Nanoscale high-throughput chemistry screen

Abstract

Methods of synthesizing and screening nanoscale high-throughput chemical libraries for compounds that modulate cellular activity. In one aspect, is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay comprising the steps of: performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent / s for the first reaction; optionally performing a second (R-II), third (R-III) and / or more reaction step in the wells; without purification of the reaction mixture, individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and assessing whether or not at least one compound in each well has a biological effect.

Description

NANOSCALE HIGH-THROUGHPUT CHEMISTRY SCREENCROSS REFERENCE

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 682,723, filed August 13, 2024, which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Provided herein methods of synthesizing and screening nanoscale high-throughput chemical libraries for compounds that modulate cellular activity.SUMMARY

[0003] Initial chemical approaches to the production of libraries by conventional techniques may be led by a random approach to structure design, computational chemistry, and predictive modeling, analogue design based on compounds with known structure and activity, medicinal chemistry intuition, or combinations of these approaches. The libraries of compounds so produced are then screened for activity against targets of interest.

[0004] The use of combinatorial chemistry to accelerate the identification of new chemical entities with desirable properties is well established. For example, in drug discovery, large collections of compounds are often synthesized very quickly using techniques collectively called “combinatorial chemistry.” These techniques can include the parallel synthesis of compound libraries using automated and non-automated methods, and may employ both solution phase and solid phase chemistry. Such libraries may be collections of discrete, individual compounds or may consist of collections of mixed or pooled compounds, which are then screened against a target of interest. Pharmaceutically useful properties may be identified through a basic screen developed to assay the ability of compounds to bind to a molecular target. If mixtures of compounds are screened, a deconvolution process is often necessary to identify the components of a mixture that are responsible for any observed activity in the screening process. This can prove difficult to achieve in practice. In many ways such high throughput screening processes have not effectively accelerated identification of new chemical entities with desirable properties.

[0005] Fragment-based drug discovery has received significant industry attention since Fesik and co-workers demonstrated that high-affinity ligands could be generated by first identifying and then combining small fragments that bound to adjacent sites on a target protein. Shuker, S. B., et al., Science 274, 1531-1534 (1996); Petros, A. M., et al., J. Med. Chem. 49, 656-663 (2006); Hajduk, P. J., et al., J. Am. Chem. Soc. 119, 5818-5827 (1997). Lead generation by fragment assembly offers an attractive complement to traditional screening: small fragments areless likely to contain interfering groups that could block an otherwise productive binding interaction, and combining prequalified fragments greatly simplifies the combinatorial search problem. Although productive techniques have been developed to identify and optimize individual fragments, the goal of merging two or more fragments to generate high-affinity compounds remains a significant challenge due to the difficulty of identifying suitable linking moieties. Erlanson, D. A., et al., J. Med. Chem. 47, 3463-3482 (2004); Jahnke, W. & Erlanson, D. A. (eds.) Fragment-based approaches in drug discovery (Wiley-VCH, Weinheim, Germany, 2006); Maly D. J., et al., Proc. Natl. Acad. Sci. U.S.A., 97(6), 2416-2424 (2000). This challenge is particularly daunting when a protein target is not amenable to structural studies, illustrating the need for simple empirical solutions to the linking problem. Tethering® with Extenders provides one such solution in which a given site on a protein is occupied by a covalently attached extender and disulfide capture is used to identify companion fragments that bind to an adjacent site. Erlanson, D. A., et al., Nat. Biotechnol. 21, 308-314. (2003); Choong, I. C., et al., J. Med. Chem. 45, 5005-5022 (2002). Initially validated using protease targets, Tethering with Extenders has recently been used to identify highly selective inhibitors of protein kinases by targeting an adaptive site adjacent to the “hinge region”. However, the high investment in protein engineering and production required to support structure-based methods or Tethering with Extenders can limit the extent to which these approaches can be routinely used.

[0006] Although such fragment-based methods have proven their utility in the development of lead candidates for medicinal chemistry optimization, there remains a need for simple, inexpensive, fast, and efficient generation of pharmacophore hits that have structures amenable to medicinal chemistry and that demonstrate affinity for or activity against biological targets of interest.

[0007] In one aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed;c) without purification of the reaction mixture of step b), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and d) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0008] In some embodiments, the biological assay is a primary assay, a secondary assay, an in vitro assay, a cellular assay, a binding assay, a functional assay, a homogenous assay or a heterogenous assay. In some embodiments, the biological assay permits identification of off- target effects or identification or compounds that agonize or antagonize specific target variants. In some embodiments, the biological assay allows for identification or avoidance of nonspecific or off-target effects by allowing screening of compounds that agonize or antagonize the same target but signal through different downstream effectors. In some embodiments, the biological assay increases the efficiency of pharmaceutical screening and understanding of biological system. In some embodiments, the biological assay allows, simultaneously or substantially simultaneously, the determination of a certain test compound’s activity against a target (e.g., a heterologous polypeptide), variants of the target, different downstream promoters that may be activated by the target, and off-target effects of the test agent simultaneously in a single well. The methods described herein are capable of such determination for thousands of compounds simultaneously. Described herein are methods and systems of screening and identifying compounds capable of regulating target activity, either positively (as an agonist) or negatively (as an antagonist).

[0009] In some embodiments, the biological assay allows for the identification of on-target effects or off-target effects with a specific biological target. In some embodiments, the biological assay is designed to assess binding to a biological target (e.g., covalent or non-covalent), binding to more than one biological target (e.g., to assess for off-target effects or to assess for proteinprotein interactions). In some embodiments, the biological assay assesses whether a test compound is capable of correcting / restoring the function of a biological target protein. In some embodiments, the biological assay assesses whether a test compound is capable of correcting / restoring the misfolding of a biological target protein.

[0010] In some embodiments, the biological assay assesses whether a test compound is capable of affecting signal transduction of at least one protein in the assay, affecting protein expression level, affecting protein folding, affecting intracellular trafficking, or affecting cell surface protein expression. In some embodiments, the biological assay assesses whether a test compound affects protein trafficking in the biological assay. In some embodiments, the biological assay assesseswhether a test compound affects membrane, such as transmembrane, protein trafficking in the biological assay. In some embodiments, the biological assay assesses whether a test compound affects protein signaling, protein expression, or protein turnover in the biological assay.

[0011] In some embodiments, the biological assay allows for the identification of the active site of a class of enzymes in normal and / or diseased cells. In some embodiments, these differential activities may lead to new strategies for combination therapies (or designed polypharmacology) and identify new molecular targets. In some embodiments, the biological assay allows for cellular compound profiling to interrogate biological mechanisms both within and outside a target protein.

[0012] In some embodiments, the biological assay in each well assesses the interactions between test compounds and biological activity more efficiently and in greater detail by showing a full array of the interactions, including whether a test compound binds to a specific heterologous polypeptide, whether the test compound binds to a variant of a heterologous polypeptide, whether certain promoters are activated through certain pathways, whether the test compound promotes toxicity in the well, or whether the test agent does not affect any biological activity. In doing so, it can be determined which test compound could be used to help treat conditions or diseases more efficiently, or which test compound could be used as a starting point for structure-activity relationship studies and for optimization into a therapeutics that could help treat conditions or diseases.

[0013] In another aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction;d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification of the reaction mixtures of step d), individually performing a biological assay with the contents of well of the plurality of wells of the microplate; and f) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0014] In yet another aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C-III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) without purification of the reaction mixtures of step e), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; andg) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0015] In a further aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C-III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) optionally repeating step e) one or more times, wherein each subsequent reaction (R- (III+n)) produces a different compound (C-(III+n)) in each well, wherein n represents the number of times step e) is repeated; g) without purification of step f), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and h) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0016] In some embodiments, the microplate comprises 96, 192, 384, 1536, 3456 or 6144 wells. In some embodiments, each reaction is performed on a nanoscale. In some embodiments, each reaction is performed on a microscale. In some embodiments, each well comprises a total volume of between 10-5000 nanoliters; or each well comprises 1-1000 nanomoles of a reactant or compound (C-(III+n)). In some embodiments, each well comprises a total volume of between 10- 5000 microliters; or each well comprise 1-1000 micromoles of a reactant or compound (C- (III+n)).

[0017] In some embodiments, the first reaction (R-I) comprises an amide coupling reaction or a sulfonamide coupling reaction. In some embodiments, the first reaction (R-I) comprises an alkylation, an alkenylation reaction, or an alkynylation reaction. In some embodiments, the reaction (R-I) comprises alkynylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises alkenylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises a deprotection, skeletal editing, atom insertion, epoxidation, aziridination, cyclopropanation, atom deletion, deoxygenation, deamination, dehalogenation, atom swapping, a sigmatropic rearrangement, a conjugate addition, an oxidation reaction, a reduction reaction, a decarb oxy lative reaction, a carboxylative reaction, a carbonylative reaction, a decarbonylative reaction, a halogenation reaction, a reductive amination, a carbonylation, an acylation, an esterification, or a de-esterification. In some embodiments, the first reaction (R-I) comprises a silylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide.

[0018] In some embodiments, reaction (R-I) comprises a borylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide. In some embodiments, the reaction (R-I) comprises arylation. In some embodiments, the reaction (R-I) comprises amination of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises carbonylation or acylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises thioesterification of an aryl halide or heteroaryl halide; optionally wherein the reaction (R-II) comprises an oxidation to provide an aryl or heteroaryl sulfone. In some embodiments, the reaction (R-I) comprises phosphorylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl or heteroaryl boronic acid. In some embodiments, the reaction (R-I) comprises a cross coupling reaction. In some embodiments, the reaction (R-I) comprises a cycloaddition reaction.

[0019] In some embodiments, at least one compound from reaction (R-I) comprises an amine and the reaction (R-II) comprises an amide coupling reaction. In some embodiments, at least onecompound from reaction (R-I) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the reaction (R-II) comprises an alkylation. In some embodiments, at least one compound from reaction (R-I) comprises an amine and the reaction (R-II) comprises a reductive amination with an aldehyde or carboxylic acid. In some embodiments, at least one compound from reaction (R-I) comprises a carboxylic acid and the reaction (R-II) comprises an esterification. In some embodiments, at least one compound from reaction (R-I) comprises an ester group and the reaction (R-II) comprises a de-esterification. In some embodiments, at least one compound from reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a cross-coupling reaction. In some embodiments, at least one compound from reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a borylation reaction. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl sulfinate and the rection (R-II) comprises an oxidation reaction.

[0020] In some embodiments, at least one compound from reaction (R-II) comprises an amine and the subsequent reaction comprises an amide coupling reaction. In some embodiments, at least one compound from reaction (R-II) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the subsequent reaction comprises an alkylation. In some embodiments, at least one compound from reaction (R-II) comprises an amine and the subsequent reaction comprises a reductive amination with an aldehyde or carboxylic acid. In some embodiments, at least one compound from reaction (R-II) comprises a carboxylic acid and the subsequent reaction comprises an esterification. In some embodiments, at least one compound from reaction (R-II) comprises an ester group and the subsequent reaction comprises a de-esterification. In some embodiments, at least one compound from reaction (R-II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequent reaction comprises a crosscoupling reaction. In some embodiments, at least one compound from reaction (R-II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequent reaction (R-III) comprises a borylation reaction to provide compound (C-III); and wherein a subsequent cross coupling reaction is performed with the borylated compound (C-III).

