WO2001020995A1 - Compounds directed against pilus biogenesis and activity in pathogenic bacteria; methods and compositions for synthesis thereof - Google Patents

Abstract

Many Gram-negative pathogens assemble adhesive structures on their surfaces that allow them to colonize host tissues and cause disease. Novel compositions which inhibit or prevent the formation of a pilus chaperone-subunit complex are disclosed. Interfering with the function of the pili chaperone negatively affects the chaperone/usher pathway which is one molecular mechanism by which Gram-negative bacteria assemble adhesive pili structures and thus prevent or inhibit pilus assembly. Also provided are methods for the treatment or prevention of diseases caused by tissue-adhering pilus-forming bacteria by inhibiting the function of pilus chaperones. Also provided are pharmaceutical preparations capable of inhibiting or preventing the formation of a pilus chaperone-subunit complex. Also provided are methods of synthesizing the N-substituted amino acid compounds and compounds useful for the synthesis thereof. In particular, novel fluorinated linker compounds and methods of synthesis are provided. Methods for using the fluorinated linker compounds in methods of solid-phase synthesis of the N-substituted amino acid compounds are also disclosed.

Description

COMPOUNDS DIRECTED AGAINST PILUS BIOGENESIS AND

ACTIVITY IN PATHOGENIC BACTERIA; METHODS AND COMPOSITIONS FOR SYNTHESIS THEREOF

This application claims priority to co-pending United States provisional patent application Ser. No. 60/155,822, filed September 23, 1999, incorporated herein by reference.

Field of Invention

The invention relates to novel compounds and compositions which inhibit or prevent bacterial growth and/or attachment by inhibiting or preventing pilus biogenesis. Further provided are fluorinated linker compositions bound to a solid support for solid phase synthesis of N-substituted amino acid compositions, as well as derivatives of carboxylic acids in general.

Background of the Invention

Many pathogenic Gram-negative bacteria such as Escherichia coli, Haemophilus influenzae, Salmonella enteriditis, Salmonella typhimurium, Bordetella pertussis, Yersinia enterocolitica, Yersinia perstis, Helicobacter pylori and Klebsiella pneumoniae assemble hair-like adhesive organelles called pili on their surfaces. Pili are thought to mediate microbial attachment, often the essential first step in the development of disease, by binding to receptors present in host tissues and may also participate in bacterial-bacterial interactions important in biofilm formation.

The prevention or inhibition of normal pilus assembly in Gram-negative bacterium impacts the pathogenicity of the bacterium by preventing the bacterium to infect host tissues. Uropathogenic strains of E. coli express type 1 and P pili that bind to receptors present in uroepithelial cells. Type 1 appear to be more common in E. coli causing cystitis whereas adhesive P pili are virulence determinants associated with pyelonephritic strains of E. coli.

Type 1 pili are adhesive fibers expressed in E. coli as well as in most of the Enterobacteriaceae family. They are composite structures in which a short tip fibrillar structure containing FimG and the FimH adhesin (and possibly the minor component FimF as well) is joined to a rod comprised predominantly of FimA subunits. The type 1 adhesin, FimH, binds D-mannose oligosaccharides present in glycolipids and glycoproteins. In uropathogenic E. coli, this binding event has been shown to play a critical role in bladder colonization and disease. Type 1 pilus biogenesis proceeds via a highly conserved chaperone/usher pathway that is involved in the assembly of over 25 adhesive organelles in Gram-negative bacteria. P pili are adhesive organelles encoded by eleven genes in the pap (pilus associated with pyelonephritis) gene cluster found on the chromosome of uropathogenic strains of E. coli. Six genes encode structural pilus subunits, PapA, PapH, PapK, PapΕ, PapF and PapG. See S . Hultgren et al., Cell 73:887 (1993). The pilus is a heteropolymeric surface fiber with an adhesive tip and consists of two major sub-assemblies, the pilus rod and the tip fibrillum. The pilus rod is a thick rigid rod made up of repeating PapA subunits arranged in a right-handed helical cylinder whereas the tip fibrillum is a thin, flexible tip fiber extending from the distal end of the pilus rod and is composed primarily of repeating PapΕ subunits arranged in an open helical configuration. Two components of the tip fibrillum, PapK and PapF, act as adaptors. PapK is thought to link the pilus rod to the base of the tip fibrillum and regulates the length of the tip fibrillum: its incorporation terminates its growth and nucleates the formation of the pilus rod. PapF is thought to join the PapG adhesin to the distal end of the flexible tip fibrillum. The biogenesis of P pili occurs via the highly conserved chaperone/usher pathway. Periplasmic chaperones are required for the assembly of these pili constructed from pilus subunits. In the absence of an interaction with the chaperone, pilus subunits aggregate and are proteolytically degraded. Two of the genes in t e pap operon, papD and papC , encode the chaperone and usher, respectively. Chaperones such as PapD in E. coli are required to bind to pilus proteins imported into the periplasmic space, partition them into assembly component complexes and prevent non-productive aggregation of the subunits in the periplasm. See Kuehn M. J. et al., Proc. Natl. Acad. Sci. USA,88:10586 (1991). PapD is a periplasmic chaperone that mediates the assembly of P pili. Detailed structural analysis has revealed that the PapD chaperone is the prototype member of a conserved family of periplasmic chaperones in Gram-negative bacteria. Periplasmic chaperones, along with outer membrane ushers, constitute a molecular mechanism necessary for guiding biogenesis of adhesive organelles in Gram-negative bacteria. These chaperones function to cap and partition interactive subunits imported into the periplasmic space into assembly competent complexes, making non-productive interactions unfavorable. PapD binds to each of the pilus subunit types as they emerge from the cytoplasmic membrane and escorts them in assembly-competent, native-like conformations from the cytoplasmic membrane to outer membrane assembly sites comprised of PapC. PapC has been termed a molecular usher since it receives chaperone-subunit complexes and incorporates, or ushers, the subunits from the chaperone complex into the growing pilus in a defined order.

The crystal structures of the PapD alone, or in complex with a peptide from the PapG adhesin, have been solved and refined to 2.5 and 3.0 A, respectively. See Holmgren and Branden, Nature. (1989) 342:248; Kuehn et al., Science. (1993) 268:1234. Recently, the structures of PapD-PapK and FimC-FimH complexes were solved by X-ray crystallography. See Sauer, et al. Science (1999) 285:1058; Choudhury et al., Science. (1999) 285:1062. The PapD molecule has two immunoglobulin-like domains oriented in an L shape to form a cleft at their interface. The chaperone cleft contains surface-exposed residues that are highly conserved. Each immunoglobulin-like domain has a β-barrel structure formed by two antiparallel β-pleated sheets with an overall topology similar to an immunoglobulin fold. All members of the periplasmic chaperone superfamily have a conserved hydrophobic core that maintains the overall features of the two domains. During pilus biogenesis, PapD binds to and caps interactive surfaces on pilus subunits and prevents their premature aggregation in the periplasm. The chaperone-subunit complexes are targeted to the outer membrane usher where subunits assemble in a specific order to form a pilus. Kuehn et al. have shown that the G, β-strand of PapD forms a β-zipper interaction with the highly conserved COOH-terminal motif of pilus subunits. See Kuehn et al., Science. (1993) 268:1234. This COOH-terminal motif also comprises at least part of a primary surface for subunit-subunit assembly interactions, indicating that the direct capping of a primary assembly surface is part of the molecular basis by which periplasmic chaperones prevent the premature oligomerization of pilus subunits. In addition, the β- zipper interaction has been proposed to facilitate the folding of the subunit into a native- like conformation via a template-mediated mechanism.

In the absence of an interaction with the chaperone, pilus subunits aggregate and are proteolytically degraded. Kolmer et al. and Jones et al. have shown that the DegP protease is greatly responsible for the degradation of pilin subunits in the absence of the chaperone (J. Bacteriol. 1996, 178:5925; BIBO 1997, 16:6394). This discovery led to the elucidation of the fate of pilus subunits expressed in the presence or absence of the chaperone using monospecific antisera in Western blots of cytosolic membrane, outer membrane and perplasmic proteins prepared according to methods known in the art.

Thus, prevention or inhibition of normal pilus assembly in Gram-negative bacterium impacts the pathogenicity of the bacterium by preventing the bacterium to infect host tissues. Moreover, changes in the binding between pilus subunits and chaperones can have a dramatic impact on the efficiency of pilus assembly, and thus on the ability of Gram-negative bacterium to adhere to and consequentially, infect host tissues. Drugs that interfere with the assembly of pili should effectively disable pathogens responsible for a wide variety of gram-negative infections, such as those responsible for bladder, kidney and middle ear infections as well as food poisoning, gastric ulcers, diarrhea, meningitis, and other illnesses. Drugs that interfere with the assembly of pili are known collectively as pilicides. Accordingly, a need exists, in general, for compounds and compositions which prevent or inhibit the interaction between pilus subunits and a chaperone as well as methods for utilizing such compounds and compositions for the treatment or prevention of bacterial infections or bacterial colonization. Combinatorial chemistry has become a powerful tool for drug discovery in the pharmaceutical and biotechnology industries. Generally, combinatorial chemistry is defined as the repetitive and systematic covalent attachment of different structural moieties to one another to produce a mixture of numerous distinct molecular entities or target molecules (i.e., combinatorial libraries); desired target molecules include peptides, oligonucleotides, and small organic molecules. Combinatorial chemistry is frequently utilized to generate a group of structurally related analogs which can then be evaluated to establish structure-activity relationships (SAR) and to optimize biological potency. See, e.g., M. A. Gallop et al., J. Med. Chem., 37:1233-1248 (1994).

Solid-phase synthesis was developed into a fast and reliable technique for synthesis of peptides almost 40 years ago. During the last ten years interest in solid-phase organic synthesis has increased substantially due to the emergence of combinatorial and parallel synthesis strategies that are now being widely applied in pharmaceutical research. This has brought about a need for adoption of synthetic organic methodology developed in solution so as to become compatible with various solid-supports. NMR spectroscopy is a well established technique in solution-phase organic chemistry and appears to be the analytical tool of choice also for solid-phase organic synthesis. However, conventional *H and ¹³C NMR spectra of substances attached to a solid support usually suffer from drawbacks such as inhomogeneous line broadening, prolonged spectral acquisition and interference of signals from the solid support. To circumvent these problems, several techniques for structural elucidation have been developed including magic angle spinning, use of selectively ¹³C enriched building blocks, presaturation of support signals, and combinations of these techniques. However, high costs and/or requirements for specialized instrumentation are drawbacks associated with these methods. An additional method used to address the problems with interference of signals from the solid support is to substitute ^!H or ¹³C for another nucleus, such as ¹⁹F, which is not part of commonly used solid-supports. ¹⁹F NMR spectroscopy is almost as sensitive as Η NMR spectroscopy since the natural abundance of ¹⁹F is 100%. Another advantage of ¹⁹F is that the large polarizability of the fluorine electron cloud makes it sensitive to remote changes in electron density, thereby spreading ¹⁹F resonances over a large range of chemical shifts. These features render fluorine well-suited as a sensor for monitoring solid- phase chemical conversions using gel-phase ¹⁹F NMR spectroscopy. The choice of the linker is an important aspect in all solid-phase synthesis and it would be an advantage if the linker can serve analytical purposes. Accordingly, fluorine-derivatized linkers or solid supports should be useful for quantitative monitoring of solid-phase organic synthesis. Accordingly, compositions and methods of synthesis are needed for synthesizing pilicidal compositions. Specifically, compositions and methods are needed which allow for the monitoring of reactions performed on solid-phase as well as determination of the structures of the resulting products while still linked to the solid support.

Summary of the Invention Accordingly, among the objects of the invention, therefore, may be noted:

(i) The provision of antibacterial compositions capable of inhibiting or preventing pilus assembly in a Gram-negative bacterium and libraries of such antibacterial compositions.

(ii) The provision of processes for synthesizing such antibacterial compounds in solution and on solid-phase.

(iii) The provision of methods of using such antibacterial compositions for treating Gram-negative infections; methods of preventing or inhibiting the attachment of Gram- negative organisms to host tissues; methods of preventing or inhibiting biofilm formation; and methods of preventing or inhibiting bacterial colonization by a Gram-negative organism.

(iv) The provision of linker compounds which may be used in the synthesis of the antibacterial compounds and method of synthesizing the linker compounds.

(v) The provision of methods of monitoring the synthesis of the antibacterial compounds and libraries of such antibacterial compounds on solid phase using the linker compounds.

(vi) The provision of complexes of the antibacterial compounds complexed to the linker compounds which are affixed to a solid support.

Briefly, therefore, the present invention is directed to a compound having the formula:

wherein each of R„ R₂ and R₃ is independently a substituted or unsubstituted alkyl (C ₀), substituted or unsubstituted acyl (C_2.15), substituted or unsubstituted aryl (C₆_ι₄), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C₇.₁₅), substituted or unsubstituted heteroarylalkyl or substituted or unsubstituted heterocycloalkyl;R₄ is a carboxy (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂), phosphonate (PO(OH)₂) or ketone (-COR) wherein R is a halogenated or unsubstituted alkyl (C,.₃); and the salts, esters and amines thereof. Another object of the invention is to provide antibacterial compounds and pharmaceutical compositions containing such antibacterial compounds which have broad specificity for a diverse group of Gram-negative bacteria. A further object of this invention is to provide compounds and methods for preventing and inhibiting biofilm formation which comprise administering an effective amount of such compounds to an environment or surface containing Gram-negative bacteria. Additionally, among other objects of the invention is to provide methods for utilizing such compounds, such as methods of treating or preventing Gram-negative infections which comprise providing to a subject an effective amount of the above compositions.

Another aspect of the present invention is to provide fluorinated linkers for use in the synthesis of the N-substituted amino acid derivatives on solid phase. Thus, the present invention is also directed to linker compounds having the formula:

wherein R', is -CO₂H, -(CH₂)_nCO₂H or -O(CH₂)_nCO₂H wherein n is between 1 and 10, preferably n is between 1 and 5, and more preferably, n is 1 or 2; and R'₂ and R'₃ is independently fluorine or hydrogen provided that when either R'₂ or R'₃ is fluorine, the other is hydrogen. Preferably, R'₂ of the linker compound is hydrogen and R'₃ of the compound is fluorine.

A related object of the present invention is to provide processes of synthesis of the above linker compounds. These processes include the steps of hydrolyzing one of the ester moieties of dimethyl-2-fluoroterephtalate, reducing the remaining ester and separating the two regioisomers. Alternatively, another process of synthesis includes (a) dealkylating a 2-fluoro-4-propoxybenzoic acid; (b) reducing the carboxylic acid of the product of step (a) thereby producing a hydroxymethylphenol compound; (c) alkylating the phenolic hydroxyl group of the hydroxymethylphenol compound; and (d) hydrolyzing the product of step (c) under basic conditions. It is another object of the invention is to provide processes for the synthesis of the antibacterial compounds in solution and on solid phase. The process of synthesis of such compounds includes the steps of condensing a compound 6 of Reaction Scheme 1 with a salicyaldehyde selected from the group consisting of the reagent compounds listed in

Table A herein. Preferably, the antibacterial compounds are synthesized on solid phase using the linker compounds of the invention. Such solid phase synthesis of the compounds include the steps of affixing a linker compound to a solid support to give a benzylic alcohol; subjecting the benzylic alcohol to acylation with bromoacetic acid; subjecting the bromoacetate to a nucleophilic substitution with an amine; acylating with ethyl malonyl chloride thereby forming a N-alkyl-N-(malonamic acid ethyl ester)-glycine derivative; condensing the N-alkyl-N-(malonamic acid ethyl ester)-glycine derivative with a salicyaldehyde; and cleaving the compound from the linker compound under acidic or basic conditions.

Another process of solid phase synthesis of the antibacterial compounds provided by the present invention includes the steps of (a) affixing a linker compound onto a solid support to give a benzylic alcohol; (b) coupling a Fmoc-protected amino acid to the benzylic alcohol thereby producing an amino acid functionalized resin; (c) removing the α-amino group of the product of step (b); (c) alkylating the α-amino group of the product of step (c) by reductive alkyation; (d) removing excess aldehyde from the amino acid functionalized resin; (e) acylating with an acid chloride thereby producing a N-(alkylated)- N-(acylated)-amino acid derivative; and (f) cleaving the compound from the linker under acidic or basic conditions. A further object of the present invention is to provide an improved method of synthesizing a combinatorial library wherein the improvement comprises affixing a linker compound of the present invention onto a solid support. This library of compounds synthesized using this improved synthesis can be used to create a library of compounds which can be screened for antibacterial activity. Yet another object of the present invention is to provide methods of monitoring solid-phase synthesis of the antibacterial compounds. The process includes the steps of affixing a linker compound onto a solid support, utilizing a means for measuring a signal originating from the linker compound and utilizing the signal as an internal reference thereby enabling the monitoring of reactions of said solid-phase synthesis of compounds. Preferably, the signal originating from the linker compounds will be a ¹⁹F resonance and is measured using ¹⁹F NMR spectroscopy.

Another related object of the present invention is to provide complexes of the antibacterial compositions complexed to the fluorine linker compounds which are affixed to a solid support. Other objects and features will be in part apparent and in part pointed out hereinafter. Description of the Figures

Figure 1 represents ^F NMR spectra showing (a) resin-bound linker 2, (b) that ~

90% conversion of 2 into 3 was obtained by acylation using 3 eq. of bromoacetic acid, (c) complete transformation of 2 into 3 was obtained after repeating the acylation with 1.5 eq. of bromoacetic acid, and (d) 8 after Knoevenagel condensation of 6 with salicylaldehyde and piperidine, indicating that -20% of the product was cleaved form the resin during the condensation.

Figure 2 represents ¹⁹F NMR spectra of resin-bond products obtained after reductive alkylation of 40 using the reaction conditions given in entries 1-3, Table D. The 19p resonance at d -115 ppm originates from the linker and shows that coupling of Fmoc- Phe-OH to 2 was quantitative. This resonance was used as internal standard. Integration of the 19p resonance originating from the -fluorobenzyl residue (d -116 ppm) showed that: (a) the reaction conditions in entry 1 resulted in -64% alkylation of 40, (b) the conditions in entry 2 led to -55% alkylation, and (c) those in entry 3 gave -78% alkylation of 40. Figure 3 is a gel-phase ^F NMR spectroscopy of resin-bound products obtained after reductive alkylation of 40, as described in entries 4 - 6, Table D, and subsequent acylation with 4-fluoronaphthoyl chloride. The line-broadening in the spectra is due to rotamers about the amide bond in 32a. Integration over the 19p resonances revealed that: (a) use of 1.5 eq. -F-benzaldehyde (entry 4) resulted in -8% dialkylation, (b) increasing the excess ofp-F-benzaldehyde to 3 eq. (entry 5) gave slightly increased formation of dialkylated 42a (-10%), whereas (c) removing excess aldehyde prior to addition of NaBH3CN (entry 6) proved to be the most efficient method, which reduced the formation of 42a to <2% and increased the overall yield of 43b to -92%.

Figure 4 is a gel-phase ^F NMR spectra of 43b obtained after reductive alkylation of 40 with 4,4,4-trifluorobutyraldehyde under the following conditions: (a) 4,4,4- trifluorobutyraldehyde (3 eq.) with direct addition of NaBH3CN gave 43b in 48%, whereas (b) removal of excess aldehyde prior to the addition of NaBH CN increased the yield of 43b to 66%. The resins were acylated with 4-fluoronaphthoyl chloride after the reductive alkylation.

Abbreviations and Definitions The terms and abbreviations have the indicated meaning as used herein. The term "alkyl" is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof. "Lower alkyl" means alkyl groups of from 1 to 8 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl, pentyl, hexyl, octyl, cyclopropylenthyl, bornyl and the like.

The "alkenyl" includes C₂-C₈ unsaturated hydrocarbons of a linear, branched, or cyclic (C₅-C₆) configuration and combinations thereof. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, c-hexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl and the like.

The term "alkynyl" includes C₂-C₈ hydrocarbons of a linear or branched configuration and combinations thereof containing at least one carbon-carbon triple bond. Examples of alkynyl groups include ethyne, propyne, butyne, pentyne, 3-methyl-l-butyne, 3, 3 -dimethyl- 1 -butyne and the like.

The term "alkoxy" refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy and the like. The term "acyl" refers to the group ~C(O)-Z', where Z is a lower alkyl. As used herein, "lower alkyl" means alkyl groups of from 1 to 8 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl, pentyl, hexyl, octyl, cyclopropylenthyl, bornyl and the like.

The term "acylamino" refers to acylamino groups of from 1 to 8 carbon atoms of a straight, branched or cyclic configuration and combinations thereof. Examples include acetylamino, butylamino, cyclohexylamino and the like.

The term "aryl" and "heteroaryl" mean a five or six-membered aromatic or heteroaromatic ring containing zero to three heteroatoms selected form O, N, and S; a bicyclic nine or ten-membered aromatic or heteroaromatic ring system containing zero to three heteroatoms selected from O, N, and S; or a tricyclic thirteen or fourteen-membered aromatic or heteroaromatic ring system containing zero to three heteroatoms selected from O, N, and S.

The term "arylalkyl" means an alkyl residue attached to an aryl ring. Examples include, e.g., benzyl, phenethyl and the like. The term "carbonyl" refers to the group -CO. Examples of organic carbonyl compounds are ketones, aldehydes, carboxylic acids and the like.

The term "heteroarylalkyl" means an alkyl residue attached to a heteroaryl ring. Examples include, pyridinylmethyl, pyrimidinylethyl and the like.

The term "heterocycloalkyl" means a cycloalkyl where one to two of the methylene (CH₂) groups is replaced by a heteroatom such as.O, NR' (wherein R' is H or alkyl), S or the like; with the proviso that when two heteroatoms are present, they must be separated by at least two carbon atoms. Examples of heterocycloalkyls include tetrahydropyranyl, piperidynl, dioxanyl and the like.

The term "hydrocarbyl" is meant a monovalent substituent containing only carbon and hydrogen which may be straight or branched chain, saturated or unsaturated, aromatic or nonaromatic or both and can be cyclic or noncyclic. An example of a hydrocarbyl alcohol of 1-lOC could include cyclopentyl ethyl alcohol, 2-pentyl alcohol, 3-butynyl alcohol, 2,4-dimethyl hexyl alcohol, benzyl alcohol and the like. The term "carboxyalkyl" means ~C(O)R", wherein R" is alkyl.

