WO2004040003A2

WO2004040003A2 - Nucleotide-binding protein-directed probes and methods of their synthesis and use

Info

Publication number: WO2004040003A2
Application number: PCT/US2003/034550
Authority: WO
Inventors: David Alan Campbell; Anna Katrin Szardenings; Kevin Robert Shreder; Juan Manuel Betancort; David Winn
Original assignee: Activx Biosciences, Inc.
Priority date: 2002-10-29
Filing date: 2003-10-29
Publication date: 2004-05-13
Also published as: WO2004040003A3; AU2003298608A1; AU2003298608A8

Abstract

The present invention provides nucleotide binding protein-directed affinity probes ('NBAPS'), and methods of their preparation and use. The subject methods and compositions can provide enhanced simplicity and accuracy in identifying changes in the presence, amount, or activity of nucleotide binding proteins in a complex protein mixture, preferably kinases, and most preferably active forms of kinases, using NBAPs that bind to target nucleotide binding protein(s). The profiling methods described herein can have a number of steps leading to the identification of target nucleotide binding protein(s) in a complex protein mixture.

Description

NUCLEOTIDE-BINDING PROTEIN-DIRECTED PROBES AND METHODS OF

THEIR SYNTHESIS AND USE

FIELD OF THE INVENTION

[0001 ] The invention relates generally to compositions and methods for labeling nucleotide-binding proteins, preferably kinases, and most preferably protein kinases.

BACKGROUND [0002] Nucleotide-binding proteins play an extremely important role as regulators of genomic and proteomic function. Examples of nucleotide binding proteins include G proteins, which act as coupling factors in association with certain receptors; protein kinases, which transfer a phosphate group to target proteins; non-protein kinases, such as hexokinase, which are involved in the metabolic pathways within cells; proteins utilizing the energy stored within nucleotide-based molecules such as ATP; etc.

[0003] Protein kinases are the enzymes responsible for catalyzing the transfer of a γ- phosphoryl group from ATP to the hydroxyl group of serine, threonine or tyrosine residues in peptides, polypeptides, and proteins in a process known as "phosphorylation." Protein phosphorylation is a ubiquitous regulatory mechanism in eukaryotic cells, where it is of central importance in controlling cell function, growth and differentiation. A protein kinase that phosphorylates, for example, tyrosine residues in its substrates is termed a protein- tyrosine.ATP phosphotransferase, or, more commonly, a tyrosine (or Tyr) kinase. The eukaryotic protein kinases make up a large superfamily of related proteins. They are related by virtue of their kinase domains (also known as catalytic domains), which consist of approximately 250-300 amino acid residues. The kinase domains that define this group of enzymes contain 12 conserved subdomains that fold into a common catalytic core structure. See, e.g., Hanks and Hunter, FASEB J. (1995) 9(8):576-96.

[0004] Eukaryotic protein kinases can be classified on the basis of their sequence, substrate specificity and regulation. One major subdivision is between Ser/Thr kinases and the Tyr kinases. Yeast have numerous Ser/Thr kinases, many of which have readily recognizable counterparts in all higher organisms, but very few dedicated Tyr kinases (an example of a yeast Tyr kinase is Swel from Saccharomyces cerevisiae and its homolog in S. pombe Weel). By contrast, many signaling pathways of multicellular organisms depend on two large and important Tyr kinase families, the receptor-Tyr kinases which have intracellular Tyr kinase domains, and the Src family of cytoplasmic Tyr kinases. There are also dual-specificity enzymes, present in both unicellular and multicellular eukaryotes, such as the mitogen-activated protein kinase kinases (MAPKKs).

[0005] Overexpression and/or mutation of certain kinases in tumor cell is believed to upregulate a number of cell cycle and anti-apoptosis pathways leading to subversion of cell cycle checkpoints and enhanced cancer cell survival and metastatic potential. Conversely, inhibition of these kinases may reverse the aberrant signaling in receptor-overexpressing cells and may result in growth arrest and/or tumor cell death. Thus, it is no surprise that kinases have been considered important targets for the identification of therapeutics. See, e.g., Bishop et al., Trends Cell Biol (2001) 11(4):167-72.

SUMMARY OF THE INVENTION [0006] The present invention provides compositions and methods for assessing profiles of nucleotide binding proteins in biological samples. In various embodiments, one or more samples, most preferably one or more complex protein mixtures as defined below, are contacted with one or more probes directed to the nucleotide binding portion of one or more nucleotide binding proteins. The binding selectivity ofthe probe(s) may be selected to allow the skilled artisan to analyze the presence, amount, and/or activity of a selected portion ofthe nucleotide binding proteins present in a sample, thereby simplifying the analysis of complex protein mixtures. [0007] The methods and compositions described herein relate to probes, referred to herein as "nucleotide binding protein-directed affinity probes" or "NBAPs" having an affinity moiety for directing the binding ofthe NBAP to one or more nucleotide binding proteins, preferably protein kinases, a reactive group for forming a covalent bond at or near the nucleotide binding site, and a TAG (e.g., a detectable label, preferably a fluorophore). [0008] One or more NBAPs are combined with a protein-containing sample under conditions for binding and reaction ofthe NBAP(s) with target nucleotide binding proteins that are present in the sample. The resulting products are then used to assess the nucleotide binding protein profile ofthe sample, and can be correlated to the presence, amount, or activity of one or more target nucleotide binding proteins present in the original complex protein mixture.

[0009] In a first aspect, the present invention relates to methods and compositions for determining an enzyme profile in a complex protein mixture. These methods comprise contacting the complex protein mixture with one or more distinct NBAPs, each of which specifically reacts with one or more target nucleotide binding proteins, most preferably target kinases. Each NBAP preferably comprises an affinity moiety conjugated to a ligand, such as a detectable label, and a reactive group that reacts with a target nucleotide binding protein when the NBAP binds to that target nucleotide binding protein, covalently binding the NBAP to the target nucleotide binding protein. The nucleotide binding protein profile can then be analyzed by the screening and/or identification methods described hereinafter. Particularly preferred are the kinase-directed NBAPs also described hereinafter. [0010] In various embodiments, the target nucleotide binding proteins can be one or more protein kinases independently selected from the group consisting of receptor tyrosine kinases, protein tyrosine kinases, serine/threonine kinases, PI kinases, and cyclin dependent kinases. [0011] In preferred embodiments, the NBAP -nucleotide binding protein conjugates can be separated from other components ofthe complex protein mixture, for example by sequestering one or more conjugates (e.g., by binding to a receptor that binds the TAG portion ofthe NBAP or by using a "tethered" NBAP), by chromatographic methods, by mass spectrographic methods, and/or by other means such as electrophoresis. [0012] In yet other embodiments, following reaction ofthe complex protein mixture with one or more NBAPs, the resulting NBAP-nucleotide binding protein conjugates may be proteolytically digested to provide NBAP-labeled peptides. This digestion may occur while the protein conjugates are sequestered to a solid phase, or while free in solution. In preferred embodiments, NBAPs are selected such that each target nucleotide binding protein forms a conjugate with a single NBAP, most preferably at a single discrete location in the target nucleotide binding protein; thus, each conjugate gives rise to a single NBAP-labeled peptide. Enrichment separation, or identification of one or more NBAP-labeled peptides may be achieved using liquid chromatography and/or electrophoresis. Additionally, mass spectrometry may be employed to identify one or more NBAP-labeled peptides by molecular weight and/or amino acid sequence. In particularly preferred embodiments, the sequence information derived from the NBAP-labeled peptide(s) is used to identify the nucleotide binding protein from which the peptide originally derived. Variations of these aspects can involve the comparison of two or more proteomes, e.g., with NBAPs having different TAGs, or, when analysis comprises mass spectrometry, having different isotopic compositions. [0013] In yet another aspect, the instant invention relates to methods for comparing the presence, amount, or activity of one or more target nucleotide binding proteins in two or more complex protein mixtures using the methods and compositions described herein. In various embodiments, these methods comprise one or more ofthe following steps: contacting one or more complex protein mixture(s) with one or more NBAPs, where the NBAP(s) specifically bind to one or more target nucleotide binding proteins present in each complex protein mixture; combining the complex protein mixtures following this contacting step to form a combined complex protein mixture; prior to and/or following this combination, removing one or more non-sequestered components ofthe complex protein mixture(s). The nucleotide binding protein profile can then be analyzed by the screening and/or identification methods described hereinafter.

[0014] In preferred embodiments, the methods and compositions described herein are applied to determining the nucleotide binding protein profiles of cancerous and other diseased tissue by obtaining one or more samples of diseased tissue, and determining the nucleotide binding protein profile ofthe tissue sample(s). In particularly preferred embodiments, the nucleotide binding protein profile of diseased tissues can be compared to that of normal tissue sample(s) to determine differences in the enzyme activity profiles ofthe two tissue samples. [0015] In still another aspect, the present invention relates to methods and compositions for detecting disease in a test sample. In preferred embodiments the test sample will be a cell or tissue sample. In particularly preferred embodiments, the tissue sample will be a neoplasmic sample and the disease is a cancer. The methods involve determining the target nucleotide binding protein profile of the test sample; comparing the enzyme profiles of the test sample with the enzyme profile of a known non-diseased sample and/or with the enzyme profile of a known diseased sample; and determining whether the test sample is in a state of disease. A "non-diseased" sample is a sample of cells or tissues that is known to not have the disease being tested for. It is preferably a normal, healthy sample ofthe cells or tissue.

[0016] In another aspect the present invention provides methods of determining the inhibitory potency of a test compound against one or more target nucleotide binding protein. The methods involve contacting one or more NBAPs with a test sample containing the test compound and the target nucleotide binding protein(s); allowing the NBAPs to react with proteins contained in the test sample; and detecting a signal that indicates the level of NBAP binding to the target nucleotide binding protein(s) in the test sample. [0017] In preferred embodiments, this level of NBAP binding is compared to the level of NBAP binding to the target nucleotide binding protein(s) in the absence ofthe test compound. By such methods, the inhibitory and/or stimulatory potency ofthe test compound against the target nucleotide binding protein(s) can be determined. The "inhibitory potency" is the extent to which the presence of the compound causes the inhibition of NBAP binding, while "stimulatory potency" is the extent to which the presence ofthe compound causes an increase in NBAP binding.

[0018] In yet another aspect, the present invention provides kits for performing the methods described. The kits contain one or more of the materials described for conducting the methods. The kits can include NBAPs in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another. By "package" is meant material enveloping a vessel containing the NBAPs. In preferred embodiments, the package can be a box or wrapping. The kit can also contain items that are not contained within the package but are attached to the outside ofthe package, for example, pipettes.

[0019] The summary ofthe invention described above is not limiting and other features and advantages ofthe invention will be apparent from the following detailed description ofthe preferred embodiments, as well as from the claims.

DETAILED DESCRIPTION OF THE INVENTION [0020] The subject methods and compositions provide enhanced simplicity and accuracy in identifying changes in the presence, amount, or activity of nucleotide binding proteins in a complex protein mixture, preferably kinases, using NBAPs that bind to target nucleotide binding protein(s). The profiling methods described herein can have a number of steps leading to the identification of, or determining the presence or amount of, target nucleotide binding protein(s) in a complex protein mixture. A complex protein mixture, and preferably two or more complex protein mixtures, e.g., a sample and a control, can be used as obtained from a natural source or as processed, e.g., to remove interfering components and/or enrich the target protein components. Each complex protein mixture to be analyzed is combined under reaction conditions with at least one NBAP to produce conjugates with target nucleotide binding protein(s). The NBAPs used in two or more complex protein mixtures can differ as to the choice of TAG moiety and/or isotopic composition in order for the labeled complex protein mixtures to be directly compared (e.g., in the same capillary of a capillary electrophoresis apparatus or lane in an electrophoresis gel, or in a mass spectrometer).

[0021 ] The analysis platforms described herein can differ as to the methods of enrichment and analysis using liquid chromatography and/or electrophoresis, and/or mass spectrometry for identification and quantitation. The choice ofthe platform is affected by the size ofthe sample, the rate of throughput ofthe samples, the mode of identification, and the need for and level of quantitation.

[0022] Of particular interest as target proteins in the present invention are protein kinases. Protein kinases are the enzymes responsible for catalyzing the transfer of a 7- phosphoryl group from ATP to the hydroxyl group of serine, threonine or tyrosine residues in peptides, polypeptides, and proteins in a process known as "phosphorylation." Protein kinases have been identified in both prokaryotes and eukaryotes, and in both plants and animals. The list of identified kinases is extensive, including the following families of proteins: cyclic nucleotide regulated protein kinase (PKA & PKG) family; diacylglycerol- activated/phospholipid-dependent protein kinase C (PKC) family; kinases that phoshorylate G protein-coupled receptors family; budding yeast AGC -related protein kinase family; kinases that phosphorylate ribosomal protein S6 family; budding yeast DBF2/20 family; flowering plant PVPK1 protein kinase homolog family; kinases regulated by Ca²⁺/CaM and close relatives family; KINl/SNFl/Niml family; cyclin-dependent kinases (CDKs) and close relatives family; ERK (MAP) kinase family; glycogen synthase kinase 3 (GSK3) family; casein kinase II family; Clk family; Src family; Tec/Atk family; Csk family; Fes (Fps) family; Abl family; Syk/ZAP70 family; Tyk2/Jakl family; Ack family; focal adhesion kinase (Fak) family; epidermal growth factor receptor family; Eph/Elk/Eck receptor family; Axl family; Tie/Tek family; platelet-derived growth factor receptor family; fibroblast growth factor receptor family; insulin receptor family; LTK/ALK family; Ros/Sevenless family; Trk/Ror family; DDR/TKT family; hepatocyte growth factor receptor family, nematode Kin 15/ 16 family; Polo family; MEK/STE7 family; PAK/STE20 family; MEKK STE11 family; NimA family; weel/mikl family; kinases involved in transcriptional control family; Raf family; activin/TGFb receptor family; flowering plant putative receptor kinases and close relatives family; PSK PTK "mixed lineage" leucine zipper domain family; casein kinase I family; and PKN prokaryotic protein kinase family.

[0023] The compositions and methods described herein find use for the most part with biological samples, which may have been subject to processing before reaction with the NBAPs. "Biological sample" intends a sample obtained from a cell, tissue, or organism. Examples of biological samples include proteins obtained from cells (e.g., mammalian cells, bacterial cells, cultured cells, human cells, plant cells, etc.), particularly as a lysate, a biological fluid, such as blood, plasma, serum, urine, bile, saliva, tears, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), a transudate or exudate (e.g. fluid obtained from an abscess or other site of infection or inflammation), a fluid obtained from a joint (e.g. synnovial fluid obtained from a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or the like. [0024] Biological samples may be obtained from any organ or tissue (including a biopsy or autopsy specimen) or may comprise cells (including primary cells, passaged or cultured primary cells, cell lines, cells conditioned by a specific medium) or medium conditioned by cells. In preferred embodiments, a biological sample is free of intact cells. If desired, the biological sample maybe subjected to prior processing, such as lysis, extraction, subcellular fractionation, and the like. See, Deutscher (ed.), 1990, Methods in Enzymology, vol. 182, pp. 147-238.

[0025] Of particular interest are samples that are "complex protein mixtures." As used herein, this phrase refers to protein mixtures having at least about 20, more usually at least about 50, even 100 or more different proteins, where the particular distribution of proteins is of interest. An example of such a complex protein mixture is a proteome, as defined hereinafter. Complex protein mixtures may be obtained from cells that are normal or abnormal in some particular, where the abnormality is informative as to treatment, status, disease, or the like, can be analyzed using the methods ofthe subject invention. [0026] The term "proteome" as used herein refers to a complex protein mixture obtained from a biological sample. Preferred proteomes comprise at least about 5% of the total repertoire of proteins present in a biological sample (e.g., the cells, tissue, organ, or organism from which a lysate is obtained; the serum or plasma, etc.), preferably at least about 10%, more preferably at least about 25%, even more preferably about 75%, and generally 90% or more, up to and including the entire repertoire of proteins obtainable from the biological sample. Thus the proteome may be obtained from an intact cell, a lysate, a microsomal fraction, an organelle, a partially extracted lysate, biological fluid, a tissue, an organ, and the like. The proteome will be a mixture of proteins, generally having at least about 20 different proteins, usually at least about 50 different proteins and in most cases 100 different proteins or more.

[0027] Generally, the sample will have at least about 1 x 10^"1 ' g of protein, and may have 1 g of protein or more, preferably at a concentration in the range of about 0.1 - 50 mg/ml. For screening applications, the sample will typically be between about 1 x 10^"1 ' g of protein and about 1 x 10^"3 g of protein, preferably between about 1 x 10^"6 g of protein and 1 x 10^"4 g of protein. For identification of labeled active target kinases, the sample will typically be between about 1 x 10^" g of protein and about 1 g of protein, preferably between about 1 x 10^"4 g of protein and 1 x 10^"1 g of protein. The term "about" in this context refers to +/- 10% ofthe amount listed.

[0028] The sample may be adjusted to the appropriate buffer concentration and pH, if desired. One or more NBAPs may then be added, each at a concentration in the range of about InM to 20mM, preferably 10 nM to ImM, most preferably 10 nm to 100 μM. After incubating the reaction mixture, generally for a time for the reaction to go substantially to completion, generally for about 0.11 - 60 minutes, at a temperature in the range of about 5 - 40°C, preferably about 10°C to about 30°C , most preferably about 20°C , the reaction may be quenched.