[0021] In some embodiments, the cross coupling reaction is a Cadiot-Chodkiewicz coupling, Castro-Stephens coupling, Corey-House synthesis, Kumada coupling, Heck reaction, Sonogashira coupling, Negishi coupling, Stille cross coupling, Suzuki reaction, Murahashi coupling, Hiyama coupling, Fukuyama coupling, Liebeskind-Srogl coupling, cross dehydrogenative coupling, decarb oxy lative cross-coupling, Ullmann-type reaction, Buchwald- Hartwig reaction, nickel-catalyzed reductive cross-coupling, or Chan-Lam coupling. In someembodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl sulfonyl halide and the reaction (R-II) comprises a Lou-Willis sulfonamide coupling reaction in each well with a different amine added to each well. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl bromide and the reaction (R-II) comprises a Suzuki reaction in each well with a different aryl or heteroaryl boronic acid added to each well, and a palladium catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl boronic acid and the reaction (R-II) comprises a Buchwald Hartwig reaction in each well with a different amine added to each well, and a palladium catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Heck reaction in each well with a different alkene added to each well, and a palladium catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Sonogashira coupling in each well with a different alkyne added to each well, and a palladium catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Stille coupling reaction in each well with a different organostannane added to each well, and a palladium catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl or heteroaryl boronic and the reaction (R-II) comprises a Chan-Lam coupling in each well with a different amine added to each well, and a copper catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl, heteroaryl, cyclic or heterocyclic amine and the reaction (R-II) comprises an amide coupling in each well with a different carboxylic acid added to each well. In some embodiments, at least one compound from reaction (R-I) comprises an aryl halide and the reaction (R-II) comprises a coupling in each well with a different aryl halide added to each well, and a nickel catalyst. In some embodiments, at least one compound from reaction (R-I) comprises an aryl halide and the reaction (R-II) comprises a coupling in each well with a different alkyl halide added to each well, and a nickel catalyst.

[0022] In some embodiments, at least one compound from reaction (R-I), (R-II), (R-III) or (R- (III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine, at least one lysine, at least one tyrosine, at least one threonine, at least one aspartate, at least one methionine, at least one glutamate, or at least one serine of the biological target. In some embodiments, at least one compound from reaction (R-I), (R-II), (R-III), or (R- (III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target. In some embodiments, at least one compound from reaction(R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one acquired cysteine of the biological target.

[0023] In some embodiments, the electrophilic warhead moiety is acrylamide, α,β-unsaturated carbonyl, α-halomethyl ketone, epoxide, haloacetamide, β-lactam, ketone, aldehyde, or boronic acid. In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target is:R1is H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or -C(=O)OR3;R2is H, halogen, substituted or unsubstituted alkyl, or -CN; and each R3is independently H or substituted or unsubstituted alkyl.

[0024] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target. In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target is

[0025] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target. In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target is

[0026] In some embodiments, the biological assay comprises a biological target of interest. In some embodiments, the biological target is a protein tyrosine phosphatase (PTP), protein tyrosine kinases (PTK), protease, small G protein, GTPase, GTPase-activating protein, G protein-coupled receptor (GPCR), ion channel, adaptor protein, transcription activator, cofactor, tumor suppressor, regulatory protein, transporter protein, enzyme, nuclear receptor protein, methyltransferase, or growth factor ligand.

[0027] Other features and advantages of the compositions, compounds, and methods described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the instant disclosure will become apparent to those skilled in the art from this detailed description.DETAILED DESCRIPTION

[0028] The present invention relates generally to methods of synthesizing and identifying compounds that exhibit enhanced properties associated with their binding affinity for a biological target. The methods also provide a means of rapidly assessing target tractability, e.g., by determining the number of hits per number of compounds screened (“hit rate”). To illustrate one advantage of an embodiment of the invention, such screening procedures may be used to leverage an affinity of the compound target complex for a target to rapidly screen and identify derivative compounds that provide enhanced affinity for the target.

[0029] The current fragment-based screening technology provides several advantages over other fragment-based approaches. For example, the methods provided herein enables rapid survey of relevant chemical space through an iterative fragment assembly approach.

[0030] The method of the invention is applicable to development of ligands for a variety of targets, including large proteins, protein complexes, and partially purified fractions. Another advantage of the method of the invention is that the three-dimensional structure of the target need not be characterized. Another advantage of the present invention is that it can be configured to provide a direct readout of inhibition of the activity of a protein or other target by the test ligands.

[0031] Other and further advantages will become apparent in the following description of methods of the invention.High-throughput screening

[0032] In one aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification of the reaction mixture of step b), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and d) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0033] In another aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification of the reaction mixtures of step d), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and f) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0034] In yet another aspect, provided herein is a and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C- III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) without purification of the reaction mixtures of step e), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and g) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0035] In a further aspect, provided herein is a synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction;b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C- III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) optionally repeating step e) one or more times, wherein each subsequent reaction (R-(III+n)) produces a different compound (C-(III+n)) in each well, wherein n represents the number of times step e) is repeated; g) without purification of step f), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and h) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

[0036] In some embodiments, each well comprises a different plurality of reactants and a solvent or a combination of solvents for the reaction (R-I). In some embodiments, each reaction (R-I) goes to completion. In some embodiments, each reaction (R-I) does not go to completion.

[0037] In some embodiments, each well comprises a different plurality of reactants and a solvent or a combination of solvents for the reaction (R-II). In some embodiments, each reaction (R-II) goes to completion. In some embodiments, each reaction (R-II) does not go to completion.

[0038] In some embodiments, each well comprises a different plurality of reactants and a solvent or a combination of solvents for the reaction (R-III). In some embodiments, each reaction (R-III) goes to completion. In some embodiments, each reaction (R-III) does not go to completion.

[0039] In some embodiments, the reaction cycle is performed n more times. In some embodiments, the reaction cycle is performed 1, 2, 3, 4 or 5 more times. In some embodiments, the reaction cycle is performed 1 more time. In some embodiments, the reaction cycle is performed 2 more times. In some embodiments, the reaction cycle is performed 3 more times.

[0040] In some embodiments, reactions (R-I), (R-II), (R-III), and (R-(III+n)) are each performed in a well of a plurality of wells of a microplate. In some embodiments, a different compound (C-I), (C-II), (C-III), and (C-(III+1)) is produced in each well of a microplate.

[0041] In some embodiments, the microplate comprises 96, 192, 384, 1536, 3456 or 6144 wells. In some embodiments, the microplate comprises 96 wells. In some embodiments, the microplate comprises 192 wells. In some embodiments, the microplate comprises 384 wells. In some embodiments, the microplate comprises 1536 wells. In some embodiments, the microplate comprises 3456 wells. In some embodiments, the microplate comprises 6144 wells.

[0042] In some embodiments, each reaction is performed on a nanoscale. In some embodiments, each well comprises a total volume of between 10-5000 nanoliters; or each well comprises 1-1000 nanomoles of a reactant or compound (C-(III+n)). In some embodiments, each well comprises a total volume of between 10-5000 nanoliters. In some embodiments, each well comprises 1-1000 nanomoles of a reactant or compound (C-(III+n)).

[0043] In some embodiments, each reaction is performed on a microscale. In some embodiments, each well comprises a total volume of between 10-5000 microliters; or each well comprise 1-1000 micromoles of a reactant or compound (C-(III+n)). In some embodiments, each well comprises a total volume of between 10-5000 microliters. In some embodiments, each well comprise 1-1000 micromoles of a reactant or compound (C-(III+n)).

[0044] In some embodiments, the compound of any of reactions (R-I), (R-II), (R-III), and (R- (III+n)) is taken into the subsequent reaction without purification. In some embodiments, reaction (R-I) is performed without further purification. In some embodiments, reaction (R-II) is performed without further purification. In some embodiments, reaction (R-III) is performed without further purification. In some embodiments, reaction (R-(III+n)) is performed without further purification.

[0045] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) is air free. In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) is conducted under an inert atmosphere.

[0046] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprises an amide coupling reaction. In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(Ill+n) comprises a sulfonamide coupling reaction.

[0047] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise an alkylation, an alkenylation reaction, or an alkynylation reaction.

[0048] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise a borylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide.

[0049] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise a silylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide.

[0050] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise a deprotection, skeletal editing, atom insertion, epoxidation, aziridination, cyclopropanation, atom deletion, deoxygenation, deamination, dehalogenation, atom swapping, a sigmatropic rearrangement, a conjugate addition, an oxidation reaction, a reduction reaction, a decarb oxy lative reaction, a carboxylative reaction, a carbonylative reaction, a decarb onylative reaction, a halogenation reaction, a reductive amination, an acylation, an esterification, or a deesterification. In some embodiments, the reaction comprises a deprotection. In some embodiments, the reaction comprises a skeletal editing, atom insertion, or atom deletion. In some embodiments, the reaction comprises an epoxidation. In some embodiments, the reaction comprises an aziridination. In some embodiments, the reaction comprises a cyclopropanation. In some embodiments, the reaction comprises a deoxygenation, deamination, or dehalogenation. In some embodiments, the reaction comprises a sigmatropic rearrangement. In some embodiments, the reaction comprises a conjugate addition. In some embodiments, the reaction comprises an oxidation reduction. In some embodiments, the reaction comprises a decarboxylative reaction. In some embodiments, the reaction comprises a carboxylative reaction. In some embodiments, the reaction comprises a carbonylative reaction. In some embodiments, the reaction comprises a decarb onylative reaction. In some embodiments, the reaction comprises an acylation. In some embodiments, the reaction comprises an esterification or a de-esterification.

[0051] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise a cycloaddition reaction. Exemplary cycloadditions include but are not limited to, a [3+2] cycloaddition, Huisgen 1,3-dipolar cycloaddition, thiol-ene reaction, Diels- Alder reaction, inverse electron demand Diels-Alder reaction, [4+1] cycloaddition between an isonitrile and a tetrazine, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), azide alkyne Huisgen cycloaddition, ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), strain-promoted azidealkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC), an inverse electron demand Diels-Alder cycloaddition (iEDDA), sydnone-alkyne cycloaddition (SPSAC), alkene and azide [3+2] cycloaddition, or alkene and tetrazine inverse-demand Diels- Alder.

[0052] In some embodiments, any of reactions (R-I), (R-II), (R-III), or (R-(III+n)) comprise a cross coupling reaction. Exemplary cross coupling reactions include a Cadiot-Chodkiewicz coupling, Castro-Stephens coupling, Corey-House synthesis, Kumada coupling, Heck reaction, Sonogashira coupling, Negishi coupling, Stille cross coupling, Suzuki reaction, Murahashi coupling, Hiyama coupling, Fukuyama coupling, Liebeskind-Srogl coupling, cross dehydrogenative coupling, decarb oxy lative cross-coupling, Ullmann-type reaction, Buchwald- Hartwig reaction, or Chan-Lam coupling. In some embodiments, the cross-coupling reaction is a Cadiot-Chodkiewicz coupling. In some embodiments, the cross-coupling reaction is a Castro- Stephens coupling. In some embodiments, the cross-coupling reaction is a Corey-House synthesis. In some embodiments, the cross coupling reaction is a Kumada coupling. In some embodiments, the cross-coupling reaction is a Heck reaction. In some embodiments, the crosscoupling reaction is a Sonogashira coupling. In some embodiments, the cross-coupling reaction is a Negishi coupling. In some embodiments, the cross-coupling reaction is a Stille cross coupling. In some embodiments, the cross-coupling reaction is Suzuki reaction. In some embodiments, the cross-coupling reaction is a Murahashi coupling. In some embodiments, the cross-coupling reaction is a Hiyama coupling. In some embodiments, the cross-coupling reaction is a Fukuyama coupling. In some embodiments, the cross-coupling reaction is a Liebeskind-Srogl coupling. In some embodiments, the cross-coupling reaction is a cross dehydrogenative coupling. In some embodiments, the cross-coupling reaction is a decarb oxy lative cross-coupling. In some embodiments, the cross-coupling reaction is an Ullmann-type reaction. In some embodiments, the cross-coupling reaction is a Buchwald-Hartwig reaction. In some embodiments, the crosscoupling reaction is a Chan-Lam coupling.

