WO2007075931A2

WO2007075931A2 - Reactive heterocyclic derivatives and methods for their synthesis and use

Info

Publication number: WO2007075931A2
Application number: PCT/US2006/048864
Authority: WO
Inventors: Joseph Barry Noar; Mariusz G. Banaszczyk; Violeta Millan; Gunars Valkirs; Kenneth F. Buechler; Brian Noland
Original assignee: Biosite Incorporated
Priority date: 2005-12-22
Filing date: 2006-12-22
Publication date: 2007-07-05
Also published as: WO2007075931A3

Abstract

The present invention describes molecules having a core structure that is an indole or an indolene that is modified to provide a functional moiety, and methods for their synthesis and use. Such functionalized heterocyclic derivatives may be used in a variety of assay formats.

Description

REACTIVE HETEROCYCLIC DERIVATIVES AND METHODS FOR THEIR

SYNTHESIS AND USE

FIELD OF THE INVENTION

[0001] The present invention relates to novel heterocyclic derivatives and to methods for their synthesis and use. Specifically disclosed are indole derivatives and indolene derivatives.

BACKGROUND OF THE INVENTION

[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0003] A variety of linkage chemistries have been described for the attachment of a particular molecule of interest, often for purposes developing binding assay (e.g., immunoassay) reagents. Thus, molecules may be coupled via a selected linkage chemistry for solid-phase immobilization, conjugation of haptens to immunogenic carrier molecules, preparation of antibody-detectable label conjugates, immunotoxins and other labeled protein and nucleic acid reagents, etc. Such linkage chemistries often provide the molecule of interest with one or more functional groups that couple to amino acid side chains of peptides. Among other characteristics, these "linkage reagents" may be classified on the basis of the following:

1. Functional group(s) and chemical specificity;

2. length and composition of the cross-bridge;

3. whether the functional group(s) react chemically or photochemically; and

4. whether the resultant linkage is cleavable.

[0004] Reactive groups that can be targeted using linkage chemistries include primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids, hi addition, many reactive groups can be coupled nonselectively using a cross-linker such as photoreactive phenyl azides.

[0005] Linkage chemistries may be provided with a variety of spacer arm (or "bridge") lengths for spacing the molecule of interest from its conjugate partner. The most apparent attribute of the bridge is its ability to deal with steric considerations of the moieties to be linked. Because steric effects dictate the distance between potential reaction sites, different lengths of bridges maybe considered for the interaction.

[0006] Each reference cited in the preceding section is hereby incorporated by reference in its entirety, including all tables, figures, and claims.

BRIEF SUMMARY OF THE INVENTION

[0007] It is an object of the invention to provide novel heterocyclic derivatives and methods for their synthesis and use.

[0008] In a first aspect, the invention provides a heterocyclic derivative, or a salt thereof, having the following structure:

wherein,

X is B-A- wherein, A comprises from 0-30 backbone atoms (including, for example, 1-30 backbone atoms), of which 0-10 of said backbone atoms are heteroatoms, and A is optionally substituted with from 1 to 6 substituents independently selected from the group consisting of Ci-₆ alkyl, halogen, oxo (=O), trihalomethyl, Ci-β alkoxy, -NO2, - NHR², -OH, -CH₂OH, -C(O)NHR², -(CH₂)o-₃-C(0)OR², and, wherein, B is a functional moiety selected from the group consisting of protected or unprotected sulfhydryl moieties, protected or unprotected amine moieties, primary amine-reactive moieties, sulfhydryl-reactive moieties, photoreactive moieties, carboxyl- reactive moieties, arginine-reactive moieties, and carbonyl-reactive moieties, protein, polypeptide, antibody, antibody fragment, single- chain variable region fragment, small molecule, nucleic acid, oligosaccharide, polysaccharide, cyclic polypeptide, peptidomimetic, aptamer, detectable label and solid phase; R¹ is absent or, when present, is selected from the group consisting of Ci_-C alkyl, -(CH₂)o-₃-CH(NHR²)C(0)OR², halogen, trihalomethyl, d.₆ alkoxy, -NO₂, -NHR², -OH, -CH₂OH, -C(O)NHR², -(CH₂)o-3-NHR², and -(CH₂)(_M-C(O)OR²; R² is, independently, H or Ci.6 alkyl; R³ is R¹, R², or-C(O)R²; and " " is a single or double bond.

[0009] Specific examples of these moieties are described hereinafter.

[0010] In some embodiments, the heterocylic derivatives of the present invention have the general formulae:

[0011] It is contemplated that, in some embodiments, R¹ is

wherein, each y=l and each R²=H.

[0012] These may be referred to as indole-3-carboxylic acid, serotonin, and tryptophan derivatives, respectively.

[0013] In other embodiments, R³ is -C(O)R² such as, for example, in the heterocyclic derivatives having the general formula:

[0014] In other embodiments, X comprises either a protected or unprotected thiol group. Variations of thiol-containing X-groups contemplated herein include, for example, the following structures:

and , wherein m = 1-4, including embodiments in which m = 1-2, and R^p refers to the protecting group, if present. For unprotected thiols, R^p is H.

[0015] Numerous protected thiol structures are known in the art, and include thioethers, thioesters, unsymmetric disulfides, and sulfenyls. See, e.g., Greene and Wutz, Protective Groups in Organic Synthesis. 3^rd Ed., Wiley-Interscience, 1999, which is hereby incorporated by reference in its entirety. Protective groups for thiols (R^p) contemplated herein include, for example:

[0016] Alternatively, a thiol may be protected by formation of a disulfide with another derivative, having the identical or a different structure. In accordance with the foregoing, protected thiol derivatives include, for example:

wherein m = 1-4 including, for example, embodiments wherein m = 1-2.

[0017] In each case, the choice of thiol protective group may optionally be replaced by another protective group, as defined above, to provide additional protected thiol derivatives. The foregoing merely illustrates some exemplary heterocyclic derivatives having protected thiol groups. Other derivatives (e.g., other thiol derivatives, indole derivatives, indolene derivatives, indole-3-carboxylic acid derivatives, serotonin derivatives, and tryptophan derivatives) can be made in accordance with the principles of this disclosure. Also, although the foregoing structures are provided with certain particularity, they are intended to be illustrative only.

[0018] In related aspects, the invention provides various compounds that are convenient intermediates, precursors, or reagents for producing the foregoing heterocyclic derivatives. These compounds may have one of the following general formulae:

; and salts thereof;

or

, and salts thereof, wherein R⁴ is H or a linkage to a protected carboxyl group; or

, and salts thereof, wherein z = 1-3. [0019] Intermediates contemplated herein include, for example, indole-3 -acetic acid, serotonin, and tryptophan intermediates.

[0020] In some embodiments, the functional moiety (B) is selected from the group consisting of a protein, polypeptide, antibody, antibody, fragment, single-chain variable region fragment, small molecule, nucleic acid, oligosaccharide, polysaccharide, cyclic polypeptide, peptidomimetic, aptamer, detectable label and solid phase. Such functional moieties may be attached to a heterocyclic derivative via a covalent bond to a constituent atom naturally present on the functional moiety. For example, the heterocyclic derivative may comprise a maleimide moiety that couples to a cysteine thiol occurring naturally in a polypeptide. Such a linkage would have the following structure formed, for example, by reaction of a sulfhydryl with a maleimide:

[0021] In alternative embodiments B can comprise a linkage chemistry introduced through chemical or other means, that is used in forming the heterocyclic derivative. For example, in the one embodiment where the heterocyclic derivative comprises a terminal S-acetyl or other thiol protective group, the derivative may be formed by deprotection of a protected thiol, followed by free thiol coupling through a maleimide moiety introduced into the target protein, polypeptide, antibody, etc.

[0022] As noted above, the heterocyclic derivatives of the present invention may be used in receptor binding assays. Assay methods include, for example, immunoassay, (e.g., competitive assays, non-competitive assays, sandwich assays, homogenous assays, etc.) and nucleic acid hybridization. Specific examples of such assays are described hereinafter.

[0023] In particular, the invention provides a method for detecting a target molecule in a fluid sample by detecting the binding of the target molecule to a heterocyclic derivative as described herein. In one particular embodiment, the method comprises:

a) contacting the heterocyclic derivative with the fluid sample under conditions suitable for binding of the heterocyclic derivative to the target molecule, wherein the functional moiety is capable of binding the target molecule; and

b) detecting the binding of the heterocyclic derivative to the target molecule.

