WO1998033783A1

WO1998033783A1 - Solid phase and combinatorial library syntheses of 3,1-benzoxazine-4-ones

Info

Publication number: WO1998033783A1
Application number: PCT/US1998/002064
Authority: WO
Inventors: Mikhail Gordeev; Dinesh Patel
Original assignee: Versicor, Inc.
Priority date: 1997-02-04
Filing date: 1998-02-04
Publication date: 1998-08-06
Also published as: AU6264898A

Abstract

The invention provides chemistry libraries containing fused 3,1-benzoxazine-4-ones. The invention also provides methods for the construction of fused 3,1-benzoxazine-4-one containing libraries. The invention further provides methods for the identification of bioactive, 3,1-benzoxazine-4-ones from those libraries.

Description

SOLID PHASE AND COMBINATORIAL LIBRARY SYNTHESES OF 3,1-

BENZOXAZINE-4-ONES

TECHNICAL FIELD This invention is directed to combinatorial chemistry libraries containing 3,1- benzoxazine-4-ones. This invention is further directed to methods for constructing combinatorial chemistry libraries containing 3,l-benzoxazine-4-ones. This invention is still further directed to methods for the identification of bioactive 3,l-benzoxazine-4-ones.

BACKGROUND ART

Modern day drug discovery is a multi-faceted endeavor. Researchers commonly delineate a biochemical pathway that is operative in a targeted pathological process. This pathway is analyzed with an eye toward determining its crucial elements: those enzymes or receptors that, if modulated, could inhibit the pathological process. An assay is constructed such that the ability of the important enzyme or receptor to function can be measured. The assay is then performed in the presence of a variety of molecules. If one of the assayed molecules modulates the enzyme or receptor in a desirable fashion, this molecule may be used directly in a pharmaceutical preparation or can be chemically modified in an attempt to augment its beneficial activity. The modified molecule that exhibits the best profile of beneficial activity may ultimately be formulated as a drug for the treatment of the targeted pathological process.

With the use of high-throughput screening techniques, one can assay the activity of tens of thousands of molecules per week. Where molecules can only be synthesized one at a time, the rate of molecule submission to an assay becomes a debilitating, limiting factor. This problem has led researchers to develop methods by which large numbers of molecules possessing diverse chemical structures can be rapidly and efficiently synthesized. One such method is the construction of chemical combinatorial libraries.

Chemical combinatorial libraries are diverse collections of molecular compounds. Gordon et al. (1995) Ace. Chem. Res. 29:144-154. These compounds are formed using a multistep synthetic route, wherein a series of different chemical modules can be inserted at any particular step in the route. By performing the synthetic route multiple times in parallel, each possible permutation of the chemical modules can be constructed. The result is the rapid synthesis of hundreds, thousands, or even millions of different structures within a chemical class.

For several reasons the initial work in combinatorial library construction focused on peptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res. 37:487-493;

Houghton et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; and Fodor et al. (1991) Science 251 :767. The rapid synthesis of discrete chemical entities is enhanced where the need to purify synthetic intermediates is minimized or eliminated; synthesis on a solid support serves this function. Construction of peptides on a solid support is well-known and well-documented.

Obtaining a large number of structurally diverse molecules through combinatorial synthesis is furthered where many different chemical modules are readily available; hundreds of amino acid modules are commercially available. Finally, many peptides are biologically active, making them interesting as a class to the pharmaceutical industry. The scope of combinatorial chemistry libraries has recently been expanded beyond peptide synthesis. Polycarbamate and N-substituted glycine libraries have been synthesized in an attempt to produce libraries containing chemical entities that are similar to peptides in structure, but possess enhanced proteolytic stability, absorption and pharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; Simon et al. (1992) Proc. Νatl. Acad. Sci. USA 89:9367-9371. Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine libraries have been synthesized, expanding combinatorial chemistry to include heterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc. 114:10997-10998; Murphy et al. (1995) J. Am. Chem. Soc. 117:7029-7030; and Gordon et al. (1995) Biorg. Medicinal Chem. Lett. 5:47-50. 3,l-Benzoxazine-4-ones (BOX) are a class of bioactive, heterocyclic molecules that have attracted considerable attention in the pharmaceutical industry. Representatives of this class of compounds have demonstrated inhibitory activity against a variety of serine proteases, including chymotrypsin, human sputum elastase, human leukocyte elastase, Clr and Cls proteases, and human neutrophile elastase. Gutschow et al. (1995) Monatshefte fur Chemie 126:1145-1149; Krantz et al. (1990) J. Med. Chem. 33:464-479; Gilmore et al.

(1996) Bioorg. & Med. Chem. Lett. 6:679-682; Edwards et al. (1994) Med. Res. Rev. j_4:127-194. Serine proteases are common drug development targets because of their role in biological pathways associated with the development of infectious, inflammatory, cardiovascular, and respiratory diseases.

Methods for the solution phase preparation of 3,l-benzoxazine-4-ones have been reported. The key transformation in these syntheses is the formation of the BOX heterocylic ring through the intramolecular cyclization of a urea substituent onto a carboxylic acid derivative. This step has been carried out using a number of different protocols: carbodiimides, acetic anhydride, acyl chlorides with base, tosyl chloride in pyridine, triphenylphosphite in pyridine, thionyl chloride, or acid. Krantz, et al. (1990) J. Med. Chem. 464-479; Cheng, et al. (1993) Heterocycles 775-789; Hauteville, et al. (1988)

J. Het. Chem. 715; Ramana, et al. (1993) Org. Prep. Proced. Int. 588; Rabilloud, et al. (1980) J. Het. Chem. 1065; GB-2262097; Papadopoulos, et al. (1982) J. Het. Chem. 269. These methods have not found application in the preparation of 3,l-benzoxazine-4-one libraries. Methods for nucleophilic aromatic substitution reactions have been described, where a fluoride substituent on an aromatic ring is displaced from a chemical intermediate that is attached to a solid support. MacDonald et al. discloses the use of a nucleophilic aromatic substitution reaction on a solid support as a step in the preparation of a quinolone library. (1996) Tetrahedron Lett. 37: 4815-4818. Phillips et al. describes the use of a nucleophilic aromatic substitution reaction in the solid phase preparation of benzimidazoles. (1996) Tetrahedron Lett. 37: 4887-4890. Shapiro et al. delineates the use of ¹⁹F NMR monitoring of a nucleophilic aromatic substitution reaction on a solid support. (1996) Tetrahedron Lett. 37: 4671-4674. None of these references either disclose or suggest the use of a nucleophilic aromatic substitution reaction in the preparation of a 3,1- benzoxazine-4-one library on a solid support.

