Scalable thiol reactivity profiling

Abstract

The application discloses the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. The application further discloses high throughput screening assays using STRP identified azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. The application further discloses an azetidinyl oxadiazole containing small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site.

Description

TSRI 2247.1PC SCALABLE THIOL REACTIVITY PROFILING CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application No. 63 / 635,007, which was filed on May 17, 2024, and which is hereby incorporated by reference in its entirety GOVERNMENT SUPPORT

[0002] This invention was made with government support under GM146865 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The application relates to the development of scalable thiol reactivity (STRP), a method which enables the facile interrogation of large chemical libraries for intrinsic reactivity with cysteine. The application further relates to high throughput screening assays using STRP identified azetidinyl oxadiazole as a moiety that selectively reacts with cysteine through a ring opening-based mechanism, capable of covalently engaging cysteine residues broadly across the human proteome. The application further relates to an azetidinyl oxadiazole containing small molecule that augments the catalytic activity of the deubiquitinase UCHL1 in vitro and in cells by covalently modifying a cysteine distal to its enzymatic active site. BACKGROUND OF THE INVENTION

[0004] Chemical moieties that react with cysteine serve essential roles as drugs, probe molecules, cross-linkers, and conjugation agents, given the intrinsic nucleophilicity of cysteine relative to other protein coding amino acids.1While becoming ever more critical to contemporary pharmacology, the use of cysteine reactive groups within FDA approved drugs, for example, has been largely restricted to acrylamides with only a handful of exceptions.2When developing covalent small molecule inhibitors, high affinity non-covalent binders are typically first optimized by medicinal chemistry and then only with complex structural information in hand are a limited number of electrophilic groups appended to endow theseTSRI 2247.1PC inhibitors with covalency. In recent years, unbiased approaches using chemical proteomics have uncovered an alternative route to covalent ligand discovery by broadly screening cellular proteomes using diverse electrophilic scout fragments.3, 4Such ‘reactivity first’ type approaches have both broadened the scope of the druggable proteome and inverted the order of operations by which chemical inhibitors are discovered. Collectively, these studies have suggested that an augmented repertoire of reactive groups targeting cysteine might enable greater access to less readily drugged sites in the proteome with molecules possessing altered selectivity, stability, and reactivity.

[0005] One potential source for new cysteine reactive groups might be the chemical matter lying ‘dormant’ in large chemical libraries. Indeed, several studies have serendipitously identified latent and unexpected covalent reactivities using high throughput screening and downstream target identification.5, 6Despite this potential opportunity, methods to directly survey large chemical libraries (>103molecules) for reactivity with cysteine are lacking. Often these approaches involve LC-MS-based or colorimetric assays, which are limited in their scalability and sensitivity.7, 8Previously, we used Nature’s electrophile sensor, the KEAP1-NRF2 pathway in mammalian cells, to identify 2-sulfonyl pyridines as a previously unappreciated cysteine reactive group, capable of being optimized as selective inhibitors to adenosine deaminase.9While this approach allows for scalable discovery of cysteine reactive groups in the context of the living cell, it can only identify electrophiles that react with the various ‘cysteine sensor’ residues of KEAP1.10

[0006] We reasoned that a potentially superior approach to identifying new electrophilic groups might involve assaying the reactivity of a given small molecule library member with cysteine directly in solution. Accordingly, we report here the development of scalable thiol reactivity profiling, a methodology inspired by the historical synthesis of luciferin that assesses the cysteine reactivity of a small molecule in vitro. We use this method to screen >10,000 small molecules, ultimately identifying the azetidnyl oxadiazole as a novel, cysteine selective reactive group that broadly reacts with cysteines across multiple protein families in the proteome and elicits modulatory effects on modified proteins. BRIEF DESCRIPTION OF THE FIGURES

[0007] Figure 1. A competitive assay for detecting the reactivity of small molecules with cysteine, inspired by the historical synthesis of luciferin.TSRI 2247.1PC

[0008] Figure 2. Scalable thiol reactivity profiling (STRP) reports on the cysteine reactivity of electrophilic compounds in miniaturized format.

[0009] Figure 3. High throughput screening with STRP identifies azetidines as cysteine selective reactive groups.

[0010] Figure 4. Alkyne probe 41 covalently engages diverse protein families across the cysteine proteome.

[0011] Figure 5. Covalent modification of C152 by 41 allosterically augments UCHL1 activity.

[0012] Figure 6. Optimization of the STRP assay and an assay to identify compounds that interfere with its activity.

[0013] Figure 7. Structures of the putative ring opened adducts of 15 with nucleophilic amino acids.

[0014] Figure 8. Determining the repertoire of azetidines that react with cysteine in the STRP assay.

[0015] Figure 9. DFT calculations reveal basis for selective reactivity of oxadiazole substituted azetidines with cysteine.

[0016] Figure 10. Reactive azetidine containing 41 covalently engages cysteines throughout the proteome.

[0017] Figure 11. Compound 41 covalently labels C152 of UCHL1. SUMMARY OF THE INVENTION

[0018] The application provides a Scalable Thiol Reactivity Profiling (STRP) assay for high-throughput determination of the ability of a test compound to covalently bind to cysteine in vitro, comprising the steps: i) incubating a test compound of with cysteine; ii) exposing the product of step i) to CHBT, or analogues thereof in Table X; iii) addition of luciferase to the product of step ii); and iv) determining the level of resultant luminesce which is inversely proportional to the reactivity of the test compound of Formula I with cysteine.TSRI 2247.1PC

[0019] The application further provides the above assay, wherein the test compound is an azetidinyl containing compound.

[0020] The application further provides the above assay, wherein the test compound is of Formula (I)wherein R1is H or halo; R2is (C6-C10) aryl, -O-(C6-C10) aryl, (C5-C10) heteroaryl, -O-(C5-C10) heteroaryl, (C3-C7) cycloalkyl, (C3-C7) heterocycloalkyl, -O-(C1-C6) alkyl, -O-(C1-C6) alkyl-(C3-C7) cycloalkyl, - S(=O)2-(C6-C10) aryl, (C5-C10) heteroaryl (C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C3-C7), -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C1-C6) alkyl, (C5- C10) heteroaryl-(C1-C6) heteroalky-(C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) alky-(C6-C10) aryl each optionally substituted with one or more halo, -(C1-C6) alkyl, -(C2-C6) alkenyl, -(C2- C6) alkynyl, or -O-(C1-C6) alkyl; or R1and R2together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl; R3and R4are each independently H or optionally substituted (C6-C10) aryl or (C5-C10) heteroaryl; or R3and R4together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl; R5is H, (C3-C7) heterocycloalkyl-(C1-C6) alkyl-(C5-C10) heteroaryl, (C3-C7) heterocycloalkyl- C(=O)2-(C5-C10) heteroaryl, -(C1-C6) alkyl-(C5-C10) heteroaryl, -(C3-C7) heterocycloalkyl- S(=O)2-(C1-C6) alkyl; including enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.TSRI 2247.1PC

[0021] The application further provides any one of the above assays, wherein the test compound is selected from the following structures:including enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

[0022] The application further provides any one of the above assays, wherein the test compound comprises an azetidinyl moiety.

[0023] The application further provides any one of the above assays, wherein the test compound comprises an azetidinyl oxadiazole moiety.

[0024] The application further provides any one of the above assays, wherein the test compound is 5-(azetidin-3-yl)-3-(4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazole or a pharmaceutically acceptable salt thereof.

[0025] The application further provides a method of labeling a cysteine residue, comprising treating a test compound having an azetidinyl moiety with a sample containing a cysteine residue, wherein the azetidinyl moiety covalently reacts with the cysteine residue.

[0026] The application further provides the above method, wherein the azetidinyl moiety is an azetidindyl oxadiazole moiety.

[0027] The application further provides the above method, wherein the test compound isTSRI 2247.1PCor a pharmaceutically acceptable salt thereof.

[0028] The application further provides the method of any one of the above embodiments, wherein the cysteine residue is distal to the deubiquitinase UCHL1 enzymatic active site.

[0029] The application further provides a compound having the structure:or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION

[0030] Firefly luciferin, a molecule that has been used extensively in numerous scalable biological assays for its capacity to luminesce when oxidized in the presence of its cognate enzyme luciferase, was of initial interest for this work.11Emil White and colleagues reported the first chemical synthesis of luciferin and its analogs in the 1960s in which a single step reaction involving the cyclization of D- or L-cysteine with 2-cyano-6-hydroxy benzothiazole (CHBT throughout) yields the enantiomeric mixture of D / L-luciferin (Figure 1A).12, 13The capacity of this reaction to spontaneously proceed in water has been exploited in recent years, as others have used solutions of CHBT and luciferase to quantify the levels of free cysteine in cells and tissues by recording the levels of produced luminescence (Figure 1B).14-16

[0031] Given the capacity of cysteine to spontaneously form luciferin in the presence of CHBT, it was reasoned that a stepwise, competitive screening assay could be generated to report on cysteine reactivity using these reagents. In this scenario, a small molecule in a screening well would first be exposed to a solution of D-cysteine for a set period, yielding either no product or a cysteine adduct, in the case of a cysteine reactive compound. Next, a solution of CHBT would be dispensed to each well, allowing the formation of D-luciferin, if no adduct had depleted the supply of reactive cysteine. Dispensing a solution of luciferaseTSRI 2247.1PC followed by luminescence recording on a plate reader would allow one to determine if the molecule within the screening well had reacted with cysteine or not, depending on the luminescence value recorded (Figure 1C).

[0032] With this framework, the Scalable Thiol Reactivity Profiling (STRP) assay for use in high throughput screening (Figure 2A) was optimized. This assay first involves the 3-hour incubation of D-cysteine (2.5 µM) in 20 µL of TRIS buffer with a given small molecule (1.5- 250 µM) delivered by pintool transfer (100 nL). A molecular excess of CHBT in 20 µL of buffer is then dispensed using an automated liquid handler followed by another 30-minute incubation. The addition of luciferase, ATP, and MgCl2 followed by shaking and luminescence measurement on a standard plate reader enables the discrimination of thiol reactive compounds (low signal) from unreactive ones (high signal). Importantly, were able to determine that a broad range of D-cysteine (1-10,000 µM), CHBT (1-1000 µM), and luciferin (0.1-1,000 µM) could be accommodated in the assay conditions, ultimately yielding a dynamic range of luminescence signal of >104-fold relative to background (Figure S1A-C). The optimized conditions were chosen such that the concentration of D-cysteine could be stoichiometrically competed with typical concentrations of screening compound (1-10 µM), although this data suggests that the STRP assay could be used in various settings with high signal to noise.

[0033] Additionally developed as a control assay to determine if a given hit compound might be decreasing signal through mechanisms other than by reacting with cysteine. This control assay involves the initial formation of D-luciferin by combining D-cysteine with CHBT, followed compound dispensing, luciferase exposure, and then luminescence recording (Figure S2D). Compounds that decrease signal in the control assay are likely inhibitors of luciferase or are non-specific modulators of the assay (e.g., aggregators).