[0024] The invention also provides a method for detecting two or more target molecules in a fluid sample by:

a) contacting an array comprising two or more different types of capture molecules with a fluid sample under conditions suitable for binding of the two or more target molecules target molecules to the capture molecules, wherein at least one type of capture molecule is a heterocyclic derivative as described herein; and

b) detecting the binding of the two or more target molecules to the capture molecules.

[0025] Any of the foregoing detection methods may further comprise measuring the amount of the binding and, optionally, relating the amount of the binding to the fluid sample as an indicator of the amount of target molecule present in the fluid sample.

[0026] In some embodiments of the foregoing methods, the heterocyclic derivative is bound to a solid support. The heterocyclic derivatives may be present in an array and, optionally, in discrete and addressable physical locations on that array.

[0027] In other embodiments, the binding of the target molecule(s) to the capture molecules (heterocyclic derivatives) may be detected using a secondary detection molecule such as an antibody or an oligonucleotide that specifically binds to the heterocyclic derivative or the target molecule. Optionally, the secondary detection molecule may be detectably labeled.

[0028] It is another object of the present invention to provide devices for performing assays. Such devices comprise a heterocyclic derivative as described herein comprising, for example, an antibody or binding fragment thereof covalently linked to a detectable label; and/or a heterocyclic derivative as described herein comprising an antibody or binding fragment thereof covalently linked to a solid phase. The devices of the present invention preferably contain a plurality of diagnostic zones (i.e., discrete and addressable physical locations), each of which is related to a particular analyte of interest. Such devices may be referred to as "arrays" or "microarrays." Following reaction of a sample with the devices, a signal may be generated from the diagnostic zone(s), which may then be correlated to the presence or amount of the analyte(s) of interest. Numerous suitable devices are known to those of skill in the art, and exemplary devices are described hereinafter.

[0029] It is another object of the present invention to provide methods for producing the heterocyclic derivatives and conjugates of the present invention. Exemplary methods are described herein.

[0030] As used herein, the term "target molecule" refers to a molecule of interest for which an assessment of presence or amount is desired.

[0031] As used herein, the term "capture molecule" refers to a molecule that binds to a target molecule and is a component of the assessment/detection methodology. For instances in which the target molecule is a protein, for example, the capture molecule may be an antibody or antibody fragment that specifically binds to the target molecule. Capture molecules of this invention further comprise a heterocyclic derivative described herein.

[0032] As used herein, the term "alkyl" refers to a saturated or unsaturated aliphatic hydrocarbon including straight chain and branched chain groups, optionally containing one or more backbone heteroatoms and may be substituted or unsubstituted. Lower alkyl groups have 1-4 backbone carbon atoms. Medium alkyl groups have 5-10 backbone carbon atoms.

[0033] The term "heteroatom" as used herein refers to non-carbon, non-hydrogen atoms such as N, O, and S.

[0034] An "alkoxy" group refers to both an -O-alkyl and an -O-cycloalkyl group; preferably an alkoxy group refers to a lower alkoxy, and most preferably methoxy or ethoxy.

[0035] The structure formula -(CH₂)o-₃-CH(NHR²)C(0)OR² refers to the following chemical structure:

[0036] The term "antibody" as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3^rd Edition, W.E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iy) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." [0037] The term "polypeptide" as used herein refers to a molecule having a sequence of amino acids linked by peptide bonds. This term includes proteins, fusion proteins, oligopeptides, cyclic peptides, and polypeptide derivatives. Antibodies and antibody derivatives are discussed above in a separate section, but antibodies and antibody derivatives are, for purposes of the invention, treated as a subclass of the polypeptides and derivatives. The term protein refers to a polypeptide that is isolated from a natural source, or produced from an isolated cDNA using recombinant DNA technology, and that has a sequence of amino acids having a length of at least about 200 amino acids.

[0038] The term "nucleic acids" as used herein shall be generic to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), to polyribonucleotides (containing D-ribose or modified forms thereof), and to any other type of polynucleotide which is an N-glycoside of purine or pyrimidine bases, or modified purine or pyrimidine bases.

[0039] The term "aptamer" as used herein is a single-stranded or double-stranded oligodeoxyribonucleotide, oligoribonucleotide or modified derivatives that specifically bind and alter the biological function of a target molecule. The target molecule is defined as a protein, peptide and derivatives thereof. The aptamer is capable of binding the target molecule under physiological conditions. An aptamer effect is distinguished from an antisense effect in that the aptameric effects are induced by binding to the protein, peptide and derivative thereof and are not induced by interaction or binding under physiological conditions with nucleic acid.

[0040] The term "polysaccharide" as used herein refers to a molecule comprising more than 10 glycosidically linked monosaccharide residues, while the term "oligosaccharide" refers to a molecule comprising from 2-10 glycosidically linked monosaccharide residues.

[0041] The term "small molecule" includes any molecule having a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, most preferably less than about 500 Da. BRIEF DESCRIPTION OF THE FIGURE

[0042] FIGURE 1 is a graph summarizing the results of the assay described in Example 10, presenting fluorescence signal (in arbitrary units) versus the log analyte concentration (in μM).

DETAILED DESCRIPTION OF THE INVENTION

[0043] This invention provides novel heterocyclic derivatives. The core nucleus of the heterocyclic derivatives is a bicyclic ring system having the following generic structure:

[0044] This generic structure is meant to encompasses both an indole ring system:

and an indolene ring system:

[0045] All of the compounds of this invention have either an indole or indolene ring system core and are collectively referred to as "heterocyclic derivatives".

The Heterocyclic Nucleus

[0046] Indole, which forms part of the side chain of the amino acid tryptophan, is present in plants, animals, and microorganisms in a number of forms. In addition to tryptophan, numerous metabolites and derivatives based upon an indole nucleus have been described, including ergotamine, ergocristine, lysergic acid amide, 3- methylindole, 3-hydroxyindole, indole-3-acetic acid, indole-3 -butyric acid, tryptamine, etc. This list is not meant to be limiting. In humans, serotonin (5- hydroxytryptamine) is involved in physiological functions such as sleep, hemostasis, and behavior regulation, and in pathological conditions such as carcinoid syndrome, thrombosis, and cardiovascular disease. 5-hydroxy-3-indoleacetic acid, a metabolite of serotonin, has been measured in urine, serum, and cerebrospinal fluid as a biomarker in a variety of these conditions.

[0047] Due to their biological importance, indole-based compounds are measured routinely in a variety of settings, and various assays, including assays based on liquid chromatography, LC-mass spectrometry, radioimmunoassay, capillary electrophoresis, etc. have been described in the art. In the case of binding assays, a particular indole-based molecule of interest will often be used to develop and identify suitable binding partners for the molecule of interest. Considering an immunoassay as an example, a small molecule such as indole is typically not immunogenic in and of itself, but instead is considered a hapten that must be coupled to an immunogenic carrier molecule for producing an immune response. Once an antibody has been selected, the indole-derived molecule may be coupled to a label for use in a competitive immunoassay format, or possibly to a solid phase for use in immobilizing a labeled antibody.

[0048] For the purposes of this invention and its various embodiments, the heterocyclic ring system of the disclosed derivatives maybe an indole or an indolene ring system. Although many of the examples and specific embodiments refer to an indole derivative, the disclosure is also applicable and encompasses an indolene ring system. The skilled artisan will understand the minor modifications necessary to effect such a substitution. Functional Moieties

[0049] Designing an indole or indolene linking reagent involves selection of the functional moiety to be employed. The choice of functional moieties is entirely dependent upon the target sites available on the species to be targeted for attachment. Some species (e.g., proteins) may present a number of available sites for targeting (e.g., lysine e-amino groups, cysteine sulfhydryl groups, glutamic acid carboxyl groups, and selection of a particular functional moiety may be made empirically in order to best preserve a biological property of interest (e.g., binding affinity of an antibody, catalytic activity of an enzyme, etc.). In addition, sites for targeting by an indole/indolene linking reagent may be introduced, for example by using a bifunctional molecule that binds to an available moiety on a protein, and provides in effect an "engineered" site for attachment. Bifunctional linkers are well known in the art, and are commercially available from companies such as Pierce Biotechnology, Inc.

