Methods for ligand discovery

(background) The drug discovery process usually begins with a large functional screening of the compound library to identify the appropriate lead for subsequent medical chemistry optimization. However, not all targets of interest can tolerate such screening. In some cases, assays that can withstand high-throughput screening are not available. In other cases, the target can have multiple binding modalities, resulting in ambiguous and difficult to interpret the results of such screenings. In still other cases, the assay conditions for a high-throughput assay are such that they are susceptible to anthropogenic results. As a result, alternative methods for ligand discovery are needed that do not necessarily rely on functional screening.

<p> (Gist of the invention) The present invention relates to a method for ligand discovery using binding techniques.</p><p> In one aspect, the invention states:</p><p><chemistry num="9"><img file="JP4391237B2_D0001.tif" /></chemistry>Concerning target-compound conjugates selected from the group consisting of. here,</p><p><chemistry num="10"><img file="JP4391237B2_D0002.tif" /></chemistry>Is the target, and R and R'are independent and unsubstituted C.₁~ C₂₀Aliphatic, permuted C₁~ C₂₀Aliphatic, unsubstituted aryl, or substituted aryl; m is 0, 1, or 2; and n is 1 or 2.</p><p> In certain embodiments, the target is a polypeptide or protein, which includes, for example, enzymes, receptors, transcription factors, ligands for receptors, growth factors, cytokines, immunoglobulins, nuclear proteins, signaling components, and allosteric enzymes. It can be selected from the group consisting of regulators. The covalent bond between the -SS-bond and the target compound may be reversible or irreversible.</p><p> In another aspect, the invention is described by each member as follows:</p><p><chemistry num="11"><img file="JP4391237B2_D0003.tif" /></chemistry>With respect to a library of compounds having a formula selected from the group consisting of, where R and R'are independent and unsubstituted C, respectively.₁~ C₂₀Aliphatic, permuted C₁~ C₂₀It is aliphatic, unsubstituted aryl, or substituted aryl; m is 0, 1, or 2; and n is 1 or 2.</p><p> The library preferably has at least about 5 members, more preferably at least about 100 members, and the atomic mass of the individual members of this library is preferably at least about 5 atomic mass units. more preferably, at least about 10 original different child mass units.</p><p> In a further aspect, the invention relates to methods including: a) Formula R that binds to the target protein^DSSR¹Steps to identify the first compound of; b) Formula R that binds to the target protein^ESSR¹Steps to identify the second compound of; c) R^DAnd R^EIn the step of forming a conjugate compound containing, here, R^DAnd R¹Are independent of each other, C₁~ C₂₀Non-replacement aliphatic, C₁~ C₂₀Are substituted aliphatic, unsubstituted aryl, and substituted aryl; and R¹Is a non-replacement C₁~ C₁₀Aliphatic, permuted C₁~ C₁₀Aliphatic, unsubstituted aryl. In certain embodiments of this method, the identification of the second compound that binds to the target is carried out in the presence of the first compound.</p><p> In another embodiment, R^DSSR¹And R^ESSR¹Are independent of the following:</p><p><chemistry num="12"><img file="JP4391237B2_D0004.tif" /></chemistry>Selected from the group consisting of, where R and R'are independent and unsubstituted C, respectively.₁~ C₂₀Aliphatic, permuted C₁~ C₂₀Aliphatic, unsubstituted aryl, or substituted aryl; m is 0, 1, or 2; and n is 1 or 2.</p><p> In a further aspect, the invention relates to a method comprising: a) The step of providing a target having an anchor group capable of forming a covalent bond at or near the site of interest or coordinating with a metal; b) The step of contacting this target with an elongation factor, thereby forming a target-elongation factor complex, where the elongation factor forms a covalent bond or coordinates to a metal. A step comprising a first functional group and a second functional group capable of forming a covalent bond, which is one of the following; c) The step of contacting this target-elongation factor complex with a candidate ligand containing a group capable of forming a covalent bond with a second functional group; d) The step of forming a covalent bond between the target elongation factor complex and the candidate ligand; e) The step of identifying a candidate ligand present in the target-elongation factor-ligand conjugate.</p><p> In certain embodiments of this method, the anchor group is selected from the group consisting of reactive electrophilic groups, reactive nucleophilic groups, and metal coordination sites.</p><p> The present invention also relates to methods including: a) The step of providing a target having a reactive nucleophile at or near the site of interest; b) The step of contacting the target with an elongation factor, thereby forming a target-elongation factor complex, where the elongation factor reacts with a nucleophile at the target to form a covalent bond. Includes a first functional group, and a second functional group capable of forming a disulfide bond; c) The step of contacting this target-elongation factor complex with a ligand candidate capable of forming a disulfide bond; d) The step of forming a disulfide bond between the target-elongation factor complex and the ligand candidate, thereby forming the target-elongation factor-ligand bond; e) The step of identifying ligand candidates present in this target-elongation factor-ligand conjugate.</p><p> The reactive nucleophile at the target can be, for example, a thiol or a masked thiol, and the elongation factor can have the following formula:</p><p><chemistry num="13"><img file="JP4391237B2_D0005.tif" /></chemistry>Where R is the unsubstituted C₁~ C₂₀Aliphatic, permuted C₁~ C₂₀Aliphatic, unsubstituted aryl, and substituted aryl; R'is H, -SR¹(Here R¹Is a non-replacement C₁~ C₁₀Aliphatic, permuted C₁~ C₁₀It is aliphatic, unsubstituted aryl, and substituted aryl); X is a leaving group, and the box in each formula represents a binding determinant.</p><p> In certain embodiments, the elongation factor is of the formula:</p><p><chemistry num="14"><img file="JP4391237B2_D0006.tif" /></chemistry>Where R'is H, -SR¹(Here R¹Is a non-replacement C₁~ C₁₀Aliphatic, permuted C₁~ C₁₀It is aliphatic, unsubstituted aryl, and substituted aryl), and the box represents a binding determinant.</p><p> In different aspects, the invention relates to a protein-elongation factor complex, wherein the protein forms a covalent bond with the extension factor, which is the first functional group capable of forming a covalent bond, and It contains a second functional group capable of forming a second covalent bond.</p><p> In another aspect, the invention relates to a protein-elongation factor complex, wherein the protein is coordinated to a metal with an elongating factor, which is the first functional group capable of coordinating to the metal. And contains a second functional group capable of forming a covalent bond.</p><p> These complexes may further contain a disulfide bond between the second functional group and a compound capable of forming a disulfide bond.</p><p> (Explanation of preferred embodiment) The present invention provides a rapid and efficient method for identifying ligands capable of binding to selected sites in a target of interest. The ligand itself identified by the methods herein can be used as a lead compound for the development of, for example, novel therapeutic agents, enzyme inhibitors, labeled compounds, diagnostic agents, affinity agents for protein purification, and the like. Find out.</p><p> Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, J. Wiley &amp; Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Chemistries and Structure 4th Edition, John Wiley &amp; Sons (New York, NY, NY) References such as 1992) provide those skilled in the art with many general guidelines for the terms used in this application.</p><p> Compounds are provided in one aspect of the invention. Unless explicitly or implicitly indicated elsewhere, these compounds can be in the form of individual enantiomers, diastereomers, geometric isomers, or mixtures thereof. If these compounds contain double bonds, these double bonds can be either Z or E, or a mixture thereof, unless otherwise indicated.</p><p> (Definition) Definitions of terms used herein include: The term "aliphatic" or "unsubstituted aliphatic" refers to straight, branched, cyclic, or polycyclic hydrocarbons, and alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl. A moiety and a cycloalkynyl moiety are included.</p><p> The term "alkyl" or "unsubstituted alkyl" refers to saturated hydrocarbons.</p><p> The term "alkenyl" or "unsubstituted alkenyl" refers to a hydrocarbon having at least one carbon-carbon double bond.</p><p> The term "alkynyl", or "unsubstituted alkynyl", refers to a hydrocarbon having at least one carbon-carbon triple bond.</p><p> The term "aryl" or "unsubstituted aryl" refers to a monocyclic or polycyclic unsaturated moiety having at least one aromatic ring. The term includes heteroaryls that contain one or more heteroatoms within at least one aromatic ring. Typical examples of aryls include: phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl. , Kinolinyl, isoquinolinyl, etc.</p><p> The term "substituted", when used to modify a moiety, refers to a substituted version of that moiety in which at least one hydrogen atom has been substituted with another group. The groups are as follows: aliphatic; aryl, alkylaryl, F, Cl, I, Br, -OH; -NO₂; -CN; -CF₃;-CH₂CF₃;-CH₂Cl; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂;-CH₂SO₂CH₃;-OR^X;-C (O) R^X;-COOR^X;-C (O) N (R)^X)₂;-OC (O) R^X;-OCOOR^X;-OC (O) N (R)^X)₂;-N (R)^X)₂;-S (O)₂R^X; And -NR^XC (O) R^XHowever, but not limited to these, here, R in each case^XAre independently hydrogen, substituted aliphatic, unsubstituted aliphatic, substituted aryl, or unsubstituted aryl. In addition, the substitution of adjacent groups in the moiety can be combined to form a cyclic group.</p><p> The term "antagonist" is used in its broadest sense, and partially or completely blocks, inhibits, or neutralizes the biological activity indicated by a target (eg, TBM). Includes any ligand. In a similar manner, the term "agonist" is used in its broadest sense and refers to the biological activity indicated by the target (eg, TBM), eg, the function or expression of such TBM, or such. By specifically altering the efficiency of TBM-mediated signal transduction, thereby altering (increasing or inhibiting) existing biological activity or mimicking it by inducing new biological activity. Includes any ligand.</p><p> The term "elongation factor" refers to a first functional group having a molecular weight of about 30 to about 1,500 daltons and capable of reacting with a group on the target, and reacting with a ligand candidate or a member of a library of ligand candidates. A molecule having a second functional group capable of forming a disulfide bond.</p><p> The term "ligand" refers to an entity that has a measurable binding affinity for a target. In general, a ligand is a K of about 100 mM or less, preferably about 10 mM or less, and more preferably about 1 mM or less._dOr K_iIt is said to have a measurable affinity when it binds to a target. In a preferred embodiment, the ligand is not a peptide and is a small molecule. Ligand is a small molecule when it is less than about 2000 daltons in size, usually less than about 1500 daltons in size. In a more preferred embodiment, the small molecule ligand is less than about 1000 daltons in size, usually less than about 750 daltons in size, and more usually less than about 500 daltons in size.</p><p> With respect to elongation factors, the term "binding determinant" refers to the portion of the elongation factor involved in binding to a target (eg, a target polypeptide).</p><p> The term "ligand candidate" is a compound that has or has been modified to have a reactive group capable of forming a covalent bond with a complementary or compatible reactive group on the target. To say. Reactive groups in either the ligand candidate or the target can be masked with, for example, protecting groups.</p><p> The term "polynucleotide", when used in the singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide that can be unmodified RNA or DNA or modified RNA or DNA. Thus, by way of example, polynucleotides as defined herein are, but are not limited to, single-stranded DNA and double-stranded DNA, DNA containing single-stranded and double-stranded regions, single-stranded. RNA and double-stranded RNA, as well as RNA containing single- and double-stranded regions, and hybrid molecules containing DNA and RNA, which are single-stranded or more typically double-stranded. However, it may include a single-strand region and a double-stranded region). In addition, the term "polynucleotide" as used herein refers to a triple chain region that includes RNA or DNA, or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. These regions may include all of one or more of the molecules, but more typically include only some regions of the molecule. One of the molecules in the triple helix region is often an oligonucleotide. The term "polynucleotide" specifically includes DNA and RNA containing one or more modified bases. Thus, a DNA or RNA having a backbone modified for stability or other reasons is a "polynucleotide" as the term is intended herein. In addition, DNA or RNA containing unusual bases (eg, inosine) or modified bases (eg, tritylated bases) is included within the scope of the term "polynucleotide" as defined herein. In general, the term "polynucleotide" includes all unmodified polynucleotides in chemically, enzymatically, and / or metabolically modified forms, as well as various viruses and cells (including simple and complex cells). ) Includes the characteristic chemical forms of DNA and RNA.</p><p> The phrase "protected (protected) thiol", as used herein, is a covalent bond that can react with a group or molecule to reduce the reactivity of the thiol and deprotect to regenerate the free thiol. A thiol that forms a bond.</p><p> The term "reversible covalent bond" as used herein refers to a covalent bond that can be disrupted, preferably under conditions that do not denature the target. Examples include, but are not limited to, disulfides, Schiff bases, thioesters, coordination complexes, boronate esters and the like.</p><p> The phrase "reactive group" is a chemical group or moiety that provides a site where a covalent bond can be formed if a compatible or complementary reactive group is provided. A typical example is that it can react with other -SH or -SS- to form disulfides-SH; activated-can react with COOH to form amides-NH.₂Can react with aldehydes or ketones to form Schiff bases-NH₂And so on.</p><p> The phrase "reactive nucleophile", as used herein, is a nucleophile capable of forming a covalent bond with a compatible functional group on another molecule under conditions that do not denature or damage the target. Refers to the nuclear group. The most suitable nucleophilic groups are thiols, alcohols, and amines. Similarly, the phrase "reactive electrophilic group", as used herein, is preferably a functional group of compatibility on another molecule, preferably under conditions that do not modify or otherwise damage the target. Refers to an electrophilic group that can form a covalent bond with. The most suitable electrophilic groups are imines, carbonyls, epoxides, aziridines, sulfonates, disulfides, activated esters, activated carbonyls, and hemiacetals.</p><p> The phrase "site of interest" refers to any site on the target to which a ligand can bind. For example, if the target is an enzyme, the site of interest may be in contact with the binding substrate, inhibitor, activator, cofactor, or allosteric modulator of this enzyme, or within about 10 angstroms (more preferably about 5 angstroms). Amino acids in the (within) position can be mentioned. If the enzyme is a protease, the sites of interest include substrate binding channels from P6 to P6', residues involved in catalytic function (eg, catalytic triads and oxyanion holes), and any cofactor (eg, Zn). Metal) binding sites such as. When the enzyme is a protein kinase, the site of interest includes a substrate binding channel in addition to the ATP binding site. When the enzyme is dehydrogenase, the sites of interest include the substrate binding region and the site occupied by NAD / NADH. When the enzyme is a hydrolase such as PDE4, the sites of interest include residues that come into contact with cAMP and residues that are involved in the binding of catalytic divalent cations.</p><p> The terms "target", "target molecule" and "TM" are interchangeably used in the broadest sense and refer to a chemical or biological entity in which ligand binding has an effect on the function of the target. The target can be a molecule, part of a molecule, or an aggregate of molecules. Ligand binding may be reversible or irreversible. Specific examples of target molecules include polypeptides or proteins (eg, enzymes (including proteases such as cysteine proteases, serine proteases, and aspartyl proteases)), receptors, transcription factors, ligands for receptors, growth factors, etc. Polynucleotides, peptides, carbohydrates, glycoproteins, glycolipids, and other polymers (eg, nucleic acid-protein complex) such as cytokines, immunoglobulins, nuclear proteins, signaling components (eg, kinases, phosphatases), allosteric enzyme regulators, etc. Examples include the body, chromatin or ribosome, lipid bilayer-containing structures (eg, membranes), or membrane-derived structures (eg, vesicles). This definition specifically includes the target biological molecule (TBM), as defined below.</p><p> A "target biological molecule" or "TBM" as used herein is a single biological molecule, or a complex that is biologically related to each other (as opposed to low). A plurality of biological molecules in which an agonist or antagonist of a molecule can form an effect on the function of TBM). In a preferred embodiment, the TBM has or has a reactive group that comprises a protein or a portion thereof, or two or more amino acids and is capable of forming a covalent bond with a compound having complementary reactive groups. It can be modified to have a group. Typical examples of TBM include: enzymes, receptors, transcription factors, ligands for receptors, growth factors, immunoglobulins, nucleoproteins, signaling components, glycoproteins, glycolipids, and other polymers ( For example, nucleic acid-protein complexes, chromatin or ribosomes), lipid bilayer-containing structures (eg, membranes), or membrane-derived structures (eg, vesicles). Targets can be obtained in a variety of modes, including isolation and purification from natural sources, chemical synthesis, recombinant production, and any combination of these and similar methods.</p><p> Erythropoietin (EPO), granulocyte colony stimulation (G-CSF) receptor, granulocyte macrophage colony stimulation (GM-CSF) receptor, thrombopoietin (TPO), interleukin (eg IL-2, IL-3) , IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), growth hormone, prolactin, human placental lactogen (LPL), CNTF, oncostatin, RANTES, MIPb, IL- 8, insulin, insulin-like growth factor 1 (IGF-1), epithelial growth factor (EGF), hiregulin-a and hiregulin-b, vascular endothelial growth factor (VEGF), placenta growth factor (PLGF), tissue growth factor (TGF) -α and TGF-β) and nerve growth factor (NGF). Other targets include various neurotrophins and their ligands, other hormones and receptors (eg, bone morphogenesis factor, follicle stimulating hormone (FSH), and luteinizing hormone (LH), CD40 ligands, apoptotic factors- 1 and apoptotic factors-2 (AP-1 and AP-2), p53, bax / bcl2, mdm2, caspase (1, 3, 8 and 9), catepsin, IL-1 / IL-1 receptor, BACE, HIV integra Lase, PDE IV, hepatitis C helicase, hepatitis C protease, rhinovirus protease, apoptosis, cPLA (cytoplasmic solphospholipase A2), CDK4, c-jun kinase, adapter (eg Grb2, GSK-3,</p><p> (Combination method) The present invention provides a novel method for ligand discovery, which is by a process called "binding". Potential ligands are covalently or "bound" to the target and subsequently identified. As previously indicated, in one aspect of the invention, the method comprises the following steps: a) The step of contacting a target containing a chemically reactive group at or near the site of interest with a compound capable of forming a covalent bond with the chemically reactive group; b) The step of forming a covalent bond between the target and the compound, thereby forming a target compound conjugate; and c) The step of identifying the target compound conjugate.