Spirocycle acrylamides that stereo- and site-selectively degrade the transcriptional and DNA-damage response protein ERCC3

Abstract

Herein described is the chemical proteomic discovery of compounds that covalently and stereoselectively bind cysteine-342 in ERCC3 and lead to the degradation of this protein in cancer cells. ERCC3 is part of the TFIIH complex that regulates transcription and DNA repair processes. The covalent compounds rapidly (within 3 h) degrade ERCC3, but not other proteins in the proteome, and this effect is observed at low-μM concentrations of compound. The enantiomers of active compounds do not engage or degrade ERCC3.

Description

TSRI 2229.1PC SPIROCYCLE ACRYLAMIDES THAT STEREO- AND SITE-SELECTIVELY DEGRADE THE TRANSCRIPTIONAL AND DNA-DAMAGE RESPONSE PROTEIN ERCC3 CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application No. 63 / 619,859 which was filed on January 11, 2024, and which is hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA231991, CA278692, and CGCATF-2021 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The application is related to the chemical proteomic discovery of covalent compounds that stereoselectively bind cysteine-342 in ERCC3 and lead to the degradation of this protein in cancer cells. ERCC3 is part of the TFIIH complex that regulates transcription and DNA repair processes. BACKGROUND OF THE INVENTION

[0004] A major goal of chemical biology is to discover small-molecule probes that can perturb the biochemical and cellular functions of human proteins1. Most human proteins, however, lack selective chemical probes, and the structural and functional diversity of proteins presents a major challenge for streamlining small-molecule screens reliant on protein-specific activity assays2. Alternative and generalizable “binding” assays have emerged to assess small molecule-protein interactions across a wide range of proteins3. Such approaches include fragment-based drug discovery4,5, DNA-encoded libraries6,7, and chemical proteomics2, 8-10.

[0005] Efforts to understand and extend the small molecule-binding potential (or ‘ligandability’) of the human proteome have also benefited from advances in compound library designs. Covalent chemistry has emerged as a particularly useful strategy forTSRI 2229.1PC addressing the ligandability of proteins that may lack traditional small-molecule binding pockets or benefit from the sustained pharmacodynamic effects afforded by irreversible protein modification4, 10, 11. The potential for covalent chemistry to furnish hit ligands, as well as selective chemical probes and drug candidates, for diverse types of proteins has been illuminated by the chemical proteomic technology activity-based protein profiling (ABPP)12, which can evaluate the interactions of electrophilic compounds with thousands of proteins in the cell through competition with broadly reactive small-molecule probes targeting nucleophilic amino acids such as cysteine, lysine, serine, and tyrosine8, 13-17.

[0006] The types of electrophilic compounds investigated to date by ABPP include fragments8, 18, natural products15, and, most recently, diversity-oriented synthesis (DOS)19- inspired sets of stereochemically defined structures (‘stereoprobes’)20-23. While fragment electrophiles provide arguably the broadest initial portrait of covalent binding sites throughout the proteome, the hit compounds emerging from fragment-based screens are often weak in potency (high-µM) and promiscuous in protein interactions and therefore challenging to use in cell biology studies without further optimization24. It was found alternatively that electrophilic stereoprobes frequently engage cysteine residues with site-specificity (only a single cysteine on a protein reacts with the stereoprobe), stereoselectivity (the enantiomer of the stereoprobe hit does not react with the cysteine), and sufficient potency (> 70% engagement at 20 µM in cells) for use in cell biological studies that include inactive enantiomeric compounds and stereoprobe-resistant cysteine mutant proteins as controls. Examples of functional stereoprobes discovered by ABPP include allosteric inhibitors of the NAD-metabolizing enzyme SARM122and the exonuclease TOE125, E3 ligase ligands that can be converted into heterobifunctional degraders21, and protein-protein interaction modulators targeting proteasomal regulatory and spliceosome complexes23.

[0007] Only a limited number of stereoprobe scaffolds have been evaluated to date by ABPP. The most extensively studied – the azetidine and tryptoline acrylamides – show strikingly little overlap in specific proteomic interactions, defined as the cysteines engaged by each scaffold with high stoichiometry and stereoselectivity20, 21, 26. These findings, as well as others27, emphasize the importance of scaffold designs in presenting recognition elements that shape the covalent interactions of electrophilic compounds across the proteome. Herein described is the synthesis and chemical proteomic analysis of a focused set of spirocycle acrylamides of defined stereochemistry and regiochemistry. The spirocycle acrylamides showed attenuated global reactivity in cysteine-directed ABPP experiments compared toTSRI 2229.1PC structurally related azetidine acrylamides, but nonetheless preferentially engaged specific cysteines on diverse protein classes, including kinases, GTPases, E3 ligases, and adaptor proteins. It was confirmed that several stereoselective and site-specific liganding events with recombinant proteins, including the transcription factor IIH (TFIIH) complex component helicase ERCC3, for which spirocycle acrylamide ligands engaging cysteine-342 (C342) were further found to cause ubiquitin-proteasome system-mediated degradation of the protein. In contrast, covalent engagement of ERCC3_C342 by the natural product triptolide did not result in the loss of ERCC3 but instead promoted collateral degradation of RNA polymerases. Finally provided was evidence that a third compound – the anti-hypertensive thioester drug spironolactone – also causes degradation of ERCC3 through engagement of C342. Our findings identify stereoselective spirocycle acrylamide-cysteine interactions on diverse proteins in human cancer cells and, through doing so, illuminate strikingly distinct functional outcomes mediated by covalent compounds engaging the same cysteine residue in ERCC3. BRIEF DESCRIPTION OF THE FIGURES

[0008] Figure 1. Design and initial profiling of spirocycle acrylamide stereoprobes.

[0009] Figure 2. Cysteine-directed ABPP of spirocycle acrylamide stereoprobes..

[0010] Figure 3. Confirmation of spirocycle acrylamide interactions with representative proteins.

[0011] Figure 4. (S)-(spiro)pyrrolidine acrylamides stereoselectively degrades ERCC3.

[0012] Figure 5. ZL-12A degrades ERCC3 by covalent modification of C342.

[0013] Figure 6. Distinct functional effects of covalent ligands engaging ERCC3_C342.

[0014] Figure 7. Cysteine-directed ABPP of spirocycle acrylamide stereoprobes.

[0015] Figure 8. Cysteine-directed ABPP data for spirocycle acrylamide-liganded proteins.