As used herein, "coumarin" shall refer to the following structure:

The term "substituted" alkyl, alkenyl, alkynyl, cycloalkyl, or heterocycloalkyl means alkyl, alkenyl, alkynyl, cycloalkyl or heterocycloalkyl wherein up to three H atoms on each C atom therein are replaced with halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxamido, cyano, carbonyl, NO₂, NR R (wherein each R' and R" is H, alkyl or arylalkyl), alkylthio, alkylthiol, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, and substituted phenyl, benzyl, heteroaryl, phenoxy, benzyloxy or heteroaryloxy.

As used herein "natural number" means a positive number including zero. In general the abbreviations used herein for designating the amino acids are based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature. See Biochemistry, 11, 1732 (1972). For instance Ala, Leu and Gly represent the amino acid residue of L-alanine, L-leucine and glycine, respectively. The term "residue" means a radical derived from the corresponding L-amino acid by eliminating the hydroxy portion of the carboxy group and a hydrogen of the α-amino group. The term "amino acid side chain" is that part of an amino acid exclusive of the ~CH(NH₂)COOH portion, as defined by K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin Inc., New York and

Amsterdam, 1966, pgs 2 and 33. Examples of amino acid side chains are -CH₂-CH(CH₃)₂ (the side chain of leucine), -H (side chain of glycine), -CH₃ (the side chain of alanine), -CH₂CONH₂ (the side chain of asparagine), -CH₂SH (the side chain of cysteine), 2- (3-indolyl)-ethyl (the side chain of tryptophan), -CH₂CH₂SCH₃ (the side chain of methionine), -CH₂OH (the side chain of serine), 4-hydroxybenzyl (the side chain of tyrosine), -CH(CH₃)₂ (the side chain of valine), benzyl (the side chain of phenylalanine), -CH(CH₃)CH₂CH₃ (the side chain of isoleucine), -CH(OH)CH₃ (the side chain of threonine) and the like. Note, therefore, that the term "amino acid side chain" includes hydrogen. The amino acids and amino acid residues are of the L or D configuration. It will be noted that the structures of the compounds of this invention include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry are included within the scope of this invention. Such isomers are obtained in substantially pure form by classical separation techniques and by sterically controlled synthesis and have arbitrarily been named as isomers L or D, respectively.

The terms "hydrophobic amino acid" and "hydrophobic amino acid residue" as used interchangeably herein means the common amino acids and amino acid residues having hydrophobic aromatic or aliphatic side chains which include tyrosine, tryptophan, phenylalanine, histidine, valine, cysteine, leucine, isoleucine, alanine, glycine, and methionine.

The terms "polar amino acid" and "polar amino acid residue" as used interchangeably herein means common amino acids and amino acid residues having polar side chains which include serine, threonine, glutamine and asparagine.

The terms "charged amino acid" and "charged amino acid residue" as used interchangeably herein means the common amino acids and amino acid residues having charged side chains which include lysine, arginine, aspartic acid and glutamic acid.

As utilized herein, the term "pilus" or "pili" relates to fibrillar heteropolymeric structures embedded in the cell envelope of many tissue-adhering pathogenic bacteria, notably pathogenic gram negative bacteria. In the present specification, the terms pilus and pili will be used interchangeably. A pilus is composed of a number of "pilus subunits" which constitute distinct functional parts of the intact pilus.

The term "chaperone" relates to a molecule in living cells which bind to pili subunits during the assembly of pili structures. Many molecular chaperones are involved in the process of pilicide biogenesis. Specialized molecular chaperones are "periplasmic chaperones" which are bacterial molecular chaperones exerting their main actions in the

"periplasmic space." The periplasmic space constitutes the space in between the inner and outer bacterial cell membrane. Periplasmic chaperones are involved in the process of correct assembly of intact pili structures. When used herein, the use of the term

"chaperone" designates a molecular, periplasmic chaperone unless otherwise indicated.

The phrase "preventing or inhibiting binding between pilus subunits and a periplasmic chaperone" indicates that the normal interaction between a chaperone and its natural ligand, i.e., the pilus subunit, is being affected either by being inhibited, expressed in another manner, or reduced to such an extent that the binding of the pilus subunit to the chaperone is measurably lower than is the case when the chaperone is interacting with the pilus subunit at conditions which are substantially identical (with regard to pH, concentration of ions, and other molecules) to the native conditions in the periplasmic space. Measurement of the degree of binding can be determined in vitro by methods known to the person skilled in the art (microcalorimetry, radioimmunoassays, enzyme based immunoassays, surface-plasmon resonance, etc.).

The term "linker" refers to any molecule containing a chain of atoms, e.g., carbon, nitrogen, oxygen, etc., that serves to link the molecules to be synthesized on the support with the support. The linker is usually attached to the support via a covalent bond, before synthesis on the support starts, and provides one or more sites for attachment of precursors of the molecules to be synthesized on the support. Various linkers can be used to attach the precursors of molecules to be synthesized to the solid phase support. The term "solid support" refers broadly to supports used in the solid phase synthesis of, for example, peptides, nucleic acids, oligonucleotides, and small organic molecules. Solid supports include, but are not limited to, polymer resins (e.g., polyethylene glycol and polystyrene), gels (e.g., polyethylene glycol gels), polyacrylamide/polyethylene glycol copolymer resins, controlled pore glass supports (e.g., the CPG supports commercially available from Millipore), and silica beads and wafers.

The term "antibodies" also includes any immuno logically reactive fragment of the immunoglobulins such as Fab, Fab' and F(ab')2 fragments as well as modified immunoreactive forms such as Fv regions, which are produced by manipulation of the relevant genes. The term "treatment" includes both prophylaxis and therapy. Thus, in treating a subject, the compounds of the invention may be administered to a subject already harboring a bacterial infection or in order to prevent such infection from occurring.

The phrase "an effective amount" means an amount of the substance in question which will in a majority of subjects have either the effect that the disease caused by the pathogenic bacteria is cured or, if the substance has been given prophylactically, the effect that the disease is prevented from manifesting itself. The term "an effective amount" also implies that the substance is given in an amount which only causes mild or no adverse effects in the subject to whom it has been administered, or that the adverse effects may be tolerated from a medical and pharmaceutical point of view in the light of the severity of the disease for which the substance has been given.

The phrase "subject in need thereof means in the present context a subject, which can be any animal, including a human being, who is infected with, or is likely to be infected with, tissue-adhering pilus-forming bacteria which are believed to be pathogenic.

Detailed Description The present invention is directed to a novel class of compounds which are effective in preventing or inhibiting pilus biogenesis and activity. The compounds of the invention may be effective in treating, preventing and inhibiting bacterial infections caused by Gram-negative organisms. Further, the present invention is directed to methods of utilizing such pilicidal compounds and to processes and compositions useful for the synthesis of such pilicidal compounds. The compounds of this invention exert their effects by interfering with the function of pilus chaperones to form pili from pilus subunits in the periplasm of the bacterium. Specifically, the compounds of the present invention inhibit or prevent the formation of the complex between PapD and PapG by binding to the

PapD chaperone, thus inhibiting or preventing the formation of the P pili which thereby reduces the capacity of piliated bacteria to attach to host tissues. Similarly, the compounds inhibit the formation of the complex between FimC and FimH, thus inhibiting or preventing the formation of the Type 1. Such interference is particularly effective since the inability of the pilus to attach to target tissues results in the loss of ability of the bacteria to infect the tissue.

Accordingly, the invention is directed generally to compounds of the formula:

wherein each of R„ R₂ and R₃ is independently a substituted or unsubstituted alkyl (C ₀), substituted or unsubstituted acyl (C₂.₁₅), substituted or unsubstituted aryl (C₆.₁₄), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C_7.15), substituted or unsubstituted heteroarylalkyl or substituted or unsubstituted heterocycloalkyl;R₄ is a carboxy (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂), phosphonate (PO(OH)₂) or ketone (-COR) wherein R is a halogenated or unsubstituted alkyl (C^); and the salts, esters and amines thereof.

The substituents on any alkyl or alkylene moiety may be selected from the group consisting of halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxamido, cyano, carbonyl, NO₂, NR R (wherein each R and R" is H, alkyl or arylalkyl), alkylthio, alkylthiol, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, and substituted phenyl, benzyl, heteroaryl, phenoxy, benzyloxy or heteroaryloxy.

Preferably, the active forms of the compounds of the invention are those wherein the chirality of the carbon at R_j is "S." The same stereochemistry is retained in the analogous compounds and derivatives although the designation of the chirality at each position may be different depending on the nature of the various substitutions made. For example, in an embodiment wherein R, is -CH₂SH, although the same stereochemistry with regard to the remainder of the molecule remains the same, the chirality would be designated "R." The invention, of course, includes racemic mixtures which include stereoisomers as well as mixtures of the various diasteriomers, as long as this particular form is included.

Included in such derivatives are the salts, especially pharmaceutically acceptable salts. Salts of carboxylic acids include those derived from inorganic bases such as the sodium, potassium, lithium, ammonium, calcium, magnesium, zinc, aluminum and iron salts and the like, as well as those derived from organic, especially nontoxic, bases such as the primary, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines and basic ion-exchange resins. Examples of such compounds capable of forming salts are isopropyl amine, trimethyl amine, triethyl amine,

2-dimethyl aminoethanol, dicyclohexyl amine, amino acids such as lysine, arginine and histidine, caffeine, procaine, betaene, theobromine, purines, piperazines, and the like.

The compounds of the present invention may also be in esterified form. Typically, the esters are prepared from a hydrocarbyl alcohol. Examples of hydrocarbyl alcohols of C,_.10 include but are not limited to, cyclopentyl ethyl alcohol, 2-pentyl alcohol, 3-butynyl alcohol, 2,4-dimethyl hexyl alcohol, benzyl alcohol. Particularly preferred are alkyl alcohols. Typical examples of alkyl alcohols include but are not limited to, methyl, ethyl, t-butyl, cyclohexyl. The alkyl esters of the compounds of the invention are particularly preferred, especially alkyl esters wherein the alcohol contains C,_₄.

In one embodiment, each R„ R₂ and R₃ is independently an amino acid residue side chain and R₄ is carboxyl or aldehyde. Preferably, the amino acid residue side chain of each R,, R₂ and R₃ is independently selected from the group consisting of hydrogen, p- hydroxybenzyl, 2-(3-indolyl)-ethyl, benzyl, 5-imidazole, isopropyl, isobutyl, 2- methylpropyl, methyl and 2-thiomethylethyl and more preferably, R_[ is hydrogen or p- hydroxybenzyl.

In another embodiment, R, is selected from side chains of amino acid residues; R₂ is substituted or unsubstituted alkyl, arylalkyl, heteroarylalkyl, and heterocycloalkyl; R₃ is substituted or unsubstituted alkyl, aryl, and heteroaryl; and R₄ is a carboxyl group (- CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂) or phosphonate (PO(OH)₂). It is preferred that the R, is selected from the side chains of hydrophobic aromatic, hydrophobic aliphatic polar and charged amino acid residues, R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl, R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, and R₄ is -CO₂H or -CHO. Another preferred embodiment include compounds wherein R, is selected from the side chains of hydrophobic aromatic, hydrophobic aliphatic, and polar and charged amino acid residues, R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl, R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl and R₄ is -CO₂H, -CONH₂ or -CHO. Yet another preferred embodiment includes compounds wherein K_{ is selected from hydrophobic aromatic and hydrophobic aliphatic amino acid residues, R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl, R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl and R₄ is - CO₂H. Examples of hydrophobic aromatic or aliphatic amino acid residues are tyrosine, tryptophan, phenylalanine, histidine, valine, leucine, isoleucine, alanine, glycine, cysteine and methionine; examples of polar amino acid residues are serine, threonine, glutamine and asparagine; and examples of charged amino acid residues are lysine, arginine, aspartic acid and glutamic acid. It is particularly preferred that R₄ is -CO₂H as those compounds containing a carboxylic acid functionality demonstrate effective antibacterial properties.

In another embodiment, R, is hydrogen, benzyl, 4-aminobutyl and p- hydroxybenzyl; R₂ is (CH₂)_mA wherein m is between 0 and 3 and A is n-butyl, 2- methoxyethyl, benzyl and 2-(3-indolyl)-ethyl; R₃ is isobutyl,

and R₄ is carboxyl.

Non-limiting preferred embodiments of the compounds of the present invention include

N-Benzyl-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-glycine (Compound 9{3, 1})

Ν-[2-(lΗ-Indol-3-yl)-ethyl]-Ν-(naphtalene-2-carbonyl)-tyrosine (Compound 19{3, 3})

N-[2-(lH-Indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-tyrosine

(Compound 17 {3, 1})

N-[2-(lH-Indol-3-yl)-ethyl]-N-(3-methyl-butyryl-carbonyl)-tyrosine

(Compound 19 {3, 4})

N-[2-(lH-Indol-3-yl)-ethyl]-N-(3-oxo-3H-naphtho[2,l-b]ρyran-2-carbonyl)-glycine

(Compound 9 {4, 2})

N-[2-(lH-Indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-lysine

(Compound 17 {4, 1})

and

N-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)-glycine (Compound 19{1, 2}).

Synthesis of Antibacterial Compounds

The compounds of the invention can be prepared using approaches illustrated by Reaction Schemes 1-3 and 5. Reaction Schemes 1-3 may be performed in solution or on solid phase. Solid-phase synthesis is employed by first coupling a linker compound to a solid support as illustrated by compound 2 in Reaction Scheme 1. This compound affixed to the solid support is used as a linker in solid phase synthesis as exemplified during the synthesis of compounds 9. REACTION SCHEME 1

6(1-4} 8(1-4, 1-5} 9(1-4, 1-5}

Reaction Scheme 1 reaction conditions: (i) Pentafluorphenol, DIC, EtOAc, TentaGel S NH₂, 0 °C to room temperature; (/ ) bromoacetic acid, DIC, HOBt, DMAP, THF, room temperature; (iii) 4{I-4), CH₃CN, 0 °C; (iv) ethyl malonyl chloride, DIPEA, CH₂C1₂, 0 °C; (v) 7 {1-5}, piperidine, CH₃CN, reflux; (vi) aquoeus 1 M LiOH, THF:H₂O:MeOH (3:1 :1).

Reaction Scheme 1 is used to synthesize N-substituted glycine derivative compounds, i.e., wherein R, is a hydrogen. As is shown in Reaction Scheme 1, acylation of the benzylic alcohol 2 with bromoacetic acid gives 3. Preferably, this acylation reaction is repeated once in order for complete coupling of bromoacetic acid to occur. Nucleophilic substitution of the bromoacetate with an amine in solution gives 5. Amidation of 5 with ethyl malonyl chloride gave a N-alkyl-N-(malonamic acid ethyl ester)-glycine 6. Condensation of 6 with a salicylaldehyde and cleavage of the product from the solid phase under basic conditions gives the N-substituted amino-acid compounds 9. Non-limiting examples of salicyaldehydes and amines which may be used in Reaction Scheme 1 are listed in Table A.

TABLE A

Preferably, Reaction Scheme 1 may be utilized to prepare the library of N- substituted glycine-derivative compounds of Table B.

Preferably, in one embodiment, N-substituted glycine-derivative compounds having the formula:

wherein R_t is hydrogen, R₂ is (CH₂)_mA wherein m is between 0 and 3 and A is selected from the group consisting of n-butyl, benzyl and 3-(2-indolyl)-ethyl, R₃ is coumarin and R₄ is carboxyl, can be synthesized using solid phase synthesis using the fluorinated linker compounds of the present invention as described herein. Preferably, solid phase synthesis of these N-substituted glycine-derivative compounds comprise the steps of: affixing a fluorinated linker compound onto a solid support to give a benzylic alcohol; subjecting the benzylic alcohol to acylation with bromoacetic acid; subjecting the bromoacetate to a nucleophilic substitution with an amine; acylating with ethyl malonyl chloride thereby forming a N-alkyl-N-(malonamic acid ethyl ester)-glycine derivative; condensing the N- alkyl-N-(malonamic acid ethyl ester)-glycine derivative with a salicyaldehyde; and cleaving the compound from the linker compound under acidic or basic conditions.

Compounds 6 may also be synthesized in solution by alkylation of amines with ethyl bromoacetate. This is illustrated for the preparation of 13 in Reaction Scheme 2.

Subsequent amidation with ethyl malonyl chloride gives 14 in Reaction Scheme 2 which is equivalent to 6 in Reaction Scheme 1.

Further, the N-alkyl-N-(malonamic acid ethyl ester)-amino acid derivative (compound 6 of Reaction Scheme 1) may be prepared in solution as described by Simon et al., Proc. Natl. Sci. USA (1992) 89:9367-9371 and Liskamp et al, Chem Eur. J. (1998) 4:1570-1580, which are both incorporated herein by reference.

REACTION SCHEME 2

Reaction Scheme 2 reaction conditions: (i) 12(7}, lib, ethyl bromoacetate, DMF, 0 °C; (t 12(2-4}, 11a, Et₃N, DMF, 70 °C; (iii) ethyl malonyl chloride, DIPEA, CH₂C1₂, 0 °C; (iv) 15(7}, piperidine, CH₃CN, reflux; (v) for 16(2-4, 7} and 18(2-5, 2-4} TFA:H₂O (2:1), room temperature; (vi) for 13(7} pentafluorophenol, DIC, 15(2}, EtOAc; for 13(2} DIC, 15(2}, EtOAc; for 13(5} DIPEA, 15(5}, CH₂C1₂, 0 °C, for 13(5} DIPEA, 15(4}, CH₂CL₂, 0 °C; (vii) for 18(7, 2} aqueous 1 M LiOH, THF:H₂O:MeOH (3:1:1); (viii) n- butylamine, DIC, CH₂C1₂, 0 °C. O 01/20995

23

T Reagents 15 (1-4}

Additional N-substituted amino acid compounds 17 and 19 wherein R, is o-amino, n-butyl, benzyl or p-hydroxybenzyl are prepared using the synthesis of Reaction Scheme 2. As can be seen in Reaction Scheme 2, compound 13 is obtained in solution from a suitable protected amino acid (e.g., Phe, Tyr, Lys) by alkylation with an alkyl halide such as 2-(3-indoyl)-ethyl bromide. Compound 13 is then acylated by a carboxylic acid such as 2-napthalene carboxylic acid, which has been activated e.g., as a pentafluorophenyl ester, with a carbodiamine derivative or an acid chloride. Deprotection of the resulting 18 by treatment with an acid, preferablytrifluoroacetic acid, followed by hydrolysis of the ester moiety then gives 19. In a preferred embodiment, compound 17 is prepared essentially as described above for the synthesis of 9 (Reaction Scheme 1) on solid phase.

Synthesis of Combinational Libraries

Various compounds similar in structure to the compounds of the present invention may be synthesized using combinational techniques. Suitable combinational techniques include those described in U.S. Patent Nos. 5,736,412, 5,840,500, 5,847,150, 5,852,028, 5,856,107, 5,856,496, 5,859,027 and 5,861,532. These techniques can be performed on solid or solution phase.

The preferred process of the present invention is a "solid phase synthesis" (SPS). Organic synthesis performed on solid phase constitutes an efficient method for preparation of large combinatorial libraries containing structurally distinct molecules. The reaction is carried out on macroscopic particles made of material insoluble in the reaction medium. A key aspect of any solid-phase synthesis is the choice of a linker compound which will be affixed to the surface of the solid support. The linker compound should be orthogonal to the required reaction conditions and allow quantitative cleavage of the product under mild conditions. Solid supports (e.g., polystyrene resin beads and silica chips) and, concomitantly, solid phase synthesis techniques are routinely utilized in generating combinatorial libraries. Each derivative is usually prepared in sufficient quantity to permit screening and analysis by conventional methods, e.g., HPLC and mass spectral analysis. Applicants have shown that ¹⁹F chemical shifts, as well as linewidths for compounds attached to polyethylene glycol grafted polystyrene resins (TentaGel resins), approach those in solution. See Svensson et al., Tetahedron Lett. 37: 7649-7652 (1996). Accordingly, gel-phase ¹⁹F NMR spectroscopy is well-suited for adoption of solution- phase chemistry to various solid supports and for optimization of reaction conditions. The insertion of a fluorine atom into a key position on the linker for use in SPS allows the linker to serve as a diagnostic marker during several of the reactions during solid phase synthesis. The fluorine atom of the linker compound allows for the monitoring and optimization of several reactions using ¹⁹F NMR spectroscopy such as the attachment of the linker to the solid phase, coupling of the first building block to the linker and cleavage of the product. Accordingly, the present invention is also directed to a novel class of fluorinated linker compounds having the formula:

wherein R', is -CO₂H, -(CH₂)_nCO₂H or -O(CH₂)_nCO₂H wherein n is between 1 and 10, preferably, n is between 1 and 5, and even more preferably, n is 1 or 2; and R'₂ and R'₃ is independently fluorine or hydrogen provided that when either R'₂ or R'₃ is fluorine, the other is hydrogen. Particularly preferred fluorinated linker compounds include, but are not limited to,

3-fluoro-4-hydroxymethylbenzoic acid (compound 22),

2-fluoro-4-hydroxymethylbenzoic acid (compound 23)

3-fluoro-4-(hydroxymethylphenyl)-propionic acid (compound 29) and

3-fluoro-4-hydroxymethyl-phenoxy-acetic acid (compound 1).