[0029] In one aspect ofthe invention, the methods and compositions provide for qualitative (e.g., relative comparison between two samples) and/or quantitative measurement of target nucleotide binding protein(s)in biological fluids, cells or tissues. Moreover, the same general strategy can be broadened to achieve the proteome-wide, qualitative and quantitative analysis of target nucleotide binding protein(s), by employing NBAPs with differing target specificities. The methods and compositions of this invention can be used to identify nucleotide binding protein(s) of low abundance that are present in complex protein mixtures and can be used to selectively analyze specific groups or classes of nucleotide binding proteins, such as membrane or cell surface kinases, or kinases contained within organelles, sub-cellular fractions, or biochemical fractions such as immunoprecipitates. Further, these methods can be applied to analyze differences in expressed nucleotide binding proteins in different cell states. For example, the methods and reagents herein can be employed in diagnostic assays for the detection ofthe presence or the absence of one or more active nucleotide binding proteins indicative of a disease state, such as cancer. [0030] The subject methods and compositions can be used for a variety of purposes, such as the diagnosis of disease, the response of cells to an external agent, e.g. a drug, staging diseases, such as neoplasia, identifying cell differentiation and maturation, identifying new proteins, screening for active drugs, determining side effects of drugs, determining selectivity of drugs, identifying responses to drugs specific to certain genotypes (e.g., allelic differences in individuals), identifying useful probes from combinatorial libraries, etc. [0031 ] The system uses NBAPs specific for the a nucleotide binding protein or a group of nucleotide binding proteins, usually directed to an active site on such proteins, and combines one or a mixture of NBAPs, depending on the specificity ofthe NBAPs and the variety in the group or groups of proteins to be assayed. In the present invention, it is not necessary that there be no reaction of an NBAP with non-target nucleotide binding protein(s). Rather, an NBAP is defined as being "specific for," as "specifically reacting with," or as "specifically binding to," target nucleotide binding protein(s) if the NBAP provides at least about twice the amount of signal from NBAP labeling of target nucleotide binding protein(s) when compared to an equivalent amount of non-target protein. Preferably the signal obtained from active target nucleotide binding protein(s) will be at least about five fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater than that obtained from an equivalent amount of non-target protein. [0032] The term "target nucleotide binding protein" as used herein refers to one or more nucleotide binding protein(s), a nucleotide-binding site of which becomes labeled by one or more NBAPs. Preferred targets are kinases generally classified under the Enzyme Commission number 2.7.1.X. Particularly preferred kinases are protein kinases, classified under the Enzyme Commission number 2.7.1.37. The reaction mixture can provide conditions under which the NBAP(s) react substantially preferentially with active target kinases. Particularly preferred target kinases include phosphorylase b kinase kinase; glycogen synthase a kinase; hydroxyalkyl-protein kinase; serine(threonine) protein kinase; A-kinase; AP50 kinase; ATP-protein transphosphorylase; βllPKC; β-andrenergic receptor kinase; calcium/phospholipid-dependent protein kinase; calcium-dependent protein kinase C; cAMP- dependent protein kinase A; cAMP-dependent protein kinase; casein kinase; casein kinase I; casein kinase II; casein kinase 2; cGMP-dependent protein kinase; CK-2; CKI; CKII; cyclic monophosphate-dependent protein kinase; cyclic AMP-dependent protein kinase; cyclic AMP-dependent protein kinase A; cyclic nucleotide-dependent protein kinase; cyclin- dependent kinase; cytidine 3',5'-cyclic monophosphate-responsive protein kinase; ε PKC; glycogen synthase kinase; Hpr kinase; hydroxyalkyl-protein kinase; protein kinase (phosphorylating); casein kinase (phosphorylating); MAPK; mitogen-activated protein kinase; mitogen-activated S6 kinase ; M phase-specific cdc2 kinase; p82 kinase; phosphorylase b kinase kinase; PKA; PKC; protein serine kinase; protein kinase A; protein kinase p58; protein phosphokinase; protein glutamyl kinase; protein serine-threonine kinase; protein kinase CK2; protein-aspartyl kinase; protein-cysteine kinase; protein-serine kinase; Raf kinase; Raf-1; ribosomal S6 protein kinase; ribosomal protein S6 kinase II; serine kinase; serine-specific protein kinase; serine protein kinase; serine/threonine protein kinase; T- antigen kinase; threonine-specific protein kinase; twitchin kinase; and type-2 casein kinase. [0033] The term "active target kinase" refers to a target kinase that is in its native conformation and is able to interact with an entity with which it normally interacts, e.g. enzyme with substrate and cofactor, receptor with ligand, etc., e.g. phosphorylated active form as compared to unphosphorylated inactive form and vice versa. In effect, the protein is in the form in which it can carry out its biological function.

[0034] The term "inactivated" as used herein refers to a sample that has been treated so that at least a portion of target nucleotide binding protein(s) that were active in the original sample are rendered inactive. An "inactive nucleotide binding protein" can result from various mechanisms such as denaturation, inhibitor binding, either covalently or non- covalently, mutation, secondary processing, e.g. phosphorylation or dephosphorylation, etc. Functional states of proteins or kinases as described herein may be distinct from the level of abundance ofthe same proteins or enzymes. Inactivated samples may be used to validate the activity-specific binding of NBAPs as described herein.

[0035] The term "untreated" as used herein refers to a sample that has not been exposed to one or more conditions as compared to a second sample not exposed to such conditions. An untreated sample may be a sample that has not been inactivated; alternatively, an untreated sample may be one not exposed to one or more molecules (e.g., drug lead compounds) in a screening assay. Thus the compositions and methods described herein may comprise comparing a complex protein mixture obtained from cell(s), tissue(s), or organism(s) treated with one or more compounds (e.g., lead compounds in drug discovery) to a complex protein mixture obtained from cell(s), tissue(s), or organism(s) not so treated. NBAP-labeled proteins and/or peptides from the two samples may be compared for relative signal intensity. Such methods may indicate alterations in active protein content due to the treatment regimen. Additionally, such methods can also differentiate between treatments that act by direct inhibition of specific proteins ("primary effects") versus treatments that affect active protein content upstream, e.g., by altering expression of protein(s) ("secondary effects").

[0036] An "active site" of a protein refers to an area on the surface of a protein, e.g., an enzyme molecule or surface membrane receptor, to which a binding molecule, e.g. substrate or reciprocal ligand, is bound and results in a change in the protein and/or ligand. For a receptor, the conformation may change, the protein may become susceptible to phosphorylation or dephosphorylation or other processing. For the most part, the active site will be the site(s) of an enzyme where the substrate and/or a cofactor bind, where the substrate and cofactor undergo a catalytic reaction; where two proteins form a complex, e.g. the site at which a G protein binds to a surface membrane receptor, two kringle structures bind, sites at which transcription factors bind to other proteins; or sites at which proteins bind to specific nucleic acid sequences, etc. [0037] Structure of NBAPs

[0038] The term "nucleotide binding protein-directed affinity probes" ("NBAPs") refer to molecules that specifically react with target proteins as compared to inactive or non- target proteins. NBAPs may be designed and synthesized using combinatorial chemistry and/or rational design methods. A detailed description of a design strategy that can be adapted to the preparation of NBAPs in which a fluorescent moiety can act as a TAG is provided in PCT Application No. PCT/US02/03808, entitled "Activity Based Probe Analysis" (Attorney Docket No. 063391-0202), filed February 5, 2002, PCT Application No. PCT/US00/34187, WO 01/77684, entitled "Proteomic Analysis," and PCT Application No. PCT/US00/34167, WO 01/77668, entitled "Proteomic Analysis," each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. Goals of a design strategy are to provide NBAPs that are able to react covalently with a targeted group of nucleotide binding protein(s), while minimizing non-specific labeling. [0039] The NBAPs ofthe present invention comprise a warhead linked to a detectable tag by a linker moiety. As will be described hereinafter, each of the warhead, the linker moiety ("L"), and the tag ("TAG") may be independently selected to provide different target specificities. Each of these components of an NBAP is described in additional detail below. [0040] The term "warhead" as used herein refers to the portion of an NBAP that is directed to and binds with an active site of an active target kinase. The warhead comprises a reactive group ("RG") and an affinity moiety ("R"). Reactive group (RG) refers to one or more chemical groups within an NBAP that specifically and covalently bond to the active site of a protein. The reactive group may, by its very structure, be directed to the active site of a target protein. Alternatively, a separate affinity moiety (R) may be provided. Affinity moiety (R) refers to a chemical group, which may be a single atom, that is conjugated to the reactive group or associated with the linker moiety that provides enhanced binding affinity for protein targets and/or changes the binding profile ofthe warhead. The affinity moiety is preferably less than 1 kilodalton in mass.

[0041 ] Exemplary RGs as used in an NBAP of the invention include an alkylating agent, acylating agent, ketone, aldehyde, sulphonate or a phosphorylating agent. Examples of particular RGs include, but are not limited to fluorophosphonyl, fluorophosphoryl, fluorosulfonyl, alpha-haloketones or aldehydes or their ketals or acetals, respectively, alpha- haloacyls, nitriles, sulfonated alkyl or aryl thiols, iodoacetylamide group, maleimides, sulfonyl halides and esters, isocyanates, isothiocyanantes, tetrafluorophenyl esters, N- hydroxysuccinimidyl esters, acid halides, acid anhydrides, unsaturated carbonyls, alkynes, hydroxamates, alpha-halomethylhydroxamates, aziridines, epoxides, or arsenates and their oxides. Sulfonyl groups may include sulfonates, sulfates, sulfinates, sulfamates, etc., in effect, any reactive group having a sulfur group bonded to two oxygen atoms. Epoxides may include aliphatic, aralkyl, cycloaliphatic and spiro epoxides. [0042] The term "linker moiety" refers to a bond or chain of atoms used to link one moiety to another, serving as a covalent linkage between two or more moieties. Since in many cases, the synthetic strategy will be able to include a functionalized site for linking, the functionality can be taken advantage of in choosing the linking moiety. The choice of linker moiety has been shown to alter the specificity of an NBAP. See, e.g., Kidd et al., Biochemistry (2001) 40: 4005-15. For example, an alkylene linker moiety and a linker moiety comprising a repeating alkyleneoxy structure (polyethylene glycols, or "PEG"), have distinct specificities and provide distinct protein profiles. Thus, one of skill in the art can select the linker moiety ofthe NBAP in order to provide additional specificity ofthe NBAP for a particular protein or protein class.

[0043] Linker moieties include among others, ethers, polyethers, diamines, ether diamines, polyether diamines, amides, polyamides, polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains (straight chain or branched and portions of which may be cyclic) aryl, diaryl or alkyl-aryl groups, having from 0 to 3 sites of aliphatic unsaturation. While normally amino acids and ohgopeptides are not preferred, when used they will normally employ amino acids of from 2 - 3 carbon atoms, i.e. glycine and alanine. Aryl groups in linker moieties can contain one or more heteroatoms (e.g., N, O or S atoms). The linker moieties, when other than a bond, will have from about 1 to 60 atoms, usually 1 to 30 atoms, where the atoms include C, N, O, S, P, etc., particularly C, N and O, and will generally have from about 1 to 12 carbon atoms and from about 0 to 8, usually 0 to 6 heteroatoms. The number of atoms referred to above are exclusive of hydrogen in referring to the number of atoms in a group, unless indicated otherwise.

[0044] Linker moieties may be varied widely depending on their function, including alkyleneoxy and polyalkyleneoxy groups, where alkylene is of from 2 - 3 carbon atoms, methylene and polymethylene, polyamide, polyester, and the like, where individual monomers will generally be of from 1 to 6, more usually 1 to 4 carbon atoms. The oligomers will generally have from about 1 to 10, more usually 1 to 8 monomeric units. The monomeric units may be amino acids, both naturally occurring and synthetic, oligonucleotides, both naturally occurring and synthetic, condensation polymer monomeric units and combinations thereof.

[0045] Linker moieties may provide a covalent linkage between a TAG and a reactive group RG, between a TAG and an affinity moiety R, or between a reactive group RG and an affinity moiety R. In addition, a linker can be "bifunctional" in that a single linker provides a covalent linkage between all three ofthe TAG, affinity moiety R, and reactive group RG portions of an NBAP. For example, the linker moiety may form a branching structure, whereby each ofthe three portions of an NBAP are bound to the same linker moiety. [0046] The term "TAG" as used herein refers to a molecule that can be used to detect and or capture the NBAP in combination with any other moieties that are bound strongly to the TAG, so as to be retained in the process ofthe reaction ofthe reactive group with the target active protein. The TAG may be added to the warhead-linker moiety combination after reaction with the target protein, to form the complete NBAP. For this purpose, the warhead- linker moiety combination will include a chemically reactive group, normally not found in proteins, that will react with a reciprocal functionality on the TAG, e.g. viccinal-diols with boronic acid, phofoactivated groups, such as diazo, azide with an alkene or alkyne, o-alkyl hydroxylamine with a ketone or aldehyde, etc. The warhead-linker moiety is then reacted with the TAG to complete the NBAP. The TAG portion permits capture ofthe conjugate of the target protein and the NBAP. The TAG may be displaced from the capture reagent by addition of a displacing TAG, which may be free TAG or a derivative ofthe TAG, or by changing solvent (e.g., solvent type or pH) or temperature or the linker may be cleaved chemically, enzymatically, thermally or photochemically to release the isolated materials (see discussion ofthe linker moiety, below).

[0047] Examples of TAGs include, but are not limited to, detectable labels such as fluorescent moieties and electrochemical labels, biotin, digoxigenin, maltose, ohgohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, a polypeptide, a metal chelate, and/or a saccharide. Examples of TAGs and their capture reagents also include but are not limited to: dethiobiotin or structurally modified biotin-based reagents, including deiminobiotin, which bind to proteins ofthe avidin/streptavidin family, which may, for example, be used in the forms of strepavidin- Agarose, oligomeric-avidin- Agarose, or monomeric-avidin-Agarose; any vicinal diols, such as 1 ,2-dihydroxyethane (HO-CH₂-CH₂-OH), and other 1,2- dihyroxyalkanes including those of cyclic alkanes, e.g., 1 ,2-dihydroxycyclohexane which bind to an alkyl or aryl boronic acid or boronic acid esters, such as phenyl-B(OH)₂ or hexyl- B(OEthyl)₂ which may be attached via the alkyl or aryl group to a solid support material, such as Agarose; maltose which binds to maltose binding protein (as well as any other sugar/sugar binding protein pair or more generally to any TAG/TAG binding protein pairs that has properties discussed above); a hapten, such as the dinitrophenyl group, to which an antibody can be generated; a TAG which binds to a transition metal, for example, an oligomeric histidine will bind to Ni(II), the transition metal capture reagent may be used in the form of a resin bound chelated transition metal, such as nitrilotriacetic acid-chelated Ni(II) or iminodiacetic acid-chelated Ni(II); glutathione which binds to glutathione-S- transferase. For the most part, the TAGs will be haptens that bind to a naturally occurring receptor, e.g. biotin and avidin, or an antibody or will be a detectable label, that is also a hapten.

[0048] One may use chemical affinity resins, e.g. metal chelates, to allow for digestion of proteins on the solid phase resin and facilitate automation. One example of this is the use of immobilized nickel (II) chelates to purify peptides that have six consecutive histidine residues (His-6 tag) (as described in the Invitrogen product brochureProBond ™ Resin (Purification) Catalog nos. R801-01, R801-15 Version D 000913 28-0076), which could be adapted to include non-peptidic chemical linkage coupling a series of imidazole- containing moieties. Alternative chemical attachments include phenyldiboronic acids (described in Bergseid, M. et al. Biotechniques (2000) 29(5), 1126-1133), and disulfide reagents (described in Daniel, SM et al., Biotechniques (1998) 24(3), 484-489). Additionally, chemical affinity tags that are useful in combinatorial synthesis could be adapted for modified peptide purification (reviewed in Porco, JA (2000) Comb. Chem. High Throughput Screening 3(2) 93-102

[0049] The term "fluorescent moiety" ("FI") refers to a TAG that can be excited by electromagnetic radiation, and that emits electromagnetic radiation in response in an amount sufficient to be detected in an assay. The skilled artisan will understand that a fluorescent moiety absorbs and emits over a number of wavelengths, referred to as an "absorbance spectrum" and an "emission spectrum." A fluorescent moiety will exhibit a peak emission wavelength that is a longer wavelength than its peak absorbance wavelength. The term "peak" refers to the highest point in the absorbance or emission spectrum. [0050] The fluorescent moiety FI may be varied widely depending upon the protocol to be used, the number of different NBAPs employed in the same assay, whether a single or plurality of lanes are used in the electrophoresis, the availability of excitation and detection devices, and the like. For the most part, the fluorescent moieties that are employed as TAG will absorb in the ultraviolet, infrared, and/or most preferably in the visible range and emit in the ultraviolet, infrared, and/or most preferably in the visible range. Absorption will generally be in the range of about 250 to 750 nm and emission will generally be in the range of about 350 to 800nm. Illustrative fluorescent moieties include xanthene dyes, naphthylamine dyes, coumarins, cyanine dyes and metal chelate dyes, such as fluorescein, rhodamine, rosamine, the BODIPY dyes (FL, TMR, and TR), dansyl, lanthanide cryptates, erbium, terbium and ruthenium chelates, e.g. squarates, and the like. Additionally, in certain embodiments, one or more fluorescent moieties can be energy transfer dyes such as those described in Waggoner et al, U.S. Patent no. 6,008,373. The literature amply describes methods for linking fluorescent moieties through a wide variety of linker moieties to other groups. The fluorescent moieties that find use will normally be under 2kDal, usually under lkDal.