</p><p> In one embodiment, multiple compounds are used, so that the method comprises the following steps: a) The step of obtaining a target containing a chemically reactive group at or near the site of interest; b) The step of matching a target with multiple compounds that can be covalently attached to a chemically reactive group, where at least one compound forms a covalent bond with the target; and c) A step of identifying a compound that has formed a covalent bond in a target-compound conjugate.</p><p> In a preferred embodiment, the target is a protein and the chemically reactive group is a thiol on a cysteine residue therein. If the site of interest does not contain naturally occurring cysteine residues, the target can be modified to contain cysteine residues at or near the site of interest. Cysteine is said to be near the site of interest if it is located within 10 Å of the site of interest, preferably within 5 Å of the site of interest. Preferred residues for modification are solvent-accessible residues. Solvent accessibility can be determined by standard numerical methods (Lee, B. &amp; Richards, FMJMol.Biol 55: 379-400 (1971); Shrake, A. &amp; Rupley, JAJMol.Biol. 79: 351-371 (1973)) or It can be calculated from a structural model using an analytical method (Connolly, MLScience 221: 709-713 (1983); Richmond, TJJ Mol.Biol. 178: 63-89 (1984)). For example, potential cysteine variants are the Lee and Richards methods (Lee, B. &amp; The combined surface area of carbon β (CB) or sulfur γ (SG) is 21 Å as calculated by Richards, FMJ Mol.Biol 55: 379-400 (1971)).²If it is larger, it is considered that the solvent is accessible. This value represents about 33% of the theoretical surface area accessible to the cysteine side chain as described by Creamer et al. (Creamer, TP et al., Biochemistry 34: 16245-16250 (1995)).</p><p> Also, residues that should be mutated to cysteine, or other thiol-containing amino acid residues, are preferably not involved in hydrogen bonding with skeletal atoms, or at most interact with the skeletal by only one hydrogen bond. .. Wild-type residues in which the side chain is involved in multiple (&gt; 1) hydrogen bonds with other side chains are also less preferred. All standard rotational isomers (-60 °, 60 °, or 180 ° χ1 angle) with any other residue of N, CA, C, O, or CB atoms. Variants that can introduce adverse steric contact are also less preferred. The unfavorable contact is defined as the interatomic distance, which is less than 80% of the total van der Waals radius of the atoms involved. In certain embodiments where the site of interest is a concave region, residues found at the edges of such sites (eg, raised regions or adjacent convex regions) are more preferred because they mutate to cysteine residues. .. Convex and concave can be calculated based on surface vectors (Duncan, BS &amp; Olson, AJ Biopolymers) 33: 219-229 (1993)) or can be calculated by determining the accessibility of water probes placed along the surface of the molecule (Nicholls, A. et al., Proteins 11: 281-296 (1991)). Brady, GP, Jr. &amp; Stouten, PFJ Comput.Aided Mol.Des. 14: 383-401 (2000)). Skeletal conformation that is nominally forbidden to L-amino acids (Ramachandran, GN et al., J. Mol. Biol. 7: 95-99 (1963); Ramachandran, GN &amp; Sasisekharahn, V.Adv.Prot.Chem.23: 283) Residues carrying -437 (1968)) are less preferred targets for modification to cysteine. Forbidden conformations are usually characterized by a positive φ angle.</p><p> Other preferred variants have been modified to cysteine and -Cys-SSR¹If combined to include, R¹It is a variant possessing a conformation that directs the atom of. Two general procedures can be used to identify these preferred variants. In the first step, the search is a single structure in Protein Databank (Berman, HM et al., Nucleic Acids Research 28: 235-242 (2000)) (Hobohm, U. et al., Protein Science 1: 409-417 (1992)). )), Identifying structural fragments containing disulfide-bonded cysteines at position j, where the skeleton atoms of fragment residues j-1, j, and j + 1 are residues of the target molecule. 0.75 Å with i-1, i, and i + 1 skeleton atoms²Can be overlaid with less than RMSD. Position i is preferred if a fragment is identified that is disulfide-bonded to cysteine at position j closer to any atom at the site of interest than the Cβ atom of residue i (when mutated to cysteine). Conceivable. In an alternative procedure, the residue at position i is computationally "mutated" to cysteine and capped with an S-methyl group via a disulfide bond.</p><p> In addition to adding one or more cysteines to the site of interest, it deletes one or more naturally occurring cysteines located outside the site of interest (and replaces them with, for example, alanine). May be desirable. These variants, in which one or more naturally occurring cysteines have been deleted or "scrambled", include another aspect of the invention. Various recombinant, chemical, synthetic and / or other techniques have been used to modify the target so that the target has the desired number of free thiol groups available for binding. Possess. Such techniques include, for example, site-directed mutagenesis of a nucleic acid sequence encoding a target polypeptide, so that the nucleic acid sequence encodes a polypeptide having a different number of cysteine residues. Polymerase Chain Reaction (PCR) Amplification (eg, US Pat. No. 4,683,195, issued July 28, 1987; and Current Protocols In Molecular). Site-directed mutagenesis using Biology, Chapter 15 (Ausubel et al., 1991) is particularly preferred. Other site-specific mutagenesis techniques are also well known in the art and are described, for example, in the following publications: Ausubel et al., Supra, Chapter 8; Molecular Cloning: A Laboratory Manual., 2nd Edition. (Sambrook et al., 1989); Zoller et al., Methods Enzymol. 100: 468-500 (1983); Zoller &amp; Smith, DNA 3: 479-488 (1984); Zoller et al., Nucl. Acids Res., 10: 6487 (1987). ); Brake et al., Proc.Natl.Acad.Sci.USA 81: 4642-4646 (1984); Botstein et al., Science 229: 1193 (1985); Kunkel et al., Methods Enzymol. 154: 367-82 (1987), Adelman Et al., DNA 2: 183 (1983); and Carter et al., Nucl. Acids Res., 13: 4331 (1986). Cassette mutagenesis (Wells et al., Gene, 34: 315 [1985]) and restricted selective mutagenesis (Wells et al., Philos.Trans.R.Soc.London SerA, 317: 415 [1986]) can also be used.</p><p> Amino acid sequence variants with more than 1 amino acid substitution can be produced in one of several methods. If the amino acids are co-located closely together in the polypeptide chain, they can be mutated simultaneously using a single oligonucleotide that encodes all of the desired amino acid substitutions. However, if the amino acids are located some distance from each other (eg, separated by more than 10 amino acids), it is more difficult to produce a single oligonucleotide that encodes all of the desired changes. is there. Alternatively, one of two alternative methods may be used. In the first method, separate oligonucleotides are produced for each amino acid to be substituted. These oligonucleotides are then simultaneously annealed to the single-stranded template DNA, and the second DNA strand synthesized from the template encodes all of the desired amino acid substitutions. Alternative methods involve two or more mutagenesis to produce the desired variant.</p><p> Once a target-compound conjugate has been formed, this conjugate can be detected using a number of methods. In one embodiment, mass spectrometry is used. The target-compound conjugate can be detected directly by mass spectrometry, or the target compound conjugate can be fragmented prior to detection. Alternatively, the compound can be released in a mass spectrometer and subsequently identified. As described in more detail below, the use of mass spectrometry to identify compounds in target-compound conjugates in such an easy and robust manner is surprising and unpredictable in the present invention. It is one of the discoveries. Both target-compound conjugates and mass spectrometers (MS) containing target-compound conjugates include aspects of the invention.</p><p> MS can detect molecules based on mass-to-charge ratio (m / z) and thus decompose molecules based on their size (Yates, Trends Genet. 16: 5-8 [2000]. (Reviewed). The mass spectrometer first converts the molecule to gas phase ions, then separates the individual ions based on the m / z ratio and finally detects them. A mass spectrometer is an integral part of a mass spectrometer that uses physical characteristics (eg, electric or magnetic fields, or time of flight [TOF]) and then strikes a particular m /. Separate z-valued ions. Mass spectrometers can generate data quickly and therefore have great potential for high throughput analysis. Mass spectrometers can be used alone or in combination with other means for detecting or identifying compounds that are covalently attached to a target. Further descriptions of mass spectrometry techniques include Fitzgerald and Siuzdak, Chemistry &amp; Biology. 3: 707-715 [1996]; Chu et al., J.Am.Chem.Soc.118: 7827-7835 [1996]; Siudzak, Proc.Natl.Acad.Sci.USA 91: 11290-11297 [1994]; Burlingame Et al., Anal.Chem.68: 599R-651R [1996]; Wu et al., Chemistry &amp; Biology 4: 653-657 [1997]; and Loo et al., Am.Reports Med.Chem.31: 319-325 [1996]. ..</p><p> Target-compound conjugates can be identified using other means. For example, various chromatographic techniques such as liquid chromatography, thin layer chromatography, etc. can be used to enhance the ability to separate the components of the reaction mixture and identify covalent molecules. Such chromatographic techniques can be used in combination with or separated from the mass spectrometer. Also, to facilitate its identification using any of the above techniques, the liberated compound may be coupled with a probe labeled (fluorescently, radioactively, or otherwise). In yet another embodiment, the formation of a new bond releases the labeled probe, which can then be monitored. Simple functional assays, such as ELISA or enzyme assays, can also be used to detect binding when binding occurs in areas where the assay is essential to measure. Other techniques that may find use in identifying organic compounds bound to a target molecule include, for example, nuclear magnetic resonance (NMR), surface plasma resonance (eg, BIACORE), capillary electrophoresis, X-ray crystallography, etc. (All of these are well known to those skilled in the art).</p><p> In another aspect of the invention, the target is a protein, and the covalent or bond is a disulfide bond. This method is as follows: a) The step of contacting a target protein capable of forming a disulfide bond with a potential ligand (which can also form a disulfide bond); b) The step of forming a disulfide bond between the target protein and the ligand candidate, thereby forming a target protein-ligand bond; and c) Target protein-the step of identifying the ligand present in the ligand conjugate, Including.</p><p> If necessary, the target protein is contacted with the ligand candidate in the presence of a reducing agent. Examples of suitable reducing agents include, but are not limited to: cysteine, cysteamine, dithiothreitol, dithioerythritol, glutathione, 2-mercaptoethanol, 3-mercaptopropionic acid, tris-( Phosphine, such as 2-carboxyethyl-phosphine (TCEP), or sodium boron hydride. In one embodiment, the reducing agent is 2-mercaptoethanol. In another embodiment, the reducing agent is cysteamine. In another embodiment, the reducing agent is glutathione, in another embodiment, the reducing agent is cysteine.</p><p> In one embodiment, the target protein carries a naturally occurring -SH group derived from cysteine that is part of the naturally occurring protein sequence. In another embodiment, the target protein carries an engineered -SH group, where mutagenicity is used to mutate a naturally occurring amino acid to a cysteine. These target proteins with non-native cysteines embrace another aspect of the invention.</p><p> In another embodiment, the target protein possesses a masked-SH in the form of a disulfide. In another embodiment, the target protein possesses a cysteine in which the thiol is masked as a disulfide. In another embodiment, the target protein possesses a cysteine in which the thiol forms a disulfide bond with another cysteine. In another embodiment, the target protein possesses a cysteine in which the thiol forms a disulfide bond with glutathione. In another embodiment, the target protein is a thiol, of formula-SSR.¹Owns the cysteine that forms the disulfide of¹Is a non-replacement C₁-C₁₀Aliphatic compound, substitution C₁-C₁₀It is an aliphatic compound, an unsubstituted aryl or a substituted aryl. In another embodiment, the target protein is a thiol, of formula-SSR.²R³Owns cysteine masked as a disulfide of, where R²Is C₁-C₅Alkyl, and R³Is NH₂, OH, or COOH. In another embodiment, the target protein is a thiol, of the formula-SSCH.₂CH₂Possess cysteine masked as a disulfide of OH. In yet another embodiment, the target protein has a thiol of formula-SSCH.₂CH₂NH₂Owns cysteine that is masked as a disulfide.</p><p> In another embodiment, the ligand candidate carries the -SH group. In another embodiment, the ligand candidate possesses a masked thiol. Ligand candidates with masked thiol groups include another aspect of the invention. In another embodiment, the ligand candidate is of formula-SSR.¹Possess a masked thiol in the form of disulfide, where R¹Is a non-replacement C₁-C₁₀Aliphatic compound, substitution C₁-C₁₀It is an aliphatic compound, an unsubstituted aryl or a substituted aryl. In another embodiment, the ligand candidate is of formula-SSR.²R³Owns a thiol masked as a disulfide of, where R²Is C₁-C₅Alkyl (preferably -CH₂-, -CH₂CH₂-Or-CH₂CH₂CH₂-) And R³Is NH₂, OH, or COOH. In another embodiment, the ligand candidate is in formula-SSCH.₂CH₂Possess a thiol masked as a disulfide of OH. In yet another embodiment, the ligand candidate is of formula-SSCH.₂CH₂NH₂Possess a masked thiol as a disulfide. Examples of example ligand candidates include:</p><p><chemistry num="15"><img file="JP4391237B2_D0007.tif" /></chemistry>Here, R and R'are independent and unsubstituted C.₁-C₂₀Aliphatic compound, substitution C₁-C₂₀It is an aliphatic compound, an unsubstituted aryl, or a substituted aryl; m is 0, 1, or 2; and n is 1 or 2.</p><p> The plurality of ligand candidates includes a library of ligand candidates. In one embodiment, the library comprises at least 5 ligand candidates. In another embodiment, the library comprises at least 20 ligand candidates. In another embodiment, the library comprises at least 100 ligand candidates. In another embodiment, the library comprises at least 500 ligand candidates. In another embodiment, the library comprises at least 1000 ligand candidates. In another embodiment, each member of the library has a different molecular weight. In another embodiment, each member of the library has a different mass than another member of the library by at least 5 atomic weight units. In another embodiment, each member of the library has a different mass than another member of the library by at least 10 atomic weight units.</p><p> The tethering method, in which the target is a protein and the covalent bond is a disulfide, is schematically shown in Figure 1. FIG. 1A shows one embodiment of a binding method in which a thiol-containing protein reacts with multiple ligand candidates (eg,&gt; 5,&gt; 20,&gt; 100,&gt; 500,&gt; 1000, etc.). In this embodiment, the ligand candidate is of formula-SSR.¹Possess a masked thiol in the form of disulfide, where R¹Is as specified above. In certain embodiments, R¹Is selected to increase the solubility of possible ligand candidates. As shown, potential ligands possessing a unique binding affinity for the target have been identified, and corresponding ligands without the disulfide moiety have been made containing the identified binding determinants (indicated by circles). Will be done.</p><p> Figure 1B outlines the theory behind coupling. The thiol-containing protein is in equilibrium with at least one disulfide-containing ligand candidate, and an equilibrium has been established between the modified and unmodified proteins. In one embodiment, the thiol-containing protein and the ligand candidate are contacted in the presence of a reducing agent. In another embodiment, the thiol-containing protein and the ligand candidate are contacted in the presence of a quasi-stoichiometric amount of reducing agent. If the ligand candidate does not have a unique binding affinity for the target protein, this equilibrium shifts towards the unmodified protein. In contrast, if the ligand candidate has a unique affinity for the protein, the equilibrium shifts towards the modified protein. Both situations are shown in Figure 1B. First, the ligand candidate R^AThe moiety has little or no binding affinity for the protein. Thus, the formation of protein-ligand conjugates is a function of the probability of forming a disulfide bond at the concentration of a given protein, ligand candidate, and reducing agent. Second, the ligand candidate R^BThe moiety possesses a unique binding affinity for the protein. As a result, once a disulfide bond is formed between the protein and the ligand candidate, this protein-ligand bond is stabilized. Therefore, this equilibrium shifts towards the formation of protein-ligand conjugates.</p><p> To further illustrate the binding, this method has been applied to thymidylate synthase (TS), an enzyme essential for virtually all living organisms. TS, along with dihydrofolate reductase (DHFR) and serine hydroxymethylase, is a biochemical functional unit, the thymidylate synthase cycle (which is from RNA-based dUMP to DNA-based thymidine 5'-monophosphate (dTMP). It provides the only new pathway for synthesis). Both TS and DHRF are targets for anti-cancer drug development. Since the TS gene is also found in many viruses, it is also a target for the development of antiparasitic, antifungal and antiviral agents.</p><p> TS is a theoretical confirmation target for several reasons. First, the large number of high-resolution crystal structures of various TS enzymes have been determined so that structural information can be incorporated into compound design. Second, a simple colorimetric assay exists to determine if a potential ligand binds to TS. This assay is 5,10-CH in the presence of dUMP₂-H₄Phorate vs. H₂Depends on the rate of phorate conversion. The second assay for binding is also spectrophotometric and relies on competition with pyridoxal-5'-phosphate (PLP) to form a complex with TS with a unique spectral sign.</p><p> The TS selected for the purposes illustrated is E. coli TS. Like all TS enzymes, E. coli TS contains naturally occurring cysteine residues in the active site (Cys146) that can be used to bind. E. coli TS contains four other cysteines, which are not conserved and hidden among other TS enzymes and are therefore inaccessible. However, if one or more of these cysteines were reactive with respect to disulfides, then mutant versions of these enzymes could be used, in which in this mutant version these cysteines would be another amino acid (eg, for example. It is mutated to alanine).</p><p> In the first experiment, wild-type TS and C146S variants (where cysteine at position 146 was mutated to serine) were mutated to cystamine (H).₂NCH₂CH₂SSCH₂CH₂NH₂). The wild-type TS enzyme reacted cleanly with 1 equivalent of cystamine, but the mutant TS did not, which indicates that cystamine reacted with Cys-146 and was selective for Cys-146. Shown.