These preferred fluorinated linker compounds 1, 22, 23 and 29 are synthesized as shown below in Reaction Schemes 3 and 4. REACTION SCHEME 3

Reaction Scheme 3 reaction conditions: (i) aqueous 1 M LiOH, THF:MeOH:H₂O (3:1:1), 0 °C to room temperature; (ii) LiBH₄, THF, 22 53%, 23 40% in two steps; (iii) aqueous Cs₂CO₃ (20%), MeOH:H₂O (10:1), then BnBr, DMS, 79%; (iv) TPAP (5 mol%), N- methylmorpholine N-oxide, 4 A molecular sieves, CH₂C1₂, 74%; (v) ΝaH, (EtO)₂P(O)CH₂CO₂C₂H₅, THF, 0 °C, 76%; (vi) Pd/C, H₂, EtOH:EtOAc (3:1) 4 atm, 88%; (vii) BH₃-DMS, (CH₃O)₃B, THF, 89%; (viii) aqueous 1 M LiOH, THF:MeOH:H₂O (3:1:1), 0 °C, 93%. As shown in Reaction Scheme 3, Linkers 22 and 28 were prepared from dimethyl-

2-fluoroterephtalate. Νon-selective, basic hydrolysis of one of the ester moieties of dimethyl-2-fluoroterephtalate, followed by reduction of the remaining ester with LiBH₄ and chromatographic separation of the two regioisomers gave the fluorinated 4- (hydroxymethyl) benzoic acids, linker compounds 22 and 23. Protection of 23 as a benzyl ester, followed by Swern oxidation and condensation of the resulting aldehyde with triethyl phosphonoacetate afforded 26 which was reduced to 27. Reduction of the carboxyl group of 27 using BH₃-DMS and (MeO)₃B followed by hydrolysis of the ethyl ester then furnished linker 29. REACTION SCHEME 4

Reaction Scheme 4 reaction conditions: (i) BBr₃, CH₂C1₂, -78 °C to room temperature, 89%; (t0 BH₃-DMS, (CH₃O)₃B, THF, 90%; (iii) BrCH₂CO₂C₂H₅, DBU, CH₃CN, reflux, 74%; (iv) aqueous 1 M LiOH, THF:MeOH:H₂O (3:1:1) 87%. Applicants employed 3-fluoro-4-hydroxymethyl-phenoxy-acetic acid, linker 1, to prepare compound 9 (4, 1}, N-[2-(lH-Indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran-3- carbonyl)-glycine which is a N-substituted glycine derivative exhibiting PapD chaperone inhibiting activity and FimC/FimH inhibiting activity, as indicated in Figure 1.

As shown in Reaction Scheme 4, linker 1 is prepared by BBr₃-induced dealkylation of 2-fluoro-4-propoxybenzoic acid, followed by reduction with BH₃-DMS and (MeO)₃B to give 3-fluoro-4-hydroxymethylphenol 32. O- Alkylation of 3-fluoro-4- hydroxymethylphenol 32 with bromoethyl acetate and DBU as a base gives ester and subsequent treatment with LiOH results in linker 1.

Alternatively, linker 1 may be used to monitor the synthesis of another class of potential pilicides consisting of N-alkylated and N-acylated amino acids which are different from the glycine derivations previously described. Preferably, such methods for monitoring solid-phase synthesis of such compound include affixing a fluorinated linker compound onto a solid support; utilizing a means for measuring a signal, preferably a ¹⁹F resonance, which originates from the linker compound; andutilizing said signal as an internal reference thereby enabling the monitoring of reactions of said solid-phase synthesis of compounds. Preferably, the ¹⁹F resonance is measured using ¹⁹F ΝMR spectroscopy.

As shown in Reaction Scheme 5 below, linker 1 is used in combination with gel- phase ¹⁹F ΝMR spectroscopy to develop conditions for solid phase synthesis of another class of potential pilicides consisting of N-alkylated and N-acylated amino acids which are different from the glycine derivatives produced in Reaction Scheme 1. Applicants utilized the fluorinated linker 1, in combination with fluorinated building blocks, to establish conditions for reductive alkylation of amino acids that could be applied to both aromatic and aliphatic aldehydes. Phenylalanine resin 40 was prepared by coupling Fmoc-Phe-OH to 2 in the presence of HOBt, DIC and DMAP, followed by removal of the N -Fmoc protecting group with 20% piperidine in DMF. The α-amino group in 40 was then alkylated with -fluorobenzaldehyde using ΝaBH CΝ as reducing agent under different conditions (See Table C of Examples). The ^F resonance originating from the linker moiety of 40 served as internal reference, and integration over the 19p resonance of the N- linked / fluorobenzyl residue enabled evaluation of the outcome of the reactions.

Preferably, this method may also be utilized for the reductive alkylation of resin- linked 40 using aliphatic aldehydes. Alkylation of 40 by treatment with 4,4,4- trifluorobutyraldehyde and ΝaBH3CΝ using the conditions of entry 5 in Table C (see Example 5), followed by acylation with 4-fluoronaphtoyl chloride resulted in formation of 43b. Alternatively, removal of excess aldehyde prior to the addition of NaBH3CN, i.e. using the conditions of entry 6 in Table C of Example 5, and subsequent acylation furnished 43b.

Reaction Scheme 5

Reaction scheme 5 reaction conditions: (i) Pentafluorophenol, DIC, TentaGel S NH2, EtOAc; Nα-Fmoc-Phe-OH, HOBt, DIC, DMAP, THF; (iii) 20% piperidine in DMF; (/v)/>-F-C6H₄CHO or F3CCH2CH2CHO, MeOH containing 1% HO Ac; then ΝaBH₃CΝ; (v) 4-fluoronaphtoyl chloride, DIPEA, CH2CI2, 0°C to room temperature; (vi) aqueous 1M LiOH, THF:MeOH:H₂ O (3:1 :1).

Accordingly, solid phase synthesis of a class of potential antibacterial compounds consisting of N-alkylated and N-acylated amino acids which are different from the glycine derivatives are also synthesized using the fluorinated linker compounds. Preferably, solid phase synthesis of such compounds include the steps of: a. affixing a fluorinated linker compound 22, 23, 29 or 1 onto a solid support to give a benzylic alcohol; b. coupling a Fmoc-protected amino acid to the benzylic alcohol thereby producing an amino acid functionalized resin; c. removing the Fmoc-protecting group from the α-amino group of the product of step (b); d. alkylating the α-amino group of the product of step (c) by reductive alkyation; e. removing excess aldehyde from the amino acid functionalized resin; f. acylating with an acid chloride thereby producing a N-(alkylated)-N- (acylated)-amino acid derivative; and g. cleaving the compound from the linker under acidic or basic conditions.

Screening Assays

The array of synthesized candidate compounds is screened into relevant assays, e.g., antichaperone or antimicrobial assays, and the compounds are further characterized according to chemical identity and purity using conventional techniques. The assay can be scored on a real-time basis and further modifications made accordingly. Antichaperone binding activity can be measured by any number of direct methods such as monitoring spectral changes in the compound and/or chaperone, determining the extent of compound binding to immobilized chaperone or vice versa, by indirect methods such as competition assays to determine the extent to which these compounds inhibit chaperone binding to target pilus subunits and/or derivatives (Soto, et al., Embo J.. (1998) 17:6155; Karlsson et al., Bioorg Med Chem. (1998) 6:2085)) and/or synthetic peptides corresponding to subunit fragments known to bind chaperones (Kuehn, et al., Science. (1993) 262:1234).

Assays to determine the extent of pilus expression in the presence of these compounds may be performed as described in Soto et al., supra, and/or by haemagglutination assays as described in Striker et al., Mol Microbiol. (1995) 16:1021.

Assays of inhibition of bacterial binding to target tissues in the presence of these compounds would be performed as described in Striker, et al., supra.

Conventional techniques, e.g., radial diffusion method against E. coli ML-35P, L. monocytogenes Strain EGD and yeast phase C. albican, may be used to evaluate the spectra of the antimicrobial activity for the novel compounds of the present invention.

Antibodies

Antibodies to the compounds of the invention may also be produced using standard immunological techniques for production of polyclonal antisera and, if desired, saving the antibody-producing cells of the immunized host for sources of monoclonal antibody production. Techniques for producing antibodies to any substance of interest are well known. The immunogenicity of the substance may be enhanced by coupling the hapten to a carrier. Carriers useful for this purpose include substances which do not themselves elicit an immune response in the subject mammal. Common carriers used include keyhole limpet hemocyanin (KLH) diptheria taxoid, serum albumin, and the viral coat protein of rotavirus, VP6. Coupling the hapten to the carrier is effected by standard techniques such as contacting the carrier with the compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide or through the use of linkers.

The compounds of the invention in immuno genie form are then injected into a suitable mammalian host and antibody titers in the serum are monitored. Polyclonal antisera may be harvested when titers are sufficiently high.

Alternatively, antibody-producing cells of the host such as spleen cells or peripheral blood lymphocytes may be harvested and immortalized. The immortalized cells are then cloned as individual colonies and screened for the production of the desired monoclonal antibodies. The genes encoding monoclonal antibodies secreted by selected hybridomas or other cells may be recovered, manipulated if desired, for example, to provide multiple epitope specificity or to encode a single-chain form and may be engineered for expression in alternative host cells.

Administration of Compounds

The antibacterial compositions of the present invention may be utilized to inhibit pili assembly by providing an effective amount of such compositions to a subject. For use as antimicrobials for treatment of animal subjects, the compounds of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, e.g., prevention, prophylaxis, therapy; the compounds are formulated in ways consonant with these parameters. A summary of such techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.

For administration to animal or human subjects, the dosage of the compounds of the invention is typically 0.1-lOOmg/kg. However, dosage levels are highly dependent on the nature of the infection, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration.

The dosage of such a substance is expected to be the dosage which is normally employed when administering antibacterial drugs to patients or animals, i.e. 1 μg - 1000 μg per kilogram of body weight per day. The dosage will depend partly on the route of administration of the substance. If the oral route is employed, the absorption of the substance will be an important factor. A low absoφtion will have the effect that in the gastro-intestinal tract higher concentrations, and thus higher dosages, will be necessary. Also, the dosage of such a substance when treating infections of the central nervous system (CNS) will be dependent on the permeability of the blood-brain barrier for the substance. As is well-known in the treatment of bacterial meningitis with penicillin, very high dosages are necessary in order to obtain effective concentrations in the CNS. It will be understood that the appropriate dosage of the substance should suitably be assessed by performing animal model tests, wherein the effective dose level (e.g. ED₅₀) and the toxic dose level (e.g. TD₅₀) as well as the lethal dose level (e.g. LD₅₀ or LD₁₀) are established in suitable and acceptable animal models. Dosage levels vary considerably depending on the nature of the infection, the condition of the patient and the frequency and method of administration. Further, if a substance has proven efficient in such animal tests, controlled clinical trials should be performed. Needless to state that such clinical trials should be performed according to the standards of Good Clinical Practice.

In general, for use in treatment, the compounds of the invention may be used alone or in combination with other antibiotics such as erythromycin, tetracycline, macrolides, for example azithromycin and the cephalosporins. Depending on the mode of administration, the compounds will be formulated into suitable compositions to permit facile delivery to the affected areas.

Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth. For oral administration, the compounds can be administered also in liposomal compositions or as microemulsions. Suitable dosage forms for oral use include tablets, dispersable powders, granules, capsules, suspensions, syrups, and elixers. Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose and talc. Tablets may also contain granulating and disintegrating agents such as starch and alginic acid, binding agents such as starch, gelatin and acacia, and lubricating agents such as magnesium stearate, stearic acid and talc. Tablets may be uncoated or may be coated by unknown techniques; e.g., to delay disintegration and absoφtion. Inert diluents and carriers which may be used in capsules include, for example, calcium carbonate, calcium phosphate and kaolin. Suspensions, syrups and elixers may contain conventional excipients, for example, methyl cellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, e.g. ethyl-p- hydroxybenzoate. Additionally, various sustained release systems for drugs have also been devised as in U.S. Patent No. 5,624,677, which claims a composition providing a relatively slow release of water-soluble drugs for delivery via the sublingual or buccal routes, for example.

Industrial Applicability

The compounds of the invention are effective in inhibiting a variety of Gram- negative bacteria and have several industrial uses, well known to those skilled in such arts, relating to their antibacterial properties. In general, these uses are carried out by bringing a biocidal or bacterial inhibitory amount of the antibacterial compositions of the present invention into contact with a surface, environment or biozone containing Gram-negative bacteria so that the composition is able to interact with and thereby interfere with the biological function of such bacteria. For example, such antibacterial compositions can be used to prevent or inhibit biofilm formation caused by Gram-negative bacteria. Compositions may be formulated as sprays, solutions, pellets, powders and in other forms of administration well known to those skilled in such arts. For use in these contexts the compounds in the invention may be supplied either as a single compound, in a mixture with several other compounds of the invention or in a mixture with additional antimicrobial agents. The following examples are intended to illustrate but not limit the invention.

EXAMPLES Example 1 : Synthesis of N-alkylated and N-acylated amino acids in solution.

N-[2-(lif-Indol-3-yl)-ethyl]-/V BOC-lysine tert-butyl ester 13(4}. Et₃N (922 μL, 6.61 mmol) was added to a stirred solution of HLys(BOC)O'BuΗCl (500 mg, 1.63 mmol) and 3-(2-bromoethyl)-indol (1.11 g, 4.96 mmol) in freshly distilled DMF (15 mL). The solution was heated at 70 °C overnight. The solution was cooled to 0 °C and water (15 mL) was added. The water phase was extracted with Et_jO (3x30 mL), and the combined organic phases were dried (MgSO₄) and concentrated. Flash chromatography (CH₂Cl₂:MeOH 100:0→99:1→98:2→95:5) gave N-[2-(lH-indol-3-yl)-ethyl]-N_ε-BOC- lysine tert-butyl ester (340 mg, 47%). Η ΝMR ( CDC1₃, 400 MHz) δ 8.22 (bs, 1H, C=CHΝH), 7.61 (d, 1Η, J=7.3 Ηz, Ar-H), 7.36 (d, 1Η, J=8.5 Ηz, Ar-H), 7.18 (dt, 1Η, J=7.5, 1.0 Ηz, Ar-H), 7.11 (dt, 1Η, J=7.5, 1.0 Ηz, Ar-H), 7.07 (d, 1Η, J=2.2 Ηz, C=CHNΗ), 4.57 (bs, 1H, NHOC(CH₃)₃), 3.13 (t, 1H, J=8.4 Hz, CH₂CHNH)₅, 3.08 (m, 2H, CH₂NHC(O)OC(CH₃)₃), 3.00-2.90 and 2.82 (m, 4H, CH₂CH₂NH), 1.73 (bs, 1H, NH), 1.60-1.50 (m, 2Η, CH₂CHNH), 1.46 (s, 9H, OC(CH₃)₃), 1.39 (s, 9Η, OC(CH₃)₃), 1.45-1.28 (m, 4Η, CH₂CH₂); ΗRMS FAB(M+Η)⁺ Calcd for C₂₅H₄₀N₃O₇446.3018, found 446.3012.

N-[2-(lH-Indol-3-yl)-ethyl]-/V-(malonamic acid ethyl ester)-N_ε-BOC-Iysine tert-butyl ester 14(4}. Ethyl malonyl chloride (205 μL, 1.62 mmol) was added to a solution of N-[2- (lH-indol-3-yl)-ethyl]-N_ε-BOC-lysine tert-butyl ester (226 mg, 0.54 mmol) and N, N'- diisopropylethylamine (278 μL, 1.62 mmol) at 0 ^°C. The reaction was stirred at 0 °C for 60 minutes and then diluted with CΗ₂C1₂ (10 mL), washed with aqueous 0.05 M HC1 (10 mL), ΝaHCO₃ aq. sat. (10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 2:1->3:2→1:1) gave N-[2-(lH- indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)-Ν_ε-BOC-lysine tert-butyl ester (275 mg, 97%). Η NMR ( CDC1₃, 400 MHz) δ 8.25 (bs, IH), 7.75 (d, lH_mιn, J=7.6 Hz, Ar-H), 7.57 (d, lΗ_maj, J=7.9 Hz, Ar-H), 7.41 (d, lΗ_maj, J=8.0 Hz, Ar-H), 7.36 (d, lΗ_mιn, J=8.0 Hz, Ar- H), 7.25-7.05 (m, 3H, Ar-H), 4.65 (bs, lΗ_maj, NHOC(CH₃)₃), 4.59 (bs, lH_mιn, NHOC(CH₃)₃) , 4.35-4.06 (m, 4H), 3.75-3.35 (m 4H), 3.09 (m, 4H), 1.50 (s, 9H,

OC(CH₃)₃), 1.42 (s, 9Η, OC(CH₃)₃), 1.49-1.43 (m, 3Η, OCH₂CH₃), 1.35 (m, 2Η); HRMS FAB(M+H)⁺ Calcd for C₃₀H₄₆N₃O₇ 560.3335, found 560.3336.

V-[2-(l^-Indol-3-yl)-ethyl]-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-N_ε-BOC-lysine tert-butyl ester 16(4, 7}. Piperidine (20 μL, 0.20 mmol) was added to a solution of N-[2- (lH-indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)-Ν_ε-BOC-lysine tert-butyl ester (89 mg, 0.15 mmol) and salicylaldehyde (53 μL, 0.50 mmol) in CΗ₃CN (12 mL). The solution was heated at reflux over night, cooled to room temperature and diluted with CH₂C1₂ (15 mL). The organic phase was washed with aqueous 0.05 M HC1 (12 mL), NaHCO₃ aq. sat. (12 mL) and brine (12 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1 % HOAc, 3 : 1 →2: 1 → 1 : 1) gave N-[2-(lH-indol-3-yl)-ethyl]- N-(2-oxo-2H-l-benzopyran-3-carbonyl)-Ν_ε-BOC-lysine tert-butyl ester (76 mg, 77%). ^!Η NMR (MeOH-</₄, 400 MHz) δ 7.47-7.65 (m, IH, Ar-H), 7.57 (m, 1Η, Ar-H), 7.45 (m, 1Η, Ar-H), 7.43-7.45 (m, 2Η, Ar-H), 7.20 (m, 1Η, Ar-H), 7.15-7.04 (m, 3Η, Ar-H), 6.78 (t, 1Η, J=7.2 Ηz, Ar-H), 6.56 (t, 1Η, J=7.4 Ηz, Ar-H), 4.23 (m, 1Η), 3.65 (m, 1Η), 3.53 (m, 1Η), 3.05 (m, 4Η), 2.10 (m, 2H), 1.56 (s, 9H, OC(CH₃)₃), 1.46 (m, 4Η), 1.40 (s, 9H, OC(CH₃)₃); ΗRMS FAB(M+Η)⁺ Calcd for C₃₅H₄₃N₃O₇ 617.3101, found 617.3091.

7V-[2-(l -Indol-3-yl)-ethyl]-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-lysine 17 (4, 7 } . TFA:H₂O (2:1, 4 mL) was added to neat N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo-2H-l- benzopyran-3-carbonyl)-Ν_ε-BOC-lysine tert-butyl ester (35 mg, 0.06 mmol). After 60 min at room temperature the solution was concentrated to dryness and the residue was concentrated three times from toluene. Flash chromatography (CΗ₂Cl₂:MeOΗ, containing 1% HOAc, 20:1:→8:1→5:1→1:1) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo-2H-l- benzopyran-3-carbonyl)-lysine (17 mg, 64%). Η ΝMR ( MeOΗ-</₄, 400 MHz) δ 8.20 (bs, lH_maj, A -H), 7.83 (bs, lH_mιn, Ar-H), 7.75 (d, lΗ_maj, J=7.6 Hz, A -H), 7.72 (d, lH^, J=7.9 Hz, Ar-H), 7.65 (m, 1Η, Ar-H), 7.40 (d, lΗ_maj, J=7.9 Hz, Ar-H), 7.35 (m, IH, Ar-H), 7.33 (d, lH_min, J=8.0 Hz, Ar-H),7.21 (m, 1Η, Ar-H), 7.12 (m, 3Η, Ar-H), 7.00 (m, 1Η, Ar-H), 6.80 (t, lΗ_maj, J=7.5 Hz), 6.52 (t, IH, J=7.4 Hz, Ar-H), 4.04 (bt, lΗ_maj, J=7.0 Hz), 3.87 (m. lH_min), 3.70-3.45 (m, 2H), 3.15-3.0 (m, 2H), 2.97 (t, IH, J=7.2 Hz), 2.84 (t, IH, J=7.1 Hz), 2.15 (m, 2H); 1.75 (m, 2H), 1.58 (m, 2H)m 1.33 (m, 2H); HRMS FAB(M+H)⁺ Calcd for C₂₆H₂₆N₃O₅Na₂ 506.1667, found 506.1658.

N-[2-(liϊ-Indol-3-yl)-ethyl]-phenylalanine tert-butyl ester 13(2}. Et₃N (2.01 mL, 14.46 mmol) was added to a stirred solution of HPheOΕu HCl (800 mg, 3.61 mmol) and 3-(2- bromoethyl)-indol (2.43 g,10.84 mmol) in freshly distilled DMF (15 mL). The solution was heated at 70 °C over night. The solution was cooled to 0 °C and water (20 mL) was added. The water phase was extracted with Et₂O (3x40 mL), and the combined organic phases were washed with brine (20 mL), dried (MgSO₄) and concentrated. Flash chromatography (CH₂Cl₂:MeOH 100:0→400:1→200:1→100:1→20:1) gave N-[2-(lH- indol-3-yl)-ethyl]-phenylalanine tert-butyl ester (914 mg, 69%). Η ΝMR (CDC1₃, 400 MHz) δ 8.24 (s, IH, C=CHΝH), 7.65 (d, 1Η, J=7.8 Ηz, Ar-H), 7.37 (dd, 1Η, J=8.0, 0.8 Ηz, Ar-H), 7.32-7.20 (m, 5Η, Ar-H), 7.15 (m, 1Η, Ar-H), 6.97 (d, 1Η, J=2.0 Ηz,

C=CHNΗ), 3.54 (IH, t, J=7.1 Hz, CHNH), 3.06-2.87 (m, 6H, -CH₂-), 1.78 (bs, 1Η, NH), 1.46 (s, 9Η, OC(CH₃)₃); ΗRMS FAB(M+Η)⁺ Calcd for C₂₃H₂₉N₂O₂ 365.2229, found 365.2229.