The cited references in the background section, and in the following sections, are herein incorporated by reference. DISCLOSURE OF THE INVENTION The present invention provides a combinatorial library that contains 3,1- benzoxazine-4-one derivatives (shown below, I):

I where R,, R₂, R₃, and R₄ can independently be a hydrogen atom, or an alkyl, aryl, heteroaryl, or electron withdrawing group, and where R₅ can independently be an alkyl, aryl, heteroaryl, alkoxy, amino, or thio group. In one embodiment, the combinatorial library contains 3,l-benzoxazine-4-one derivatives that are substituted at the 2-position.

In another embodiment, the combinatorial library contains fused 3,1-benzoxazine- 4-one derivatives that are substituted at the 2-position with an alkyl, aryl, or heteroaryl group. In another embodiment, the combinatorial library contains fused 3,1-benzoxazine-

4-one derivatives that are substituted at the 2-position with an alkoxy, amino, or thio group.

In another embodiment, the combinatorial library contains fused 3,1-benzoxazine- 4-one derivatives that are substituted at the 2-position with an amino acid derivative. The present invention also provides a combinatorial library that contains 3,1- benzoxazine-4-one derivatives that are attached to a solid support.

The present invention also provides a method of producing a combinatorial library that contains 3,l-benzoxazine-4-one derivatives.

In one embodiment, the method of combinatorial library production involves the construction of 3 , 1 -benzoxazine-4-one derivatives on a solid support. In another embodiment, the method of combinatorial library production involves the cleavage of the 3,l-benzoxazine-4-one derivatives from the solid support upon the intramolecular cyclization forming the heterocyclic ring.

In another embodiment, the method of combinatorial library production involves the modification of the aromatic ring of the 3,l-benzoxazine-4-one derivatives through a nucleophilic aromatic substitution.

In another embodiment, the method of combinatorial library production involves the modification of the aromatic ring of the 3,l-benzoxazine-4-one derivatives through the displacement of fluoride in a nucleophilic aromatic substitution reaction on a suitable chemical intermediate.

The present invention also provides a method of screening a library that contains 3,l-benzoxazine-4-one derivatives.

In one embodiment, the 3,l-benzoxazine-4-one derivatives are screened for inhibitory activity against a serine or cysteine protease. The present invention also provides a method of screening a library that contains

3,l-benzoxazine-4-one derivatives attached to a solid support.

In one embodiment, the 3,l-benzoxazine-4-one derivatives attached to a solid support are screened for inhibitory activity against a serine or cysteine protease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the general solid phase synthesis of 3,l-benzoxazine-4-one derivatives from immobilized amine reagents.

FIG. 2 illustrates the general solid phase synthesis of 3,l-benzoxazine-4-one derivatives from immobilized anthranilic acid derived reagents. FIG. 3 illustrates the general solid phase synthesis of 3,l-benzoxazine-4-one derivatives from o-nitro-p-fluorobenzoates.

FIG. 4 illustrates the general solid phase synthesis of 3,l-benzoxazine-4-one derivatives, where a substituent is introduced at the 7-position through a nucleophilic aromatic substitution reaction. FIG. 5 illustrates the general solid phase synthesis of 3,l-benzoxazine-4-one derivatives from immobilized amino acids. FIG. 6 illustrates a specific solid phase synthesis of 3,l-benzoxazine-4-one derivatives, where a substituent is introduced at the 7-position through the nucleophilic aromatic substitution of a fluoride substituent.

BEST MODE FOR CARRYING OUT THE INVENTION

Definitions

"Bioactive" molecule refers to a molecule that exhibits a dissociation constant of 10^'6 or less when combined with a targeted cellular ligand, including but not limited to, enzymes and receptors. "Chemical library" or "array" is an intentionally created collection of differing molecules which can be prepared synthetically and screened for biological activity in a variety of different formats (e.g., libraries of soluble molecules, libraries of molecules bound to a solid support).

"Alkyl" refers to a cyclic, branched, or straight chain chemical group containing only carbon and hydrogen, such as methyl, pentyl, and adamantyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, amino, hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or other functionality that may be suitably blocked, if necessary for purposes of the invention, with a protecting group. Alkyl groups can be saturated or unsaturated (e.g., containing -C=C- or -C≡C- subunits), at one or several positions. Typically, alkyl groups will comprise 1 to

12 carbon atoms, preferably 1 to 10, and more preferably 1 to 8 carbon atoms.

"Amino acid" refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers), synthetic analogs and derivatives thereof, α- Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain". The side chains of naturally occurring amino acids are well known in the art and include, for example, hydrogen (e.g., as in glycine), alkyl (e.g., as in alanine, valine, leucine, isoleucine, proline), substituted alkyl (e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan), substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g., as in histidine). See, e.g., Harper et al. (1977) Review of Physiological Chemistry, 16th Ed., Lange Medical Publications, pp. 21-24. One of skill in the art will appreciate that the term "amino acid" also includes β- γ-, δ-, and ω-amino acids, and the like. Unnatural amino acids are also known in the art, as set forth in, for example, Williams (ed.), Synthesis of Optically Active a-Amino Acids, Pergamon Press (1989); Evans et al, J. Amer. Chem. Soc, 112:4011^1030 (1990); Pu et al., J. Amer.

Chem. Soc, 56:1280-1283 (1991); Williams et al, J. Amer. Chem. Soc, 113:9276-9286 (1991); and all references cited therein.