[0053] In some embodiments, the first reaction (R-I) comprises an amide coupling reaction or a sulfonamide coupling reaction. In some embodiments, the first reaction (R-I) comprises an amide coupling. In some embodiments, the first reaction (R-I) comprises a sulfonamide coupling reaction.

[0054] In some embodiments, the first reaction (R-I) comprises an alkylation, an alkenylation reaction, or an alkynylation reaction. In some embodiments, the first reaction (R-I) comprises an alkylation. In some embodiments, the first reaction (R-I) comprises an alkenylation reaction. In some embodiments, the first reaction (R-I) comprises an alkynylation reaction.

[0055] In some embodiments, the reaction (R-I) comprises alkynylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises alkynylation of an aryl halide. In some embodiments, the aryl halide is phenyl chloride or bromide. In some embodiments, the reaction (R-I) comprises alkynylation of a heteroaryl halide.

[0056] In some embodiments, the reaction (R-I) comprises alkenylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises alkenylation of an aryl halide. In some embodiments, the reaction (R-I) comprises alkenylation of a heteroaryl halide.

[0057] In some embodiments, the reaction (R-I) comprises a deprotection, skeletal editing, atom insertion, epoxidation, aziridination, cyclopropanation, atom deletion, deoxygenation, deamination, dehalogenation, atom swapping, a sigmatropic rearrangement, a conjugate addition, an oxidation reaction, a reduction reaction, a decarb oxy lative reaction, a carboxylative reaction, a decarb onylative reaction, a halogenation reaction, a reductive amination, a carbonylation, an acylation, an esterification, or a de-esterification.

[0058] In some embodiments, the first reaction (R-I) comprises a silylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide. In some embodiments, the first reaction (R-I) comprises a silylation reaction of an aryl halide. In some embodiments, the aryl halide is phenyl chloride or phenyl bromide. In some embodiments, the first reaction (R-I) comprises a silylation reaction of a heteroaryl halide. In some embodiments, the heteroaryl is pyridine, pyrimidine, or triazine. In some embodiments, the first reaction (R-I) comprises a silylation reaction of an alkenyl halide. In some embodiments, the first reaction (R-I) comprises a silylation reaction of an alkyl halide.

[0059] In some embodiments, reaction (R-I) comprises a borylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide. In some embodiments, reaction (R-I) comprises a borylation reaction of an aryl halide. In some embodiments, the aryl halide is phenyl chloride or phenyl bromide. In some embodiments, reaction (R-I) comprises a borylation reaction of a heteroaryl halide. In some embodiments, reaction (R-I) comprises a borylation reaction of an alkenyl halide. In some embodiments, reaction (R-I) comprises a borylation reaction of an alkyl halide.

[0060] In some embodiments, the reaction (R-I) comprises arylation. In some embodiments, the reaction (R-I) comprises amination of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises amination of an aryl halide. In some embodiments, the reaction (R-I) comprises amination of a heteroaryl halide.

[0061] In some embodiments, the reaction (R-I) comprises carbonylation or acylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises carbonylation or acylation of an aryl halide. In some embodiments, the reaction (R-I) comprises carbonylation or acylation of a heteroaryl halide.

[0062] In some embodiments, the reaction (R-I) comprises thioesterification of an aryl halide or heteroaryl halide; optionally wherein the reaction (R-II) comprises an oxidation to provide anaryl or heteroaryl sulfone. In some embodiments, the reaction (R-I) comprises thioesterification of an aryl halide. In some embodiments, the reaction (R-I) comprises thioesterification of a heteroaryl halide.

[0063] In some embodiments, the reaction (R-I) comprises phosphorylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises phosphorylation of an aryl halide. In some embodiments, the reaction (R-I) comprises phosphorylation of a heteroaryl halide.

[0064] In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl halide or heteroaryl halide. In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl halide. In some embodiments, the reaction (R-I) comprises a sulfinylation of a heteroaryl halide.

[0065] In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl or heteroaryl boronic acid. In some embodiments, the reaction (R-I) comprises a sulfinylation of an aryl boronic acid. In some embodiments, the reaction (R-I) comprises a sulfinylation of a heteroaryl boronic acid.

[0066] In some embodiments, the reaction (R-I) comprises a multicomponent reaction (MCR).

[0067] In some embodiments, the multicomponent reaction comprises an Asinger 4-component reaction, alkyne trimerization, aldehyde-alkyne-amine reaction, Biginelli 3 -component reaction (B-3CR), Bucherer-Bergs reaction, Gewald 3-component reaction (G-3CR), Groebke- Blackbum-Bienayme 3-component reaction (GBB-3CR), Grieco 3-component coupling, Hantzsch pyridine synthesis, Hantzsch dihydropyridine synthesis, Kabachnik-Fields reaction, Mannich 3-component reaction, Passerini 3-component reaction (P-3CR), Passerini-reaction- Amine-Deprotection-Acyl-Migration (PADAM), Pauson-Khand 3-component reaction, Petasis 3-component reaction, Strecker 3-component reaction, Staudinger 3-component reaction, Strecker amino acid synthesis, Ugi 3-component reaction (U-3CR), Ugi 4-component reaction (U-4CR), Ugi-Deprotection-Cyclization, Van Leusen reaction, or any combination thereof.

[0068] In some embodiments, the multicomponent reaction is an aldehyde-alkyne-amine reaction, wherein each well comprises a different aldehyde, a different alkyne, and a different amine to provide a compound (C-I) comprising a propargylamine. In some embodiments, the multicomponent reaction is a Biginelli 3-component reaction (B-3CR), wherein each well comprises a different aryl aldehyde, ethyl acetoacetate, and urea to provide a compound (C-I) comprising a 3,4-dihydropyrimidin-2(lH)-one. In some embodiments, the multicomponent reaction is a Bucherer-Bergs reaction, wherein each well comprises aldehyde or ketone, ammonium carbonate, and potassium cyanide to provide a compound (C-I) comprising a hydantoin. In some embodiments, the multicomponent reaction is a Bucherer-Bergs reaction,wherein each well comprises a different cyanohydrin, ammonium carbonate, and potassium cyanide to give to provide a compound (C-I) comprising a hydantoin. In some embodiments, the multicomponent reaction is a Gewald 3-component reaction (G-3CR), wherein each well comprises a different ketone or aldehyde, a different α-cyanoester, elemental sulfur, and base to provide a compound (C-I) comprising a 2-amino-thiophene. In some embodiments, the multicomponent reaction is a Groebke-Blackbum-Bienayme 3-component reaction (GBB-3CR), wherein each well comprises: a different amidine, a different aldehyde, and a different isocyanide to provide a compound (C-I) comprising an imidazo[l,2-a]azine. In some embodiments, the multicomponent reaction is a Grieco 3-component coupling, wherein each well comprises a different aldehyde, a different aniline, a different electron-rich alkene, and trifluoroacetic acid or Lewis acid to provide a compound (C-I) comprising a six-member heterocycle. In some embodiments, the multicomponent reaction is a Hantzsch dihydropyridine synthesis, wherein each well comprises a different aldehyde, a different β-keto ester, and ammonium acetate to provide a compound (C-I) comprising a dihydropyridine. In some embodiments, the multicomponent reaction is a Kabachnik-Fields reaction, wherein each well comprises a different amine, a different aldehyde or ketone, and a different dialkylphosphite to provide a compound (C-I) comprising an α-aminomethylphosphonate. In some embodiments, the multicomponent reaction is a Mannich 3-component reaction, wherein each well comprises a different ketone, a different primary or secondary amine, and formaldehyde to provide a compound (C-I) comprising a β-amino-carbonyl. In some embodiments, the multicomponent reaction is a Passerini 3-component reaction (P-3CR), wherein each well comprises a different isocyanide, a different aldehyde or ketone, and a different carboxylic acid to provide a compound (C-I) comprising an α-acyloxy amide. In some embodiments, the multicomponent reaction is a Passerini-reaction-Amine-Deprotection-Acyl-Migration (PADAM), wherein each well comprises a different isocyanide, a different N-protected α-aminoaldehyde, and a different carboxylic acid to provide a compound (C-I) comprising an α-hydroxy-β-amino acid. In some embodiments, the multicomponent reaction is a Pauson-Khand 3-component reaction, wherein each well comprises a different olefin, a different alkyne, and carbon monoxide to provide a compound (C-I) comprising an α,β-cyclopentenone. In some embodiments, the multicomponent reaction is a Petasis 3-component reaction, wherein each well comprises a different aldehyde, a different amine, and a different boronic acid to provide a compound (C-I) comprising a tertiary amine. In some embodiments, the multicomponent reaction is a Strecker 3-component reaction, wherein each well comprises a different amine, a different aldehyde or ketone, and a cyanide salt to provide a compound (C-I) comprising an α-amino nitrile. In some embodiments, themulticomponent reaction is a Staudinger 3-component reaction, wherein each well comprises a different diazoketone, a different aldehyde, and a different amine to provide a compound (C-I) comprising a β-lactam. In some embodiments, the multicomponent reaction is a Strecker amino acid synthesis, wherein each well comprises a different aldehyde, cyanide, and ammonia, to provide α-aminonitriles followed by hydrolysis with acid to provide a compound (C-I) comprising an amino acid. In some embodiments, the multicomponent reaction is an Ugi-4CR, wherein each well comprises a different primary amine, a different aldehyde or ketone, a different carboxylic acid, and a different isocyanide to provide a compound (C-I) comprising a bis-amide. In some embodiments, the multicomponent reaction is an Ugi-deprotection- cyclization (UDC) to provide a compound (C-I) comprising isoindolone, pyrolidindione, di-, tri-, or tetra-cyclic scaffolds. In some embodiments, the multicomponent reaction is a Van Leusen reaction, wherein each well comprises a different a ketone, and toluenesulfonylmethyl isocyanide to provide a compound (C-I) comprising a nitrile; or a Van Leusen reaction, wherein each well comprises a different aldehyde, and toluenesulfonylmethyl isocyanide to provide a compound (C-I) comprising an oxazole or imidazole.

[0069] In some embodiments, the reaction (R-I) comprises a cycloaddition reaction, wherein each well comprises a different azide and a different alkyne to provide compound (C-I) comprising a triazole.

[0070] In some embodiments, the reaction (R-I) comprises a cycloaddition reaction, wherein each well comprises a different azide, a different alkyne and a copper catalyst to provide compound (C-I) comprising a triazole.

[0071] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an amine and the reaction (R-II) comprises an amide coupling reaction.

[0072] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the reaction (R-II) comprises an alkylation. In some embodiments, the compound (C-I) of the reaction (R-I) comprises a primary amide group. In some embodiments, the compound (C-I) of the reaction (R- I) comprises a secondary amide group and the reaction (R-II) comprises an alkylation. In some embodiments, the compound (C-I) of the reaction (R-I) comprises a secondary amide group and the reaction (R-II) comprises an alkylation. In some embodiments, the compound (C-I) of the reaction (R-I) comprises a hydroxy group and the reaction (R-II) comprises an alkylation.

[0073] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an amine and the reaction (R-II) comprises a reductive amination with an aldehyde. In some embodiments,the compound (C-I) of the reaction (R-I) comprises an amine and the reaction (R-II) comprises a reductive amination with a carboxylic acid.

[0074] In some embodiments, the compound (C-I) of the reaction (R-I) comprises a carboxylic acid and the reaction (R-II) comprises an esterification.

[0075] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an ester group and the reaction (R-II) comprises a de-esterification.

[0076] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a cross-coupling reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl halide and the reaction (R-II) comprises a cross-coupling reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises a heteroaryl halide and the reaction (R-II) comprises a cross-coupling reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises an alkenyl halide and the reaction (R-II) comprises a cross-coupling reaction.

[0077] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a borylation reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl halide and the reaction (R-II) comprises a borylation reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises a heteroaryl halide and the reaction (R-II) comprises a borylation reaction. In some embodiments, the compound (C-I) of the reaction (R-I) comprises an alkenyl halide and the reaction (R-II) comprises a borylation reaction.