[0016] Figure 9. Evolutionary conservation of NEK9_C623 and RAB30_C168.

[0017] Figure 10. (S)-(spiro)pyrrolidine acrylamide ZL-12A stereoselectively degrades ERCC3.

[0018] Figure 11. Distinct functional effects of covalent ligands engaging ERCC3_C342.

[0019] Figure 12. Evidence that spironolactone degrades ERCC3 by covalent modification of C342.TSRI 2229.1PC

[0020] Figure 13. Cancer Dependency Map profiles for TFIIH complex and POLR2 members. SUMMARY OF THE INVENTION

[0021] Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Herein describes is a set of stereo- and regio-chemically defined spirocycle acrylamides and their analysis in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamides, spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound ZL-12A stereoselectively promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL- 12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation, but instead caused collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another’s protein degradation profiles. Finally, it was shown that the anti-hypertension drug spironolactone — previously found to promote ERCC3 degradation through an enigmatic mechanism — also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

[0022] The application provides a method of covalently binding to cysteine-342 in ERCC3 which leads to subsequent degradation of ERCC3 using the following spirocycle acrylamides of Formula I or II:IIIwherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaryl ring system;TSRI 2229.1PC including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salt thereof.

[0023] The application provides the above method, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one.

[0024] The application further provides a compound of Formula I selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one.

[0025] The application further provides a composition comprising the above compounds of Formulae I-II, admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0026] The application further provides the above composition, further comprising one or more therapeutic compounds or compositions.

[0027] The application further provides the above composition, wherein the one or more therapeutic compounds or compositions is a second anti-cancer compound or composition.TSRI 2229.1PC

[0028] The application further provides a method of preventing, ameliorating, or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the compound of of Formulae I-II, compositions thereof, or combination compositions thereof.

[0029] The application further provides an ERCC3 protein covalently bound to a compound, wherein the compound is covalently bound to cysteine residue 342 of the ERCC3 protein.

[0030] The application further provides an ERCC3 protein covalently bound to a compound, wherein the compound is covalently stereoselectively bound to cysteine residue 342 of the ERCC3 protein.

[0031] The application further provides an ERCC3 protein covalently bound to a compound, wherein the compound covalently bound to cysteine residue 342 of ERCC3 has the structure of the following spirocycle acrylamides of Formula I or II:wherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaromatic ring system; including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof. DETAILED DESCRIPTION OF THE INVENTION

[0032] ABPP studies performed on DOS-inspired azetidine and tryptoline stereoprobes have identified diverse liganding events in the human proteome20-23, 25, 26. It is interpreted that the limited overlap in proteins liganded by azetidine and tryptoline stereoprobes as evidenceTSRI 2229.1PC that their distinct stereochemically defined, entropically constrained cores bestow each compound family with a select number of productive protein interactions. The stereoselectivity and site-specificity of these stereoprobe-cysteine interactions further provide stringent filters to enable streamlined transition to functional studies in cells, but also emphasize the need to explore additional stereoprobe scaffolds to address as-of-yet unliganded proteins.

[0033] Spirocyclic compounds contain two rings that are connected via a single atom. These motifs are abundant in natural products and are of growing interest to the medicinal chemistry community, enabled by emergent synthetic methods to access and accommodate diverse spirocyclic systems28, 29. Spirocyclic compounds offer well-defined structures, conformational rigidity, high fractional sp3 content (Fsp3), and empirically improved physiochemical and pharmacokinetic properties (e.g., solubility, metabolic stability)30. It was then intended to explore electrophilic spirocycle scaffolds by ABPP and selected azetidine- pyrrolidine acrylamides as an initial test set (Figure 1A). These compounds contain two endocyclic nitrogens, each of which can serve as a site for installation of an electrophilic acrylamide that is positioned proximal to the bridging carbon and stereocenter. The azetidine- pyrrolidine spirocycle further perturbs the conformational structure of the azetidine ring, accesses distinct stereochemically defined exit vectors compared to original azetidine acrylamide stereoprobes, and facilitates comparison between two sets of regioisomers ((spiro)azetidine and (spiro)pyrrolidine acrylamides) (Figure 1A).

[0034] A focused library of spirocycle acrylamides was then synthesized containing four sets of regio- and stereo-isomers (16 members total) (Figure 1B). Appendage diversity focused on (hetero)aromatic substituents, given their prevalence in advanced chemical probes and FDA-approved drugs31. One stereoisomeric set was appended with a (prop-2-yn-1- yloxy)benzene group to yield alkynylated stereoprobes for additional evaluation of protein- directed gel- or mass spectrometry (MS)-ABPP experiments23, 26using copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry32, 33with azide reporter tags34. Initial gel- ABPP experiments were then performed to benchmark the proteome-wide reactivity of the alkynylated spirocycle acrylamides ZL-13A / B / / 14A / B in comparison to previously described azetidine acrylamide stereoprobes MY-11A / B / / 12A / B21. Exposure of the Ramos human B- cell cancer line to alkynylated stereoprobes (20 or 50 µM, 1 or 3 h), followed by lysis and CuAAC conjugation of stereoprobe-labeled proteins to an azide-rhodamine reporter tag and in-gel fluorescence scanning, revealed that ZL-13A / B / / 14A / B displayed much lower overallTSRI 2229.1PC proteomic reactivity compared MY-11A / B / / 12A / B (Figure 1C). The spirocycle acrylamides showed a similar reduction in reactivity with glutathione in comparison to azetidine acrylamides (Supplementary Table 1). Despite their tempered reactivity, spirocycle acrylamides displayed several stereoselective protein interactions at both test concentrations and time points of incubation (marked with red asterisks for the 50 µM, 3 h condition in Figure 1C). Based on these initial gel-ABPP data, a condition of 50 µM stereoprobe, 3 h treatment time for evaluating the spirocycle acrylamides by MS-ABPP was selected.