"Aryl" or "Ar" refers to a monovalent unsaturated aromatic carbocyclic group having a single-ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can be optionally unsubstituted or substituted with amino, hydroxyl, lower alkyl, alkoxy, chloro, halo, mercapto, and other substituents.

"Electron withdrawing group" refers to a substituent that draws electrons to itself more than a hydrogen atom would if it occupied the same position in a molecule. Examples of electron withdrawing groups include — R₂, — COOH, — OR, — SR₂, — F, —COR, — C 1 , — SH, NO₂, — Br, — SR, — SO₂R, —I, —OH, — CN, — C=CR, — COOR,

— Ar, — CH=CR₂, where R is akyl, aryl, arylalkyl, or heteroaryl.

"Heteroaryl" or "HetAr" refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., pyrridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl) and having at least one hetero atom, such as N, O, or S, within the ring, which can optionally be unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, halo, mercapto, and other substituents.

"Protecting group" refers to a chemical group that exhibits the following characteristics: (1) reacts selectively with the desired functionality in good yield to give a protected substrate that is stable to the projected reactions for which protection is desired; 2) is selectively removable from the protected substrate to yield the desired functionality; and 3) is removable in good yield by reagents compatible with the other functional group(s) generated in such protected reactions. Examples of protecting groups can be found in Greene et al. (1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley & Sons, Inc., New York). 3, l-Benzoxazine-4-one Libraries

The present invention provides a combinatorial library containing 3,1-benzoxazine- 4-one derivatives. These 3,l-benzoxazine-4-one derivatives are compounds of the following general structure:

I R„ R₂, R₃, and R₄ can independently be a hydrogen atom, or an alkyl, aryl, heteroaryl, or electron withdrawing group. R₅ can independently be an alkyl, aryl, heteroaryl, alkoxy, amino or thio group.

The 3,l-benzoxazine-4-one derivatives are constructed on a solid support. Prior to cleavage from the solid support, these derivatives, or synthetic intermediates for the construction of these derivatives, are attached to the solid support through a bridge to a single substituent: R,, R₂, R₃, R₄, or R₅. Attachment to R₅, for instance, is illustrated in

Figures 1 and 4; attachment to R₃ is illustrated in Figure 3.

Overview of Combinatorial Synthesis Combinatorial library synthesis is typically performed on a solid support. See, for example, Lam et al. (1991) Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86. A large number of beads or particles are suspended in a suitable carrier (such as a solvent) in a parent container. The beads, for example, are provided with a functionalized point of attachment for a chemical module. The beads are then divided and placed in various separate reaction vessels. The first chemical module is attached to the bead, providing a variety of differently substituted solid supports. Where the first chemical module includes 3 different members, the resulting substituted beads can be represented as A,, A₂, and A₃ The beads are washed to remove excess reagents and subsequently remixed in the parent container. This bead mixture is again divided and placed into various separate reaction vessels. The second chemical module is coupled to the first chemical module.

Where the second chemical module includes 3 different members, B„ B₂, and B₃, 9 differently substituted beads result: A,B,, A,B₂, A,B₃, A₂B„ A₂B₂, A₂B₃ , A₃B„ A₃B₂, and A₃B₃. Each bead will have only a single type of molecule attached to its surface. The remixing/redivision synthetic process can be repeated until each of the different chemical modules has been incorporated into the molecule attached to the solid support. Through this method, large numbers of individual compounds can be rapidly and efficiently synthesized. For instance, where there are 4 different chemical modules, and where each chemical module contains 20 members, 160,000 beads of different molecular substitution can be produced. Combinatorial library synthesis can be performed either manually or through the use of an automated process. For the manual construction of a combinatorial library, a scientist would perform the various chemical manipulations. For the construction of a combinatorial library through an automated process, the various chemical manipulations will typically be performed robotically. For example, see U.S. Patent No. 5,463,564. Solid Supports

The synthesis of a 3,l-benzoxazine-4-one library can be performed on a solid support. "Solid support" includes an insoluble substrate that has been appropriately derivatized such that a chemical module can be attached to the surface of the substrate through standard chemical methods. Solid supports include, but are not limited to, beads and particles, such as peptide synthesis resins. For example, see Merrifield (1963) J. Am.

Chem. Soc. 85:2149-2154; U.S. Patent No. 4,631,211; and Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 8k3998-4002.

Solid supports can consist of many materials, limited primarily by the capacity of the material to be functionalized through synthetic methods. Examples of such materials include, but are not limited to, polymers, plastics, resins, polysaccharides, silicon or silica based materials, carbon, metals, inorganic glasses and membranes. Preferred resins include Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland), and TentaGel S AC, TentaGel PHB, TentaGel SAM, or TentaGel S NH₂ resin (polystyrene-polyethylene glycol copolymer resins available from Rapp Polymere, Tubingen, Germany). The solid support can be purchased with suitable functionality already present such that a chemical module can be attached to the support surface (e.g., Novabiochem, Bachem Bioscience, Rapp Polymere). Alternatively, the solid support can be chemically modified such that a chemical module can be attached to the support surface. Grant (1992) Synthetic Peptides. A User's Guide, W.H. Freeman and Co; Hermkens et al. (1996) Tetrahedron 52:4527-4554. The choice of functionality used for attaching a molecule to the solid support will depend on the nature of the compound to be synthesized and the type of solid support. Examples of functionality present on the solid support that can be used to attach a chemical module, include, but are not limited to, alkyl or aryl halides, aldehydes, alcohols, ketones, amines, sulfides, carboxyl groups, aldehyde groups, and sulfonyl groups. Preferably, the functional group on the solid support that permits the attachment of a chemical module will be an alcohol, an amine, a thiol, an ester, a silyl, or an amide group. Gordon et al. (1994) J. Med. Chem. 37:1385-1401; Hermkens et al. (1996) Tetrahedron 52:4527-4554.