[0051 ] Preferred fluorescent moieties FI can include elaborated conjugated pyran molecules, including xanthenes. Such molecules include eosin, erythrosin, fluorescein, Oregon green, and various commercially available Alexa Fluor ® dyes (Molecular Probes, Inc.). Structural examples of such dyes include:

[0052] Particularly preferred fluorescent moieties are the rhodamine dyes. These molecules typically have the general structure:

[0053] Where K is -CO₂H, or -S0₃H; Y is -H, -CH₃, or together with R forms a six- membered ring; Z is -H or together with R forms a six-membered ring; and R is -H, -CH₃, - CH₂CH₃, or together with Y or Z forms a six-membered ring. Rhodamine molecules such as tetramethylrhodamine, 5 -carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, carboxyrhodamine-6G, rhodamine-B sulfonyl chloride, rhodamine-red-X, and carboxy-X- rhodamine are well known to those of skill in the art. See, e.g., Handbook of Fluorescent Probes and Research Products, Molecular Probes, Inc., 2001, which is hereby incorporated by reference in its entirety. Advantageous properties of rhodamines include high quantum yields, low sensitivity of fluorescence over a pH range of from about pH 3 to about pH 8, advantageous water solubility, good photostability, and absorption of light in the visible spectrum. Particularly preferred fluorescers are 5 -carboxytetramethylrhodamine and 6- carboxytetramethylrhodamine. [0054] Other preferred fluorescent moieties FI include the BODIPY dyes, which are elaborations of a 4-bora-3a,4a-diaza-5-indacene structure. Exemplary structures are provided below:

[0055] Yet other preferred fluorescent moieties include the cyanine dyes, conjugated structures comprising a polymethine chain terminating in nitrogen atoms. Typically, the nitrogens are themselves part of a conjugated heterocycle. An exemplary structures is provided below:

[0056] Also of interest for use as TAGs are matched dyes as described in U.S. Patent

No. 6,127,134, which is hereby incorporated by reference in its entirety, including all tables, figures, and claims, which is concerned with labeling proteins with dyes that have different emissions, but have little or no effect on relative migration of labeled proteins in an electrophoretic separation. Of particular interest are the cyanine dyes disclosed therein, being selected in '134 because of their positive charge, which matches the lysine to which the cyanine dyes bind. In addition there is the opportunity to vary the polyene spacer between cyclic ends, while keeping the molecular weight about the same with the introduction of an alkyl group in the shorter polyene chain dye to offset the longer polyene. Also described are the BODIPY dyes, which lack a charge. The advantage of having two dyes that similarly affect the migration ofthe protein would be present when comparing the native and inactived samples, although this would require that in the inactivated sample at least a portion ofthe protein is monosubstituted.

[0057] In each ofthe foregoing examples of preferred fluorescent moieties, carboxyl groups can provide convenient attachment sites for linker moieties. In the particularly preferred 5- and 6-carboxyrhodamine molecules, the 5- or 6- carboxyl is particularly preferred as an attachment site:

While the following preferred embodiments and exemplified compounds are generally described using only the 5-carboxyrhodamine molecules for the sake of brevity, in each case the 6-carboxyrhodamine version ofthe indicated molecule, or a mixture ofthe 5- and 6- carboxyrhodamine molecules should also be considered as an exemplified preferred embodiment.

[0058] In general, any affinity label-capture reagent commonly used for affinity enrichment, which meets the suitability criteria discussed above, can be used in the method of the invention. Biotin and biotin-based affinity TAGs are particularly illustrated herein. Of particular interest are structurally modified biotins, such as deiminobiotin or dethiobiotin, which will elute from avidin or streptavidin (strept/avidin) columns with biotin or under solvent conditions compatible with ESI-MS analysis, such as dilute acids containing 10-20% organic solvent. For example, deiminobiotin tagged compounds will elute in solvents below about pH 4.

[0059] In certain embodiments, NBAPs can be immobilized on a solid phase to form a "tethered" NBAP. In preferred embodiments, a plurality of different NBAPs may be tethered to different regions of one or more solid phases to form a patterned array. Such a patterned array having two or more regions comprising NBAPs that differ in structure and/or reactivities from each other could be used to simultaneously measure the presence, amount, or activity of a plurality of target nucleotide binding proteins. 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, 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. Diamandis 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, glass, 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); Gottschhng et al, Bioorg. And Medicinal Chem. Lett. 11 : 2997 (2001). [0060] The NBAP(s) employed will have an affinity for an active site, which may be specific for a particular active site or generally shared by a plurality of related proteins. The affinity may be affected by the choice of the reactive group, the linker moiety, the binding moiety, the TAG, or a combination thereof. One or more NBAPs may be designed that exhibit specificity for a single target protein, or that exhibit specificity for a plurality of targets that may be structurally or functionally related. [0061] 4-Phenylamino quinazoline-related NBAPs

[0062] In one embodiment, the NBAP(s) ofthe present invention are 4-phenylamino quinazoline-related compounds. Such compounds preferably label receptor tyrosine kinase family members, e.g., kinase insert domain receptor (KDR), EGFR, etc. The NBAP(s) have one ofthe following general formula:

[0063] Preferably, each Ri is independently selected from the group consisting of -F,

-Br, -CI, -SCH₃, -OH, -CH₂OH, -C(0)N(R)(R), -CN, -NO₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Cι-₆ alkyl straight or branched chain;

[0064] n is between 0 and 5 inclusive;

[0065] each W is independently carbon or nitrogen;

[0066] TAG is a detectable label;

[0067] RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; and

[0068] L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -

C(R)(R)-, where each R is independently H or -Cι-₆ alkyl straight or branched chain, or optionally form an optionally substituted fused carbocyclic or heterocyclic ring structure.

[0069] The person of ordinary skill will realize that pharmaceutically acceptable salt or complexes of these compounds are also useful and are also contemplated within the scope ofthe invention.

[0070] In preferred embodiments, a reactive group RG is preferably selected from the following:

o , — . o o . — . o o , — , o o

_F-S- J- p-ji- - i- ^i' - yi.. _x l_yy

6 ^=/ OR ^=/ o ^=/

o o V o o -s- — S — _ 1.- o OR R o o

where X = CI, Br, F, or I.

Such RG groups may be linked in various ways to the present probes, e.g., directly to a linear portion of a linker or to a branch from a linker or other moiety ofthe probe. For example, a group RG may link through a N, C, or other single atom or through a short chain, e.g., 2-6 linked atoms. [0071] A preferred group of linking moieties Li and L₂ fall within the following formulae:

[0072] where n and m are independently in the range of 0 to 8, and X is RG, TAG, or a covalent linkage through linking moiety L to both RG and TAG; and most preferably TAG is a fluorescent moiety, FI.

[0073] When present, a preferred group of linking moieties L come within the following formulae:

[0074] where each n is independently in the range of of 0 to 8, and R is H or Cl-6 alkyl. Most preferably, TAG is a fluorescent moiety, FI. [0075] The fluorescent moiety TAGs of particular interest come within the following formulae:

where the exemplified 5-carboxyrhodamine or 5-carboxyfluorescein may also be the equivalent 6-substituted molecule or a mixture of 5- and 6-substituted molecules.

[0076] Staurosporine related NBAPs

[0077] Staurosporine is a nonselective and low nanomolar protein kinase C inhibitor that blocks ATP binding. Staurosporine competes with ATP, but not with peptide substrates, for binding to protem kinases. The present invention discloses NBAPs that are staurosporine related compounds.

[0078] Preferably, staurosporine-related NBAPs have the following structure:

[0079] Where W is carbon, oxygen, sulfur, or nitrogen; Rl and R2 are independently

H or OH or Rl and R2 together are =O. and preferably Rl and R2 are each H; Rl is OH and

R2 is H; Rl is H and R2 is OH;

[0080] TAG is a detectable label;

[0081] RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; and

[0082] L and Li are optionally present and are independently an alkyl or heteroalkyl group of 1 -20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -

C(R)(R)-, where each R is independently H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure.

[0083] The person of ordinary skill will realize that pharmaceutically acceptable salt or complexes of these compounds are also useful and are also contemplated within the scope of the invention. [0084] In preferred embodiments, linking moieties Li and L, reactive groups RG, and

TAGs fall within the formulae described above for 4-phenylamino quinazoline-based

NBAPs.

[0085] Preferred linking moieties L fall within the following formulae:

where n and m are independently in the range of 0 to 8, and X is a covalent linkage through linking moiety L to both RG and TAG; and most preferably TAG is a fluorescent moiety, FI. [0086] A preferred group of linking moieties "L" fall within the following formulae:

[0087] where each n is independently in the range of 0 to 8, and R is H or Cl-6 alkyl.

Most preferably TAG is a fluorescent moiety, FI. [0088] In related aspects, bis-indole maleimides may exhibit different kinase selectivity profiles than staurosporine. NBAPs based on such bis-indole maleimides analogues preferably have the general structure:

[0089] Preferably, Ri, R₂, R₃ R₄, and R₅ are each independently selected from the group consisting of-H, -OH, Ri and R₂ together are =O, and R₃ and R₄ together are =0; [0090] Arl and Ar2 are optionally present and are independently selected from the group consisting -N(R)-, -O-, -S- or -C(R)(R)-, aryl, or heteroaryl, where each R is independently H or -Cι-₆ alkyl straight or branched chain, wherein if Ari or Ar₂ are aryl or heteroaryl, the aryl or heteroaryl group optionally forms a fused carbocyclic or heterocyclic ring structure with the maleimide ring;

[0091] L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of-N(R)-, -0-, -S- or - C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure; [0092] TAG is a detectable label;

[0093] and RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group. [0094] The person of ordinary skill in the art will realize that pharmaceutically acceptable salts or complexes ofthe compounds may also be useful and are contemplated as within the scope ofthe disclosure.

[0095] In preferred embodiments, the reactive group RG, TAGs and linker moieties

L, Li and L₂ fall within the formulae described above for 4-phenylamino quinazoline-related

NBAPs. Additional preferred reactive group RG, TAGs and linker moieties L, Li and L₂ are described below in preferred embodiments.

[0096] In yet other related aspects, additional NBAP compounds preferably have one of the following general structures:

[0097] where W is carbon, oxygen, sulfur, or nitrogen; Ri, R₂, R₃ and R-. are independently -H, -OH, or together =O; R6 is H or OH; and R7, R8, R9, and R10 are independently -H, -OH, -C alkyl, or -00(0)-^ alkyl; and where one of R7, R8, R9 or R10 comprises a linker moiety L- covalently linked to a linker moiety L that links each of TAG and RG, or two of R7, R8, R9 or R10 comprise linker moieties Li to a reactive group RG and L₂ to a TAG; or

[0098] where R_n, R,₃ and R₁₄ are independently -F, -Br, -CI, -SCH₃, -OH, -CH₂OH, -

C(O)N(R)(R), -CN, -N0₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Ci- ₆ alkyl straight or branched chain; and

[0099] L, Li , and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -O-, -S- or -C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure; or

[0100] where R₁₅ and R_]6 are independently -F, -Br, -CI, -SCH₃, -OH, -CH₂OH, -

C(O)N(R)(R), -CN, -NO₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Ci-

₆ alkyl straight or branched chain; and

[0101 ] L, L), and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -O-,

-S- or -C(R)(R)-, where R is H or -Cι-6 alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure.

[0102] In each ofthe foregoing aspects, TAG is a detectable label; and

[0103] RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group. [0104] In preferred embodiments, the reactive groups RG, TAGs and linker moieties

L, Li and L₂ fall within the formulae described above for 4-phenylamino quinazoline-related NBAPs or for the first staurosporine-related NBAPs. Additional preferred reactive group RG, TAGs and linker moieties L, Li and L₂ are described below in preferred embodiments. [0105] In particularly preferred embodiments, the NBAPs of the present invention have one ofthe following structures:

37

38 where the exemplified 5-carboxyrhodamine may also be the equivalent 6-substituted molecule or a mixture of 5- and 6-substituted molecules. Pyrido[2,3-d]pyrimidine analogue-related NBAPs

[0106] In additional aspects, the present invention also provides pyrido[2,3- d]pyrimidine related NBAPs ofthe general formula:

[0107] Preferably each R* is independently selected from the group consisting of -F, -

Br, -CI, -SCH₃, -OH, -CH₂OH, -C(O)N(R)(R), -CN, -NO₂, -N(R)(R), acetoxy, -C(R)(R)(R), -

OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Cι-₆ alkyl straight or branched chain;

[0108] R₂ is Co-6 alkyl;

[0109] n is between 0 and 5 inclusive; [0110] each W is independently carbon, or nitrogen;

[0111] TAG is a detectable label;

[0112] RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene to an activating group, and halogen oc to an activating group; and

[0113] L, Li and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1 -20 backbone atoms selected from the group consisting of -N(R)-, -O-,

-S- or -C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure.

[0114] In preferred embodiments, the reactive group RG, TAGs and linking moieties

L, Li and L₂ fall within the formulae described above for 4-phenylamino quinazoline-based

NBAPs.

[0115] As noted above, many variations of the molecule are possible. In preferred embodiments, L, Li and/or L₂ is selected from the group consisting of: NH₂, NH(CH₂)₃NEt₂,

NH(CH₃)₄NEt₂, NMe(CH2) ₃NMe₂, NHCH₂CMe₂CH2NMe₂, NH(CH₂)₃ (morpholin-1-yl),

NH(CH₂)₃ (2-methylpiperidin-l-yl), NH(CH₂)₃ (N-methylpiperazin-1-yl), H₂NCO, EtNHCO, allylNHCO, t-PrNHCO, t-BuNHCO, n-ocytlNHCO, benzylNHCO, cyclohexylNHCO, adamantylNHCO, BOCHN(CH₂) ₂NHCO, Me₂N(CH2) ₂NHCO, Et₂NCO, PhNHCO, 4-

ClPhNHCO, 4-BrPhNHCO, 4-CF₃PhNHCO, 3,4-(Cl) ₂PhNHCO, 4-MePhNHCO, 2-

MeOPhNHCO, 3-MeOPhNHCO, 4-MeOPhNHCO, 1-napthylNHCO; and R₂ is selected from the following groups: H, H₂NCO, EtNHCO, allylNHCO, t-PrNHCO, t-BuNHCO, n- octylNHCO, benzylNHCO, cyclohexylNHCO, adamantylNHCO, BOCNH(CH₂) ₂NHCO,

Me₂NCH₂) ₂NHCO, Et2NCO, PhNHCO, 4-ClPhNHCO, -BrPhNHCO, 4-CF₃PhNHCO, 3,4-

(Cl)2PhNHCO, 4-MePhNHCO, 2-MeOPhNHCO, 3-MeOPhNHCO, 4-MeOPhNHCO, 1- napthylNHCO, t-BuCH₂CO, Me2NCH, EtNHCS, EtNHCNH, (morpholin-l-yl)(CH₂) ₃NHCS, EtCH₂CO, t-Bu-CH₂CO, t-BuNHCO, PhCH₂CO, PhSO₂, PhNHCNH, t-PrNHCN-t- Pr, t-BuNHCS, PhNHCS. Additional preferred reactive group RG, TAGs and linker moieties Li and L₂ are described below in preferred embodiments.

[0116] In particularly preferred embodiments the NBAPs of the present invention have the following structure:

[0117] Adenine-related Activity Probes

[0118] The cyclin-dependent kinases are a conserved family of serine/threonine protein kinases that play an important role in cell cycle control. Inhibitors ofthe cyclin- dependent kinases can provide effective anti-cancer drugs with anti-proliferative activity.

[0119] The present invention provides adenine-related compounds having the general structure

[0120] Preferably each W is independently carbon, or nitrogen;

[0121] Ri and R₂ are independently selected from the group consisting of -F, -Br, -CI,

-SCH₃, -OH, -CH₂OH, -C(O)N(R)(R), -CN, -N0₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -

OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Cι-₆ alkyl straight or branched chain;

[0122] N is 0 to 3;

[0123] TAG is a detectable label;

[0124] RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino, the reactive group being a single reactive group or a combination of reactive groups selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen β to an activating group; and

[0125] L, L* and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -O-, -S- or -C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure. [0126] In preferred embodiments, the reactive group RG, TAGs and linking moieties

L, L| and L₂ fall within the formulae described above for 4-phenylamino quinazoline-based NBAPs. Additional preferred reactive group RG, TAGs and linker moieties L, L* and L₂ are described below in preferred embodiments. Preferably, R₂ is Cι-₆ alkyl straight or branched chain or C -₆ cycloalkyl or aryl, and most preferably cyclopentyl, isopropyl, or ethyl. [0127] Analysis of samples with NBAPs

[0128] After the reaction between the complex protein mixture and the NBAP(s) is completed, the conjugates ofthe NBAPs and protein targets will be analyzed. Preferably, the NBAPs ofthe present invention comprise a TAG that allows for manipulation ofthe conjugates, either for sequestering the conjugates or detecting the conjugates or both. The NBAPs may be analyzed by separating into components, e.g., by electrophoresis, for example gel electrophoresis, capillary electrophoresis or microfluidic electrophoresis; mass spectrometry, e.g., MALDI-TOF, microcapillary liquid chromatography-electrospray tandem MS, or other technique. To enhance the analysis, the conjugates may be deglycosylated using an appropriate glycosidase, such as PGNaseF, under conventional deglycosylation conditions indicated by the enzyme supplier. Labeled active target proteins can be identified based on a variety of physical criteria, such as apparent molecular weight, peptide sequence composition, enzymatic activity (e.g., serine hydrolase activity), or a combination of such criteria.

[0129] The term "separating" as used herein refers to methods that enrich the concentration of a molecule of interest in a particular location or container relative to other molecules originally present. For example, gel electrophoresis enriches the concentration of molecules that migrate at a particular rate relative to other molecules originally present that migrate at different rates; sequestration methods enrich the concentration of molecules capable of being sequestered (e.g., by binding to a receptor) relative to other molecules not so capable (e.g., removed by washing out molecules that do not bind to a receptor). Numerous additional analytical procedures are known to the artisan for separating and analyzing complex protein mixtures (e.g., chromatographic methods such as HPLC, FPLC, ion exchange, size exclusion; mass spectrometry; differential centrifugation). [0130] In preferred embodiments, the NBAP products are analyzed by electrophoresis, e.g., slab gel, capillary or microfluidic, optionally using a gel for separation ofthe different components. In particularly preferred embodiments, SDS-PAGE is used, including 2D PAGE. The sample composition may be preliminarily separated using isoelectric focusing, followed by using bands or regions for further electrophoretic separation. Conventional conditions can be employed for the electrophoresis, using a denaturing medium, so that the active sample and the inactivated sample are both denatured in the gel. Numerous patents have issued for performing electrophoresis for the separation of proteins. See, e.g., U.S. Patent Nos. 4,415,655; 4,481,094; 4,865,707; and 4,946,794. Texts describing procedures include Laemmli, Nature 227:680-685 (1970); Sambrook et al., "Molecular Cloning: A Laboratory Manual." 3^rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY. (2001).