</p><p> Wild-type TS was subjected to several binding experiments with different pools of ligand candidates. Figure 2 illustrates two typical binding experiments, where the ligand candidates are given the following equations:</p><p><chemistry num="16"><img file="JP4391237B2_D0008.tif" /></chemistry>Is.</p><p> This is the expression RSSR¹Is a particular embodiment of the genus of ligand candidates for, where R is R.^CC (= O) NHCH₂CH₂-Corresponds to, and R¹Is-CH₂CH₂NH₂Corresponds to. This is also the formula RSSR²R³Is a particular embodiment of the genus of ligand candidates for, where R is R.^CC (= O) NHCH₂CH₂-Corresponds to, and R²R³Together-CH₂CH₂NH₂Corresponds to. R^CIs a non-replacement C₁~ C₁₀Alkylate, substituted C₁~ C₁₀It is an alkyl, unsubstituted aryl or substituted aryl, and is a variable portion of the library members between this pool.</p><p> FIG. 2A is an inversely superimposed integral mass spectrum of the reaction of TS with a pool of 10 different ligand candidates with little or no binding affinity for TS. In the absence of any binding interactions, the equilibrium in the disulfide exchange reaction between TS and individual ligand candidates is towards the unmodified enzyme. This is schematically illustrated by the following equation.</p><p><chemistry num="17"><img file="JP4391237B2_D0009.tif" /></chemistry> As expected, the peak corresponding to the unmodified enzyme is one of the two most prominent peaks in the spectrum. The other prominent peak is TS in which the thiol of Cys146 is modified with cysteamine. This species does not form to a significant extent for any individual library member, but its peak is due to the cumulative effect of equilibrium reactions for each member of the library pool. If the reaction is carried out in the presence of a thiol-containing reducing agent (eg, 2-mercaptoethanol), the active site cysteine can also be modified with that reducing agent. Since cysteamine and 2-mercaptoethanol have similar molecular weights, their respective disulfide-bonded TS enzymes are indistinguishable under the conditions used in this experiment. The small peak to the right of it corresponds to another (discreet) library member. Notably, none of these peaks are prominent. FIG. 2A is characterized by a spectrum in which none of the ligand candidates has a unique binding affinity for the target.</p><p> FIG. 2B is an inversely superimposed mass spectrum of the TS reaction with a pool of 10 different ligand candidates. Here, one of the ligand candidates has a unique binding affinity for the enzyme. As can be seen, the most prominent peak is the peak corresponding to TS in which the thiol of Cys146 is modified with an N-tosyl-D-proline compound. This peak makes all other peaks appear small, including the peak corresponding to the unmodified enzyme and TS (the thiol of Cys146 is modified with cysteamine). FIG. 2B is an example of a mass spectrum that captures a moiety where the bond has a unique strong binding affinity for the desired site.</p><p> If the binding occurs in the presence of a reducing agent, this process is more thermodynamically driven and balanced. FIG. 3 illustrates this phenomenon, in which TS is reacted with the same library pool containing the selected compound N-tosyl-D-proline compound in the presence of an increasing concentration of reducing agent (2-mercaptoethanol). Three experiments are shown.</p><p> FIG. 3A is an inversely superimposed mass spectrum when the reaction is carried out without 2-mercaptoethanol. The most prominent peak corresponds to cysteamine-modified TS. However, the peak corresponding to N-tosyl-D-proline is nevertheless reasonably selected for other ligand candidates. FIG. 3B is an inversely superimposed mass spectrum of the reaction in the presence of 0.2 mM 2-mercaptoethanol. In contrast, for the spectrum in FIG. 3A, the peak corresponding to N-tosyl-D-proline is the most prominent peak and is therefore strongly selected for other ligand candidates. Finally, Figure 3C is an inversely superimposed mass spectrum of the reaction in the presence of 20 mM 2-mercaptoethanol. Not surprisingly, the most prominent peak under such strong reducing conditions is the unmodified enzyme. Nevertheless, the peak corresponding to N-tosyl-D-proline is still selected for the peaks of other ligand candidates in the library pool.</p><p> Figure 3 emphasizes the fact that the degree of cysteine modification in the target protein by a particular ligand candidate with a unique affinity for the target is partly a function of the reducing agent concentration. In general, the higher the binding affinity of a ligand candidate for a target protein, the higher the concentration of reducing agent that can be used, and the stronger the selection. As a result, the concentration of reducing agent used in binding screening can be used as a surrogate for binding affinity and to set the lower bound of binding affinity for which ligand candidates must be strongly selected.</p><p> In one aspect, this method involves the following steps: a) A step of contacting a target protein capable of forming a disulfide bond with a ligand candidate that can also form a disulfide bond; b) The step of forming a disulfide bond between this target protein and the ligand candidate, thereby forming a target protein-ligand bond; c) The step of contacting this target protein-ligand conjugate with the reducing agent; and d) The step of determining the concentration of reducing agent to reduce the amount of this target protein-ligand conjugate to the desired amount.</p><p> The concentration of reducing agent required to reduce the amount of target protein-ligand conjugate is then used as a surrogate for the binding affinity of the target protein for the ligand candidate.</p><p> Alternatively, this method can be used to calibrate the binding experiment. An example of such calibration is as follows. A first binding experiment is performed on multiple ligand candidates, where the more strongly selected ligand candidates are identified. Alternatively, a known substrate with a particular affinity is modified, for example, by the addition of a disulfide. The identified ligand candidates (or calibration compounds) are then used to calibrate the experimental conditions necessary to select only ligand candidates with a particular minimal binding affinity. In one embodiment, this calibration is the concentration of reducing agent, the calibration compound is used in a series of binding tests, where a constant concentration range of reducing agent is used. An example is as follows, and this method involves the following steps: a) The step of contacting a target protein capable of forming a disulfide bond with a calibration compound which can also form a disulfide bond; b) The step of forming a disulfide bond between this target protein and the calibration compound, thereby forming a target protein-calibration compound conjugate; c) The step of contacting this target protein-calibration compound conjugate with the reducing agent; and d) The step of determining the concentration of reducing agent required to reduce the amount of this target protein-ligand conjugate to the desired amount.</p><p> In general, lower concentrations of reducing agents result in higher proportions of targets being modified with calibration compounds and vice versa. In one embodiment, the desired amount is 50%. In other words, about 50% of the target protein is in unmodified form and the remaining about 50% is present as a target protein-calibration compound conjugate. Therefore, the concentration of reducing agent associated with the desired amount (in this case, about 50%) is used in subsequent binding experiments where the ligand candidate needs to have a somewhat lower level of binding affinity of choice. Will be done. Examples of other desired amounts that can be used depending on the desired lower level of binding affinity include about 20%, 25%, 30%, 40%, 60%, 75% and the like.</p><p> As previously mentioned, the binding method can be used with a single ligand candidate or with multiple ligand candidates. In a preferred embodiment, this binding method is used to screen multiple ligand candidates (eg, 5, 20, 100, 500, 1000, and even&gt; 1000) to maximize throughput and efficiency. To. FIG. 4 shows the experimental results in which the number of ligand candidates in the library pool was changed. This experiment shows that N-tosyl-D-proline is strongly selected even when its pool contains 100 ligand candidates, but a larger number of ligand candidates (eg, about&gt; 500,&gt; Libraries containing 750,&gt; 1000) are now commonly used.</p><p> The structure-activity relationship (SAR) can be developed using information from binding experiments in exactly the same way that SAR was developed using traditional assays. For example, R on the left side of the schematic diagram below^CLigand candidates with E. coli TS were strongly selected for E. coli TS, but on the right side R^CLigand candidates with the above were not selected.</p><p><chemistry num="18"><img file="JP4391237B2_D0010.tif" /></chemistry> Based on data from screening of about 1200 compounds, it was determined that a phenyl-sulfonamide core and a proline ring are essential. For example, TS is fairly flexible around the phenyl ring, which may be substituted or substituted in a range of certain groups including methyl, t-butyl, and halogen. It seems to adapt, but its presence is necessary for choice. Similarly, the proline ring appears to be essential, as no compound substituted with phenylalanine, phenylglycine or pyrrole was selected.</p><p> In addition to the above, further experiments were performed to ensure that the compounds selected from the binding corresponded to compounds having a binding affinity for the target. In one example, the binding experiment is performed in the presence of a known substrate. If the selected ligand candidate has a unique binding affinity for the target, it is resistant to substrate substitution. In contrast, ligand candidates lacking a unique binding affinity or cysteamine are readily replaced by the substrate. Another example is a traditional enzyme assay for unbound analogs. For example, the R of the ligand fragment^CThe part is below:</p><p><chemistry num="19"><img file="JP4391237B2_D0011.tif" /></chemistry>Affinity was determined using Michaelis-Mention dynamics. Free acid 1 K_iWas 1.1 ± 0.25 mM. It should be noted that the free acid competed with the natural substrate dUMP. Therefore, N-tosyl-D-proline 1 is a weak but competitive inhibitor of TS.</p><p> In another embodiment, a naturally occurring cysteine residue in the active site was mutated to serine (C146S) and another cysteine was introduced (L143C or H147C). Binding using the C146S / L143C mutant produced results similar to wild-type enzymes. It should be noted that the N-tosyl-D-proline analog was strongly selected. In contrast, C146S / H147C did not select the N-tosyl-D-proline analog, but selected some other molecules. These results are believed to reflect differences in the local binding environment surrounding reactive cysteines and the geometric constraints of the disulfide linker.</p><p> X-ray crystallography was used to elucidate the three-dimensional structure of native enzymes and some complexes, confirming that the information obtained from the binding could correlate with abundant binding to the target. Table 1 details the crystallographic data and refinement parameters. One complex is a complex of free acids of N-tosyl-D-proline bound to TS (4th description in Table 1). Another complex is a complex of N-tosyl-D-proline derivatives bound to the active site cysteine (Cys-146) (second description in Table 1). Yet another complex is a complex of N-tosyl-D-proline derivatives bound to the C146S / L143C mutant (3rd description in Table 1).</p><p><tables num="1"><img file="JP4391237B2_D0012.tif" /></tables> This is not a "true" free R factor, as the starting model was a completely refined structure. However, the set of free R factors for the reflex was kept constant for each of the above subdivisions. * 12₁Three crystals contain one monomer per asymmetric unit. P6₃Morphology includes biologically related homodimers.^†The value in parentheses is the highest resolution bin.^$R_sym(l) = Σ_hki| l_hki(l_hki) | / Σ_hkil_hki(Here l_hkiIs a reflection_hkiIs the strength of)^§R_cryst= Σ_hki|| F_obs|-| F_calc|| / | F_obs| (Here F_obsAnd F_calcAre the observed and calculated structural factors for the data used in the subdivision, respectively)^¶R_tree= Is Σ_hki|| F_obs|-| F_calc|| / | F_obs| (Here F_obsAnd F_calcAre the observed and calculated structural factors for 10% of the data omitted from the subdivision, respectively) Importantly, the positions of the N-tosyl-D-proline moieties are very similar in all three cases (0.55 to 1.88 compared to 0.11 to 0.56 Å for all Cα carbons in the protein). Å RMSD). The fact that the N-tosyl-D-proline substituents are tightly overlapped while the alkyl-disulfide bond is concentrated in this moiety derived from a different cysteine residue is the fact that the N-tosyl-D-proline moiety ( Supports the notion that (rather than binding) is a binding determinant.</p><p> As can be seen, binding is a potent method that can identify ligands that bind to the site of interest in the target. Bindings can be used alone or in combination with other medicinal chemistry methods to identify and optimize drug candidates.</p><p> In one aspect of the invention, binding is a binding determinant (eg, R).^C), And then traditional medicinal chemistry is used to make higher affinity compounds containing the identified binding determinants or variations thereof. In one embodiment, binding can be used both to identify binding determinants and to assess whether a compound binds to a target with higher affinity. For example, binding is an alternative to traditional binding experiments where either functional assays are not available or are susceptible to artifacts. This approach is schematically illustrated in Figure 5. As can be seen, the binding is a binding determinant R^DUsed to identify. Once such binding factors have been identified, traditional medicinal chemistry approaches have been used to R in the modified library.^DVariants are synthesized. The modified library of ligand candidates is R^DIncludes variants of (eg, its isostere and homologues). The modified library is also R^DAlternatively, it may include a variant thereof, as well as an "expanded" compound containing other binding determinants that may utilize adjacent binding regions. FIG. 5 illustrates selected compounds from a modified library, where the original binding determinant R^DIs R^D'The selected compound was modified to the second binding determinant R.^Eincluding. Example 6 further illustrates this method for an attempt to optimize TS low μM affinity compounds (2 and 3) identified from the optimization of compound 1, which is a low μM compound.</p><p> In another aspect of the invention, methods are provided for identifying two binding determinants that are substantially bound together. In general, this method involves the following steps: a) Steps to identify the first compound that binds to the target protein; b) Steps to identify a second compound that binds to this target protein; c) The step of binding the first compound and the second compound via a linker element to form a conjugate molecule that binds to the target protein. In a preferred embodiment, the conjugate molecule binds to the target protein with a higher binding affinity than either the first compound or the second compound alone.</p><p> In one embodiment, the first compound is of formula R^DSSR¹The second compound is of formula R^ESSR¹Compounds (where R and R¹Is as described above, R^DAnd R^EAre independent of each other, C₁~ C₂₀Aliphatic, C₁~ C₂₀It is a substituted aliphatic, unsubstituted aryl, or substituted aryl), and the first and second compounds bind to the target protein via a disulfide bond. FIG. 6 is a schematic illustration of this method, where the binding determinant R is used using two separate binding experiments.^DAnd R^ETo identify. These binding determinants are then combined together to form a conjugate molecule that binds to the target protein.</p><p> In another embodiment, the binding determinant R^DAnd R^EThis binding experiment to identify is occurring at the same time. In this way, the two identified binding determinants are confirmed to bind to the target protein at non-overlapping sites. Therefore, this method involves the following steps: a) Steps to identify the first compound that binds to the target protein; b) The step of identifying a second compound that binds to this target protein in the presence of the first compound that binds to this target protein; c) The step of binding the first compound and the second compound via a linker element to form a conjugate molecule that binds to this target protein. FIG. 7 is a schematic example of this method. In the first binding experiment, the binding determinant R^DIs identified. Once R^DOnce identified, either a second reactive cysteine is introduced or the mask is removed and the binding determinant R^EBinding experiments to identify binding determinants R^DOccurs in the presence of. These two binding determinants R^DAnd R^EIs then bound to form a conjugate molecule that binds to its target protein.</p><p> In another embodiment, the first compound is identified using binding and the second compound is identified through a non-binding method. In one embodiment, this non-binding method comprises rational drug design and traditional medicinal chemistry. The crystal structure of N-tosyl-D-proline bound to TS revealed that its tosyl group was in approximately the same position and orientation as the benzamide moiety of methylenetetrahydrofolic acid (a natural cofactor of the TS enzyme). .. In conclusion, the glutamic acid moiety of methylenetetrahydrofolic acid was grafted to compound 1. Table 2 shows a selected number of these compounds.</p><p><tables num="2"><img file="JP4391237B2_D0013.tif" /></tables> There is significant selectivity for D-enantiomer (Compound 5) of proline over L-enantiomer (Compound 4), and the α-carboxylate of glutamate residues is important. This is because removing α-carboxylate (Compound 12) or converting it to a primary amide (Compound 10) correlates with a significant loss of binding affinity.</p><p> In another aspect of the invention, variations on the binding method are provided for use in making and optimizing compounds. In general, this method involves the following steps: a) The step of providing a target having an anchoring group capable of forming a covalent bond, or coordinating a metal at or near the site of interest; b) The step of contacting this target with an extender to form a target-extender complex, where the extender either forms a covalent bond or coordinates a metal. A step comprising a first functional group and a second functional group capable of forming a covalent bond; c) The step of contacting this target-extender complex with a candidate ligand containing a group capable of forming a covalent bond with a second functional group; d) The step of forming a covalent bond between this target-extender complex and this candidate ligand; e) The step of identifying candidate ligands present in this target-extender-ligand bond.</p><p> In one embodiment, the stationary group in this target is a reactive nucleophilic or electrophilic group, forming an irreversible covalent bond with the first functional group of the extender. In another embodiment, the stationary group in the target is a reactive nucleophilic or electrophilic group, forming an irreversible covalent bond with the first functional group of the extender. In another embodiment, the fixing group in the target is a metal coordination site, and the fixing group is combined with the first functional group to form a metal coordination site. Examples of suitable metals that can bind to such sites include Cd, Hg, As, Zn, Fe, Cu, Ni, Co and Ca. In another embodiment, the second functional group is a reactive nucleophile or reactive electrophile.