[0050] The following discussion of functional moieties that may be included in the derivatives of the present invention and/or introduced into a target protein, polypeptide, antibody, antibody fragment, single-chain variable region fragment, small molecule, nucleic acid, oligosaccharide, polysaccharide, cyclic polypeptide, peptidomimetic, aptamer, detectable label or solid phase is not intended to be limiting.

1. Coupling through Amine Groups

[0051] Imidoester and N-hydroxysuccinimidyl ("NHS") esters are typically employed as amine-specific functional moieties. NHS esters yield stable products upon reaction with primary or secondary amines. Coupling is efficient at physiological pH, and NHS-ester cross-linkers are more stable in solution than their imidate counterparts. Homobifunctional NHS-ester conjugations are commonly used to cross-link amine-containing proteins in either one-step or two-step reactions. Primary amines are the principle targets for NHS-esters. Accessible oamine groups present on the N-termini of proteins react with NHS-esters to form amides. However, because cϋ-amines on a protein are not always available, the reaction with side chains of amino acids become important. While five amino acids have nitrogen in their side chains, only the e-amino group of lysine reacts significantly with NHS-esters. A covalent amide bond is formed when the NHS-ester agent reacts with primary amines, releasing N-hydroxysuccinimide.

2. Coupling through Sulfhydryl Groups

[0052] Maleimides, alkyl and aryl halides, ohaloacyls, and pyridyl disulfides are typically employed as sulfhydryl-specific functional moieties. The maleimide group is specific for sulfhydryl groups when the pH of the reaction mixture is kept between pH 6.5 and 7.5. At pH 7, the reaction of the maleimides with sulfhydryls is 1000-fold faster than with amines. Maleimides do not react with tyrosines, histidines or methionines.

[0053] When free sulfhydryls are not present in sufficient quantities, they can often be generated by reduction of available disulfide bonds. Alternatively, molecules such as N-succinirnidyl-S-acetylthioacetate (SATA) and N-succinimidyl-S- acetylthiopropionate (SATP) may be used to introduce a protected sulfhydryl into a target molecule through reaction with a primary amine. Protected sulfhrdryls may also be introduced into the heterocyclic derivatives of the present invention as described herein, and used to couple through, for example, maleimides, alkyl and aryl halides, α-haloacyls, and pyridyl disulfides present in the target to which the indole is to be attached.

3. Coupling Through Carboxyl Groups

[0054] Carbodiimides couple carboxyls to primary amines or hydrazides, resulting in formation of amide or hydrazone bonds. Carbodiimides are unlike other conjugation reactions in that no cross-bridge is formed between the carbodiimide and the molecules being coupled; rather, a peptide bond is formed between an available carboxyl group and an available amine group. Carboxy termini of proteins can be targeted, as well as glutamic and aspartic acid side chains. 4. Nonselective Labeling

[0055] A photoaffinity reagent is a compound that is chemically inert but becomes reactive when exposed to ultraviolet or visible light. Arylazides are photoaffinity reagents that are photolyzed at wavelengths between 250-460 nm, forming a reactive aryl nitrene. The aryl nitrene reacts nonselectively to form a covalent bond. Reducing agents must be used with caution because they can reduce the azido group.

5. Arginine Specific Cross-linkers

[0056] Glyoxals are useful compounds for targeting the guanidinyl portion of arginine residues. Glyoxals will target arginines at mildly alkaline pH. There is some cross-reactivity (the greatest at higher pH) with lysines.

6. Carbonyl Specific Cross-Linkers

[0057] Carbonyls (aldehydes and ketones) react with amines and hydrazides at pH 5-7. The reaction with hydrazides is faster than with amines, making this useful for site-specific cross-linking. Carbonyls do not readily exist in proteins; however, mild oxidation of sugar moieties using sodium metaperiodate will convert vicinal hydroxyls to aldehydes or ketones.

Protective Groups

[0058] Heterocyclic derivatives may be prepared that comprise either protected or unprotected reactive functionalities. The term "protected" as used herein refers to modification of a reactive group to preclude undesired reactions at the protected site until removal of the protecting moiety in a deprotection reaction. Lists of appropriate protective groups may be found in standard reference works such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd edition, John Wiley & Sons Inc., 1999, which is hereby incorporated by reference in its entirety. As described therein, and as recognized in the art, suitable thiol protective groups include thioesters. thioethers, unsymrnetrical disulfides, and sulfenyls; suitable amino protecting groups include acyl groups such as 9-fiuorenylmethoxycarbonyl (FMOC), formyl and acetyl, and arylalkyl groups such as benzyl; suitable hydroxy protecting groups include ether forming groups such as methyl and ethyl, and acyl groups such as acetyl and tert- butoxycarbonyl (TBOC); and suitable carboxylic acids generally are protected as esters, for example, 2,2,2-trichloroethyl and benzyl.

Applications for Use of Reactive Heterocyclic Derivatives

1. Carrier Protein-Hapten/Peptide/Polypeptide Conjugates for Use as Immunogens

[0059] Numerous companies offer commercially available products in this area of immunological research. There are many cross-linkers used for the production of these conjugates, and the best choice is dependent on the reactive groups present on the hapten and the ability of the hapten-carrier conjugate to function successfully as an immunogen after its injection. Carbodiimides are good choices for producing peptide carrier conjugates because both proteins and peptides usually contain several carboxyls and primary amines.

[0060] Other heterobifunctional cross-linkers can also be used to make immunogen conjugates. Often peptides are synthesized with terminal cysteines to allow for their attachment to supports or to carrier proteins through a part of the molecule that is not important for activity or recognition. Sulfhydryl-reactive, heterobifunctional cross-linkers can be coupled to carrier proteins through their other functional group and then can be linked to an available cysteine in the molecule to be linked {e.g., the exemplary indole derivatives of the present invention). This method can be very efficient and yield an immunogen that is capable of eliciting a good response upon injection.

2. Solid-Phase Immobilization

[0061] Proteins, peptides and other molecules can be immobilized on solid-phase matrices for use as affinity supports or for sample analysis. The term "solid phase" as used herein refers to a wide variety of materials including solids, semi-solids, gels, films, membranes, meshes, felts, composites, particles, papers and the like typically used by those of skill in the art to sequester molecules. The solid phase can be non- porous or porous. Suitable solid phases include those developed and/or used as solid phases in solid phase binding assays. See, e.g., chapter 9 of Immunoassay, E. P. Dianiandis and T. K. Christopoulos eds., Academic Press: New York, 1996, hereby incorporated by reference. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. See, e.g., Leon et al., Bioorg. Med. Chem. Lett. 8: 2997, 1998; Kessler et al., Agnew. Chem. Int. Ed. 40: 165, 2001; Smith et al., J. Comb. Med. 1: 326, 1999; Orain et al., Tetrahedron Lett. 42: 515, 2001; Papanikos et al., J. Am. Chem. Soc. 123: 2176, 2001; Gottschling et al., Bioorg. Med. Chem. Lett. 11: 2997, 2001.

[0062] Surfaces such as those described above may be modified to provide linkage sites, for example by bromoacetylation, silation, addition of amino groups using nitric acid, and attachment of intermediary proteins, dendrimers and/or star polymers. This list is not meant to be limiting, and any method known to those of skill in the art may be employed.

3. Detectable Label Derivatives

[0063] One of the most widely used applications of linkage chemistry is the production of detectable label heterocyclic derivatives. Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate an enzyme, fluorophore, radionuclide, or other molecule to a protein, nucleic acid, small molecule, etc. that has affinity for one of the components in the biological system being studied. Antibody-enzyme conjugates (primary or secondary antibodies) and hapten-enzyme conjugates are among the most common conjugates used. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

[0064] Particularly preferred detectable labels are fluorescent latex particles such as those described in U.S. Patents 5,763,189, 6,238,931, and 6,251,687; and International Publication WO95/08772, each of which is hereby incorporated by reference in its entirety. Exemplary conjugation to such particles is described hereinafter.

Use of Heterocyclic Derivatives in Receptor Binding Assays

[0065] In another aspect, the invention relates to the use of the heterocyclic derivatives described herein in various detection assays. In one embodiment, the heterocyclic derivatives of the present invention may be advantageously used to covalently link solid phases or detectable labels to proteins, polypeptides, antibodies, small molecules, nucleic acids, oligosaccharides, polysaccharides, cyclic polypeptides, peptidomimetics, and aptamers. This list is not meant to be limiting. In some embodiments, these conjugates are used in receptor binding assays. Receptor binding assays include any assay in which a signal is dependent upon specific binding of an analyte (including drugs and other small molecule therapeutics and metabolites) to a cognate receptor, and include immunoassays, ligand-receptor assays, and nucleic acid hybridization assays.