[0131] Using the NBAPs of the present invention, labeled target nucleotide binding protein(s) may be identified by excitation and detection of light emitted upon excitation of the fluorescent moiety, e.g., in electrophoresis gels. In certain embodiments, such as when the NBAP labels a plurality of target nucleotide binding proteins or when the identity of a labeled target nucleotide binding protein is unknown, the labeled target nucleotide binding protein(s) present in various electophoretic bands may be further assayed to identify the specific proteins to which the NBAP(s) bound, e.g., by fragmentation and mass spectrometric analysis. In particular, the sequence of proteins can be determined using tandem MS (MSⁿ) techniques. By application of sequence database searching techniques, the protein from which a sequenced peptide originated can be identified.

[0132] In designing a gel-based analysis system, the artisan may balance various considerations, such as speed, resolution, sample volume, choice of fluorophore, detection methods, etc., in order to arrive at an optimal solution. For example, for simple screening analysis (i.e., when gel bands are not to be identified by means of eluting proteins from the gel matrix for further analysis), very thin gels may be run quickly. Additionally, such thin gels are amenable to the use of laser-induced fluorescence scanning systems and narrow gel lanes, as laser focusing and confocal detection optics permit the detection of very small amounts of NBAP in a sample. Conversely, thicker gels may be advantageous in protein identification analysis, as a sufficient amount of material must be obtained from a gel band to permit further manipulations.

[0133] For rapid screening analysis, a suitable gel electrophoresis platform would consist of a glass sandwich gel format of from 15-40 cm in width, 20-40 cm in length, and from 0.6 to 0.2 cm in thickness. A partciularly preferred format is from about 30-35 cm in width, about 25-30 cm in length, and about 0.4 mm in thickness. The term "about" in this context refers to +/- 10% of a given dimension. The gel format is preferably combined with a laser-induced fluorescence detector apparatus comprising detection optics that permit sampling ofthe gel without removal from the gel plates, as such thin gels may be extremely fragile. Typically, such an instrument uses confocal optics for detection. By matching the thickness ofthe gel to the thickness ofthe confocal "slice," signal detection can be matched to a minimal amount of sample.

[0134] The spacing between sample wells is limited only by the amount of sample necessary to obtain a sufficient signal for measurement. Appropriate spacings are between 1 and 4 mm, most preferably about 2.25-3 mm. The term "about" in this context refers to +/- 10% ofthe spacing between wells. Selecting a spacing between wells of about 2.25 mm as an example, a gel platform 25 cm in width could accommodate as many as 96 individual samples.

[0135] After completing the electropherogram, the bands may then be read using any convenient detection means (e.g., a fluorescent reader, e.g., Hitachi FMbio Flatbed Fluorescence Scanner, when the NBAP comprises a fluorescent moiety), where the intensity of each band may be transferred to a data processor for processing. Depending on whether one or more lanes are involved with the analysis, the data may be compiled from a single or multiple lanes to establish the bands associated with active target proteins that are absent with the inactive sample, the different target proteins that reacted with different NBAPs as evidenced by the different fluorescence emission for each ofthe NBAPs, and any cross- reactivity between the NBAPs. The bands that are obtained in the gel are sharp and provide for excellent resolution. Particularly, much better resolution and sensitivity may be obtained than when biotin-labeled NBAPs are used, followed by complex formation with labeled avidin, and Western blotting.

[0136] The results obtained from analyzing the nucleotide binding protein profiles may then be organized in a manner that allows for ready comparisons and differentiation between samples. One technique that finds utility is cluster analysis. One applies a hierarchical clustering algorithm to the samples using the Pearson coπelation coefficient as the measure of similarity and average linking clustering (Cluster program: Ross et al., Nat. Genet. 24:227-35 (2000); Eisen et al., Proc. Natl. Acad. Sci. USA 95: 14863-68 (1998)). For each enzyme activity, averaged cell sample values are compared to identify the cell sample that expressed the highest level of a particular enzyme activity. The activity levels may then be expressed as a percentage of this highest activity to normalize the data sets. As data sets are built up from cell samples, the cluster analysis can be modified in light of new data that provides a new maximum for a particular enzyme, so that one may have cluster analysis within a given group of samples as well as cluster analysis extending over many samples and groups of samples. Cluster analysis can also be applied as to the individual fractions and pair-wise combinations, so as to maximize information from the cell samples in relating the samples to each other and standards. For large numbers of samples, clustergrams can be used to rapidly identify the similarities between samples, for example, in terms of origin ofthe cells, aggressiveness and invasiveness, diagnosis, prognosis, preferential therapies and how the tumor has responded to a course of treatment.

[0137] Following NBAP labeling of target nucleotide binding protein(s), protein digestion may be employed to produce both unlabeled and NBAP-labeled peptides. The digestion may be performed while the proteins are in solution or when the conjugates are sequestered, e.g., by receptors bound to a solid support. Digestion preferably employs only one protease; however, two or more, usually not more than three, proteases may be used. The proteases may be in solution or bound to a surface. The proteases may be combined in the same reaction mixture, or the sample may be divided into aliquots and each ofthe aliquots treated with a different protease. Digestion may also occur before binding to the conjugate to a support and/or a after the conjugates are bound to a solid support. Enzymes that find use include, but are not limited to, trypsin, chymotrypsin, bromelain, papain, carboxypeptidase A, B and Y, proteinase A and K, chymopapain, plasmin, subtilisin, clostripain etc. [0138] In particularly preferred embodiments, additional steps can be used to reduce the complexity ofthe analysis to be performed. For example, the complex protein mixture can be denatured following labeling, e.g., by the addition of urea, guanidinium salts, detergents, organic solvents, etc., in order to reduce or eliminate unwanted proteolysis from endogenous proteases present in the mixture. Additionally, cysteine residues can be reduced and alkylated to maintain the homogeneity of cysteine-containing peptides and to prevent refolding of endogenous proteases following removal ofthe denaturant. Moreover, proteases can be combined with additional enzymes, such as glycosidases, phosphatases, sulfatases, etc., that can act to remove post-translational modifications from proteins. Examples of such post-translational modifications include, but are not limited to, glycosylations, phosphorylations, sulfations, prenylations, methylations, amidations, and myristolations. Such steps can be mixed and matched by the skilled artisan, depending on the requirements of a particular analysis.

[0139] Prior to digestion, a buffer exchange step may be employed, e.g., by gel filtration, dialysis, etc. This step may be used to remove excess NBAPs, to remove denaturant, and/or to provide suitable buffer conditions for digestion. In particularly preferred embodiments, buffer exchange is performed by gravity flow gel filtration. [0140] Digestion will be carried out in an aqueous buffered medium, generally at a pH in the range of about 4 to 10, depending on the requirements ofthe protease. The concentration ofthe protease will generally be in the range of about 6 x 10^"8 M to about 6 x 10^"6 M, more preferably in the range of about 1.8 x 10^"8 M to about 2 x 10^"7 M, and most preferably about 6 x 10^"7 M (e.g., 150 ng / 10 μL). The term "about" in this context means +/- 10% of a givem measurement. The time for the digestion will be sufficient to go to at least substantial completion, so that at least substantially all ofthe protein will have been digested. Digests may be performed at a temperature that is compatible with the protease(s) employed, preferably from 20°C to 40°C, most preferably about 37°C. Where the digestion takes place in solution, the protease may be quenched by any convenient means, including heating or acidification ofthe sample. Alternatively, quenching can be achieved by sequestering the fragment conjugates with a receptor for the TAG bound to a surface, or by addition of a protease inhibitor (e.g., E64, DIFP, PMSF, etc.). Where the proteins are bound to a surface, the proteases may be washed away before the bound digested protein is released. [0141] Following protein digestion, peptides can be sequestered, e.g., by binding to receptors for the TAG of one or more NBAP-labeled peptides. Preferably, sequestration relies on receptors bound to a solid support that can be easily manipulated during wash steps. The support may be beads, including paramagnetic beads, prepared from various materials, such as Bioglas, polystyrene, polyacrylate, polymethylmethacrylate, polyethylene, polysaccharides, such as Agarose, cellulose, amylose, etc., polyurethane, and the like. Desirably, the support surface will not interfere with the binding of TAG to its cognate receptor, and the receptor may be linked to the support by a hydrophilic bridge that allows for the receptor to be removed from the surface. When beads are employed, the beads will generally have a cross-dimension in the range of about 5 to lOOμ. Instead of beads, one may use solid supports, such as slides, the walls of vessels, e.g. microtiter well walls, capillaries, etc. There is an extensive literature of receptor bound supports that is readily applicable to this invention, since the sequestering step is conventional. The sample is contacted with the support for sufficient time, usually about 5 to 60 min, to allow all ofthe conjugate to become bound to the surface. At this time, all ofthe non-specifically bound components from the sample may be washed away, greatly enriching the target proteins as compared to the rest of the sample.

[0142] Following separation by sequestration, NBAP-labeled peptides may then be released from the receptor. The particular method of release will depend upon the TAG- receptor pair. In some instances, one may use an analog of the TAG as a "releasing agent" to release the conjugate. This is illustrated by the use of deimino- or dethiobiotin as the TAG and biotin as the releasing agent. Where this is not convenient, as in the case of many fluorescent moieties as TAGs where there may not be a convenient analog, conditions such as high salt concentrations, chaeotropic agents (e.g., isothiocyanate or urea) low pH, detergents, organic solvents, etc., may be used to effect release. Once the conjugate has been released, dialysis, ion exchange resins, precipitation, or the like may be used to prepare the conjugate solution for the next stage.

[0143] Where the migration rates in various separation procedures provide the necessary identification ofthe peptide(s) generated and, therefore, the protein from which they are obtained, no further analysis may be required. However, where further identification is desired or the earlier results do not provide certainty as to the identification and amount of a particular component, an identification method using mass spectrometry (MS) can be employed. See, for example, WO 00/11208. The use of mass spectrometry will be described below. Such identification methods potentially provide greater information, but requires greater sample size in comparison to, for example, capillary electrohoresis, and has a lower throughput.

[0144] Chromatographic and/or electrophoretic separation methods as described herein may be used to simplify the mixtures introduced into the mass spectrometer, allowing for a more accurate analysis. For NBAP-labeled peptides, the use of fluorescent moieties as NBAP TAGs can permit the use of an online fluorescence detector to trigger ESI-MS data collection or fraction collection for subsequent analysis, e.g., providing sample on a MALDI plate. In this way, only fractions and bands that contain NBAP-labeled peptides will be selected for further processing, thereby avoiding using the MS with certain fractions. [0145] In particularly preferred embodiments, the identification methods described herein can be combined with one or more separation methods to develop a "separation profile" that can be used to identify peptides without the need for MS analysis. In these methods, a sample (e.g., material from a chromatography column) is divided into at least two portions; one portion is used for MS analysis, and the other portion(s) are used for one or more separation methods (e.g., a single CE run, or two or more CE runs using different separation conditions). The peptide identification obtained from the MS analysis can be assigned to the observed separation profile (e.g., the elution time ofthe peptide observed in the CE run(s)). Observation of this separation profile in subsequent samples can then be correlated to the peptide known to exhibit that separation profile. [0146] The identification methods described herein may also utilize NBAPs that differ isotopically in order to enhance the information obtained from MS procedures. For example, using automated multistage MS, the mass spectrometer may be operated in a dual mode in which it alternates in successive scans between measuring the relative quantities of peptides obtained from the prior fractionation and recording the sequence information ofthe peptides. Peptides may be quantified by measuring in the MS mode the relative signal intensities for pairs of peptide ions of identical sequence that are tagged with the isotopically light or heavy forms ofthe reagent, respectively, and which therefore differ in mass by the mass differential encoded with the NBAP. Peptide sequence information may be automatically generated by selecting peptide ions of a particular mass-to-charge (m/z) ratio for collision-induced dissociation (CID) in the mass spectrometer operating in the MS" mode. (Link, et al., (1997) Electrophoresis 18:1314-34; Gygi, et al., (1999) idid 20:310-9; and Gygi et al., (1999) Mol. Cell. Biol. 19: 1720-30). The resulting CID spectra may be then automatically correlated with sequence databases to identify the protein from which the sequenced peptide originated. Combination ofthe results generated by MS and MS" analyses of affinity tagged and differentially labeled peptide samples allows the determination ofthe relative quantities as well as the sequence identities ofthe components of protein mixtures. [0147] Protein identification by MS" may be accomplished by correlating the sequence contained in the CID mass spectrum with one or more sequence databases, e.g., using computer searching algorithms (Eng. et al. (1994) J. Am. Soc. Mass Spectrom. 5:976- 89; Mann, et al., (1994) Anal. Chem. 66:4390-99; Qin, et al., (1997) ibid 69:3995-4001; Clauser, et al., (1995) Proc. Natl. Acad. Sci. USA 92:5072-76). Pairs of identical peptides tagged with the light and heavy affinity tagged reagents, respectively (or in analysis of more than two samples, sets of identical tagged peptides in which each set member is differentially isotopically labeled) are chemically identical and therefore serve as mutual internal standards for accurate quantitation. The MS measurement readily differentiates between peptides originating from different samples, representing different cell states or other parameter, because ofthe difference between isotopically distinct reagents attached to the peptides. The ratios between the intensities ofthe differing weight components of these pairs or sets of peaks provide an accurate measure ofthe relative abundance ofthe peptides and the correlative proteins because the MS intensity response to a given peptide is independent of the isotopic composition ofthe reagents. The use of isotopically labeled internal standards is standard practice in quantitative mass spectrometry (De Leenheer, et al., (1992) Mass Spectrom. Rev. 11:249-307).

[0148] The following examples are offered by way of illustration and not by way of limitation.

[0149] In the following examples, Η-NMR spectra were recorded using deuterated chloroform (CDC1₃; δ = 7.26 ppm) as the solvent unless otherwise indicated. Preparative HPLC was carried out on a reverse phase Polaris Cι₈ column (5 μ column; 150 mm x 21 mm; Metachem/Ansys; Torrance, CA) using a binary system of water and acetonitrile with TFA as a modifier (water 0.1%, acetonitrile 0.1%). Analytical LC-MS was carried out on a Polaris C18 column (5 μ column; 50 mm x 4.6 mm; Metachem/Ansys; Torrance, CA) using a binary system of water and acetonitrile with TFA as a modifier (water 0.1%, acetonitrile 0.1%). All compounds were obtained from the Aldrich Chemical Company (Milwaukee, WI) unless indicated otherwise. Fmoc-4-(aminomethyl)benzoic acid was obtained from Advanced ChemTech (Louisville, Kentucky); the mixed 5- and 6-succinimidyl ester of tetramethylrhodamine was obtained from Molecular Probes (TAMRA-SE; Eugene, OR); and fluoroacetyl fluoride was obtained from ProChem, Inc (Rockford, IL).

[0150] Example 1 - Preparation of 4-Phenylamino quinazoline-related NBAPs

[0151] The following exemplary reaction scheme is depicted in Fig. 1.

[0152] (3-Chloro-4-fluoro-phenyl)-(7-fluoro-6-nitro-quinazolin-4-yl)-amine (1).

[0153] The title compound was prepared according to literature procedures. Smaill et al., J. Med. Chem. (2000) 43, 1380-1397.

[0154] 2-[2-(2-{2-[4-(3-Chloro-4-fluoro-phenylamino)-6-nitro-quinazolin-7-yloxy]- ethoxy} -ethoxy)-ethoxy] -ethanol (2).

[0155] In a 250 ml two neck round bottom flask fitted with a dean stark trap and a septum, 1 eq of pulverized KOH (119 mg, 2.98 mmol) was suspended in 80 ml of benzene and 20 ml of tetraethylene glycol. To this mixture was added a suspension of 18-crown-6 ether (79 mg, 0.3 mmol) in 20 ml of benzene. The mixture was stirred at room temperature.

Then a solution of 1 (1.0 g, 2.98 mmol) in benzene (20 ml) was added and the mixture was refluxed. The progress ofthe reaction was monitored by HPLC. The solvents were then removed under vacuum. The oily residue was dissolved in EtOAc (150 ml) and washed with water (100 ml). The organic phase was dried with Na₂S0₄ and the solvents removed under vacuum. The oily residue was used without further purification in the next step.

[0156] [7-(2- {2-[2-(2-Azido-ethoxy)-ethoxy]-ethoxy} -ethoxy)-6-nitro-quinazolin-4- yl]-(3-chloro-4-fluoro-phenyl)-amine (3).

[0157] 0.300 g of 2 (0.6 mmol) were dissolved in 2 ml of anhydrous dichloromethane and kept under a nitrogen atmosphere. To this light yellow colored solution 2 equivalents of triethylamine (167 μl, 1.2 mmol) where added and the reaction was cooled to 0 °C. Then 1.1 equivalents of methanesulfonyl choride (51 μl, 0.7 mmol) were added. The reaction was monitored by HPLC up to 85% of completion. The solvents were removed under vacuum to total dryness. The crude obtained was dissolved in 2 ml of DMF and 4 eq of NaN₃ (39 mg, 2.4 mmol) were added. The reaction mixture was heated at 80 °C under pressure overnight. The DMF was then removed under high vacuum and the oily residue was partitioned between EtOAc (5 ml) and water (5 ml). The organic layer was dried with Na₂S0₄ and purified by silica gel column chromatography to afford 3 in 75% yield (240 mg, 0.45 mmol). Η NMR (400 MHz, CDC1₃) δ 8.80 (s, IH), 8.73 (bs, IH), 8.69 (s, IH), 8.05-8.03 (m, IH), 7.71-7.67 (m, IH), 7.17 (dd, J = 8.8, 8.8 Hz, IH), 6.93 (s, IH), 4.05-4.02 (m, 2H), 3.87-3.81 (m, 6H), 3.75-3.72 (m, 2H), 3.63-3.61 (m, 2H), 3.49 (dd, J = 5.2, 5.2 Hz, 2H), 3.18 (dd, J = 5.0, 5.0 Hz, 2H).