</p><p> In a preferred embodiment, the extender comprises a binding determinant comprising the first and second functional groups described above and having a unique binding affinity for the target. If the binding determinants do not already contain the first and second functional groups, they can be modified to contain them. In one method, binding is a binding determinant R^CUsed to identify, then this R^CIs modified to include the first and second functional groups. Alternatively, the binding determinant is obtained from a known substrate or fragment thereof of the target.</p><p> In another embodiment, the fixed group in the target is a reactive nucleophile and the extender is a first functional group capable of forming a covalent bond with the nucleophile and a second functional group capable of forming a disulfide bond. Includes groups. This method involves the following steps: a) The step of providing a target having a reactive nucleophile at or near the site of interest; and b) The step of contacting the target with the extender, thereby forming a target-extender complex, where the extender reacts with the nucleophilic group in the target to form a covalent bond. A step comprising a first functional group to form and a second functional group capable of forming a disulfide bond; c) The step of contacting this target-extender complex with a ligand candidate capable of forming a disulfide bond; d) The step of forming a disulfide bond between this target-extender complex and this ligand candidate, thereby forming a target-extender-ligand bond; e) The step of identifying ligand candidates present in this target-extender-ligand bond. If necessary, the target is contacted with the ligand candidate in the presence of a reducing agent.</p><p> Examples of suitable reducing agents include, but are not limited to: cysteine, cysteamine, dithiothreitol, dithiothreitol, glutathione, 2-mercaptoethanol, 3-mercaptopropionic acid, phosphine (eg, tris-). (2-carboxyethyl-phosphine) (TCEP), or sodium borohydride. In one embodiment, the reducing agent is 2-mercaptoethanol. In another embodiment, the reducing agent is cysteamine. In another embodiment, the reducing agent is glutathione. In another embodiment, the reducing agent is cysteine.</p><p> In one embodiment, the target comprises -OH as a reactive nucleophile, the extender of which is a first functional group and a disulfide bond capable of forming a covalent bond with the reactive nucleophile on the target. Contains a second functional group capable of forming. In another embodiment, the reactive nucleophile on this target is -OH derived from serine, threonine, or tyrosine, which is part of a naturally occurring protein sequence. In another embodiment, the reactive nucleophile on this target is an engineered -OH group. Here, mutagenesis is used to mutate naturally occurring amino acids into serine, threonine, or tyrosine. In another embodiment, the first functional group of the extender is boronic acid and the second functional group is -SH or masked -SH. An example of masked-SH is the expression-SSR¹(Here R¹Is a disulfide (as described above).</p><p> In another embodiment, the target comprises -SH as a reactive nucleophile and the extender has a first functional group and a disulfide bond capable of forming a covalent bond with the reactive nucleophile on this target. Contains a second functional group that can be formed. In one embodiment, the reactive nucleophile on this target is naturally occurring-SH, which is derived from cysteine, which is part of the naturally occurring protein sequence. In another embodiment, the reactive nucleophile on this target is an engineered-SH group. Here, mutagenesis was used to mutate naturally occurring amino acids to cysteine.</p><p> In another embodiment, the target protein has -SH masked in the form of a disulfide as a reactive nucleophile. In another embodiment, the target protein comprises cysteine, where the thiol is masked as a disulfide. In another embodiment, the target protein comprises cysteine, where the thiol is masked as a disulfide bond with another cysteine. In another embodiment, the target protein comprises cysteine, where the thiol is masked as a disulfide bond with glutathione. In another embodiment, the target protein comprises cysteine, where its thiol is of formula-SSR.¹(Here R¹Is masked as a disulfide (as described above).</p><p> In one embodiment, the first and second functional groups of the extender are each independently -SH or masked -SH. An example of a masked thiol is the formula-SSR¹(Here R¹Is a disulfide (as described above). In this embodiment, the covalent bond formed between the target and the extender is a disulfide bond and thus a reversible covalent bond. In one variation of this method, the target is contacted with an extender, after which the target-extender complex is contacted with one or more ligand candidates. In another variation, the target is contacted with an extender and a pool containing one or more ligand candidates.</p><p> In another embodiment, the first functional group is a group capable of forming an irreversible covalent bond with the reactive nucleophile of the target under conditions that do not denature the target, and the second functional group is-. SH or masked-SH. In the first embodiment, the first functional group is a group capable of undergoing SN2-like addition. Examples of such extenders include (i) α-haloic acid:</p><p><chemistry num="20"><img file="JP4391237B2_D0014.tif" /></chemistry>(ii) For example, the following fluorophosphonates:</p><p><chemistry num="21"><img file="JP4391237B2_D0015.tif" /></chemistry>(iii) For example, the following epoxides:</p><p><chemistry num="22"><img file="JP4391237B2_D0016.tif" /></chemistry>(iv) For example, the following aziridine:</p><p><chemistry num="23"><img file="JP4391237B2_D0017.tif" /></chemistry>(v) For example, the following thirane:</p><p><chemistry num="24"><img file="JP4391237B2_D0018.tif" /></chemistry>(vi) For example, the following halomethyl ketone / amide:</p><p><chemistry num="25"><img file="JP4391237B2_D0019.tif" /></chemistry>Where R is unsubstituted C₁~ C₂₀Aliphatic, permuted C₁~ C₂₀Aliphatic, unsubstituted aryl, and substituted aryl; R^’Is H, -SR¹And here R¹Is as defined above; and X is a leaving group. Examples are halogen, N₂, OR, -P (= O) Ar2, -NO (C = O) R,-(C = O) R, -SR and vinyl sulfone. In these and other structures exemplified below, the box indicates a binding determinant within a small molecule extender (SME), i.e., a portion of the SME having a binding affinity for the target.</p><p> In another embodiment, the first functional group is a group capable of undergoing SNaryl-like additions. Examples of suitable groups are 7-halo-2,1,3-benzoxadiazaole, and, for example, ortho / paranitro-substituted halobenzenes such as:</p><p><chemistry num="26"><img file="JP4391237B2_D0020.tif" /></chemistry>(Here R^’And X are as specified above).</p><p> In another embodiment, the first functional group is a group that is capable of receiving Michael-type additions. Examples of suitable groups are electron withdrawing systems (eg, carbonyl, imine, kinin, CN, NO).₂, And any part containing a double or triple bond adjacent to -S (= O)-). Examples of such extenders include:</p><p><chemistry num="27"><img file="JP4391237B2_D0021.tif" /></chemistry>Here R^’Is as specified above.</p><p> Extenders are often tailored to a particular target or family of targets. Examples of kinase-specific extenders include:</p><p><chemistry num="28"><img file="JP4391237B2_D0022.tif" /></chemistry>Here R^a, R^{b b}, R^c, R^d, R^e, And R^fIndependently, hydrogen, C₁~ C₅Alkyl, C₁~ C₅Alkylamines, and aryls (where at least one R group on the extender is the Michael acceptor and the other R group is-(CH).₂)_n-SR'; -C (= O)-(CH₂)_n-SR'; -O- (CH₂)_n-SR';-(CH₂)_n-Selected from the group consisting of SR'; and thiol protecting groups, where R'is as described above. Examples of suitable Michael acceptors include:</p><p><chemistry num="29"><img file="JP4391237B2_D0023.tif" /></chemistry> Examples of protease-specific extenders include:</p><p><chemistry num="30"><img file="JP4391237B2_D0024.tif" /></chemistry> The first functional group in these extenders is the metal coordination site and the second functional group is -SSCH.₂CH₂NH₂A thiol masked in the form of-SSR¹Can be in the form of, where R¹Is as described above. These extenders bind to serine proteases only in the presence of zinc (Katz et al., Nature 391: 608-12 (1998); Katz and Luong, J. Mol. Biol. 292: 669-84 (1999); Janc. Et al., Biochemistry 39: 4792-800 (2000)). A version of this compound lacking a second functional group binds to the active site of serine proteases through the active sites histidine and serine, as shown below.</p><p><chemistry num="31"><img file="JP4391237B2_D0025.tif" /></chemistry> FIG. 8 shows one embodiment of a binding method using an extender. As shown, the target containing the reactive nucleophile-SH has a first functional group X capable of forming a covalent bond with this reactive nucleophile-SR and a second functional group-SR capable of forming a disulfide bond.^1’(Here R^1’Is the R specified above¹Is contacted with an extender containing). A bond-extender complex is formed, which is then contacted with multiple ligand candidates. This extender provides one binding determinant (circle), the ligand candidate provides a second binding determinant (square), and the resulting binding determinants bind together. Form a conjugate compound.</p><p> Form reversible or irreversible covalent bonds between reactive groups and ligands on the target, between targets and elongation factors, between target-elongation factor complexes and ligands, or between two ligands. Synthetic methods for this are well known in the art and are basic textbooks (eg, March, Advanced Organic Chemistry, John Wiley &amp; Sons, New). It is described in York, 4th edition, 1992). Reductive aminations between aldehydes, ketones and amines are described, for example, in March et al. (Supra), pp. 898-900; alternative methods for preparing amines are described in pp. 1276; hydrazone and hydrazone derivatives ( For example, the reaction between aldehydes, ketones and hydrazine derivatives to obtain (semicarbazone) is on pages 904-906; amide bond formation is on page 1275; urea formation is on page 1299; thiocarbamate. The formation is on page 892; the formation of carbamate is on page 1280; the formation of sulfonamide is on page 1296; the formation of thioether is on page 1297; the formation of disulfide is on page 1284; the formation of ether is on page 1285. On page; ester formation on page 1281; epoxide addition on page 368; azilysin addition on page 368; acetal and ketal formation on page 1269; carbonate formation on page 392; The formation of denamines is on page 1264; the compound decomposition of alkens is on pages 1146-1148 (see also Grubbs et al., Acc. Chem. Res. 28: 446-453 [1995]); alcans and acetylenes. The transition metal-catalyzed coupling of aryl halides and sulfonates with, for example, the Heck reaction, is described on pages 717-178; Reaction of aryl halides and sulfonates with organic metal reagents (eg, organic boron reagents). On page 662 (see also Miyaura et al., Chem. Rev. 95: 2457 [1995]); Organic tin and organic zinc reagents, formation of oxazolidine (Ede et al., Tetrahedron Letts. 28: 7119-7122 [ 1997]); Formation of thiazolidine (Patek et al., Tetrahedron Letts. 36: 2227-2230 [1995 [); Amine linked via amidin group by coupling of amine via imide ester (Davies et al., Canadian J. Biochem) ..</p><p> To further illustrate the binding method using elongation factors, this method was applied to the anti-apoptotic target caspase-3 (a member of the cysteine aspartyl protease family). Currently, about a dozen members of the caspase family are known, many of which are involved in the inhibition or transmission of the apoptotic cascade. Caspases have a variety of therapeutic indications, including excessive or abnormal levels of programmed cell death (eg, seizures, traumatic brain injury, spinal cord injury, Alzheimer's disease, Huntington's disease, Parkinson's disease, cardiovascular disease, liver). It is a potential drug target for (deficiency, and sepsis). In addition, caspase-3 contains naturally occurring cysteine residues in the active site and is well characterized both functionally and crystallographically.</p><p> Suitable elongation factors for use in the caspase-3 active site were designed using the fact that small aspartyl-based arylacyloxymethyl ketones are known to react irreversibly with cysteine in the active site. .. Examples 7-10 and 14 describe the synthesis of five representative elongation factors. These elongation factors can also be used in binding experiments with other caspase targets (eg, caspase-1 and caspase-7). The two elongation factors described in more detail are compounds 13 and 14.</p><p><chemistry num="32"><img file="JP4391237B2_D0026.tif" /></chemistry> As can be seen, compounds 13 and 14 include an aspartic acid moiety as a binding determinant. In particular, the carbonyl of this aspartic acid moiety is also part of the first functional group (arylacyloxymethylketone moiety) that forms a covalent bond with the thiol of the active site cysteine. Elongation factors 13 and 14 also contain a second functional group, masked in the form of thioester-SH, which can be unmasked at the appropriate time. For example, this thioester can be converted to a free thiol by treating the target-extension factor complex with hydroxylamine.</p><p> Both elongation factors have been shown to selectively modify caspase-3 in the active site cysteine, and treatment with hydroxylamine produced the following target-extension factor complexes:</p><p><chemistry num="33"><img file="JP4391237B2_D0027.tif" /></chemistry> Example 11 describes in more detail the procedure for modifying caspase-3 with elongating factor 13 to produce the target-elongating factor complex 13'.</p><p> Target-elongation factor complexes 13'and 14'were used in methods of binding to a library of approximately 10,000 ligand candidates, respectively. Typical examples of selected ligand candidates using the target-elongation factor complex 13'are:</p><p><chemistry num="34"><img file="JP4391237B2_D0028.tif" /></chemistry> Typical examples of selected ligand candidates using the target-elongation factor complex 14'are:</p><p><chemistry num="35"><img file="JP4391237B2_D0029.tif" /></chemistry> In particular, ligand candidate 15 was not selected by target-elongation factor complex 14', and ligand candidate 16 was not selected by target-elongation factor complex 13'. The structure-activity relationship between the selected compounds was also clear. For example, Ligand Candidate 17:</p><p><chemistry num="36"><img file="JP4391237B2_D0030.tif" /></chemistry>(It is identical to Ligand Candidate 15 except that it lacks a hydroxyl group) was not selected by either the target-elongation factor complex 13'or 14'.</p><p> Two structures of the target-elongation factor ligand complex were determined to evaluate how the elongation factor and the selected ligand candidate bind to the target. General crystallographic procedures are further described in Example 12. The first structure was a conjugate formed when the target-elongation factor complex 13'was contacted with ligand candidate 15. The second structure was a conjugate formed when the target-elongation factor complex 14'was contacted with ligand candidate 16. Table 3 summarizes selected crystallographic data for these structures.</p><p><tables num="3"><img file="JP4391237B2_D0031.tif" /></tables> In particular, the aspartic acid portion of both elongation factors could overlap with aspartic residues in known tetrapeptide substrates. With respect to the binding determinants of Ligand Candidate 15, salicylic acid sulfonamides make numerous contacts with proteins containing four hydrogen bonds. This salicylic acid moiety occupies the P4 pocket of the enzyme that preferentially recognizes aspartic acid in caspase-3. With respect to the binding determinants of Ligand Candidate 16, this sulfone makes some of the same contacts as salicylic acid.</p><p> Considering that binding determinants from elongation factors and ligand candidates create generative contacts with the active site of caspase-3, we designed compounds in which disulfides were replaced by more stable bonds. Furthermore, a derivative for probing the SAR of the binding determinant was prepared. For conjugates containing elongation factor 13 and ligand candidate 15, target-extension factor ligand conjugates include:</p><p><chemistry num="37"><img file="JP4391237B2_D0032.tif" /></chemistry> From this conjugate, a class of potent caspase-3 inhibitors was created, including the following moieties:</p><p><chemistry num="38"><img file="JP4391237B2_D0033.tif" /></chemistry> Table 4 shows four representative examples of compounds made based on conjugates for both optimization and SAR.</p><p><tables num="4"><img file="JP4391237B2_D0034.tif" /></tables> As can be seen, a conservative approach was taken in which the two sulfur atoms were replaced with two methylene units and the arylacyloxymethyl ketone (first functional group) was replaced with a simple aldehyde, with compound 18 (2.8 μM). K_iA potent inhibitor of caspase-3) was obtained. Removal of the hydroxyl group to give compound 19 reduced the affinity by a factor of 5, confirming the SAR observed on the binding screen. The entire binding affinity was eliminated by removing both the hydroxyl group and the acid moiety to give compound 20. Modeling studies suggest that replacing the methylene linker with a rigid aminobenzyl moiety effectively crosslinks the distance between the aspartyl group and salicylic acid, while reducing the entropy cost of the linker. In fact, as can be seen, compound 21 is 10 times greater than compound 18 K._iHave.</p><p> Similarly, a novel class of caspase-3 inhibitors arose from target-elongation factor ligand conjugates, including elongation factor 14 and ligand candidate 16.</p><p><chemistry num="39"><img file="JP4391237B2_D0035.tif" /></chemistry> In one embodiment, the compound comprises:</p><p><chemistry num="40"><img file="JP4391237B2_D0036.tif" /></chemistry> In another embodiment, the compound is a compound having the following structure:</p><p><chemistry num="41"><img file="JP4391237B2_D0037.tif" /></chemistry>Where X is CH₂, S, SO, SO₂, And R⁵Is an unsubstituted or substituted aryl. In another embodiment, R⁴Is an unsubstituted heteroaryl or a substituted heteroaryl. A typical example of this class of compounds is 0.33 μM K._iIt is a compound 22 having.</p><p> Examples 13 and 15-21 describe further details of the best-selling caspase-3 inhibitors synthesized based on the use of bindings using elongation factors 13 and 14.</p><p> The salicylic acid sulfonamide-containing compounds of the present invention are even more valuable. Identification of salicylic acid sulfonamides as suitable P4 binding fragments has not been found using conventional medical science. Using compound 21 as an example, the salicylic acid sulfonamide-free version of compound 21 is about 28 μM K._iSo, it inhibits caspase-3. Addition of salicylic acid sulfonamide to this fragment improved binding by about 200-fold and about 0.16 μM K._iProduces compound 21 having. In contrast, when a known tripeptide (eg, Compound I) that binds to the P1-P3 site of caspase-3 is used as a starting point, the binding affinity is reduced.</p><p><chemistry num="42"><img file="JP4391237B2_D0038.tif" /></chemistry> As can be seen, compound I is 0.051 μM K_iAnd addition of a salicylic acid sulfonamide moiety to this compound yields Compound II, which exhibits a reduction of about 300-fold in binding affinity. Due to this dramatic reduction, investigating P4 binding to tripeptides did not result in the identification of salicylic acid sulfonamides as suitable P4 binding fragments. However, compounds with this fragment available for binding to P4 are potent inhibitors. As a result, this example emphasizes the binding force for identifying important fragments that may not be found using conventional methods. As seen in the case of caspase-3, these fragments can be combined together to form a potent antagonist or agonist of the target of interest.</p><p> The present invention will be further described by the following non-limiting re-executions.</p>