[0035] Cysteine-directed ABPP of three sets of spirocycle acrylamides – ZL- 7A / B / / 10A / B, ZL-8A / B / / 11A / B, and ZL-9A / B / / 12A / B (50 µM, 3 h) – in Ramos cells were performed following described protocols that assign stereoprobe engagement through blockade of cysteine reactivity with an iodoacetamide desthiobiotin (IA-DTB) probe35. Cysteine-stereoprobe interactions are quantified by multiplexed tandem mass tagging (TMT) MS, where stereoprobe-treated samples are normalized to a DMSO control (10plex, two replicates per condition; Figure 7A). Stereoprobe-liganded cysteines were defined as those showing > 60% cysteine engagement by one or more stereoprobes, and stereoselective liganding events were further assigned as those displaying > two-fold differences in engagement between enantiomeric stereoprobes. In aggregate, ~8500-15,000 cysteines were quantified per spirocycle acrylamide set, of which 32 cysteines were liganded (Figure 7B-D and Supplementary Dataset 1). Fourteen of these liganded cysteines were stereoselectively engaged by one or more pairs of spirocycle acrylamides (Figure 2A). These stereoselectively liganded cysteines generally showed robust regioselectivity as well (Figure 2A), and additional structure-activity relationship (SAR) information was extractable from substituent effects for a subset of liganded cysteines (e.g., RAB30_168; ZC4H2_124).

[0036] The stereoselectively liganded cysteines were found in diverse protein classes, including kinases (NEK9_C623; PIK3CA_C242), E3 ligases (CRBN_C287), GTPases (RAB30_C168), helicases (ERCC3_C342), adaptors (WASHC5_C385), and zinc finger proteins (ZC4H2_C124; ZFC3H1_C1778). While some of these cysteines, such as CRBN_C287 and GSTO1_C32, have also been found to stereoselectively react with azetidine acrylamides21(Figure 8A), a substantial number were exclusively liganded by spirocycle acrylamides (Figure 2A, C and Supplementary Dataset 1). Among the spirocycle acrylamide-preferring cysteines, C242 of PIK3CA is located near the interface of binding to KRAS (Figure 2D), and covalent ligands have recently described in the patent literature thatTSRI 2229.1PC perturb interactions of PIK3CA with KRAS through engagement of this cysteine36(Figure 8B).

[0037] It was next intended to confirm the liganding of representative proteins by spirocycle acrylamides. NEK9 – a member of the Never In Mitosis Gene A (NIMA)-related kinase (NEK) family – was stereoselectively liganded at C623 by each (S)-(spiro)pyrrolidine acrylamide (Figure 2A). The NEK family contains eleven members that regulate mitotic, DNA damage response, and programmed cell death processes37. NEK9 has been implicated in cancer38, and mutations in this protein cause nevus comedonicus, a rare form of severe acne39. To our knowledge, selective NEK9 ligands are lacking. C623 is located in RCC1 repeat domain of NEK9 that has autoinhibitory function40and harbors several disease- relevant mutations39(Figure 3A), but has not yet been structurally determined. C623 is generally conserved across vertebrate NEK9 orthologs (Figure 9A) and is paralog-restricted (not found in other NEK family members; Figure 9B). Several cysteines in NEK9 were quantified in our ABPP experiments, but only C623 was stereoselectively liganded by (S)- (spiro)pyrrolidine acrylamides (Figure 3B), suggesting that this interaction is site-specific on NEK9. Consistent with this conclusion, protein-directed MS-ABPP experiments comparing the enrichment profiles of alkyne stereoprobes ZL-13A / B / / 14A / B revealed stereoselective enrichment of NEK9 with (S)-(spiro) pyrrolidine acrylamide ZL-14A (Figure 3C). A similar stereoselective engagement profile for ZL-14A by gel-ABPP performed with recombinantly expressed wild type (WT)-NEK9 protein in HEK293T cells was observed, while a C623A- NEK9 mutant showed greatly attenuated reactivity with ZL-14A (Figure 3D). The ZL-14A- WT-NEK9 interaction was stereoselectively blocked by pre-treatment with ZL-10A (Figure 3E), matching the profile of engagement observed for endogenous NEK9_C623 in cysteine- directed ABPP experiments (Figure 2B, 3B).

[0038] RAB30 – a small-molecular-weight guanosine triphosphatase (GTPase) implicated in anti-microbial autophagy41– was stereoselectively liganded at C168 by (spiro)azetidine acrylamide ZL-8B, but not other tested spirocycle acrylamides including additional (spiro)azetidine acrylamides ZL-7B and ZL-9B (Figure 2A). C168 is conserved across vertebrate RAB orthologs (Figure 9C), but not found in most other RAB proteins with the exception of RAB33A (Figure 9D). C168 is located on the opposite face of RAB30 from the GTP-binding site in proximity to a predicted small molecule-binding pocket (Figure 3F). A second quantified cysteine in RAB30 (C93) was not engaged by ZL-8B in our cysteine- directed ABPP experiments (Figure 3G), and, consistent with a site-specific interaction, itTSRI 2229.1PC was found that the corresponding alkyne stereoprobe ZL-13B reacted with recombinant WT- RAB30 to a much greater extent than a C168A-RAB30 mutant in HEK293T cells (Figure 3H). While ZL-13B did not react enantioselectively with recombinant WT-RAB30 (Figure 3H), this interaction was stereoselectively blocked by ZL-8B (Figure 3I), matching the profile of engagement observed for endogenous RAB30_C168 in cysteine-directed ABPP experiments (Figures 2b and 3G).

[0039] Our studies with recombinant NEK9 and RAB30 proteins supported the site- specific and stereoselective liganding assignments for spirocycle acrylamides derived from cysteine-directed ABPP experiments. Specific biochemical and cellular assays for determining the effects of spirocycle acrylamides on NEK9 and RAB30 activity, however, are limited, and alternative ways to functionally characterize spirocycle acrylamide-protein interactions in cells were therefore considered.

[0040] ZL-12A stereoselectively degrades ERCC3 by covalently modifying C342. In the course of performing protein-directed ABPP experiments with representative alkyne spirocycle acrylamides in Ramos cells (Figure 10A), it was noticed that the (S)- (spiro)pyrrolidine acrylamide-liganded protein ERCC3 displayed a peculiar profile where robust stereoselective enrichment by ZL-14A was observed at 1 h, but not 3 h post-treatment (Figure 4A). This loss in stereoselective enrichment coincided with lower relative ERCC3 signals in ZL-14A-treated cells at 1 vs 3 h (Figure 4A), a profile that contrasted with those of other stereoselectively engaged proteins, which uniformly showed preserved or greater enrichment at 3 vs 1 h (Figure 4B). The atypical time-dependent loss in stereoselective enrichment of ERCC3 was also observed in a second human cancer cell line (the prostate cancer line 22Rv1; Figure 4B).