[0158] 7-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy} -ethoxy)-N4-(3-chloro-4- fluoro-phenyl)-quinazoline-4, 6-diamine (4).

[0159] 0.1 g of 3 (0.2 mmol) were dissolved in 4 ml of acetic acid. Iron powder (45 mg, 0.8 mmol) was added in one portion and the mixture was refluxed at 80 °C for 4 hr. Once the reduction was completed, 2 ml of THF were added and the mixture was filtered through a small pad of celite. The acetic acid and THF were removed under high vacuum. The crude residue was purified by HPLC to afford 4 in 50% yield (48 mg, 0.1 mmol). Η NMR (400 MHz, CDC1₃) δ 10.24 (s), 8.23-8.14 (m), 8.01 (d, J = 8.0 Hz), 7.75-7.71 (m), 7.66 (s), 7.33 (s), 7.14 (dd, J = 8.0, 8.0 Hz), 4.24-4.20 (m), 3.86-3.78 (m), 3.71-3.62 (m), 3.17-2.70 (m). MS m/z: 479 (M). [0160] Compound s.

[0161] To a solution of TAMRA-SE (13 mg, 0.025 mmol) in DMF (0.7 ml) was added TEA (9 μL, 0.063 mmol). The purple solution was then cooled in an ice/water bath and a solution of 4 (10 mg, 0.021 mmol) in DMF (0.25 ml) was added dropwise. The reaction mixture was stirred for 5 h and then placed under high vacuum to remove the solvent. The residue was purified by HPLC to afford 5 in 55% yield as a purple solid. Η NMR (400 MHz, CDC1₃) δ 10.65 (bs), 8.97-8.93 (m), 8.67 (d, J = 6.4 Hz), 8.30 (d, J = 8.4 Hz), 8.02-7.98 (m), 7.71-7.63 (m), 7.59-7.47 (m), 7.14 (s), 7.05-6.90 (m), 4.34-4.30 (m), 3.92-3.88 (m), 3.67-2.90 (m). MS m/z: 892 (M+H). [0162] Compound 6.

[0163] To a solution of Et₃N (2 μl, 14.1 μmol) in dry THF (0.5 ml) under nitrogen cooled to 0 °C was added acrylic acid (0.3 μl, 4.7 μmol). Then 2,4,6-trichlorobenzoyl chloride (0.8 μl, 4.7 μmol) was added dropwise. The resulting mixture was stirred for 15 min. A solution of 5 (4.2 mg, 4.7 μmol) and DMAP (cat) in THF (0.5 ml) under nitrogen was added dropwise. The resulting purple solution was stined at room temperature overnight. The solvents were removed under high vacuum and the residue purify by HPLC to afford 6 as a purple solid in 21% yield (1.0 mg, 1 μmol). MS m/z: 968 (M+Na), 946 (M+H). [0164] Example 2 - Preparation of Adenine-Related Activity NBAPs

[0165] The following exemplary reaction scheme is depicted in Fig. 2.

[0166] {2-[2-(2- {4-[(9H-Fluoren-9-ylmethoxycarbonylamino)-methyl]- benzoylamino}-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester (7). [0167] To a solution of {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl} -carbamic acid tert- butyl ester (Zuckermanet al., J. Med. Chem. (1994) 37, 2678-2685) (1.4 g, 5.6 mmol), Fmoc- 4-(aminomethyl)benzoic acid (2.1 g, 5.6 mmol), HOBt (0.16 g, 1.2 mmol) in DMF (8 mL) was added ED AC (1.4 g, 7.3 mmol) and the resulting solution was stirred for 16 hours. Solvent was removed using rotary evaporation. The resulting residue was taken up in ethyl acetate (50 mL) and washed with 1 N HCI (2 x 25 mL), saturated NaHCO₃ (2 x 25 mL), and brine (1 x 25 mL). The organic layer was dried with Na₂SO₄, decanted, and concentrated using rotary evaporation. The resulting residue was purified on a column of silica gel using ethyl acetate to yield the title compound 7 as an oil (2.3 g, 68%). Η NMR (300 MHz): δ 1.43 (s, 9 H); 3.25 (quad, 2H, J= 5.4 Hz); 3.51 (t, 2H, J= 5.1 Hz); 3.60 (m, 4H); 3.65 (m, 4H); 4.71 (m, 2H); 4.21 (t, IH, J= 6.6 Hz); 4.38 (d, 2H, J= 5.1 Hz); 4.45 (d, 2H, J= 6.6 Hz); 5.02 (b, IH), 5.60 (b, IH), 6.80 (b, IH), 7.28 (d, 2H); 7.39 (t, 4H); 7.59 (d, 2H); 7.75 (d, 4H). ESMS: 504.3 (M - Boc + H⁺), 626.3 (M + Na⁺).

[0168] (2- {2-[2-(4-Aminomethyl-benzoylamino)-ethoxy]-ethoxy} -ethyl)-carbamic acid tert-butyl ester (8).

[0169] A solution of compound 7 (0.52 g, 0.86 mmol) in a 1:1 solution of dichloromethane/piperidine (2 mL) was allowed to stand at ambient temperature for 1 hour. The solvent was removed using rotary evaporation and remaining residual solvent was removed under high vacuum. The resulting residue was purified on a short column of silica gel using a gradient of 10-15% methanol/dichloromethane to yield the title compound as an oil (0.19 g, 56%). Η NMR (300 MHz): δ 1.42 (s, 9 H); 3.26 (quad, 2H); 3.53 (t, 2H); 3.63 (m, 4H); 3.66 (m, 4H); 3.96 (s, 2H); 5.04 (b, IH), 6.88 (b, IH), 7.28 (s, IH); 7.40 (d, 2H); 7.75 (d, 2H). ESMS: 382.3 (M + H⁺), 404.2 (M + Na⁺).

[0170] {2-[2-(2- {4-[(2-Chloro-9-cyclopentyl-9H-purin-6-ylamino)-methyl]- benzoylamino}-ethoxy)-ethoxy]-ethyl} -carbamic acid tert-butyl ester (9). [0171] A solution of 2,6-dichloro-9-cyclopentyl-9H-purine (0.13 g, 0.51 mmol), compound 8 (0.19 g, 0.50 mmol), and N,N-diisopropylethylamine (0.4 mL, 2.3 mmol) in EtOH (6 mL) were refluxed for 16 hours. Solvent was removed using rotary evaporation. The resulting residue was purified on a column of silica gel using a gradient of 2-4% methanol dichloromethane to yield the title compound as a tan solid (0.24 g, 80%). Η NMR (300 MHz): δ 1.41 (s, 9 H); 1.76 (m, 2 H); 1.88 (m, 4 H); 2.24 (m, 2 H); 3.27 (quad, 2H, J = 5.1 Hz); 3.52 (t, 2H, J= 5.1 Hz); 3.61 (m, 4H); 3.65 (m, 4H); 4.86 (m, 2H); 5.02 (b, IH); 6.79 (b, IH); 7.40 (d, 2H, J= 8.1 Hz); 7.65 (s, IH); 7.74 (d, 2H, J= 7.2 Hz). ESMS: 502.3 (M - Boc + H⁺), 602.3 (M + H⁺), 624.3 (M + Na⁺). [0172] (2- {2-[2-(4- { [2-(2-Amino-ethylamino)-9-cyclopentyl-9H-purin-6-ylamino]- methyl}-benzoylamino)-ethoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl ester (10). [0173] A solution of compound 9 (0.25 g, 0.42 mmol) in ethylenediamine (0.4 mL) was stirred at 100 °C in a sealed tube for 16 hours. The resulting residue was taken up in dichloromethane (10 mL) and washed with water (3 x 5 mL) followed by brine (2 x 5 mL). The aqueous layers were combined and extracted with dichloromethane (2 x 5 mL). The organic layers were combined, dried with Na₂S0₄, decanted, and concentrated using rotary evaporation. The resulting residue was dried under high vacuum to yield the title compound as a tan solid in quantitative yield. Η NMR (300 MHz): δ 1.42 (s, 9 H); 1.75 (m, 2 H); 1.95 (m, 4 H); 2.19 (m, 2 H); 2.90 (t, 2 H, J= 5.4 Hz); 3.28 (quad, 2H, J= 5.4 Hz); 3.48 (m, 2H); 3.53 (t, 2H, J= 5.1 Hz); 3.62 (m, 4H); 3.67 (m, 4H); 4.71 (m, 2H); 4.80 (b, IH); 5.12 (b, IH); 5.30 (b, IH); 6.59 (b, IH); 6.95 (b, IH); 7.38 (d, 2H, J= 8.4 Hz); 7.50 (s, IH); 7.72 (b d, 2H). ESMS: 626.4 (M + H⁺).

[0174] [2-(2- {2-[4-( {9-Cyclopentyl-2-[2-(4-fluorosulfonyl-benzoylamino)- ethylamino]-9H-purin-6-ylamino}-methyl)-benzoylamino]-ethoxy}-ethoxy)-ethyl]-carbamic acid tert-butyl ester (11).

[0175] To a stirred solution of compound 10 (19 mg, 0.030 mmol) and N,N- diisopropylethylamine (6 μL, 0.03 mmol) in dichloromethane (1 mL) at ambient temperature was added 4-(fluorosulfonyl)benzoyl chloride (FSB-Cl, 7 mg, 0.03 mmol). After 1 hour, the reaction was loaded onto a silica gel column and purified using a gradient of 0-5% methanol/dichloromethane to yield the title compound as a tan solid (23 mg, 93%). Η NMR (300 MHz): δ 1.42 (s, 9 H); 1.70 (m, 2 H); 1.86 (m, 4 H); 2.09 (m, 2 H); 3.26 (quad, 2H, J = 5.4 Hz); 3.52 (t, 2H, J= 4.8 Hz); 3.63 (m, 4H); 3.66 (m, 4H); 3.70 (m, 4H); 4.59 (m, IH); 4.73 (b, 2H); 5.00 (b, IH); 5.30 (b, IH); 6.36 (b, IH); 6.73 (b, IH); 7.34 (d, 2H, J= 7.5 Hz); 7.53 (s, IH); 7.70 (b d, 2H); 7.80 (b, IH), 7.85 (d, 2H, J= 8.4 Hz); 7.95 (d, 2H, J= 8.1 Hz). ESMS: 812.4 (M + H⁺), 834.4 (M + Na⁺).

[0176] 4- {2-[6-(4- {2-[2-(2-Amino-ethoxy)-ethoxy]-ethylcarbamoyl} -benzylamino)-

9-cyclopentyl-9H-purin-2-ylamino]- ethylcarbamoyl}-benzenesulfonyl fluoride (12). [0177] A solution of compound 11 (11 mg, 0.014 mmol) in 1:1 trifluoroacetic acid/dichloromethane (1 mL) was allowed to stand at ambient temperature for 40 minutes. Solvent was removed using rotary evaporation and the resulting residue was dried under high vacuum to yield a clear solid. This material was taken on directly to the next reaction without further purification. Analytical LC/MS analysis at 254 nm indicated the presence of a single major peak (96%) with the expected mass: ESMS: 356.6 (M + 2H⁺), 712.4 (M + it), 734.3 (M + Na⁺).

[0178] Compound 13.

[0179] To a stined solution of TAMRA-SE (7.5 mg, 0.014 mmol) and N, N- diisopropylethylamine (10 μL, 0.058 mmol) in dimethylformamide (DMF, 1 mL) cooled in an ice bath was added compound 12 (0.014 mmol) in DMF (1 mL) dropwise over 10 minutes. The reaction was allowed to warm to ambient temperature then stirred for an additional 2 hours. Solvent was removed using rotary evaporation. The resulting residue was purified via preparative HPLC using a flow rate of 30 mL/minute and a 90 minute gradient of 0.08% TFA/acetonitrile:0.1% TF A/water (2-98%) to yield the title compound (mixed isomer conjugate) as a bright purple solid (5.6 mg, 38%). Η NMR (300 MHz, CD₃CN): δ 1.71 (b, 2 H); 1.87 (m, 4 H); 2.16 (b, 2 H); 3.20 (m, 12H); 3.47 (quad, 2H, J= 5.3 Hz); 3.57 (b s, 4H); 3.61 (m, 4H); 3.64 (b s, 4H); 3.66 (m, 2H); 4.71 (b, 2H); 5.24 (b, IH); 6.7-8.6 (aromatic H, 20H). ESMS: 562.8 (M + 2H⁺), 1124.5 (M + H⁺), 1146.6 (M + Na⁺). [0180] (2-{2-[2-(4-{[2-(3-Amino-propylamino)-9-cyclopentyl-9H-purin-6-ylamino]- methyl}-benzoylamino)-ethoxy]-ethoxy}-ethyl)-carbamic acid tert-butyl ester (14). [0181] A solution of compound 9 (0.1 g, 0.16 mmol) and 1 ,3-diaminopropane ( 15 mg, 0.20 mmol) in THF (0.5 mL) was stined at 100 °C in a sealed tube for 3 days. The resulting residue was loaded onto a silica gel column and purified using a gradient of 0-20% methanol/dichloromethane to yield the title compound as a tan solid (31 mg, 29%). Η NMR (400 MHz): δ 1.43 (s, 9 H); 1.71 (m, 2 H); 1.76 (m, 4 H); 1.94 (m, 2 H); 2.22 (m, 2 H, J= 7.6 Hz); 2.77 (b, 2H); 3.29 (quad, 2H, J = 4.8 Hz); 3.49 (quad, 2H, J= 6.4 Hz); 3.54 (t, 2H, J = 5.2 Hz); 3.63 (m, 4H); 3.67 (m, 4H); 4.72 (m, IH); 4.82 (b, 2H); 4.92 (b, IH); 5.05 (b, IH); 6.10 (b, IH); 6.92 (b, IH); 7.41 (d, 2H, J= 7.6 Hz); 7.49 (s, IH); 7.74 (b d, 2H). ESMS: 640.4 (M + H⁺), 662.3 (M + Na⁺).

[0182] [2-(2-{2-[4-({2-[3-(2-Chloro-acetylamino)-propylamino]-9-cyclopentyl-9 H- purin-6-ylamino} -methyl)-benzoylamino]-ethoxy} -ethoxy)-ethyl]-carbamic acid tert-butyl ester (15).

[0183] To a stined solution of compound 14 (19 mg, 0.030 mmol) and N,N- diisopropylethylamine (11 μL, 0.06 mmol) in dichloromethane (1 mL) at ambient temperature was added chloroacetyl chloride (4 mg, 0.036 mmol). After 1 hour, the reaction was loaded onto a silica gel column and purified using 10% methanol/dichloromethane to yield the title compound as a tan solid (5 mg, 23%). Η NMR (400 MHz): δ 1.42 (s, 9 H); 1.78 (b, 6 H); 1.92 (m, 2 H); 2.21 (b m, 2 H); 3.29 (b, 2H); 3.38 (quad, 2H, J= 6.4 Hz); 3.48 (quad, 2H, J= 6.4 Hz); 3.53 (t, 2H, J= 5.2 Hz); 3.63 (m, 4H); 3.67 (m, 4H); 4.01 (m, IH); 4.76 (b m, IH); 4.82 (b, 2H); 5.01 (b, IH); 6.72 (b, IH); 6.89 (b, IH); 7.42 (d, 2H, J= 7.6 Hz); 7.52 (s, 1H); 7.73 (b d, 2H). ESMS: 716.3 (M + H⁺).

[0184] N- {2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl} -4-( {2-[3-(2-chloro-acetylamino) propylamino]-9-cyclopentyl-9H-purin-6-ylamino}-methyl)-benzamide (16). [0185] A solution of compound 15 (5 mg, 0.007 mmol) in 1 :1 trifluoroacetic acid/dichloromethane (1 mL) was stirred at ambient temperature for 20 minutes. Solvent was removed using rotary evaporation and the resulting residue was dried under high vacuum to yield a clear solid. This material was taken on directly to the next reaction without further purification. Analytical LC/MS analysis at 254 nm indicated the presence of a single major peak (>95%) with the expected mass: ESMS: 308.7 (M + 2H⁺), 616.2 (M + H⁺), 638.2 (M + Na⁺).

[0186] Compound 17.

[0187] To a stined solution of TAMRA-SE (3.7 mg, 0.007 mmol) and N, N- diisopropylethylamine (2.4 μL, 0.014 mmol) in DMSO (0.4 mL) was added compound 16 (0.007 mmol) in DMSO (0.4 mL) dropwise over 10 minutes. The reaction was stined for 2 hours. The reaction was purified via preparative HPLC using a flow rate of 30 mL/minute and a 90 minute gradient of 0.1% TFA/acetonitrile:0.1% TF A/water (2-98%) to yield the title compound (mixed isomer conjugate) as a bright purple solid (1 mg, 14%). Η NMR (400 MHz, CD₃CN): δ 1.77 (b, 2 H); 1.90 (m, 4 H); 2.11 (m, 2 H); 3.24 (m, 12H); 3.35 (m, 2H); 3.49 (m, 2H); 3.61 (b s, 4H); 3.65 (m, 8H); 3.69 (t, 2H); 4.72 (b, 2H); 6.8-8.6 (aromatic H, 20H). ESMS: 514.7 (M + 2H⁺).