(Example 1) Several variants of the unmodified or "wild-type" E. coli TS enzyme were made, overexpressed and purified in E. coli strain χ2913 (with the TS gene eliminated). This χ2913 strain requires thymidine supplementation. This is because the (deleted) TS gene is essential for survival. The first mutant is the active site cysteine replaced with serine (abbreviated as C146S). The second and third mutants contain, in addition to the C146S mutation, a non-native cysteine introduced into the active site. The second variant contains cysteine instead of leucine at residue 143 and is shown as C146S / L143C. The third variant contains cysteine instead of histidine at residue 147 and is shown as C146S / H147C. Other variants included D169C, W83C, and I79C, which maintained the active site cysteine (C146).

(Example 2) Disulfide-containing library members from commercially available carboxylic acids and mono-N- (tert-butoxycarbonyl) -protected cystamines (mono-BOC-cystamines) to Parlow and collaborators (Mol. Diversity). It was made by applying the method of 1: 266-269 (1995)). Briefly, 260 μmol of each carboxylic acid on 130 μmo 1 equivalent 4-hydroxy-3-nitrobenzophenone on polystyrene resin, 1,3-diisopropylcarbodiimide (DMF) in N, N-dimethylformamide (DMF). Fixed using DIC "). After 4 hours at room temperature, the resin was rinsed with DMF (2x), dichloromethane (DCM, 3x), and tetrahydrofuran ("THF", 1x) to remove unbound acid and DIC. This acid was cleaved from the resin via amide formation with 66 μmol of mono-BOC protected cystamine in THF. After a 12 hour reaction at ambient temperature, the solvent was evaporated and the BOC groups were removed from the unbound halves of each disulfide using 80% trifluoroacetic acid (TFA) in DCM. This product was characterized by HPLC-MS, and a substantially pure product was used without further purification. A total of 530 compounds were made using this methodology.