[0078] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl or heteroaryl sulfinate and the rection (R-II) comprises an oxidation reaction.

[0079] In some embodiments, the compound (C-II) of the reaction (R-II) comprises an amine and the subsequent reaction comprises an amide coupling reaction.

[0080] In some embodiments, the compound (C-II) of reaction (R-II) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the subsequent reaction comprises an alkylation. In some embodiments, the compound (C-II) of reaction (R-II) comprises a primary amide group and the subsequent reaction comprises an alkylation. In some embodiments, the compound (C-II) of reaction (R-II) comprises a secondary amide group and the subsequent reaction comprises an alkylation. In some embodiments, the compound (C-II) of reaction (R-II) comprises an amine group and the subsequent reaction comprises an alkylation. In some embodiments, the compound (C-II) of reaction (R-II) comprises a hydroxy group and the subsequent reaction comprises an alkylation.

[0081] In some embodiments, the compound (C-II) of the reaction (R-II) comprises an amine and the subsequent reaction comprises a reductive amination with an aldehyde. In some embodiments, the compound (C-II) of the reaction (R-II) comprises an amine and the subsequent reaction comprises a reductive amination with a carboxylic acid.

[0082] In some embodiments, the compound (C-II) of the reaction (R-II) comprises a carboxylic acid and the subsequent reaction comprises an esterification.

[0083] In some embodiments, the compound (C-II) of the reaction (R-II) comprises an ester group and the subsequent reaction comprises a de-esterification.

[0084] In some embodiments, the compound (C-II) of the reaction (R-II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequent reaction comprises a cross- coupling reaction. In some embodiments, the compound (C-II) of the reaction (R-II) comprises an aryl halide and the subsequent reaction comprises a cross-coupling reaction. In some embodiments, the compound (C-II) of the reaction (R-II) comprises a heteroaryl halide and the subsequent reaction comprises a cross-coupling reaction. In some embodiments, the compound (C-II) of the reaction (R-II) comprises an alkenyl halide and the subsequent reaction comprises a cross-coupling reaction.

[0085] In some embodiments, the compound (C-II) of second reaction (R-II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequent reaction (R-III) comprises a borylation reaction to provide compound (C-III); and wherein a subsequent cross coupling reaction is performed with the borylated compound (C-III). In some embodiments, the compound (C-II) of second reaction (R-II) comprises a phenyl halide, pyridyl halide, or pyrimidyl halide and the subsequent reaction (R-III) comprises a borylation reaction to provide compound (C-III); and wherein a subsequent cross coupling reaction is performed with the borylated compound (C- III).

[0086] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl sulfonyl halide and the reaction (R-II) comprises a Lou-Willis sulfonamide coupling reaction in each well with a different amine added to each well. In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises a phenyl sulfonyl halide and the reaction (R-II) comprises a Lou-Willis sulfonamide coupling reaction in each well with a different amine added to each well.

[0087] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl bromide and the reaction (R-II) comprises a Suzuki reaction in each well with a different aryl or heteroaryl boronic acid added to each well, and a palladium catalyst.

[0088] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl boronic acid and the reaction (R-II) comprises a Buchwald Hartwig reaction in each well with a different amine added to each well, and a palladium catalyst.

[0089] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Heck reaction in each well with a different alkene added to each well, and a palladium catalyst.

[0090] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Sonogashira coupling in each well with a different alkyne added to each well, and a palladium catalyst.

[0091] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Stille coupling reaction in each well with a different organostannane added to each well, and a palladium catalyst.

[0092] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl boronic and the reaction (R-II) comprises a Chan-Lam coupling in each well with a different amine added to each well, and a copper catalyst.

[0093] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl, heteroaryl, cyclic or heterocyclic amine and the reaction (R-II) comprises an amide coupling in each well with a different carboxylic acid added to each well.

[0094] In some embodiments, the compound (C-I) in each well of the reaction (R-I) comprises an aryl halide and the reaction (R-II) comprises a cross-coupling in each well with a different aryl or alkyl halide added to each well, and a nickel catalyst.

[0095] For example, in some embodiments, the reaction (R-I) comprises an alkylation. In some embodiments, the reaction (R-II) comprises a borylation reaction. In some embodiments, the reaction (R-III) comprises an amide coupling reaction.

[0096] In other embodiments, the reaction (R-I) comprises an amide coupling reaction. In some embodiments, the reaction (R-II) comprises an alkylation reaction. In some embodiments, the reaction (R-III) comprises a borylation reaction.

[0097] In some embodiments, the subsequent reaction (R-(III+n)), comprises a cross coupling reaction. In some embodiments, n is 1.

[0098] In other embodiments, the reaction (R-I) comprises a borylation reaction. In some embodiments, the reaction (R-II) comprises an amide coupling reaction. In some embodiments, the reaction (R-III) comprises a cross coupling reaction.

[0099] In some embodiments, the compound (C-I) of the reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a borylationreaction. In some embodiments, the compound (C-II) of the reaction (R-II) comprises an amine and the subsequent reaction comprises an amide coupling reaction.

[0100] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III) or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine, at least one lysine, at least one tyrosine, at least one threonine, at least one aspartate, at least one methionine, at least one glutamate of the biological target, or at least one serine.

[0101] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target.

[0102] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one acquired cysteine of the biological target.

[0103] In some embodiments, the electrophilic warhead moiety is acrylamide, α,β-unsaturated carbonyl, α-halomethyl ketone, epoxide, haloacetamide, β-lactam, ketone, aldehyde, or boronic acid. In some embodiments, the electrophilic warhead moiety is acrylamide. In some embodiments, the electrophilic warhead moiety is α,β-unsaturated carbonyl. In some embodiments, the electrophilic warhead moiety is α-halomethyl ketone. In some embodiments, the electrophilic warhead moiety is epoxide. In some embodiments, the electrophilic warhead is haloacetamide. In some embodiments, the electrophilic warhead moiety is β-lactam. In some embodiments, the electrophilic warhead moiety is ketone or aldehyde. In some embodiments, the electrophilic warhead moiety is boronic acid.

[0104] In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target is:wherein:R1is H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or -C(=O)OR3;R2is H, halogen, substituted or unsubstituted alkyl, or -CN; and each R3is independently H or substituted or unsubstituted alkyl.

[0105] In some embodiments, R1is H, halogen, methyl, ethyl, propyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,

[0106] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target.

[0107] In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target isIn some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target is

[0108] In some embodiments, the compound of the reaction (R-I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target.

[0109] In some embodiments, the electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target is

[0110] In some embodiments, the biological target is a protein tyrosine phosphatase (FTP), protein tyrosine kinases (PTK), protease, small G protein, GTPase, GTPase-activating protein, G protein-coupled receptor (GPCR), ion channel, adaptor protein, transcription activator, cofactor, tumor suppressor, regulatory protein, transporter protein, enzyme, nuclear receptor protein, methyltransferase, or growth factor ligand. In some embodiments, the biological target is a protein tyrosine phosphatase (PTP). In some embodiments, the biological target is a protein tyrosine kinases (PTK). In some embodiments, the biological target is a protease. In some embodiments, the biological target is a small G protein. In some embodiments, the biological target is a GTPase. In some embodiments, the biological target is a GTPase-activating protein. In some embodiments, the biological target is a transcription activator. In some embodiments, the biological target is a cofactor. In some embodiments, the biological target is a tumor suppressor. In some embodiments, the biological target is a regulatory protein. In some embodiments, the biological target is a transporter protein. In some embodiments, the biological target is an enzyme. In some embodiments, the biological target is a nuclear receptor protein. In some embodiments, the biological target is a methyltransferase. In some embodiments, the biological target is a growth factor ligand. In some embodiments, the biological target is a G protein- coupled receptor (GPCR). In some embodiments, the biological target is an ion channel. In some embodiments, the biological target is an adaptor protein.Definitions

[0111] Unless otherwise stated, the following terms used in this application have the definitions given below. The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0112] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0113] The term “aliphatic”, as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched) or branched aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, the term “alkyl” includes straight and branched alkyl groups. An analogous convention applies to other generic terms such as “alkenyl”, “alkynyl” and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”, “alkynyl” and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, “lower alkyl” is used to indicate those alkyl groups (substituted, unsubstituted, branched or unbranched) having about 1-6 carbon atoms.

[0114] The term “alicyclic”, as used herein, refers to compounds which combine the properties of aliphatic and cyclic compounds and include but are not limited to cyclic, or polycyclic aliphatic hydrocarbons and bridged cycloalkyl compounds, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “alicyclic” is intended herein to include, but is not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties, which are optionally substituted with one or more functional groups. Illustrative alicyclic groups thus include, but are not limited to, for example, cyclopropyl, -CH2- cyclopropyl, cyclobutyl, -CH2-cyclobutyl, cyclopentyl, -CH2-cyclopentyl-n, cyclohexyl, -CH2- cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norbornyl moieties and the like, which again, may bear one or more substituents.

[0115] The term “cycloalkyl”, as used herein, refers specifically to cyclic alkyl groups having three to seven, preferably three to ten carbon atoms. Suitable cycloalkyls include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of aliphatic, heteroaliphatic or heterocyclic moieties, may optionally be substituted. An analogous convention applies to other generic terms such as “cycloalkenyl”, “cycloalkynyl” and the like.

[0116] The term “heteroaliphatic”, as used herein, refers to aliphatic moieties in which one or more carbon atoms in the main chain have been substituted with a heteroatom. Thus, a heteroaliphatic group refers to an aliphatic chain which contains one or more oxygen, sulfur, nitrogen, phosphorus or silicon atoms, i.e., in place of carbon atoms. Thus, a 1-6 atom heteroaliphatic linker having at least one N atom in the heteroaliphatic main chain, as used herein, refers to a C1-6aliphatic chain wherein at least one carbon atom is replaced with a nitrogen atom, and wherein any one or more of the remaining 5 carbon atoms may be replaced by an oxygen, sulfur, nitrogen, phosphorus or silicon atom. As used herein, a 1-atom heteroaliphaticlinker having at least one N atom in the heteroaliphatic main chain refers to -NH- or -NR- where R is aliphatic, heteroaliphatic, acyl, aromatic, heteroaromatic or a nitrogen protecting group. Heteroaliphatic moieties may be branched or linear unbranched. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, any of the substituents described above.

[0117] The term “heteroalicyclic”, “heterocycloalkyl” or “heterocyclic”, as used herein, refers to compounds which combine the properties of heteroaliphatic and cyclic compounds and include but are not limited to saturated and unsaturated mono- or polycyclic heterocycles such as morpholino, pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl, etc., which are optionally substituted with one or more functional groups, as defined herein. In certain embodiments, the term “heterocyclic” refers to a non-aromatic 5-, 6- or 7-membered ring or a polycyclic group, including, but not limited to a bi- or tri-cyclic group comprising fused six-membered rings having between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 2 double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iii) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above heterocyclic rings may be fused to an aryl or heteroaryl ring. Representative heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0118] In general, the term “aromatic moiety”, as used herein, refers to stable substituted or unsubstituted unsaturated mono- or poly-cyclic hydrocarbon moieties having preferably 3-14 carbon atoms, comprising at least one ring satisfying the Huckel rule for aromaticity. Examples of aromatic moieties include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthryl, and anthracyl.

[0119] The term “aromatic moiety”, as used herein, refers to stable substituted or unsubstituted unsaturated mono- or poly-cyclic hydrocarbon moieties having preferably 3-14 carbon atoms, comprising at least one ring satisfying the Huckel rule for aromaticity. Examples of aromatic moieties include, but are not limited to, phenyl, indanyl, indenyl, naphthyl, phenanthryl, and anthracyl.