[0188] [2-(2- {2-[4-( {9-Cyclopentyl-2-[3-(2-fluoro-acetylamino)-propylamino]-9H- purin-6-ylamino}-methyl)-benzoylamino]-ethoxy}-ethoxy)-ethyl]-carbamic acid tert-butyl ester (18).

[0189] To a stined solution of compound 14 (30 mg, 0.048 mmol) and N,N- diisopropylethylamine (17 μL, 0.096 mmol) in dichloromethane (1 mL) at ambient temperature was added fluoroacetyl chloride (5.5 mg, 0.057 mmol). After 1 hour, the reaction was loaded onto a silica gel column and purified using a gradient of 0-10% methanol/dichloromethane to yield the title compound as a tan solid (8 mg, 24%). Η NMR (400 MHz): δ 1.41 (s, 9 H); 1.78 (b, 6 H); 1.92 (m, 2 H); 2.20 (b m, 2 H); 3.11 (m, 2H); 3.29 (m, 2H); 3.50 (m, 2H); 3.55 (t, 2H); 3.64 (m, 4H); 3.68 (m, 4H); 4.73 (s, IH); 4.85 (s, IH); 5.03 (b, IH); 5.01 (b, IH); 6.71 (b, IH); 7.03 (b, IH); 7.43 (d, 2H); 7.53 (s, IH); 7.74 (b d,

2H). ESMS: 700.4 (M + H⁺), 722.3 (M + Na⁺).

[0190] N-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-4-({9-cyclopentyl-2-[3-(2-fluoro- acetylamino)-propylamino]-9H-purin-6-ylamino}-methyl)-benzamide (19).

[0191] A solution of compound 18 (8 mg, 0.011 mmol) in 1 :1 trifluoroacetic acid/dichloromethane (1 mL) was stined at ambient temperature for 30 minutes. Solvent was removed using rotary evaporation and the resulting residue was dried under high vacuum to yield a clear solid. This material was taken on directly to the next reaction without further purification. Analytical LC/MS analysis at 254 nm indicated the presence of a single major peak (>95%) with the expected mass: ESMS: 300.7 (M + 2H⁺), 600.3 (M + H⁺), 622.3 (M +

Na⁺).

[0192] Compound 20.

[0193] To a stined solution of TAMRA-SE (6 mg, 0.011 mmol) and N, N- diisopropylethylamine (8 μL, 0.046 mmol) in dimethylformamide (DMF, 0.3 mL) was added compound 19 (0.011 mmol) in DMF (0.5 mL) dropwise over 10 minutes. The reaction was allowed to warm to ambient temperature then stined for 6 hours. Solvent was removed using rotary evaporation. The resulting residue was purified via preparative HPLC using a flow rate of 30 mL/minute and a 60 minute gradient of 0.1% TFA/acetonitrile:0.1% TF A/water (2-

98%) to yield the title compound (mixed isomer conjugate) as a bright purple solid (4 mg,

38%). Η NMR (400 MHz, CD₃CN): δ 1.79 (m, 2 H); 1.91 (m, 4 H); 2.13 (b, 2 H); 3.24 (m,

12H); 3.40 (m, 2H), 3.50 (quad, 2H, J= 5.2 Hz); 3.60 (b s, 4H); 3.63 (m, 4H); 3.66 (b s, 4H);

3.70 (m, 2H); 4.66 (b, IH); 4.77 (b, 2H); 5.26 (b, IH); 6.7-8.6 (aromatic H, 20H). ESMS:

506.8 (M + 2H⁺), 1012.5 (M + H⁺).

[0194] Example 3 - Preparation of Staurosporine-related NBAPs

[0195] The following exemplary reaction scheme is depicted in Fig. 3. [0196] 1 -[6-(Tetrahydro-pyran-2-yloxy)-hexyl]- 1 H-indole (22).

[0197] To a solution of indole 21 ( 120 mg, 1.02 mmol) in DMF (9 mL) cooled to 0

°C is added NaH (62 mg, 1.55 mmol, 60% mineral oil dispersion). The reaction mixture was allowed to warm to room temperature and stir for 45 min. It was then cooled with an ice/water bath and a solution of methanesulfonic acid 6-(tetrahydro-pyran-2-yloxy)-hexyl ester (302 mg, 1.08 mmol) in DMF (2 mL) was added dropwise. The reaction was stined overnight. The reaction was quenched by addition of saturated NEL-Cl (10 mL) and extracted with CH₂C1₂ (3 x 5 mL). The organic layers were dried with Na₂SO and filtered. The solvent was removed in vacuo and the residue purified by column chromatography to afford 22 in 98%> yield (303 mg, 1.0 mmol).

[0198] Oxo-{l-[6-(tetrahydro-pyran-2-yloxy)-hexyl]-lH-indol-3-yl}-acetic acid methyl ester (23).

[0199] To a solution of 22 (303 mg, 1.0 mmol) in Et₂O (9 mL) cooled in an ice/water bath was added dropwise oxalyl chloride (99 μL, 1.1 mmol). The resultant mixture was stirred for 30 min and then cooled to -65 °C. A 25 wt % solution of sodium methoxide in MeOH (0.52 mL, 2.2 mmol) was added dropwise. The reaction was allowed to warm to room temperature and quenched by addition of water (5 mL). The organic layer was dried with Na₂SO₄ and filtered. The solvent was removed in vacuo and the residue purified by column chromatography to afford 23 in 93% yield (362 mg, 0.93 mmol). Η NMR (400 MHz, CDC1₃) δ 8.46-8.43 (m, IH), 8.38 (s, IH), 7.40-7.33 (m, 3H), 4.55-4.53 (m, IH), 4.18 (dd, J = 7.4, 7.4 Hz, 2H), 3.95 (s, 3H), 3.87-3.81 (m, IH), 3.72 (ddd, J= 9.6, 6.4, 6.4 Hz, IH), 3.51- 3.45 (m, IH), 3.37 (ddd, J= 9.6, 6.4, 6.4 Hz, IH), 1.96-1.89 (m, 2H), 1.83-1.75 (m, IH), 1.73-1.65 (m, IH), 1.61-1.48 (m, 6H), 1.45-1.37 (m, 4H). [0200] 2-(l -Methyl- lH-indol-3-yl)-acetamide (26). [0201 ] The title compound was prepared according to literature procedures (Faul, M.

M.; Winneroski, L. L.; Krumrich, C. A. J. Org. Chem. (1998) 63, 6053-6058) [0202] 3-[ 1 -(6-Hydroxy-hexyl)- 1 H-indol-3-yl]-4-( 1 -methyl- 1 H-indol-3-yl)-pyrrole-

2,5-dione (27).

[0203] To a solution of 23 (93 mg, 0.24 mmol) and 26 (45 mg, 0.24 mmol) in THF (3 mL) at 0 °C was added 1.0 M KO'Bu (0.96 mL, 0.96 mmol) in THF. The reaction was allowed to come to room temperature and stined overnight. The reaction was quenched with concentrated HCI (37%, 0.3 mL) and diluted with EtOAc (3 mL). The organic layers were washed with water (3 mL), dried with Na₂SO and filtered. Purification was achieved by column chromatography to provide 27 in 66% yield as a red solid (70.1 mg, 0.16 mmol) and the conesponding tetrahydropyranyl protected alcohol 28 in 29% yield (45.0 mg). Η NMR (400 MHz, CDC1₃) δ 7.72 (s, IH), 7.63 (s, IH), 7.37 (bs, IH), 7.30-7.26 (m, 2H), 7.11-7.06 (m, 2H), 7.02 (d, J= 8.0 Hz, IH), 6.82 (d, J= 8.0 Hz, IH), 6.78-6.74 (m, IH), 6.70-6.66 (m, IH), 4.14 (dd, J= 7.2, 7.2 Hz, 2H), 3.86 (s, 3H), 3.63 (dd, J= 6.4, 6.4 Hz, 2H), 1.88-1.80 (m, 2H), 1.59-1.52 (m, 2H), 1.43-1.30 (m, 4H). MS m/z: 464 (M+23), 442 (M+H). [0204] 6-tert-Butoxycarbonylamino-2-(9H-fluoren-9-ylmethoxycarbonylamino)- hexanoic acid-6-{3-[4-(l-methyl-lH-indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pyπol-3-yl]- indol-1-yl}- hexyl ester (29).

[0205] To a solution of 27 (70 mg, 0.16 mmol) and Fmoc-Lys(Boc)-OH (223 mg,

0.48 mmol) in CH₂C1₂ (2 mL) under N₂ at room temperature was added DMAP (19.4 mg, 0.16 mmol). The reaction was then cooled in an ice/water bath and N,N'- dicyclohexylcarbodiimide (0.48 mL, 0.48 mmol) was added. After stirring for 45 min, the reaction mixture was diluted with CH₂C1₂ (2 mL) and washed with water (3 x 2 mL). The organic layer was dried over anhydrous Na₂SO₄ and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography to afford 29 in 70% yield as a red solid (134 mg, 0.15 mmol). Η NMR (400 MHz, CDC1₃) δ 7.75 (d, J= 7.6 Hz), 7.71 (s), 7.62-7.58 (m), 7.53 (bs), 7.42-7.35 (m), 7.32-7.25 (m), 7.08 (dd, J= 7.6, 7.6 Hz), 7.02- 6.99 (m), 6.82 (d, J= 8.0 Hz), 6.77-6.73 (m), 6.69-6.65 (m), 5.43-5.40 (m), 4.63-4.58 (m), 4.41-4.32 (m), 4.22 (dd, J= 6.8, 6.8 Hz), 4.15-4.10 (m), 4.08-4.03 (m), 3.84 (s), 3.52-3.45 (m), 3.13-3.07 (m), 1.96-1.90 (m), 1.87-1.77 (m),1.73-1.65 (m), 1.64-1.57 (m), 1.44 (s), 1.40- 1.23 (m), 1.20-1.04 (m). MS m/z: 914 (M+Na), 792 (M-Boc).

[0206] 2-Amino-6-tert-butoxycarbonylamino-hexanoic acid 6-{3-[4-(l-methyl-lH- indol-3-yl)-2,5-dioxo-2,5-dihydro- 1 H-pynol-3-yl]-indol- 1 -yl} -hexyl ester (30). [0207] To a solution of 29 (34 mg, 0.038 mmol) in CH₂C1₂ (1 mL) cooled in an ice/water bath was added Et₂NH (1 mL). The reaction was allowed to warm to room temperature and stined for 4h. The solvents were removed in vacuo and the red residue was purified by column chromatography to afford 30 in 69% yield (17.6 mg, 0.026 mmol). MS m/z: 692 (M+Na), 670 (M+H).

[0208] 6-tert-Butoxycarbonylamino-2-(2-chloro-acetylamino)-hexanoic acid 6-{3-[4-

(l-methyl-lH-indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl]-indol-l-yl}-hexyl ester (31).

[0209] To a solution of 30 ( 17 mg, 0.025 mmol) in DMF (2 mL) was added TEA

(10.6 μL, 0.075 mmol). The reaction was then cooled in an ice/water bath and chloroacetyl chloride (2.5 μL, 0.03 mmol) was added. The reaction was stirred at 0 °C for 4 h. The reaction mixture was diluted with CH₂C1₂ (2 mL) and washed with water (2 mL). The organic layer was dried over anhydrous Na₂S0₄ and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography to afford 31 in 70% yield as a red solid (13.3 mg, 0.018 mmol). Η NMR (400 MHz, CDC1₃) δ 7.73 (s, IH), 7.64 (s, IH), 7.46 (bs, IH), 7.33-7.22 (m, 2H), 7.10-7.06 (m, 2H), 7.01-6.98 (m, IH), 6.84-6.81 (m, IH), 6.78- 6.72 (m, IH), 6.69-6.65 (m, IH), 4.60-4.57 (m, 2H), 4.20-4.12 (m, 3H), 4.06 (s, 2H), 3.86 (s, 3H), 3.43-3.35 (m, 3H), 3.12-3.08 (m, 2H), 1.90-1.21 (m, 14H). MS m/z: 768 (M+Na), 646

(M-Boc).

[0210] 6-Amino-2-(2-chloro-acetylamino)-hexanoic acid 6- {3-[4-(l -methyl- 1H- indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl]-indol-l-yl}-hexyl ester (32).

[0211] Compound 31 (6.8 mg, 0.009 mmol) was dissolved in neat trifluoroacetic acid

(0.5 mL) and stirred at room temperature for 10 seconds. The violet-brown solution was then placed under high vacuum to remove the solvent and use without further purification to yield

32. MS m/z: 668 (M+Na), 646 (M+H).

[0212] Compound 33.

[0213] To a solution of TAMRA-SE (5.8 mg, 0.011 mmol) in DMF (0.7 mL) was added TEA (2.5 μL, 0.018 mmol). The purple solution was then cooled in an ice/water bath and a solution of crude 32 in DMF (0.25 mL) was added dropwise. The reaction mixture was stined for 1.5 h and then placed under high vacuum to remove the solvent. The residue was purified by HPLC to afford 33 in 66% yield from 31 as a purple solid. Η NMR (400 MHz,

CDC1₃) δ 8.66 (s), 8.40-8.33 (bs), 8.27-8.23 (m), 8.18-8.15 (m), 8.07-8.04 (m), 7.87-7.80 (m),

7.68-7.57 (m), 7.42-7.39 (m), 7.29-7.26 (m), 7.17-7.13 (m), 7.10-7.04 (m), 7.01-6.97 (m),

6.85-6.65 (m), 4.54-4.41 (m), 4.16-4.05 (m), 3.83 (s), 3.82 (s), 3.75-3.72 (m), 3.55-3.53 (m),

3.49 (dd, J= 6.8, 6.8 Hz), 3.24 (s), 3.19 (s), 1.83-1.51 (m), 1.48-1.20 (m). MS m/z 1080

(M+Na), 1058 (M+H).

[0214] 2-Acryloylamino-6-tert-butoxycarbonylamino-hexanoic acid 6-{3-[4-(l- methyl-lH-indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pynol-3-yl]-indol-l-yl}-hexyl ester (34).

[0215] The procedure described above to synthesize 31 was followed using 30 (15 mg, 0.022 mmol) and acryloyl chloride (2.7 μL, 0.034 mmol) instead of chloroacetyl chloride. The residue was purified by flash column chromatography to afford 34 in 53% yield as a red solid (8.6 mg, 0.012 mmol). Η NMR (400 MHz, CDC1₃) δ 7.73 (s, IH), 7.68 (bs, IH), 7.64 (s, IH), 7.30-7.262 (m, 2H), 7.110-7.06 (m, 2H), 7.01 (d, J= 8.0 Hz, IH), 6.82 (d,

J= 8.0 Hz, IH), 6.77-6.73 (m, IH), 6.69-6.65 (m, IH), 6.33 (m, IH), 6.32 (dd, J= 17.0, 1.4

Hz, lH), 6.14 (dd, J= 17.0, 10.2 Hz, IH), 5.64 (dd, J= 10.2, 1.4 Hz, IH), 4.67-4.63 (m, 2H),

4.16-4.04 (m, 4H), 3.85 (s, 3H), 3.12-3.07 (m, 2H), 1.92-1.80 (m, 3H), 1.75-1.69 (m, IH),

1.66-1.57 (m, 4H), 1.44 (s, 9H), 1.54-1.30 (m, 6H). MS m/z: 746 (M+Na), 624 (M-Boc).

[0216] 2-Acryloylamino-6-amino-hexanoic acid 6-{3-[4-(l-methyl-lH-indol-3-yl)-

2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl]-indol-l-yl}-hexyl ester (35).

[0217] The procedure described above to synthesize 32 was followed using 34 (8.6 mg, 0.012 mmol). The residue was use without further purification in the next step. MS m/z:

646 (M+23), 624 (M+H).

[0218] Compound 36.

[0219] The procedure described above to synthesize 33 was followed using crude 35.

The residue was purified by HPLC to afford 36 in 41% yield (5.1 mg, 0.005 mmol) from 34 as a purple solid. Η NMR (400 MHz, CDC1₃) δ 8.66 (s), 8.43-8.37 (bs), 8.32-8.28 (s), 8.25

(dd, J= 7.6 Hz), 8.17 (dd, J= 8.0 Hz), 8.11-8.06 (m), 7.99-7.96 (m), 7.80 (bs), 7.64-7.57 (m),

7.24-7.26 (m), 7.19-7.12 (m), 7.11-7.04 (m), 7.00-6.95 (m), 6.86-6.63 (m), 6.28-6.24 (m),

6.18-6.16 (m), 5.57-5.54 (m), 5.46-5.43 (m), 4.57-4.47 (m), 4.13-4.04 (m), 3.82 (s), 3.81 (s),

3.61-3.35 (m), 3.23 (s), 3.18 (s), 1.89-1.71 (m), 1.70-1.44 (m), 1.42-1.23 (m). MS m/z: 1058

(M+Na), 1036 (M+H).

[0220] 6-tert-Butoxycarbonylamino-2-(4-fluorosulfonyl-benzoylamino)-hexanoic acid 6-{3-[4-(l-methyl-lH-indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl]-indol-l-yl}- hexyl ester (37).