[0041] One potential explanation for the time-dependent loss of ERCC3 enrichment would be if covalent reaction with (S)-(spiro)pyrrolidine acrylamides led to degradation of the protein. Consistent with this hypothesis, western blotting and MS-based proteomic experiments revealed concentration-dependent stereoselective decreases in ERCC3 (Figure 4C, D and Figure 10B), but not other proteins (Figure 4D), in cancer cells treated with ZL- 12A (50 µM, 3 h). IC50 values of 5.5 and 2.7 µM were calculated for ZL-12A-induced ERCC3 degradation in 22Rv1 cells at 3 and 12 h treatments, respectively (Figure 4C and Figure 10C), and this degradation effect was fully blocked by the Neddylation inhibitor MLN4924 and partly blocked by the proteasome inhibitors carfilzomib (CFZ) and MG132 as measured by western blotting (Figure 4E) or MS-based proteomics (Figure 10D, 4E). It wasTSRI 2229.1PC not fully understood why proteasome inhibitors failed to fully prevent ZL-12A-mediated degradation of ERCC3, but interpret the complete protection displayed by MLN4924 to support the involvement of a Cullin-RING ubiquitin ligase (CRL) system.

[0042] The SAR for spirocycle acrylamide-induced degradation of ERCC3 correlated with apparent engagement of C342 of ERCC3 in our cysteine-directed ABPP experiments (Figure 5A). Recognizing, however, that stereoprobe-induced degradation might technically confound the assignment of ligandable cysteines in ERCC3, the cysteine-directed ABPP experiments both in situ and in vitro and in the presence of MLN4924 were repeated. These experiments revealed robust stereoselective blockade of IA-DTB labeling of ERCC3_C342 by ZL-12A in all test conditions (in situ and in vitro + / - MLN4924) (Figure 5B, C). Further evidence for direct engagement of ERCC3 by (S)-(spiro)pyrrolidine acrylamides using cell models engineered to express WT or a C342T mutant of ERCC3 against a backdrop of CRISPR-Cas9-mediated genetic deletion of endogenous ERCC3 was obtained. These cell models revealed that WT-ERCC3, but not C342T-ERCC3 stereoselectively reacted with ZL- 14A (Figure 5D) and the ZL-14A-WT-ERCC3 reaction was blocked by pre-treatment with ZL-12A, but not ZL-12B (Figure 5E). Finally, it was found that ZL-12A promoted the stereoselective degradation of WT-ERCC3, but not C342T-ERCC3 (Figure 5F). These data collectively indicate that (S)-(spiro)pyrrolidine acrylamides such as ZL-12A stereoselectively and site-specifically engage C342 of ERCC3, leading to rapid ubiquitin-proteasome system- mediated degradation of the protein.

[0043] ERCC3 is a DNA helicase component of the general transcription and DNA repair factor IIH (TFIIH) complex, which is made up of ten subunits and plays key roles in transcription and nucleotide excision repair (NER) processes42. ERCC3 opens DNA promoters during transcriptional initiation via ATP-dependent DNA translocase activity43, 44and, during NER, anchors the TFIIH complex to chromatin45and facilitates DNA strand separation around the site of damage46, 47. Interestingly, C342 of ERCC3 has also been identified as a site of covalent modification by the diterpene triepoxide natural product triptolide, which inhibits the DNA-dependent ATPase activity of ERCC3, suppresses RNA polymerase II-mediated transcription48-50, and triggers degradation of associated RNA polymerase subunits (e.g., POLR2A)51. However, triptolide has not, to our knowledge, been found to lead to the direct degradation of ERCC3.

[0044] Curious about the different apparent functional effects of ZL-12A and triptolide on ERCC3, 22Rv1 cells to triptolide (1 µM, 1 h) followed by treatment with ZL-12A or ZL-TSRI 2229.1PC 12B (50 µM, 3 h) were first exposed. This experiment revealed that triptolide pre-treatment did not independently affect ERCC3 abundance, but completely prevented ZL-12A-mediated ERCC3 degradation (Figure 6A). Conversely, it was found that pre-treatment with ZL-12A, but not ZL-12B (50 µM, 3 h), substantially blocked triptolide (1 µM, 3 h)-dependent loss of POLR2A and other RNA polymerase II subunits as measured by MS-based proteomics (Figure 6B, Figure 11A, 5B, and Supplementary Dataset 1) or western blotting (Figure 6C). While the mechanistic basis for triptolide-mediated reductions in POLR2 proteins remains poorly understood, it was found that this effect was mitigated by co-treatment with MLN4924 (Figure 11C, 5D), pointing to a degradation process involving a CRL and the ubiquitin- proteasome system. The bidirectional antagonism displayed by ZL-12A and triptolide (Figure 6A-C) provides strong evidence that these compounds, despite binding the same cysteine residue (C342) in ERCC3, produce distinct functional outcomes, with ZL-12A leading to direct ERCC3 degradation and triptolide sparing ERCC3 loss in exchange for promoting collateral loss of associated RNA polymerases.

[0045] The degradation of ERCC3 mediated by ZL-12A resembled a pharmacological effect reported previously for the sterol derivative spironolactone in a drug repositioning screen for inhibitors of NER52. Spironolactone is an antihypertensive drug that acts as a mineralocorticoid receptor antagonist53, and the molecular mechanism by which it promotes ERCC3 degradation has remained elusive, but assumed to occur indirectly through binding another protein target54-56. While only limited SAR information is available for spironolactone-mediated ERCC3 degradation, it was noted that spironolactone analogs lacking an electrophilic thioacetyl group are inactive54. Based on our discovery that ZL-12A promotes ERCC3 degradation through covalent binding to C342, it was considered whether spironolactone might act similarly. Consistent with this hypothesis, spironolactone (10 µM, 3 h) blocked IA-DTB reactivity of ERCC3_C342 with high stoichiometry (Figure 6D) and good proteome-wide selectivity (Figure 6D and Supplementary Dataset 1) in cysteine- directed ABPP experiments. Additionally, it was found that spironolactone promoted the degradation of WT-ERCC3, but not a C342T-ERCC3 mutant (Figure 6E, Figure 12A, and Supplementary Dataset 1), and this degradation of WT-ERCC3 was blocked by MLN4924 (Figure 12B), CFZ (Figure 12B), and triptolide (Figure 12C). Finally, previous studies have found that the CRL FBXL18 is involved in spironolactone-mediated degradation of ERCC354, and these findings were confirmed and extended to show that CRISPR / Cas9- medited genetic disruption of FBXL18 also blocked ZL-12A-mediated ERCC3 degradationTSRI 2229.1PC (Figure 6F). On the other hand, the collateral degradation of POLR2 proteins caused by triptolide was unaffected by genetic disruption of FBXL18 (Figure 12D), suggesting the involvement of a distinct CRL system. These results, taken together, indicate that spironolactone, like ZL-12A, promotes ERCC3 degradation through direct covalent binding to C342, which, in the case of spironolactone, it is interpreted to involve a trans- thioacetylation event.