[0066] In immuno-based assays, the presence or amount of an analyte is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), including a sandwich ELISA, radioimmunoassays (RIAs), competitive binding assays, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, and the like. [0067] Numerous methods and devices are well known to the skilled artisan for the practice of receptor binding assays. See, e.g., U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. These devices and methods can utilize detectably labeled molecules and antibody solid phases in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman Access, Abbott AxSym, Roche ElecSys, Dade Behring Stratus systems are among the immunoassay analyzers that are capable of performing such immunoassays. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Patents 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. In such an assay the cross-linkers of the present invention could be used to increase the mass of material binding to the assay surface for detection.

[0068] In its simplest form, an assay device may be a solid surface comprising receptor(s) that specifically bind one or more analytes of interest. For example, antibodies may be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material or membrane (such as plastic, nylon, paper), and the like using the cross-linkers of the present invention.

[0069] The analysis of a plurality of analytes may be carried out separately or simultaneously with one test sample. For separate or sequential assay of markers, suitable apparatuses include clinical laboratory analyzers such as the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), the ADVIA® CENTAUR® (Bayer) immunoassay systems, the NICHOLS ADVANTAGE® (Nichols Institute) immunoassay system, etc. Preferred apparatuses or protein chips perform simultaneous assays of a plurality of analytes on a single surface. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes. Such formats include protein microarrays, or "protein chips" (see, e.g., Ng and Hag, J. Cell MoI. Med. 6: 329-340 (2002)) and certain capillary devices (see, e.g., U.S. Patent No. 6,019,944). In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more analyte(s) (e.g., a marker) for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one analyte (e.g., a marker) for detection.

[0070] In particular, the invention provides a method for detecting a target molecule in a fluid sample by detecting the binding of the target molecule to a heterocyclic derivative as described herein. In one particular embodiment, the method comprises:

b) detecting the binding of the heterocyclic derivative to the target molecule.

[0071 ] The invention also provides a method for detecting two or more target molecules in a fluid sample by:

b) detecting the binding of the two or more target molecules to the capture molecules. [0072] Optionally, the amount of binding may be measured and related to the original fluid sample as an indicator of the amount of the target molecule(s) present in the fluid sample. Measuring the amount of binding may be done directly (e.g., by measuring the amount of bound target molecules) or indirectly in, for example, a competitive binding assay format. The principles of competitive binding assays are well known in the art. In one variation, for example, the target molecules are detectably labeled and the competing ligands are not. Typically one of the ligands (either the target molecule or the competing ligand) is titrated against a fixed concentration of the other in order to generate signal vs. concentration curves. Most commonly, the target molecules, whether detectably labeled or not, are "pre-bound" to the capture molecules. The target molecules are then "competed off' using increasing concentrations of the competing ligand. For assays in which the target molecules carry the detectable label, a reduction in the level of binding is measured with increasing concentrations of competing ligand. The opposite is true when the competing ligand is carrying the detectable label. As noted previously, these assays may be performed with the capture molecules (heterocyclic derivatives) attached to a solid support (e.g., an array) or in solution.

Examples

[0073] The following examples serve to illustrate the present invention. These examples are in no way intended to limit the scope of the invention.

Example 1. Preparation of methyl 2-(5-Bromo-lH-indol-3-yl)acetate (1)

[0074] Chlorotrimethylsilane (6 mL) was added to methanol (50 mL) and cooled to —20⁰C, then combined immediately with another solution comprising methanol (50 mL) and 5 -Bromoindole-3 -acetic acid (1.04 g). The reaction mixture was stirred overnight under argon. The solvents were evaporated under vacuum and the crude product dried overnight providing methyl 2-(5-Bromo-lH-indol-3-yl)acetate. The crude methyl ester was used in the next step without any further purification.

Example 2. Preparation of methyl 2-(5-cvano-lH-indol-3-yl^')acetate (2)

[0075] Copper (I) cyanide (0.77 g) was added to compound (1) (1.07 g) in 10 mL l-methyl-2-pyrrolidone, and the mixture purged with argon. The mixture was heated at 150⁰C for 6 hours, and then cooled to room temperature. The solvent was evaporated under vacuum, and the residue partitioned in 200 mL ethyl acetate and 50 mL H₂O containing 12 mmol sodium citrate. The organic layer was removed and rinsed with 3x50 mL sodium citrate solution (12 mmol in 150 mL H₂O), lx50mL sodium EDTA (0.1 N), 3x50 mL saturated sodium chloride, and dried overnight with anhydrous magnesium sulfate. The organic layer was filtered and evaporated to dryness. The product was extracted from the oily residue with ethyl ether (200 mL). The organic layer was filtered through a plug of aluminum oxide and evaporated to dryness under vacuum providing compound (2), which was used in a next step without further purification. An additional amount of compound (2) can be recovered from the oily residue by repeated extractions with ethyl ether.

Example 3. Preparation of 2-f5-(aminomethv0-lH-indol-3-yl')acetic acid hydrochloride (3)

[0076] Compound (2) dissolved in ethanol containing sodium hydroxide was hydrogenated in a Parr hydrogenation apparatus at 42 psi over Raney nickel catalyst at room temperature overnight. The catalyst was then removed by filtration and the solution evaporated to dryness under vacuum. The residue was dissolved in H₂O (10 mL), and the pH adjusted to 2.8 with 4N HCl. The formed white precipitate of sodium chloride was collected and rinsed with ethanol (3x5 mL) and ethyl ether (3x 1O mL). The organic filtrates were combined and evaporated to dryness. The residue was suspended in ethanol and the solids (NaCl) removed by filtration. The filtrate was again evaporated under vacuum. Finally, the crude product was pre-dried azeotropically with ethanol (2 x 10 mL) and dried under vacuum over P₂O₅ to provide (3):

Example 4. Preparation of 2-(5-((2-(tert-butoxycarbonylamino)acetamido)methyl)- lH-indol-3-yl)acetic acid (4)

[0077] N-(N-ot-Boc-glycyloxy)succinimide (280 mg) was dissolved in acetonitrile (5 mL), and a solution comprising compound (3) (206 mg), lithium hydroxide (72 mg) and methanol (5 mL) was added dropwise. The mixture was stirred overnight under argon, and then evaporated to dryness. The residue was re- suspended in ethyl acetate (50 mL) and rinsed first with a phosphate-sodium chloride buffer (3 x 50 mL) comprising of equal volume of saturated sodium chloride and 20 mM phosphate buffer pH 3 and then finally with saturated sodium chloride solution (2 x 50 mL). The organic layer was dried with anhydrous magnesium sulfate, filtered, and evaporated to dryness. The residue was than further dried under vacuum over P₂O₅ to provide (4):

Example 5. Preparation of 2-(5-(f2-f3- facetylthio)propanamido)acetamido)methyl)-lH-indol-3-yl^')acetic acid (5)

[0078] Under acidic conditions (chlorotrimethylsilane (1 mL), methanol (20 mL) and compound (4) (240 mg) were stirred for 2 hours at room temperature to remove the tBOC protective group, thereby forming the methyl ester. The volatile products were evaporated under vacuum and the resulting product was dried under vacuum over P₂O₅. The crude product and lithium hydroxide (60 mg) were dissolved in methanol (5 mL) and stirred under argon at room temperature overnight. After about 1 day, an additional 60 mg lithium hydroxide was added, and the mixture was stirred under argon for an additional three hours to complete the hydrolysis of the methyl ester. Excess lithium hydroxide was neutralized with glacial acetic acid (65 μL). The resulting 5-aminomethylene indole-3 -acetic acid solution was added in a dropwise fashion to a solution of SATP (196 mg) dissolved in dry acetonitrile (2 mL). An additional 10 mL of methanol was used to completely transfer 5-aminomethylene indole-3-acetic acid. More glacial acetic acid (65 μL) was added, and the reaction allowed to proceed for 3 hours, at which time 46 mg SATP was added. The reaction was stirred overnight under argon. Glacial acetic acid (130 μL) was added, and the mixture was evaporated to dryness under vacuum. The residue was suspended in ethyl acetate (50 mL) and an aqueous solution of equal parts saturated NaCl and 0.02N HCl (50 mL). The organic layer was washed three times in this fashion, followed by a wash with saturated NaCl (2x50 mL), then dried with anhydrous magnesium sulfate. The ethyl acetate layer was filtered, and the drying agent washed with 3x5 mL ethyl acetate, and the ethyl acetate portions combined and evaporated under vacuum. The crude product was dried under vacuum over P₂O₅. Excess SATP was removed by suspending the residue in dry acetonitrile (2 mL), stirring for 1 hour, then filtering and washing the solids with dry acetonitrile (3x1 mL). Finally, the solid product was dried under vacuum over P₂O₅, affording (5):