[0120] The term “heteroaromatic moiety”, as used herein, refers to stable substituted or unsubstituted unsaturated mono-heterocyclic or polyheterocyclic moieties having preferably 3-14 carbon atoms and 1-8 heteroatoms, comprising at least one ring satisfying the Huckel rule foraromaticity. Common heteroatoms include O, N, S, P, and Si. Examples of heteroaromatic moieties include, but are not limited to, pyridyl, quinolinyl, dihydroquinolinyl, isoquinolinyl, quinazolinyl, dihydroquinazolyl, and tetrahydroquinazolyl.

[0121] It will also be appreciated that aromatic and heteroaromatic moieties, as defined herein, may be attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety to provide moieties such as -(aliphatic)aromatic, -(heteroaliphatic)aromatic, - (aliphatic)heteroaromatic, -(heteroaliphatic)heteroaromatic, -(alkyl)aromatic, - (heteroalkyl)aromatic, -(alkyl)heteroaromatic, and -(heteroalkyl)heteroaromatic moieties. Substituents of these moieties include, but are not limited to, any of the previously mentioned substituents resulting in the formation of a stable compound.

[0122] The term “aryl” refers to aromatic moieties, as described above, excluding those attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety. In certain embodiments of the present invention, “aryl” refers to a mono- or bicyclic carbocyclic ring system having one or two rings satisfying the Huckel rule for aromaticity, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

[0123] Similarly, the term “heteroaryl” refers to heteroaromatic moieties, as described above, excluding those attached via an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl) moiety. In certain embodiments of the present invention, the term “heteroaryl”, as used herein, refers to a cyclic unsaturated radical having from about five to about ten ring atoms of which one ring atom is selected from S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from S, O and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

[0124] Substituents for aryl and heteroaryl moieties include, but are not limited to, any of the previously mentioned substituents, i.e., the substituents recited for aliphatic moieties, or for other moieties as disclosed herein, resulting in the formation of a stable compound.

[0125] The terms “alkoxy” (or “alkyloxy”), and “thioalkyl” as used herein refers to an alkyl group, as previously defined, attached to the parent molecular moiety through an oxygen atom (“alkoxy”) or through a sulfur atom (“thioalkyl”). In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphaticcarbon atoms. Examples of alkoxy groups, include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl groups include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n- butylthio, and the like.

[0126] The term “amine” refers to a group having the structure — N(R)2wherein each occurrence of R is independently hydrogen, or an aliphatic, heteroaliphatic, aromatic, heteroaromatic, -(alkyl)aromatic, -(heteroalkyl)aromatic, -(heteroalkyl)heteroaromatic, or - (heteroalkyl)heteroaromatic moiety, or the R groups, taken together with the nitrogen to which they are attached, may form a heterocyclic moiety.

[0127] The term “alkylamino” refers to a group having the structure -NHR' wherein R' is alkyl, as defined herein. The term “aminoalkyl” refers to a group having the structure NH2R'-, wherein R' is alkyl, as defined herein. In certain embodiments, the alkyl group contains about 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains about 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain about 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains about 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains about 1-4 aliphatic carbon atoms. Examples of alkylamino include, but are not limited to, methylamino, ethylamino, isopropylamino and the like.

[0128] The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0129] The term “haloalkyl” denotes an alkyl group, as defined above, having one, two, or three halogen atoms attached thereto and is exemplified by such groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

[0130] The term “acyloxy”, as used herein, does not substantially differ from the common meaning of this term in the art, and refers to a moiety of structure -OC(O)Rx, wherein Rx is a substituted or unsubstituted aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.

[0131] The term “acyl”, as used herein, does not substantially differ from the common meaning of this term in the art, and refers to a moiety of structure -C(O)Rx, wherein Rx is a substituted or unsubstituted, aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.

[0132] The term “imino”, as used herein, does not substantially differ from the common meaning of this term in the art, and refers to a moiety of structure -C(=NRx)RY, wherein Rx is hydrogen or an optionally substituted aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl orheteroaryl moiety; and Ry is an optionally substituted aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety.

[0133] The term “C1-C6alkylene”, as used herein, refers to a substituted or unsubstituted, linear or branched saturated divalent radical consisting solely of carbon and hydrogen atoms, having from one to six carbon atoms, having a free valence at both ends of the radical.

[0134] The term “C2-C6alkenylene”, as used herein, refers to a substituted or unsubstituted, linear or branched unsaturated divalent radical consisting solely of carbon and hydrogen atoms, having from two to six carbon atoms, having a free valence at both ends of the radical, and wherein the unsaturation is present only as double bonds and wherein a double bond can exist between the first carbon of the chain and the rest of the molecule.

[0135] As used herein, the terms “aliphatic”, “heteroaliphatic”, “alkyl”, “alkenyl”, “alkynyl”, “heteroalkyl”, “heteroalkenyl”, “heteroalkynyl”, and the like encompass substituted and unsubstituted, saturated and unsaturated, and linear and branched groups. Similarly, the terms “alicyclic”, “heterocyclic”, “heterocycloalkyl”, “heterocycle” and the like encompass substituted and unsubstituted, and saturated and unsaturated groups. Additionally, the terms “cycloalkyl”, “cycloalkenyl”, “cycloalkynyl”, “heterocycloalkyl”, “heterocycloalkenyl”, “heterocycloalkynyl”, “aromatic”, “heteroaromatic”, “aryl”, “heteroaryl”, and the like, used alone or as part of a larger moiety, encompass both substituted and unsubstituted groups

[0136] By “structurally related analog”, “analog” and the like, of a given fragment is meant a fragment that has substantially the same chemical structure as a given fragment except that the analog has a different chemically reactive functionality than does the given fragment. The analog of the fragment may also optionally possess or lack one or more substituents that are either lacking or present, respectively, on the fragments identified provided that the presence or absence of those substituents does not substantially alter the compounds ability to bind to the target. An analog may differ from a reference compound by replacement of one atom by an atom of a different element or replacement of one functional group by another.

[0137] “ Isostere” as used herein refers to any two pharmacophores with similar properties. Bioisosteres are pharmacophores that fit the broadest definition for isosteres and have the same type of biological activity (e.g., see Nicholas A. Meanwell, “Applications of Bioisosteres in the Design of Biologically Active Compounds”, Journal of Agricultural and Food Chemistry 2023 71 (47), 18087-18122).Biological Assays

[0138] In some embodiments, the biological assay is a primary assay, a secondary assay, an in vitro assay, a cellular assay, a binding assay, a functional assay, a homogenous assay or aheterogenous assay. In some embodiments, the biological assay permits identification of off- target effects or identification or compounds that agonize or antagonize specific target variants. In some embodiments, the biological assay allows for identification or avoidance of nonspecific or off-target effects by allowing screening of compounds that agonize or antagonize the same target but signal through different downstream effectors. In some embodiments, the biological assay increases the efficiency of pharmaceutical screening and understanding of biological system. In some embodiments, the biological assay allows, simultaneously or substantially simultaneously, the determination of a certain test compound’s activity against a target (e.g., a heterologous polypeptide), variants of the target, different downstream promoters that may be activated by the target, and off-target effects of the test agent simultaneously in a single well. The methods described herein are capable of such determination for thousands of compounds simultaneously. Described herein are methods and systems of screening and identifying compounds capable of regulating target activity, either positively (as an agonist) or negatively (as an antagonist).

[0139] In some embodiments, the biological assay allows for the identification of on-target effects or off-target effects with a specific biological target. In some embodiments, the biological assay is designed to assess binding to a biological target (e.g., covalent, non-covalent, reversible, irreversible), binding to more than one biological target (e.g., to assess for off-target effects or to assess for protein-protein interactions). In some embodiments, the biological assay assesses whether a test compound is capable of correcting / restoring the function of a biological target protein. In some embodiments, the biological assay assesses whether a test compound is capable of correcting / restoring the misfolding of a biological target protein.

[0140] In some embodiments, the biological assay allows for the identification of the active site of a class of enzymes in normal and / or diseased cells. In some embodiments, these differential activities may lead to new strategies for combination therapies (or designed polypharmacology) and identify new molecular targets. In some embodiments, the biological assay allows for cellular compound profiling to interrogate biological mechanisms both within and outside a target protein.

[0141] In some embodiments, the biological assay in each well assesses the interactions between test compounds and biological activity more efficiently and in greater detail by showing a full array of the interactions, including whether a test compound binds to a specific heterologous polypeptide, whether the test compound binds to a variant of a heterologous polypeptide, whether certain promoters are activated through certain pathways, whether the test compound promotes toxicity in the well, or whether the test agent does not affect any biologicalactivity. In doing so, it can be determined which test compound could be used to help treat conditions or diseases more efficiently, or which test compound could be used as a starting point for structure-activity relationship studies and for optimization into a therapeutics that could help treat conditions or diseases.

[0142] Representative assays are known in the art, e.g., see Eric M Jones, et al., “Structural and functional characterization of G protein-coupled receptors with deep mutational scanning”, eLife 9:e54895 (2020); R Chong, et al., “A multiplexed assay for exon recognition reveals that an unappreciated fraction of rare genetic variants cause large-effect splicing disruptions,” Molecular cell 73 (1), 183-194. e8 (2019); Sriram Kosuri, et al., “Composability of regulatory sequences controlling transcription and translation in Escherichia coli,” PNAS, 110 (34) 14024-14029 (2013); EM Jones, et al., “A scalable, multiplexed assay for decoding GPCR-ligand interactions with RNA sequencing,” Cell systems 8 (3), 254-260. e6 (2019); EM Jones, et al., “A Scalable, Multiplexed Assay for Decoding Receptor-Ligand Interactions,” bioRxiv, 358739 (2018); S Kosuri, E Jones, “Multiplexed receptor-ligand interaction screens,” WG2019010270Peter J. Greasley, et al., “G-protein-coupled receptor screening technologies,” Drug Discovery Today: Technologies, Volume 2, Issue 2, 2005, Pages 163-170; Kota, S., Hou, S., Guerrant, W. et al. “A novel three-dimensional high-throughput screening approach identifies inducers of a mutant KRAS selective lethal phenotype”. Oncogene 37, 4372-4384 (2018); Multiplexed Receptor- Ligand Interaction Screen, WO 2019 / 010270A1; System for Protein-Protein Interaction Screening, WO 2020 / 118198A1; Transcriptional Relay System, WO 2020 / 243164A1; and Systems and Methods for Measuring Cell Signaling Protein Activity, WO2022 / 169767A1. All of the references cited in this section are hereby expressly incorporated by reference for all that they disclose.

[0143] As used herein “measurable” in reference to binding affinity or other affinity parameter means that a value for the affinity parameter is reliably detectable for a ligand of the target. The skilled person will understand that different affinity parameters may be measured with different degrees of precision and accuracy. Ideally, the, precision, accuracy, and dynamic range of an assay will easily accommodate a range of values, so that ligands exhibiting a wide range of measured values for the affinity parameter can be studied. The skilled person will often establish thresholds against which a given test result may be said to be meaningful. For example, in an assay of enzyme inhibition, the concentration of a putative inhibitor of the enzyme may be required to be below a preselected concentration to be considered to be meaningful.

[0144] The term “antagonist” is used in the broadest sense and includes any ligand that partially or fully blocks, inhibits or neutralizes a biological activity exhibited by a target.

[0145] The term “agonist” is used in the broadest sense and includes any ligand that mimics a biological activity exhibited by a target, such as a target, for example, by specifically changing the function or expression of such target, or the efficiency of signaling through such target, thereby altering (increasing or inhibiting) an already existing biological activity or triggering a new biological activity.

[0146] “Active” or “activity” means a measurable, quantitative biological and / or immunological property. Examples of biological activities for protein targets include proteinprotein binding and catalytic activity of enzymes.