The library was also constructed from mono-BOC-protected cystamines and various sulfonyl chlorides, isocyanates, and isothiocyanates. For sulfonyl chlorides, 10 μmol of each sulfonyl chloride is coupled with 10.5 μmol of mono-BOC protected cystamine in THF (containing 2% diisopropylethylamine) in the presence of 15 milligrams of poly (4-vinylpyridine). It was. After 48 hours, the poly (4-vinylpyridine) was removed by filtration and the solvent was evaporated. BOC groups were removed using 50% TFA in DCM. For iso (thio) cyanates, 10 μmol of each isocyanate or isothiocyanate was coupled with 10.5 μmol of mono-BOC-protected cystamine in THF. After a 12 hour reaction at ambient temperature, the solvent was evaporated and the BOC groups were removed using 50% TFA in DCM. A total of 212 compounds were made using this methodology.

Finally, an oxime-based library, 10 μmol of a particular aldehyde or ketone, 10.5 μmol of HO (CH) in 1: 1 methanol: chloroform (with 2% acetic acid added).₂)₂SS (CH)₂)₂ONH₂And, it was constructed by reacting at ambient temperature for 12 hours to obtain an oxime product. A total of 448 compounds were made using this methodology.

Individual library members were dissolved in either acetonitrile or dimethyl sulfoxide to a final concentration of 50 mM or 100 mM. Each of these aliquots is then pooled into groups of 8-15 distinct compounds, with each member of this pool having a unique molecular weight.

(Example 3) The N-tosyl-proline derivative was synthesized as follows. Proline methyl ester hydrochloride was reacted with 4- (chlorosulfonyl) benzoic acid and sodium carbonate in water. The product was converted to pentafluorophenyl ester by reacting with trifluoroacetate pentafluorophenyl and pyridine in N, N-dimethylformamide and purified by flash chromatography. The activated ester is then reacted with a methyl ester of glutamate (or any other amino acid tested) in the presence of triethylamine and dichloromethane, the product is purified by flash chromatography and the methyl. The ester was hydrolyzed with lithium hydroxide in water. The final product was purified by reverse phase HPLC and lyophilized.

Alternatively, the above procedure was started with proline t-butyl ester. After coupling the amino ester to benzoic acid, this t-butyl ester was removed with triethylsilane as a scavenger with 50% TFA in DCM. The free acid was then converted to pentafluorophenyl ester as described above and reacted with the appropriate amine. The methyl ester was hydrolyzed in water with lithium hydroxide and the final product was purified by reverse phase HPLC and lyophilized.

(Example 4) The disulfide library screening was performed as follows. In a representative experiment, 1 μl DMSO solution containing a library of 8-15 disulfide-containing compounds was added to 49 μl protein-containing buffer. These compounds were selected so that each had a unique molecular weight. Ideally, these molecular weights differ by at least 10 atomic weight units (amu), as the analysis is clear. A pool of 8-15 disulfide-containing compounds was typically used for ease of analysis, but larger pools may be used. The protein is present at a concentration of about 15 μM and each of the disulfide library members is present at about 0.2 mM, so the total concentration of all disulfide library members is about 2 mM. Screening was performed in buffer containing 25 mM potassium phosphate (pH 7.5) and 1 mM 2-mercaptoethanol, but other buffers and reducing agents may be used. These reactions were equilibrated at ambient temperature for at least 30 minutes. These conditions can vary considerably depending on the ease with which the protein is ionized in the mass spectrometry system (see below), the reactivity of a particular cysteine, and the like. In the case of TS, it was found that the above conditions were satisfactory. No special effort was made to eliminate oxygen or foreign metal ions; at the time scale of these reactions, there are sufficient free thiols to facilitate disulfide exchange.

After equilibration, the reaction was injected into HP1100 HPLC and chromatographically separated on a C18 column mounted on a mass spectrometer (Finnigan MAT LCQ). Multiple charged ions from the protein were analyzed using available software (Xcalibur) to reveal the mass of this protein. The identification of any library member bound to this protein via a disulfide bond was then readily determined by subtracting the observed mass from the known mass of the unmodified protein. This process assumes that the attachment of library members does not dramatically change the ionization properties of the protein itself. This is a conservative assumption, often due to the fact that proteins are at least 20 times larger than any given library member. This assumption was confirmed by demonstrating that small molecules selected by one protein are not selected by another protein.

(Example 5) Crystals were grown as previously described in Perry et al., Proteins 8: 315-333 (1990), with the exception of including 1 mM compound for non-covalent complexes in the crystallization buffer. It was. Crystals are 70% saturated (NH) before data collection₄)₂SO₄, 20% glycerol, 50 mM K₂HPO₄Transferred to a solution containing (pH 7.0). For the non-covalent N-tosyl-D-proline complex, 10 mM compound was added to the immersion solution; for other complexes, 1 mM compound was included. Diffraction data were collected at -170 ° C using a Rigaku RU-3R generator and R-axis-IV detector and processed using d * TREK. Since these crystals were isomorphic to the previously described structures (PDB code 1TJS for I2 13 morphology and 2TSC for P63 morphology), purification was initiated by rigid body purification using REFMAC (CCP4). This protein model is referred to as INSIGHT-II (MSI, San). Prepared using the compound model constructed in Diego), and a PROTIN (CCP4) dictionary was made using MAKEDIC (CCP4). Purification with position factors and individual isotropic temperature factors was performed with REFMAC (CCP4) using all reflections in the shown resolution range. Solvent molecules were automatically placed using ARPP (CCP4) and purification was continued until no intervening features remained in the Fo-Fc difference map. PDB registration numbers are native C146-bound N-tosyl-D-proline, L143C-bound N-tosyl-D-proline, N-tosyl-D-proline free acid immersion, glutamate-N-tosyl-D-proline immersion, and For glutamate-N-tosyl-D-proline β-alanine crystals, 1F4B, 1F4C, 1F4D, 1F4E, 1F4F, respectively.

(Example 6) Selected N-tosyl-D-proline compounds were optimized and tested as a series of ligand candidates using binding. Based on the crystal structure of N-tosyl-D-proline bound to TS, the methyl group away from the phenyl ring was in a promising position for use as a derivatization point. Scheme 1 is 88 different aldehydes (where R⁵(Selected from unsubstituted or substituted aryl) and six different linkers are used to describe the general methods used to synthesize derivatives.

<chemistry num="43"><img file="JP4391237B2_D0039.tif" /></chemistry> The inhibition constants of the unbound version of the selected ligand candidate were determined. Two of the best compounds were:

<chemistry num="44"><img file="JP4391237B2_D0040.tif" /></chemistry>Compound 2 K_iWas determined to be about 55 μM, and K of compound 3_iWas determined to be about 40 μM.

(Example 7) This example describes one embodiment for the synthesis of compound 13. The general reaction scheme is outlined in Scheme 2.

<chemistry num="45"><img file="JP4391237B2_D0041.tif" /></chemistry> (2- (2-Acetylsulfanyl-Acetylamino) -Succinic Acid 4-tert-Butyl Ester 24) Acetylsulfanyl-acetate pentafluorophenyl ester (1.6 g, 5.3 mmol) and H-Asp (OtBu) -OH (1 g, 5.3 mmol) were mixed in 20 ml of dry dichloromethane (DCM). Then 1.6 ml of triethylamine (11.5 mmol) was added and the reaction was allowed to proceed at ambient temperature for 3.5 hours. The organic layer was then extracted with 3 x 15 ml 1M sodium carbonate and the combined aqueous fraction was acidified with 100 ml 1M sodium hydrogensulfate and extracted with 3 x 30 ml ethyl acetate. The combined organic fractions were then rinsed with 30 ml 1M sodium hydrogensulfate, 30 ml 5M NaCl, dried over sodium sulfate, filtered and evaporated under reduced pressure to add 1.97 g of 24 to a nearly colorless syrup. And used without further purification. MW = 305 (measured value 306, M + l).

(3- (2-Acetylsulfanyl-Acetylamino) -5-chloro-4-oxo-pentanoic acid tert-butyl ester 25) Free acid 24 was dissolved in 10 ml dry tetrahydrofuran (THF), cooled to 0 ° C. and treated with 0.58 ml N-methyl-morpholine (5.3 mmol) and 0.69 ml isobutyl chloroformate. A thick white precipitate formed immediately, and after 30 minutes, the reaction was filtered through a glass frit and transferred to a new flask containing an additional 10 ml THF. On the other hand, diazomethane by reacting 1-methyl-3-nitro-1-nitrosoguanidine (2.3 g, 15.6 mmol) with 7.4 ml of 40% aqueous KOH and 25 ml of diethyl ether at 0 ° C for 45 minutes. Was prepared. The yellow ether layer was then decanted into the reactants containing the mixed anhydride, and the reaction proceeded with slow warming to ambient temperature over 165 minutes. The reaction was cooled to 8 ° C and 4N in 1.5 ml dioxane. HCl (6 mmol total) was added dropwise. This created a significant amount of air bubbles, and the yellow solution became colorless. The reaction was allowed to proceed for 2 hours, gradually warming to ambient temperature, then quenched with 1 ml glacial acetic acid. The solvent is removed under reduced pressure and the residue is redissolved in 75 ml ethyl acetate, rinsed with 2 x 50 ml saturated sodium bicarbonate, 50 ml 5M NaCl, dried over sodium sulfate, filtered and It was evaporated to dryness and then purified by flash chromatography using 90:10 chloroform: ethyl acetate to give 0.747 g of 25 as a pale yellow oil (2.2 mmol, 23-42%). Calculated value MW = 337.7, measured value 338 (M + 1).

(2,6-dichloro-benzoic acid 3- (2-acetylsulfanyl-acetylamino) -4-tert-butoxycarbonyl-2-oxo-butyl ester 26) Chloromethylketone 25 (0.25 g, 0.74 mmol) was dissolved in 5 ml of dry N, N-dimethylformamide (DMF), to which 0.17 g of 2,6-dichlorobenzoic acid (0.89 mmol) and 0.107 g. KF (1.84 mmol) was added. The reaction was allowed to proceed at ambient temperature for 19 hours, at which point it was diluted with 75 ml ethyl acetate and rinsed with 2 x 50 ml saturated sodium bicarbonate, 50 ml 1M sodium hydrogensulfate, 50 ml 5M NaCl. It was dried over sodium sulphate, filtered, and dried under reduced pressure to give a yellow syrup, which was found by HPLC-MS to be about 75% product 26 and 25% unreacted 25. .. It was used without further purification. Calculated MW = 492.37, measured 493 (M + l).

(2,6-dichloro-benzoic acid 3- (2-acetylsulfanyl-acetylamino) -4-carboxy-2-oxo-butyl ester 13) Product 26 was dissolved in 10 ml of dry DCM, cooled to 0 ° C. and treated with 9 ml of trifluoroacetic acid (TFA). The reaction was then removed from the ice bath and warmed to ambient temperature for 1 hour. The solvent was removed under reduced pressure and the residue was redissolved and evaporated into DCM twice to remove residual TFA. The crude product 13 was purified by reverse phase high performance liquid chromatography to give 101.9 mg (0.234 mmol, 25-32%) of white hygroscopic powder. Calculated value MW = 436.37, measured value 437 (M + 1). This was dissolved in dimethyl sulfoxide (DMSO) to give a 50 mM stock solution.

(Example 8) This example describes one embodiment for a particular elongation factor (Compound 32, which was used in a binding experiment for caspase 3). The general scheme is described in Scheme 3.

<chemistry num="46"><img file="JP4391237B2_D0042.tif" /></chemistry> a) 125 mL of deionized (DI) K in water₂CO₃3-Mercaptopropionic acid (4 g, 37.69 mmol) was added to the degassed solution (15.63 g, 113 mmol) under nitrogen. The solution was then cooled to 0 ° C. and acetic anhydride (3.56 ml, 37.69 mmol) was added dropwise. The reaction is stirred for 15 minutes and 2 x 50 mL Et.₂Washed with O and acidified to pH 2 with 1 M HCL. The aqueous layer was then extracted with 3 x 25 mL ethyl acetate (" EtOAc"). The combined organic layers are washed with brine and anhydrous Na.₂SO₄Dry through, filter, and remove the solvent under reduced pressure to give compound 27 (5.19 g, 35 mmol), 93%, ES (+) MS m / e = 148 (M + H). Used without further purification.

b) Compound 27 (2.36 g, 15.94 mmol) was dissolved in 50 mL anhydrous tetrahydrofuran (THF) and pyridine (1.35 mL, 16.74 mmol) followed by pentafluorophenyltrifluoroacetate (2.71 mL, 15.78 mmol). ) Was added. The solution was stirred at ambient temperature for 2 hours. THF is removed under reduced pressure and the residue is redissolved in 75 mL EtOAc, 2 x 25 mL 1M HCl, 25 mL saturated LVDS.₃, Wash with 25 mL brine, anhydrous Na₂SO₄Dry through, filter, and remove solvent under reduced pressure to give compound 28 (3.77 g, 12 mmol, 75%), ES (+) MS m / e = 314 (M + H), which is further purified. Was used without.

c) Compound 28 (3.77 g, 11.99 mmol), H₂N-Asp (OtBu)-CO₂It was mixed with H (2.27 g, 11.99 mmol) and suspended in 40 mL dry DCM. Then triethylamine (2.9 ml, 20.8 mmol) was added and the solution was stirred for 16 hours, at which point 100 mL of EtOAc was poured into it and 2 x 50 ml of 1 M NaHSO.₄And rinse with 50 mL brine, and anhydrous Na₂SO₄Dry through, filter, and remove the solvent under reduced pressure to give the product, which is referred to as 94: 5: 1 CHCl.₃: Methanol: Purification by flash chromatography with acetic acid to give compound 29 (2.62 g, 8.2 mmol, 68% yield, ES (+) MS m / e = 264 ((M-tBu) + H)) ).

d) Compound 29 (2.62 g, 8.2 mmol) was dissolved in 25 mL anhydrous THF and cooled to 0 ° C. To this solution was added N-methylmorpholine (1.88 mL, 17.06 mmol) followed by isobutylcurloformate (2.15 mL, 16.56 mmol). The resulting suspension was stirred for an additional 2 hours and the mixture was filtered. This solution was poured into an ether-like diazomethane solution at 0 ° C. The dark yellow solution was warmed to room temperature overnight. Nitrogen was bubbled for 30 minutes through this dark orange solution. Half of this solution was cooled to 0 ° C. and 4M HCl (3.8 mL, 15 mmol) was added dropwise and the solution was stirred at 0 ° C. for 1 hour. The solvent was removed under reduced pressure and the residue was redissolved in 50 mL of EtOAc. 2 x 25 mL saturated LVDS of organic layer₃, Wash with 25 mL brine, anhydrous Na₂SO₄Dry and concentrate through, and 95: 5 CHCl₃Purification by flash column chromatography using: EtOAc gave compound 30 (0.198 g, 0.562 mmol, 14%), ES (+) MS m / e = 296 ((M-tBu) + H)).

e) Compound 30 (50 mg, 0.143 mmol) dissolved in 1 ml dimethylformamide anhydride (DMF) and a mixture of 2,6-dichlorobenzoic acid (33 mg, 0.172 mmol) and KF (21 mg, 0.358 mmol). Was added. The solution is stirred at ambient temperature for 16 hours, then 20 mL of EtOAc is poured and 2 x 10 mL of saturated acrylamide is added.₃Rinse with 10 mL brine and anhydrous Na₂SO₄The mixture was dried through, filtered, and the solvent was removed under reduced pressure to give compound 31 (48 mg, 0.0948 mmol, 67%), ES (+) MS m / e = 451 ((M-tBu) + H). ).

f) Compound 31 was dissolved in 5 mL of dichloromethane (DCM), cooled to 0 ° C., 5 mL of trifluoroacetic acid (TFA) was added, and the solution was stirred for 30 minutes. The solvent was removed under reduced pressure and the crude residue was purified by reverse phase preparative HPLC to give compound 32 (0.006 g, 0.013 mmol, 14%) ES (+) MS: m / e = 450.29 (M +). 1).