For certain combinatorial libraries, one can purchase a solid support with an existing, protected chemical module already attached. An example of such a support is

FmocGly Sasrin, which is commercially available from Rapp Polymere. Typically, however, the first step of the combinatorial library synthesis is the attachment of a chemical module to the solid support through the existing functionality on the support surface. Examples of chemical reactions that can be used to attach a chemical module to the support include, but are not limited to, nucleophilic displacement of a halide or other leaving group, etherification of an alcohol, esterification of an alcohol, amidation of an amine, acetalization of an aldehyde, and ketalization of a ketone. Hermkens et al. (1996) Tetrahedron 52:4527-4554.

Preferably, the reaction used to attach the chemical module to the solid support will be an esterification of an alcohol, an amidation of an amine, or the nucleophilic aromatic substitution of fluoride. For example, see Hermkens et al. (1996) Tetrahedron 52:4527- 4554.

For the attachment of certain chemical modules to the solid support, masking of functionality that is not involved in the attachment process, but that is incompatible with the mode of attachment, may be necessary. A non-limiting example of this type of process is the esterification of an alcohol functionalized solid support, using a hydroxyl-substituted carboxylic acid as the coupling partner. Prior to the esterification reaction, the hydroxyl group of the carboxylic acid would be "protected" through alkylation, silylation, acetylation, or through some other standard method. Strategies for the use of masking or protecting groups have been well-described in the art, such as in Green (1985) Protecting

Groups in Organic Synthesis, Wiley.

Nucleophilic Aromatic Substitution Reactions

Nucleophilic substitution reactions at aromatic carbon centers typically proceed too slowly to be of synthetic utility. Under 4 different scenarios, however, there are exceptions to this rule: 1) where an electron withdrawing group is either ortho or para to the leaving group; 2) where a strong base forms an aryne intermediate that is subject to nucleophilic attack; 3) where the nucleophile can donate an electron through a transfer mechanism; 4) where a diazonium salt is replaced, see March, "Advanced Organic Chemistry," John Wiley & Sons, New York, 1985. Of these 4 mechanisms, the first is the most utilized form.

The displacement of a fluoride substituent from an aromatic ring that contains a para carbonyl group is a version of the first nucleophilic aromatic substitution reaction mechanism. The carbonyl group can be an ester, ketone, heterocyclic vinylogous amide, or heterocyclic conjugated ketone. Luo et al. (1994) J. Org. Chem. 1761-1765; Berge et al.

(1994) Synlett 187-188; Inoue et al. (1994) J. Med. Chem. 586-592; Cecchetti et al. (1993) J. Het. Chem. 1143-1148. Although 1 carbonyl group is sufficient to promote nucleophilic aromatic substitution, this reaction can be facilitated by the addition of a second or third electron withdrawing group to the aromatic ring. Examples of such electron withdrawing groups include, but are not limited to, NO₂, F, Cl, Br, C(O)R, CN, SO₂R, S(O)R. A variety of nucleophiles can be used to displace fluoride from an aromatic ring containing a para carbonyl group. Suitable nucleophilic reagents include, but are not limited to, amines, hydrazines, hydroxylamines, NH-heterocycles, alcohols, and thiols. Inoue et al. (1994) J. Med. Chem. 586-592; Cecchetti et al. (1993) J. Het. Chem. 1143- 1148; Kalindjian et al. (1991) Synlett 803-804; Ziegler et al. (1988) J. Het. Chem. 1543; Cooper et al. (1992) J. Med. Chem. 1392-1398; Luo et al. (1994) J. Org. Chem. 1761- 1765; Stabler et al. (1994) Synth. Commun. 123-129; Yeager et al. (1991) Synthesis 63- 68; Berge et al. (1994) Syntlett 187-188; Guggenheim (1987) Tetrahedron Lett. 6139; Gornostaev et al. (1992) Zh. Org. Khim. 2291-2293.

Synthetic Routes to Fused 3, 1 -Benzoxazine-4-one Libraries

A general strategy for the construction of 3,l-benzoxazine-4-one libraries, delineating three possible synthetic routes involving the use of immobilized amine reagents, is shown in Figure 1. The first route employs the addition of an anthranilic acid derivative to an immobilized amine derivative. The second route employs the addition of an immobilized amine derivative to an anthranilic acid derivative containing an isocyanate and an acid chloride or functionally activated ester. The third route employs the addition of an immobilized amine derivative to an anthranilic acid derivative containing an isocyanate and an ester. To construct a 3,l-benzoxazine-4-one library through the first route shown in

Figure 1 , a chemical module containing a terminal amine, or protected terminal amine, is attached to a functionalized resin. Where the terminal amine of the chemical module is protected, the synthetic route proceeds through the deprotection of the terminal amine. The terminal amine is acylated to provide an activated carbamate. A urea is formed upon condensation of the activated carbonate with an anthranilic acid derivative. This urea is treated to effect an intramolecular cyclization, forming the solid support bound 3,1- benzoxazine-4-one derivative.

To construct a 3,1-benzoxazine library through the second route shown in Figure 1, a functionalized resin containing a terminal amine is either purchased or constructed as described above. The immobilized amine is condensed with an anthranilic acid derivative that contains both an isocyanate group and an activated acid. Treatment of the resultant urea with base then facilitates the intramolecular cyclization, forming the solid support bound 3,l-benzoxazine-4-one derivative.

To construct a 3,1-benzoxazine library through the third route shown in Figure 1, a functionalized resin containing a terminal amine is condensed with an anthranilic acid derivative that contains both an isocyanate group and an alkyl or aryl ester to form a urea.

The alkyl or aryl ester is deprotected under standard conditions to provide the corresponding acid. This acid is then treated under conditions to induce the intramolecular cyclization, forming the solid support bound 3,l-benzoxazine-4-one derivative.

A general strategy for the construction of 3,l-benzoxazine-4-one libraries, delineating three possible synthetic routes involving the use of immobilized anthranilic acid derivatives, is shown in Figure 2. The first route employs the addition of an activated carbamate to an immobilized anthranilic acid derivative. The second route employs the addition of an amine to an immobilized anthranilic acid derivative that contains both an isocyanate group and an activated carboxylic acid. The third route employs the addition of an amine to an immobilized anthranilic acid derivative that contains both an isocyanate group and an alkyl or aryl ester. The isocyanate group in the reactions listed above can be replaced, as will be understood by one of ordinary skill in the art, by comparably reactive functional groups such as -nitrophenyl carbamoyl group.