[0034] It was next sought to understand if the STRP assay could accurately and dose dependently measure the cysteine reactivity of a ‘training set’ of twelve diverse, electrophilic small molecules that have been previously reported to react with cysteine in the literature. This compound set included methylsulfonyl benzothiazole17(MSBT, 1; IC50= 19.9 µM), 4- Chloro-7-nitrobenzofurazan18(NBD-Cl, 2 IC50 = 8.3 µM), dimethyl fumarate19(DMF, 3; IC50= 13.4 µM); monomethyl fumarate19(MMF, 4; IC50= 15.4 µM), n-ethyl maleimide20(NEM, 5; IC50= 6.3 µM), ethyl vinyl sulfone21(EVS, 6; IC50= 13.6 µM), phenylmethylsulfonyl fluoride22(PMSF, 7; IC50 = 50.5 µM), 3-(Chloromethyl)-4-ethyl-4H- 1,2,4-triazole (CMET, 8; IC50 = 31.4 µM), chloroacetamide23(CA, 9; IC50 = 114.7 µM),TSRI 2247.1PC iodoacetamide24(IA, 10; IC50= 20.7 µM), acrylamide23(11, IC50= 54.5 µM), and methylglyoxal5(MGX, 12, IC50 = 85 µM); of which all were found to inhibit STRP assay luminescence signal concentration dependently (Figure 2B,C). Notably, this assay could accurately predict which of two similar molecules with established reactivities was more reactive. For example, dimethyl fumarate was found to be more potent than monomethyl fumarate (13.4 vs.15.4 µM respectively), and iodoacetamide was found to be considerably more potent than chloroacetamide (20.7 vs 114.7 µM, respectively). Importantly, only two of these molecules, 1 and 2, were found to show any inhibitory activity in the control assay at concentrations less than 250 µM, and the IC50s (cIC50s = 129 and 211 µM respectively) were considerably higher than those of obtained with STRP assay (Figure 2D). Collectively, these data suggest that the STRP assay can report on the cysteine reactivity of diverse electrophilic chemicals across a broad range of concentrations. High throughput screening identifies azetidinyl oxadiazoles as cysteine reactive groups.

[0035] As proof of concept that STRP can identify novel cysteine reactive groups, a high throughput screening campaign was engaged in, surveying a commercial library of 10,561 small molecules largely devoid of common electrophiles (e.g., acrylamides, chloroacetamides, etc.) for SRTP inhibitory activity (Figure 3A). Strikingly, we found that among the top 100 scoring hits, 11 bore azetidines within their structures, prompting us to inquire whether this moiety might possess a previously unappreciated reactivity towards cysteine. Fresh material for 3 related hits was obtained, compounds 13, 14, and 15, which all bear azetidinyl oxadiazoles (Figure 3B). The dose responsive inhibitory activity for these compounds was confirmed (13, IC50 = 113; 14, IC50 = 102; 15, IC50 =45), all of which do not display inhibitory activity in the control STRP assay (IC50 > 250 µM; Figure 3C, D).

[0036] It was next sought to understand the mechanism by which the azetidnyl oxadiazole might react with cysteine to form a covalent adduct. From model in vitro reactions with 15 and N-Acetyl-L-cysteine methyl ester (Ac-Cys-OMe) in phosphate buffer (pH 8), we found strong evidence for the formation of opening of the azetidine ring via cysteine attack at C2. We were able to trap the ring opened product of 15 with Ac-Cys-OMe via Boc protection (Figure 3E), enabling larger scale reactions, purification, and confirmation of the structure of the purified product by NMR (Figure 3F and Supporting Information). We additionally confirmed that among putatively nucleophilic amino acids besides cysteine (lysine, serine, threonine, histidine, and tyrosine), only cysteine showed robust evidence of productTSRI 2247.1PC formation from in vitro reactions with these protected amino acids with 15 (Figure 3G, Figure S2).

[0037] Next performed was a structure activity relationship study to determine, what, if any, other azetidine containing molecules might also possess reactivity to cysteine. From a library of 24 additional commercially available C3 substituted azetidine fragments, it was found that only 3 retained cysteine reactivity (IC50 <250 µM), as determined by inhibitory activity in the STRP assay (Figure S2). Notably, 39 also contains an oxadiazole like 13, 14, and 15 and exhibited similar potency in the STRP assay (IC50= 102 µM; Figure S2). Methoxyphenyl substituted 17 and pyrrolidine substituted 35 also displayed inhibitory activity in this assay (Figure S2).

[0038] To gain a potential mechanistic explanation for the increased reactivity of oxadiazole substituted azetidines, density functional theory (DFT)-based calculations comparing 15 to 19, an imidazole substituted azetidine with no SRTP inhibitory activity, were performed. It was proposed that this reaction proceeds through thiolate attack at the carbon of position 2 of the azetidine ring through a concerted SN2-like reaction (Figure S4). The transition state of 19 with ethanethiol was found to be of a lower energetic barrier (27.72 kcal / mol) relative to that of 15 (32.41 kcal / mol). Decreased energy required to achieve the transition state was attributed to the potential of the azetidine of 19 to make favorable H- bonding interactions with the oxadiazole nitrogen, as well as the natural bond orbital (NBO) analysis of the oxadiazole 15 suggested a relatively favorable NBO charge on the azetidine carbon (-0.15 for 15 vs. -0.171 for 19). Azetidnyl oxadiazoles broadly engage cysteines throughout the proteome.

[0039] It was next sought to understand if the azetidinyl oxadiazole group could covalently ligand cysteines across the proteome in live cells. Accordingly, 41, a derivative of 16 bearing an alkyne moiety for use in affinity tagging and enrichment studies (Figure 4A), was synthesized.41 retained similar inhibitory potency in the STRP assay (34 µM vs.45 µM for 16) and did not interfere in the control STRP assay (cIC > 250 µM; Figure 4B). Notably, it was found that exposure of HEK293T cells to increasing concentrations of 41 (1- 1000 µM) for one hour resulted in the dose dependent labeling of many observable bands across a broad molecular weight range (Figure S5A, B) after lysates were subjected to click reaction-based conjugation with rhodamine azide. Additionally, it was found that the vast majority of rhodamine labeled bands could be competed away when iodoacetamide (10 mM)TSRI 2247.1PC was administered to cells 1 hour before exposure to 41 (Figure 4C, Figure S5C), indicative that 41 likely only labels cysteine residues in live cells.

[0040] Chemical proteomic enrichment studies were next performed to identify the proteins modified by 41. Here, HEK293T cells were exposed to 41 (1 mM) for one hour and lysates subjected to click chemistry reactions to affix biotin azide to labeled proteins. Streptavidin enrichment in denaturing conditions coupled to shotgun AP-MS / MS proteomic analysis identified 546 proteins that were statistically enriched in these conditions (Figure 2D). Most of these protein targets have not been drugged (81%), as determined by DrugBank annotations (Figure 4E).25Likewise, these liganded proteins correspond to a broad array of protein functional classes including transporters, enzymes, chaperones, and others (Figure 4F). These protein targets conformed to several protein classes as assessed by GO term enrichment, including RNA binding proteins, ubiquitin conjugation machinery, and isopeptide hydrolases (Figure 4G). These classes were represented among the most enriched proteins, which included E3 ligases, cysteine protein hydrolases, and RNA binders (Figure 4D). Attention was next focused on two highly enriched E3 ligases, UBR7 (ubiquitin protein ligase E3 component n-recognin 7) and UBE2O (ubiquitin conjugating enzyme E2 O), and two highly enriched deubiquitinases, USP11 (ubiquitin specific peptidase 11) and UCHL1 (ubiquitin c-terminal hydrolase L1). From experiments in which these four proteins were overexpressed as FLAG-tagged transgenes in HEK293T cells, it was found that a modest concentration of 41 (20 µM) resulted in the robust labeling of all four transgenes after one hour of exposure, as determined by rhodamine positivity of anti-FLAG immunoprecipitated material (Figure S5D), confirming the accuracy of our AP-MS chemoproteomic profiling. Collectively, these data suggest that 41 and likely the azetidinyl oxadiazole can covalently label a substantial portion of the cysteine proteome, targeting many proteins that have yet to be drugged with small molecule ligands. Discovery of an activator of UCHL1.

[0041] Lastly, it was sought to demonstrate the utility of the identified reactive group by characterizing the mechanism by which an azetidinyl oxadiazole containing compound might modulate the function of a covalently modified protein. Efforts were therefore focused on the interaction of 41 with UCHL1, a thiol peptidase that cleaves C-terminally conjugated ubiquitin molecules from client proteins central to the maintenance of neuronal cellular populations, where it is most highly expressed.26The nucleophilicity of the active siteTSRI 2247.1PC cysteine has enabled the discovery of several covalent inhibitors of the catalytic activity of UCHL1 in the literature.27, 28

[0042] That 41 covalently labels UCHL1 in a dose dependent manner was first confirmed from experiments in which FLAG tagged transgene was overexpressed in HEK293T cells (Figure S6A). Surprisingly, 41 was not found to label the nucleophilic active site cysteine (C90), but instead labeled C152, a residue distal to the active site. Experiments in which C90 or C125 of the FLAG tagged transgene were mutated to serine or alanine confirmed that only labeling was abrogated in the context of C152 mutation (Figure 5A, Figure S6B). The nature of this labeling event was additionally confirmed, as one could detect the ring opened tryptic peptide fragment adduct containing C152 by MS / MS (Figure 5B, Figure S6C) from cells overexpressing FLAG tagged UCHL1 treated with 41 (20 µM).

[0043] UCHL1 bears one of the most complex protein folds yet discovered for a eukaryotic protein with five internal backbone crossings forming a Gordian knot.26In addition, UCHL1 possesses a ‘crossover loop’ which, upon its movement, enables access to the active site for protein substrates (Figure 5C). C152 occupies a site near the middle of the active site crossover loop, suggesting that its modification might modulate the catalytic activity of the protein. Indeed, previous work has shown that C152 can be modified by the endogenous lipophilic electrophilic compound, 5-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), a modification which, in neuronal cells, promotes aggregation of the protein and cellular death.29Accordingly, it was sought to understand how modification of C152 by 41 might also modulate the enzymatic activity of UCHL1. Again, surprisingly, it was found that treating recombinant preparations with 10 µM 41 increased the rate of reaction by nearly two-fold (Figure 5D). It was additionally found that FLAG tagged protein isolated from HEK293T cells labeled with 41 (10 µM) in situ additionally displayed increased rates of reaction, albeit to a lesser degree (Figure 5E). UCHL1 has been shown, at least in part, to promote cellular survival by deubiquitinating components of the TGF-beta signaling pathway, SMAD2 and ALK5, resulting in augmented signaling through this pathway.30It was found that treatment with 41 in HEK293T cells increased the capacity of myc tagged UCHL1 to further deubiquitinate both FLAG tagged SMAD2 and ALK5 as assessed by anti-HA (measuring ubiquitination) Western blotting of FLAG immunoprecipitated material (Figure 5F, G). These data indicate that covalent modification of C152 by 41 functionally increases the isopeptidase activity of UCHL1 in vitro and in cells.TSRI 2247.1PC

[0044] Thus, herein, a simple and scalable assay has been described for measuring the intrinsic reactivity of small molecules with cysteine. The STRP assay enables the unbiased evaluation of cysteine reactivity in solution, as STRP accurately reports on the covalent reactivity of known cysteine reactive electrophiles across several reaction types (SNAr, Michael addition, etc.). Additionally, the STRP assay can produce measurable luminescence signal across several orders of magnitude of reactant concentrations, suggesting that even weakly cysteine reactive molecules might be able to be identified in an appropriately tuned assay condition. The work described herein was performed in 384-well format using typical equipment found in an academic laboratory; however, given the robustness of this assay, it is highly likely that it could be further miniaturized (e.g., 1536-well format) to screen larger libraries (>106compounds) with automated screening equipment.

[0045] While STRP has been used in this work to pan for new reactive groups that covalently modify cysteine, STRP will likely find utility in additional applications. One key concern in the development of reversible small molecule drugs is the potential for reactivity with glutathione (GSH) thiol, either intrinsically or after metabolism by endogenous oxidases.31It is conceivable that compound libraries, either in pharmaceutical companies or in academic screening centers, could be profiled for inherent thiol reactivity risk using STRP, either alone or in the presence of relevant P450 enzymes (e.g., CYP3A4) to generate a potential reactivity risk profile for each compound. Such a map would enable potentially labile molecules to be appropriately flagged at a much earlier stage in the drug development process.