Example 6. Preparation of 2-(5-((3-(acetyltMo)propanamido)methylMH-indol-3- vDacetic acid derivative (6^*)

[0079] Lithium hydroxide (80 mg) and (2) (268 mg) were combined in methanol (10 mL) and the contents heated gently to dissolve the solids. The resulting solution was added in a dropwise fashion to a solution of SATP (352 mg) dissolved in dry acetonitrile (10 mL). The mixture was stirred overnight under argon. The solvents were removed under vacuum, and the residue dissolved in acetonitrile (20 mL). The precipitate formed was recovered and washed with acetonitrile and dried under vacuum, affording (6):

Example 7. Preparation of indole derivative-solid phase conjugates

[0080] Bovine serum albumin ("BSA") and polystyrene latex particles (Interfacial Dynamics) were incubated at 25 ⁰C for 30 minutes at 1-10 mg BSA per mL of latex slurry at 1-10% solids in 25 mM citrate buffer, pH approximately 4. The solution was then brought to approximately neutral pH with 150 mM potassium phosphate/30 mM potassium borate, and incubated for an additional 2 hours at 25 ⁰C. The suspension was washed three times by resuspension in 50 mM potassium phosphate/10 mM potassium borate/150 mM sodium chloride at approximately neutral pH followed by centrifugation.

[0081] An N-hydroxysuccinimide/maleimide bifunctional poly(ethylene glycol) crosslinker as described in U.S. Patent 6,887,952 was added at 5-500 mg/mL in deionized water to the BSA-latex particles at 1-10% solids. The crosslinker was incubated with the BSA-latex particles at room temperature for 2 hours. Excess crosslinker was removed by centrifugation and resuspension in PBS of the now maleimide-functionalized BSA-latex particles.

[0082] An S-acetyl-functionalized indole derivative was deprotected by base hydrolysis in the following way. The derivative (4-8 mg) was dissolved in 0.8 mL DMF-water solution (70:30 v/v) and 200 μL of 1 M KOH, and was incubated for 10 minutes at room temperature. Then the excess of the base was neutralized with a phosphate/hydrochloric acid buffer to pH 7. The maleimide-functionalized BSA- latex particles were added to the solution containing deprotected indole derivative in the presence of 0.1 mM EDTA, and the mixture was incubated at room temperature overnight. KOH was added to maintain the pH at about 7.0. The reaction was stopped in two steps. First by addition of 0.2 mM 0-mercaptoethanol and incubation for 30 at room temperature and then by addition of 6 mM N-(hydroxyethyl)maleimide and additional incubation for 30 minutes at room temperature. The indole derivative- conjugated latex particles were purified by centrifugation and resuspension in PBS.

Example 8. Synthesis of KLH-SMCC

[0083] Keyhole Limpet Hemocyanin (KLH, Calbiochem #374817, 50 mg/mL in glycerol) was passed through a 4OmL GH25 column equilibrated in 0.1 M potassium phosphate, 0.1 M borate, 0.15M sodium chloride buffer, pH 7.5 to remove glycerol. A 1.5-fold molar excess of N-ethylmaleimide was added, and the mixture incubated 30 minutes at room temperature. A 200-fold molar excess of sulfo-SMCC (Pierce #22322) from a 5OmM stock in distilled water was added while vortexing. Vortexing was continued for another 30 seconds, followed by incubation for 10 minutes at room temperature. A 100-fold molar excess of SMCC (Pierce #22360 ) from an 8OmM stock in acetonitrile was added while vortexing. IM KOH was added to maintain a pH of between 7.2 and 7.4. The mixture was stirred at room temperature for 90 minutes. After 90 minutes incubation, KLH-SMCC was purified by gel filtration using a GH25 column equilibrated in 0.1M potassium phosphate, 0.02M borate, 0.15M sodium chloride buffer, pH 7.0.

Example 9. Indole Derivative Conjugates

[0084] S-acetyl-functionalized indole derivatives were conjugated to KLH-SMCC as follows. First, an S-acetyl-functionalized indole derivative was deprotected by base hydrolysis to provide free thiol. The derivative (4-8 mg) was dissolved in 0.8 mL DMF-water solution (70:30 v/v) and 200 μL of 1 M KOH, and was incubated for 10 minutes at room temperature. The excess of the base was neutralized with a phosphate/hydrochloric acid buffer and pH brought to 7. Then, a 2-fold molar excess of derivative (based on the concentration of SMCC in a particular batch of KLH- SMCC) was added to KLH-SMCC, and the mixture stirred for 90 minutes at room temperature. Conjugates were purified by exhaustive dialysis in PBS.

Example 10. Immunoassay for detecting 5-hvdroxy-3-indoleacetic acid (5-HIAA)

[0085] A competitive assay for detecting 5-hydroxy-3-indoleacetic acid (5-HIAA) in blood and serum was developed using microfluidic devices manufactured at Biosite Incorporated essentially as described in WO98/43739, WO98/08606, WO98/21563, and WO93/24231. An anti-5-HIAA antibody was developed by phage display using a hapten-KLH conjugate as immunogen. This antibody was conjugated to a 0.13 μm maleimide-functionalized latex particle via a free cysteine residue on the antibody. The detection reagent consisted of a 0.50 μm fluorescence energy transfer latex particle (essentially as described in U.S. Patents 5,763,189, 6,238,931, and 6,251,687; and International Publication WO95/08772) made according to Example 6. 130 nL (comprising 0.22% solids) of the antibody-particle conjugate was spotted onto the diagnostic lane of the microfluidic device, and 170 nL (comprising 0.4% solids) of the 5-HIAA-particle conjugate was applied to the device reaction chamber. Analytes were dissolved in deionized water, then diluted into pools of human plasma to achieve the desired final concentration. 210 μL of sample was applied to the device sample addition zone and allowed to run > 15 minutes prior to reading the fluorescence in the TRIAGE (Biosite Incorporated) meter. A fluorescent signal was obtained by integrating the fluorescence as a function of distance from the device origin.

[0086] The results of this assay are presented in Fig. 1 , which plots fluorescence signal (in arbitrary units) on the Y-axis versus the log analyte concentration (in μM) on the X-axis. Each point represents the average of 6 individual replicates and the error bars indicate one standard deviation from the mean. The solid line through the 5-HIAA data points represents a non-linear least-squares fit to a simple competitive model. As indicated in the figure, the assay detects 5-HIAA, but does not appreciably crossreact with the closely related indoles serotonin, melatonin, or L-tryptophan.

Example 11. Preparation of Ethyl 2-(5-(aminomethyl)-lH-indoI-3-yl)acetate (7)

[0087] Chlorotrimethylsilane (476 μL, 3.77 mmol) was added dropwise to absolute ethanol (4 mL) in a dry ice / isopropanol bath with stirring, at such a rate that the temperature did not rise above -200⁰C. The resulting solution was then added to a solution of 2-(5-(aminomethyl)-lH-indol-3-yl)acetic acid (75 mg, 0.314 mmol) in absolute ethanol (4 mL) which had been cooled to -200⁰C. The resulting solution was allowed to warm up to room temperature and stirred, under argon, at room temperature for 4 hrs. HPLC showed complete reaction. The solvent was evaporated in vacuo and the residue dried under vacuum overnight when it crystallized to a light brown solid.

(7) Example 12. Preparation of N-bromoacetyl-L-HCTL (8)

[0088] To a stirring solution of bromoacetic acid (1.39g, 10 mmol) and L- homocysteiethiolactone hydrochloride (1.54g, 10 mmol) in dry DMF (50 mL) was added pyridine (1.70 mL, 21 mmol). The solution was then treated with EDC hydrochloride (2.1 Ig, 11 mmol) and allowed to stir, under argon, at room temperature for 16 hrs. The DMF was evaporated in vacuo and the thick oily yellow residue treated with water (20 mL) to afford a white suspension which was filtered off washing with water (20 mL). The white solid product was dried under vacuum and weighed 1.02g. HPLC showed pure product.