[0147] “Derivative” as used herein means a compound obtained from another compound (i.e., a “parent” compound) and containing essential elements of the parent compound, or is a compound related structurally to such parent compound. “Derivative” encompasses compounds that may be obtained directly from the parent compound, or that may be obtained from a common intermediate thereto using analogous chemical methods. For example, adenine is a derivative of purine.

[0148] The term “reactive nucleophile” as used herein refers to a nucleophile that is capable of forming a covalent bond through reaction with a compatible functional group, typically an electrophilic group, on another molecule. In certain embodiments, reactive nucleophiles form a covalent bond through reaction with an electrophile under conditions that do not denature or damage the target. Exemplary reactive nucleophiles include, without limitation, thiols, alcohols, activated carbonyls, epoxides, aziridines, aromatic sulfonates, hemiacetals, and amines.

[0149] Similarly, the term “reactive electrophile” as used herein refers to an electrophile that is capable of forming a covalent bond with a compatible functional group, typically a nucleophilic group, on another molecule. In certain embodiments, reactive electrophiles form a covalent bond through reaction with a nucleophile under conditions that do not denature or otherwise damage the target. Exemplary reactive electrophiles include, without limitation, imines, carbonyls, epoxides, aziridines, sulfonates, and hemiacetals.

[0150] The phrases “nucleophile-reactive group” and “electrophile-reactive group,” as used herein, mean functional groups that can form a covalent bond through reaction with a corresponding compatible functional group, i.e., an electrophile or nucleophile, respectively. In certain embodiments, a nucleophile-reactive group or electrophile-reactive group forms a covalent bond through reaction with a corresponding compatible functional group, i.e., an electrophile or nucleophile, respectively, under conditions that do not denature or otherwise damage the target.

[0151] The phrase “reversible covalent bond” as used herein means a covalent bond which can be broken, generally under conditions that do not denature the target. Examples include, without limitation, disulfides, Schiff-bases, thioesters, and the like.

[0152] The term “target” means a chemical or biological entity for which a ligand has intrinsic binding affinity. The target can be a molecule, a portion of a molecule, or an aggregate of molecules. Specific examples of targets include polypeptides, proteins, ligands for receptors, allosteric enzyme regulators, immunoglobulins, polynucleotides, carbohydrates, glycolipids, and other macromolecules, such as protein complexes, nucleic acid-protein complexes, chromatin, ribosomes, lipid bilayer-containing structures, such as membranes, or structures derived from membranes, such as vesicles.

[0153] A “site” on a target refers to a site to which a specific ligand binds, which may include a specific sequence of monomeric subunits, e.g., amino acid residues, or nucleotides, and may have a characterized three-dimensional structure. Typically, the molecular interactions between the ligand and the site of interest on the target are non-covalent, and include hydrogen bonds, van der Waals interactions and electrostatic interactions. In the case of polypeptides a site of interest broadly includes the amino acid residues involved in binding of the target to a molecule with which it forms a natural complex in vivo or in vitro.

[0154] When, for example, the target is a protein that exerts its biological effect through binding to another protein, such as with hormones, cytokines or other proteins involved in signaling, it may form a natural complex in vivo with one or more other proteins. In this case, the site of interest is defined as the critical contact residues involved in a particular protein: protein binding interface. Critical contact residues are defined as those amino acids on a first protein that make direct contact with amino acids on a second protein, and when mutated to alanine decrease the binding affinity by at least 10-fold, alternately at least 20-fold, as measured with a direct binding or competition assay (e.g., ELISA or RIA).

[0155] The term “compound-biological target complex” refers to the complex the compound of any of reactions (R-II), (R-II), ( R- III), or (R-(III+n)) and the biological target. In some embodiments, the complex formation is through a covalent bond. In some embodiments, the bond is reversible or irreversible bond. . In some embodiments, the complex formation is through an electrostatic van der Waals interaction.

[0156] “Binding”, “binds”, or “selectively binds” as used herein in the context of a ligand, e.g., a test ligand, binding to a target (e.g., a protein) means that the dissociation constant of the ligand for the target is at least 10-fold lower than the dissociation constant of the ligand for another biological molecule(s) being used as a reference. For example, if a ligand “selectively binds”Aurora- A over Aurora-B, it binds Aurora- A with at least a 10-fold lower dissociation constant than its corresponding dissociation constant for Aurora-B.

[0157] The term “modulate” as used herein, means to interact with a target either directly or indirectly so as to alter the activity of the target, including, by way of example only, to enhance the activity of the target, to inhibit the activity of the target, to limit the activity of the target, or to extend the activity of the target.

[0158] The term “modulator” as used herein, refers to a molecule that interacts with a target either directly or indirectly. The interactions include, but are not limited to, the interactions of an agonist, partial agonist, an inverse agonist, antagonist, allosteric modulator, degrader, or combinations thereof. In some embodiments, a modulator is an agonist.

[0159] “Selectively modulates” as used herein in the context of a molecule modulating a given functional or structural property of a target (e.g., a protein), means that the molecule measurably alters that property, either positively or negatively, to a greater degree than the modulation of the property by a ligand of another biological molecule(s) being used as a reference.

[0160] As used herein, “protein” means any molecule comprising two or more peptide units, each comprising an amino acid residue, arranged in a linear chain and joined together by peptide bonds. Protein chains comprising more than 30 amino acid residues may be referred to as polypeptides. Protein chains of 30 amino acid residues or fewer may be referred to as oligopeptides. Proteins include, but are not limited to, enzymes (e.g., cysteine protease, serine protease, and aspartyl proteases), receptors, transcription factors, growth factors, cytokines, immunoglobulins, nuclear proteins, signal transduction components (e.g., kinases, phosphatases), and glycoproteins.

[0161] “Polynucleotide,” as used herein in singular or plural, means any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double- stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” includes DNAs and RNAs that contain one or more modified bases.Thus, for example, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, are included within the term “polynucleotides” as defined herein. In general, the term “polynucleotide” embraces all chemically, enzymatically and / or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cell.

[0162] A “ligand” as defined herein is a molecule that has an intrinsic binding affinity for the target. Ligands are typically small organic molecules that have an intrinsic binding affinity for the target, but may also be other sequence-specific binding molecules, such as peptides (D-, L-, or a mixture of D- and L-), peptidomimetics, complex carbohydrates, or other oligomeric molecules that bind specifically to the target.

[0163] The phrases “modified to contain” and “modified to possess” are used interchangeably, and refer to making a mutant, variant or derivative of the target, or the reactive nucleophile or electrophile, including but not limited to chemical modifications. For example, in a protein one can substitute an amino acid residue having a side chain containing a nucleophile or electrophile for a wild-type residue. Another example is the conversion of the thiol group of a cysteine residue to an amine group.EXAMPLES

[0164] Various chemistries may be employed for chemically reacting the any of compounds (C-I), (C-II), (C-II), or (C-(III+n)) with the biological assay. Chemistries available for forming a reversible or irreversible covalent bond between a compound of (C-I), (C-II), (C-III), or (C- (III+n)) and a test fragment are known in the art, and are described in basic textbooks, such as, e.g., March, Advanced Organic Chemistry, John Wiley & Sons, New York, 4thedition, 1992. The chemistries include, for example, reductive aminations between aldehydes or ketones and amines are described, for example, in March et al., supra, at pp. 898-900; alternative methods for preparing amines at p. 1276; reactions between aldehydes or ketones and hydrazide derivatives to give hydrazones and hydrazone derivatives such as semicarbazones at pp. 904-906; amide bond formation at p. 1275; formation of ureas at p. 1299; formation of thiocarbamates at p. 892; formation of carbamates at p. 1280; formation of sulfonamides at p. 1296; formation of thioethers at p. 1297; formation of disulfides at p. 1284; formation of ethers at p. 1285; formation of esters at p. 1281; additions to epoxides at p. 368; additions to aziridines at p. 368; formation of acetals and ketals at p. 1269; formation of carbonates at p. 392; formation of enamines at p. 1264;Metathesis of alkenes at pp. 1146-1148 (see also Grubbs et al., Acc. Chem. Res. 28:446-453

[1995] ); transition metal-catalyzed couplings of aryl halides and sulfonates with alkanes and acetylenes, e.g., Heck reactions, at p.p. 717-178; the reaction of aryl halides and sulfonates with organometallic reagents, such as organoboron reagents, at p. 662 (see also Miyaura et al., Chem. Rev. 95:2457

[1995] ); organotin, and organozinc reagents, formation of oxazolidines (Ede et al., Tetrahedron Letts. 28:7119-7122

[1997] ); formation of thiazolidines (Patek et al., Tetrahedron Letts. 36:2227-2230

[1995] ); amines linked through amidine groups by coupling amines through imidoesters (Davies et al., Canadian J. Biochem. c50:416-422

[1972] ), reactions between aldehydes or ketones and O-alkyl-hydroxylamine derivatives to give oximes (Maly et al., Proc. Nat. Acad. Set. USA 97:2419-2424

[2000] ); and the like. Additionally, the Huisgen 1,3- dipolar cycloaddition of azides and acetylenes can give 1,2,3-triazoles (Lewis et al., Angew.Chem. Int. Ed. Engl. 41 : 1053-1047

[2002] ). In particular, disulfide-containing small molecule libraries may be made from commercially available carboxylic acids and protected cysteamine (e.g., mono-BOC-cysteamine) by adapting the method of Parlow et al., Mol. Diversity 1 :266-269 (1995). All of the references cited in this section are hereby expressly incorporated by reference for all that they disclose.

[0165] The following examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.Example 1: Amide libraries using carboxylic acid core and amine fragments

[0166] Primary (1 Am) and secondary (2 Am) amine fragments (300 nL, 50 mM in DMA, 15 nmol, 1.5 equiv) were dispensed onto 1,536-well library plates using the Access Laboratory Workstation / Beckman Echo(R) Liquid Handler (11,709 wells across 9 plates). A set of 96 “QC fragments per fragment class (viz. 1 Am and 2Am, respectively) was also dispensed onto each of the library plates for benchmarking the reaction efficiency and consistency across multiple plates. Separately, a stock solution of carboxylic acid core (0.2 mmol, 1 equiv), base (5 equiv), and activator, (1.5 equiv) in DMA (4 mL, 50 mM w.r.t. core) was prepared freshly, and the mixture was dispensed (200 nL per well) onto the library plates using Dispendix I.DOT liquid handler.An “activator” as used herein, refers to a reagent that promotes amide bond formation by way of an activated ester. Exemplary activators include but are not limited to HATU, HBTU, HOBt, DMTMM, EDC, DCC, PyAOP, PyBOP, CITU, BOP, and PyOxim. The library plates were sealed, spun down with a centrifuge, and loaded onto a plate shaker, where it was shaken for an overnight (16 h) at ambient temperature (with the option to heat up to 70 °C). After that, the plates were spun down again with a centrifuge, unsealed, and bulk-filled with DMSO (3 μL per well). The resulting assembled library with 9 plates was then handed over to the Screening team, and ready for the preparation of Assay -Ready Plates.

[0167] In some embodiments, each R’ and R” is independently substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is independently substituted with one to four halogen, -OH, -NH2, -CN, - C(O)OH, -C(O)NH2, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, each R’ and R” is independently substituted or unsubstituted C1-C6alkyl. In some embodiments, each R’ and R” is substituted or unsubstituted C3-C10cycloalkyl, or substituted or unsubstituted C3-C10heterocycloalkyl. In some embodiments, each R’ and R” is independently substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl. In some embodiments, each R’ and R” is independently substituted or unsubstituted phenyl. In some embodiments, R’ and R” together with the atoms to which they are attached form a substituted or unsubstituted C3-C10heterocycloalkyl.