To construct a 3,l-benzoxazine-4-one library through the first route shown in Figure 2, an anthranilic acid derivative is attached to a solid support. The aniline moiety of the attached anthranilic acid derivative is acylated with an activated carbamate. Intramolecular cyclization of the resultant urea completes the synthesis of the solid support bound 3,l-benzoxazine-4-one derivative.

To construct a 3,1-benzoxazine library through the second route shown in Figure 2, an anthranilic acid derivative is attached to a solid support. The aniline moiety of the attached anthranilic acid derivative is converted into an isocyanate group. The carboxylic acid moiety of the attached anthranilic acid derivative is activated by conversion to an acyl halide or a reactive ester. An amine is condensed with the isocyanate group of the derivatized anthranilic acid forming a urea. This urea is treated with base to form the solid support bound 3,1 -benzoxazine-4-one derivative through an intramolecular cyclization reaction. To construct a 3,1-benzoxazine library through the third route shown in Figure 2, an anthranilic acid derivative is attached to a solid support. The aniline moiety of the attached anthranilic acid derivative is converted into an isocyanate group. The carboxylic acid moiety of the attached anthranilic acid derivative is converted into an alkyl or aryl ester. An amine is condensed with the isocyanate group of the derivatized anthranilic acid forming a urea. The resultant urea is subjected to intramolecular cyclization conditions, forming the 3,l-benzoxazine-4-one derivative.

A synthetic strategy for the construction of 3,l-benzoxazine-4-one containing libraries, where an anthranilic acid derivative is attached to a solid support through the nucleophilic displacement of fluoride from the anthranilic acid derivative, is shown in

Figure 3. A solid support bound amine, thiol, or alcohol is reacted with an anthranilic acid derivative containing an ester, a nitro group, and a fluoride substituent para to the ester. The fluoride substituent is displaced in the process forming an attachment between the solid support and the anthranilic acid derivative. The nitro group is reduced to the corresponding amine using tin(II) chloride or other appropriate reagent. This amine is condensed with an activated carbamate equivalent, such as an isocyanate, forming a urea. Deprotection of the ester functionality of the anthranilic acid derivative provides a carboxylic acid. Intramolecular cyclization of the urea on the carboxylic acid is effected upon treatment with a reagent such as tosyl chloride in pyridine. The resultant 3,1- benzoxazine-4-one derivative can then be cleaved from the solid support using chemical or physical means.

A synthetic strategy for the construction of 3,l-benzoxazine-4-one containing libraries, where the substituent at the 7-position of the 3,l-benzoxazine-4-one derivatives can be altered through the nucleophilic displacement of fluoride, is shown in Figure 4. A solid support bound amine reagent is condensed with an anthranilic acid derivative containing an activated carbamate, an alkyl ester, and a fluoride substituent para to the ester. The resultant urea is treated with an amine, alcohol, or thiol that displaces the fluoride substituent in a nucleophilic process. Deprotection of the ester functionality of the anthranilic acid derivative provides a carboxylic acid. Intramolecular cyclization of the urea onto the carboxylic acid is effected upon treatment with a reagent such as tosyl chloride in pyridine. The resultant 3,l-benzoxazine-4-one derivative can then be cleaved from the solid support using chemical or physical means.

A specific embodiment of the invention is shown in Figure 5, where the 3,1- benzoxazine-4-one derivatives are constructed using an immobilized amino acid route. The solid support is purchased with an attached amino acid, or an amino acid is chemically attached to the solid support. The amine group is acylated to provide an activated carbamate or equivalent. An anthranilic acid derivative is condensed with the activated carbamate. The resultant urea is subjected to conditions such as tosyl chloride, pyridine to effect an intramolecular cyclization. Liberation of the solid support attached 3,1- benzoxazine-4-one derivative can be carried out upon treatment with chemical or physical means.

A specific embodiment of the invention is shown in Figure 6, where the 3,1- benzoxazine-4-one derivatives are constructed using an immobilized amino acid route. An amino acid functionalized resin is condensed with an anthranilic acid derivative containing a functionally activated carbamate and a fluoride substituent para to a methyl ester. The resultant urea is reacted with 1 -methylpiperazine to displace the fluoride substituent. Treatment of the methyl ester with potassium trimethylsilanolate provides the corresponding carboxylic acid. The acid is cyclized to a solid support bound 3,1- benzoxazine-4-one derivative upon reaction with tosyl chloride in pyridine. Cleavage of the 3,l-benzoxazine-4-one from the solid support is effected by treatment with trifluoroacetic acid.

A solid support bound, fused 3,l-benzoxazine-4-one library can be recovered through conventional methods such as filtration or centrifugation. Confirmation that the solid support contains the desired fused 3,l-benzoxazine-4-one compound can be accomplished by cleaving the 3,l-benzoxazine-4-one from a small portion of the solid support, and then subjecting the cleaved product to conventional analysis. Examples of commonly used analytical methods include, but are not limited to, nuclear magnetic resonance spectroscopy and high performance liquid chromatography.

Methods of Cleavage In one embodiment of the invention, the 3,l-benzoxazine-4-one library is bound to a solid support. In another embodiment of the invention, the 3,l-benzoxazine-4-one is cleaved from the solid support to produce soluble 3,l-benzoxazine-4-one libraries. Soluble libraries can be advantageous for a variety of purposes, including assaying the biological activity of compounds and performing structural analysis of compounds.

The cleavage of compounds from a solid support to produce a soluble chemical library can be accomplished using a variety of methods. For example, a compound can be photolytically cleaved from a solid support (Wang et al. (1976) J. Org. Chem. 41 :3258; Rich et al. (1975) J. Am. Chem. Soc. 97:1575-1579), and through nucleophilic attack (U.S. Patent No. 5,549,974), or through hydrolysis (Hutchins et al. (1994) Tetrahedron Lett.