[0046] Previous studies have indicated that spironolactone is much less disruptive to cancer cell transcription than triptolide57, suggesting somewhat paradoxically that ERCC3 inhibition causes greater impairments to cancer cell biology than ERCC3 degradation. In agreement with this model, it was found that both ZL-12A and spironolactone, at concentrations producing near-complete degradation of ERCC3, only marginally impaired cancer cell growth compared to control compound ZL-12B (Figure 6G). Triptolide, on the other hand, completely blocked cancer cell growth (Figure 6G). It was observed sustained loss of ERCC3 in ZL-12A-treated cells across the 72 h time period measuring cell proliferation (Figure 6H), excluding the possibility that recovery of ERCC3 expression at later time points may have obscured a more substantial effect of ZL-12A on cancer cell growth. Interestingly, this time course analysis also revealed that, at later time points (12-72 h), other subunits of the TFIIH complex showed reductions in abundance (12-72 h; Figure 6H, right blots). While not yet fully understanding the mechanistic basis for the delayed loss of additional TFIIH complex proteins in ZL-12A-treated cells, it is possible that ERCC3 is required for efficient formation, but not maintenance of TFIIH complexes in the cells. It was finally speculated that the higher cytotoxicity of triptolide compared to ZL-12A or spironolactone could reflect the greater essentiality of POLR2 proteins compared to the TFIIH complex. Indeed, a review of the Cancer Dependency Map58 shows that most POLR2 proteins register as essential for cancer cell growth following either total (CRISPR / Cas9) or partial (RNAi) genetic disruption, while partial disruption of TFIIH factors does not appear to substantially impact cancer cell growth (Figure 13).

[0047] Herein described is a set of spirocycle acrylamide stereoprobes and their chemical proteomic analysis in human cancer cells, leading the discovery of novel covalent small molecule-protein interactions not observed with original DOS-inspired azetidine (Figure 2) and tryptoline acrylamides23, 26. The preferential engagement of such proteins by spirocycle acrylamides occurred against a backdrop of lower chemical (GSH; Supplementary Table 1; and ref.21) and proteomic (Figure 1C) reactivity for this class of electrophilic stereoprobes,TSRI 2229.1PC emphasizing the importance of the spirocycle core as a productive binding element supporting specific protein interactions. While having focused mainly on evaluating the stereo- and regio-chemical effects of spirocycle acrylamide interactions with the proteome, the synthetic routes for preparing these stereoprobes should allow for incorporation of a range of appendages to provide additional sources of structural diversity.

[0048] One of the key advantages of ABPP for ligand discovery is the breadth of proteins that can be simultaneously evaluated in native biological systems. Additional studies, however, are often needed to infer how such covalent small-molecule binding events impact protein function. A way to infer the functional effects of covalent ligands on protein complexes in human cells by integrating ABPP with proteome-wide size-exclusion chromatography23have recently been described. Herein, it is shown how global measurement of protein expression can offer a complementary method to deduce functional effects for covalent ligands. Time-dependent measurements of proteins enriched by spirocycle acrylamides flagged an atypical profile for ERCC3 that was found to reflect rapid and direct ligand-induced protein degradation in cancer cells. Determining the functional consequences of additional stereoselective and site-specific liganding events displayed by the spirocycle acrylamides is an important future goal. It is encouraging that, for a subset of cysteines engaged by the spirocycle acrylamides, other covalent ligands have been described in the patent literature that impact protein-protein interactions (PIKC3A_C342)36and allosterically modulate E3 ligase activity (CRBN_C287)59. If the spirocycle acrylamide ligands identified for proteins like NEK9 and RAB30 are also found to be functional, they would have the additional attribute of targeting paralog-restricted cysteines, which can be a source for improvements in selectivity for covalent ligands60-62.

[0049] Our finding that electrophilic compounds engaging the same cysteine on ERCC3 can produce either direct (ZL-12A, spironolactone) or collateral (triptolide) protein degradation underscores the diversity of functional effects that can be exhibited by chemical probes, even when engaging a common site. This phenomenon is well-appreciated in receptor pharmacology, where ligands binding the same pocket can act as agonists or antagonists63, and subtle structural changes have also been found to convert protein-protein interaction inhibitors into stabilizers64. While it is not yet fully understood how different covalent ligands engaging ERCC3_C342 promote direct versus collateral protein degradation, our data, building on previous work54, indicate the involvement of distinct CRL systems. Our findings further address a longstanding mystery surrounding the mechanism for spironolactone-TSRI 2229.1PC induced degradation of ERCC3. This effect had been assumed to involve the action of another spironolactone-binding protein possibly serving to bridge FBXL18 to ERCC354-56, but our ABPP and mutagenesis results indicate a much simpler mechanism wherein spironolactone directly binds to ERCC3 to promote its degradation. Considering previous studies have found that ERCC3 degradation requires the thioacetate group of spironolactone54, it is hypothesized that trans-thioacetylation of ERCC3_C342 may initiate degradation, although one cannot exclude the possibility that spironolactone binds reversibly to ERCC3 in a manner that is sensitive to mutation of C342.