[0168] In some embodiments, R is substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is substituted with one to four halogen, -OH, -NH2, -CN, -C(O)OH, -C(O)NH2, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, R is substituted or unsubstituted C1-C6alkyl. In some embodiments, R is substituted or unsubstituted C3-C10cycloalkyl or substituted or unsubstituted C3-C10heterocycloalkyl. In some embodiments, substituted or unsubstituted C6-C10aryl or substituted or unsubstituted C5-C10heteroaryl. In some embodiments, R is substituted or unsubstituted phenyl.Example 2. Formal cross-electrophile coupling of aryl bromides (2-step borylation-Suzuki coupling)Part A - Borylation in Batch

[0169] A reaction vessel (such as a culture tube or a scintillation vial) was charged with a stirrer bar, an aryl halide (bromide or chloride) core (0.05 mmol, 1 equiv), XPhos-Pd-G4 (5 mol%), XPhos (10 mol%), B2(OH)4 (1.5 equiv), potassium 2-ethylhexanoate (K2EH) (3 equiv), and ethylene glycol (3 equiv). The vessel was evacuated and back-filled with N2 for three times before degassed ethanol (0.25 mL, 0.2 M w.r.t. core) was added. The reaction mixture was heated to 80 °C for 1 h, which was then cooled down to ambient temperature, diluted with 3 parts of degassed DMA (0.75 mL) under N2. The diluted mixture with filtered using a syringe filter(PTFE, 015 mm, 0.22 pm) into a sealed tube, and degassed for high-throughput step (use of glove box is default, but optional).Part B - High-Throughput Suzuki Coupling

[0170] Aryl bromide (ABr) fragments (220 nL, 50 mM in DMA, 1.1 equiv w.r.t. aryl halide core) were dispensed onto 1,536-well library plates using the Access LaboratoryWorkstation / Beckman Echo(R) Liquid Handler (2,444 wells across 2 plates - subject to expansion, plus 96 QC fragments per plate). *(Optional) The plates were frozen at -80 °C for 30 min before being brought into the glove box* Aqueous K3PO4 [600 mM] (50 nL, 30 nmol, 3equiv), XPhos-Pd-G4 (20 nL, 25 mM in DMA, 0.5 nmol, 5 mol%), and the diluted, filtered reaction crude [50 mM] (200 nL) (10 nmol, 1 equiv w.r.t. aryl halide core) from Part A were dispensed sequentially using Dispendix I.DOT. The library plates were sealed and spun down with a centrifuge, which were then loaded into a vacuum oven. The vacuum oven was sealed, evacuated and back-filled with N2 for 3-6 times. The plates were heated under N2at 80-110 °C for 16-24 h, after which the plates were removed from the oven and allowed to cool down to ambient temperature. The plates were unsealed and bulk-filled with DMSO (3 μL per well), before they were sealed again and shaken on a plate shaker at 2500 rpm or above for at least 10 min to ensure the reaction crudes were thoroughly mixed with DMSO. The assembled library comprised of 2 plates was then handed over to the Screening team for the preparation of Assay- Ready Plate.

[0171] To determine reaction efficiencies, samples (1 μL) were transferred out from the “QC” wells to a 384-well plate, and bulk-filled with a diluent (25-40 μL per well) comprised of 95% water, 5% acetonitrile, 0.1% formic acid and an internal standard (1 pM of 5- carboxytetramethylrhodamine (CAS# 91809-66-4) in DMSO). The samples were then injected into an Agilent 1290 UHPLC system coupled with Sciex X500R QTOF high-resolution mass spectrometer. Extracted ion chromatograms for targeted mass-to-charge ratio (m / z) were analyzed and the area under the curve (AUCs) for targeted peaks were normalized against internal standard.

[0172] In some embodiments, R1is -CN, -NRaRb, -OH, -C(O)Ra, -NHC(O)Ra, haloalkyl, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is substituted with one to four halogen, - OH, -NO2, -CN, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl., wherein Rais hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Rbis alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0173] In some embodiments, R2is -CN, -NRaRb, -OH, -C(O)Ra, -NHC(O)Ra, haloalkyl, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is substituted with one to four halogen, -OH, -NO2, -CN, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl., wherein Rais hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Rbis alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.Example 3. Three-Component Amide-Suzuki Coupling Library SynthesisPart A - High-Throughput Amide Coupling

[0174] Primary (1 Am) and secondary (2Am) amine fragments (SM-III) (250 nL, 50 mM inDMA, 12.5 nmol, 1.1 equiv) were dispensed onto 1,536-well library plates using the AccessLaboratory Workstation / Beckman Echo(R) Liquid Handler (11,709 wells across 9 plates). A set of 96 “QC” fragments per fragment class (viz. 1 Am and 2Am, respectively) was also dispensed onto each of the library plates for benchmarking the reaction efficiency and consistency acrossmultiple plates. Separately, a stock solution of bifunctional carboxylic acid linker (SM-II) (0.3 mmol) and DMTMM (as activator) (0.45 mmol) in DMA (6 mL) was prepared freshly. The mixture (250 nL per well) and aqueous K3PO4[500 mM] (75 nL, 37.5 nmol, 3 equiv) was dispensed sequentially onto the library plates using Dispendix I.DOT liquid handler. The library plates were sealed, spun down with a centrifuge, and loaded onto a plate shaker, where it was shaken for an overnight (16 h) at ambient temperature (with the option to heat up to 70 °C). After that, the plates containing intermediate amides (C-I) were spun down again with a centrifuge for Part C - High-Throughput Suzuki Coupling.Part B - Borylation in Batch

[0175] A reaction vessel (such as a culture tube or a scintillation vial) was charged with a stirrer bar, an aryl halide (bromide or chloride) core (SM-I) (0.2 mmol, 1 equiv), XPhos-Pd-G4 (5 mol%), XPhos (10 mol%), B2(OH)4(1.5 equiv), potassium 2-ethylhexanoate (K2EH) (3 equiv), and ethylene glycol (3 equiv). The vessel was evacuated and back-filled with N2for three times before degassed ethanol (1 mL, 0.2 M w.r.t. core) was added. The reaction mixture was heated to 80 °C for 1 h, which was then cooled down to ambient temperature, diluted with 3 parts of degassed DMA (3 mL) under N2. The diluted mixture with filtered using a syringe filter (PTFE, 015 mm, 0.22 μm) into a sealed tube, and degassed for high-throughput step (use of glove box is default, but optional).Part C - High-Throughput Suzuki Coupling

[0176] Library plates from Part A containing intermediate amides (C-I) were frozen at -80 °C for 30 min before being brought into the glove box. Aqueous K3PO4[500 mM] (90 nL, 45 nmol, 4 equiv), XPhos-Pd-G4 (25 nL, 22.5 mM in DMA, 0.56 nmol, 5 mol%), and the diluted, filtered reaction crude [50 mM] (225 nL) (11.3 nmol, 1 equiv w.r.t. aryl halide core) from Part B were dispensed sequentially using Dispendix I.DOT. The library plates were sealed and spun down with a centrifuge, which were then loaded into a vacuum oven. The vacuum oven was sealed, evacuated and back-filled with N2 for 3-6 times. The plates were heated under N2at 110 °C for 16 h, after which the plates were removed from the oven and allowed to cool down to ambient temperature. The plates were unsealed and bulk-filled with DMSO (3 μL per well), before they were sealed again and shaken on a plate shaker at 2500 rpm or above for at least 10 min to ensure the reaction crudes were thoroughly mixed with DMSO. The assembled library comprised of 9 plates was then handed over to the Screening team for the preparation of Assay -Ready Plate.

[0177] To determine reaction efficiencies, samples (1 μL) were transferred out from the “QC” wells to a 384-well plate, and bulk-filled with a diluent (25-40 pL per well) comprised of 95% water, 5% acetonitrile, 0.1% formic acid and an internal standard (1 μM of 5-carboxytetramethylrhodamine (CAS# 91809-66-4) in DMSO). The samples were then injected into an Agilent 1290 UHPLC system coupled with Sciex X500R QTOF high-resolution mass spectrometer. Extracted ion chromatograms for targeted mass-to-charge ratio (m / z) were analyzed and the area under the curve (AUCs) for targeted peaks were normalized against internal standard.

[0178] In some embodiments, each R’ and R” is independently substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is independently substituted with one to four halogen, -OH, -NH2, -CN, - C(O)OH, -C(O)NH2, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, each R’ and R” is independently substituted or unsubstituted C1-C6alkyl. In some embodiments, each R’ and R” is independently substituted or unsubstituted C3-C10cycloalkyl, or substituted or unsubstituted C3-C10heterocycloalkyl. In some embodiments, each R’ and R” is independently substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl. In some embodiments, each R’ and R” is independently substituted or unsubstituted phenyl. In some embodiments, R’ and R” together with the atoms to which they are attached form a substituted or unsubstituted C3-C10heterocycloalkyl.

[0179] In some embodiments, R is substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C2-C6alkenyl, substituted or unsubstituted C2-C6alkynyl, substituted or unsubstituted C3-C10cycloalkyl, substituted or unsubstituted C3-C10heterocycloalkyl, substituted or unsubstituted C6-C10aryl, or substituted or unsubstituted C5-C10heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl when substituted is substituted with one to four halogen, -OH, -NH2, -CN, -C(O)OH, -C(O)NH2, alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In some embodiments, R is substituted or unsubstituted C1-C6alkyl. In some embodiments, R is substituted or unsubstituted C3-C10cycloalkyl or substituted or unsubstituted C3-C10heterocycloalkyl. In some embodiments, substituted or unsubstituted C6-C10aryl or substituted or unsubstituted C5-C10heteroaryl. In some embodiments, R is substituted or unsubstituted phenyl.

[0180] A representative non-limiting 4-step and 4-component library was synthesized by the methods of Examples 1-3 by the following steps: (1) an alkylation reaction, (2) a borylation, (3) an amide coupling, and (4) a cross-coupling reaction, e.g., a Suzuki reaction. Each successive intermediate is carried into the next step without purification. The representative steps are interchangeable and can be performed in any order. For example, Step 1 may be an amide coupling and Step 2 maybe be a cross-coupling reaction, and so on, to assemble a different compound library.

[0181] In some embodiments, the pro-Core is

[0182] In some embodiments,IS

[0183] In some embodiments, the bifunctional linker is selected from

[0184] In some embodiments,is selected fromExample 4. Representative Biological Assessment of a Representative Library

[0185] Echo qualified 1536-well cyclic olefin copolymer (COC) library plates containing the crude mixtures of synthesized compounds made through the methods of Examples 1-3 were centrifuged at 500 ref for 10 s. A Beckman Echo 650 acoustic dispenser was used to transfer each compound from their source library plates into the destination wells of empty 1536-well white CulturPlates (the assay ready plates). The transfer volume of each compound was calculated to achieve a concentration of 50 μM in the final assay well volume of 3 μL for the high-throughput screen. DMSO was dispensed into each well to achieve a 1% DMSO v / v in the final assay volume. The plates were sealed on an Agilent PlateLoc Thermal Microplate Sealer, centrifuged at 500 ref for 10 s, and stored at room temperature.

[0186] The H1299 cell line was engineered to contain the TP53-R273H mutation under Tet inducible expression with a firefly luciferase reporter expressed off a p53 response element. The cells were cultured in RPMI media containing 10% dialyzed FBS. Cells were washed once with PBS buffer before TrypLE treatment to dissociate them from the tissue culture flask surface. Once cells were detached, an equal volume of RPMI media was added to the cells, transferred to conical tubes, and centrifuged for 300 ref for 3 min. The supernatant was discarded, and the cells were resuspended into fresh RPMI media. Cells were counted on a Countess 3 cell counter, and additional RPMI media was added to achieve a final cell concentration of 106cells per mL. Doxycycline was added to the media for a final concentration of 2 μM to induce expression of the p53 mutant protein.