(8)

Example 13. Preparation of 4,4'-disulfanedi ylbis(2-(2-(( 3-f 2-ethoxy-2-oxoethylV lH-indol-5-yl)methylamino)acetamido)butanoic acid) (9)

[0089] A solution of (7) (84 mg, 0.314 mmol) in DMF was treated with (8) (82 mg, 0.345 mmol) followed by diisopropylethylamine (180 uL, 1.036 mmol) with stirring. The resulting solution was allowed to stir at room temperature under argon, monitoring the reaction by HPLC. After two weeks the reaction was virtually complete and the solvent was removed in vacuo to ultimately afford a thick red oil. The oil was treated with methylene chloride (1 mL), briefly sonicated and applied to two 20x20 cm silica (lOOOμm thickness) TLC plates which were subsequently eluted with methylene chloride - methanol 10: 1. After allowing to dry, the three major UV absorbing bands were removed, treated with methanol, sonicated to a fine suspension and filtered, washing the filtered silica with a little methanol. The filtrates were evaporated, the residues re-dissolved in a little methanol (1 mL) and filtered through glass wool plugged pipettes into weighed vials, after which time they were evaporated and dried in vacuo. The residue from the lowest running product band was isolated as a beige solid and weighed 33 mg.

Example 14. Preparation of 5-f3(tritylthio) propanamido methyl indole] (10)

[0090] 5-Aminomethylene-indole (1.47 g, 10.0 mmol), Tr-S-CH₂CH₂COOH (3.6g, 10.00 mmol) and solid EDC (2.0 g, 1 LOO mmol) were added to a 100 mL round bottom flask. 40 mL of pyridine was added to the flask and the reaction mixture was sonicated for 1 hour. The reaction mixture was stirred under argon overnight. The solution was evaporated in vacuo to give the crude active indole. The crude active indole was dissolved in 200 mL OfCH₂Cl₂ and washed with 2OmM phosphoric acid buffer pH3 (3 x 100 mL), DI water (2 x 100 mL), 4% sodium carbonate (3 x 50 mL) and saturated NaCl solution (1 x 100 mL). The organic layer was separated and dried over anhydrous MgSO₄. The solvent was removed in vacuo to give 4.6 g (9.65 mmol, 98%) of desired product as a white solid. TLC (silica Rf =0.84, IPA/NH3/H2O, 10:2:1 v/v/v). IH NMR (DMSO): <5: 2.28 (m, 2H,CH2), 2.30 (m, 2H, CH2), 4.3 (d, 2H, CH2), 6.3 (s, IH, CH), 6.9 (d,lH, CH), 7.2 (m, 4H, AR), 7.31 (m, 16H, Ar), 8.6 (m, IH, NH), 11.1 (s, IH, NH).

Example 15. 5-F3 (tritylthio) propanamido 3-N diethyl methylene indole! (11)

[0091 ] A solution of ( 10) (2g, 4.20 mmol) in 10 mL THF was added dropwi se to an ice cooled mixture of 10 mL THF, 4.2 mL acetic acid, 1.4 mL diethylamine, and 918 μL formaldehyde. The clear solution was stirred under ice for two hours. It was left stirring at room temperature overnight. The solution was evaporated in vacuo to give the crude active indole as a foamy residue. The crude active indole was dissolved in 10 mL of 5 N HCl and washed with CH₂Cl₂ (3 x 50 mL). The organic layer was separated and dried over anhydrous MgSO₄. The solvent was removed in vacuo to give 2.31g (4.11 mmol, 98.1%) of the desired product. TLC (silica, R_f =0.76, IPA/NH3/H20, 10:2:1 v/v/v).

Example 16. 5F3 Ctritylthio) propanamide 3-cvano methylene indole^"! (12)

[0092] A solution of dimethyl sulfate (428.70 uL, 4.50 mmol) in 15 mL THF was slowly added to a solution of (11) (2.3 g, 4.09 mmol) in 15 mL THF. The mixture was left stirring at room temperature overnight. A solution of sodium cyanide (1.0 g, 20.47 mmol) in 5 mL DI water was added to the mixture and refluxed for an hour. The solvents were evaporated in vacuo to give the crude indole as an oil. The crude active indole was dissolved in 100 mL CH2CI₂ and washed with 5OmM phosphoric acid buffer pH 3 ( 3 x 100 mL). The organic layer was separated and dried over anhydrous MgSCU. The solvent was removed in vacuo and the residue was purified by flash chromatography over silica gel using hexane and a gradient of ethyl acetate (10-100%) to yield 0.490 g (0.950 mmol, 94.1%) of the desired product.

Example 17. NEM Blocked Thiol Indole Derivative Cl 3)

[0093] Amide of Indole 3-Cyanoacetyl 5-Carboxylic Acid and Cystamine (26.2 mg, 0.05 mmol) and NaKEU (115.1 mg, 3.04 mmol) were dissolved in 2 mL IPA and stirred under argon overnight. The white precipitate was filtered and rinsed with IPA and the filtrate was evaporated in vacuo. The residue was dissolved in 10 mL DI water/MeOH v/v and adjusted the pH to 3 with 5N NaOH. A solution of N- ethylmaleimide (6.98 mg, 0.06 mmol) in 1 mL methanol was added to the mixture. The pH was adjusted to 7 with 1.2 N HCl and additional N-ethylmaleimide (0.70 mg, 0.01 mmol) was added. The solution was evaporated in vacuo to give the crude derivative. The crude derivative was dissolved in DI water and washed with CH₂Cl₂. The organic layer was separated and dried over anhydrous MgSCV The solvent was removed in vacuo to give 11.9 mg (4.57 mmol, 100%) of desired product as a white solid. ¹H NMR (DMSO): δl .05 (t, 3H, CH₃),

[0094] 2.89 (m, 2H₅ CH₂), 3.01 (m, 2H, CH₂), 3.19 (m, 2H, CH₂), 3.41 (m, 2H, CH₂), 3.51 (m, 2H, CH₂) 4.06 (s, 2H, CH₂), 7.43 ( t, IH, Ar) 7.67 (t, IH, Ar) 8.18 (d, IH, Ar), 8.53 (t, IH, NH), 11.36 (s, IH, NH).

Example 17. Iodoacetamide Blocked Thiol Indole Derivative (14)

[0095] Amide of Indole 3-Cyanoacetyl 5-Carboxylic Acid and Cystamine (100.0 mg, 0.19 mmol) and NaBH₄ (659.0 mg, 17.42 mmol) were dissolved in 20 mL IPA/MeOH v/v, and stirred under argon overnight. The solution was evaporated in vacuo and dissolved in 10 mL DI water. The pH was adjusted to 3.5 with concentrated H₃PO₄ incubated for 10 minutes and adjusted to pH 8 with NaOH. A solution of Iodoacetamide (358.08 mg, 1.94 mmol) in 10 mL methanol was added. After two hours of incubation the solution was evaporated in vacuo and the residue suspended in DI water. The white precipitate was filtered and dried to give 70 mg (22.15 mmol, 88.6%) of desired product. ¹H NMR (DMSO): δ: 2.78 (t, 2H, CH₂), 3.14 (s, 2H, CH₂), 3.49 (m, 2H, CH₂), 4.07 (s, 2H, CH₂), 7.44 (m, 3H, Ar), 7.68 (m, IH, Ar), 8.18 (s, IH, Ar), 8.50 (t, IH, NH), 11.35 (s, IH, NH).