[0046] From screening a moderately sized library of ~10,000 diverse small molecules for STRP inhibitory activity, it was herein identified that azetidines, namely azetidinyl oxadiazoles, possess previously unreported reactivity with cysteine. This ring opening reaction proceeds through a SN2-like mechanism, one that is believed to be accelerated by intramolecular interactions between the azetidine nitrogen and the oxadiazole. Interestingly, azetidines have been lauded in the medicinal chemistry literature for their physical properties, namely their stability in biological systems and their ability to induce rigidity into small molecules without adding significant molecular weight.32In contrast, this work suggests that certain azetidines might pose a potential cysteine reactivity risk and should be profiled appropriately before their introduction into a medicinal chemistry campaign. While it has been shown that oxadiazole substituted azetidines possess increased reactivity relative to other molecules evaluated here, there are likely an expanded repertoire of azetidines thatTSRI 2247.1PC possess good reactivity with cysteine. Indeed, the SAR campaign in this work was more limited, and future mechanistic studies will be required to determine the scope of reactive azetidines that might be harnessed for use as cysteine targeting reactive groups.

[0047] Chemical proteomics were performed with an alkyne derivatized azetidinyl oxadiazole, 41, discovering that hundreds of proteins in live cells across various protein classes could be covalently modified by this chemotype. This result indicates that this novel reactive group may provide an unprecedented capacity to target cysteines in certain protein classes or domains, as most proteins identified here have not been drugged before. The utility of this reactive group was demonstrated by studying the effect of 41 on modifying UCHL1, a deubiquitinase that is essential to neuronal survival. It was found that 41 does not modify the presumably more nucleophilic active site cysteine, C90, but instead modifies C152, a residue on a flexible loop covering the active site, movement of which is required for access of ubiquitin substrates.26 15, 33, 34. One such mutation, E7A, results in early onset neurodegeneration by decreasing catalytic activity ofThis glutamate makes key H- bonding contacts with residues Q151 and R153 (on either side of C152) to stabilize the crossover active site loop when bound to ubiquitin. As noted, C152 has also been the site of covalent modification by endogenous electrophilic chemicals and has been reported to be susceptible to s-nitrosylation.35Given these observations, it is conceivable that C152 may act as a sensor switch to control the level of activity of UCHL1 and that modification of C152 by 41 may promote a more constitutively active state of the protein. Clearly, future work will be required to fully delineate the role of C152 in the regulating the catalysis of UCHL1. Nevertheless, 41 will likely serve as a useful mechanistic tool to aid in these efforts. Likewise, it will be interesting to understand if molecules like 41 can be optimized by medicinal chemistry to increase UCHL1 activity in the context of neurodegenerative disease. Projecting forward, it is anticipated that the azetidinyl oxadiazole, and, more broadly, the azetidine, will provide a novel chemotype from which to design new covalent inhibitors and to target the cysteine proteome more effectively. References: [1] Bak, D. W., Bechtel, T. J., Falco, J. A., and Weerapana, E. (2019) Cysteine reactivity across the subcellular universe, Curr Opin Chem Biol 48, 96-105. [2] Boike, L., Henning, N. J., and Nomura, D. K. (2022) Advances in covalent drug discovery, Nat Rev Drug Discov 21, 881-898.TSRI 2247.1PC [3] Backus, K. M., Correia, B. E., Lum, K. M., Forli, S., Horning, B. D., Gonzalez-Paez, G. E., Chatterjee, S., Lanning, B. R., Teijaro, J. R., Olson, A. J., Wolan, D. W., and Cravatt, B. F. (2016) Proteome-wide covalent ligand discovery in native biological systems, Nature 534, 570-574. [4] Weerapana, E., Wang, C., Simon, G. M., Richter, F., Khare, S., Dillon, M. B., Bachovchin, D. A., Mowen, K., Baker, D., and Cravatt, B. F. (2010) Quantitative reactivity profiling predicts functional cysteines in proteomes, Nature 468, 790-795. [5] Bollong, M. J., Lee, G., Coukos, J. S., Yun, H., Zambaldo, C., Chang, J. W., Chin, E. N., Ahmad, I., Chatterjee, A. K., Lairson, L. L., Schultz, P. G., and Moellering, R. E. (2018) A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling, Nature 562, 600-604. [6] Boskovic, Z. V., Kemp, M. M., Freedy, A. M., Viswanathan, V. S., Pop, M. S., Fuller, J. H., Martinez, N. M., Figueroa Lazu, S. O., Hong, J. A., Lewis, T. A., Calarese, D., Love, J. D., Vetere, A., Almo, S. C., Schreiber, S. L., and Koehler, A. N. (2016) Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile, ACS Chem Biol 11, 1844-1851. [7] Maurais, A. J., and Weerapana, E. (2019) Reactive-cysteine profiling for drug discovery, Curr Opin Chem Biol 50, 29-36. [8] Resnick, E., Bradley, A., Gan, J., Douangamath, A., Krojer, T., Sethi, R., Geurink, P. P., Aimon, A., Amitai, G., Bellini, D., Bennett, J., Fairhead, M., Fedorov, O., Gabizon, R., Gan, J., Guo, J., Plotnikov, A., Reznik, N., Ruda, G. F., Diaz-Saez, L., Straub, V. M., Szommer, T., Velupillai, S., Zaidman, D., Zhang, Y., Coker, A. R., Dowson, C. G., Barr, H. M., Wang, C., Huber, K. V. M., Brennan, P. E., Ovaa, H., von Delft, F., and London, N. (2019) Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening, J Am Chem Soc 141, 8951-8968. [9] Zambaldo, C., Vinogradova, E. V., Qi, X., Iaconelli, J., Suciu, R. M., Koh, M., Senkane, K., Chadwick, S. R., Sanchez, B. B., Chen, J. S., Chatterjee, A. K., Liu, P., Schultz, P. G., Cravatt, B. F., and Bollong, M. J. (2020) 2-Sulfonylpyridines as Tunable, Cysteine- Reactive Electrophiles, J Am Chem Soc 142, 8972-8979.TSRI 2247.1PC

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[0035] Kumar, R., Jangir, D. K., Verma, G., Shekhar, S., Hanpude, P., Kumar, S., Kumari, R., Singh, N., Sarovar Bhavesh, N., Ranjan Jana, N., and Kanti Maiti, T. (2017) S- nitrosylation of UCHL1 induces its structural instability and promotes alpha-synuclein aggregation, Sci Rep 7, 44558.TSRI 2247.1PC Embodiments

[0048] Embodiment 1. A Scalable Thiol Reactivity Profiling (STRP) assay for high- throughput determination of the ability of a test compound to covalently bind to cysteine in vitro, comprising the steps: v) incubating a test compound of with cysteine; vi) exposing the product of step i) to CHBT; vii) addition of luciferase to the product of step ii); and viii) determining the level of resultant luminesce which is inversely proportional to the reactivity of the test compound of Formula I with cysteine.

[0049] Embodiment 2. The assay of Embodiment 1, wherein the test compound is a small molecule, protein, or peptide.

[0050] Embodiment 3. The assay of Embodiment 1 or Embodiment 2, wherein the test compound is an azetidinyl containing compound.

[0051] Embodiment 4. The assay of Embodiment 3, wherein the test compound is of Formula (I)wherein R1is H or halo; R2is (C6-C10) aryl, -O-(C6-C10) aryl, (C5-C10) heteroaryl, -O-(C5-C10) heteroaryl, (C3-C7) cycloalkyl, (C3-C7) heterocycloalkyl, -O-(C1-C6) alkyl, -O-(C1-C6) alkyl-(C3-C7) cycloalkyl, - S(=O)2-(C6-C10) aryl, (C5-C10) heteroaryl (C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C3-C7), -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C1-C6) alkyl, (C5- C10) heteroaryl-(C1-C6) heteroalky-(C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) alky-(C6-C10) aryl each optionally substituted with one or more halo, -(C1-C6) alkyl, -(C2-C6) alkenyl, -(C2- C6) alkynyl, or -O-(C1-C6) alkyl;TSRI 2247.1PC or R1and R2together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl; R3and R4are each independently H or optionally substituted (C6-C10) aryl or (C5-C10) heteroaryl; or R3and R4together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl; R5is H, (C3-C7) heterocycloalkyl-(C1-C6) alkyl-(C5-C10) heteroaryl, (C3-C7) heterocycloalkyl- C(=O)2-(C5-C10) heteroaryl, -(C1-C6) alkyl-(C5-C10) heteroaryl, -(C3-C7) heterocycloalkyl- S(=O)2-(C1-C6) alkyl; including enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

[0052] Embodiment 5. The assay of any one of Embodiments 1-4, wherein the test compound is selected from the following structures:including enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.TSRI 2247.1PC

[0053] Embodiment 6. The assay of any one of Embodiments 1-5, wherein the test compound comprises an azetidinyl oxadiazole moiety.

[0054] Embodiment 7. The assay of any one of Embodiments 1-6, wherein the test compound is 5-(azetidin-3-yl)-3-(4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazole or a pharmaceutically acceptable salt thereof.

[0055] Embodiment 8. A control assay for detecting small molecules which interfere with the STRP assay of any one of Embodiments 1-7, comprising: i) incubation of cysteine with CHBT, or analogue thereof in Table X, to enable formation of D-luciferin; ii) addition of a a test compound of Formula I to determine its interference with CHBT, or analogue thereof in Table X, for luciferase activity; iii) addition of luciferase to promote luminescence; and iv) measuring resultant luminescence.

[0056] Embodiment 9. An assay for profiling test compounds in a chemical library for thiol reactivity liabilities, comprising measuring each test compound’s ability for covalent adduction with glutathione.

[0057] Embodiment 10. The assay of Embodiment 9, wherein the GSH / thiol / cysteine reactivity risk profile for a given test compound is generated to expose potential liabilities associate with a given test molecule in the early stages of development.

[0058] Embodiment 11. A kit providing the assays of any one of Embodiments 1-10 for use in an STRP assay for high-throughput determination of the ability of a test compound to covalently bind to cysteine in vitro.

[0059] Embodiment 12. The assay of embodiment 1, comprising the following step: i) incubating a test compound with cysteine for approximately 3 hours at room temperature.

[0060] Embodiment 13. The assay of embodiment 12, further comprising the following step: ii) exposing the product of step i) to CHBT, or analogue thereof in Table X, for approximately 30 minutes at room temperature.TSRI 2247.1PC

[0061] Embodiment 14. The assay of embodiment 13, further comprising the following step: iii) addition of ATP-Mg2+luciferase to the product of step ii).

[0062] Embodiment 15. The assay of embodiment 14, further comprising the following step: iv) determining the level of resultant luminesce in the product of step iii).

[0063] Embodiment 16. A method of labeling a cysteine residue, comprising treating a test compound having an azetidinyl moiety with a sample containing a cysteine residue, wherein the azetidinyl moiety covalently reacts with the cysteine residue.

[0064] Embodiment 17. The method of Embodiment 16, wherein the azetidinyl moiety is an azetidindyl oxadiazole moiety.

[0065] Embodiment 18. The method of Embodiment 17, wherein the test compound isor a pharmaceutically acceptable salt thereof.

[0066] Embodiment 19. The method of any one of Embodiments 16-18, wherein the cysteine residue is distal to the deubiquitinase UCHL1 enzymatic active site.

[0067] Embodiment 20. A compound having the structure:or a pharmaceutically acceptable salt thereof. Definitions

[0068] The phrase “a” or “an” entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.TSRI 2247.1PC

[0069] The phrase "as defined herein above" refers to the broadest definition for each group as provided in the Summary of the Invention, the Detailed Description of the Invention, the Experimentals, or the broadest claim. In all other embodiments provided below, substituents which can be present in each embodiment and which are not explicitly defined retain the broadest definition provided in the Summary of the Invention.