[0187] Using a BioTek MultiFlo bulk dispenser, 3 μL of the cell solution was seeded into each well of the assay ready plates. The cells were incubated with the unpurified compounds for 18hours in a 37 °C incubator. The Promega Bright-Glo reagent was prepared according to the manufacturer’s instructions, and 3 μL was dispensed into each well of the plate. The plate was centrifuged at 150 ref for 1 min, and the luciferase luminescence signal was read out on a SpectraMax i3x microplate reader. Assay results were quantified as luminescence fold change vs. control and reported in Table 1.Table 1.

[0188] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification of the reaction mixture of step b), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate assay with the compounds in each well; and d) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

2. A synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed;e) without purification of the reaction mixtures of step d), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate well; and f) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

3. A synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C-III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) without purification of the reaction mixtures of step e), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and g) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

4. A synthesis and high-throughput screening method for a compound that affects at least one biological property in a biological assay designed to assess such biological properties comprising the steps of: a) performing a first reaction (R-I) in each well of a plurality of wells of a microplate to produce a different compound (C-I) in each well, wherein each well of the plurality of wells of the microplate comprises a different plurality of reactants and a solvent or a combination of solvents for the first reaction; b) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the first reaction was performed; c) without purification, performing a second reaction (R-II) in each well of the plurality of wells of the microplate in step b) to produce a different compound (C-II) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step b) and a different plurality of reactants for the second reaction (R-II) and a solvent or a combination of solvents for the second reaction; d) optionally removing the solvent or the combination of solvents from the plurality of wells of the microplate in which the second reaction was performed; e) without purification, optionally performing at least a subsequent reaction (R-III) in each well of the plurality of wells of the microplate from step d) to produce a different compound (C-III) in each well, wherein each well of the plurality of wells of the microplate comprises the reaction mixture of step d) and a different plurality of reactants for the subsequent reaction (R-III) and a different plurality of reactants and a solvent or a combination of solvents for the subsequent reaction; optionally removing the solvent or combination of solvents from the plurality of wells of the microplate in which the subsequent reaction was performed; f) optionally repeating step e) one or more times, wherein each subsequent reaction (R- (III+n)) produces a different compound (C-(III+n)) in each well, wherein n represents the number of times step e) is repeated; g) without purification of step f), individually performing a biological assay with the contents of each well of the plurality of wells of the microplate; and h) assessing whether or not at least one compound in each well has a biological effect in the biological assay.

5. The method of any one of claims 1-4, wherein the microplate comprises 96, 192, 384, 1536, 3456, or 6144 wells.

6. The method of any one of claims 1-5, wherein each reaction is performed on a nanoscale.

7. The method of any one of claims 1-6, wherein each well comprises a total volume of between 10-5000 nanoliters; or each well comprises 1-1000 nanomoles of a reactant or compound (C-(III+n)).

8. The method of any one of claims 1-5, wherein each reaction is performed on a microscale.

9. The method of any one of claims 1-5 or 8, wherein each well comprises a total volume of between 10-5000 microliters; or each well comprise 1-1000 micromoles of a reactant or compound (C-(III+n)).

10. The method of any one of claims 1-9, wherein the reaction (R-I) comprises an amide coupling reaction or a sulfonamide coupling reaction.

11. The method of any one of claims 1-9, wherein the reaction (R-I) comprises an alkylation, an alkenylation reaction, or an alkynylation reaction.

12. The method of any one of claims 1-9, wherein the reaction (R-I) comprises alkynylation of an aryl halide or heteroaryl halide.

13. The method of any one of claims 1-9, wherein the reaction (R-I) comprises alkenylation of an aryl halide or heteroaryl halide.

14. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a deprotection, skeletal editing, atom insertion, epoxidation, aziridination, cyclopropanation, atom deletion, deoxygenation, deamination, dehalogenation, atom swapping, a sigmatropic rearrangement, a conjugate addition, an oxidation reaction, a reduction reaction, a decarboxylative reaction, a carboxylative reaction, a carbonylative reaction, a decarb onylative reaction, a halogenation reaction, a reductive amination, a carbonylation, an acylation, an esterification, or a de-esterification.

15. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a silylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide.

16. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a borylation reaction of an aryl halide, heteroaryl halide, alkenyl halide, or alkyl halide.

17. The method of any one of claims 1-9, wherein the reaction (R-I) comprises arylation.

18. The method of any one of claims 1-9, wherein the reaction (R-I) comprises amination of an aryl halide or heteroaryl halide.

19. The method of any one of claims 1-9, wherein the reaction (R-I) comprises carbonylation or acylation of an aryl halide or heteroaryl halide.

20. The method of any one of claims 1-9, wherein the reaction (R-I) comprises thioesterification of an aryl halide or heteroaryl halide; optionally wherein the reaction (R-II) comprises an oxidation to provide an aryl or heteroaryl sulfone.

21. The method of any one of claims 1-9, wherein the reaction (R-I) comprises phosphorylation of an aryl halide or heteroaryl halide.

22. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a sulfinylation of an aryl halide or heteroaryl halide.

23. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a sulfinylation of an aryl or heteroaryl boronic acid.

24. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a cross coupling reaction.

25. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a cycloaddition reaction.

26. The method of claim 25, wherein the reaction (R-I) comprises [3+2] cycloaddition, Huisgen 1,3-dipolar cycloaddition, thiol-ene reaction, Diels-Alder reaction, inverse electron demand Diels- Alder reaction, [4+1] cycloaddition between an isonitrile and a tetrazine, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), azide alkyne Huisgen cycloaddition, ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC), strain- promoted azide-alkyne cycloaddition (SPAAC), strain-promoted alkyne-nitrone cycloaddition (SPANC), an inverse electron demand Diels-Alder cycloaddition (iEDDA), sydnone-alkyne cycloaddition (SPSAC), alkene and azide [3+2] cycloaddition, or alkene and tetrazine inverse-demand Diels-Alder.

27. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a cycloaddition reaction, wherein each well comprises a different azide and a different alkyne to provide compound (C-I) comprising a triazole.

28. The method of any one of claims 1-9, wherein the reaction (R-I) comprises a cycloaddition reaction, wherein each well comprises a different azide, a different alkyne and a copper catalyst to provide compound (C-I) comprising a triazole.

29. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an amine and the reaction (R-II) comprises an amide coupling reaction.

30. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the reaction (R-II) comprises an alkylation.

31. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an amine and the reaction (R-II) comprises a reductive amination with an aldehyde or carboxylic acid.

32. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises a carboxylic acid and the reaction (R-II) comprises an esterification.

33. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an ester group and the reaction (R-II) comprises a de-esterification.

34. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a cross-coupling reaction.

35. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the reaction (R-II) comprises a borylation reaction.

36. The method of any one of claims 1-28, wherein the compound (C-I) of the reaction (R-I) comprises an aryl or heteroaryl sulfinate and the rection (R-II) comprises an oxidation reaction.

37. The method of any one of claims 1-36, wherein the compound (C-II) of the reaction (R- II) comprises an amine and the subsequent reaction comprises an amide coupling reaction.

38. The method of any one of claims 1-36, wherein the compound (C-II) of reaction (R-II) comprises an amine group, a primary amide group, a secondary amide group, or hydroxy group and the subsequent reaction comprises an alkylation.

39. The method of any one of claims 1-36, wherein the compound (C-II) of the reaction (R- II) comprises an amine and the subsequent reaction comprises a reductive amination with an aldehyde or carboxylic acid.

40. The method of any one of claims 1-36, wherein the compound (C-II) of the reaction (R- II) comprises a carboxylic acid and the subsequent reaction comprises an esterification.

41. The method of any one of claims 1-36, wherein the compound (C-II) of the reaction (R- II) comprises an ester group and the subsequent reaction comprises a de-esterification.

42. The method of any one of claims 1-36, wherein the compound (C-II) of the reaction (R- II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequent reaction comprises a cross-coupling reaction.

43. The method of any one of claims 1-36, wherein the compound (C-II) of second reaction (R-II) comprises an aryl halide, heteroaryl halide, or alkenyl halide and the subsequentreaction (R-III) comprises a borylation reaction to provide compound (C-III); and wherein a subsequent cross coupling reaction is performed with the borylated compound (C-III).

44. The method of claim 32, 41, or 42, wherein the cross coupling reaction is a Cadiot- Chodkiewicz coupling, Castro-Stephens coupling, Corey-House synthesis, Kumada coupling, Heck reaction, Sonogashira coupling, Negishi coupling, Stille cross coupling, Suzuki reaction, Murahashi coupling, Hiyama coupling, Fukuyama coupling, Liebeskind- Srogl coupling, cross dehydrogenative coupling, decarb oxy lative cross-coupling, Ullmann-type reaction, Buchwald-Hartwig reaction, or Chan-Lam coupling.

45. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl sulfonyl halide and the reaction (R-II) comprises a Lou-Willis sulfonamide coupling reaction in each well with a different amine added to each well.

46. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl bromide and the reaction (R-II) comprises a Suzuki reaction in each well with a different aryl or heteroaryl boronic acid added to each well, and a palladium catalyst.

47. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl boronic acid and the reaction (R-II) comprises a Buchwald Hartwig reaction in each well with a different amine added to each well, and a palladium catalyst.

48. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Heck reaction in each well with a different alkene added to each well, and a palladium catalyst.

49. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Sonogashira coupling in each well with a different alkyne added to each well, and a palladium catalyst.

50. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl halide and the reaction (R-II) comprises a Stille coupling reaction in each well with a different organostannane added to each well, and a palladium catalyst.

51. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl or heteroaryl boronic and the reaction (R-II) comprises a Chan-Lam coupling in each well with a different amine added to each well, and a copper catalyst.

52. The method of any one of claims 1-28, wherein the compound (C-I) in each well of the reaction (R-I) comprises an aryl, heteroaryl, cyclic or heterocyclic amine and the reaction (R-II) comprises an amide coupling in each well with a different carboxylic acid added to each well.

53. The method of any one of claims 1-51, wherein at least one of the reaction steps is air free.

54. The method of any one of claims 1-52, wherein at least one compound from reaction (R- I), (R-II), (R-III) or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine, at least one lysine, at least one tyrosine, at least one threonine, at least one aspartate, at least one methionine, at least one glutamate, or at least one serine of the biological target.

55. The method of any one of claims 1-52, wherein at least one compound from reaction (R- I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target.

56. The method of any one of claims 1-52, wherein at least one compound from reaction (R- I), (R-II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one acquired cysteine of the biological target57. The method of any one of claims 53-55, wherein the electrophilic warhead moiety is acrylamide, α,β-unsaturated carbonyl, α-halomethyl ketone, epoxide, haloacetamide, β- lactam, ketone, aldehyde, or boronic acid.

58. The method of claim 54 or 55, wherein the electrophilic warhead moiety capable of covalently reacting with at least one cysteine of the biological target is:R1is H, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or -C(=O)OR3;R2is H, halogen, substituted or unsubstituted alkyl, or -CN; and each R3is independently H or substituted or unsubstituted alkyl.

59. The method of claim 58, wherein R1is H, halogen, methyl, ethyl, propyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,60. The method of any one of claims 1-52, wherein the compound of the reaction (R-I), (R- II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target.

61. The method of claim 60, wherein the electrophilic warhead moiety capable of covalently reacting with at least one lysine of the biological target is62. The method of any one of claims 1-52, wherein the compound of the reaction (R-I), (R- II), (R-III), or (R-(III+n)) comprises an electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target.

63. The method of claim 62, wherein the electrophilic warhead moiety capable of covalently reacting with at least one aspartate or at least one glutamate of the biological target is64. The method of any one of claims 1-63, wherein the biological target is a protein tyrosine phosphatase (PTP), protein tyrosine kinases (PTK), protease, small G protein, GTPase,GTPase-activating protein, G protein-coupled receptor (GPCR), ion channel, adaptor protein, transcription activator, transcription factor, cofactor, tumor suppressor, regulatory protein, transporter protein, enzyme, nuclear receptor protein, methyltransferase, or growth factor ligand.

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