[0050] Projecting forward, it is believed that the spirocycle acrylamide degraders of ERCC3 such as ZL-12A will offer valuable tool compounds for studying the biological functions of this protein and the TFIIH complex. So far, ZL-12A appears to share pharmacological effects with spironolactone in terms of promoting FBXL18-dependent ERCC3 degradation without causing substantial effects on cancer cell growth, a property that, for spironolactone, correlates with minimal effects on transcription in comparison to triptolide57. The much greater impact of triptolide on cancer cell transcription and proliferation is presumably caused by the collateral loss of RNA polymerases (Figure 6B and ref.46). Spironolactone has, on the other hand, been found to inhibit NER52and homology directed repair65, as well as enhance platinum agent cytotoxicity in cancer cells52, 66. Future studies can test whether spirocycle acrylamides have similar effects on DNA repair processes, while presumably lacking the mineralocorticoid receptor cross-reactivity of spironolactone. Also of interest is understanding how ZL-12A leads to the time-dependent, partial reduction in other components of the TFIIH complex, and whether this outcome reflects an instability in TFIIH complexes lacking ERCC3 or impairments in the formation of new complexes in the absence of this protein. These types of more in-depth cellular studies may benefit from further improvements in the potency of ZL-12A, which is a low-µM degrader of ERCC3 in cancer cells. More generally, our findings, along with other recent work20, 21, 23, 26, 27, highlight the utility of combining focused sets of stereochemically defined electrophilic compounds with chemical proteomics for the discovery of covalent ligands that impact protein function in diverse ways in human cells. 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Bull. 2022, 45, 669-674. 64. Ramharter, J.; Kessler, D.; Ettmayer, P.; Hofmann, M. H.; Gerstberger, T.; Gmachl, M.; Wunberg, T.; Kofink, C.; Sanderson, M.; Arnhof, H.; Bader, G.; Rumpel, K.; Zophel, A.; Schnitzer, R.; Bottcher, J.; O'Connell, J. C.; Mendes, R. L.; Richard, D.; Pototschnig, N.; Weiner, I.; Hela, W.; Hauer, K.; Haering, D.; Lamarre, L.; Wolkerstorfer, B.; Salamon, C.; Werni, P.; Munico-Martinez, S.; Meyer, R.; Kennedy, M. D.; Kraut, N.; McConnell, D. B., One Atom Makes All the Difference: Getting a Foot in the Door between SOS1 and KRAS. J. Med. Chem.2021, 64, 6569- 6580. 65. Shahar, O. D.; Kalousi, A.; Eini, L.; Fisher, B.; Weiss, A.; Darr, J.; Mazina, O.; Bramson, S.; Kupiec, M.; Eden, A.; Meshorer, E.; Mazin, A. V.; Brino, L.; Goldberg, M.; Soutoglou, E., A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair. 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[0051] Embodiment 1. A method of covalently binding to cysteine-342 in ERCC3 which leads to subsequent degradation of ERCC3 using the following spirocycle acrylamides of Formula I or II:wherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaromatic ring system; including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

[0052] Embodiment 2. The method of Embodiment 1, wherein R is 1H-indole.

[0053] Embodiment 3. The method of Embodiment 1, wherein R is quinoline.

[0054] Embodiment 4. The method of Embodiment 1, wherein R is substituted phenyl.

[0055] Embodiment 5. The method of Embodiment 4, wherein R is Ph-OMe.

[0056] Embodiment 6. The method of Embodiment 1, wherein R is (prop-2-yn-1- yloxy)benzene.

[0057] Embodiment 7. The method of Embodiment 1, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one;TSRI 2229.1PC (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one.

[0058] Embodiment 8. The method of Embodiment 1, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one.

[0059] Embodiment 9. The method of Embodiment 1, wherein the compound of Formula I is (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4]octan-6-yl)prop-2-en-1- one.

[0060] Embodiment 10. A compound of Formula I selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one;TSRI 2229.1PC (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one.

[0061] Embodiment 11. A compound of Formula II selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one. (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one.

[0062] Embodiment 12. A compound selected from the group consisting of: tert-Butyl (S)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate; and tert-Butyl (R)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate.

[0063] Embodiment 13. A composition comprising the compound of any one of Embodiments 10-12, admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

[0064] Embodiment 14. The composition of Embodiment 13, further comprising one or more therapeutic compounds or compositions.TSRI 2229.1PC

[0065] Embodiment 15. The composition of Embodiment 14, wherein the one or more therapeutic compounds or compositions is a second anti-cancer compound or composition.

[0066] Embodiment 16. A method of preventing, ameliorating, or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the compound of any one of Embodiments 10-12 or composition of any one of embodiments 13-15.

[0067] Embodiment 17. The method of Embodiment 16, further comprising combination with one or more therapeutic compounds or compositions.

[0068] Embodiment 18. The method of Embodiment 17, wherein the one or more therapeutic compounds or compositions is another anti-cancer compound or composition.

[0069] Embodiment 19. The method of Embodiment 180 wherein at least one of the one or more therapeutic compounds or compositions is another degrader of ERCC3.

[0070] Embodiment 20. An ERCC3 protein covalently bound to a compound, wherein the compound is covalently bound to cysteine residue 342 of the ERCC3 protein.

[0071] Embodiment 21. The ERCC3 protein of Embodiment 20, wherein the compound is covalently and stereoselectively bound to cysteine residue 342 of the ERCC3 protein.

[0072] Embodiment 22. The ERCC3 protein of Embodiment 20 or Embodiment 21, wherein the compound covalently bound to cysteine residue 342 of ERCC3 has the structure of the following spirocycle acrylamides of Formula I or II:wherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaromatic ring system; including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.TSRI 2229.1PC

[0073] Embodiment 23. The ERCC3 protein of Embodiment 22, wherein R is 1H-indole.

[0074] Embodiment 24. The ERCC3 protein of Embodiment 22, wherein R is quinoline.

[0075] Embodiment 25. The ERCC3 protein of Embodiment 22, wherein R is substituted phenyl.

[0076] Embodiment 26. The ERCC3 protein of Embodiment 22, wherein R is Ph-OMe.

[0077] Embodiment 27. The ERCC3 protein of Embodiment 22, wherein R is (prop-2- yn-1-yloxy)benzene.

[0078] Embodiment 28. The ERCC3 protein of Embodiment 22, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one.

[0079] Embodiment 29. The ERCC3 protein of Embodiment 22, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one;TSRI 2229.1PC (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one.

[0080] Embodiment 30. The ERCC3 protein of Embodiment 22, wherein the compound of Formula I is (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop- 2-en-1-one.

[0081] Embodiment 31. The ERCC3 protein of Embodiment 22, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one.

[0082] Embodiment 32. The ERCC3 protein of Embodiment 22, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one;TSRI 2229.1PC (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one.

[0083] Embodiment 33. The ERCC3 protein of Embodiment 22, wherein the compound is selected from the group consisting of: tert-Butyl (S)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate; and tert-Butyl (R)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate.