[0070] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open- ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0071] As used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and / or" and not the "exclusive" sense of "either / or".

[0072] The term "independently" is used herein to indicate that a variable is applied in any one instance without regard to the presence or absence of a variable having that same or a different definition within the same compound. Thus, in a compound in which “R” appears twice and is defined as "independently selected from” means that each instance of that R group is separately identified as one member of the set which follows in the definition of that R group. For example, “each R1and R2is independently selected from carbon and nitrogen" means that both R1and R2can be carbon, both R1and R2can be nitrogen, or R1or R2can be carbon and the other nitrogen or vice versa.

[0073] When any variable occurs more than one time in any moiety or formula depicting and describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and / or variables are permissible only if such compounds result in stable compounds.

[0074] The symbols "*" at the end of a bond or a line drawn through a bond or “~~~~” drawn through a bond each refer to the point of attachment of a functional group or other chemical moiety to the rest of the molecule of which it is a part.

[0075] A bond drawn into ring system (as opposed to connected at a distinct vertex) indicates that the bond may be attached to any of the suitable ring atoms.TSRI 2247.1PC

[0076] The term “optional” or “optionally” as used herein means that a subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted” means that the “optionally substituted” moiety may incorporate a hydrogen or a substituent.

[0077] The phrase “optional bond” means that the bond may or may not be present, and that the description includes single, double, or triple bonds. If a substituent is designated to be a "bond" or "absent", the atoms linked to the substituents are then directly connected.

[0078] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0079] Certain compounds disclosed herein may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertable species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium and attempts to isolate an individual tautomers usually produce a mixture whose chemical and physical properties are consistent with a mixture of compounds. The position of the equilibrium is dependent on chemical features within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates while; in phenols, the enol form predominates. Common prototropic tautomers include keto / enol (-C(=O)-CH- -C(-OH)=CH-), amide / imidic acid (-C(=O)- NH- -C(-OH)=N-) and amidine (-C(=NR)-NH--C(-NHR)=N-) tautomers. The latter two are particularly common in heteroaryl and heterocyclic rings and the present invention encompasses all tautomeric forms of the compounds.

[0080] In this disclosure, a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate,TSRI 2247.1PC hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3- naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0081] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10thEd., McGraw Hill Companies Inc., New York (2001). Any suitable materials and / or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.

[0082] The definitions described herein may be appended to form chemically-relevant combinations, such as “heteroalkylaryl,” “haloalkylheteroaryl,” “arylalkylheterocyclyl,” “alkylcarbonyl,” “alkoxyalkyl,” and the like. When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically-named group. Thus, for example, “phenylalkyl” refers to an alkyl group having one to two phenyl substituents, and thus includes benzyl, phenylethyl, and biphenyl. An “alkylaminoalkyl” is an alkyl group having one to two alkylamino substituents. “Hydroxyalkyl" includes 2-hydroxyethyl, 2-hydroxypropyl, 1-(hydroxymethyl)-2- methylpropyl, 2-hydroxybutyl, 2,3-dihydroxybutyl, 2-(hydroxymethyl), 3-hydroxypropyl, and so forth. Accordingly, as used herein, the term “hydroxyalkyl” is used to define a subset of heteroalkyl groups defined below. The term -(ar)alkyl refers to either an unsubstitutedTSRI 2247.1PC alkyl or an aralkyl group. The term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group.

[0083] The term “acyl” as used herein denotes a group of formula -C(=O)R wherein R is hydrogen or lower alkyl as defined herein. The term or "alkylcarbonyl" as used herein denotes a group of formula C(=O)R wherein R is alkyl as defined herein. The term C1-6acyl refers to a group -C(=O)R contain 6 carbon atoms. The term "arylcarbonyl" as used herein means a group of formula C(=O)R wherein R is an aryl group; the term "benzoyl" as used herein an "arylcarbonyl" group wherein R is phenyl.

[0084] The term “alkyl” as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 12 carbon atoms. The term “lower alkyl” or “C1-C6alkyl” as used herein denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms. "C1-12 alkyl" as used herein refers to an alkyl composed of 1 to 12 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

[0085] When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically- named group. Thus, for example, “phenylalkyl” denotes the radical R'R"-, wherein R' is a phenyl radical, and R" is an alkylene radical as defined herein with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene radical. Examples of arylalkyl radicals include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl. The terms “arylalkyl” or "aralkyl" are interpreted similarly except R' is an aryl radical. The terms "(het)arylalkyl" or "(het)aralkyl" are interpreted similarly except R' is optionally an aryl or a heteroaryl radical.

[0086] When a range of values is listed, it is intended to encompass each value and sub– range within the range. For example, “C1–6alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1–6, C1–5, C1–4, C1–3, C1–2, C2–6, C2–5, C2–4, C2–3, C3–6, C3–5, C3–4, C4–6, C4–5, and C5–6alkyl.

[0087] “Alkyl” refers to a radical of a straight–chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1–20alkyl”). In some embodiments, an alkyl group has 1 to 15 carbon atoms (“C1–15 alkyl”). In some embodiments, an alkyl group has 1 to 14 carbon atoms (“C1–14 alkyl”). In some embodiments, an alkyl group has 1 to 13 carbonTSRI 2247.1PC atoms (“C1–13alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1–12alkyl”). In some embodiments, an alkyl group has 1 to 11 carbon atoms (“C1–11 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1–10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1–9alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1–8alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1–7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1–6alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1–5alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1–4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1–3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1–2alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2–6 alkyl”). Examples of C1–6 alkyl groups include methyl (C1), ethyl (C2), n–propyl (C3), isopropyl (C3), n–butyl (C4), tert–butyl (C4), sec–butyl (C4), iso–butyl (C4), n– pentyl (C5), 3–pentanyl (C5), amyl (C5), neopentyl (C5), 3–methyl–2–butanyl (C5), tertiary amyl (C5), and n–hexyl (C6). Additional examples of alkyl groups include n–heptyl (C7), n– octyl (C8) and the like.

[0088] “Alkenyl” or “olefin” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 10 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds (“C2–10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2–9alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2–8alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2–7alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2–6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2–5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2–4alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2–3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon– carbon double bonds can be internal (such as in 2–butenyl) or terminal (such as in 1–butenyl). Examples of C2–4alkenyl groups include ethenyl (C2), 1–propenyl (C3), 2–propenyl (C3), 1– butenyl (C4), 2–butenyl (C4), butadienyl (C4), and the like. Examples of C2–6 alkenyl groups include the aforementioned C2–4alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.TSRI 2247.1PC

[0089] “Alkynyl” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C2–10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2–9alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2–8alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2–7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2–6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2–5alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2–4alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2–3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2alkynyl”). The one or more carbon– carbon triple bonds can be internal (such as in 2–butynyl) or terminal (such as in 1–butynyl). Examples of C2–4 alkynyl groups include, without limitation, ethynyl (C2), 1–propynyl (C3), 2–propynyl (C3), 1–butynyl (C4), 2–butynyl (C4), and the like. Examples of C2–6 alkenyl groups include the aforementioned C2–4alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.

[0090] The terms “haloalkyl” or “halo-lower alkyl” or “lower haloalkyl” refers to a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms wherein one or more carbon atoms are substituted with one or more halogen atoms.

[0091] The term "alkylene" or "alkylenyl" as used herein denotes a divalent saturated linear hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH2)n)or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., -CHMe- or -CH2CH(i-Pr)CH2-), unless otherwise indicated. Except in the case of methylene, the open valences of an alkylene group are not attached to the same atom. Examples of alkylene radicals include, but are not limited to, methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, butylene, 2-ethylbutylene.

[0092] The term "alkoxy" as used herein means an -O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t- butyloxy, pentyloxy, hexyloxy, including their isomers. "Lower alkoxy" as used herein denotes an alkoxy group with a "lower alkyl" group as previously defined. "C1-10alkoxy" as used herein refers to an-O-alkyl wherein alkyl is C1-10.TSRI 2247.1PC

[0093] The term "hydroxyalkyl" as used herein denotes an alkyl radical as herein defined wherein one to three hydrogen atoms on different carbon atoms is / are replaced by hydroxyl groups.

[0094] The terms "alkylsulfonyl" and "arylsulfonyl" as used herein refers to a group of formula -S(=O)2R wherein R is alkyl or aryl respectively and alkyl and aryl are as defined herein. The term “heteroalkylsulfonyl” as used herein refers herein denotes a group of formula -S(=O)2R wherein R is “heteroalkyl” as defined herein.

[0095] The terms "alkylsulfonylamino" and "arylsulfonylamino"as used herein refers to a group of formula -NR'S(=O)2R wherein R is alkyl or aryl respectively, R' is hydrogen or C1-3 alkyl, and alkyl and aryl are as defined herein.

[0096] The term “cycloalkyl” as used herein refers to a saturated carbocyclic ring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. "C3-7 cycloalkyl" as used herein refers to an cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring.

[0097] The term carboxy-alkyl as used herein refers to an alkyl moiety wherein one, hydrogen atom has been replaced with a carboxyl with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom. The term “carboxy” or “carboxyl” refers to a –CO2H moiety.

[0098] The term "heteroaryl” or "heteroaromatic" as used herein means a monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing four to eight atoms per ring, incorporating one or more N, O, or S heteroatoms, the remaining ring atoms being carbon, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring. As well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group need only have some degree of aromatic character. Examples of heteroaryl moieties include monocyclic aromatic heterocycles having 5 to 6 ring atoms and 1 to 3 heteroatoms include, but is not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazol, isoxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadiaxoline which can optionally be substituted with one or more, preferably one or two substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halo, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino,dialkylamino, aminoalkyl, alkylaminoalkyl, and dialkylaminoalkyl, nitro, alkoxycarbonyl and carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, alkylcarbonylamino andTSRI 2247.1PC arylcarbonylamino. Examples of bicyclic moieties include, but are not limited to, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzoxazole, benzisoxazole, benzothiazole and benzisothiazole. Bicyclic moieties can be optionally substituted on either ring; however the point of attachment is on a ring containing a heteroatom.

[0099] The term "heterocyclyl", “heterocycloalkyl” or "heterocycle" as used herein denotes a monovalent saturated cyclic radical, consisting of one or more rings, preferably one to two rings, including spirocyclic ring systems, of three to eight atoms per ring, incorporating one or more ring heteroatoms (chosen from N,O or S(O)0-2), and which can optionally be independently substituted with one or more, preferably one or two substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, unless otherwise indicated. Examples of heterocyclic radicals include, but are not limited to, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl and imidazolinyl.

[0100] “Heterocyclyl” or “heterocyclic” refers to a group or radical of a 3– to 14– membered non–aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3–14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon– carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.TSRI 2247.1PC

[0101] In some embodiments, a heterocyclyl group is a 5–10 membered non–aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5–8 membered non–aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5–6 membered non–aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–6 membered heterocyclyl”). In some embodiments, the 5–6 membered heterocyclyl has 1–3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heterocyclyl has 1–2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0102] Exemplary 3–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5–membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl–2,5–dione. Exemplary 5– membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6–membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6–membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinanyl. Exemplary 7–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,TSRI 2247.1PC decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro–1,8–naphthyridinyl, octahydropyrrolo[3,2–b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H–benzo[e][1,4]diazepinyl, 1,4,5,7–tetrahydropyrano[3,4–b]pyrrolyl, 5,6–dihydro–4H–furo[3,2–b]pyrrolyl, 6,7–dihydro– 5H–furo[3,2–b]pyranyl, 5,7–dihydro–4H–thieno[2,3–c]pyranyl, 2,3–dihydro–1H– pyrrolo[2,3–b]pyridinyl, 2,3–dihydrofuro[2,3–b]pyridinyl, 4,5,6,7–tetrahydro–1H–pyrrolo- [2,3–b]pyridinyl, 4,5,6,7–tetrahydrofuro[3,2–c]pyridinyl, 4,5,6,7–tetrahydrothieno[3,2– b]pyridinyl, 1,2,3,4–tetrahydro–1,6–naphthyridinyl, and the like.