3^:4055-4058).

Preferably, the cleavage of compounds from a solid support to produce a soluble chemical library is accomplished using hydrolytic conditions, such as through the addition of dilute trifluoroacetic acid.

Screening

The present invention is directed toward the generation of 3,l-benzoxazine-4-one libraries. These libraries are used to select one or more 3,l-benzoxazine-4-one species that demonstrate a specific interaction with a targeted cellular ligand including, but not limited to, enzymes or receptors. A cellular ligand is targeted when it is believed that the ligand is of importance in the modulation of a disease. Examples of disease states for which 3,1- benzoxazine-4-one libraries can be screened include, but are not limited to, inflammation, infection, respiratory diseases, and cardiovascular disorders.

Several methods have been developed in recent years to screen libraries of compounds to identify the compounds having the desired characteristics. Typically, where a compound exhibits a dissociation constant of 10^"6 or less when combined with the targeted enzyme or receptor, the compound is thought to demonstrate a specific interaction with the enzyme or receptor. Methods for isolating library compound species that demonstrate desirable affinity for a receptor or enzyme are well-known in the art. For example, an enzyme solution may be mixed with a solution of the compounds of a particular combinatorial library under conditions favorable to enzyme-ligand binding. Specific binding of library compounds to the enzyme may be detected by any of the numerous enzyme inhibition assays which are well known in the art. Compounds which are bound to the enzyme may be readily separated from compounds which remain free in solution by applying the solution to a Sephadex G-25 gel filtration column. Free enzyme and enzyme-ligand complexes will pass through the column quickly, while free library compounds will be retarded in their progress through the column. The mixture of enzyme- ligand complex and free enzyme can then be treated with a powerful denaturing agent, such as guanidinium hydrochloride or urea, to cause release of the ligand from the enzyme. The solution can then be injected onto an HPLC column (for example, a Vydac C-4 reverse-phase column, eluted with a gradient of water and acetonitrile ranging from 0% acetonitrile to 80% acetonitrile). Diode array detection can provide discrimination of the compounds of the combinatorial library from the enzyme. The compound peaks can then collected and subjected to mass spectrometry for identification.

An alternate manner of identifying compounds that inhibit an enzyme is to divide the library into separate sublibraries where one step in the synthesis is unique to each sublibrary. To generate a combinatorial library, reactants are mixed together during a step to generate a wide mixture of compounds. At a certain step in the synthesis, however, the resin bearing the synthetic intermediates can be divided into several portions, with each portion then undergoing a unique transformation. The resin portions are then (separately) subjected to the rest of the synthetic steps in the combinatorial synthetic method. Each individual resin portion thus constitutes a separate sublibrary. When testing the compounds, if a given sublibrary shows more activity than the other sublibraries, the unique step of that sublibrary may then be held fixed. The sublibrary then becomes the new library, with that step fixed, and forms the basis for another round of sublibrary synthesis, where a different step in the synthesis is optimized. This procedure can be executed at each step until a final compound is arrived at. The aforementioned method is the generalization of the method described in Geysen, WO 86/00991, for determining peptide "mimotopes," to the synthetic method of this invention.

Finding a compound that inhibits an enzyme is most readily performed with free compound in solution. The compounds can also be screened while still bound to the resin used for synthesis; in some applications, this may be the preferable mode of finding compounds with the desired characteristics. For example, if a compound that binds to a specific antibody is desired, the resin-bound library of compounds may be contacted with an antibody solution under conditions favoring a stable antibody-compound-resin complex. A fluorescently labeled second antibody that binds to the constant region of the first antibody may then be contacted with the antibody-compound-resin complex. This will allow identification of a specific bead as carrying the compound recognized by the first antibody binding site. The bead can then be physically removed from the resin mixture and subjected to mass spectral analysis. If the synthesis has been conducted in a manner such that only one compound is likely to be synthesized on a particular bead, then the binding compound has been identified . If the synthesis has been carried out so that many compounds are present on a single bead, the information derived from analysis can be utilized to narrow the synthetic choices for the next round of synthesis and identification. The enzyme, antibody, or receptor target need not be in solution either. Antibody or enzyme may be immobilized on a column. The library of compounds may then be passed over the column, resulting in the retention of strongly binding compounds on the column after weaker-binding and non-binding compounds are washed away. The column can then be washed under conditions that dissociate protein-ligand binding, which will remove the compounds retained in the initial step. These compounds can then be analyzed, and synthesized separately in quantity for further testing. Similarly, cells bearing surface receptors can be expressed on a cell surface may be contacted with a solution of library compounds. The cells bearing bound compounds can be readily separated from the solution containing non-binding compounds. The cells can then be washed with a solution which will dissociate the bound ligand from the cell surface receptor. Again, the cells can be separated from the solution, and the solution which now contains the ligands bound in the initial step can be analyzed.

The following examples are provided to illustrate, but not limit, the invention.

EXAMPLES General Methods Reagents were purchased from Aldrich, Sigma, Bachem Biosciences and Rapp

Polymere and used without further purification. Immobilized N-Fmoc-protected amino acids were prepared from commercial Fmoc-amino acids using standard coupling protocols [Grant (1992) Synthetic Peptides. A User 's Guide. W.H. Freeman and Co.] or purchased from Bachem Bioscience. Tentagel SAM was purchased from Rapp Polymere.

Concentration of solutions after workup was performed by reduced pressure rotary evaporation, or using the Savant's SpeedVac instrument.

NMR spectra were recorded on a Varian Gemini 300 Mhz instrument with CDC1, as solvent unless noted. Η NMR data are reported as follows: chemical shifts relative to tetramethylsilane (0.00 ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplex), coupling, and integration. Mass-spectra were obtained using ESI technique.

HPLC analysis and purification were performed using Beckman System Gold^R; detection at 220 nm. Analytical HPLC was performed on Rainin Microsorb C18 (4.6 mm x 150 mm) reverse phase column (gradient from 100% of the aq. 0.1% TFA to 100% of 0.1% TFA in MeCN over 35 min, flow rate 1.0 mL/min).