Example 18. Disulfide of Indole Acetic Acid Ethyl Ester 5-Methylene (3 ' Thiopropiono Amide) (15)

[0096] 3,3-Dithiodipropionic acid (0.9 g, 4.20 mmol) and EDC (2.7g, 14.30 mmol) were added to the solution of Indole 3-CH₂COOEt-S-CH₂NH₂-HCl (2.3 g, 8.41 mmol) in 30 mL pyridine. The reaction was stirred at room temperature overnight. The solution was evaporated in vacuo and the crude product was dissolved in 200 mL ethyl acetate. This mixture was washed with IN HCl/saturated NaCl (3 x 200 mL; 1:1 v/v), IM sodium carbonate/saturated NaCl (3 x 50 mL; 1:1 v/v), and a final rinse with saturated NaCl (3 x 100 mL). The organic layer was separated and dried over anhydrous MgSO₄. The solvent was removed in vacuo to give 2.06 g (3.22 mmol, 79.2%) of desired product as a white solid. ¹H NMR (DMSO) δ: 1.36 (m, 3H, 2 CH₃), 2.53 (m, 2H, 2 CH₂), 2.93 (ra, 2H, 2 CH₂), 3.32 (s, 2H, CH₂), 3.69 (s, 2H, CH₂), 4.04 (m, 2H, 2 CH₂), 4.32 (d, 2H, 2 CH₂), 6.99 (t, IH, Ar), 7.22 (d, IH, Ar), 7.28 (d, IH, Ar), 7.34 (s, IH, Ar), 8.37 (t, IH, NH), 10.89 (s, IH, NH).

Example 19. Disulfide of Indole Acetic Acid Ethyl Ester 5-Methylene (3 ' Thiopropiono Amide) (16)

[0097] A solution OfNaBH₄ (3.7 g, 96.74 mmol) in water was added to a solution of disulfide of 5-amino methylene indole 3-acetic acid (2.1 g, 3.22 mmol) and NaOH (0.3g, 7.09 mmol) in 35 mL ethanol. The mixture was stirred under argon overnight. 8.38 mL NaOH and 17.5 mL methanol were added to the reaction mixture, along with 10 mL water and 10 mL concentrated HCl. Extreme heat was generated. The solvent was removed in vacuo to give 1.56 g (5.34 mmol, 57.7%) of desired product.

Example 20. 5-Amino N-acetyl Indole 3-Acetic Acid (17)

[0098] Palladium (O.lg) was added to the solution of 5-Nitro-N-acetylindole 3- acetic acid (1.0 g, 3.78 mmol) in 10 mL methanol and stirred under hydrogen atmosphere for two hours. The solution was evaporated in vacuo to give a gray solid. The gray solid was dissolved in 20 mL DI water and adjusted the pH to 12 with 5 N NaOH. The precipitate was filtered and the filtrate was adjusted to pH 4 with 1 N HCl. The solvent was evaporated in vacuo to give 1.14g (4.86 mmol, 98%) of desired product. ¹H NMR (DMSO) δ: 2.11 (s, 3H, CH₃), 2.55 (m. 2H₅ CH2), 2.72 (m, 2H, CH2), 3.66 (m, 2H, CH2), 3.75 (m, IH, CH), 4.27 (m, 2H, CH2), 6.78 (d, IH, Ar), 6.87 (s, IH, Ar), 7.90 (d, IH, Ar).

Example 2 ϊ . 5-Aminomethylene Indole 3-Acetic Acid SATP Derivative (18)

[0099] Sodium Cyanoborohydride (128.97 mg, 2.05 mmol) was added to the solution of 5-aminomethylene indole 3-acetic acid thiol (300 mg, 1.03 mmol) in 5 mL acetic acid, and stirred at room temperature overnight. 1 mL concentrated HCl was added and a white precipitate was observed. The precipitate was filtered and the filtrate was evaporated in vacuo. The residue was dissolved in 3.28 mL 5N NaOH and 1.55 mL acetic anhydride. The pH was adjusted to 3 with concentrated HCl and the solution was evaporated in vacuo to give a white solid. The solid was dissolved in 10 mL ethanol and filtered. The filtrate was evaporated in vacuo to give 548 mg (1.44 mmol, 86.3%) of desired product as a white solid. ¹H NMR (DMSO) δ: 2.14 (s, 3H, NOCH₃), 2.31 (s, 3H, SOCH₃). 2.44 (m, 2H, CH₂), 3.01 (t, 2H, CH₂), 3.4 (m, 2H, CH₂), 4.24 (m, 2H, CH₂), 6.98 (m, IH Ar), 7.98 (m, IH, Ar), 8.41 (m, IH, NH).

Example 22. l-Acetyl-5(2- Acetyl thiopropionvD Amino Indoline 3-Acetic Acid (19)

[00100] 1,1 Carbonyldiimidazole (0.84 g, 5.21 mmol) was added in portions to a solution of ATP (0.82 g, 5.55 mmol) in 4 mL of THF and stirred for 45 minutes at room temperature. In another flask, imidazole (0.36 g, 5.35 mmol) was added to a solution of 5-amino N-acetyl indoline (1.14 g, 4.87 mmol) in 20 mL anhydrous THF. The ATP/CDI mixture was added to the indoline-imidazole solution and stirred under argon overnight. 20 mL DMF was added and the mixture stirred for six hours. The solvents were evaporated in vacuo and the residue dissolved in 10 mL iced DI water and adjusted pH to 3 with 2.7 mL iced 6N HCl. The water was washed with CH₂Cl₂ (4 x 10 mL), and the organic layer was separated and dried over anhydrous MgSO-j. The solvent was removed in vacuo to give 1.31 g of oily brown product. The oily brown product was purified by flash chromatography over a C18 silica using phosphoric acid pH3 buffer and a gradient of acetonitrile (1-10%) to yield 129mg (35.4 mmol, 97%) of the desired product as a white solid. ¹H NMR (DMSO) δ: 2.1 (s,3H, NOCH₃), 2.3 (s, 3H, SOCH₃), 2.55 ( m, 2H, CH₂), 3.1 (m, 2H, CH₂), 3.77 (m, 3H, CH₂), 7.29 (d, IH, Ar), 7.53 (s, IH, Ar), 7.93 (d, IH, Ar), 9.91 (s, IH, NH).

Example 23. 5- Amino Methyl Gramine (20)

[00101] Raney Nickel (2.86 g, 48.68 mmol) and NaOH (1.95 g, 49 mmol) was added to a solution of 5-cyano gramine (4.85g, 24.34 mmol) in 30 mL ethanol and left under a hydrogen atmosphere overnight. The nickel was filtered and the solvent was evaporated in vacuo. The residue was dissolved in 150 mL DI water and washed with CH₂Cl₂ (3 x 150 mL) and saturated NaCl (3 x 150 mL). The organic layer was separated and dried over NaSO₄ and filtered through basic activated aluminum oxide. The solvent and the water washes were evaporated to dryness to give 2.42 g (11.90 mmol, 72%) of desired product.

Example 24. CBz protected 5-Methyl Amino Gramine (21)

[00102] N-(Benzyloxycarbonyl) succinimide (3.26 g, 13.10 mmol) and DIEA (6.2 mL, 35.71 mmol) were added to a solution of 5-methyl amino gramine (2.42 g, 11.90 mmol) and stirred at room temperature overnight. The solution was evaporated in vacuo and the residue was dissolved in 100 mL OfCHaCl₂ and washed with K3PO4 pHl 1 buffer (3 x 100 mL). The organic layer was separated and dried over NaSO₄. The solvent was removed in vacuo to give 800 mg (2.37 mmol, 32%) of the product.

Example 25. 5 Cvano Gramine Derivative f22)

[00103] Acetic acid (50 mL), formaldehyde (4.06 mL), and dimethylamine (9.01 mL) were added to a solution of 5-Cyanoindole (7. H g, 50.00 mmol) in 101.52 mL dioxane. The solution was stirred at room temperature overnight. The solution was evaporated in vacuo to give the crude active gramine. The crude active gramine was dissolved in water and adjusted pH to 11.0 with 5N NaOH. The product initially separated as oil but it solidified as a fine powder which was later collected by filtration, to give 9.67g (48.53 mmol, 94%) of product. TLC (silica R_f= 0.63, IPA/NH₃/H₂0, 10:2:1 v/v/v). ¹H NMR (DMSO) δ: 2.14 (s, 6H, 2CH₃), 3.55 (s, 2H, CH₂), 7.41 (m, 2H, Ar)₅ 7.53 (d, IH, Ar), 8.09 (s, IH, Ar), 11.50 (s, IH, NH).