N-[2-(l//-Indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)-phenylalanine tert-butyl ester 14 (2} . Ethyl malonyl chloride (260 μL, 2.06 mmol) was added to a solution of N-[2- (lH-indol-3-yl)-ethyl]-phenylalanine tert-butyl ester (250 mg, 0.68 mmol) and N, N'- diisopropylethylamine (352 μL, 2.06 mmol) in CΗ₂C1₂ (20 mL) at 0 °C. The reaction was stirred at 0 °C for 40 min and then diluted with CH₂C1₂ (10 mL), washed with aqueous 0.05 M HC1 (10 mL), ΝaHCO₃ aq. sat. (10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane :EtO Ac, containing 1% HOAc,

3:1→2:1→1 :1) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)- phenylalanine tert-butyl ester (304 mg, 93%). Η ΝMR (CDCl₃,δ 400 MHz) δ 8.26 (bs, IH, C=CHΝH), 7.45 (d, 1Η, J=7.8 Ηz), 7.36 (m, 1Η, Ar-H), 7.32-7.15 (m, 5Η, Ar-H), 7.12 (dt, IH, J=7.4, 1.0 Hz, Ar-H), 6.95 (d, 1Η, J=2.2 Ηz), 4.21 (q, 2Η, OCH₂CH₃), 4.12 (m, IH, CH₂CHN), 3.47-3.27 (m 5Η, -CH₂-), 3.01-2.85 (m, 2Η, -CH₂-), 2.76 (m, 1Η, - CH₂-), 1.50 (s, 9Η, OC(CH₃)₃), 1.44 (m, 3Η, OCH₂CH₃); ΗRMS FAB(M+Η)⁺ Calcd for C₂₈H₃₅N₃O₅ 479.2545, found 479.2541.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-phenylaIanine tert-butyl ester 16(2, 7}. Piperidine (43 μL, 0.43 mmol) was added to a solution of N-[2- (lH-indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)-phenylalanine tert-butyl ester (172 mg, 0.36 mmol) and salicylaldehyde (113 μL, 1.07 mmol) in CΗ₃CΝ (15 mL). The solution was heated at reflux over night, cooled to room temperature and diluted with CH₂C1₂ (20 mL). The organic phase was washed with aqueous 0.05 M HC1 (15 mL), NaHCO₃ aq. sat. (15 mL) and brine (15 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 3:1→2:1→1 :1) gave N-[2-(lH- indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-phenylalanine tert-butyl ester (104 mg, 54%). Η ΝMR (MeOΗ-d₄, 400 MHz) δ 7.82 (bd, IH, J=7.5 Hz, Ar-H) 7.50 (t, 1Η, J=7.5 Ηz, Ar-H), 7.39 (m, 3Η, Ar-H), 7.30 (m, 2Η, Ar-H), 7.26-7.15 (m, 3Η, Ar-H), 7.13-7.00 (m, 2Η, Ar-H), 6.93 (d, 1Η, J=7.8 Ηz, Ar-H), 6.86 (bs, 1Η, Ar-H), 6.80 (t, 1Η, J=7.5 Ηz, Ar-H), 6.56 (t, 1Η, J=7.5 Ηz, Ar-H), 4.41 (dd, lΗ_min, J=8.9, 5.8 Hz, CH₂CHΝ), 4.33 (dd, lΗ_maj, J=8.9, 5.8 Hz, CH₂CHN), 3.40 (m, 3Η, -CH₂-), 2.90-2.75 (m, 3Η, -CH₂-), 1.55 (s, 9Η, OC(CH₃)₃); ΗRMS FAB(M+Η)⁺ Calcd for C₃₃H₃₃N₂O₅ 537.2389, found 537.2391.

7V-[2-(liϊ-Indol-3-yl)-ethyl]-/V-(2-oxo-2H-l-benzopyran-3-carbonyl)-phenylalanine

17(2, 7}. TFA:H₂O (2:1, 4 ml) was added to neat N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo- 2H-l-benzopyran-3-carbonyl)-phenylalanine tert-butyl ester (36 mg, 0.07 mmol). After 90 min at room temperature the solution was concentrated to dryness and the residue was concentrated three times from toluene. Flash chromatography (toluene: EtOAc :ΗO Ac) 85:10:5→80:15:5→60:35:5) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran- 3-carbonyl)-phenylalanine (24 mg,74%). Η ΝMR (CDC1₃, 400 MHz) δ 8.58 (bs, lH^, C=CH), 8.21 (bs, lΗ_min, C=CH), 7.40-7.29 (m, 5Η, Ar-H), 7.24-7.14 (m, 2H, Ar-H), 7.05- 6.95 (m, 3H, Ar-H), 6.85 (bs, 1Η, Ar-H), 6.79 (bs, 1Η, Ar-H), 6.72 (t, 1Η, J=7.5 Ηz, Ar- H), 6.58 (t, 1Η, J=7.4 Ηz, Ar-H), 4.23 (m, 1Η, ArCΗ₂CH), 3.56 (m 2Η, ΝCH₂CH₂), 3.47 (m, IH, ArCH₂CH), 3.23-3.05 (m, IH, ArCH₂CH), 2.86 (m, IH, NCH₂CH₂), 2.75 (m, 1Η, NCΗ₂CH₂); ΗRMS FAB(M+Η)⁺ Calcd for C₂₉H₂₅N₂O₅ 481.1763, found 481,1758.

7V-[2-(lH-Indol-3-yl)-ethyl]-O'Bu-tyrosine tert-butyl ester 13(5}. Et₃N (888 μL, 6.37 mmol) was added to a stirred solution of HTyr('Bu)O'BuΗCl (500 mg, 1.59 mmol) and 3- (2-bromoethyl)-indol (1.07 g, 4.77 mmol) in freshly distilled DMF (15 mL). The solution was heated at 70 °C over night. The solution was cooled to 0 °C and water (15 mL) was added. The water phase was extracted with Et₂O (3x40 mL), and the combined organic phases were dried (MgSO₄) and concentrated. Flash chromatography (toluene:EtOH 30:1→10:1→10:2) gave N-[2-(lH-indol-3-yl)-ethyl]-OT3u-tyrosine tert-butyl ester (319 mg, 46%). Η ΝMR (CDC1₃, 400 MHz) δ 8.10 (bs, IH, C=CHΝH), 7.61 (d, 1Η, J=7.4 Ηz, Ar-H), 7.35 (d, 1Η, J=8.1 Ηz, Ar-H), 7.19 (m, 1Η, Ar-H), 7.13-7.04 (m, 3Η, Ar-H), 6.98 (d, IH, J=2.2 Hz, C=CH), 6.87 (dd, 1Η, J=6.6, 1.9 Ηz, Ar-H), 3.45 (dd, 1Η, J=8.3, 6.3 Ηz, ArCΗ₂CH), 3.00-2.75 (m, 6Η, ArCH₂CH, NCH₂CH₂ and NCH₂CH₂), 1.33 (s, 9Η, OC(CH₃)₃), 1.28 (s, 9Η, OC(CH₃)₃); ΗRMS FAB(M+Η)⁺ Calcd for C₂₇H₃₇N₂O₃ 437.2804, found 437.2808.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(malonamic acid ethyl ester)-O'Bu-tyrosine tert-butyl ester 14(5}. Ethyl malonyl chloride (181 μL, 0.14 mmol) was added to a solution of N-[2- (lH-indol-3-yl)-ethyl]-O'Bu-tyrosine tert-butyl ester (210 mg, 0.47 mmol) and N, N'- diisopropylethylamine (246 μL, 0.14 mmol) in CΗ₂C1₂ (15 mL) at 0 °C. The reaction was stirred at 0 "C for 50 min and then diluted with CH₂C1₂ (15 mL), washed with aqueous 0.05 M HC1 (15 mL), ΝaHCO₃ aq. sat. (15 mL) and brine (15 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 4:1→3:1→2:1→1:1) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)- OΕu-tyrosine tert-butyl ester (147 mg, 56%). Η ΝMR (CDC1₃, 400 MHz) δ 8.16 (bs, IH, C=CHΝH), 7.44 (d, 1Η, J=7.8 Ηz, Ar-H), 7.35 (m, 1Η, Ar-H), 7.19 (m, 1Η, Ar-H), 7.12 (m, 3Η, Ar-H), 6.92 (m, 3H, Ar-H), 4.38 (t, lΗ_mιn, J=7.32 Hz, ArCH₂CH), 4.27-4.17 (m, 2Η, OCH₂CH₃), 4.06 (dd, lHL^, J=8.9, 6.3 Hz, ArCH₂CH), 3.45-3.25 (m, 5Η, -CH₂-), 2.96-2.85 (m, 2Η, -CH₂-), 2.80-2.73 (m, 1Η, -CH₂-), 1.50 (s, 9Η, OC(CH₃)₃), 1.26 (m, 12 Η, OC(CH₃)₃ and OCH₂CΗ₃); HRMS FAB(M+H)⁺ Calcd for C₃₂H₄₃N₂O₆ 551.3121, found 551.3109.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-O'Bu-tyrosine tert-butyl ester 16(5, 7}. Piperidine (26 μL, 0.26 mmol) was added to a solution ofN-[2- (lH-indol-3-yl)-ethyl]-N-(malonamic acid ethyl ester)-O'Bu-tyrosine tert-butyl ester (121 mg, 0.21 mmol) and salicylaldehyde (69 μL, 0.65 mmol) in CΗ₃CΝ (15 mL). The solution was heated at reflux over night, cooled to room temperature and diluted with CH₂C1₂ (35 mL). The organic phase was washed with aqueous 0.05 M HC1 (15 mL), NaHCO₃ aq. sat. (15 mL) and brine (15 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 4:1→3:1→2:1→1 :1) gave N-[2-(lH-indol-3-yl)- ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-O⁽Bu-tyrosine tert-butyl ester (77 mg, 58%). Η ΝMR (CDCl_j, 400 MHz) δ 8.05 (bs, lH_mιn, Ar-H), 7.98 (bs, lΗ^ Ar-H), 7.60- 7.50 (m, 1Η, Ar-H), 7.37 (m, 1Η, Ar-H), 7.30-7.10 (m, 7Η, Ar-H), 6.99 (m, 2H, Ar-H), 6.87 (m, 1Η, Ar-H), 6.80 (dt, 1Η, J=7.5, 0.9 Ηz, Ar-H), 4.28 (t, lΗ_mιn, J=7.3, ArCH₂CH), 4.19 (t, lΗ,^, J=7.4, ArCΗ₂CH), 3.50-3.15 (m, 3Η, -CH₂-), 3.05-2.75 (m, 3Η, -CH₂-), 1.55 (s, 9Η_maj, OC(CH₃)₃), 1.44 (s, 91^ O(CH₃)₃), 1.34 (s, 9H_mιn, O(CH₃)₃), 1.30 (s, 911^, OC(CH₃)₃); HRMS FAB(M+H)⁺ Calcd for C₃₇H₄₀Ν₂O₆ 608.2886, found 608.2878 N-[2-(l#-Indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-tyrosine 17(5, 7} . TFA:H₂O (2:1, 6 ml) was added to neat N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo-2H-l- benzopyran-3-carbonyl)-O'Bu-tyrosine tert-butyl ester (54 mg, 0.08 mmol). After 80 min at room temperature the solution was concentrated to dryness and the residue was concentrated three times from toluene. Flash chromatography (toluene:EtOAc:ΗOAc) 85:10:5→60:35:5- 35:60:5) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(2-oxo-2H-l-benzopyran- 3-carbonyl)-tyrosine (42 mg, 95%). Η ΝMR (MeOΗ-rf₄, 500 MHz) δ 7.80 (d, lH_maj, J=7.9 Hz, Ar-H), 7.70 (d, lΗ_min, Ar-H), 7.64 (m, lΗ^, Ar-H), 7.55 (m, lΗ_majι Ar-H), 7.30 (m, IH, Ar-H), 7.27 (m, 1Η, Ar-H), 7.26 (m, 2Η, Ar-H), 7.19 (m, 1Η, Ar-H), 7.02 (m, 1Η, Ar- H), 6.98 (m, 1Η, Ar-H), 6.78 (m, 2Η, Ar-H), 6.70 (m, 1Η, Ar-H), 6.58 (m, 1Η, Ar-H), 4.32 (bs, 1Η, CΗ₂CHΝ), 3.51 (m, 2Η, NCH₂CH₂), 3.39 (m, IH, PhCH₂CH), 3.35 (m, IH, PhCH₂CH), 2.85 (m, IH, NCH₂CH₂), 2.78 (m, 1Η, NCΗ₂CH₂),; ¹³C NMR (MeOΗ-tf₄, 125 MHz) δ 159.7, 157.3, 155.0, 144.0, 137.9, 134.8, 133.8, 132.0, 131.6, 130.5, 130.4, 130.3, 128.3, 126.2, 126.0, 125.2, 124.7, 123.6, 122.4, 120.0, 119.7, 119.6, 119.3, 118.3, 117.6, 117.3, 116.8, 116.5, 112.4, 112.3, 65.2, 52.5, 53.1, 36.5, 35.0, 25.3; HRMS FAB(M+H)⁺ Calcd for C₂₉H₂₅N₂O₆ 497.1712, found 497.1208.

7V-[2-(lH-Indol-3-yl)-ethyl]-glycine ethyl ester 13(7}. Ethyl bromoacetate (99 μL, 0.89 mmol) was added to a solution of tryptamine (431 mg, 2.69 mmol) in freshly distilled DMF (5 mL) at 0 °C. The solution was stirred at 0 °C for lh 45 min and then water (10 mL) was added. The water phase was extracted with Et_jO (3x15mL) and the combined organic phases were dried (MgSO₄) and concentrated. Flash chromatography (CH₂Cl₂:MeOH 98:2->96:4) gave N-[2-(lH-indol-3-yl)-ethyl]-glycine ethyl ester (216 mg, 98%). 'Η ΝMR (CDC1₃, 400 MHz) δ 8.20 (bs, IH, C=CHΝH), 7.63 (d, 1Η, J=7.8 Ηz, Ar- H), 7.37 (d, 1Η, J=8.1, Ar-H), 7.19 (dt, 1Η, J=7.6, 1.1 Ηz, Ar-H), 7.12 (dt, 1Η, J= 7.4, 1.0 Ηz, Ar-H), 7.10 (d, 1Η, J=2.3 Ηz, C=CHNΗ), 4.16 (q, 2H, J=7.1 Hz, OCH₂CH₃), 3.43 (s, 2H, CH₂NH); 2 98 (m, 4H, CH₂CH₂NH), 1.24 (t, 3H, J=7.1 Hz, OCH₂CH₃); ΗRMS FAB(M+Η)⁺ Calcd for C₁₄H₁₉N₂O₂ 247.1446, found 247.1444.

7V-[2-(l -^'-Indol-3-yl)-ethyl]-7V-(naphtalene-2-carbonyl)-glycine ethyl ester 18(7, 2}. N, N'-Diisopropylcarbodiimide (188 μL, 1.21 mmol) was added to a solution of pentafluorophenol (231 mg, 1.25 mmol) in EtOAc (10 mL) at 0 "C. After 30 min 2- naphtoic acid (436 mg, 2.43 mmol) was added and the mixture was stirred for 60 min at 0 °C then N-[2-(lH-indol-3-yl)-ethyl]-glycine ethyl ester (100 mg, 0.40 mmol) was added. The mixture was stirred over night at room temperature. The precipitate, N, N'- diisopropylurea, was removed by filtration and the solvent was diluted with EtOAc (20 mL) and washed with aqueous 0.05 M HC1 (2x10 mL), NaHCO₃ aq. sat. (2x10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 4:1→3:1→2:1→1:1→1:2) gave N-[2-(lH-indol-3-yl)-ethyl]-N- (naphtalene-2-carbonyl)-glycine ethyl ester (70 mg, 43%). Η ΝMR (CDC1₃, 400 MHz) δ 8.53 (bs, IH, C=CHΝH), 7.89-7.65 (m, 4Η, Ar-H), 7.56 (m, 2Η, Ar-H), 7.38 (bd, IH, J=8.1 Hz, Ar-H), 7.26 (m, 1Η, Ar-H), 7.20 (m, 1Η, Ar-H), 7.07 (m, 1Η, Ar-H), 6.95 (bd, 1Η, J=8.1 Ηz, Ar-H), 6.85 (s, 1Η, Ar-H), 6.75 (t, 1Η, J=7.3 Ηz, Ar-H), 4.36 (s, 211^, (O)CCH₂N), 4.30 (q, 2Η_maj, J=7.0 Hz, OCH₂CH₃), 4.16 (q, 2H_mιn, J=7.1 Hz, OCH₂CH₃), 3.94 (s, 2H_mιn, (O)CCH₂N), 3.84 (m, 2Η_mιn, -CH₂-), 3.68 (t, 2Η_mιn, J=7.3 Hz, -CH₂-), 3.24 (t, 2Η_mιn, J=7.0 Hz, -CH₂-), 3.00 (t, 2Η_maj, J=7.3 Hz, -CH₂-), 1.35 (t, 3Η_maj, J=7.1 Hz, OCH₂CH₃), 1.22 (t, 3Η_mιn, J=7.1 Hz, OCH₂CH₃).

7V-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)-glycine 19(7, 2}. Aqueous LiOΗ 1 M (300 μL, 0.29 mmol) was added to a solution of N-[2-(lH-indol-3-yl)-ethyl]-N- (naphtalene-2-carbonyl)-glycine ethyl ester (60 mg, 0.14 mmol) in TΗF:MeOΗ:Η₂O (3:1:1, 5 mL). After 40 min the solution was cooled to 0 ^°C and acidified with aqueous 1 M HC1 (400 μL). The solution was diluted with EtOAc (10 mL) and a few mL brine added. The organic phase was separated, dried (MgSO₄) and concentrated. Flash chromatography (heptane:EtOAc, containing 1% HOAc, 1:1→T :2→1:4) gave N-[2-(lH- indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)-glycine (quant, yeild). Η ΝMR (MeOΗ-rf₄, 400 MHz) δ 7.89 (m, IH, Ar-H), 7.84 (m, 1Η, Ar-H), 7.76 (d, 1Η, J=8.4 Ηz, Ar-H), 7.68 (m, 1Η, Ar-H), 7.55-7.43 (m, 3Η, Ar-H), 7.32 (2xd, 1Η, J=8.1 Ηz, Ar-H), 7.21 (dd, 1Η, J=8.4, 1.5 Ηz, Ar-H), 7.07 (2xt, 1Η, J=7.4 Ηz, Ar-H), 6.95 (m, 1Η, Ar-H), 6.81 (d, 1Η, J=7.9 Ηz, Ar-H), 6.54 (dt, 1Η, J=7.5, 0.8 Ηz, Ar-H), 4.36 (s, 2Η_ma)) (O)CCH₂Ν); 3.89 (t, 2Η_mιn, J=7.5 Hz, -CH₂-), 3.82 (s, 2Η_mιn, (O)CCH₂N), 3.67 (t, 2Η_maj, J=7.0 Hz, -CH₂-), 3.20 (t, 2Η_mιn, J=7.3 Hz, -CH₂-), 3.00 (t, 21^, J=6.8 Ηz, -CH₂-); ΗRMS FAB(M+Η)⁺ Calcd for C₂₃H₂₁N₂O₃ 373.1552, found 373.1546.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(naphtalene-2-carbonyl)-phenylalanine tert-butyl ester

18(2, 2}. N, N'-Diisopropylcarbodiimide (40 μL, 0.26 mmol) was added to a solution of N-[2-(lH-indol-3-yl)-ethyl]-phenylalanine tert-butyl ester (50 mg, 0.13 mmol), 2-naphtoic acid (49 mg, 0.27 mmol) and 1-hydroxybenzotriazole (56 mg, 0.41 mmol) in TΗF (5 mL) at 0 °C. The mixture was stirred over night at room temperature and then concentrated. The residue was diluted with EtOAc (5 ml), the precipitate, N, N'-diisopropylurea, was removed by filtration and the solvent was diluted with EtOAc (20 mL). The organic phase was washed with aqueous 0.05 M ΗC1 (2x10 mL), ΝaΗCO₃ aq. sat. (2x10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography (heptane: EtOAc, containing 1% HOAc, 4:1) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)- phenylalanine tert-butyl ester (21 mg, 30%). Η ΝMR (CDC1₃, 400 MHz) δ 8.15 (s, IH, Ar-H), 7.96-7.69 (m, 4Η, Ar-H), 7.55 (m, 2Η, Ar-H), 7.39 (m, 3H, Ar-H), 7.26 (m, 3Η, Ar-H), 7.18 (m, 2H, Ar-H), 6.98 (m, 1Η, Ar-H), 6.65 (m, 1Η, Ar-H), 6.55 (m, 1Η, Ar-H), 4.61 (m, lΗ_mιn), 4.30 (m, lH_maj), 3.94 (m, lH_min), 3.71 (m, lH_maj), 3.65-3.40 (m, 2H) 3.25- 3.09 (m, IH), 2.98 (m, IH), 2.70 (m, IH), 1.62 (s, 911^, OC(CH₃)₃), 1.58 (s, 9I ,, OC(CH₃)₃); HRMS FAB(M+H)⁺ Calcd for C₃₄H₃₅Ν₂O₃519.2647, found 519.2638.

7V-[2-(lH-Indol-3-yl)-ethyl]-/V-(naphtalene-2-carbonyl)-phenylalanine 19 (2, 2} . TFA:H₂O (2:1, 6 mL) was added to neat N-[2-(lH-indol-3-yl)-ethyl]-N-(naphtalene-2- carbonyl)-phenylalanine tert-butyl ester (16 mg, 0.003 mmol). After 1.5 h at room temperature the solution was concentrated to dryness, and then concentrated three times from toluene. Flash chromatography (toluene:EtOAc:ΗOAc 90:8:2→85:10:5→60:35:5) gave N-[2-(lH-indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)-phenylalanine (10 mg, 70%). 'Η ΝMR (MeOΗ-</₄, 400 MHz) δ 7.86 (m, 2H, Ar-H), 7.81 (m, 2Η, Ar-H), 7.67 (s, 1Η, Ar-H), 7.54 (m, 3Η, Ar-H), 7.38 (m, 4Η, Ar-H), 7.30-7.05 (m, 8H, Ar-H), 6.87 (d, 1Η, J=6.9 Ηz, Ar-H), 6.81 (dt, 1Η, J=7.4, 1.1 Ηz, Ar-H), 6.74 (s, 1Η, Ar-H), 6.35 (m, 1Η, Ar- H), 4.69 (dd, lΗ_min, J=10.4, 3.7 Hz, CH₂CHΝ), 4.60 (dd, lΗ_maj, J=10.9, 5.1 Hz, CH₂CHN), 3.60 (dd, 1Η, J=13.7, 10.8 Ηz, -CH₂-), 3.50 (dd, 1Η, J=14.0, 5.0 Ηz, -CH₂-), 3.39 (m, 1Η, -CH₂-), 2.95 (m, 1Η, -CH₂-), 2.68 (m, 2Η, -CH₂-); ΗRMS FAB(M+Η)⁺ Calcd for C₃₀H₂₇N₂O₃ 463.2021, found 463.2010.