[0084] Embodiment 34. Any compound, composition, method, or ERCC3 protein as described herein. Definitions

[0085] 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.

[0086] 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.

[0087] 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 termTSRI 2229.1PC "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0088] 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".

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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. InTSRI 2229.1PC general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0096] 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 bound 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.

[0097] 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, 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.TSRI 2229.1PC

[0098] 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.

[0099] 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 unsubstituted alkyl or an aralkyl group. The term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group.

[0100] 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.

[0101] 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 chainTSRI 2229.1PC hydrocarbon residue containing 1 to 6 carbon atoms. "C1-12alkyl" 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.

[0102] 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.

[0103] When a range of values is listed, it is intended to encompass each value and sub– range within the range. For example, “C1–6 alkyl” 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.

[0104] “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 (“C1–15alkyl”). 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 carbon 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 (“C1–11alkyl”). 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 (“C1–9 alkyl”). 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 (“C1–6 alkyl”). 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 (“C1–3alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1–2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1alkyl”). In some embodiments, an alkyl group has 2 toTSRI 2229.1PC 6 carbon atoms (“C2–6alkyl”). Examples of C1–6alkyl 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.

[0105] “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 (“C2–10alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2–9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2–8 alkenyl”). 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 (“C2–6alkenyl”). 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 (“C2–3alkenyl”). 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 C2–4 alkenyl 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.

[0106] “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) (“C2–10alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2–9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2–8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2–7alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2–6alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2–5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2–4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2–3alkynyl”). 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 C2–4alkynyl groups include, without limitation, ethynyl (C2), 1–propynyl (C3),TSRI 2229.1PC 2–propynyl (C3), 1–butynyl (C4), 2–butynyl (C4), and the like. Examples of C2–6alkenyl groups include the aforementioned C2–4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.

[0107] 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.

[0108] 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.

[0109] 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. "C1-10 alkoxy" as used herein refers to an-O-alkyl wherein alkyl is C1-10.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.TSRI 2229.1PC

[0114] 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.

[0115] 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 and 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.

[0116] 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,TSRI 2229.1PC azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl and imidazolinyl.

[0117] “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.

[0118] 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.TSRI 2229.1PC

[0119] 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, 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.

[0120] “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 (“C10aryl”; e.g.,TSRI 2229.1PC 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.

[0121] “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 the 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).

[0122] 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 selectedTSRI 2229.1PC 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.

[0123] 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, 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.

[0124] “Saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.

[0125] 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 asTSRI 2229.1PC 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.

[0126] 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, 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, C1–10alkyl, C1–10perhaloalkyl, C2–10alkenyl, C2–10alkynyl, C3–14carbocyclyl, 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, C1–10 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 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,TSRI 2229.1PC 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(C1–6 alkyl), –NHC(=O)( C1–6 alkyl), –N(C1–6 alkyl)C(=O)( C1–6 alkyl), – 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(C1–6alkyl), –SO2N(C1–6alkyl)2, –SO2NH(C1–6alkyl), –SO2NH2,–SO2C1–6alkyl, - 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.

[0127] “Halo” or “halogen” refers to fluorine (fluoro, –F), chlorine (chloro, –Cl), bromine (bromo, –Br), or iodine (iodo, –I).

[0128] 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.

[0129] “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 with 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,TSRI 2229.1PC 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.

[0130] 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.

[0131] 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.

[0132] 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.TSRI 2229.1PC

[0133] 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.

[0134] 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 stereoisomers 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

[0135] Figure 1. Design and initial profiling of spirocycle acrylamide stereoprobes. (A), (B) Comparison of structures of azetidine21and spirocyclic acrylamide stereoprobes (A) where red and blue R1 designations refer to substituents on (spiro)azetidine and (spiro)pyrrolidine acrylamides, respectively (B). (C) Gel-ABPP data for Ramos cells treated with alkyne stereoprobes (20 or 50 µM, 1 or 3 h). Stereoprobe-reactive proteins wereTSRI 2229.1PC visualized by CuAAC conjugation to an azide-rhodamine reporter group, SDS-PAGE, and in gel fluorescence scanning. Red asterisks mark representative proteins that were stereoselectively engaged by spirocycle acrylamides (shown for 50 µM, 3 h conditions). Data are from a single experiment representative of three independent experiments.

[0136] Figure 2. Cysteine-directed ABPP of spirocycle acrylamide stereoprobes. (A) Heat map showing cysteines that were stereoselectively liganded in Ramos cells treated with spirocycle acrylamides (50 µM, 3 h). (B) Bar graphs showing cysteine-directed ABPP data for NEK9_C623, RAB30_C168, and ERCC3_C342, each of which was steresoelectively liganded by spirocycle acrylamides, but not azetidine acrylamides (20 µM, 3 h, Ramos cells). (C), (D) Cysteine-directed ABPP data for PIK3CA_C242 (C) and location of this cysteine in a model of a PIK3CA-KRAS complex generated by structural alignment of PIK3CA (PDB: 7PG5) with KRAS-PIK3CG complex (PDB: 1HE8). The RAS binding domain on PIK3CA is highlighted in cyan. a-c, data are average values from two independent cysteine-directed ABPP experiments each with two replicates. B, C average values ± SD.

[0137] Figure 3. Confirmation of spirocycle acrylamide interactions with representative proteins. (A) Domain map of NEK9 with C623 highlighted in red and human disease-relevant mutations in blue. (B) Dot plot showing stereoselective and site-specific engagement of NEK_C623 by ZL-10A. Data are from two independent cysteine-directed ABPP experiments in Ramos cells (50 µM, 3h) each with two technical replicates. (C) Bar graph showing the stereoselective enrichment of NEK9 by ZL-14A. Data are average values ± SD from a single experiment with two technical replicates representative of two independent experiments. (D) Gel-ABPP data demonstrating stereoselective engagement of WT-NEK9, but not the C623A- NEK9 mutant, by ZL-14A. HEK293T cells recombinantly expressing FLAG epitope-tagged WT-NEK9 or C623A-NEK9 mutant were treated with the indicated alkyne stereoprobes (10 µM, 1 h), after which gel-ABPP signals were measured by following our established protocol as described in Figure 1C. (E) Left, gel-ABPP showing the stereoselective blockade of ZL- 14A-WT-NEK9 interactions by pre-treatment with ZL-10A (50 µM, 3 h). Right, quantification of gel-ABPP data (average values ± SD for three independent experiments). (F) Left, x-ray structure of RAB30 (PDB: 2EW1). Right, location of RAB30_C168 highlighted in yellow and a small molecule-binding pocket in teal predicted from a computational binding-site search of a crystal structure of RAB30 (PDB: 2EW1). (G) Bar graph showing stereoselective and site-specific engagement of RAB30_C168 by ZL-8B. Data are from two independent cysteine-directed ABPP experiments in Ramos cells (50 µM, 3h)TSRI 2229.1PC each with two technical replicates. (H), (I) Gel-ABPP data demonstrating engagement of recombinant WT-RAB30, but not C168A-RAB30 by ZL-13B (10 µM, 1 h) (H) and stereoselective blockade of this engagement by pre-treatment with ZL-8B (50 µM, 3 h) (I, left). Quantification of gel-ABPP data (average values ± SD for three independent experiments). (I, right). For D, E, H, and I, data are from a single experiment representative of three independent experiments. IB = immunoblot, UT = untransfected cells.