[0221] The procedure described above to synthesize 31 was followed using 30 (14.3 mg, 0.021 mmol) and 4-fluorosulfonylbenzoyl chloride (6.3 mg, 0.025 mmol) instead of chloroacetyl chloride. The residue was purified by HPLC to afford 37 in 28% yield as a red solid (5.1 mg, 0.006 mmol). Η NMR (400 MHz, CDC1₃) δ 8.05 (m), 7.73 (s), 7.64 (s), 7.52 (bs), 7.29-7.26 (m), 7.09-7.05 (m), 6.99-6.97 (m), 6.81 (d, J= 8.0 Hz), 6.76-6.72 (m), 6.69- 6.64 (m), 4.74-4.63 (m), 4.19-4.10 (m), 3.86 (s), 3.68-3.40 (bs), 3.15-3.08 (m), 2.10-1.81 (m), 1.69-1.63 (m), 1.55-1.35 (m), 1.36 (s). MS m/z: 878 (M+Na), 756 (M-Boc). [0222] 6-Amino-2-(4-fluorosulfonyl-benzoylamino)-hexanoic acid 6- {3 -[4-( 1 - methyl-lH-indol-3-yl)-2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl]-indol-l-yl}-hexyl ester (38). [0223] The procedure described above to synthesize 32 was followed using 37 (5.1 mg, 0.006 mmol). The residue was use without further purification in the next step. MS m/z 778 (M+Na), 756 (M+H). [0224] Compound 39.

[0225] The procedure described above to synthesize 33 was followed using crude 38.

The residue was purified by HPLC to afford 39 in 40% yield (2.7 mg, 0.003 mmol) from 37 as a purple solid. Η NMR (400 MHz, CDC1₃) δ 8.73 (s), 8.21-8.17 (m), 7.96-7.94 (m), 7.64 (s), 7.28-7.26 (m), 7.12-7.04 (m), 6.95-6.91 (m), 6.88-6.81 (m), 6.74-6.69 (m), 4.70-4.61 (m), 4.18-4.12 (m), 4.10-4.06 (m), 3.82 (s), 3.56-3.52 (m), 3.27 (s), 2.11-1.98 (m), 1.84-1.72 (m), 1.70-1.55 (m), 1.41-1.27 (m). MS m/z: 1168 (M+H). [0226] 4'-N-[6-tert-Butoxycarbonylamino-2-(9H-fluoren-9- ylmethoxycarbonylamino)-hexanoyl]-staurosporine (41).

[0227] To a solution of staurosporine 40 (1.0 mg, 2.1 μmol) and Fmoc-Lys(Boc)-OH

(3.0 mg, 6.4 μmol) in DMF (0.7 mL) under N₂ at room temperature was added 1- hydroxybenzotriazole (0.9 mg, 6.4 μmol). The reaction was then cooled in an ice/water bath and N,N'-dicyclohexylcarbodiimide (6.5 μL, 6.4 μmol) was added. The reaction was stined for 4 days at room temperature. It was then placed under high vacuum to remove the solvent. The residue was taken up in CH₂C1₂ (1 mL) and water (1 mL). The organic layer was separated, dried over anhydrous Na₂SO and the solvents were removed under reduced pressure. The residue was purified by HPLC to afford 41 in 76% yield (1.5 mg, 1.64 μmol). Η NMR (400 MHz, CDC1₃) δ 9.31 (d, J= 6.8 Hz), 7.93 (d, J= 7.2 Hz), 7.79 (d, J= 7.6 Hz), 7.61 (d, J= 7.6 Hz), 7.44-7.31 (m), 6.81-6.77 (m), 5.65 (d, J= 8.4 Hz), 5.14-5.07 (m), 4.71- 4.56 ( ), 4.42-4.31 (m), 4.05-3.97 (m), 3.21-3.11 (m), 2.98 (s), 2.66-2.62 (m), 2.49 (s), 2.35- 2.31 (m), 2.13-1.85 (m), 1.69-1.15 (m). MS m/z: 939 (M+Na), 917 (M+H), 817 (M-Boc). [0228] 4'-N-[2-Amino-6-tert-butoxycarbonylamino-hexanoyl]-staurosporine (42).

[0229] The procedure described above to synthesize 30 was followed using 41 (1.5 mg, 1.64 μmol). The residue was purified by HPLC to afford 42 in 79% yield (0.9 mg, 1.3 μmol). MS m/z: 111 (M+Na), 695 (M+H).

[0230] 4'-N-[6-tert-Butoxycarbonylamino-2-(4-fluorosulfonyl-benzoylamino)- hexanoyl] -staurosporine (43).

[0231 ] The procedure described above to synthesize 31 was followed using 42 (0.9 mg, 1.3 μmol) and 4-fluorosulfonylbenzoyl chloride (0.4 mg, 1.6 μmol) instead of chloroacetyl chloride. The residue was purified by HPLC to afford 43 in 73% yield (0.8 mg,

0.95 μmol). MS m/z: 903 (M+Na), 881 (M+H), 781 (M-Boc).

[0232] 4'-N-[6-Amino-2-(4-fluorosulfonyl-benzoylamino)-hexanoyl]-staurosporine

(44).

[0233] To a solution of 43 (0.8 mg, 0.95 μmol) in CH₂C1₂ (0.8 mL) cooled to 0 °C was added TFA (0.15 mL). The reaction was allowed to warm to room temperature. Once the reaction was finished, the solvents were removed by passing a stream of N₂ over the solution.

The residue was used without further purification in the next step. MS m z: 781 (M+H).

[0234] Compound 45.

[0235] The procedure described above to synthesize 33 was followed using crude 44.

The residue was purified by HPLC to afford 45 in 70% yield (0.8 mg, 0.7 μmol) from 43 as a purple solid. Η NMR (400 MHz, acetone-d₆) δ 9.44-9.40 (m), 8.67-8.60 (m), 8.49-8.40 (m), 8.37-8.12 (m), 8.11-8.05 (m), 7.97-7.91 (m), 7.86 (bs), 7.67-7.61 (m), 7.59-7.41 (m), 7.39-

7.17 (m), 6.85-6.68 (m), 6.62-6.50 (m), 6.46-6.38 (m), 5.36-5.32 (m), 5.13-4.95 (m), 4.31-

4.25 (m), 3.78-3.74 (m), 3.30-2.00 (m), 1.81-1.52 (m), 1.35-0.90 (m). MS m/z: 1193 (M+H).

[0236] The structures ofthe following Compounds 46-48 are shown in Fig. 4.

[0237] Compound 46

[0238] The overall synthetic sequence described above to synthesize 45 was followed using Nβ-Fmoc-Nω-Boc-L-β-homolysine instead of Fmoc-Lys(Boc)-OH and using chloroacetyl chloride instead of 4-fluorosulfonyl chloride. Compound 46 (AX7569): MS m/z: 11 19 (M+Na), 1097 (M+H).

[0239] Compound 47

[0240] The overall synthetic sequence described above to synthesize 45 was followed using (S)-N-4-Fmoc-N-8-Boc-diaminooctanoic acid instead of Fmoc-Lys(Boc)-OH and using chloroacetyl chloride instead of 4-fluorosulfonyl chloride. Compound 47 (AX7570): MS m/z: 1133 (M+Na), 1111 (M+H).

[0241] Compound 48

[0242] The overall synthetic sequence described above to synthesize 45 was followed using 3-(N-6-Boc-N-2-Fmoc-diaminohexanoyl)-3-aminopropionic acid instead of Fmoc-

Lys(Boc)-OH and using chloroacetyl chloride instead of 4-fluorosulfonyl chloride.

Compound 48 (AX7619): MS m/z: 1154 (M+H).

[0243] Example 4 - Preparation of 4-Phenylamino quiazoline-related NBAPs

[0244] The following exemplary reaction scheme is depicted in Fig. 5.

[0245] Materials and Methods. All solvents and reagents were obtained from the

Aldrich Chemical Company (Milwaukee, WI) unless otherwise indicated; the mixed 5- and

6-succinimidyl ester of tetramethylrhodamine was obtained from Molecular Probes

(TAMRA-SE; Eugene, OR). Proton nuclear magnetic resonance (Η NMR) spectra were recorded on a Bruker 400 MHz NMR spectrometer using the residual peaks in the deuterated solvents as internal standards. Samples were dissolved in deuterated dimethylsulfoxide (de- DMSO) unless otherwise indicated. Preparative HPLC was carried out on reverse phase Polaris Cι₈ or Cβ columns (5 μ column; 150 mm x 21 mm; Varian; Tonance, CA) using a binary system of water and acetonitrile with 0.1% trifluoroacetic acid as a modifier. Analytical LC/MS samples were carried out on a Polaris C-iβ column (5 μ column; 50 x 4.6 mm; Varian; Tonance, CA) using a binary system of water and acetonitrile with 0.1% trifluoroacetic acid as a modifier. HPLC purity was determined using the LC/MS at 254 nm. [0246] N-(7-Benzyloxy-6-methyoxy-quinazolin-4-yl)-benzene-l,3-diamine (50). A solution of 7-benzyloxy-4-chloro-6-methoxy-quinazoline (Julia et al. Bull. Soc. Chim. 1965, 5, 1417; Hennequin et al. J. Med. Chem. 1999, 42, 5369) (compound 49, 350 mg, 1.16 mmol) and 1,3-phenylenediamine (377 mg, 3.49 mmol) in iPrOH (10.0 mL) was refluxed in a sealed tube at 95 °C for 45 minutes. The solution was allowed to cool to room temperature and the precipitate was collected using vacuum filtration. The filtered solid was dried under high vacuum, yielding compound 50 as a dark green-blue solid (410 mg, 95% yield). Η- NMR δ: 10.70 (bs, IH), 8.69 (s, IH), 8.11 (s, IH), 7.51 (d, 2H, J= 4.0 Hz), 7.41 (m, 5H), 7.11 (t, IH, J= 8.0 Hz), 6.91 (s, IH), 6.84 (d, IH, J= 8.0 Hz), 6.54 (d, 1H, J= 8.0 Hz), 6.41 (s, IH), 5.31 (s, 2H), 3.97 (s, 3H). ESMS: 373.1 [M + H]⁺.

[0247] 4-(3-Amino-phenylamino)-6-methoxy-quinazolin-7-ol (51). A solution of compound 50 (400 mg, 1.07 mmol) in trifluoroacetic acid (10.0 mL) was refluxed in a sealed tube for 2 hours at 90 °C. The acid was removed under vacuum and the solid was washed with ether, yielding a black solid. The solid was dissolved in a minimal amount of DMSO and purified by reverse phase chromatography on a C₈ column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 51 as a tan solid (40 mg, 10% yield). Η-NMR δ: 10.83 (s, IH), 8.74 (s, IH), 8.03 (s, IH), 7.16 (m, 2H), 6.86 (bs, 2H), 6.62 (m, IH), 3.96 (s, 3H). ESMS: 283.1 [M + H]⁺.

[0248] 4-(3-Azido-phenylamino)-6-methoxy-quinazolin-7-ol (52). To a solution of compound 51 (141 mg, 0.50 mmol) in 80% acetic acid (5.0 mL) cooled in an ice bath was added sodium nitrite (38 mg, 0.55 mmol). After 5 minutes, sodium azide (36 mg, 0.55 mmol) was added and the reaction was stined for 2 hours. The solvent was removed under vacuum and the resulting solid was dissolved in a minimal amount of DMSO and purified on a Cι₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 52 as a pale yellow powder (31 mg, 20% yield). Η-NMR δ: 10.84 (bs, IH), 8.77 (s, IH), 8.01 (s, IH), 7.49 (m, 3H), 7.18 (s, IH), 7.065 (m, IH). ESMS: 281.1 [M + H - 28]⁺, 309.1 [M + H]⁺. [0249] Benzenesulfonic acid 2-{2-[2-N,N-bis(2-tert-butoxycarbonyl)amino- ethoxy)-ethoxy]-ethoxy}-ethyl ester (53). A solution of di-tert-butyl iminodicarboxylate (9.51 g, 43.8 mmol), di(ethylene glycol) di-^-tosylate (20.0 g, 39.8 mmol), and K₂CO₃ (5.50 g, 39.8 mmol) in anhydrous DMF (10.0 mL) was heated at 60 °C. After 2 hours, EtOAc (50 mL) and H₂O (30 mL) were added. The organic layer was washed with 1 N HCI (2 x 20 mL), H₂O (2 x 15 mL), and brine (2 x 15 mL). The organic layer was dried over Mg₂S0 and concentrated to dryness. The solid was redissolved in a minimal amount of CH₂Cl₂, loaded onto a silica gel column, and eluted using a gradient of 10-30% EtOAc Hexanes. The product was dried via rotary evaporation, affording compound 53 as a colorless oil (8.50 g, 39% yield). Η-NMR δ (CD₃CN): 7.78 (d, 2H, J= 8.4 Hz), 7.44 (d, 2H, J= 8.0 Hz), 4.08 (m, 2H), 3.66 (t, 2H, J= 6.0 Hz), 3.57 (m, 2H), 3.48 (t, 2H, J= 5.6 Hz), 2.45 (s, 3H), 1.45 (s, 18H). ESMS: 348.1 [M + H - 200]⁺, 570.2 [M + H + 23]⁺.

[0250] {2-[2-(2-{2-[4-(3-Azido-phenylamino)-6-methoxy-quinazolin-7-yloxy]- ethoxy}-ethoxy)-ethoxy]-ethyl}-dicarbamic acid tert-butyl ester (54). A solution of compound 53 (39 mg, 0.07 mmol) in DMF (1.0 mL) was added dropwise to a stined suspension of compound 52 (20 mg, 0.06 mmol) and K₂C0 (18 mg, 0.13 mmol) in anhydrous DMF (1.0 mL) under N₂ gas at ambient temperature. The reaction was heated to 60 °C for 16 hours. The cooled solution was filtered through a syringe filter (0.45 μm) and purified on a Cι₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 54 as a yellow hydroscopic solid (25 mg, 57% yield). Η-NMR δ: 10.64 (bs, IH), 8.77 (s, IH), 8.00 (s, IH), 7.51 (m, 3H), 7.24 (s, IH), 7.04 (d, 1H, J= 6.8 Hz), 4.30 (m, 2H), 3.98 (s, 3H), 3.83 (m, 2H) 3.53 (m, 10H), 1.41 (s, 18H). ESMS: 684.2 [M + H]⁺, 706.2 [M + H + 23]⁺.

[0251] Probe 1 (55). To a vigorously stirred solution of compound 54 (16 mg, 0.02 mmol) in CH₂C1₂ (1.5 mL) was added trifluoroacetic acid (100 μL) and the reaction was stined at ambient temperature for 3 hours. The solvent was removed using a steady stream of N₂ gas without heating, affording a yellow oil. A solution ofthe yellow oil in anhydrous DMSO (0.5 mL) was added dropwise to a stirring solution of TAMRA-SE (12 mg, 0.02 mmol) and diisopropylethylamine (8 μL) in anhydrous DMSO (0.5 mL). The reaction was stirred for 16 hours at ambient temperature, and then purified on a Cι₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 55 as a red powder (4 mg, 20% yield). Η-NMR δ (CD₃CN): 8.46 (d, 2H, J= 20.8 Hz), 8.11 (d, IH, J= 7.6 Hz), 7.56 (m, 3H), 7.32 (m, 3H), 6.90 (m, 3H), 6.68 (d, 2H,J= 9.2 Hz), 6.53 (s, 2H), 4.29 (bs, 2H), 3.88 (bs, 5H), 3.66 (m, 12H), 3.15 (s, 12H). ESMS: 448.7 [M + 2H]²⁺, 896.2 [M + H]⁺. [0252] Example 5 - Preparation of 4-Phenylamino quiazoline-related NBAPs

[0253] The following exemplary reaction scheme is depicted in Fig. 6. [0254] Materials and Methods. All solvents and reagents were obtained from the

Aldrich Chemical Company (Milwaukee, WI) unless otherwise indicated; the mixed 5- and 6-succinimidyl ester of tetramethylrhodamine was obtained from Molecular Probes (TAMRA-SE; Eugene, OR). Proton nuclear magnetic resonance (Η NMR) spectra were recorded on a Bruker 400 MHz NMR spectrometer using the residual peaks in the deuterated solvents as internal standards. Samples were dissolved in deuterated dimethylsulfoxide (dβ- DMSO) unless otherwise indicated. Preparative HPLC was carried out on reverse phase Polaris Ci₈ or Cβ columns (5 μ column; 150 mm x 21 mm; Varian; Tonance, CA) using a binary system of water and acetonitrile with 0.1% trifluoroacetic acid as a modifier. Analytical LC/MS samples were carried out on a Polaris de column (5 μ column; 50 x 4.6 mm; Varian; Tonance, CA) using a binary system of water and acetonitrile with 0.1% trifluoroacetic acid as a modifier. HPLC purity was determined using the LC/MS at 254 nm. [0255] (4-Azido-phenyl)-(7-benzyloxy-6-methoxy-quinazolin-4-yI)-amine (56). A solution of 7-benzyloxy-4-chloro-6-methoxy-quinazoline (compound 49, 300 mg, 1.00 mmol) and 4-azidoaniline hydrochloride (187 mg, 1.10 mmol) in iPrOH (10.0 mL) was refluxed in a sealed tube at 95 °C for 3 hours. The solution was allowed to cool to room temperature and the precipitate was collected using vacuum filtration. The filtered solid was dried under high vacuum, yielding compound 56 as a tan solid (290 mg, 73% yield). Η- NMR δ: 11.37 (s, IH), 8.81 (s, IH), 8.28 (s, IH), 7.73 (d, 2H, J= 12.0 Hz), 7.52 (d, 2H, J= 4.0 Hz), 7.42 (m, 4H), 7.24 (d, 2H, J= 8.0 Hz), 5.33 (s, 2H), 4.00 (s, 3H). ESMS: 371.1 [M + H - 28]⁺, 399.1 [M + H]⁺.

[0256] 4-(4-Amino-phenylamino)-6-methoxy-quinazolin-7-ol (57). A solution of compound 56 (280 mg, 0.70 mmol) in MeOH (5.0 mL) and iPrOH (5.0 mL) was flushed for 10 minutes with N₂ gas and then charged with 5% Pd/C (100 mg). The flask was degassed for 5 minutes and then stined under 1 atm of H₂ gas for 64 hours at ambient temperature. The reaction mixture was filtered through a bed of Celite and the filtrate was concentrated to dryness yielding compound 57 as a colorless oil (quantitative yield). Η-NMR δ: 11.51 (bs, IH), 10.81 (s, IH), 8.68 (s, IH), 8.00 (s, IH), 7.29 (d, 2H, J= 8.0 Hz), 7.13 (s, IH), 6.76 (d, 2H, J= 8.0 Hz). ESMS: 283.1 [M + H]⁺.