7V-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)-O'Bu-tyrosine tert-butyl ester

18(5, 5}. 2-Naρhtoylchloride (18 mg, 0.09 mmol) in CH₂C1₂ (1 mL) was added to an ice cooled solution of N-[2-(lH-indol-3-yl)-ethyl]-O'Bu-tyrosine tert-butyl ester (14 mg, 0.03 mmol) and DIPEA (16 μL, 0.09 mmol) in CΗ₂C1₂ (5 mL). The solution was stirred at 0 ^°C for lh 15 min, allowed to reach room temperature and then diluted with CH₂C1₂ (10 mL). The organic phase was washed with aqueous 0.05 M HC1, ΝaHCO₃ aq. sat. and brine (10 mL each), dried (MgSO₄) and concentrated. Flash chromatorgraphy (heptane:EtOAc, containing 1% HOAc, 4:1→1:1) gave N-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2- carbony -CBu-tyrosine tert-butyl ester (14 mg, 74%). Η ΝMR (CDC1₃, 400 MHz) δ 8.15 (bs, IH), 7.95-7.73 (m, 5H, Ar-H), 7.55 (m, 2Η, Ar-H), 7.40 (bd, 1Η, J=7.9 Ηz, Ar-H), 7.19 (m, 3Η, Ar-H), 7.00 (bd, 1Η, J=7.8 Ηz, Ar-H), 6.85 (m, 1Η, Ar-H), 6.63 (m, 1Η, Ar- H), 4.60 (m, lΗ_min, CH₂CHΝ), 4.27 (m, lΗ_maj, CH₂CHN), 3.93 (m, 1Η, -CH₂-), 3.71 (m, 1Η, -CH₂-), 3.58 (m, 1Η, -CH₂-), 3.43 (m, 2Η, -CH₂-), 3.20 (m, 1Η, -CH₂-), 3.00 (m, 2Η, - CH₂-), 2.67 (m, 2Η, -CH₂-), 1.60 (m, 9Η, OC(CH₃)₃), 1.31 (m, 9H, OC(CH₃)₃). N-[2-(li7-Indol-3-yl)-ethyl]-N-(naphtaIene-2-carbonyl)-tyrosine 19 (5, 5} . TFA:H₂O (2:1, 5 mL) was added to neat N-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2- carbonyO-OΕu-tyrosine tert-butyl ester (14 mg, 0.02 mmol). The solution was stirred at room temperature for lh and 30 min, concentrated to dryness, and then concentrated three times from toluene. Flash chromatography (toluene:EtOAc:ΗOAc,

90:8:2→85:10:5→60:35:5) gave N-[2-(lH-Indol-3-yl)-ethyl]-N-(naphtalene-2-carbonyl)- tyrosine (10 mg, 87%). Η ΝMR (MeOΗ-rf₄, 400 MHz) δ 7.85-7.65 (m, 4H, Ar-H), 7.54 (m, 2Η, Ar-H), 7.35-7.05 (m, 6Η, Ar-H), 6.85-6.54 (m, 4H, Ar-H), 6.40 (m, IH, Ar-H), 4.56 (m, lH_maj+min, CH₂CHΝ), 4.00-2.60 (m, 6Η, -CH₂-).

2-Oxo-2//-l-benzopyran-3-carboxylic acid butylamide (10).

N, N'-Diisopropylcarbodiimide (81 μL, 0.52 mmol) was added to a solution of coumarine- 3-carboxylic acid (100 mg, 0.52 mmol) and w-butylamine (104 μL, 1.05 mmol) in CΗ₂C1₂ (5 mL) and the solution was stirred at room temperature over night. The precipitate was removed by filtration, the solvent diluted with CH₂C1₂ (25 mL) and washed with aqueous 0.05 M HCl (2x10 mL), ΝaHCO₃ aq.sat. (2x10 mL) and brine (10 mL), dried (MgSO₄) and concentrated. Flash chromatography(heptane: EtOAc, containing 1% HOAc, 5:1) gave 2-oxo-2H-l-benzopyran-3-carboxylic acid butylamide (26 mg, 20%). Η NMR (MeOΗ-rf₄, 400 MHz) δ 8.80 (s, IH, C=CH), 7.80 (m, IH, Ar-H), 7.71 (m, 1Η, Ar-H), 7.41 (m, 1Η, Ar-H), 3.41 (t, 2Η, J=7.0 Hz, CH₂CH₂N), 1.60 (m, 2Η, -CH₂-), 1.43 (m, 2Η, -CH₂-), 0.96 (t, 2Η, J=7.3 Hz, CH₂CH₃); ΗRMS FAB(M+Η)⁺ Calcd for C₁₄H₁₆NO₃ 24.1130, found 246.1133.

Example 2: Synthesis of Fluorinated Linkers

General. TLC was performed on Silica Gel 60 F₂₅₄ (Merck). Flash column chromatography employed Grace Amicon silica gel 60 A (30-60 μm) with distilled solvents. CH₂C1₂ and CH₃CN were distilled from calcium hydride immediately before use; THF was distilled from sodium-benzophenone ketyl and ethanol was dried over 4 A molecular sieves. Reactions in these solvents were performed under an atmosphere of nitrogen; solvents, reactant solutions and liquid reagents being transferred via oven-dried syringes. Η and ¹³C NMR spectra were obtained on a Bruker DRX-400 spectrometer for solutions in CDC1₃ [residual CHC1₃ (δ_H 7.27 ppm) and CDC1₃ (δ_c 77.23 ppm) as internal standard] and MeOH-<7₄ [residual CH₂DOD (δ_H 3.31 ppm) and CD₃OD (δ_c 49.00 ppm) as internal standard] at 295 K. High-resolution mass spectra [HRMS (EI+)] were recorded on a Jeol JMS-SX 102 spectrometer. 3-Fluoro-4-hydroxymethylbenzoic acid (22) and 2-fluoro-4-hydroxymethylbenzoic acid (23). Aqueous LiOH (1 M, 1.55 mL, 1.55 mmol) was added to a stirred solution of dimethyl 2-fluoroterephthalate (300 mg, 1.41 mmol) in THF:MeOH:H₂O (3:1:1, 4 mL) at 0 °C. The resultant solution was allowed to reach room temperature and was then stirred over night. The solution was diluted with EtOAc (10 mL), the water phase was separated and cooled to 0 °C, acidified with aqueous 1 M HCl and extracted with EtOAc (4x20 mL). The combined organic phases were dried (Na^G,), and concentrated. Flash chromatography (heptane: EtOAc 7:1→6:1, containing 1% HOAc) gave a mixture of terephtalic acid mono-methyl esters (215 mg, 77%), as well as terephtalic acid (70 mg). The mixture of terephtalic acid mono-methyl esters (80 mg, 0.494 mmol) was dissolved in THF (0.8 mL) and added to a stirred solution of LiBH₄ (28 mg, 1.21 mmol) in THF (0.8 mL). The solution was stirred at ambient temperature for 30 min, then ethanol (3.2 mL) was added droppwise, and the resultant slurry was stirred over night. The reaction mixture was cooled to 0 °C and acetone (0.2 mL) followed by aqueous 1 M HCl (1.2 mL) were added to give a clear solution. The water phase was extracted with EtOAc (4x10 mL) and the combined organic phases were washed once with brine (10 mL), dried (Na^O and concentrated. Flash chromatography (heptane: EtOAc 4:1→1:1, containing 1% HOAc) of the residue gave 22 (36 mg, 53%) and 23 (28 mg, 40%). Compound 22 had Η NMR (MeOH-</₄, 400 MHz) δ 7.94 (dd, IH, J=7.9, 1.4 Hz, Ar-H), 7.64 (dd, 1Η, J=10.6, 1.4 Ηz, Ar-H), 7.58 (t, 1Η, J=7.6 Ηz, Ar-H), 4.73 (s, 2Η, ArCH₂OH); ¹³C NMR (MeOH-rf₄, 100 MHz) δ 168.6, 162.6, 160.1, 135.1, 130.2, 126.8, 117.2, 58.7; HRMS (EI+) Calcd for C₈H₇O₃F 170.0379, found 170.0374. Compound 23 had Η NMR (MeOH- ₄, 400 MHz) δ 7.90 (t, IH, J=7.9, 1.4 Hz, Ar-H), 7.21 (m, 2Η, Ar-H), 4.65 (s, 2Η, ArCH₂OH); ¹³C NMR (MeOH-</₄, 100 MHz) δ 167.5, 165.05, 162.5, 151.5, 133.5, 122.9, 115.7, 64.0; HRMS (EI+) Calcd for C₈H₇O₃F 170.0379, found 170.0374.

Benzyl 2-fluoro-4-hydroxymethylbenzoate (24). A solution of 23 (492 mg, 2.89 mmol) in MeOH:H₂O (10:1, 13.2 mL) was titrated to pH 7 with aqueous Cs₂CO₃ (20%, -3.5 mL). The resultant solution was concentrated to dryness and the residue was concentrated twice from freshly distilled DMF (2x8 mL). Benzyl bromide (0.411 mL, 3.47 mmol) was added to a slurry of the solid cesiumsalt in DMF (8 mL), and the reaction solution was stirred over night at ambient temperature. The mixture was concentrated and water was added to the residue. The water phase was extracted with EtOAc (4x50 mL) and the combined organic phases were dried (Na^O^ and concentrated. Flash chromatography (heptane:EtOAc 4:1→1:1) of the residue gave 24 (588 mg, 79%). Η NMR (CDC1₃, 400 MHz) δ 7.95 (t, IH, J=7.8 Hz, Ar-H), 7.45 (m, 2Η, Ar-H), 7.38 (m, 3Η, Ar-H), 7.17 (m, 2Η, Ar-H), 5.35 (s, 2Η, PhCH₂O), 4.76 (d, 2Η, J=6 Hz, ArCH₂OH); ¹³ C NMR (CDC1₃, 400 MHz) δ 164.3, 164.2, 163.8, 161.2, 148.9, 148.8, 135.9, 132.5, 128.8, 128.4, 128.3, 121.8, 121.7, 117.6, 117.5, 115.0, 114.8, 67.1, 64.1; HRMS (EI+) Calcd for C₁₅H_I3O₃F 260.0848, found 260.0846.

Benzyl 4-((E)-2-ethoxycarbonyl-vinyl)-2-fluorobenzoate (26). Tetrapropylammonium perruthenate (TPAP, 5 mg, 0.013 mmol, 5 mol%) was added in one portion to a stirred slurry of 24 (70 mg, 0.269 mmol), 4-methylmoφholine N-oxide (47 mg, 0.403 mmol) and 4 A molecular sieves (134 mg) in CH₂C1₂ (5 mL). The resultant slurry was stirred at ambient temperature for 30 min and then filtrated through a pad of silica gel eluted with CH₂C1₂ (50 mL). Concentration of the solution and flash chromatography (heptane:EtOAc 7:1) of the residue gave the aldehyde 25 (51 mg, 74%) as a colorless oil. Η ΝMR (CDC1₃, 400 MHz) δ 10.05 (d, IH, J=1.5 Hz, ArCHO), 8.12 (t, 1Η, J=7.3 Ηz, Ar-H), 7.74 (dd, 1Η, J=7.9, 1.3 Ηz, Ar-H), 7.64 (dd, 1Η, J=10.1, 1.3 Hz, Ar-H), 7.48-7.33 (m, 4H, Ar-H), 5.45 (s, 2Η ArCH₂O); ΗRMS (EI+) Calcd for C₁₅Η_πO₃F 258.0692, found 258.0693. (EtO)₂P(O)CH₂CO₂C₂H₅ (397 mg, 1.77 mmol) was added to a slurry of ΝaH (55-65%, 62 mg, 1.55 mmol) in THF (5mL) at 0 °C. After a few minutes the mixture became clear and was allowed to reach room temperature after which 25 (286 mg, 1.10 mmol) dissolved in THF (5 mL) was added. After stirring the mixture for lh, H₂O (20 mL) was added and the solution was poured into Et₂O (30 mL). The water phase was extracted with Et₂O (4x30 mL) and the combined organic layers were washed with H₂O (15 mL) and brine (2x1 OmL). The organic phase was dried (MgSO₄), concentrated and the residue was purified by flash chromatography (heptane:EtOAc 8:1) to give 26 (276 mg, 76%). Η ΝMR (CDC1₃, 400 MHz) δ 7.95 (t, IH, J=7.7 Hz, Ar-H), 7.62 (d, 1Η, J=16.0 Ηz, Ar-H), 7.46 (m, 2Η, Ar-H), 7.41-7.35 (m, 4Η, Ar-H), 7.27 (m, 1Η, ArCH=CΗ), 6.49 (d, IH, J=16.0 Hz, CH=CHCO₂Et), 5.38 (s, 2Η PhCH₂O), 4.28 (q, 2Η, J=7.1 Hz, CO₂CH₂CH₃), 1.34 (t, 3H, J=7.1 Hz, CO₂CH₂CH₃); ¹³C ΝMR (CDC1₃, 100 MHz) δ 166.1, 163.6, 160.8, 141.7, 140.9, 135.6, 132.7, 128.6, 128.3, 128.1, 123.4, 119.6, 116.0, 115.8, 67.1, 60.9, 14.2; HRMS (EI+) Calcd for C₁₉H₁₇O₄F 328.1110, found 328.1111.

4-(2-Ethoxycarbonylethyl)-2-fluorobenzoic acid (27). A small amount of 10% Pd-C was added to a solution of 26 (254 mg; 0.774 mmol) in dry EtOH:EtOAc (3:1, 8 mL) and the resultant mixture was hydrogenated at 4 atm for 15 h. The mixture was filtered through a pad of Celite, concentrated and the residue was purified by flash chromatography (heptane:EtOAc 3:1→1:1, containing 1% HOAc) to give 27 (164 mg, 88%). Η ΝMR (CDCl_j, 400 MHz) δ 9.94 (bs, IH, CO₂H), 7.93 (bs, 1Η, Ar-H), 7.05 (d, 1Η, J=7.7, Ar-H), 7.01 (d, 1Η, J=l 1.4 Ηz, Ar-H), 4.15 (q, 2Η, J=7.1 Hz, CO₂CH₂CH₃), 3.00 (t, 2H, J=7.6 Hz, ArCH₂CH₂), 2.65 (t, 2H, J=7.6 Hz, ArCH₂CH₂), 1.24 (t, 3Η, J=7.1 Hz, CO₂CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.5, 161.8, 159.4, 142.7, 129.5, 124. 1, 115.3, 60.5, 59.3, 35.5, 30.4, 14.1; HRMS (EI+) Calcd for C₁₂H₁₃O₄F) 240.0798, found 240.0801.

Ethyl 3-(3-fluoro-4-hydroxymethylphenyl)-propionate (28). BH₃-DMS (92 mg, 1.21 mmol) was added to a solution of 27 (145 mg, 0.604 mmol) and (CH₃O)₃B (251 mg, 2.41 mmol) in THF (8 mL) was added. The resultant mixture was stirred at ambient temperature until 27 was consumed (TLC) and a clear solution was obtained. MeOH (1 mL) was added and the mixture was concentrated to dryness. The residue was dissolved in Et₂O (10 mL) and washed with water (5 mL) and NaHCO₃ (2x3 mL). The organic solution was dried (MgSO₄), concentrated and the residue was purified by flash chromatography (heptane:EtOAc 4: 1) to give 28 (106 mg, 89%). ^]H NMR (CDC1₃, 400 MHz) δ 7.32 (t, IH, J=7.8 Hz, Ar-H), 6.99 (dd, 1Η, J=7.7, 1.6 Ηz, Ar-H), 6.91 (dd, 1Η, J=11.0, 1.5 Ηz, Ar-H), 4.72 (d, 2Η, J=6.0 Hz, ArCH₂OH), 4.12 (q, 2H, J=7.1 Hz, CO₂CH₂CH₃), 2.93 (t, 2H, J=7.7 Hz, ArCH₂CH₂), 2.60 (t, 2H, J=7.7 Hz, ArCH₂CH₂), 1.80 (bt, 1Η, J=6.0 Ηz, ArCΗ₂OH), 1.23 (t, 3Η, J=7.1 Hz,

CO₂CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.5, 162.3, 159.8, 143.1, 129.9, 124.6, 115.7, 60.9, 59.7, 35.9, 30.8, 14.6; HRMS (EI+) Calcd for C₁₂H₁₅O₃F 226.1005, found 260.1002.

3-(3-Fluoro-4-hydroxymethylphenyl)-propionic acid (29). Aqueous LiOH (1 M, 0.448 mL, 0.448 mmol) was added to a stirred solution of 28 (84.5 mg, 0.373 mmol) in

THF:MeOH:H₂O (3:1:1, 1.5 mL) at 0 °C. After 30 min the solution was allowed to reach room temperature and was then stirred for 4.5 h. The solution was then recooled to 0 °C, acidified with aqueous 1 M HCl and poured into EtOAc:H₂O (8:2, 10 mL). The water phase was extracted with EtOAc (2x5 mL) and the combined organic phases were washed once with brine (6 mL), dried (MgSO₄) and concentrated. The residue was purified by flash chromatography (heptane: EtOAc 4:1→3:1, containing 1% HOAc) to give 29 (69 mg, 93%). Η NMR (MeOH-J₄, 400 MHz) δ 7.35 (t, IH, J=7.8 Hz, Ar-H), 7.04 (dd, IH, J=7.8, 1.5 Hz, Ar-H), 6.96 (dd, IH, J=l 1.2, 1.5 Hz, Ar-H), 4.62 (s, 2H ArCH₂OH), 2.91 (t, 2H, J-7.5 Hz, ArCH₂CH₂), 2.60 (t, 2H, J=7.5 Hz, ArCH₂CH₂); ¹³C NMR (CDC1₃, 100 MHz) δ 175.4, 162.0, 159.6, 143.2, 143.1, 129.6, 129.2, 129.1, 126.1, 124.2, 124.1, 115.0, 114.8, 109.0, 57.7, 35.3, 30.4; HRMS (EI+) Calcd for C₁₀H„O₃F 198.0692, found 198.0692.

2-Fluoro-4-hydroxybenzoic acid (31). 2-Fluoro-4-propoxybenzoic acid (30, 1.21 g, 6.09 mmol) was dissolved in CH₂C1₂ (16 mL), the solution was cooled to -78 °C and BBr₃ (1 M in CH₂C1₂, 18.3 mL, 18.3 mmol) was added. The solution was slowly allowed to reach room temperature and was then stirred at ambient temperature over night. H₂O (35 mL) was added and the resultant mixture was poured into Et₂O (20 mL). The water phase was extracted with Et₂O (4x40 mL) and the combined organic phases were dried (MgSO₄). Concentration and flash chromatography (heptane: EtOAc 2:1→1 :2, containing 1% HOAc) of the residue gave 31 (850 mg, 89%). Η NMR (MeOH-rf₄, 400 MHz) δ 7.80 (t, IH, J=8.7 Hz, Ar-H), 6.54 (dd, IH, J=8.7, 2.3 Hz, Ar-H), 6.54 (dd, IH, J=12.9, 2.3 Hz, Ar-H); ¹³C NMR (MeOΗ-rf₄, 100 MHz) δ 167.7, 166.7, 165.1, 164.1, 135.0, 112.6, 104.6; HRMS (EI+) Calcd for C₇H₅O₃F 156.0222, found 156.0221.

3-Fluoro-4-hydroxymethylphenol (32). Compound 31 (679 mg, 4.35 mmol) in THF (20 mL) was added to a stirred solution of (CH₃O)₃B (3.62 g, 34.8 mmol) and BH₃-DMS (1.32 g, 17.4 mmol) in THF (50 mL). The mixture was stirred at ambient temperature until 31 was consumed (TLC) and a clear solution was obtained. MeOH (26 mL) was added, the mixture was concentrated, and the residue was co-concentrated from MeOH (3x50 mL). The residue was flash chromatographed (heptane: EtOAc 2:1- 1:1) to give 32 (558 mg, 90%). Η NMR (MeOH-</₄, 400 MHz) δ 7.21 (t, IH, J=8.6 Hz, Ar-H), 6.57 (dd, 1Η, J=8.4, 2.4 Hz, Ar-H), 6.49 (dd, 1Η, J=l 1.8, 2.4 Ηz, Ar-H), 4.53 (s, 2Η, ArCH₂OH); ¹³C NMR (MeOH- ₄, 100 MHz) δ 164.1, 161.7, 160.1, 131.9, 120.1, 112.3, 103.5, 58.8; HRMS (EI+) Calcd for C₇H₇O₂F 142.0430, found 142.0427.

Ethyl (3-fluoro-4-hydroxymethylphenoxy)-acetate (33). DBU (777 mg, 5.10 mmol) was added to a stirred solution of 32 (558 mg, 3.93 mmol) and ethyl a-bromoacetate (1.18 g, 7.07 mmol) in CH₃CN (50 mL). The solution was refluxed over night and then cooled to room temperature. The solution was poured into Et₂O (100 mL) and washed with aqueous 0.05 M HCl (2x50 mL) and brine (40 mL), dried (Na_jSO and concentrated. The residue was flash chromatographed (toluene: EtOAc 6:1→3:1→1:1) to give 33 (108 mg, 74%). Η NMR (CDC1₃, 400 MHz) δ 7.31 (t, IH, J=8.5 Hz, Ar-H), 6.68 (dd, 1Η, J=8.3, 2.8 Ηz, Ar- H), 6.63 (dd, 1Η, J=l 1.5, 2.5 Ηz, Ar-H), 4.66 (s, 2Η, ArCH₂OH), 4.60 (s, 2H, OCH₂CO₂H), 4.27 (q, 2H, J=7.1 Hz, OCH₂CH₃), 1.98 (bs, IH, OH), 1.30 (t, 3Η, J=7.1 Hz, OCH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 168.7, 162.6, 160.1, 158.9, 130.5, 121.3, 110.3, 102.9, 65.7, 61.7, 59.1, 14.3; HRMS (EI+) Calcd for C_πH₁₃O₄F 228.0797, found 228.0795.