[0103] “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6–14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6–14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1–naphthyl (α-naphthyl) and 2–naphthyl (β-naphthyl)). In some embodiments, an aryl group has 14 ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.

[0104] “Heteroaryl” refers to a radical of a 5–14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in theTSRI 2247.1PC fused polycyclic (aryl / heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2–indolyl) or the ring that does not contain a heteroatom (e.g., 5–indolyl).

[0105] In some embodiments, a heteroaryl group is a 5–10 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5–8 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5–6 membered aromatic ring system having ring carbon atoms and 1–4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5–6 membered heteroaryl”). In some embodiments, the 5–6 membered heteroaryl has 1–3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heteroaryl has 1–2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5–6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0106] Exemplary 5–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5–membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5–membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5–membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6–membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6–membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7–membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6– bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,TSRI 2247.1PC benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6–bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

[0107] “Saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.

[0108] Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may be optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted. In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a non-hydrogen substituent, and which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogen substituents and / or non-hydrogen substituents which satisfy the valencies of the heteroatoms and results in the formation of a stable compound.

[0109] Exemplary non-hydrogen substituents wherein a moiety is “optionally substituted” as used herein means the moiety may be substituted with any additional moiety selected from, but not limited to, the group consisting of halogen, –CN, –NO2, –N3, –SO2H, –SO3H, – OH, –ORaa, –N(Rbb)2, –N(ORcc)Rbb, –SH, –SRaa, –C(=O)Raa, –CO2H, –CHO, –CO2Raa, – OC(=O)Raa, –OCO2Raa, –C(=O)N(Rbb)2, –OC(=O)N(Rbb)2, –NRbbC(=O)Raa, –NRbbCO2Raa, – NRbbC(=O)N(Rbb)2, –C(=NRbb)Raa, –C(=NRbb)ORaa, –OC(=NRbb)Raa, –OC(=NRbb)ORaa, – C(=NRbb)N(Rbb)2, –OC(=NRbb)N(Rbb)2, –NRbbC(=NRbb)N(Rbb)2, –C(=O)NRbbSO2Raa, – NRbbSO2Raa, –SO2N(Rbb)2, –SO2Raa, –S(=O)Raa, –OS(=O)Raa, -B(ORcc)2, C1–10alkyl, C2–10alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14 aryl, and 5– to 14- membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups, or two geminal hydrogens on a carbon atom are replaced with the group =O; each instance of Raais, independently, selected from the group consisting of C1–10 alkyl, C1–10 perhaloalkyl, C2–10 alkenyl, C2–10alkynyl, C3–14carbocyclyl, 3– to 14- membered heterocyclyl, C6–14aryl, and 5– to 14- membered heteroaryl, or two Raagroups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4,TSRI 2247.1PC or 5 Rddgroups; each instance of Rbbis, independently, selected from the group consisting of hydrogen, –OH, –ORaa, –N(Rcc)2, –CN, –C(=O)Raa, –C(=O)N(Rcc)2, –CO2Raa, –SO2Raa, – SO2N(Rcc)2, –SORaa, C1–10 alkyl, C1–10 perhaloalkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14aryl, and 5– to 14- membered heteroaryl, or two Rbbgroups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; each instance of Rccis, independently, selected from the group consisting of hydrogen, C1–10alkyl, C1–10 perhaloalkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14aryl, and 5– to 14- membered heteroaryl, or two Rccgroups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups; and each instance of Rddis, independently, selected from the group consisting of halogen, –CN, –NO2, –N3, –SO2H, –SO3H, –OH, – OC1–6 alkyl, –ON(C1–6 alkyl)2, –N(C1–6 alkyl)2, –N(OC1–6 alkyl)(C1–6 alkyl), –N(OH)(C1–6 alkyl), –NH(OH), –SH, –SC1–6 alkyl, –C(=O)(C1–6 alkyl), –CO2H, –CO2(C1–6 alkyl), – OC(=O)(C1–6alkyl), –OCO2(C1–6alkyl), –C(=O)NH2, –C(=O)N(C1–6alkyl)2, – OC(=O)NH(C1–6alkyl), –NHC(=O)( C1–6alkyl), –N(C1–6alkyl)C(=O)( C1–6alkyl), – NHCO2(C1–6 alkyl), –NHC(=O)N(C1–6 alkyl)2, –NHC(=O)NH(C1–6 alkyl), –NHC(=O)NH2, –C(=NH)O(C1–6alkyl),–OC(=NH)(C1–6alkyl), –OC(=NH)OC1–6alkyl, –C(=NH)N(C1–6alkyl)2, –C(=NH)NH(C1–6alkyl), –C(=NH)NH2, –OC(=NH)N(C1–6alkyl)2, – OC(NH)NH(C1–6 alkyl), –OC(NH)NH2, –NHC(NH)N(C1–6 alkyl)2, –NHC(=NH)NH2, – NHSO2(C1–6 alkyl), –SO2N(C1–6 alkyl)2, –SO2NH(C1–6 alkyl), –SO2NH2,–SO2C1–6 alkyl, - B(OH)2, -B(OC1–6alkyl)2,C1–6alkyl, C1–6perhaloalkyl, C2–6alkenyl, C2–6alkynyl, C3–10carbocyclyl, C6–10 aryl, 3–to 10- membered heterocyclyl, and 5- to 10- membered heteroaryl; or two geminal Rddsubstituents on a carbon atom may be joined to form =O.

[0110] “Halo” or “halogen” refers to fluorine (fluoro, –F), chlorine (chloro, –Cl), bromine (bromo, –Br), or iodine (iodo, –I).

[0111] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients.

[0112] “Salt” includes any and all salts. “Pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact withTSRI 2247.1PC the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts include those derived from inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2–hydroxy–ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2– naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3–phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p–toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1–4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0113] Unless otherwise indicated, compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC). Compounds described herein can be in the form of individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.TSRI 2247.1PC

[0114] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of19F with18F, replacement of a carbon by a13C- or14C- enriched carbon, and / or replacement of an oxygen atom with18O, are within the scope of the disclosure. Other examples of isotopes include15N,18O,17O,31P,32P,35S,18F,36Cl and123I. Compounds with such isotopically enriched atoms are useful, for example, as analytical tools or probes in biological assays.

[0115] Certain isotopically-labelled compounds (e.g., those labeled with3H and14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon-14 (i.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability.

[0116] Certain isotopically-labelled compounds of Formula (I) can be useful for medical imaging purposes, for example, those labeled with positron-emitting isotopes like11C or18F can be useful for application in Positron Emission Tomography (PET) and those labeled with gamma ray emitting isotopes like123I can be useful for application in Single Photon Emission Computed Tomography (SPECT). Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and hence, may be preferred in some circumstances. Additionally, isotopic substitution at a site where epimerization occurs may slow or reduce the epimerization process and thereby retain the more active or efficacious form of the compound for a longer period of time. Isotopically labeled compounds of Formula (I), in particular those containing isotopes with longer half- lives (t1 / 2>1 day), can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

[0117] If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomersTSRI 2247.1PC of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them. DETAILED DESCRIPTION OF THE FIGURES

[0118] Fig.1A-C. A competitive assay for detecting the reactivity of small molecules with cysteine, inspired by the historical synthesis of luciferin. A) The first synthetic route to luciferin and its analogs described in the literature by Emil White and colleagues. B) Work from Ohimya using CHBT to detect L-cysteine levels in situ. C) Scheme depicting this work. A competitive assay based on the spontaneous formation of D-luciferin in vitro. Small molecules are first incubated with D-cysteine, either allowing D-cysteine to form D-luciferin or a non-productive covalent adduct with cysteine. Addition of luciferase allows the detection of cysteine in the case of non-reactive compounds or decreased luminescence in the case of reactive small molecules.

[0119] Fig.2A-D. Scalable thiol reactivity profiling (STRP) reports on the cysteine reactivity of electrophilic compounds in miniaturized format. A) Schematic depicting the steps and timing of the miniaturized Scalable Thiol Reactivity Profiling (STRP) assay. B) Structures and summary of activities of inhibitory potencies of a training set of known cysteine-reactive small molecule fragments in the STRP (C) and control (D) assays.

[0120] Fig.3A-G. High throughput screening with STRP identifies azetidines as cysteine selective reactive groups. A) Z scores of luminescence signal from a high throughput screen of 10,561 diverse small molecules assayed for STRP inhibitory activity with azetidine containing hit compounds colored in peach. Structures and summary of activities (B), dose responsive inhibitory activity in the STRP (C) and control (D) assays of three commercially available azetidine containing hit compounds (13, 14, 15). E) Scheme depicting the steps to form the ring opened, Boc protected cysteine adduct of 15 in vitro. F)1H NMR spectra of 15 (bottom, gray) and the ring opened cysteine adduct of 15 (top, green). Protons noted on structures to the right as colored circles correspond to circles noted on spectra (left). G) Extracted ion chromatograms (EICs) for the adducted, ring opened species with each of the protected nucleophilic amino acids. Structures of each predicted adduct are depicted in Figure 8.TSRI 2247.1PC

[0121] Fig.4A-G. Alkyne probe 41 covalently engages diverse protein families across the cysteine proteome. A) Structure and summary of activities of 41. B) STRP assay and control assay of the indicated concentrations of alkyne probe 41. C) Rhodamine based fluorescence scan of SDS-PAGE resolved proteomes from HEK293T cells exposed to the indicated concentrations of 41 for 1 hour with or without pre-treatment with iodoacetamide (IA, 10 mM) for 1 hour. D) Fold enrichment vs -log10P values for covalently modified proteins identified by MS / MS-based proteomics after streptavidin enrichment from HEK293T cells exposed to 41 (1 mM) for 1 hour. E) Fraction of identified targets that have been liganded according to DrugBank. F) Fraction of identified targets associated with the indicated protein functions. G) -log10P values of the top GO term associations for statistically enriched proteins.

[0122] Fig.5A-G. Covalent modification of C152 by 41 allosterically augments UCHL1 activity. A) Rhodamine fluorescence scan and Coomassie staining of anti-FLAG immunoprecipitated content of the indicated FLAG tagged UCHL1 transgenes overexpressed in HEK293T cells and exposed to 41 (20 µM) for 1 hour. B) MS / MS spectra of UCHL1 peptide containing modified C152 from anti-FLAG immunoprecipitated material from HEK293T cells expressing UCHL1-FLAG and exposed to 41 (20 µM) for 1 hour. C) Image of apo crystal structure of UCHL1 (PDB: 2ETL) indicating the catalytic triad H161, D176, and C90 along with C152 (green) within the flexible crossover active site loop (gray). D) Activity of recombinant UCHL1 exposed to 41 (10µM) for 1 hour. E) Activity of anti-FLAG immunoprecipitated UCHL1 from HEK293T cells exposed to 41 (10 µM) for 1 hour. Anti- HA Western blotting for ubiquitinated anti-FLAG immunoprecipitated SMAD2 (F) and ALK5 (G) after exposure of HEK293T cells expressing UCHL1 to 41 (100 µM) for 1 hour.

[0123] Fig.6A-D. Optimization of the STRP assay and an assay to identify compounds that interfere with its activity. Titration of CHBT (A), D-cysteine (B), or Luciferin (C) allows for an optimized assay for detecting the spontaneous detection of luciferin formation. D) Schematic depicting the control assay for detecting small molecules which interfere with STRP assay.

