on a suitable stationary phase such as normal phase silica gel and eluted with an appropriate mobile phase such as hexanes / ethyl acetate mixtures, to afford the desired compound (15.7 g, 85%). A portion is repurified to yield a sample for analysis.
• a suitable stationary phase such as normal phase silica gel and eluted with an appropriate mobile phase such as hexanes / ethyl acetate mixtures
• a 10 x molar excess of aqueous 1.0 ⁇ ⁇ Cl is added, and stirring continues at 50 °C for 4 hours.
• the solvent is partially evaporated by rotary evaporation.
• the residual is dissolved in a suitable solvent such as methylene chloride or diethyl ether, extracted with saturated aqueous ⁇ a ⁇ CO 3 (100 mL) to neutralize the acid, followed by brine ( 100 mL), then dried over MgSO 4 .
• the solvent is removed on a rotary evaporator and vacuum pump to yield a solid (8.16 g, 80%). A portion is recrystallized to afford a sample for analysis.
• This example describes preparation of a coating by a ring-opening reaction followed by Michael-addition.
• NMR nuclear magnetic resonance
• FTIR Fourier transform infrared reflection
• FTIR azlactone CO band at 1820 cm - 1 absent; strong amide bands present in 1670 - 1700 cm - 1 region.
• This example describes preparation of an affinity coating from compound (III) as prepared in the previous example.
• the silica contained the following attached groups:
• Bovine Serum Albumin in the accompanying instructions (Technical Note No. 4151) from Chromatochem Inc., Missoula, MT.
• the measured capacity of the packing was 12 mg IgG per ml of column volume.
• a suitable experimental procedure is as follows.
• the azlactone-functional support is slurried in a suitable solvent, such as CHCl 3 , and cooled to 0°C.
• An amount of the bifunctional nucleophile equivalent on a molar basis to the total number of surface azlactone groups present, is dissolved in the same solvent and added with shaking.
• the mixture is then shaken at 0°C for 6 hours, allowed to come to room temperature, and shaken at room temperature overnight.
• the support is collected by filtration, washed with fresh solvent, re-slurried in an appropriate solvent and one equivalent of vinylazlactone, dissolved in the same solvent, is added thereto.
• the mixture is then shaken, heated to 70°C and held at this temperature for 12 hours. At the end of this time, the mixture is cooled and the support collected by filtration.
• the support is then washed thoroughly with fresh solvent and dried in vacuo.
• EXAMPLE 26 is
• the functional beads prepared as above are suspended in pH 7.5 aqueous phosphate buffer.
• a solution of protein A (Repligen) in 10 mM phosphate buffer (pH 7.0) and at a concentration of 10 mg/900 Ul is added, and the mixture is then gently shaken at room temperature for 3 hours.
• the beads are concentrated by centrifugation, the supernate decanted off and the beads washed five times with pH 7.5 aqueous phosphate buffer.
• the beads are then loaded into a 0.46 cm inner-diameter glass column and used to purify human IgG from serum using standard affinity purification techniques.

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