General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones

Method A. From Immobilized Activated Carbamates. An appropriate N-Fmoc- protected amino acid resin [0.06 mmol, ca. 100 mg for the Sasrin support immobilized amines) was deprotected with 20% piperidine in dimethylformamide for 30 min. The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. The amine resin thus obtained was agitated with p-nitrophenyl chloroformate (85 mg, 0.42 mmol) and organic base (such as 2,6-lutidine, 0.15 ml) in CH₂C1₂ (1.5 ml) for 1-4 h (until a negative ninhydrine test indicated the absence of a free amine on a solid phase). The resultant p-nitrophenylcarbamate resin was filtered, washed liberally with CH₂C1₂, dried under vacuum (r.t, 0.5 Torr). An appropriate anthranilic acid

(1 mmol) and a solution of organic base such as 10% pyridine or 2,6-lutidine in dimethylformamide (2 ml) was added, and the mixture agitated at 20-70 °C for 8-24 h (typically, this reaction with anthranilic acid was essentially completed overnight at r.t.). The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. The resultant immobilized urea was agitated at 20-70 °C with a solution of the cyclizing reagent, such as N,N'-diisopropylcarbodiimide (DIC, 20% v/v in tetrahydrofurane, 2 ml), acetic anhydride (2 ml), or p-toluenesulfonylchloride (TsCl, 0.3 M solution in pyridine, 2 ml), over 3-24 h (this cyclization for the urea intermediate generated from L-Phe-Wang resin and anthranilic acid was essentially complete for each of the cyclizing reagents over ca. 15 h at r.t.). The resin was filtered, washed liberally with dimethylformamide, acetone, and CH₂C1₂, and dried under vacuum. The resulted benzoxazinone products were cleaved from supports with 1-40% TFA in CH₂C1₂ for 0.5-2 h. Thus, the Sasrin resin immobilized products were typically released from support with 1-3% TFA in CH₂C1₂ (30 min), whereas the Wang resin tethered compounds were cleaved with 40% TFA in CH₂C1₂ for 0.5-2 h. The crude products were lyophilized and analyzed by NMR, MS, and HPLC. The results obtained are depicted in Table 1.

Table 1. SPS of 3,l-Benzoxazine-4-ones

Structures are in agreement with H NMR and ESI MS data. ^bDetection at 220 nm. ^cContains a ¹³C-2 label.

These data demonstrate that BOX products can indeed be efficiently synthesized on a solid phase in good yield and purity.

Another example shown in Fig. 6 demonstrates the synthesis of 7-substituted benzoxazinones on a solid support utilizing aromatic nucleophilic substitution (cf. with Fig. 4). Method B. From Immobilized Isocyanates. An appropriate amine resin (such as immobilized amino acid reagents, see above, Method A; 0.06 mmol, ca. 100 mg for Sasrin support) was agitated with triphosgene (60 mg, 0.19 mmol) and organic base (such as 2,6- lutidine, 0.3 ml) in CH₂C1₂ (1.5 ml) for 0.5-1.5 h (until a negative ninhydrine test indicated the absence of a free amine on a solid phase). Alternatively, 1.9 M phosgene in toluene

(0.18 ml, 0.34 mmol) was added dropwise with stirring to the above amine resin in CH₂C1₂ (1.5 ml) containing organic base (such as 2,6-lutidine, 0.33 ml) at ca. -10 - 0 °C, and the mixture was allowed to warm up to r.t. over 2-3 h. In both cases, the resulted isocyanate resin was washed liberally with CH₂C1₂, and dried (r.t., 0.5 Torr). An appropriate anthranilic acid (1 mmol) and a solution of organic base such as 10% pyridine or 2,6- lutidine in dimethylformamide (2 ml) was added, and the mixture agitated at 20-70 °C for 4-24 h. The resultant immobilized ureas were cyclized into respective benzoxazinones and isolated as described above in Method A.

2-[(S)- 1 -Isopropyl- 1 -carboxy-'³C-methyl1amino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Val-Sasrin resin with anthranilic acid according to the Method A of the General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. Fast gel-phase ¹ C NMR for the Sasrin resin immobilized product in C₅D₆ (δ, ppm): 58.8. Cleaved from the Sasrin resin with 3% TFA in CD₂C1₂. Mass-spectrum (m/z): 265 (M+H)⁺.

2-[(S)- 1 -Benzyl- 1 -carboxymethyllamino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with anthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of 3,1 - Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using either DIC in tetrahydrofuran, acetic anhydride, or p-toluenesulfonyl chloride in pyridine (all three reactions were performed over 15 h at r.t. and produced essentially similar results). 'H NMR in CDC1₃ (δ, ppm): 3.08-3.19 (m, 1 H), 3.47-3.57 (m, 1 H), 4.86 (m, 1 H), 7.15- 7.42 (m, 7 H), 7.80 (m, 1 H), 8.00 (d, J = 8.1 Hz, 1 H). Mass-spectrum (m/z): 311 (M+H)⁺.

6,7-Dimethoxy-2-[(S)- 1 -Benzyl- 1 -carboxymethyllamino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with 4,5- dimethoxyanthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. 'H NMR in CDC1₃ (δ, ppm): 3.16 (m, 1 H), 3.45-3.48 (m, 1 H), 3.91 (s, 3 H), 4.00 (s, 3 H), 4.83 (m, 1 H), 7.10-7.37 (m, 7 H).

Mass-spectrum (m/z): 371 (M+H)⁺.

7-Fluoro-2-[(S)-l -Benzyl- l-carboxymethyl]amino-3.1-benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with 4-fluoroanthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. Mass-spectrum (m z): 329 (M+H)⁺. 6.7-Difluoro-2-[(S)- 1 -Benzyl- 1 -carboxymethyllamino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with 4,5- difiuoroanthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. Mass-spectrum (m/z): 347 (M+H)⁺.

6-Hydroxy-2- (S)- 1 -Benzyl- 1 -carboxymethyl]amino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with 5- hydroxyanthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of 3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. Mass-spectrum (m z): 327 (M+H)⁺.