3-Fluoro-4-(hydroxymethyl-phenoxy)-acetic acid (1). Aqueous LiOH (1 M, 1.03 mL, 1.03 mmol) was added to a stirred solution of 33 (157 mg, 0.691 mmol) in THF:MeOH:H₂O (3:1:1, 10 mL) at 0 ^°C. After 30 min the solution was allowed to reach room temperature and was then stirred at ambient temperature for 1.5 h. The solution was recooled to 0 °C, acidified with aqueous 1 M HCl and poured into EtOAc (30 mL). The water phase was extracted with EtOAc (3x10 mL) and the combined organic phases were washed with brine (15 mL), dried (Na^O and concentrated. The residue was flash chromatographed (heptane: EtOAc 1:2, containing 1% HOAc) to give 1 (119 mg, 87%). Η NMR (MeOH-</₄, 400 MHz) δ 7.34 (t, IH, J=8.6 Hz, Ar-H), 6.76 (ddd, 1Η, J=8.3, 2.5, 0.8 Ηz, Ar-H), 6.70 (dd, 1Η, J=l 1.8, 2.5 Ηz, Ar-H), 4.66 (s, 2Η, ArCH₂OH), 4.58 (s 2H, OCH₂OH); ¹³C NMR (MeOH-</₄, 100 MHz) δ 172.4, 163.8, 161.4, 160.4, 131.6, 122.5, 111.4, 103.5, 66.2, 58.6; HRMS (EI+) Calcd for C₉H₉O₄F 200.0484, found 200.0486.

Example 3: Use of Fluorinated Linkers to Monitor Solid-Phase Synthesis

Applicants have previously shown that 19p chemical shifts, as well as linewidths for compounds attached to polyethylene glycol grafted polystyrene resins (TentaGel resins), approach those in solution. This makes gel-phase 19p NMR spectroscopy well suited for adoption of solution-phase chemistry to various solid supports and for optimization of reaction conditions. Linker 1 was now attached to an amino functionalized TentaGel resin (TentaGel S NH2), and to amino functionalized polystyrene, after which the primary hydroxyl group of the linker was acylated with j-fluorobenzoyl chloride. ^F NMR spectroscopy of the two functionalized resins revealed that the l^F line widths were 2-3 times larger for the polystyrene resin, as compared to the TentaGel resin, when CDCI3, DMSO-dg, benzene-dg, or pyridine-d5 were used to swell the resins. This result is in good agreement with previous studies of the influence of the resin on the quality of ^H NMR spectra of solid-supported compounds.

As shown in Reaction Scheme 1, attachment of 1 to a TentaGel S NH2 resin using l-hydroxy-7-azabenzotriazole (HOAt) and N,N'-diisopropylcarbodiimide (DIC) for activation, was also found to result in some coupling of the activated linker to the hydroxyl group of 2. This was indicated in the ^F ΝMR spectrum which showed a peak for acylated linker at d -115 ppm in addition to the peak at d -117 ppm which originates from linker-resin 2 (peak ratio 1 :3). Employing milder reaction conditions, i.e. coupling of the linker activated as the pentafluorophenyl ester, circumvented this 0-acylation (Figure la), but still allowed complete coupling of 1 to the resin as revealed by monitoring with bromophenol blue. Acylation of 2 with bromoacetic acid (3 eq.) in the presence of 1- hydroxybenzotriazole (HOBt), DIC and a catalytic amount of NJV'- dimethylaminopyridine (DMAP), did not give complete conversion into 3 (Figure lb). As judged by ^F ΝMR spectroscopy, the conversion of 2 into 3 was improved from 90% to 100% by repeating the acylation using 1.5 eq. of bromoacetic acid (Figure lc). It should be pointed out that high quality spectra were obtained within minutes for samples of resin (~ 100 mg) in an ordinary ΝMR tube using a standard ΝMR spectrometer.

Nucleophilic substitution of the α-brominated ester 3 with n-butylamine, followed by amidation of 5 with ethyl malonyl chloride and NN'-diisopropylethylamine (DIPEA), did not induce any change in the ^F ΝMR chemical shift. The 19p ΝMR spectrum of 8 showed that Knoevenagel condensation of 6 with salicylaldehyde in CH3CΝ using piperidine as base, was accompanied by a side-reaction (cf Figure Id). An additional peak having a shift identical to that of 2 indicated that some cleavage (-20%) of 9 from the solid support had occurred. Different reaction conditions were explored, but did not lead to any improvement. Pilicide 9 was finally cleaved from the resin using either aqueous LiOH in THF:MeOH:H2θ or the optimized conditions based on TFA as described above. In both cases 9 was isolated in 48% yield based on the overall capacity of the resin.

Example 4: Solid-Phase Synthesis of N-Alkylated and N-Acylated Glycine Derivatives

Methods. TLC was performed on Silica Gel 60 F₂₅₄ (Merck). Flash column chromatography employed Grace Amicon silica gel 60 A (30-60 μm) with distilled solvents. CH₂C1₂, DMF and CH₃CN were distilled from calcium hydride immediately before use, and THF were distilled from sodium-benzophenone ketyl. Reactions in these solvents were performed under an atmosphere of nitrogen; solvents, reactant solutions and liquid reagents being transferred via oven-dried syringes. Solid phase synthesis of 9(7-4, 7-5} was performed on TentaGel S NH₂ resin (130 μm, 0.224 mmol g). ^!H and ¹³C NMR spectra were obtained on a Bruker DRX-400 spectrometer for solutions in CDC1₃ [residual CHC1₃ (δ_H 7.27) and CDC1₃ (δ_c 77.23) as internal standard] and MeOH- /₄ [residual CHD₂OD (δ_H 3.31) and CD₃OD (δ_c 49.00) as internal standard] at 295 K. Proton resonances were assigned from COSY experiments. The peaks were assigned from mixtures of rotamers. Gel-phase ¹⁹F NMR spectra were recorded with a Bruker ARX-400 spectrometer for solutions in CDC1₃, MeOH- d₄ or DMSO- ₆ with CC1₃F (δ_F 0.0 ppm) as internal standard. Positive ion fast-atom bombardment [HRMS, FAB(M+H)⁺] and electron impact mass spectroscopy [HRMS, (EI+)] were recorded on a Jeol JMS-SX 102 spectrometer.

Attachment of the linker (3-fluoro-4-hvdroxymethylphenoxyVacetic acid. N, N'- Diisopropylcarbodiimide (DIC, 835 μL , 5.40 mmol) was added to an ice-cold solution of pentafluorophenol (1.99 g, 10.80 mmol) in EtOAc (30 mL). After 30 min (3-fluoro-4- hydroxymethylphenoxy)-acetic acid (1.13 g, 5.67 mmol) was added and the solution was stirred at 0 °C for 60 min. The mixture was then added to the resin (10 g, 2.70 mmol, pre- swollen in EtOAc) and the mixture was agitated at ambient temperature for 12 h. The resin was washed with EtOAc (100 mL), MeOH (80 mL), THF (80 mL) and dry THF (40 mL) and dried under vacuum.

General procedure for preparation of Compounds 9(7-4. 7-5}:

Resin-bound bromoacetic acid (3). DIC (1.04 mL, 6.75 mmol) was added to a solution of bromoacetic acid (1.13 g, 8.10 mmol) and 1-hydroxybenzotriazole (HOBt, 729 mg, 5.40 mmol) in THF (30 mL) and the solution was stirred at ambient temperature for 60 min. The mixture and a catalytic amount of N, N'-dimethylaminopyridme (DMAP, 108 mg, 0.89 mmol) in THF (10 mL) were then added to 2 (2.70 mmol, pre-swollen in dry THF) and the mixture was agitated over night. The resin was washed with THF, MeOH, THF (each 100 mL) of and dry THF (20 mL) and dried under vacuum. In order to obtain complete coupling of bromoacetic acid to 2 the reaction was repeated once.

Resin-bound 7V-alkyl-glycine 5(7-4}. A solution of each amine 4(7-4} (3 eq, 8.10 mmol) in freshly distilled CH₃CΝ (30 mL), for tryptamine freshly distilled DMF was used as solvent, was added to 3 (2.70 mmol, pre-swollen in dry CH₃CN or DMF) at 0 °C. The resin was agitated at 0 °C for 90 min and then washed with CH₃CN, MeOH, THF (100 mL each) and dry THF (20 mL) before being dried under vacuum.

Resin-bound 7V-alkyl-7V-(malonamic acid ethyl ester)-glycine 6(7-4}. Ethyl malonyl chloride (1.02 mL, 8.10 mmol) dissolved in CH₂C1₂ (10 mL) was added to a suspension of 5(7-4} (2.7 mmol) andN, N'-diisopropylethylamine (DIPEA, 1.38 mL, 8.10mmol) inCH₂Cl₂ (20 mL) at 0 °C and the resin was agitated at 0 °C for 60 min. The resin was washed with CH₂C1₂, MeOH, THF (100 mL each) and dry THF (20 mL), and then dried under vacuum.

Resin-bound 8(7-4, 7-5}. Each amine of the resins 6(7-4} was split into five portions (each approximately 1.9 g resin, 0.54 mmol) which were reacted with the five different salicylaldehydes 7(7-5}. A solution of each salicyaldehyde (3 eq., 1.54 mmol) in freshly distilled CH₃CΝ (7 mL) was added to 6(7-4} which had been pre-swollen in CH₃CN. The mixture was heated to reflux as piperidine (1.2 eq., 61 μL , 0.62 mmol) in CH₃CN (1 mL) was added. After refluxing over night the resin allowed to reach room temperature before being washed with CH₃CN, MeOH, THF (each 50 mL) and dry THF (10 mL). Then the resin was dried under vacuum.

7V-aIkyl- V-(2-oxo-2Jϊ-l-benzopyran-3-carbonyl)-glycine 9(7-4, 7-5} . Aqueous LiOH (1 M, 5 mL, 5 mmol) was added to 8(7-4, 7-5} (approximately 0.54 mmol) in THF:H₂O:MeOH (3 : 1 : 1 , 40 mL) at 0 °C. After 2.5 h at ambient temperature the resin was filtered of and washed with THF, acetic acid and THF (each 50 mL). The filtrate was concentrated almost to dryness and then concentrated from toluene (2x50 mL). The residue was dissolved in a mixture of EtOAc (30 mL) and aqueous HCl (0.05 M, 10 mL), the organic layer was separated and washed with aqueous HCl (0.05 M, 2x10 mL). The organic phase was dried (Na^O and concentrated. The crude products were purified by flash column chromatography as stated below for each compound.

7V-Butyl-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9(7, 7}. Flash column chromatography with toluene:EtOAc:HO Ac 80:15:5→60:35:5; 55% yield; 'HNMR(CDC1₃, 400 MHz) δ 7.97 (s, lH_maj+mιn, C=CH), 7.60 (m, 2H_maj+mιn, Ar-H), 7.36 (m, 2Η_raaj+mιn, Ar-H), 4.26 (s, 2Η_maj, (O)CCH₂N), 4.06 (s, 2Η_mm, (O)CCH₂N), 3.35 (t, 2Η_mιn, J=7.4 Hz, NCH₂CH₂), 3.33 (t, 2H_maj,J=7.7Hz,NCH₂CH₂), 1.61 (m, 2H_maj+mιn, NCH₂CH₂), 1.41 (m, 2Η_mιn, CH₂CΗ₃), 1.26 (m, 2H_maj, CH₂CH₃), 0.96 (t, 3H_mιn, J=7.3 Hz, CH₂CH₃), 0.85 (t, 3Η_maj, J=7.3 Hz, CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.8, 172.5, 166.4, 166.0, 158.5, 158.1, 154.3, 144.2, 143.5, 133.3, 133.2, 129.2, 129.1, 125.3, 125.2, 124.7, 124.5, 118.4, 118.3, 117.1, 116.9, 50.7, 50.1, 47.5, 47.1, 30.3, 29.2, 20.2, 19.9, 13.9, 13.8; HRMS (EI+) Calcd for C₁₆H₁₇NO₅ 303.1106, found 303.1104.

7V-ButyI-N-(3-oxo-3H-naphtho[2,l-6]pyran-2-carbonyl)-glycine 9(7, 2}. Flash column chromatography with toluene:EtOAc:HO Ac 70:25:5; 30% yield; ΗNMR (CDC1₃, 400 MHZ) δ 8.82 (s, lH_maj, C=CH), 8.80 (s, lΗ_mιn, C=CH), 8.29 (d, lΗ_maj, J=8.4 Hz, Ar-H), 8.18 (d, lΗ_mιn,J=8.1 Hz, Ar-H), 8.04 (d, lΗ_maj,J=9.1 Hz, Ar-H), 7.97 (d, lΗ_mm,J=8.9 Hz, Ar-H), 7.91 (d, lΗ_maj,J=8.1 Hz, Ar-H), 7.84 (d, lΗ_mιn, J=7.8 Ηz, Ar-H), 7.71 (bt, lΗ_maj, J=7.4 Ηz, Ar-H), 7.60 (m, 2Η_maj+mιn, Ar-H), 7.45 (d, lΗ_maj, J=8.9 Hz, Ar-H), 7.40 (d, lΗ_mιn, J=8.9 Hz, Ar-H), 6.95 (bs, lΗ_maj+mιn, CO₂H), 4.26 (s, 2Η_maj, (O)CH₂N), 4.09 (s, 2Η_mιn, (O)CH₂N), 3.55 (bt, 2Η_mιn, J=7.6 Hz, NCH₂CH₂), 3.34 (bt, 2H_maj, J=7.7 Hz, NCH₂CH₂), 1.61 (m, 2H_maj+mm, NCH₂CH₂), 1.38 (m, 2Η_mιn, CH₂CH₃), 1.22 (m, 2H_maj, CH₂CH₃), 0.94 (t, 311^ J=13 Hz, CH₂CH₃), 0.82 (t, 3Η_maj, J=7.3 Hz, CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.3, 166.9, 166.5, 158.5, 158.2, 154.4, 140.8, 140.1, 134.8, 134.7, 130.5, 130.4, 129.3, 129.2, 129.0, 128.9, 126.7, 126.6, 123.2, 123.0, 121.9, 116.8, 116.7, 112.9, 112.8, 50.7, 47.6, 47.3, 30.3, 29.2, 20.2, 19.9, 14.0, 13.8; HRMS (EI+) Calcd for C₂₀H₁₉NO₅ 353.1263, found 353.1263.

7V-Butyl-7V-(8-fluoro-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9(7, 5} . Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 19% yield; ΗNMR(CDC1₃, 400 MHz) δ 7.97 s (lH_ma)+mιn, C=CH), 738 (m, 2Η, Ar-H), 7.30 (m, 2Η, Ar-H), 4.24 (s, 2Η_maj, (O)CCH₂N), 4.06 (s, 2Η_mm(O)CCH₂N), 3.55 (t, 2Η_mιn, J=7.6 Hz, NCH₂CH₂), 3.32 (t, 2H_maj, J=7.8 Hz, NCH₂CH₂), 1.60 (m, 2H_maj+mιn, NCH₂CH₂), 1.40 (m, 21^, CH₂CΗ₃), 1.26 (m, 2^, CH₂CH₃), 0.96 (t, 3H_mιn, J=7.3 Hz, CH₂CH₃), 0.85 (t, 3Η_maj, J=7.3 Hz, CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.1, 165.9, 156.7, 142.9, 142.8, 125.5, 125.2, 125.1, 124.1, 124.0, 120.1, 119.6, 119.4, 50.7, 47.2, 30.3, 29.2, 20.2, 20.0, 14.0, 13.8; HRMS (EI+) Calcd for C₁₆H₁₆NO₅F 321.1012, found 321.1014.

7V-Butyl-7V-(7-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9(7,4}. Flash column chromatography with toluene:EtOAc:HO Ac 80:15:5; 22% yield; 'HNMR(CDC1₃, 400 MHZ) δ 7.93 (s, lH_maj+mιn, C=CH), 7.48 (m, 2Η_maj+mm, Ar-H), 6.91 (dd, lΗ_maj+mιn,J=8.7, 2.3 Hz, Ar- H), 6.85 (m, 2H_maj+mιn, Ar-H), 4.23 (bs, 21^, (O)CCH₂N), 4.05 (bs, 2Η_mιn, (O)CCH₂N), 3.91 (s, 3Η_mιn, OCH₃), 3.83 (s, 3Η_mm, OCH₃), 3.53 (bt, 2Η_mιn, J=7.2 Hz, NCH₂CH₂), 3.33 (t, 2H_maj, J=7.7Hz, NCH₂CH₂), 1.59 (m, 2H_maj+mιn, NCH₂CH₂), 1.38 (m, TH^ CH₂CH₃), 1.23 (m, 21^, CH₂CH₃), 0.94 (t, 3H_mιn, J=7.3 Hz, CH₂CH₃), 0.84 (t, 3Η_maj, J=7.3 Hz, CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.5, 166.9, 166.5, 164.1, 164.0, 158.8, 158.5, 156.3, 144.7, 144.0, 130.2, 130.0, 120.7, 120.5, 113.6, 112.1, 111.9, 100.8, 100.7, 56.1, 50.7, 47.6, 47.1, 30.3, 29.1, 22.8, 20.2, 19.9, 14.3, 14.0, 13.8; HRMS (EI+) Calcd for C₁₇H₁₉NO₆ 333.1212, found 333.1212.

7V-(6-Bromo-8-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-7V-butyl-glycine 9 (7, 5} . Flash column chromatography with heptane:EtOAc containing 1% HOAc 3:1—^2:1, 45% yield; 'HNMR (CDC1₃, 400 MHz) δ 7.85 (s, lH^^, C=CH), 7.29-7.15 (m, 2H_maj+mιn, Ar-H), 4.21 (bs, 2Η_maj, (O)CCH₂N), 4.01 (bs, 2Η_mιn, (O)CCH₂N), 3.96 (s, 3Η_maj, OCH₃), 3.93 (s, 3Η_mιn, OCH₃), 3.50 (bt, 2Η_mιn, J=7.5 Hz, NCH₂CH₂), 3.28 (bt, 2H_maj, J=7.7 Hz, NCH₂CH₂), 1.57 (m, 2H_maj+mιn, NCH₂CH₂), 1.40-1.15 (m, 2Η_maj+mιn, CH₂CH₃), 0.93 (bt, 3H_mιn, J=7.3 Hz, CH₂CH₃), 0.83 (bt, 3Η_maj, J=7.3 Hz, CH₂CH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.4, 172.3, 165.9, 165.8, 157.3, 157.0, 147.9, 147.8, 142.9, 142.3, 129.2, 128.3, 125.9, 125.7, 125.5, 125.4, 122.3, 122.1, 120.0, 119.8, 117.9, 117.8, 117.5, 117.4, 56.7, 50.6, 47.2, 32.0, 30.0, 29.1, 29.1, 22.8, 20.1, 19.9, 14.3, 14.0, 13.8; HRMS (EI+) Calcd forC₁₇H₁₈NO₆Br411.0317, found 411.0315.

7V-(2-Methoxy-ethyl)-N-(2-oxo-2H^r-l-benzopyran-3-carbonyl)-gIycine 9 {2, I}. Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 40% yield; 'HNMR (MeOH-</₄, 400MHz) δ 8.12 (s, lH_maj, C=CH), 8.04 (s, lΗ_mιn, C=CH), 7.69 (m, 2Η_maj+mιn, Ar- H), 7.39 (m, 2H_maj+mm, Ar-H), 4.32 (s, 2Η_maj, (O)CH₂N), 4.19 (s, 2Η_mιn, (O)CH₂N), 3.74 (t, lΗ_mιn,J=5.2 Ηz,NCH₂CΗ₂), 3.62 (t, lΗ_mm,J=5.2 Ηz,NCΗ₂CH₂), 3.57 (m, lΗ_maj,NCH₂CΗ₂), 3.53 (m, lH_maj, NCH₂CH₂), 3.34 (s, 3Η_mιn, OCH₃), 3.30 (s, 3Η_maj, OCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.9, 166.5, 166.4, 158.4, 158.3, 154.2, 144.3, 144.1, 133.3, 133.2, 129.1, 128.9, 125.3, 125.2, 124.5, 124.4, 118.4, 118.3, 117.0, 116.9, 71.4, 71.3, 59.0, 58.8, 52.0, 50.4, 48.6, 47.7; HRMS FAB(M+H)⁺ Calcd for C₁₅H₁₆NO₆ 306.0977, found 306.0979

7V-(2-Methoxy-ethyl)-7V-(3-oxo-3^-^r-naphtho[2,l- ]pyran-2-carbonyl)-glycine 9(2, 2} . Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 35% yield; Η NMR (CDC1₃, 400 MHz) δ 8.83 (s, lH_maj, C=CH), 8.77 (s, lΗ_mιn, C=CH), 8.22 (m, 1Η, Ar-H), 7.96 (t, 1Η, J=9.8 Ηz, Ar-H), 7.85 (m, 1Η, Ar-H), 7.63 (m, 1Η, Ar-H), 7.55 (m, 1Η, Ar-H), 7.37 (m, 1Η, Ar-H), 4.33 (bs, 2Η_maj, (O)CH₂N), 4.20 (bs, 2Η_mιn, (O)CH₂N), 3.80 (m, lH_mιn, NCH₂CH₂), 3.67 (m, lH_mιn, NCH₂CH₂), 3.54 (bs, 2H_maj, NCH₂CH₂ and NCH₂CH₂), 3.32 (s, 3H_mιn, OCH₃), 3.27 (s, 3H_maj, OCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 173.2, 167.0, 158.6, 154.3, 140.5, 134.6, 130.4, 129.3, 129.2, 129.1, 129.0, 128.4, 126.7, 123.1, 122.7, 121.9, 116.7, 112.9, 109.3, 71.2, 70.9, 59.0, 58.8, 50.4, 48.8, 47.7; HRMS FAB(M+H)⁺ Calcd for C₁₉H₁₈NO₆ 356.1134, found 356.1126.

7V-(8-Fluoro-2-oxo-2H-l-benzopyran-3-carbonyl)-7V-(2-methoxy-ethyl)-glycine 9 (2, } . Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 13% yield; Η NMR (CDCI₃, 400 MHz) δ 8.02 (s, lH_maj, C=CH), 7.98 (s, lH_mιn, C=CH), 7.37 (m, 2Η_maj+mιn, Ar-H), 7.25 (m, lΗ_maj+mιn, Ar-H), 4.36 (s, 2Η_maj, (O)CH₂N), 4.18 (s, 2Η_mιn, (O)CH₂N), 3.80 (t, lΗ_mιn, J=4.6 Hz, NCH₂CH₂), 3.69 (t, lH_mm, J=4.5 Hz, NCH₂CH₂), 3.56 (bs, 21^, NCH₂CΗ₂ and NCH₂CH₂), 3.36 (s, 3Η_mιn, OCH₃), 3.30 (s, 3Η_maj, OCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 173.0, 166.4, 166.2, 157.3, 157.2, 150.8, 148.3, 143.7, 143.6, 142.5, 142.4, 125.6, 125.4, 125.3, 124.4, 124.3, 120.5, 120.4, 119.7, 119.5, 71.4, 71.1, 59.2, 59.1, 50.6, 48.8, 47.8; HRMS FAB(M+H)⁺ Calcd for C₁₅H₁₅NO₆F 324.0883, found 324.0888.

iV-(2-Methoxy-ethyl)-N-(7-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9 (2, 4} .

Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 23% yield; Η NMR (CDC1₃, 400 MHz) δ 7.99 (s, ll ^, C=CH), 7.95 (s, lΗ^, C=CH), 7.47 (t, lΗ_maj+mιn, J=8.1 Hz, Ar-H), 6.89 (m, lΗ_maj+mm, Ar-H), 6.81 (m, lΗ_maj+mιn, Ar-H), 4.35 (s, 2H_maj, (O)CH₂N), 4.20 (bs, 2Η_mιn, (O)CH₂N), 3.91 (s, 3Η_mιn, ArOCH₃), 3.89 (s, 3Η_mιn, ArOCH₃), 3.78 (m, lΗ_mm, NCH₂CH₂), 3.68 (m, lH_mm, NCH₂CH₂), 3.55 (bs, 2Η_maj, NCH₂CH₂ andNCH₂CH₂), 3.36 (s, 3Η_mιn, OCH₃), 3.29 (s, 3Η_maj, OCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 173.0, 167.3, 167.1, 164.3, 164.2, 159.0, 156.5, 144.9, 144.8, 130.4, 130.3, 120.8, 120.6, 113.8, 112.4, 112.3, 101.0, 100.9, 71.5, 71.4, 59.1, 59.0, 56.3, 52.4, 50.6, 49.0, 47.8; HRMS FAB(M+H)⁺ Calcd for C₁₆H₁₈NO₇ 336.1083, found 336.1082.

7V-(6-Bromo-8-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-7V-(2-methoxy-ethyl)- glycine 9 {2, 5}. Flash column chromatography with heptane:EtOAc, containing 1 % HOAc, 1:1→1:3, 21% yield; Η NMR (CDC1₃, 400 MHz) δ 7.87 (s, lH_maj, C=CH), 7.85 (s, lΗ^, C=CH), 7.27 (m, lΗ_maj+mιn, Ar-H), 7.19 (dd, lH_maj+mιn, J=l l.l, 2.0, Ar-H), 4.35 (s, 21^, (O)CCH₂N), 4.15 (s, 2Η_mιn, (O)CCH₂N), 3.96 (s,- 31^, ArOCH₃), 3.94 (s, 3Η_mιn, ArOCH₃), 3.75 (t, lΗ_mιn, J=4.8 Hz, NCH₂CH₂), 3.63, (t, lH^, J=4.8 Hz, NCH₂CH₂), 3.53 (m, 2Η_maj, NCH₂CH₂ and NCH₂CH₂), 3.31 (s, 3Η_mιn, OCH₃), 3.28 (s, 3Η_maj, OCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172. 5, 166.1, 166.0, 157.2, 157.2, 147.9, 147.8, 143.1, 143.0, 142.9, 125.8, 125.7, 122.3, 122.1, 120.0, 119.9, 117.9, 117.5, 117.4, 117.3, 71.3, 71.2, 58.9, 58.8, 56.7, 51.8, 50.3, 48.4, 47.7; HRMS FAB(M+H)⁺ Calcd for C₁₆H₁₇NO₇Br 414.0188, found 414.0178.

7V-Benzyl-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9(5, 7}. Flash column chromatography with toluene:EtOAc:HO Ac 80:15:5→60:35:5; 44% yield; ΗNMR(CDC1₃, 400 MHz) δ 8.03 (s, lH_mιn, C=CH), 7.97 (s, lH_maj, C=CH), 7.57 (m, 4H_maj+mιn, Ar-H), 7.33 (m, 5H_maj+mιn, Ar-H), 4.84 (bs 2Η_mιn, ArCH₂N), 4.60 (bs 2Η_maj, ArCH₂N), 4.14 (bs, 211^, (O)CH₂N), 3.96 (bs, 2Η_mιn, (O)CH₂N); ¹³C NMR (CDC1₃, 100 MHz) δ 172.6, 166.8, 166.5, 158.2, 154.3, 154.2, 144.3, 143.4, 135.4, 134.6, 133.3, 129.1, 129.0, 128.0, 128.5, 128.4, 128.2, 128.8, 125.2, 124.3, 124.2, 118.4, 118.2, 117.0, 116.9, 54.0, 49.4, 46.1; HRMS FAB(M+H)⁺ Calcd for C₁₉H₁₆NO₅ 338.1028, found 338.1031.

7V-Benzyl-7V-(3-oxo-3H-naphtho[2,l-6]pyran-2-carbonyl)-glycine 9 {3, 2}. Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5→35:60:5; 55%yield; (CDC1₃, 400 MHz) δ 8.86 (s, IH,,,,,,, C=CH), 8.77 (s, llL^, C=CH), 8.19 (m, 1Η, Ar-H), 8.02 (m, 1Η, Ar-H), 7.89 (m, 1Η, Ar-H), 7.62 (m, 2Η, Ar-H), 7.45-7.23 (m, 6Η, Ar-H), 4.90 (s, 2Η,,,,,,, ArCH₂N), 4.66 (s, 2Η_maj, ArCH₂N), 4.19 (s, lΑ^, (O)CH₂N), 4.03 (s, 2Η™, (O)CH₂N); ^,3C NMR (CDC1₃, 100 MHz) 172.8, 167.3, 166.9, 158.6, 154.3, 154.2, 140.4, 139.5, 135.8,

135.3, 134.6, 134.5, 130.6, 130.3, 130.2, 129.5, 129.1, 129.0, 128.9, 128.4, 128.2, 127.9, 127.8, 127.0, 126.6, 126.5, 122.9, 122.5, 121.9, 121.8, 116.5, 112.9, 112.6, 54.0, 49.5, 47.3;

HRMS FAB(M+H)⁺ Calcd for C₂₃H₁₈NO₅ 388.1184, found 388.1183.

7V-Benzyl-7V-(8-fluoro-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9 (5, 5} . Flash column chromatography with toluene: EtOAc:HOAc 80:15:5→60:35:5; 23%yield; 'HNMR(CDC1₃, 400 MHz) δ 8.02 (s, lH_mιn, C=CH), 7.95 (s, lΗ_maj, C=CH), 7.37-728 (m, (Η_maj+mm, Ar-H), 4.83 (s, 2H , ArCH₂N), 4.60 (s, 2Η_maj, ArCH₂N), 4.12 (s, 2Η_maj, (O)CCH₂N), 3.94 (s, 2Η_mm, (O)CCH₂N); ¹³C NMR(CDC1₃, 100 MHz) δ 172.7, 166.6, 166.3, 157.4, 157.3, 150.8, 148.3, 143.6, 142.9, 142.5, 142.4, 135.7, 135.0, 129.4, 129.2, 129.1, 128.7, 128.6, 128.3, 128.2, 125.5, 125.4, 125.3, 124.5, 124.3, 120.4, 120.2, 119.8, 119.6, 54.2, 49.7, 46.9; HRMS FAB(M+H)⁺ Calcd for C₁₉H₁₅NO₅F 356.0934, found 356.0931.

7V-Benzyl-7V-(7-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9(5, 4}. Flash column chromatography with toluene:EtOAc:HO Ac 80:15:5→60:35:5; 34% yield; 'HNMR (CDC1₃, 400 MHz) δ 7.99 (s, lH_mιn, C=CH), 7.93 (s, lΗ_maj, C=CH), 7.45 (m, 1Η, Ar-H), 7.37-7.32 (m, 4Η, Ar-H), 7.18 (m, 1Η, Ar-H), 6.88 (m, 1Η, Ar-H), 6.81 (m, 1Η, Ar-H), 6.05 (bs, 1Η, CO₂H), 4.83 (bs 2Η_mιn, ArCH₂N), 4.60 (bs 2Η_maj, ArCH₂N), 4.12 (bs, 2Η_maj, (O)CH₂N), 3.95 (bs, 2Η_mιn, (O)CH₂N), 3.88 (s, 3Η_maj+mιn, ArOCH₃); ¹³C NMR (CDC1₃, 100 MHz) δ 172.4, 167.3, 167.0, 164.1, 158.8, 158.7, 156.4, 156.3, 144.8, 143.9, 135.6, 135.0, 130.2, 130.0, 129.2, 129.1, 129.0, 128.4, 128.3, 128.2, 127.9, 120.3, 120.2, 113.6, 112.1, 111.9, 100.8, 100.7, 56.1, 54.0, 49.5, 49.3, 46.5; HRMS FAB(M+H)⁺ Calcd for C₂₀H₁₈NO₆ 368.1134, found 368.1140.

7V-Benzyl-iV-(6-bromo-8-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9 (5, 5} .

Flash column chromatography with heptane:EtOAc, containing 1% HOAc 2:1→1:3, 23% yield; Η NMR (MeOH- ₄, 400 MHz) δ 8.00 (s, H ^, C=CH), 7.98 (s, lΗ^, C=CH), 7.42- 7.17 (m, 7Η_maj+mιn, Ar-H), 4.78 (bs, 2Η_mm, ArCH₂N), 4.65 (bs, 2Η_maj, ArCH₂N), 4.08 (bs, 2H_min, (O)CCH₂N), 3.99 (bs, 2Η_maj, (O)CCH₂N), 3.95 (s, 3Η_maj, ArOCH₃), 3.94 (s, 31^, ArOCH₃); ¹³C NMR (MeOΗ- ₄, 100 MHz) δ 172.4, 171.5, 168.0, 167.9, 163.7, 158.8, 149.2, 144.2, 144.1, 143.2, 143.1, 138.5, 137.0, 136.8, 131.6, 131.5, 129.8, 129.3, 129.1, 128.8, 128.7, 128.6, 126,7, 123.7, 123.3, 122.3, 121.3, 119.0, 118.2, 115.1, 111.3, 111.2, 103.3, 103.1, 99.0, 66.1, 58.5, 58.2, 57.2, 56.8, 56. 7, 54.8; HRMS FAB(M+H)⁺ Calcd for C₂₀H₁₇NO₆Br 446.0239, found 446.0236.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(2-oxo-2H-l-benzopyran-3-carbonyl)-glycine 9 {4, 7} . Flash column chromatography with toluene:EtOAc:HOAc 80: 15:5→60:35:5; 54% yield; Η NMR (CDC1₃, 400 MHz) δ 8.49 (s, lH_maj, C=CH), 8.35 (s, lH_min, C=CH), 7.46 (m, lΗ,Ar-H), 7.20-7.07 (m 4Η, Ar-H), 7.00 (m, 1Η, Ar-H), 6.93 (s, 1Η, Ar-H), 6.83 (bt, 1Η, J=7.9 Ηz, Ar- 77), 6.68 (bt, 1Η, J=7.9 Ηz, Ar-H), 4.29 (bs, 2Η_ma), (O)CCH₂), 3.89 (bs, 2Η_πώl, (O)CCH₂, and 2Η , NCH₂CH₂), 3.69 (m, 2H_maj, NCH₂CH₂), 3.17 (m, 2H_min, NCH₂CH₂), 3.00 (m, 213^, NCΗ₂CH₂); ¹³C NMR (CDC1₃, 100 MHz) δ 171.7, 167.3, 158.4, 153.6, 144.0, 136.1, 132.7, 129.3, 127.0, 124.6, 124.3, 122.7, 122.1, 119.4, 118.8, 117.7, 117.5, 116.3, 111.6, 110.8, 51.3, 48.9, 32.0, 29.2, 24.3, 22.8; HRMS FAB(M+H)⁺ Calcd for C₂₂H₁₉N₂O₅ 391.1239, found 391.1300.

7V-[2-(lH-Indol-3-yl)-ethyl]-iV-(3-oxo-3H-naphtho[2,l-/ ]pyran-2-carbonyl)-glycine9{4,

2}. Flash column chromatography with toluene:EtOAc:HO Ac 80: 15:5→60:35:5; 36% yield; 'HNMR (CDCI₃, 400 MHz) δ 8.46 (s, lH_min), 8.37 (s, lH_maj), 8.03 (s, IH, Ar-H), 7.93 (d, 1Η, J=8.2 Ηz, Ar-H), 7.81 (t, 2Η, J=8.3 Hz, Ar-H), 7.60 (t, 2Η, J=7.8 Hz, Ar-H), 7.53 (t, 2Η, J=7.6 Hz, Ar-H), 7.12 (m, 4H, Ar-H), 6.50 (m, 1Η, Ar-H), 6.22 (m, 1Η, Ar-H), 4.33 (s, lΗ_maj, (O)CH₂N), 4.00 (s, lΗ_min, (O)CH₂N), 3.92 (s, llL^, NCH₂CΗ₂), 3.81 (s, lH^, NCH₂CH₂), 3.19 (bs, lH_min,NCH₂CH₂), 2.96 (s, lΗ_maj,NCΗ₂CH₂); ¹³CNMR(CDC1₃, 100MΗz,) δ 171.8, 167.9, 158.3, 154.0, 141.2. 135.6, 134.3, 130.1, 129.3, 128.8, 128.6, 126.8, 126.4, 124.3, 121.8, 121.5, 121.4, 119.1, 117.0, 116.3, 112.6, 111.3, 111.1, 51.9, 50.4, 32.0, 29.2, 24.6, 22.9, 14.3; HRMS FAB(M+H)⁺ Calcd for C₂₆H₂₁N₂O₅ 441.1450, found 441.1452.

7V-(8-Fluoro-2-oxo-2^-l-benzopyran-3-carbonyl)-7V-[2-(lH-Indol-3-yl)-ethyl]-glycine

9(4, 5}. Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 36% yield; Η NMR (MeOH-</₄, 400 MHz) δ 7.45-7.25 (m, IH, Ar-H), 7.17 (m, 2Η, Ar-H), 7.10 (m, 2Η, Ar-H), 7.00 (m, 2Η, A -H), 6.75 (m, IH, Ar-H), 6.63 (m, IH, Ar-H), 4.28 (bs, 2Η_maj, (O)CCH₂), 3.95 (bs, 2Η_min, (O)CCH₂), 3.81 (t, 2Η_min, J=7.1 Hz, NCH₂CH₂), 3.71 (t, 211^, J=6.0 Hz, NCH₂CH₂), 3.12 (t, 2H_min, J=7.3 Hz, NCH₂CH₂), 2.98 (t, 21^, J=6.0 Ηz, NCΗ₂CH₂); ¹³C NMR (MeOΗ-</₄, 100 MHz) δ 172.1, 167.8, 158.1, 151.4, 148.9, 143.9, 142.9, 142.8, 138.2, 137.9, 130.0, 129.3, 128.4, 126.4, 126.3, 126.2, 125.9, 125.8, 125.7, 125.9, 125.1, 124.1, 122.5, 121.1, 119.8, 119.7, 119.6, 119.4, 118.4, 112.5, 112.4, 112.2, 52.2, 49.8, 25.1, 24.1; HRMS FAB(M+H)⁺ Calcd for C₂₂H₁₈N₂O₅F 409.1199, found 409.1297.

7V-[2-(lH-Indol-3-yl)-ethyl]-7V-(7-methoxy-2-oxo-2H-l-benzopyran-3-carbonyl)-gIycine

9(4, 4}. Flash column chromatography with toluene:EtOAc:HOAc 80:15:5→60:35:5; 34% yield; Η NMR (CDC1₃, 400 MHz), δ 8.49 (s, lH_maj), 8.38 (s, lH_mιn), 7.19 (m, 3H, Ar-H), 7.07 (m, 1Η, Ar-H), 6.85 (m, 3Η, Ar-H), 6.70 (m, 2H, Ar-H), 6.60 (d, 1Η, J=2.2 Ηz, Ar-H), 4.27 (s, lΗ_maj, (O)CH₂N), 3.92 (s, lΗ_mιnj, (O)CH₂N), 3.82 (m, 3Η, ArOCH₃, and 2Η_mιn, NCH₂CH₂), 3.70 (bs, 2H_maj, NCH₂CH₂), 3.15 (bs, 2H_mιn, NCH₂CH₂), 2.98 (bs, 2Η_maj, NCH₂CH₂); ¹³C NMR (CDC1₃, 100 MHz) δ 171.8, 167.8, 163.7, 158.8, 155.7, 144.3, 138.0, 136.2, 130.3, 129.2, 128.4, 127.0, 125.4, 124.3, 121.9, 119.3, 118.9, 117.6, 113.0, 111.6, 111.5, 110.9, 100.2, 55.9, 51.4, 49.1, 24.3, 21.6; HRMS FAB(M+H)⁺ Calcd for C₂₃H₂₁N₂O₆421.1399, found 421.1389.

N-(6-Bromo-8-methoxy-2-oxo-2Z/-l-benzopyran-3-carbonyl)-7V-[2-(l/-^r-Indol-3-yI)- ethyl]-glycine 9(4, 5}. Flash column chromatography with heptane:EtOAc, containing 1% HOAc, 3:1→2:1→1:2, 50% yield; Η NMR (CDC1₃, 400 MHz), δ 8.68 (s, lH_maj), 8.60 (s, lH_mιn), 7.20-7.03 (m, 5H, Ar-H), 6.94 (d, lΗ, J=1.8 Ηz, Ar-H), 6.82 (t, lΗ,J=7.6 Ηz, Ar-H), 6.64 (t, 1Η. J=7.5 Ηz, Ar-H), 6.48 (s, 1Η, Ar-H), 6.44 (d, 1Η,J=1.7 Hz, Ar-H), 4.25 (s, lΗ_maj, (O)CH₂N), 3.90 (s, lΗ_mιn, (O)CH₂N), 3.82 (m, 3Η, ArOCH₃, and 2Η_mιn, NCH₂CH₂), 3.65 (bs, 2H_maj, NCH₂CH₂), 3.12 (m, 2H_mιn, NCH₂CH₂), 2.90 (m, bs 2Η_maj, NCH₂CH₂); ¹³C NMR (CDC1₃, 100 MHz) δ 171.5, 166.9, 157.3, 147.2, 142.8, 142.2, 136.1, 129.2, 128.4, 127.0, 124.7, 123.8, 122.7, 121.9, 119.3, 119.1, 117.4, 117.2, 116.7, 111.6, 110.7, 56.6, 51.2, 48.9, 24.1, 22.8; HRMS FAB(M+H)⁺ Calcd for C₂₃H₂₀N₂O₆Br 499.0504, found 499.0502.

Example 5: Solid-Phase Synthesis of N-Alkylated and N-Acylated amino acids

Resin-bound phenylalanine (23). DIC (201 μL, 1.30 mmol) was added to a solution of Fmoc-Phe-OH (604 mg, 1.56 mmol) and HOBt (140 mg, 1.04 mmol) in dry THF (4 mL). After 1 h, the activated amino acid and DMAP (21 mg, 0.17 mmol, dissolved in dry THF, 1 mL) were added to 2 (2.0 g, 0.52 mmol, pre-swollen in dry THF) and the mixture was agitated at ambient temperature over night. After filtration the resin was washed with THF, MeOH and THF (50 mL of each solvent), and dried over vacuum. A solution of 20% piperidine in DMF was added to the resin (pre-swollen in DMF) and the mixture was agitated for 30 min. After filtration the resin was washed with portions of DMF, MeOH, THF and dry THF, and then dried under vacuum. Solid-phase reductive alkylation of 40. Resin 40 was split into portions and subjected to different conditions for reductive alkylation. These, and the results from the reductive alkylation, are summarized under entries 1-6 in Table B, and described in detail below.

Table C. Reductive alkylation of 40 with j-fluorobenzaldehyde using NaBH3CN as reducing agent.

Entry Reaction conditions Yield (°/op

Solvent Ratio 23:/ F-PhCHO: Reaction time 24a/25a 2^"5a 26a

^a Conversion of 40 as revealed by gel-phase ^F NMR spectroscopy. ^ TMOF = Trimethylortho formate. ^c The aldehyde was allowed to react with 40 for 1 h, excess aldehyde was then removed by filtration prior to addition of NaBH3CN.

Entry 1. / Fluorobenzaldehyde (42 μL, 390 μmol) was added to 40 [150 mg, 39 μmol, pre- swollen in dry trimethyl orthoformiate (TMOF)] in TMOF (2 mL), and the mixture was agitated for40 min. NaB^CN (25 mg, 390 μmol) dissolved in TMOF (1 mL) andHOAc (18 μL) was added and the mixture was agitated for 10 min. After filtration the resin was washed with portions of TMOF, MeOH and THF (2x10 mL of each solvent) and dried under vacuum.

Entry 2. ?-Fluorobenzaldehyde (5 μL, 47 μmol) was added to 40 [150 mg, 39 μmol, pre- swollen in THF:HOAc:H₂O (90:5:5)] in THF:HOAc:H₂O (90:5:5, 1 mL), and the mixture was agitated for 10 min. A solution of NaBH3CN (35 μL, of a 1 M solution in THF, 35 μmol) was added and the mixture was agitated for 3 h. After filtration, the resin was washed with portions of THF, H2O, MeOH and THF (10 mL of each solvent), and dried under vacuum.

Entry 5./?-Fluorobenzaldehyde (4.6 μL, 43 μmol) was added to a suspension of 40 (150 mg, 39 μmol, pre-swollen in MeOH containing 1% HOAc) in MeOH containing 1% HOAc (1 mL), and the mixture was agitated for 60 min. NaBH3CN (12 mg, 195 μmol) dissolved in MeOH (200 μL) was added and the mixture was agitated for 3 h. After filtration, the resin was washed with MeOH, H2O, MeOH and THF (10 mL of each solvent), and dried under vacuum.

Entries 4 and 5. Reductive alkylation was performed as described for entry 3 using 40 (100 mg, 26 μmol), but with larger amounts of/ fluorobenzaldehyde (4.2 μL, 39 μmol for entry 4; 8.4 μL, 78 μmol for entry 5) and NaBH3CN (8 mg, 130 μmol for entry 4; 11 mg, 182 μmol for entry 5).

Entry 6. ?-Fluorobenzaldehyde (8.4 μL, 78 μmol) was added to a suspension of 40 (100 mg, 26 μmol, pre-swollen in MeOH containing 1% HOAc) in MeOH containing 1% HOAc (1 mL), and the mixture was agitated for 60 min. The solution was removed by filtration and additional MeOH containing 1% HOAc (1 mL) was added followed by NaBH3CN (11 mg, 182 μmol) dissolved in MeOH (200 μL). The mixture was agitated for 3 h after which the resin was filtered and washed with MeOH, H2O, MeOH and THF (10 mL of each solvent), and dried under vacuum.