[0124] Fig.7. Structures of the putative ring opened adducts of compound 15 with nucleophilic amino acids. Structures and masses of potential adducts with cysteine (Ac-Cys- OMe, top left), lysine (Boc-Lys, top middle), serine (Boc-Ser-OTbu, top right), tyrosine (Boc-Tyr-OMe, bottom left), histidine (Ac-His, bottom middle), and threonine (Thr-OMe, bottom right) are shown.TSRI 2247.1PC

[0125] Fig.8A-B. Determining the repertoire of azetidines that react with cysteine in the STRP assay. A) Structure and summary of activities of 16-40 in the STRP (B, left) and control (B, right) assays.

[0126] Fig.9. DFT calculations reveal basis for selective reactivity of oxadiazole substituted azetidines with cysteine. Computed energy profile (left) of the reactive azetidine 15 (A) vs the unreactive azetidine 19 (B). Gibbs free energies and enthalpies, kcal / mol; distances (in black), angstrom; red numbers, natural population analysis (NPA) charge of indicated atoms.

[0127] Fig.10A-D. Reactive azetidine containing 41 covalently engages cysteines throughout the proteome. A) Representative fluorescence scan (left) and Coomassie loading control (right) of SDS-PAGE resolved proteomes after exposure of HEK293T cells to the indicated concentrations of 41 for 1 hour followed by click reactions with rhodamine azide. B) Densitometry-based quantification of labeling signal in A. C) Coomassie stained loading control of SDS-PAGE resolved lysates from Fig 4C. D) Rhodamine fluorescence scan and anti-FLAG Western blotting of anti-FLAG immunoprecipitated content of the indicated FLAG tagged deubiquitnase overexpressed in HEK293T cells and exposed to 41 (20 µM) for 1 hour.

[0128] Fig.11A-C.41 covalently labels C152 of UCHL1. A) Rhodamine fluorescence scan and Coomassie staining of anti-FLAG immunoprecipitated content of WT UCHL1- FLAG overexpressed in HEK293T cells and exposed to the indicated concentrations of 41 for 1 hour. B) Rhodamine fluorescence scan and Coomassie staining of anti-FLAG immunoprecipitated FLAG tagged UCHL1 transgenes overexpressed in HEK293T cells and exposed to 41 (20 µM) for 1 hour. C) b and y ion designations from the covalently modified tryptic UCHL1 peptide containing C152, as shown in Figure 5B.

[0129] Table X. Structural analogues of CHBT:TSRI 2247.1PCTSRI 2247.1PCEXAMPLES Abbreviations

[0130] Commonly used abbreviations include: acetyl (Ac), azo-bis-isobutyrylnitrile (AIBN), atmospheres (Atm), 9-borabicyclo[3.3.1]nonane (9-BBN or BBN), tert- butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), benzyl (Bn), butyl (Bu), Chemical Abstracts Registration Number (CASRN), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole (CDI), 1,4-diazabicyclo[2.2.2]octane (DABCO), diethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N'-dicyclohexylcarbodiimide (DCC), 1,2- dichloroethane (DCE), dichloromethane (DCM), diethyl azodicarboxylate (DEAD), di-iso- propylazodicarboxylate (DIAD), di-iso-butylaluminumhydride (DIBAL or DIBAL-H), 1,3- Diisopropylcarbodiimide (DIC), di-iso-propylethylamine (DIPEA), N,N-dimethyl acetamide (DMA), 4-N,N-dimethylaminopyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,1'-bis-(diphenylphosphino)ethane (dppe), 1,1'-bis- (diphenylphosphino)ferrocene (dppf), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH), 2-ethoxy-2H- quinoline-1-carboxylic acid ethyl ester (EEDQ), diethyl ether (Et2O), O-(7-azabenzotriazole- 1-yl)-N, N,N’N’-tetramethyluronium hexafluorophosphate acetic acid (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC),TSRI 2247.1PC iso-propanol (IPA), lithium hexamethyl disilazane (LiHMDS), methanol (MeOH), melting point (mp), MeSO2- (mesyl or Ms), , methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrum (ms), methyl t-butyl ether (MTBE), N-bromosuccinimide (NBS), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), phenyl (Ph), propyl (Pr), iso-propyl (i-Pr), pounds per square inch (psi), pyridine (pyr), room temperature (rt or RT), tert-butyldimethylsilyl or t-BuMe2Si (TBDMS), triethylamine (TEA or Et3N), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), triflate or CF3SO2- (Tf), trifluoroacetic acid (TFA), 1,1'-bis-2,2,6,6-tetramethylheptane-2,6-dione (TMHD), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), thin layer chromatography (TLC), tetrahydrofuran (THF), trimethylsilyl or Me3Si (TMS), p- toluenesulfonic acid monohydrate (TsOH or pTsOH), 4-Me-C6H4SO2- or tosyl (Ts), N- urethane-N-carboxyanhydride (UNCA),. Conventional nomenclature including the prefixes normal (n), iso (i-), secondary (sec-), tertiary (tert-) and neo have their customary meaning when used with an alkyl moiety. (J. Rigaudy and D. P. Klesney, Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.). Methods. Miniaturized assay for performing Scalable Thiol Reactivity Profiling

[0131] 20 µL of 2.5 µM D-Cysteine in 0.1 M Tris-HCl (pH 8) was added to white 384- well plates (Corning) using a Multidrop dispenser (Thermo). Subsequently, 100 nL of electrophilic compounds were transferred using a Bravo Liquid Handling instrument (Agilent) affixed with a pintool head (V&P Scientific) to achieve final assay concentrations ranging from 1.5 µM to 250 µM. After a 3-hour incubation period at room temperature, 20 µL of 95 µM CHBT in 0.1 M Tris-HCl (pH 8) was added to each well. The mixture was then incubated for an additional 30 minutes at room temperature.40 µL of a luciferase containing solution (0.1 µg / mL luciferase (Sigma), 1 mM ATP (Sigma), 2.5 mM MgCl2 in 0.1 M Tris- HCl) was added to each well and shaken for one minute using an orbital shaker. Luminescence values were then immediately recorded using an Envision plate reader (PerkinElmer). Control assay for Scalable Thiol Reactivity ProfilingTSRI 2247.1PC

[0132] 20 µL of 2.5 µM D-Cysteine in 0.1 M Tris-HCl (pH 8) was added to white 384- well plates followed by addition of 20 µL of 95 µM CHBT in 0.1 M Tris-HCl (pH 8) using a Multidrop dispenser. After a 30-min incubation period, 100 nL of electrophilic compounds were transferred via pintool, as above. After a 3-hour incubation period, 40 µL of a luciferase solution (0.1 µg / mL luciferase, 1 mM ATP, 2.5 mM MgCl2in 0.1 M Tris-HCl) was added to each well and shaken for one minute using an orbital shaker. Luminescence values were then immediately captured on an Envision plate reader. High Throughput Screening

[0133] For high throughput screening, compounds were assayed as above at a final assay concentration of 25 µM. Iodoacetamide (1 mM final concentration) and DMSO ere used as positive and neutral stimulation controls respectively. The chemical library (3D- Pharmacophore Based Diversity Library) was obtained from ChemDiv. Plasmids

[0134] pCMV6-UCHL1-FLAG and pCMV6-UBR7-FLAG was purchased from OriGene Technologies. pCMV6-UCHL1 C90S-FLAG, pCMV6-UCHL1 C152S-FLAG, pCMV6- UCHL1 C152A-FLAG, and pCMV6-UCHL1-Myc were generated by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit per the manufacturer’s instructions (New England Biolabs). pcDNA3.1-UBE2O-FLAG (#105718), pQFlag-USP11 (#46747), pRK5F-SMAD2 (#12623), and pcDNA3-ALK5 (#80876), and pcDNA3-Ubiquitin-HA (#18712) were obtained from Addgene. Cell culture

[0135] HEK293T cells were purchased from American Type Culture Collection and maintained in Dulbecco's Modified Eagle Medium (DMEM, Corning) supplemented 10% with fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Pen-Strep, Gibco). Immunoblotting

[0136] HEK293T cells were collected by scraping into 250 μL ice cold 1x RIPA buffer (EMD Millipore). Lysates were sonicated using a tip sonicator (Branson) followed by centrifugation to remove insoluble material. The protein concentration of the supernatants was determined by absorbance measurements using a Nanodrop instrument.50 μg of lysate was run on each lane of a Bis-Tris 4-12% SDS-PAGE gel (Invitrogen) and then transferred toTSRI 2247.1PC PVDF membrane (Bio-Rad). Membranes were blocked in 5% non-fat dry milk (BioRad) in TBS-T (Tris-buffered saline with 0.1% Tween 20) for 1 hr at room temperature. The membrane was incubated with primary antibody in milk overnight at 4°C. Primary antibodies used here anti-FLAG (1:2000), anti-HA (1:1000), and anti-Myc (1:1000). After 6x TBST washes for 10 minutes, the membrane was incubated with HRP-conjugated (1:3000, ThermoFisher) secondary antibody for 1 hr and then washed again with TBS-T before visualization on a ChemiDoc MP Imager (BioRad). Proteomics studies

[0137] For proteomics target identification studies, confluent HEK293T cells in a 10-cm tissue culture dish were washed with DMEM (Gibco) twice. The cells were treated with indicated concentrations of compound or with DMSO in DMEM without FBS and incubated at 37 °C for 1 h. The cells were washed with PBS twice, scraped into 1 mL PBS, and then lysed by sonication. Insoluble materials were removed by centrifugation. Two 1.5 mL tubes per biological sample containing 0.5 mL of 2 mg / mL lysate were incubated with Click reagent mix (30 µL of 1.7 mM TBTA in tBuOH:DMSO 4:1, 10 µL of 50 mM CuSO4 in H2O, 10 µL of 50 mM TCEP in H2O, 2.5 µL of 20 mM biotin-PEG3-N3) at room temperature for 1 h. The reaction mixtures were precipitated in ice cold methanol, combined, and the pellets were sent to the Sanford Burnham Prebys proteomics core for streptavidin pulldown and MS- based identification of proteins. Proteome labeling studies

[0138] For labeling studies in wild-type cells, HEK293T cells (5 × 106) were seeded per well of six-well plates. After 48 h, cells were treated with indicated concentrations of compound in DMEM medium without FBS for 1 h. For competition experiments, cells were pre-treated with indicated concentrations of iodoacetamide for 1 h before treatment with probe. Cells were washed twice with PBS and then scraped into 250 µL of ice-cold PBS and lysed by sonication. Insoluble materials were removed by centrifugation and protein concentration were then quantified by absorbance measurements using a Nanodrop instrument.1 mg of lysate in 250 µL of PBS was subject to click reaction (described previously) and incubated at room temperature for 1 h. The reaction mixture was precipitated in ice cold methanol and the pellet was redissolved in 50 µL SDS-PAGE sample buffer with 10% beta-mercaptoethanol. Immunoprecipitated samples were run on an SDS-PAGE gel andTSRI 2247.1PC analyzed by rhodamine fluorescent scan on a ChemiDoc MP imager (Bio-Rad) and by Coomassie staining. Protein labeling studies

[0139] For labeling studies with epitope-tagged transgenes, HEK293T cells (5 × 106) were transfected with 2 µg of each plasmid per well of six-well plates using 100 µL of OptiMEM medium (Gibco) containing 8 µL of FuGENE HD transfection reagent (Promega). After 24 h, growth medium was washed and replaced with fresh medium and cells were grown another 24 h.48 h after transfection, cells were treated with indicated concentrations of compound in DMEM medium without FBS for 1 h. Cells were washed twice with PBS and then scraped into 250 µL of ice-cold RIPA buffer (EMD Millipore) and lysed by sonication. Insoluble materials were removed by centrifugation and protein concentration were then quantified by absorbance measurements using a Nanodrop instrument.1 mg of lysate in 1 mL of RIPA was incubated overnight at 4 °C in the presence of 20 µL of anti- FLAG M2 magnetic bead slurry (Sigma). After three washes with 300 µl of RIPA, immunoprecipitated material was eluted with 250 µg / mL of FLAG peptide in PBS (DYKDDDDK, Sino Biological). Eluted material from immunoprecipitations were then subject to click reaction (described previously) and incubated at room temperature for 1 h. The reaction mixture was precipitated in ice cold methanol and the pellet was redissolved in 50 µL SDS-PAGE sample buffer with 10% beta-mercaptoethanol. Immunoprecipitated samples were run on an SDS-PAGE gel and analyzed by rhodamine fluorescent scan on a ChemiDoc MP imager and by Coomassie staining. MS / MS-based site of labeling