5-Chloro-2-r(S)- 1 -Benzyl- 1 -carboxymethyllamino-3.1 -benzoxazine-4-one

The compound has been prepared from the Fmoc-Phe-Wang resin with 6-chloroanthranilic acid according to the Method B of the General Procedures for Solid Phase Preparations of

3,l-Benzoxazine-4-ones. Heterocyclization of the intermediate urea was effected using DIC in tetrahydrofuran at r.t. Mass-spectrum (m/z): 345 (M+H)⁺. 2-(N- Amidomethyl-N-methyl)amino-6-fluoro-7-(4-methyl)piperazino-3 , 1 -benzoxazine-4- one

N-Fmoc-Protected sarcosine (0.149 g, 0.48 mmol) was pre-activated with diisopropylcarbodiimide (0.038 ml, 0.24 mmol) in CH₂C1₂ (2 ml) and N- methylpyrrolidine-2-one (1 ml) for 20 min at r.t. The coupling solution was added to the resin Tentagel S AM (0.500 g, ca. 0.12 mmol), and mixture agitated at r.t. for 2 h (until negative ninhydrine reaction indicated a complete amine acylation). The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. The Fmoc-sarcosine-resin thus obtained (0.06 g, ca. 0.012 mmol) was deprotected with

20%) piperidine in dimethylformamide for 30 min (1 ml) filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. The resulted amine resin was agitated with methyl 2-(p-nitrophenyl)carbamoyl-4,5-difluorobenzoate (0.015 g, 0.04 mmol) in 10% pyridine in N,N-dimethyformamide (0.5 ml) for ca. 48 h at r.t. The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. The resulted urea resin was agitated with 0.5 M 1-methylpiperazine in N-methylpyrrolidine-2-one (2 ml) for 4 h at 75 °C. The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, and dried under vacuum. Next, the ester deprotection was performed by stirring the resin with 0.6 M KOTMS in tetrahydrofuran (1.5 ml; 4.5 h at r.t.). Cyclization of the penultimate immobilized intermediate thus obtained into the benzoxazinone product was achieved by treatment of the resin with p-toluenesulfonyl chloride (0.080 g, 0.42 mmol) in pyridine (1.5 ml) for 16 h at r.t. The resin was filtered, washed liberally with dimethylformamide, MeOH, and CH₂C1₂, dried under vacuum, and then cleaved with 40% trifluoracetic acid in CH₂C1₂. Solvent was removed in vacuo to afford the expected benzoxazinone product. Mass- spectrum (m z): 350 (M+H)⁺.

3-[(S)- 1 -Benzyl- 1 -carboxymethyl]- 1 -benzyl-2,4-( 1 H,3H)quinazolinedione.

Wang resin immobilized 2-[(S)-l-benzyl-l-carboxymethyl]amino-3.1-benzoxazine-4-one prepared as described above (30 mg, ca. 0.026 mmol) was agitated with benzyl chloride (0.062 ml, 0.54 mmol) and tetramethylguanidine (0.068 ml, 0.54 mmol) in N- methylpyrrolidine-2-one for 14 h at r.t. The resin was filtered, washed liberally with methanol and CH₂C1₂, and dried under vacuum. The products was cleaved from the resin with 40% TFA in CH₂C1₂ (1.0 ml, r.t., 1 h), lyophilized, and analyzed by HPLC and spectral data. HPLC purity for crude material ca. 70%. The compound can be further purified by RP HPLC. Analytical data are identical to that of the authentic quinazolinedione prepared according to [Gordeev and Patel (1996), Solid Phase and Combinatorial Syntheses of Fused 2,4-Pyrimidinediones, Serial No. 08/740,103]. Mass- spectrum (m/z): 401 (M+H)⁺.

Although the forgoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practical. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

Claims

1. A combinatorial library comprising derivatives of 3 , 1 -benzoxazine-4-one.

2. The combinatorial library of claim 1, wherein the 3,l-benzoxazine-4-one derivatives are substituted at the 2-position.

3. The combinatorial library of claim 2, wherein the substituent at the 2- position is selected from the group consisting of alkyl, aryl, or heteroaryl.

4. The combinatorial library of claim 2, wherein the substituent at the 2- position is selected from the group consisting of alkoxy, amino, thio.

5. The combinatorial library of claim 4, wherein the amino group is an amino acid derivative.

6. A combinatorial library comprising derivatives of 3,l-benzoxazine-4-one attached to a solid support.

7. A method for synthesizing a combinatorial library comprising derivatives of

3 , 1 -benzoxazine-4-one.

8. The method of claim 7, wherein the 3 , 1 -benzoxazine-4-one derivatives are constructed on a solid support.

9. The method of claim 8, wherein the 3,l-benzoxazine-4-one derivatives are cleaved from the solid support upon the intramolecular cyclization forming the heterocyclic ring.

10. The method of claim 7, wherein the aromatic portion of the 3,1- benzoxazine-4-one derivatives is modified through a nucleophilic aromatic substitution reaction.

11. The method of claim 10, wherein fluoride is displaced from the 3 , 1 - benzoxazine-4-one derivatives in a nucleophilic aromatic substitution reaction on a suitable precursor.

12. A method for screening the library of claim 1 comprising: a) contacting the 3 , 1 -benzoxazine-4-one derivatives with a targeted receptor of enzyme under conditions conducive to specific binding; and b) isolating the 3,l-benzoxazine-4-one derivative that specifically binds to the targeted receptor or enzyme.

13. The method of claim 12, wherein the targeted receptor or enzyme is a serine or cysteine protease.

14. A method for screening the library of claim 6 comprising: a) cleaving the 3,l-benzoxazine-4-one derivatives from the solid support; b) contacting the 3,l-benzoxazine-4-one derivatives with a targeted receptor or enzyme under conditions conducive to specific binding; and c) isolating the 3,l-benzoxazine-4-one derivative that specifically binds to the targeted receptor or enzyme.

15. The method of claim 14, wherein the targeted receptor or enzyme is a serine or cysteine protease.