Gel-phase ^F NMR spectroscopy of the resins was recorded as described in general methods and materials.

Acylation of resins obtained by reductive alkylation of 40. Resins obtained by reductive alkylation as described under entries 4 - 6 above (each 100 mg, 26 μmol) were suspended in dry CH2CI2 (1 mL). DIPEA (13 μL, 78 μmol) was added, followed by 4- fluoronaphtoyl chloride (16 mg, 78 μmol) dissolved in dry CH2CI2 (200 μL), after which the mixture was agitated at ambient temperature for 2 h. The solution was filtered off, and the resin was washed with CH2CI2, MeOH and THF (20 mL portions of each solvent). The resin was then dried under vacuum. Gel-phase ^ NMR spectroscopy of the resins was recorded as described in general methods and materials.

N-(4-Fluorobenzyl)-JV-(4-fluoronaphtoyl)-phenylalanine (44). Solid-phase reductive alkylation of 40 was performed as described in entry 6 above by treatment of resin 40 (1.0 g, 0.26 mmol) with/j-fluorobenzaldehyde (84 μL, 0.78 mmol) and NaB^CN (114 mg, 1.82 mmol). Acylation of the resulting resin was accomplished using 4-fluoronaphtoyl chloride (162 mg, 0.78 mmol) and DIPEA (130 μL, 0.78 mmol) as described above. Compound 44 was cleaved from the resin using aqueous LiOH (1 M, 4 mL) in THF:MeOH:H2θ (3:1:1; 40 mL) at ambient temperature for 2 h. After filtration and subsequent washing of the resin with HOAc and THF (80 mL of each solvent), the filtrate was concentrated and finally co-concentrated from toluene (3x50 mL). The residue was dissolved in a mixture of EtOAc (30 mL) and aqueous HCl (0.05 M, 10 mL). The water phase was separated and acidified with aqueous HCl (1 M) and extracted with EtOAc (2x30 mL). The combined organic layers were dried (Na2SO4) and concentrated. The crude product was purified by flash column chromatography (heptane:EtOAc 4:102:1, containing 1% HOAc) to give 44 (88 mg, 76% yield, based on the initial loading capacity of the resin). !H NMR (OMSO-d , 300 MHz, 420 K) 8.07 (d, IH, J=8.2 Hz, Ar-H), 7.84 (d, IH, J=8.0 Hz, Ar-H), 7.66-7.50 (dt, 2H, J=7.6, 1.2 Hz, Ar-H), 7.26-7.05 (m, 9 H, Ar- H), 6.90 (t, 2 H, J=8.7 Hz, Ar-H), 4.63 (dd, IH, J=8.3, 6.2 Hz, Phe-H₀), 4.55 (m, IH, Phe- H_β), 4.27 (bd, IH, J=15.6 Hz, Phe-H_β), 3.43 (dd, IH, J=14.0, 6.1 Hz, ArCH₂N), 3.26 (m, IH, ArCH₂N); ¹³C NMR (CDC1₃, 100 MHz) 174.8, 171.7, 163.8, 161.3, 160.7, 158.2, 138.0, 130.5, 129.6, 129.5, 129.2, 129.0, 128.7, 128.6, 128.4, 127.3, 127.2, 125.2, 121.1, 115.7, 115.5, 108.8, 60.6, 54.8, 54.8, 34.9; HRMS (EI+) Calcd for C27H22NO3F2 446.1567, found 446.1573.

Example 6 : Direct Binding Assay

The affinity of the low molecular weight compounds as synthesized in the above Examples, for periplasmic chaperones PapD and FimC were investigated using a direct binding assay on BIACORE 3000.

Methods

PapD (50 μg/mL in 10 mM NaAc pH 5.5) and FimC (50 μg/mL in 10 mM NaAc pH 5.5) were immobilized on Sensor Chip CM5 using a standard thiol coupling procedure. This procedure was also employed for coupling of non-target proteins. Immobilization levels of 6-8 000 RU were obtained. Unmodified dextrane in one of the flow cells was used as reference surface.

The compounds were prepared as described in Examples 1 and 2 and diluted from 10 mM DMSO stock solutions to a final concentration of 100 μM or 10 μM in running buffer (67 mM phosphate buffer (9.6 g Na₂HPO₄ •2H₂O, 1.7 g KH2PO4, 4.1 g NaCl, 1000 mL H₂O), 3.4 mM EDTA, 0.01% Tween, 5% DMSO, pH 7.4.) so that the concentrations of DMSO and buffer substances were carefully matched. The compounds were injected (flow rate was 30 μl/min at 25 °C) and the binding of the compounds to the immobilized chaperone proteins was observed on real time. The surface was regenerated by injection of 10 mM glycine^»HCl, pH 2.0. To avoid carry over, the flow system was washed with a 1 : 1 mixture of DMSO and H2O.

For screening of the affinity of the compounds for PapD and FimC, the compounds were injected (flow rate was 30 μl/min at 25 °C) at a concentration of 100 μM in duplicate or triplicate and in random order. The surface was regenerated by injection of 10 mM glycine*HCl, pH 2.0. To avoid carry over, the flow system was washed with a 1:1 mixture of DMSO and H2O. Reference chemicals were used as negative controls.

For the estimation of Krj, the compounds were injected in concentration series of 1.0 μM-100 μM, and association and dissociation kinetics were studied. Selectivity data was obtained by injecting the compounds over both target and non-target, i.e. Protein A, Streptavidin, and Anti-myoglobin mAb, surfaces.

Results

Table D: Affinity of Compounds for PapD and FimC.

Compound Response Units PapD FimC N-[2-(lH-Indol-3-yl)-ethyl]-N- 110 120

(naphtalene-2-carbonyl)-tyrosine 19 (3,3}

N-[2-(lH-Indol-3-yl)-ethyl]-N-(2-oxo- 40 48 2H- 1 -benzopyran-3 -carbonyl)-tyrosine 17(5, 7}.

N-[2-(lH-Indol-3-yl)-ethyl]-N-(3-oxo- 30 38 3H-naphtho[2, 1 -b]pyran-2-carbonyl)- glycine 9 (4, 2} .

N-[2-(lH-Indol-3-yl)-ethyl]-N-(2-oxo- 20 17 2H- 1 -benzopyran-3-carbonyl)-lysine 17(4, 7}.

N-[2-(lH-Indol-3-yl)-ethyl]-N-(3- 18 methyl-butyryl-carbonyl)-tyrosine 19 {3,4} N-[2-(lH-Indol-3-yl)-ethyl]-N- 17 22

(naphtalene-2-carbonyl)-glycine 19(7, 2).

For compounds showing strong and medium affinity for PapD and FimC (Ν-[2- (lΗ-Indol-3-yl)-ethyl]-N-(naphalene-2-carbonyl)-tyrosine 19 (3,3 } ,N-[2-(lH-indol-3-yl)- ethyl]-N-(2-oxo-2H-l-benzopyran-3-carbonyl)-tyrosine 17(5, 7}, KJJ was estimated to 1- 100 μM.

No significant non-specific binding of the compounds to the non-target proteins were observed.

Example 7: Inhibition Assay Using FimCΗ Reconstitution Assay In order to conclusively identify the compounds that inhibit a pilus chaperone, Applicants conducted a reconstitution assay using FimCH and PapDG.

Methods. The FimCH complex was brought to 3M Urea in 20mM MES pH 6.8 and injected onto a 1ml Source 15S Pharmacia column (lml/min flow rate) and pure FimH was collected in the Flow Through. The PapDG complex was brought to 5M Urea in 20mM MES pH 6.8 and injected onto a 1ml Source 15S Pharmacia column (lml min flow rate) and pure Pap D was collected in the Flow Through. For wild type control assay, PapG in 5M Urea was diluted lOfold volume into

20mM MES pH 5.8 containing an equimolar amount of PapD. This was reinjected onto the 1ml Source 15S column and the reconstituted PapDG complex was eluted using 40mM NaCl and the excess PapD was eluted using 65mM NaCl. Using the Unicorn program for the Pharmacia AKTA, the area under the PapDG peak was integrated and this was calculated to be 100% reconstitution. The same process was repeated for the wild type control assay for FimCH except that FimH was in 3M Urea was diluted lOfold volume into 20mM MES pH 5.8 containing an equimolar amount of PapD.

The candidate compounds were synthesized as described in Examples 1 and 2. For the tested compounds, a 38 M excess was preincubated with the PapD (or FimC) for 15 minutes and the same reconstitution and PapDG (or FimCH) separation was performed. Binding of the compounds to the chaperones in the column were detected using UV light (UVA 280). Peak areas were calculated as a percent of wild type.

Results

Table E represents the library of compounds evaluated for the inhibition of formation of the complex between PapD and PapG; the % inhibition of the complex between PapD and PapG at an inhibitor/PapD ratio of 38. Compound 9(4, 1} was also evaluated as an inhibitor of FimCH complex formation. It gives 18% inhibition at a 69 fold excess as compared to FimC and 60% inhibition at a 207 fold excess. TABLE E

Other features, objects and advantages of the present invention will be apparent to those skilled in the art. The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the invention, its principles, and its practical application. Those skilled in the art may adapt and apply the invention in its numerous forms, as may be best suited to the requirements of a particular use. Accordingly, the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the present invention.

Claims

WE CLAIM:

1. A compound of the formula:

R ₂

wherein each of R_l5 R₂ and R₃ is independently a substituted or unsubstituted alkyl (C,_.10), substituted or unsubstituted acyl (C₂.₁₅), substituted or unsubstituted aryl

(C_6.14), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C_7.15), substituted or unsubstituted heteroarylalkyl or substituted or unsubstituted heterocycloalkyl;

R₄ is a carboxy (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂), phosphonate (PO(OH)₂) or ketone (-COR) wherein R is a halogenated or unsubstituted alkyl (C,_₃); and the salts, esters and amines thereof.

2. The compound of claim 1 wherein substituents on any alkyl or alkylene moiety is selected from the group consisting of halogen, hydroxy, loweralkoxy, carboxy, carboalkoxy, carboxamido, cyano, carbonyl, NO₂, alkylthio, alkylthiol, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy, substituted phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, heteroaryloxy and NR R wherein each R^' and R"is independently H, alkyl or arylalkyl.

3. The compound of claim 2 wherein R, is selected from side chains of amino acid residues; R₂ is substituted or unsubstituted alkyl, arylalkyl, heteroarylalkyl, or heterocycloalkyl; R₃ is substituted or unsubstituted alkyl, aryl, or heteroaryl; and R₄ is a carboxyl group (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂) or phosphonate (PO(OH)₂).

4. The compound of claim 3 wherein R¹ is selected from the side chains of hydrophobic aromatic, hydrophobic aliphatic, polar and charged amino acid residues; R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl; R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; and R₄ is -CO₂H, -CONH₂ or -CHO.

The compound of claim 4 wherein R, is selected from the side chains of hydrophobic aromatic, hydrophobic aliphatic and polar amino acid residues; R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl; R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; and R₄ is -CO₂H or -CHO.

The compound of claim 5 wherein R_j is selected from hydrophobic aromatic and hydrophobic aliphatic amino acid residues; R₂ is substituted or unsubstituted arylalkyl or substituted or unsubstituted heteroarylalkyl; R₃ is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; and R₄ is -CO₂H.

The compound of claim 6 wherein R^ is hydrogen, benzyl, 4-aminobutyl or p- hydroxy-benzyl; R₂ is (CH₂)_mA wherein m is between 0 and 3 and A is n-butyl, phenyl and 3-indolyl; R₃ is isobutyl,

and R₄ is carboxyl.

8. The compound of claim 7 wherein said compound is

9. The compound of claim 7 wherein said compound is

10. The compound of claim 7 wherein said compound is

11. The compound of claim 7 wherein said compound is

12. The compound of claim 7 wherein said compound is

13. The compound of claim 7 wherein said compound is

14. The compound of claim 7 wherein said compound is

15. The compound of claim 7 wherein said compound is

16. The compound of claim 1 wherein each R„ R₂ and R₃ is independently an amino acid residue side chain and R₄ is carboxyl or aldehyde.

17. The compound of claim 16 wherein the amino acid residue side chain is selected from the group consisting of hydrogen, p-hydroxybenzyl, 2-(3-indolyl)-ethyl, benzyl, 5-imidazole, isopropyl, isobutyl, 2-methylpropyl, methyl and 2- thiomethylethyl.

18. The compound of claim 17 wherein R, is hydrogen or p-hydroxybenzyl.

19. The compound of claim 18 wherein R_j is p-hydroxybenzyl.

20. The compound of claim 18 wherein R_x is hydrogen.

21. The compound of any one of claims 1-20 contained in a mixture of its stereoisomers.

22. The compound of any one of claims 1 -20 in optically pure form.

23. The compound of any one of claims 1-20 wherein said compound exhibits antibacterial activity against a Gram-negative bacterium selected from the group consisting of Escherichia coli, Haemophilus influenzae, Salmonella enteriditis, Salmonella typhimurium, B or detella pertussis, Yersinia pestis, Yersinia enterocolitica, Helicobacter pylori and Klebsiella pneumoniae.

24. The compound of claim 23 wherein said compound exhibits antibacterial activity by binding to a pilus chaperone thereby exhibiting antibacterial activity against a Gram-negative bacterium.

25. The compound of claim 24 wherein the pilus chaperone is a PapD chaperone.

26. The compound of claim 24 wherein the pilus chaperone is a FimC chaperone.

27. A method of treating a Gram-negative infection in a subject which method comprises administering to a subject in need thereof an effective amount of the compound of claim 23.

28. A method of preventing or inhibiting the attachment of a Gram-negative organism to host tissues in a mammal, said method comprising administering an effective amount of the compound of claim 23 to said mammal thereby inhibiting pili assembly.

29. A method of preventing or inhibiting biofilm formation, said method comprising administering a compound of claim 23 to an environment or surface containing Gram-negative bacteria.

30. A method of preventing or inhibiting bacterial colonization by a Gram-negative organism in a mammal, said method comprising administering the compound of claim 23 to said mammal.

31. A pharmaceutical composition comprising the compound of claim 23 in combination with a pharmaceutically acceptable carrier.

32. A process for the preparation of a compound of the formula

wherein R_t is hydrogen; R₂ is (CH₂)_mA wherein m is between 0 and 3 and A is n- butyl, 2-methoxyethyl, benzyl and 2-(3-indolyl)-ethyl; R₃ is coumarin; and R₄ is carboxyl, said method comprising condensing a compound 6 of Reaction Scheme 1 with a salicyaldehyde selected from the group consisting of the compounds listed in Table A.

33. The process of claim 32 wherein the process occurs in solution.

34. A linker compound of the formula:

wherein R', is -CO₂H, -(CH₂)_nCO₂H or -O(CH₂)_nCO₂H wherein n is between 1 and 10; and

R'₂ and R'₃ is independently fluorine or hydrogen provided that when either R'₂ or R'₃ is fluorine, the other is hydrogen.

35. The linker compound of claim 34 wherein R'₂ is hydrogen and R'₃ is fluorine.

36. The linker compound of claim 35 wherein n is between 1 and 5.

37. The linker compound of claim 36 wherein n is 1 or 2.

38. The linker compound of claim 37 wherein the linker compound is

39. The linker compound of claim 37 wherein the linker compound is

40. The linker compound of claim 37 wherein the linker compound is

41. The linker compound of claim 34 wherein the linker compound is

42. A process of synthesizing a linker compound, said process comprising: a. hydrolyzing one of the ester moieties of dimethyl-2-fluoroterephtalate; b. reducing the remaining ester; and c. separating the two regioisomers.

43. The linker compound produced by the process of claim 42.

44. The process of claim 42 wherein the remaining ester is reduced with LiBH₄.

45. The process of claim 42 further comprising: d. protecting a fluorinated 4-(hydroxymethyl) benzoic acid produced by the process of claim 42 thereby forming a benzyl ester; e. oxidizing the hydroxymethyl group and condensing the resulting aldehyde with an ethyl acetate compound; f. reducing the product of step (e); g. reducing the carboxyl group of the product of step (f); and h. hydrolyzing the ethyl ester.

46. The compound produced by the process of claim 45.

47. A process of synthesizing a linker compound, said process comprising: a. dealkylating a 2-fluoro-4-propoxybenzoic acid; b. reducing the carboxylic acid of the product of step (a) thereby producing a hydroxymethylphenol compound; c. alkylating the phenolic hydroxyl group of the hydroxymethylphenol compound; and d. hydrolyzing the product of step (c) under basic conditions.

48. The linker compound produced by the process of claim 47.

49. The process of claim 32 wherein the process occurs on solid phase whereby a linker compound of any one of claims 38-41, 43, 45 or 48 is interposed between a solid support and said compound 6 of Reaction Scheme 1.

50. A process for the solid-phase synthesis of a compound of the formula:

wherein R, is hydrogen, R₂ is (CH₂)_mA wherein m is between 0 and 3 and A is selected from the group consisting of n-butyl, benzyl and 2-(3-indolyl)-ethyl, R₃ is coumarin and R₄ is carboxyl, said process comprising the steps of: a. affixing a linker compound of any one of claims 38-41, 43, 45 or 48 onto a solid support to give a benzylic alcohol; b. subjecting the benzylic alcohol to acylation with bromoacetic acid; c. subjecting the bromoacetate to a nucleophilic substitution with an amine; d. acylating with ethyl malonyl chloride thereby forming a N-alkyl-N- (malonamic acid ethyl ester)-glycine derivative; e. condensing the N-alkyl-N-(malonamic acid ethyl ester)-glycine derivative with a salicyaldehyde; and f. cleaving the compound from the linker compound under acidic or basic conditions.

51. The process of claim 50 wherein step (b) is repeated once.

52. A process for the solid-phase synthesis of a compound of the formula

wherein R_[ is selected from side chains of any amino acid residue provided that R, is not hydrogen, R₂ and R₃ is independently a substituted or unsubstituted alkyl (C ₁₀), substituted or unsubstituted acyl (C_2.15), substituted or unsubstituted aryl (C₆.₁₄), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C₇_

₁₅), substituted or unsubstituted heteroarylalkyl or substituted or unsubstituted heterocycloalkyl and R₄ is carboxyl, said process comprising the steps of: a. affixing a linker compound of any one of claims 38-42, 43, 45 or 48 onto a solid support to give a benzylic alcohol; b. coupling a Fmoc-protected amino acid to the benzylic alcohol thereby producing a amino acid functionalized resin; c. removing the Fmoc-protecting group from the α-amino acid group of the product of step (b); d. alkylating the α-amino group of the product of step (c) by reductive alkyation; e. removing excess aldehyde from the amino acid functionalized resin; f. acylating with an acid chloride thereby producing a N-(alkylated)-N- (acylated)-amino acid derivative; and g. cleaving the compound from the linker under acidic or basic conditions.

53. In a method of synthesizing a combinatorial library, an improvement comprising affixing a linker compound of any one of claims 38-41, 43, 45 or 48 onto a solid support.

54. A library of compounds which are candidates targeted for antibacterial properties comprising at least two different compounds having the formula:

R ₂

I

wherein each of R„ R₂ and R₃ is independently a substituted or unsubstituted alkyl (C,_.10), substituted or unsubstituted acyl (C₂.₁₅), substituted or unsubstituted aryl (C_6.14), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C_7.15), substituted or unsubstituted heteroarylalkyl or substituted or unsubstituted heterocycloalkyl; and

R₄ is a carboxy (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂), phosphonate (PO(OH)₂) and ketone (-COR) wherein R is a halogenated or unsubstituted alkyl (C^), wherein any of the candidate compounds are retrievable and analyzable for said antibacterial properties.

55. The library of claim 54 wherein each R, is independently hydrogen, p- hydroxybenzyl, benzyl or 2-(3-indolyl)-ethyl.

56. The library of claim 55 wherein each R, is hydrogen.

57. The library of claim 55 wherein each R, is p-hydroxybenzyl.

58. The library of claim 56 or 57 wherein the library comprises at least five different compounds.

59. The library of claim 56 or 57 wherein the library comprises at least ten different compounds.

60. The library of any one of claims 55-59 wherein said library of compounds exhibit antibacterial activity against a Gram-negative bacterium selected from the group consisting of Escherichia coli, Haemophilus influenzae, Salmonella enteriditis, Salmonella typhimurium, Bordetella pertussis, Yersinia pestis, Yersinia enterocolitica, Helicobacter pylori and Klebsiella pneumoniae.

61. The library of claim 60 wherein said library of compounds exhibits antibacterial activity by binding to a pilus chaperone thereby inhibiting pili assembly.

62. The library of claim 61 wherein the pilus chaperone is a PapD chaperone.

63. The library of claim 61 wherein the pilus chaperone is a FimC chaperone.

64. A method for monitoring solid-phase synthesis of a compound having the formula:

R ₂

wherein each of R„ R₂ and R₃ is independently a substituted or unsubstituted alkyl (C,_.10), substituted or unsubstituted acyl (C₂.₁₅), substituted or unsubstituted aryl (C_6.14), substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl (C_7.l5), substituted or unsubstituted heteroarylalkyl and substituted or unsubstituted heterocycloalkyl; and

R₄ is a carboxy (-CO₂H), carboxamide (-CONH₂), aldehyde (-CHO), boronate (-B(OH)₂), phosphonate (PO(OH)₂) and ketone (-COR) wherein R is a halogenated or unsubstituted alkyl (C^), wherein said method comprises: a. affixing a linker compound of any one of claims 38-41 , 43, 45 or 48 onto a solid support; b. utilizing a means for measuring a signal originating from the linker compound; and c. utilizing said signal as an internal reference thereby enabling the monitoring of reactions of said solid-phase synthesis of compounds.

65. The method of claim 64 wherein said signal originating from said linker compound is a ^I9F resonance.

66. The method of claim 65 wherein said means for measuring the ¹⁹F resonance is ¹⁹F NMR spectroscopy.

67. The method of claim 64 wherein said solid support comprises polystyrene resin beads, silica chips and polyethylene glycol resins.

68. The method of claim 67 wherein said solid support is polyethylene glycol grafted polystyrene resin.

69. A complex comprising the compound of any one of claims 1-20 complexed with a linker compound of any one of claims 38-41, 43, 45 or 48 wherein said linker compound is affixed to a solid support.