[0257] 4-(4-Azido-phenylamino)-6-methoxy-quinazolin-7-ol (58). A solution of compound 57 (130 mg, 0.46 mmol) in 80% acetic acid (10.0 mL) was cooled in an ice bath whereupon sodium nitrite (35 mg, 0.51 mmol) was added. After 5 minutes of stirring, sodium azide (33 mg, 0.51 mmol) was added and the reaction was stined for 1 hour. The solvent was removed under vacuum and the resulting solid was dissolved in a minimal amount of DMSO and purified on a Cι₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 58 as a fluffy yellow powder (54 mg, 38% yield). Η-NMR δ: 11.49 (bs, IH), 10.81 (bs, IH), 8.74 (s, IH), 8.01 (s, IH), 7.68 (d, 2H, J= 8.8 Hz), 7.24 (d, 2H, J= 8.8 Hz), 7.16 (s, lH), 3.97 (s, 3H). ESMS: 281.1 [M + H - 28]⁺, 309.1 [M + H]⁺.

[0258] {2-[2-(2-{2-[4-(4-Azido-phenylamino)-6-methoxy-quinazolin-7-yloxy]- ethoxy}-ethoxy)-ethoxy]-ethyl}-dicarbamic acid tert-butyl ester (59). [0259] A solution of compound 7 (35 mg, 0.06 mmol) in DMF (2.0 mL) was added dropwise to a stined suspension of compound 58 (18 mg, 0.06 mmol) and K₂CO (16 mg, 0.12 mmol) in anhydrous DMF (1.0 mL) under N₂ gas at ambient temperature. The reaction was heated to 60 °C for 16 hours. The cooled solution was filtered through a syringe filter (0.45 μm) and purified on a C*₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 59 as an orange oil (30 mg, 75% yield). Η-NMR δ: 10.72 (bs, IH), 8.74 (s, IH), 8.01 (s, IH), 7.71 (d, 2H, J= 8.8 Hz), 7.24 (m, 3H), 4.29 (t, 2H, J = 4.8 Hz), 3.98 (s, 3H), 3.84 (t, 2H, J= 4.0 Hz), 3.52 (m, 12H), 1.41 (s, 18H). ESMS: 684.2 [M + H]⁺.

[0260] Probe 2 (60). To a vigorously stined solution of compound 59 (6 mg, 0.01 mmol) in CH₂C1₂ (1 0 mL) was added trifluoroacetic acid (100 μL) and the reaction was stined at ambient temperature for 1.5 hours. The solvent was removed using a steady stream of N₂ gas without heating, affording a yellow oil. A solution ofthe yellow oil in anhydrous DMSO (0.2 mL) was added dropwise to a stirring solution of TAMRA-SE (4 mg, 0.01 mmol) and diisopropylethylamine (3 μL) in anhydrous DMSO (0.6 mL). The reaction was stined for 16 hours at ambient temperature, and then purified on a Cj₈ reverse phase chromatography column. The fractions containing the desired product in >95% purity by HPLC were collected and lyophilized overnight affording compound 60 as a red powder (4 mg, 81% yield). Η-NMR δ (CD₃CN): 10.22 (bs, IH), 8.42 (d, 2H, J= 10.4 Hz), 8.11 (d, IH, J= 8.0 Hz), 7.61 (m, 4H), 7.32 (d, IH, J= 8.0 Hz), 6.95 (m, 4H), 6.72 (d, 2H, J= 9.2 Hz), 6.59 (s, 2H), 4.26 (bs, 2H), 3.87 (s, 5H), 3.65 (m, 12H), 3.17 (s, 12H). ESMS: 448.6 [M + 2H]²⁺, 896.1 [M + H]⁺.

[0261 ] The invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by prefened embodiments and optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0262] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents. [0263] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by prefened embodiments and optional features, modification and variation ofthe inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0264] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description ofthe invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0265] In addition, where features or aspects ofthe invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members ofthe Markush group. [0266] Other embodiments are set forth within the following claims.

Claims

We claim:

1. A nucleotide binding protein-directed probe having the structure,

wherein each Ri is independently selected from the group consisting of -F, -Br, -CI, -SCH₃, -OH, - CH₂OH, -C(0)N(R)(R), -CN, -NO₂, -N(R)(R), N₃, acetoxy, -C(R)(R)(R), -OCH₃, - OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Cι-₆ alkyl straight or branched chain; n is 0 to 5; each W is independently carbon, or nitrogen;

TAG is a detectable label;

RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; and L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of -N(R)-, -0-, -S- or -C(R)(R)-, where each R is independently H or -Cι-₆ alkyl straight or branched chain, or optionally form an optionally substituted fused carbocyclic or heterocyclic ring structure; or a pharmaceutically acceptable salt or complex thereof.

2. A nucleotide binding protein-directed probe of claim 1 wherein RG is selected from the group consisting of:

where X = CI, Br, I. 3. A nucleotide binding protein-directed probe of claim 1 wherein L₂ is selected from the group consisting of:

wherein is in the range of 0 to 8.

4. A nucleotide binding protein-directed probe of claim 1 wherein Li is selected from the group consisting of:

and L is selected from the group consisting of

-N-_™ , - °-yχxx_H.. -TAG

(CH₂)n

(CH₂)n NH NH

RG

x y -_N...._TAG

(CH₂)n NH

RG

wherein each m is independently 0 to 8, and wherein R is H or Cι-₆ alkyl.

5. A nucleotide binding protein-directed probe of claim 1 wherein the TAG is selected from the group consisting of:

wherein 5-substituted carboxyrhodamme or 5-substituted carboxyfluorescein may be replaced with 6-carboxyrhodamine or 6-carboxyfluorescein, or with a mixture of 5- and 6- substituted carboxyrhodamme or carboxyfluorescein.

A nucleotide binding protein-directed probe having the structure:

A nucleotide binding protein-directed probe having the structure;

wherein W is carbon, oxygen, sulfur, or nitrogen;

Ri and R₂ are independently H or OH or Ri and R₂ together are =O;

TAG is a detectable label;

RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; and

L and Li are each independently an optionally present alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -C(R)(R)-, where each R is independently H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure. 8. A nucleotide binding protein-directed probe according to claim 7, wherein Ri and R₂ are each H; Ri is OH and R₂ is H; or Ri is H and R₂ is OH.

A nucleotide binding protein-directed probe according to claim 7, wherein Li is selected from the group consisting of:

and L is selected from the group consisting of

- - -TAG ^■ ftγO - -TAG

(CH₂)n

(CH₂)n NH NH

RG

wherein each m is independently 0 to 8 and wherein R is H or Cι-₆ alkyl.

0. A nucleotide binding protein-directed probe of claim 7 wherein RG is selected from the group consisting of:

where X = CI, Br, F, or I.

11. A nucleotide binding protein-directed probe according to claim 7 wherein the TAG is selected from the group consisting of:

wherein 5-substituted carboxyrhodamine or 5-substituted carboxyfluorescein may be replaced with 6-carboxyrhodamine or 6-carboxyfluorescein, or with a mixture of 5- and 6- substituted carboxyrhodamine or carboxyfluorescein. 12. A nucleotide binding protein-directed probe having the structure;

wherein Ri, R₂, R₃ R , and R₅ are each independently selected from the group consisting of-H, -OH, Ri and R₂ together are =O and R₃ and R-_t together are =O; Ari and Ar₂ are independently selected from the group consisting of -N(R)-, -O-, -S- or

-C(R)(R)-, aryl, or heteroaryl, where each R is independently H or -Cι.₆ alkyl straight or branched chain, wherein if Ari or Ar₂ are aryl or heteroaryl, the aryl or heteroaryl group optionally forms a fused carbocyclic or heterocyclic ring structure with the maleimide ring;

L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -C(R)(R)-, where R is H or -Cι_6 alkyl straight or branched chain, and optionally form an optionally substituted fused carbocyclic or heterocyclic ring structure;

TAG is a detectable label; and RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; or a pharmaceutically acceptable salt or complex thereof.

13. A nucleotide binding protein-directed probe of claim 12 wherein RG is selected from the group consisting of:

,NHf \ — / -

v rx

where X = CI, Br, F, or I. 14. A nucleotide binding protein-directed probe according to claim 12 wherein L₂ is selected from the group consisting of:

wherein n and m are independently in the range of 0 to 8.

15. A nucleotide binding protein-directed probe of claim 12 wherein L] is selected from the group consisting of:

L-N(- °-- «---^N. y₀— °-

H L- N

_L-_Nft-

and L is selected from the group consisting of

wherein each m is independently 0 to 8 and wherein R is H or Cι-₆ alkyl.

16. A nucleotide binding protein-directed probe according to claim 12 wherein TAG is selected from the group consisting of:

wherein 5-substituted carboxyrhodamine or 5-substituted carboxyfluorescein may be replaced with 6-carboxyrhodamine or 6-carboxyfluorescein, or with a mixture of 5- and 6- substituted carboxyrhodamine or carboxyfluorescein. 17. A nucleotide binding protein-directed probe having the structure

wherein W is carbon, oxygen, sulfur, or nitrogen; Ri, R₂, R₃ and } are independently selected from the group consisting of -H, -OH, Rj and R₂ together are =O, and R₃ and R» together are =O;

R_ό is H or OH;

R₇, Rg, R , and R]₀ are independently -H, -OH, -C_M alkyl, or -CO(O)-CM alkyl, and one R₇,

R₈, R , or Rio comprises a linker moiety Li that forms a covalent linkage through linker moiety L for each of TAG and RG, or two of R₇, R₈, R , or Rio comprise linker moieties Lj to a reactive group RG and L₂ to a TAG;

L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain, and optionally form an optionally substituted fused carbocyclic or heterocyclic ring structure;

TAG is a detectable label; and RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; or a pharmaceutically acceptable salt or complex thereof. 18. A nucleotide binding protein-directed probe according to claim 17, wherein Li is selected from the group consisting of:

and L is selected from the group consisting of

wherein each m is independently 0 to 8 and wherein R is H or Cι-₆ alkyl.

19. A nucleotide binding protein-directed probe according to claim 17, wherein L] and L₂ have the structure

wherein n and m are 0 to 8, and X is RG or TAG.

0. A nucleotide binding protein-directed probe of claim 17 wherein RG is selected from the group consisting of:

where X = CI, Br, F, or I.

21. A nucleotide binding protein-directed probe according to claim 17 wherein TAG is selected from the group consisting of:

wherein 5-substituted carboxyrhodamine or 5-substituted carboxyfluorescein may be replaced with 6-carboxyrhodamine or 6-carboxyfluorescein, or with a mixture of 5- and 6- substituted carboxyrhodamine or carboxyfluorescein.

22. A nucleotide binding protein-directed probe having the structure

wherein R₎₂, R_]3 and R₁₄ are independently -F, -Br, -CI, -SCH₃, -OH, -CH₂OH, - C(O)N(R)(R), -CN, -NO₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Ci. ₆ alkyl straight or branched chain; n is 0 to 4;

L, Li, and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -0-, -S- or - C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure;

TAG is a detectable label; and RG is a reactive group capable of reacting with at least one of thiol, hydroxyl, carboxyl or amino selected from the group consisting of fluorosulfonyl, fluorophosphonyl ester, halogen, epoxide, ethylene α to an activating group, and halogen α to an activating group; or a pharmaceutically acceptable salt or complex thereof. 23. A nucleotide binding protein-directed probe according to claim 22, wherein Lj is selected from the group consisting of:

and L is selected from the group consisting of

wherein each m is independently 0 to 8 and wherein R is H or Cι-₆ alkyl.

24. A nucleotide binding protein-directed probe according to claim 22, wherein linker moieties Li and L₂ have the structure

-rft- - ^x^° m wherein m is independently 0 to 8, and X is RG or TAG.

5. A nucleotide binding protein-directed probe of claim 22 wherein RG is selected from the group consisting of:

-'Y_'-_' '- [> R-

where X = CI, Br, F, or I.

26. A nucleotide binding protein-directed probe according to claim 22 wherein TAG is selected from the group consisting of:

27. A nucleotide binding protein-directed probe having the structure:

wherein Rι₅ and Rι₆ are independently -F, -Br, -CI, -SCH₃, -OH, -CH₂OH, -C(O)N(R)(R), - CN, -N0₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Ci-₆ alkyl straight or branched chain; n is 0 to 5;

L, Li and L₂ are optionally present and are independently alkyl or heteroalkyl groups of 1-20 backbone atoms selected from the group consisting of -N(R)-, -O-, -S- or -C(R)(R)-, where R is H or -Cι.₆ alkyl straight or branched chain, and optionally form an optionally substituted fused carbocyclic or heterocyclic ring structure;

28. A nucleotide binding protein-directed probe according to claim 27, wherein Li is selected from the group consisting of:

and L is selected from the group consisting of

wherein each m is independently 0 to 8 and wherein R is H or Cι.₆ alkyl. 29. A nucleotide binding protein-directed probe according to claim 27, wherein linker moieties L] and L₂ have the structure

wherein m is independently 0 to 8, and X is RG or TAG.

0. A nucleotide binding protein-directed probe of claim 29 wherein RG is selected from the group consisting of:

CI

NH ,N-- N-N

S=C=N- - θ=C=N- - N _/)— N- -

MeO A A-

0₂N' "Nθ₂ CI

where X = CI, Br, F, or I.

31. A nucleotide binding protein-directed probe according to claim 29 wherein TAG is selected from the group consisting of:

2. A nucleotide binding protein-directed probe having the structure:

or

33. A nucleotide binding protein-directed probe having the structure;

wherein each Ri is independently selected from the group consisting of -F, -Br, -CI, -SCH₃, -OH, - CH₂OH, -C(O)N(R)(R), -CN, -N0₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, -OCH₂CH₃, methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Ci-₆ alkyl straight or branched chain;

R₂ is Co-6 alkyl; n is 0 to 5; each W is independently carbon, or nitrogen;

TAG is a detectable label;

L, Li, and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -0-, -S- or - C(R)(R)-, where R is H or -Ci-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure; or a pharmaceutically acceptable salt or complex thereof.

34. A nucleotide binding protein-directed probe of claim 33 wherein RG is selected from the group consisting of:

p p

^■AA- «J~ - -s- •— s-- vr ό OR ό

where X = CI, Br, F, or I. 35. A nucleotide binding protein-directed probe of claim 37 wherein Li is selected from the group consisting of:

wherein m is in the range of 1 to 3.

36. A nucleotide binding protein-directed probe of claim 37 wherein L₂ is selected from the group consisting of:

wherein each m is independently in the range of 1 to 3. 37. A nucleotide binding protein-directed probe of claim 37 wherein the TAG is selected from the group consisting of:

38. A nucleotide binding protein-directed probe having the structure

wherein each W is independently carbon, or nitrogen;

R_t and R₂ are independently selected from the group consisting of -F, -Br, -CI, -SCH₃, -OH, -CH₂OH, -C(O)N(R)(R), -CN, -NO₂, -N(R)(R), acetoxy, -C(R)(R)(R), -OCH₃, - OCH₂CH , methylene dioxy, trihalomethyl, trihalomethoxy, and an aliphatic or aryl ring of 5 or 6 ring atoms optionally containing one or more ring heteroatoms, where each R is independently -H or -Cι.₆ alkyl straight or branched chain; n is 0-3;

TAG is a detectable label;

L, Li, and L₂ are optionally present and are independently an alkyl or heteroalkyl group of 1-20 backbone atoms selected from the group consisting of-N(R)-, -O-, -S- or -

10 C(R)(R)-, where R is H or -Cι-₆ alkyl straight or branched chain or optionally forms an unsubstituted or substituted fused carbocyclic or heterocyclic ring structure; or a pharmaceutically acceptable salt or complex thereof.

I l l

9. A nucleotide binding protein-directed probe of claim 38 wherein RG is selected from the group consisting of:

o o

F-S- V F-P— '/ X // v X 6 OR x ό

OγNγθ

where X = CI, Br, F, or I.

40. A nucleotide binding protein-directed probe of claim 38 wherein Li and L₂ are independently selected from the group consisting of:

wherein m is independently 0 to 8, and X is RG or TAG.

41. A nucleotide binding protein-directed probe of claim 38, wherein Li is selected from the group consisting of:

H -_NA-~ L -N

and L is selected from the group consisting of

wherein each m is independently 0 to 8 and wherein R is H or Cι-₆ alkyl.

42. A nucleotide binding protein-directed probe of claim 42 wherein the TAG is selected from the group consisting of:

43. A nucleotide binding protein-directed probe having the structure:

wherein X is CI.

44. A method for determining the enzyme profile of one or more target nucleotide binding proteins in a complex protein mixture, employing one or more probes comprising an affinity moiety that specifically binds to nucleotide binding protein(s) covalently attached to a TAG through a linking moiety, and a reactive functionality that reacts with an amino acid functionality of said nucleotide binding protein(s) when said probe is bound to said nucleotide binding protein(s), said method comprising: combining in a reaction medium said probe(s) and said complex protein mixture under conditions of reaction of said probe(s) with said nucleotide binding protein(s), whereby a conjugate of said probes and said nucleotide binding protein(s) is formed; and determining said enzyme profile by generating a signal from one or more conjugates formed thereby; wherein said probe(s) are selected from the nucleotide binding protein- directed probes of one of claims 1-43.

45. A method according to Claim 44, wherein said reactive functionality is selected from the group consisting of fluorosulfonyl, vinylsulfonyl, acryl, and chloroacetyl.

46. A method according to Claim 44, wherein said probe binds to a plurality of target nucleotide binding proteins.