Example 25. 5 Cvano Tryptophan Nitro Ethyl Ester (23~)

[00104] Ethyl Nitroacetate (1.6 g, 12.05 mmol) and DIEA (6.6 mL, 37.14 mmol), were added to a solution of (22) ( 2.0 g, 10.04 mmol) in 50 mL toluene and refluxed under argon overnight. The solution was evaporated in vacuo to give a brown residue. This residue was dissolved in CH₂CI2 and purified by flash chromatography over silica gel using CH₂Cl₂ and a gradient of IPA (10-100%) to yield 1.57 g (5.46 mmol, 89%) of the desired product. IH NMR (DMSO) δ: 1.14 (t, 3H, CH3), 3.57 (m, IH, CH2), 3.65 (m, IH, CH2), 4.19 (m, 2H, CH2), 6.02 (IH, Ar), 6.86 (m, 2H, Ar), 7.52 (IH, Ar), 8.25 (IH, NH).

Example 26. 3-Tritylthio propionic Acid (24)

[00105] Triphenylmethanol (26.03 g, 100.0 mmol), and 3-mercaptopropionic acid (9.6 mL, 110.0 mmol) were dissolved in 200 mL TFA and stirred at room temperature overnight. The solution was evaporated in vacuo dissolved in IL ethanol and refluxed for 3 hours. A white precipitate was observed and filtered while the solution was still hot to give 24.08 g (69.10 mmol) of desired product as a white solid. IH NMR (DMSO) δ: 2.16 (m, 2H, CH2), 2.27 (m, 2H, CH2), 7.25 (m 3H, Ar), 7.33 (m, 12H- Ar).

Example 27. 3-Tritylthio propionic Acid (25)

[00106] Indole 3-acetic acid (1.97 g, 10 mmol) and sodium cyanoborohydride (1.26 g, 20 mmol) were dissolved in 30 mL acetic acid. After two hours of stirring 10 mL of concentrated HCL was added and a white precipitate was observed which was later removed by filtration. The solution was evaporated in vacuo and dissolved in 35 mL of IN NaOH. 3.77 mL of acetic anhydride was added and a white precipitate was observed. 20 mL of 1 N NaOH was added, followed by 8 mL 5N NaOH to bring the pH to 5.2. Another 3.77 mL acetic anhydride was added and the solution was stirred for two hours. After two hours of stirring it was acidified to pH 3 with concentrated HCl. A white precipitate was observed which was filtered and dried under vacuum. The white solid was dissolved in 22 mL of acetic acid and 11 mL of fuming nitric acid was added. The solution was stirred for 1 hour and then poured over 51 grams of ice and filtered to give 1.29 g, (4.9 mmol, 96%) of the desired product.

(25) [00107] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[00108] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[00109] Other embodiments are set forth within the following claims.

Claims

We claim:

1. A heterocyclic derivative, or a salt thereof, having the following structure:

wherein,

X is B-A- wherein, A comprises from 0-30 backbone atoms, of which 0-10 of said backbone atoms are heteroatoms, and A is optionally substituted with from 1 to 6 substituents independently selected from the group consisting of C₁-_O alkyl, halogen, oxo, trihalomethyl, C₁^ alkoxy, -NO₂, -NHR², -OH, -CH₂OH₅ -C(O)NHR², -(CH₂)₀-₃-C(O)OR², and, wherein, B a functional moiety selected from the group consisting of protected or unprotected sulfhydryl moieties, protected or unprotected amine moieties, primary amine-reactive moieties, sulfhydryl-reactive moieties, photoreactive moieties, carboxyl- reactive moieties, arginine-reactive moieties, and carbonyl-reactive moieties, protein, polypeptide, antibody, antibody fragment, single- chain variable region fragment, small molecule, nucleic acid, oligosaccharide, polysaccharide, cyclic polypeptide, peptidomimetic, aptamer, detectable label and solid phase; R is absent or, when present, is selected from the group consisting OfC_1-^ alkyl, -(CH₂)_0-3-CH(NHR²)C(O)OR², halogen, trihalomethyl, C_1-6 alkoxy, -NO₂, -NHR², -OH, -CH₂OH, -C(O)NHR², -(CH₂)o-₃-NHR², and

R² is, independently, H or C₁^ alkyl; R³ is R¹, R², or -C(O)R²; and " " is a single or double bond.

2. A heterocyclic derivative according to claim 1 selected from the group consisting of:

wherein, each R² is independently H or d-β alkyl; y=l-3; and

R³ is H Or-C(O)R².

3. A heterocyclic derivative according to claim 2 having the following structure:

wherein, y=l; each R² is H; and

R³ is H or -C(O)R².

4. A heterocyclic derivative according to claim 2 having the following structure:

wherein, y=l; each R² is H; and

R³ is H or -C(O)R².

5. A heterocyclic derivative according to claim 1, wherein X comprises a protected or unprotected sulfhydryl moiety.

6. A heterocyclic derivative according to claim 5, wherein R¹ is selected from the group consisting of

X is selected from the group consisting of

y = i-3; m = 1-4; and R^p is selected from the group consisting of

H,

and

7. A heterocyclic derivative according to claim 6, wherein

y=i; m=l-2;

R-₂ is H; and

R^p is selected from the group consisting of

H₃

and

8. A heterocyclic derivative according to claim 6, wherein

y=i; m = 1-2;

R₂ is H; and

R^p is selected from the group consisting of

9. A heterocyclic derivative according to claim 1, wherein " " is a single bond.

10. A heterocyclic derivative according to claim 1, wherein " " is a double bond.

11 A heterocyclic derivative according to claim 1, wherein A is an alkyl comprising from 0-30 backbone atoms, of which 0-10 of said backbone atoms are heteroatoms, and said alkyl is optionally substituted with from 1 to 6 substituents independently selected from the group consisting of Ci_e alkyl, halogen, oxo, trihalomethyl, d.₆ alkoxy, -NO₂, -NHR², -OH, -CH₂OH, -C(O)NHR², -(CH₂)_O-3-C(O)OR².

12. A heterocyclic derivative according to claim 1, wherein functional moiety (B) is selected from the group consisting of a protein, polypeptide, antibody, antibody fragment, single-chain variable region fragment, small molecule, nucleic acid, oligosaccharide, polysaccharide, cyclic polypeptide, peptidomimetic, aptamer, detectable label and solid phase.

13. A heterocyclic derivative according to claim 12, wherein the covalent linkage between B and^" the heterocycle comprises the following structure formed by reaction of a sulfhydryl with a maleimide:

14. A heterocyclic derivative according to claim 12, wherein R¹ is selected from the group consisting of

X is selected from the group consisting of

y = 1-3; and m = l-4.

15. A heterocyclic derivative according to claim 12, wherein

y=l; m — 1-2; and

R₂ is H.

16. A heterocyclic derivative according to claim 12, wherein

y=i; m = 1-2; and R₂ is H.

17. A heterocyclic derivative according to claim 12 wherein B comprises a latex particle.

18. A heterocyclic derivative according to claim 17, wherein said latex particle is covalently or noncovalently bound to a protein, and wherein said heterocyclic derivative is covalently linked to said protein.

19. A heterocyclic derivative according to claim 17, wherein said latex particle comprises one or more fluorescently detectable molecules covalently bound on or within said latex particle.

20. A heterocyclic derivative according to claim 19, wherein said one or more fluorescently detectable molecules emit a signal through fluorescence energy transfer.

21. A method for detecting a target molecule in a fluid sample, said method comprising: a) contacting a heterocyclic derivative of claim 1 with said fluid sample under conditions suitable for binding of said heterocyclic derivative to said target molecule, wherein said functional moiety is capable of binding said target molecule; and b) detecting the binding of said heterocyclic derivative to said target molecule.

22. The method according to claim 21, wherein said heterocyclic derivative is bound to a solid support.

23. The method according to claim 22, wherein said solid support is an array of said heterocyclic derivatives.

24. A method for detecting two or more target molecules in a fluid sample, said method comprising: a) contacting an array comprising two or more different types of capture molecules with said fluid sample under conditions suitable for binding of said two or more target molecules target molecules to said capture molecules, wherein at least one type of said capture molecules is a heterocyclic derivative of claim 1 ; and b) detecting the binding of said two or more target molecules to said capture molecules.

25. The method according to claim 24, wherein each of said types of capture molecules are present is discrete and addressable physical locations on said array.

26. A method for detecting a target molecule in a fluid sample, said method comprising detecting the binding of said target molecule to a heterocyclic derivative of claim 1.

27. The method according to claim 26, wherein the amount of said binding is measured and related to said fluid sample as an indicator of the amount of said target molecule present in said fluid sample.