[0140] For MS / MS analysis of probe site of labeling of UCHL1, UCHL1 WT-FLAG was overexpressed in HEK293T cells and immunoprecipitated as described above. Excised bands from SDS-PAGE gels of immunoprecipitated samples were sent to the Scripps Research proteomics core for MS / MS-based identification of UCHL1 residues with probe mass addition. UCHL1 activity assays

[0141] For recombinant UCHL1 activity assays, 5nM UCHL1 was diluted in 1X PBS was treated with indicated concentrations of compound for 1 h at 37ºC.49µL of treated UCHL1 was added to each well of a black 384-well plate and 1µL of 50µM ubiquitin-TSRI 2247.1PC rhodamine 110 was added to begin the assay. The fluorescence signal was measured for 10 minutes at an interval of 60 seconds using a SpectraMax iD3 microplate reader with excitation and emission filter set of 485 / 20 and 535 / 20 nm, respectively. For in vitro UCHL1 assays, UCHL1-FLAG was overexpressed in HEK293T cells and immunoprecipitated as described above. Eluted protein was then diluted 1:50,000 before being treated and assayed as described above. Recombinant UCHL1 was purchased from Sino Biological and Ubiquitin-rhodamine 110 was purchased from BPS Bioscience. Synthetic procedures

[0142] For chemical syntheses, solvents and reagents were purchased from commercial vendors and used directly without further purification. Unless otherwise stated, all reactions were carried out using anhydrous solvents under a N2atmosphere. Analytical thin layer chromatography (TLC) was performed using 0.20 mm SiliCycle silica plates (F254). Visualization was carried out with short-wave UV light, and Ninhydrin or KMnO4 were used with heat as developing agents. LC-MS analysis of reaction mixtures was carried out on an Agilent 6135 Quadrupole LC / MS, using an Agilent ZORBAX 300SB-C85 μm, (4.6 X 50 mm) column. High-resolution mass spectra (HRMS) were recorded on an Agilent Mass spectrometer using ESI-TOF (electrospray ionization-time of flight). Automated flash chromatography was performed on Teledyne ISCO CombiFlash NextGen 300+. NMR analysis

[0143] All1H NMR spectra were acquired on a Bruker AV III HD 600 MHz or Bruker AV NEO 500 MHz NMR instruments in the specified deuterated solvents.13C NMR spectra were acquired on a Bruker AV NEO 500 MHz or Bruker AV III HD 600 MHz NMR spectrometer. Chemical shifts are given in ppm with respect to residual undeuterated solvent signal as internal standard. Coupling constants are reported as J-values in Hertz (Hz). The following abbreviations were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet, bs = broad singlet, br = broad. Spectral data were processed with MestreNova software. Synthesis of 5-(azetidin-3-yl)-3-(2-methoxyphenyl)-1,2,4-oxadiazole hydrochlorideTSRI 2247.1PC

[0144] A suspension was prepared by combining 2-methoxybenzonitrile (5.00 g, 37.6 mmol), hydroxylamine hydrochloride (5.25 g, 75.6 mmol), and NaHCO3 (7.90 g, 94.0 mmol) in a mixture of water (7.5 mL) and ethanol (150 mL). The reaction mixture was stirred in a round-bottom flask at 90 °C for 4 hours. After cooling to room temperature, water was added, and the product was extracted with EtOAc. The organic layer was subsequently washed twice with brine, dried over Na2SO4, and concentrated to yield N-hydroxy-2- methoxybenzimidamide (4.00 g, 64%) without further purification. LC-MS [M+H]+= 167.1.

[0145] Next, N-hydroxy-2-methoxybenzimidamide (4.00 g, 24.1 mmol), 1-(tert- butoxycarbonyl)azetidine-3-carboxylic acid (5.76 g, 28.9 mmol), and HATU (10.96 g, 28.8 mmol) were combined in CH2Cl2(100 mL). DIPEA (12.56 mL, 72.1 mmol) was added to the mixture, which was then stirred at room temperature overnight. The reaction mixture was washed with water, followed by brine, dried over Na2SO4, and evaporated. The resulting residue was dissolved in N-methyl-2-pyrrolidone (NMP, 100 mL) and stirred at 130 °C overnight. After evaporation, the product was purified by flash column chromatography (30% EtOAc / hexanes) and further purified through recrystallization in EtOAc, resulting in tert- butyl 3-(3-(2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)azetidine-1-carboxylate as a white solid (4.20 g, combined yield of 52%). LC-MS [M-56+H]+= 276.0.

[0146] Finally, tert-butyl 3-(3-(2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)azetidine-1- carboxylate (2.20 g, 6.6 mmol) in EtOAc (50 mL) was treated with a solution of HCl in EtOAc (4 M, 75 mL), and the reaction was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure and then recrystallized using a mixture of MeCN and MeOH to yield 1.60 g (90%) of the HCl salt of 5-(azetidin-3-yl)-3-(2- methoxyphenyl)-1,2,4-oxadiazole as a white solid. LC-MS [M+H]+= 232.1.1H NMR (500 MHz, DMSO) δ 9.52 (s, 2H), 7.86 (dd, J = 7.6, 1.8 Hz, 1H), 7.58 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 7.13 (td, J = 7.5, 1.0 Hz, 1H), 4.53 – 4.46 (m, 1H), 4.38 – 4.34 (m, 2H), 4.31 – 4.27 (m, 2H), 3.88 (s, 3H).13C{1H} NMR (126 MHz, DMSO) δ 176.8, 166.1, 157.7, 132.8, 130.7, 120.6, 114.9, 112.4, 55.9, 48.6, 28.0.TSRI 2247.1PC HRMS (ESI-TOF) calc’d for C12H13N3O2[M+H]+:232.1081, found: 232.1085 Synthesis of 5-(azetidin-3-yl)-3-(4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazole hydrochloride

[0147] A suspension was prepared by combining 4-bromo-2-methoxybenzonitrile (2.80 g, 13.2 mmol), hydroxylamine hydrochloride (1.82 g, 26.2 mmol), and NaHCO3 (2.74 g, 32.6 mmol) in a mixture of water (53 mL) and ethanol (2.7 mL). The reaction mixture was stirred in a round-bottom flask at 90°C for 4 hours. After reaching room temperature, water was added, and the product was extracted using EtOAc. The organic layer was washed twice with brine, dried over Na2SO4, and concentrated to yield 4-bromo-N-hydroxy-2- methoxybenzimidamide (2.50 g, 77%) without further purification. LC-MS [M+H]+= 245.0.

[0148] Next, a mixture of 4-bromo-N-hydroxy-2-methoxybenzimidamide (2.50 g, 10.2 mmol), 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid (2.80 g, 14.0 mmol), and HATU (5.40 g, 14.2 mmol) in CH2Cl2 (50 mL) was treated with DIPEA (6.20 mL, 35.6 mmol). The reaction mixture was stirred at room temperature overnight. Afterward, it was washed with water, followed by brine, dried over Na2SO4, and evaporated. The residue was dissolved in N-methyl-2-pyrrolidone (NMP, 50 mL) and stirred at 130°C overnight. The solvent was evaporated using nitrogen gas flow and the residue was purified by flash column chromatography (30% EtOAc / hexanes), resulting in tert-butyl (Z)-3-(((4-bromo-2- methoxyphenyl)(hydroxyimino)methyl)carbamoyl)azetidine-1-carboxylate as a white solid (2.00 g, combined yield of 48%). LC-MS [M-56+H]+= 354.0.

[0149] Tert-butyl (Z)-3-(((4-bromo-2- methoxyphenyl)(hydroxyimino)methyl)carbamoyl)azetidine-1-carboxylate (2.00 g, 4.9 mmol) was dissolved in triethylamine (2.6 mL) and DMF (20 mL). The mixture was added via cannula transfer to a second flask containing PdCl2(PPh3)2(690 mg, 1.0 mmol) and CuI (90 mg, 0.5 mmol), followed by the addition of ethynyltrimethylsilane (1.50 mL, 10.8 mmol). The reaction mixture was heated to 50°C. After 5 hours, it was cooled to room temperatureTSRI 2247.1PC and stirred overnight. Water was then added, and the mixture was extracted with CH2Cl2. The combined organic fraction was dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (30% EtOAc / hexanes) to obtain tert-butyl 3-(3-(2-methoxy-4-((trimethylsilyl)ethynyl)phenyl)- 1,2,4-oxadiazol-5-yl)azetidine-1-carboxylate (1.10 g, 52%). LC-MS [M-56+H]+= 372.1.

[0150] To a solution of tert-butyl 3-(3-(2-methoxy-4-((trimethylsilyl)ethynyl)phenyl)- 1,2,4-oxadiazol-5-yl)azetidine-1-carboxylate (1.10 g, 2.6 mmol) in methanol (24 mL) and EtOAc (1.8 mL) was added potassium carbonate (1.05 g, 7.6 mmol). The reaction mixture was stirred at room temperature for 1 h and then poured over 10 mL of 1 M KHSO4. The mixture was extracted with CH2Cl2, and the combined organic fraction was dried with anhydrous Na2SO4, and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography (20% EtOAc / hexanes) to afford 640 mg (70%) of tert- butyl 3-(3-(4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)azetidine-1-carboxylate. LC- MS [M-56+H]+= 300.1.

[0151] Finally, tert-butyl 3-(3-(4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazol-5- yl)azetidine-1-carboxylate (200 mg, 0.56 mmol) in EtOAc (4 mL) was treated with a solution of HCl in EtOAc (4 M, 16 mL), and the reaction was stirred at room temperature overnight. The reaction mixture was subsequently concentrated under reduced pressure. The salt was then precipitated by adding EtOAc, filtered, and the HCl salt of of 5-(azetidin-3-yl)-3-(4- ethynyl-2-methoxyphenyl)-1,2,4-oxadiazole (160 mg, 97%) was collected as a pale yellow solid. LC-MS [M+H]+= 256.1.1H NMR (500 MHz, DMSO) δ 9.53 (d, J = 136.5 Hz, 2H), 7.89 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 1.4 Hz, 1H), 7.25 (dd, J = 7.9, 1.4 Hz, 1H), 4.53 – 4.46 (m, 1H), 4.45 (s, 1H), 4.38 – 4.27 (m, 4H), 3.91 (s, 3H).13C{1H} NMR (151 MHz, DMSO) δ 177.0, 165.6, 157.5, 131.0, 125.9, 124.0, 115.6, 115.4, 83.0, 82.8, 56.2, 48.4, 28.0. HRMS (ESI-TOF) calc’d for C14H13N3O2[M+H]+:256.1081, found: 256.1086. Synthesis of methyl N-acetyl-S-(3-((tert-butoxycarbonyl)amino)-2-(3-(2- methoxyphenyl)-1,2,4-oxadiazol-5-yl)propyl)-L-cysteinateTSRI 2247.1PC