(Example 9) This example describes one embodiment for the synthesis of compound 14. The general reaction scheme is outlined in Scheme 4.

<chemistry num="47"><img file="JP4391237B2_D0043.tif" /></chemistry> a) Using Z-ASP (OtBu) -OH, compound 33 was obtained in the same manner as compound 30 in Example 8. ES (+) MS m / e = 344 ((M-tBu) + H).

b) Compound 34 was prepared according to the procedure of Example 8e (88%), except starting with compound 33 instead of compound 30. ES (+) MS m / e = 454 ((M-tBu) + H)).

c) Compound 34 (0.5 g, 0.9 mmol) was dissolved in 10 mL of MeOH and cooled to 0 ° C. Then NaBH₄(0.074 g, 1.96 mmol) was added in part and the reaction was stirred for 1.5 hours. 25 mL of 1M HCl is poured into this reaction and extracted with 3 x 10 mL DCM, anhydrous Na.₂SO₄The mixture was dried through, filtered, and the solvent was removed under reduced pressure to give compound 35 (0.297 g, 0.058 mmol, 60%), ES (+) MS m / e = 456 ((M-tBu) + H. )).

d) Compound 35 (0.297 g, 0.579 mmol) was dissolved in 5 mL of MeOH, then the solution was sprayed with nitrogen and wet Pd / C (10% weight / weight, Aldrich, 0.123 g) was added. The solution was then stirred under a hydrogen-filled balloon for 30 minutes. The reaction was then filtered through Celite and the solvent was removed under reduced pressure to give compound 36 (0.188 g, 0.497 mmol, 86%), ES (+) MS m / e = 292 ((M-). tBu) + H)).

e) 40 mL CCl₄A solution of meta-toluenesulfonyl chloride (6.8 g, 35.67 mmol), N-bromosuccinimide (6.35 g, 35.67 mmol), and benzoyl peroxide (0.670 g, 3.07 mmol) in was refluxed for 2 hours. After cooling to room temperature, the mixture was filtered, the solvent was removed under reduced pressure and the product was purified by flash chromatography with 9.5: 0.5 hexane: EtOAc to give compound 37 (3.43 g, 12.7 mmol). , 36%), ES (+) MS m / e = 213 ((M-) + H)).

f) Compound 36 (0.188 g, 0.497 mmol) was dissolved in 2 mL of DCM and diisopropylethylemine (0.173 mL, 0.994 mmol) was added, then the solution was dissolved in 20 mL of DCM. It was added dropwise to 37 (0.670 g, 2.49 mmol). After 20 minutes of stirring at room temperature, the DCM is removed under reduced pressure and the residue is redissolved in 20 mL of EtOAc and 2 x 10 mL of 1 M NaHSO.₄, 10 mL saturated LVDS₃Rinse with 10 mL brine and anhydrous Na₂SO₄The product was obtained by drying through, filtering, and removing the solvent under reduced pressure, and the product was purified by flash chromatography with 4: 1 hexane: EtOAc to give compound 38 (0.068 g, 0.111 mmol, 22%), ES (+) MS m / e = 555 ((M-tBu) + H)).

g) Compound 38 (0.068 g, 0.111 g) was dissolved in 1 mL DMF and potassium thioacetate (0.013 g, 0.111 mmol) was added. The reaction is stirred at ambient temperature for 1 hour, then 10 mL DCM is poured and 2 x 5 mL 1M NaHSO.₄, 5 mL saturated LVDS₃And wash with 5 mL brine, anhydrous Na₂SO₄The mixture was dried through, filtered, and the solvent was removed under reduced pressure to give compound 39 (0.044 g, 0.073 mmol, 66%), ES (+) MS m / e = 550 ((M-tBu) + H). )).

h) Compound 39 (0.044 g, 0.073 mmol) was dissolved in 2 mL of DCM and Dess-Martin periodinane (0.046 g, 0.108 mmol) was added. The reaction was stirred at room temperature for 30 minutes and the reaction was filtered. 5 mL of DCM was added and the solution was cooled to 0 ° C. and 7 mL of TFA was added. The reaction was stirred for 30 minutes and the solvent was removed under reduced pressure. The crude residue was purified by reverse phase preparative HPLC to give compound 14 (0.005 g, 0.008 mmol, 11%) ES (+) MS: m / e = 548.41 (M + 1).

(Example 10) This example describes one embodiment for the synthesis of elongation factor 40 used in binding to caspase 3, where the thiol is directed to the prime site of this enzyme. The general reaction scheme is outlined in Scheme 5.

<chemistry num="48"><img file="JP4391237B2_D0044.tif" /></chemistry> Cbz-Asp (OtBu) -OH (7.778 g, 24.1 mmol) was dissolved in 65 ml THF, cooled in an ice water bath, and N-methyl-morpholine (2.6 ml, 23.6 mmol) and isobutylchloroformate (2.6 ml, 23.6 mmol). 3.1 ml, 23.9 mmol) was added. The reaction is stirred on ice for 20 minutes, during which time N, O-dimethylhydroxyamine hydrochloride (3.51 g, 36 mmol) and potassium carbonate (7 g, 51 mmol) are suspended in 24 ml THF and 1 ml water. It was turbid and vigorously stirred at ambient temperature for 20 minutes, then filtered directly through a filter paper into the above carbonate solution and 20 ml of THF was added. After 40 minutes, 200 ml EtOAc is poured into the reaction, rinsed with 3 x 75 ml 1N HCl, 75 ml saturated sodium hydrogen carbonate, and 75 ml brine, dried over sodium sulfate, filtered, evaporated and colorless. A syrup was obtained and used without further purification (9 g, 24.1 mmol, 100%, ES (+) MS m / z = 389 (M + Na)).

Dissolve the amide (8.8 g, 24 mmol) in dry THF (100 ml), cool to -5 ° C in an ice-brine bath under nitrogen, and add 10 1M aluminum hydroxide in THF (12 ml, 12 mmol). Added in minutes. The reaction was stirred on ice for 40 minutes, then 75 ml saturated sodium hydrogensulfate and 250 ml diethyl ether were added and stirred on ice for 15 minutes. The ether layer was removed and dried over sodium sulphate, filtered and evaporated to give the aldehyde, which was used without further purification (8.3 g, 24 mmol, 100%, ES (+) MS m). / z = 348 (M + Na + H)₂O)).

Aldehyde (8.3 g, 24 mmol) was dissolved in dry THF (100 ml), cooled in a dry ice / acetone bath, and 1 M vinyl magnesium bromide in THF (30 ml, 30 mmol) was added. After 1 hour, another 20 ml Grignard was added, followed by 2 hours later another 20 ml Grignard. After 4 hours, the reaction was warmed to ambient temperature and continued for 90 minutes. At 90 minutes, cool in an ice-water bath, add 100 ml saturated sodium hydrogen sulphate, drain the aqueous layer, and rinse the organic layer with 75 ml 1N HCl, 75 ml saturated sodium hydrogen carbonate, and 75 ml brine. And then dried over sodium sulphate, evaporated and dried, and purified on silica gel using flash chromatography (first using 80:20 hexane: EtOAc, then 70:30 hexane: EtOAc) to produce. A product alcohol (2.5 g, 7.45 mmol , 31%, ES (+) MS m / z = 358 (M + Na)) was produced.

Alcohol (2.5 g, 7.45 mmol) is dissolved in dry DCM (40 ml), cooled in an ice-water bath, and metachloroperoxy (perxoy) benzoic acid (mCPBA, 10 g, 44.6 mmol) and another 40 ml dried. Processed with DCM. The reaction was continued for 19 hours, at which time 75 ml saturated sodium hydrogen carbonate was added with another 100 ml DCM. Drain the aqueous layer, rinse the organic layer with 75 ml saturated sodium hydrogen carbonate, 2 x 100 ml 20% saturated sodium hydrogen carbonate, 75 ml brine, dry with sodium sulphate, filter, evaporate and dry, and flash chromatograph. Purified using (first using 70:30 hexane: EtOAc, then 50:50 hexane: EtOAc) and product epoxide (0.828 g, 2.36 mmol, 32%, ES (+) MS m / z = 352 (M + H)) was obtained.

Epoxide (0.132 g, 0.376 mmol) is dissolved in dry methanol (2 ml), to which thiourea (52.3 mg, 0.687 mmol) and 3 ml additional methanol are added. The reaction is then sprayed and kept under nitrogen for 2 days. Then pour 50 ml EtOAc into the reaction, rinse with 2 x 25 ml 1 M sodium bisulfite, 2 x 25 ml sodium bisulfite, 25 ml brine, dry with sodium sulphate, filter, evaporate and dry, and flash chromatography. Purified with (first using 80:20 hexane: EtOAc, then 70:30 hexane: EtOAc) and product thiirane (35 mg, 0.095 mmol, 25%, ES (+) MS m / z = 390 (M +) Na)) was obtained.

Tyran (35 mg, 0.095 mmol) is dissolved in dry DCM (0.5 ml), and Dess-Martin peryodinane (43.3 mg, 0.102 mmol) is added, followed by another 0.5 ml dry DCM. After 30 minutes, the reaction is diluted with 7 ml DCM, filtered through a 0.45 μ filter and purified by flash chromatography (using 80:20 hexane: EtOAc) to produce the product (17 mg, 0.047 mmol, 49%, ES). (+) MS m / z = 388 (M + Na)) was produced.

Tyran (17 mg, 0.047 mmol) was dissolved in dry DCM (5 ml), cooled in an ice-water bath and treated with 5 ml trifluoroacetic acid. The reaction was continued on ice for 40 minutes, at 40 minutes it was evaporated to dryness and purified using reverse phase HPLC to give compound 40 (1.8 mg, 0.0058 mmol, 13%, ES (+)) as a white solid. MS m / z = 332 (M + Na)) was produced. This material is not stable in DMSO, but is stable for 1 month as a solution in methanol maintained at -20 ° C. The conjugate reaction of this elongation factor to the active site thiol of the caspase is generally preferably carried out for only 2-5 minutes at pH 6 and with low stoichiometry (1-3 equals) for the enzyme.

(Example 11) This example describes the modification of caspase 3 with elongation factor 13. Caspase 3 is cloned, overexpressed and purified using standard techniques. 10 μl of 50 mM compound 13 was added to 2 ml of 0.2 mg / ml solution and the reaction was continued at ambient temperature for 3.5 hours. Mass spectrometry at 3.5 hours showed complete modification of the three major caspase subunits (MW 16861, calculated 16860). Thioester was deprotected by adding 0.2 ml of 0.5 M hydroxylamine buffered in PBS buffer and the reaction was continued for 18 hours. At 18 hours, the large subunit had a mass of 16819 (calculated value 16818). Protein was concentrated in an Ultrafree 5 MWCO unit and buffer was exchanged for 0.1 MTES pH 7.5 using a Nap-5 column.

(Example 12) Crystals of caspase 3 were grown at 20 ° C. using the hanging drop vapor diffusion method. Equal volume of protein solution (5-10 mg / ml previously modified protein in 10 mM Tris pH 8.5) containing 100 mM sodium citrate, pH 5.9, 4% glycerol, 10-20% PEG6000 and 10 mM DTT Mix with. Small rhombic plates usually appear after 1-2 weeks. The rhombic plate reaches a maximum size of about 200 x 200 x 20 μm after 2 months. Prior to collecting data, the crystals are briefly immersed in a storage solution containing 25% glycerol and then flash frozen in liquid nitrogen.

Diffraction data of the two bound compounds were collected at 100K using the Rigaku (Tokyo) RU-3R generator, R-axis-IV detector, and D.^*Processed with Trek. Protein Data Bank Entry 1 Analyze the structure by molecular substitution as executed in the program AmoRe (Navaza, J., Acta Crystallogr.Sect.A, A50: 157 ~ 163 (1994)) using the coordinates of CP3. A compound model was constructed in Pymol (DeLano, WL, World Wide Web URL: http://www.pymol.org), and the model was programmed in Program O (Jones, TA et al., Acta Cryst., A47: 110-119 ( Adjust using 1991)) and refine using the program Refmac (CCP4).

(Example 13) This example describes one embodiment for the synthesis of compound 50. The general reaction scheme is outlined in Scheme 6.

<chemistry num="49"><img file="JP4391237B2_D0045.tif" /></chemistry> a) In 500 ml DI water, mix 3- (chlorosulfonyl) benzoic acid (10.38 g, 47.04 mmol) with H-ASP (OtBu) -OMe (10.25 g, 42.76 mmol) and sodium carbonate (14.05 g, 133 mmol). And the reaction was stirred at room temperature for 16 hours. Filter the solution and then 1M NaHSO₄Was acidified to pH 2. Extract the aqueous solution with 3 x 300 mL EtOAc. The combined organic layers were then washed with 250 mL brine and anhydrous Na.₂SO₄Dry with, filter, and remove solvent under reduced pressure to compound 41 (7.07 g, 18.25 mmol, 39%), ES (+) MS m / e = 331 ((M-tBu) + H)). Was produced and used without further purification.

b) Compound 41 (7.07 g, 18.25 mmol) is suspended in 90 ml dry THF under a nitrogen atmosphere and cooled to 0 ° C. Isobutylchloroformic acid (2.49 ml, 19.16 mmol) is added via syringe, followed by N-methylmorpholin (2.21 mL, 20 mmol). The reaction was stirred at 0 ° C for 30 minutes and then poured into a solution of sodium hydrogenboride (2.4 g, 63.88 mmol) in 182 mL THF and 63 mL mL MeOH at -78 ° C. The reaction is stirred at -78 ° C for 2 hours, then most of the THF is removed under reduced pressure. Pour 200 mL EtOAc into the residue, 2 x 75 mL 1M NaHSO₄, 75ml saturated LVDS₃, And rinse with 75 ml brine and anhydrous Na₂SO₄Dry above, filter, and remove solvent under reduced pressure to give compound 42 (6.80 g, 18.21 mmol, 100%), ES (+) MS m / e = 317 ((M-tBu)) as a white solid. ) + H)) was produced and used without further purification.

c) Dissolve compound 42 (6.80 g, 18.21 mmol) in 100 ml dry DCM under a nitrogen atmosphere and cool the solution to 0 ° C. Triethylamine (5.34 mL, 38.33 mmol) is added, then methaneshonyl chloride (1.55 mL, 20.08 mmol) is added dropwise. The reaction is stirred at 0 ° C for 1 hour, then 2 x 35 mL 1M NaHSO.₄Rinse with 40 mL brine and anhydrous Na₂SO₄It was dried in, filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography (using 3: 2 hexane: EtOAc) and compound 43 (6.69 g, 14.82 mmol, 83%, ES (+) MS m / e = 395 ((M-tBu) + H). )) Was produced.

d) Compound A was prepared by the method of Example 13c, except starting from Fmoc-β-alaninol (5.14 g, 17.29 mmol) instead of compound 42 (93%) as shown below.

<chemistry num="50"><img file="JP4391237B2_D0046.tif" /></chemistry>ES (+) MS m / e = 375 (M + 1) This was used without further purification.

e) Compound B was prepared by the method of Example 9g, except starting from compound A instead of compound 38 (91%) as shown below.

<chemistry num="51"><img file="JP4391237B2_D0047.tif" /></chemistry>ES (+) MS m / e = 355 (M + 1) This was used without further purification.

f) Compound B (5.12 g, 14.4 mmol) was dissolved in 10 mL DCM and 50 mL MeOH was added. Nitrogen is bubbled through the solution for 15 minutes, then hydroxylamine (50% in water, 4.42 mL, 72 mmol) is added, followed by TCEP (4.13 g, 14.4 mmol), and the reaction is applied for 4 hours, Stirred in a nitrogen atmosphere. The solvent is then removed under reduced pressure and the residue is dissolved in 100 mL EtOAc and 50 mL saturated LVDS.₃Wash with, and wash with 50 mL brine, anhydrous Na₂SO₄It was dried in and filtered. The solvent was removed under reduced pressure and the residue was purified by flash chromatography using 4: 1 hexane: EtOAc to give compound 44 (3.32 g, 10.6 mmol, 74%). ES (+) MS m / e = 313 (M + 1).

g) Compound 43 (2.29 g, 5.07 mmol) was dissolved in 25 mL DMF, potassium iodide (1.68 g, 10.15 mmol) was added, and the mixture was stirred for 15 minutes at room temperature. Compound 44 (1.59 g, 5.07 mmol) was added, followed by sodium bicarbonate (0.426 g, 5.07 mmol). The reaction was purged with nitrogen and stirred at ambient temperature for 20 hours. Then, 100 mL EtOAc was poured into this reaction solution, and 2 × 50 mL 1M NaHSO was added.₄, 50mL saturated LVDS₃, And rinse with 50 mL brine, anhydrous Na₂SO₄Dry with, filter, and remove the solvent under reduced pressure to give the product, which is CHCl.₃: 2M NH in MeOH₃ Purification by flash chromatography using 95: 5 gave compound 45 (1.38 g, 2.06 mmol, 41% yield). ES (+) MS m / e = 612 ((M-tBu) + H).

h) Compound 45 (1.38 g, 2.06 mmol) was dissolved in 10 mL DCM. Then 10 mL diethylamine was added. The reaction is stirred for 16 hours at ambient temperature, the solvent is removed under reduced pressure, and the residue is CHCl.₃: 2M NH in MeOH₃ Purification by flash chromatography using 95: 5 gave compound 46 (0.723 g, 1.62 mmol, 79% yield). ES (+) MS m / e = 390 ((M-tBu) + H) i) Compound 47 was prepared according to the procedure of Example 8b, except starting with 5- (methanesulfonyl) thiophen-2-carboxylic acid instead of compound 27 (97%). ES (+) MS m / e = 372 (M + H).

j) Compound 46 (0.320 g, 0.717 mmol) was dissolved in 5 mL DCM, compound 47 (0.401 g, 1.08 mmol) was added, followed by DIEA (0.249 mL, 1.43 mmol). The residue was stirred for 16 hours at ambient temperature and the solvent was removed under reduced pressure. This reaction was redissolved in 20 mL EtOAc and 2 x 5 mL 1M NaHSO.₄, Wash with 5 mL brine, anhydrous Na₂SO₄Dry, filter, and remove solvent under reduced pressure to give the product, which is purified by flash chromatography using DCM: EtOAc 4: 1 to compound 48 (0.126 g, 0.198 mmol). , 28% yield) was obtained. ES (+) MS m / e = 578 ((M-tBu) + H).

k) Compound 48 (0.062 g, 0.098 mmol) was dissolved in 0.5 mL anhydrous THF. Lithium borohydride (0.003 g, 0.121 mmol) in 1 mL ethyl ether was added to this solution. The reaction is stirred for 45 minutes at room temperature, then inflowing 10 mL EtOAc and 5 mL saturated LVDS.₃, And rinse with 5 mL brine, anhydrous Na₂SO₄It was dried in, filtered, and the solvent was removed under reduced pressure to give compound 49 (0.058 g, 0.096 mmol, 98%). ES (+) MS m / e = 550 ((M-tBu) + H).

l) Compound 49 (0.058 g, 0.098 mmol) was dissolved in 1 mL DMSO and IBX was added (0.082 g, 0.294 mmol). The reaction is stirred for 5 hours at ambient temperature, then poured with 10 mL EtOAc and 5 mL saturated LVDS.₃, And wash with 5 mL brine and anhydrous Na₂SO₄It was dried on, filtered, and the solvent was removed under reduced pressure to give a yellow solid, which was then dissolved in 5 mL DCM and cooled to 0 ° C. 5 mL of TFA was added and the reaction was stirred for 30 minutes. After removing the solvent under reduced pressure, the crude product residue was purified by reverse phase preliminary HPLC to give compound 50 (0.009 g, 0.016 mmol, 17%). ES (+) MS m / e = 548.68 (M + H).

(Example 14) This example shows an embodiment for the synthesis of compound 51 shown below.

<chemistry num="52"><img file="JP4391237B2_D0048.tif" /></chemistry> Compound 51 was prepared according to the procedure of Examples 9a-h, except that meta-toluenesulfonyl chloride was replaced with para-toluenesulfonyl chloride. ES (+) MS: m / e = 548.41 (M + 1).

(Example 15) This example shows an embodiment for the synthesis of compound 54. A general reaction scheme is outlined in Figure 7.

<chemistry num="53"><img file="JP4391237B2_D0049.tif" /></chemistry> a) Compound 48 (0.063 g, 0.099 mmol) was dissolved in 5 mL MeOH and hydrogen peroxide (0.026 mL, 0.297 mmol, 30% in water) was added. The reaction was heated to 50 ° C. for 16 hours and the solvent was removed under reduced pressure to give compound 52 (0.063 g, 0.097 mmol, 98%). ES (+) MS m / e = 594 ((M-tBu) + H).

b) Compound 53 was prepared according to the procedure of Example 13k, except that compound 48 was replaced with compound 52. ES (+) MS m / e = 566 ((M-tBu) + H).

c) Compound 54 was prepared according to the procedure of Example 9h, except that compound 39 (0.005 g, 0.009 mmol, 11%) was replaced with compound 53. ES (+) MS m / e = 564.68 (M + 1).

(Example 16) This example shows an embodiment for the synthesis of compound 56. A general reaction scheme is outlined in Figure 8.

<chemistry num="54"><img file="JP4391237B2_D0050.tif" /></chemistry>a) Compound 48 (0.150 g, 0.236 mmol) was dissolved in 5 mL MeOH, acetic acid (5 mL) was added, followed by hydrogen peroxide (0.77 mL, 10 mmol, 35% in water). The reaction was heated to 80 ° C. for 16 hours and the solvent was removed under reduced pressure to give compound 55 (0.157 g, 0.236 mmol, 100%). ES (+) MS m / e = 610 ((M-tBu) + H).

b) Compound 56 was prepared according to the procedure of Example 13k followed by Example 9h, except that compound 56 (0.005 g, 0.0086 mmol, 36%) was used as a starting point. ES (+) MS m / e = 580 (M + 1).

(Example 17) This example describes an embodiment for the synthesis of compound 57 shown below.

<chemistry num="55"><img file="JP4391237B2_D0051.tif" /></chemistry> Compound 57 was prepared according to the procedure of Examples 13a-l, except that 5- (methanesulfonyl) thiophen-2-carboxylic acid was replaced with 4- (methylsulfonyl) benzoic acid. ES (+) MS: m / e = 543 (M + 1).

(Example 18) This example describes an embodiment for the synthesis of compound 58 shown below.

<chemistry num="56"><img file="JP4391237B2_D0052.tif" /></chemistry> Compound 58 was prepared according to the procedure of Examples 13a-j, except that 5- (methanesulfonyl) thiophen-2-carboxylic acid was replaced with 4- (methylsulfonyl) benzoic acid, followed by Examples 15a-. Prepared according to step c. ES (+) MS: m / e = 559 (M + 1).

(Example 19) This example describes an embodiment for the synthesis of compound 59 shown below.

<chemistry num="57"><img file="JP4391237B2_D0053.tif" /></chemistry> Compound 59 was prepared according to the procedure of Examples 13a-j, except that 5- (methanesulfonyl) thiophen-2-carboxylic acid was replaced with 45-chloro-6-hydroxynicotinic acid, followed by Example 16a. Prepared according to the procedure of ~ b. ES (+) MS: m / e = 548 (M + 1).

(Example 20) This example describes an embodiment for the synthesis of compound 60 shown below.

<chemistry num="58"><img file="JP4391237B2_D0054.tif" /></chemistry> Compound 60 was prepared according to the procedure of Examples 13a-l, except that 5- (methanesulfonyl) thiophen-2-carboxylic acid was replaced with benzothiazole-6-carboxylic acid. ES (+) MS: m / e = 522 (M + 1).

(Example 21) This example describes an embodiment for the synthesis of compound 61 shown below.

<chemistry num="59"><img file="JP4391237B2_D0055.tif" /></chemistry> Compound 61 was prepared according to the procedure of Examples 13a-j, except that 5- (methanesulfonyl) thiophen-2-carboxylic acid was replaced with a benzothiazole-6-carboxylic acid, followed by Examples 15a-c. It was prepared according to the procedure of. ES (+) MS: m / e = 538 (M + 1).

All references cited throughout this specification are expressly incorporated herein by reference. Although the present invention has been described with reference to that particular embodiment, it is relevant that various variations can be made and that equivalents can be replaced without departing from the spirit and scope of the invention. Should be understood by those skilled in the art. In addition, many modifications can be made to suit a particular situation, substance, composition of substance, process, etc. All such modifications are within the scope of the claims attached herein.

<figref num="1">FIG. 1A is a schematic diagram of one embodiment of the coupling method. The thiol-containing protein reacts with multiple ligand candidates. Ligand candidates with unique binding affinities for this target are identified, and the ligand is made to contain an identified binding determinant (represented by a circle) that does not contain a disulfide moiety. Figure 1B is a schematic diagram of the theory behind the bond. When the thiol-containing protein is in equilibrium with at least one disulfide-containing ligand candidate (most preferably in the presence of a reducing agent), an equilibrium between the modified and unmodified proteins is established. If the ligand candidate does not have a unique binding affinity for the target protein, this equilibrium shifts towards the unmodified protein. In contrast, if the ligand candidate does not have a unique affinity for the protein, this equilibrium shifts towards the modified protein.</figref><figref num="2">2A and 2B are typical examples of binding experiments. FIG. 2A is an analyzed mass spectrum of the reaction of thymidylate synthase (TS) with a pool of 10 different ligand candidates with little or no binding affinity for TS. FIG. 2B is an analyzed mass spectrum of TS's reaction to a pool of 10 different ligand candidates when one of the ligand candidates has a unique binding affinity for this enzyme.</figref><figref num="3">Figures 3A, 3B, and 3C show the effect of reducing agent concentration on typical binding experiments. FIG. 3A is the analyzed mass spectrum when the reaction was carried out without 2-mercaptoethanol. FIG. 3B is the analyzed mass spectrum when the same reaction was performed in the presence of 0.2 mM 2-mercaptoethanol. Figure 3C is the analyzed mass spectrum when the same reaction was performed in the presence of 20 mM 2-mercaptoethanol.</figref><figref num="4">Figures 4A, 4B, and 4C show the effect of the number of ligand candidates in the library in a typical binding experiment. FIG. 4A is a binding experiment using a library pool containing 20 ligand candidates. FIG. 4B is a binding experiment using a library pool containing 50 ligand candidates. Figure 4C is a binding experiment using a library pool containing 100 ligand candidates.</figref><figref num="5">Figure 5 shows the initially selected binding determinant R.^DUsing R^DIt is a schematic diagram when the library of the compound containing and the variant thereof was prepared. In this figure, the modified library is a variant R of the first binding determinant.^D', And the second binding determinant R^EThe binding experiment is shown when the compound containing is contained. As shown, these two binding determinants are subsequently bound together to form a disulfide-deficient conjugate molecule.</figref><figref num="6">Figure 6 shows two binding determinants (R).^DAnd R^EThese are the embodiments of two binding experiments used to identify (which subsequently bind together to form a conjugate molecule).</figref><figref num="7">Figure 7 shows the second binding determinant R^EBut R^DIt is a schematic of two binding experiments when identified in the presence of binding. Once identified, these two binding determinants combine to form a conjugate molecule.</figref><figref num="8">FIG. 8 is a schematic diagram of one embodiment of the binding method when an extender containing a first functional group and a second functional group is used. As shown, the thiol-containing target is the first functional group X, which can form a covalent bond with the reactive thiol, and the second functional group-SR.^1’It is contacted with elongation factors, including (which can form disulfide bonds). A binding-elongation factor complex is formed, which is then contacted with multiple ligand candidates. This elongation factor provides one binding determinant (circle), and this ligand candidate provides a second binding determinant (square), and the resulting binding determinants bind together. , Form a conjugate compound.</figref>