[0152] A mixture of N-Acetyl-L-cysteine methyl ester (1.00 g, 5.6 mmol) and 5- (azetidin-3-yl)-3-(2-methoxyphenyl)-1,2,4-oxadiazole hydrochloride (631 mg, 2.4 mmol) in degassed phosphate buffer pH=7.4 (40 mL) was stirred at 37oC and the reaction progress monitored with LCMS. After 4 days, the reaction mixture was cooled to room temperature. di-tert-butyl dicarbonate (1.60 g, 7.3 mmol) and MeCN (30 mL) was added to the mixture and stirred overnight. The product was then extracted with EtOAc. The organic layer was subsequently washed with brine, dried over Na2SO4, and concentrated. The crude residue was purified by silica gel flash chromatography with an EtOAc / hexanes gradient solvent system where the desired product came out with 100% EtOAc. LC-MS [M+H]+= 509.2.1H NMR (600 MHz, DMSO) δ 8.37 (d, J = 7.8 Hz, 1H), 7.85 (ddd, J = 7.7, 3.4, 1.8 Hz, 1H), 7.56-7.53 (m, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.15 – 7.04 (m, 2H), 4.45 (td, J = 8.0, 5.3 Hz, 1H), 3.86 (s, 3H), 3.61 (d, J = 10.5 Hz, 3H), 3.52 – 3.46 (m, 1H), 3.45 – 3.39 (m, 1H), 3.35 – 3.33 (m, 1H), 3.0 – 2.94 (m, 2H), 2.91 – 2.86 (m, 1H), 2.79 – 2.74 (m, 1H), 1.85 (d, J = 5.9 Hz, 3H), 1.31 (s, 9H).13C{1H} NMR (151 MHz, DMSO) δ 178.2, 171.2, 169.4, 165.7, 157.6, 155.5, 132.5, 130.8, 120.4, 115.3, 112.3, 78.0, 55.8, 52.0, 51.9, 42.2, 39.3, 33.0, 31.9, 28.1, 22.3. HRMS (ESI-TOF) calc’d for C23H32N4O7S [M+Na]+:531.1884, found: 531.1885. Computational Methods

[0153] Density functional theory (DFT) calculations for the mechanistic energy profile were performed using the Jaguar 9.1 suite of ab initio quantum chemistry programs.[1] The B3LYP functional including Grimme’s D3 dispersion correction was employed for all conformations.[2-7] Geometry optimizations, transition state calculation, and solvation energy calculations were performed with the 6-31G** basis set. For geometry optimization of the structures, the solvation model was implemented to maximize the sum of the electronicTSRI 2247.1PC energy along with the solvation correction employing the dielectric constant of water, ε = 78.4.[8] The self-consistent reaction field (SCRF) approach was treated for the molecular system in the aqueous environment.[9-11] For continuum models, atomic radii for generating the solute surface were subject to empirical parameterization and the standard set of optimized radii for each atoms used in this study were; H (1.150 Å), C (1.900 Å), N (1.600 Å), O (1.600 Å), and S (1.900 Å) as implemented in Jaguar 9.1.

[0012] Based on the optimized geometries, single-point calculations were conducted with the triple-ζ quality of the basis set, cc-pVTZ(-f), to re-evaluate the electronic energies of each molecule.

[0013] Vibrational calculations were computed at the identical level of the structure optimizations to obtain the zero-point energy (ZPE) and entropy (S). The solution-phase Gibbs free energies [G(sol)] were calculated from the energy components as follows: G(sol) = G(gas) + G(solv) G(gas) = H(gas) – TS(gas) H(gas) = ZPE + E(SCF) ΔG(sol) = ΣG(sol) for products − ΣG(sol) for reactants G(gas) is the gas phase free energy; G(solv) is the free energy of solvation; H(gas) is the gas phase enthalpy; T is the temperature (37 °C, 310.15 K); S(gas) is the entropy in gas phase which refers to the vibrational, rotational, and translational entropy of the solute; E(SCF) is the self-consistent field converged electronic energy; ZPE is the vibrational zero-point energy. References: 1. Bochevarov, A. D.; Harder, E.; Hughes, T. F.; Greenwood, J. R.; Braden, D. A.; Philipp, D. M.; Rinaldo, D.; Halls, M. D.; Zhang, J.; Friesner, R. A. Jaguar: A High‐ performance Quantum Chemistry Software Program with Strengths in Life and Materials Sciences. Int. J. Quantum Chem.2013, 113, 2110. 2. Slater, J. C. Quantum Theory of Molecules and Solids, Vol.4: The Self-consistent Field for Molecules and Solids. McGraw-Hill: New York, 1974. 3. Vosko, S. H.; Wilk, L.; Nusair, M. Accurate Spin-dependent Electron Liquid Correlation Energies for Local Spin Density Calculations: A Critical Analysis. Can. J. Phys. 1980, 58, 12001211.TSRI 2247.1PC 4. Becke, A. D. Density-functional Exchange-energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A 1988, 38, 3098–3100. 5. Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785–789. 6. Becke, A. D. Density‐functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys.1993, 98, 5648–5652. 7. Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, S. A Consistent and Accurate ab initio Parametrization of Density Functional Dispersion Correction (DFT-D) for the 94 Elements H-Pu. J. Chem. Phys.2010, 132, 154104–154119. 8. Ditchfield, R.; Hehre, W. J.; Pople, J. A. Self-Consistent Molecular-Orbital Methods. IX. An Extended Gaussian-type Basis for Molecular-orbital Studies of Organic Molecules. J. Chem. Phys.1971, 54, 724−728. 9. Marten, B.; Kim, K.; Cortis, C.; Friesner, R. A.; Murphy, R. B.; Ringnalda, M. N.; Sitkoff, D.; Honig, B. New Model for Calculation of Solvation Free Energies:  Correction of Self-Consistent Reaction Field Continuum Dielectric Theory for Short-range Hydrogen- bonding Effects. J. Phys. Chem.1996, 100, 11775. 10. Edinger, S. R.; Cortis, C.; Shenkin, P. S.; Friesner, R. A. Solvation Free Energies of Peptides:  Comparison of Approximate Continuum Solvation Models with Accurate Solution of the Poisson−Boltzmann Equation. J. Phys. Chem. B 1997, 101, 1190. 11. Friedrichs, M.; Zhou, R.; Edinger, S. R.; Friesner, R. A. Poisson−Boltzmann Analytical Gradients for Molecular Modeling Calculations. J. Phys. Chem. B 1999, 103, 3057. 12. Rashin, A.A.; Honig, B. Reevaluation of the Born Model of Ion Hydration. J. Phys. Chem.1985, 89, 5588. 13. Dunning, T. H., Jr. Gaussian Basis Sets for Use in Correlated Molecular Calculations. I. The Atoms Boron through Neon and Hydrogen. J. Chem. Phys.1989, 90, 1007.

[0154] The foregoing disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appendedTSRI 2247.1PC claims. Therefore, it is to be understood that the above description is intended to be illustrative and not restrictive. The scope of the disclosure should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled.

[0155] This application refers to various issued patents, published patent applications, journal articles, and other publications, each of which are incorporated herein by reference.

Claims

TSRI 2247.1PC WHAT IS CLAIMED IS:

1. A Scalable Thiol Reactivity Profiling (STRP) assay for high-throughput determination of the ability of a test compound to covalently bind to cysteine in vitro, comprising the steps: i) incubating a test compound of with cysteine; ii) exposing the product of step i) to CHBT, or analogues thereof in Table X; iii) addition of luciferase to the product of step ii); and iv) determining the level of resultant luminesce which is inversely proportional to the reactivity of the test compound of Formula I with cysteine.

2. The assay of Claim 1, wherein the test compound is a small molecule, protein, or peptide.

3. The assay of Claim 1 or Claim 2, wherein the test compound is an azetidinyl containing compound.

4. The assay of Claim 3, wherein the test compound is of Formula (I)wherein R1is H or halo; R2is (C6-C10) aryl, -O-(C6-C10) aryl, (C5-C10) heteroaryl, -O-(C5-C10) heteroaryl, (C3-C7) cycloalkyl, (C3-C7) heterocycloalkyl, -O-(C1-C6) alkyl, -O-(C1-C6) alkyl-(C3-C7) cycloalkyl, - S(=O)2-(C6-C10) aryl, (C5-C10) heteroaryl (C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C3-C7), -(C5-C10) heteroaryl-(C1-C6) heteroalky-C(=O)(C1-C6) alkyl, (C5- C10) heteroaryl-(C1-C6) heteroalky-(C6-C10) aryl, -(C5-C10) heteroaryl-(C1-C6) alky-(C6-C10) aryl each optionally substituted with one or more halo, -(C1-C6) alkyl, -(C2-C6) alkenyl, -(C2- C6) alkynyl, or -O-(C1-C6) alkyl; or R1and R2together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl;TSRI 2247.1PC R3and R4are each independently H or optionally substituted (C6-C10) aryl or (C5-C10) heteroaryl; or R3and R4together form (C3-C7) heterocycloalkyl or (C3-C7) cycloalkyl, each optionally substituted with one or more halo, -(C1-C6) alkyl, or -O-(C1-C6) alkyl; R5is H, (C3-C7) heterocycloalkyl-(C1-C6) alkyl-(C5-C10) heteroaryl, (C3-C7) heterocycloalkyl- C(=O)2-(C5-C10) heteroaryl, -(C1-C6) alkyl-(C5-C10) heteroaryl, -(C3-C7) heterocycloalkyl- S(=O)2-(C1-C6) alkyl; including enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

5. The assay of any one of Claims 1-4, wherein the test compound is selected from theincluding enantiomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

6. The assay of any one of Claims 1-5, wherein the test compound comprises an azetidinyl oxadiazole moiety.

7. The assay of any one of Claims 1-6, wherein the test compound is 5-(azetidin-3-yl)-3- (4-ethynyl-2-methoxyphenyl)-1,2,4-oxadiazole or a pharmaceutically acceptable salt thereof.TSRI 2247.1PC 8. A control assay for detecting small molecules which interfere with the STRP assay of any one of Claims 1-7, comprising: i) incubation of cysteine with CHBT or analogue thereof in Table X, to enable formation of D-luciferin; ii) addition of a a test compound of Formula I to determine its interference with CHBT or analogue thereof in Table X, for luciferase activity; iii) addition of luciferase to promote luminescence; and iv) measuring resultant luminescence.

9. An assay for profiling test compounds in a chemical library for thiol reactivity liabilities, comprising measuring each test compound’s ability for covalent adduction with glutathione.

10. The assay of Claim 9, wherein the GSH / thiol / cysteine reactivity risk profile for a given test compound is generated to expose potential liabilities associate with a given test molecule in the early stages of development.

11. A kit providing the assays of any one of Claims 1-10 for use in an STRP assay for high-throughput determination of the ability of a test compound to covalently bind to cysteine in vitro.

12. The assay of claim 1, comprising the following step: v) incubating a test compound with cysteine for approximately 3 hours at room temperature.

13. The assay of claim 12, further comprising the following step: vi) exposing the product of step i) to CHBT, or analogue thereof in Table X, for approximately 30 minutes at room temperature.

14. The assay of claim 13, further comprising the following step: vii) addition of ATP-Mg2+luciferase to the product of step ii).

15. The assay of claim 14, further comprising the following step: viii) determining the level of resultant luminesce in the product of step iii).

16. A method of labeling a cysteine residue, comprising treating a test compound having an azetidinyl moiety with a sample containing a cysteine residue, wherein the azetidinyl moiety covalently reacts with the cysteine residue.

17. The method of Claim 16, wherein the azetidinyl moiety is an azetidindyl oxadiazole moiety.TSRI 2247.1PC 18. The method of Claim 17, wherein the test comound isor a pharmaceutically acceptable salt thereof.

19. The method of any one of Claims 16-18, wherein the cysteine residue is distal to the deubiquitinase UCHL1 enzymatic active site.

20. A compound having the structure:or a pharmaceutically acceptable salt thereof.

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