[0138] Figure 4. (S)-(spiro)pyrrolidine acrylamides stereoselectively degrades ERCC3. (A) Protein-directed MS-ABPP data showing the time-dependent stereoprobe enrichment profile for ERCC3. The relative signal intensity for ERCC3 is presented across each treatment condition, and the decrease in enrichment of ERCC3 in ZL-14A-treated Ramos cells in the 50 µM, 1 h vs 50 µM, 3 h conditions is noted. (B) Scatter plot showing the enantioselective enrichment values of stereoselectively enriched proteins at 1 h (x-axis) versus the fold-change in enrichment for these proteins with the preferred stereoprobe at 1 vs 3 h treatment (y-axis) for Ramos (left) or 22Rv1 cells (right) treated with spirocycle acrylamides ZL-13A / B / / 14A / B. Proteins stereoselectively enriched by ZL-14A or other spirocycle acrylamides (ZL-13A, ZL-13B, and ZL14B) are marked in blue and gray, respectively. Data are average values ± SD from a single protein-directed MS-ABPP experiment with two technical replicates. (C) Left, western blots showing concentration- dependent, stereoselective effects of ZL-12A on ERCC3 abundance in 22Rv1 cells (3 h). Right, quantification of ERCC3 signals from which an IC50value of 5.5 µM (95% confidence interval of 4.0-7.5 µM) was calculated for ZL-12A-induced decreases in ERCC3. (D) Volcano plot of MS-based proteomic data comparing protein abundance measurements of 22Rv1 cells treated with DMSO or ZL-12A (50 µM, 3 h). ERCC3 is highlighted in red. Data are from a single experiment representative of two independent experiments each with two technical replicates. (E) Left, western blots showing effects of proteasome (Carfilzomib) and Neddylation (MLN4924) inhibitors on ZL-12A-dependent degradation of ERCC3.22Rv1 cells were treated with ZL-12B or ZL-12A (10 µM) either with or without Carfilzomib (2 µM, 1 h pre-treatment) or MLN4924 (10 µM) for 3 h. Right, quantification of relative ERCC3 abundance in the indicated treatment groups. Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing ZL-12A- and ZL-12A / CFZ-treated cells. *P < 0.05, **P < 0.01. For graphs in C and E, data are average values ± SD for three independent experiments.TSRI 2229.1PC

[0139] Figure 5. ZL-12A degrades ERCC3 by covalent modification of C342. (A) Left, Western blots showing effects of representative spirocycle acrylamides on ERCC3 degradation in 22Rv1 cells (10 µM, 3 h). Middle, quantification of relative ERCC3 abundance in the indicated treatment groups. Right, bar graph showing ERCC3_C342 signals for 22Rv1 cells treated with the indicated spirocycle acrylamides in cysteine-directed ABPP experiments. (B), (C) Cysteine-directed ABPP data for ERCC3_C342 from 22Rv1 cells (in situ, B) or 22Rv1 cell lysates (in vitro, C) treated with ZL-12A or ZL-12B (50 µM, 3 h), or DMSO, with or without co-treatment with MLN4924 (10 µM). Data are average values ± SD for two independent experiments. (D) Gel-ABPP data demonstrating stereoselective engagement of WT-ERCC3, but not the C342T-ERCC3 mutant by ZL-14A.22Rv1 cells with CRISPR / Cas9-disrupted endogenous ERCC3 (∆ERCC3) and stable expression of recombinant WT-ERCC3-HA or C342T-ERCC3-HA were treated with ZL-14A or ZL-14B (20 µM, 1 h) in the presence of MLN4924 (10 µM), followed by immunoprecipitation with anti-HA magnetic beads and analysis by gel-ABPP. Rh, rhodamine. (E) Gel-ABPP data demonstrating blockade of ZL-14A-WT-ERCC3 interactions (20 µM, 1 h) by pre-treatment with ZL-12A, but not ZL-12B (ZL-12A or ZL-12B, 20 µM, 3 h, followed by ZL-14A (10 µM, 1 h)). All experiments were performed in the presence of MLN4924 (10 µM). (F) Left, western blots comparing effects of ZL-12A and ZL-12B (50 µM, 3 h) on abundance of WT- versus C342T-ERCC3 in 22Rv1 cells. Right, quantification of relative ERCC3 abundance in the indicated treatment groups. For graphs in A and F, data are average values ± SD from three independent experiments except for cysteine-directed ABPP data, which are average values ± SD for two independent experiments each with two replicates. For D and E, data are from a single experiment with two technical replicates representative of two independent experiments.

[0140] Figure 6. Distinct functional effects of covalent ligands engaging ERCC3_C342. (A) Left, western blots showing the blockade of ZL-12A-induced ERCC3 degradation by triptolide.22Rv1 cells were pre-treated with triptolide (1 µM, 1 h) followed by washing with PBS and treatment with ZL-12A or ZL-12B (50 µM, 3 h). Right, structure of triptolide. (B) Quantification of representative RNA polymerase II proteins by MS-based proteomics from 22Rv1 cells treated with triptolide (1 µM, 3 h) with or without pre-treatment with ZL-12A or ZL-12B (50 µM, 3 h). Data from average values ± SD for two-three independent experiments. (C) Western blots showing blockade of triptolide (1 µM, 3 h)-induced RNA polymerase II degradation by pre-treatment with ZL-12A, but not ZL-12B (50 µM, 3 h). (D)TSRI 2229.1PC Left, quantification of engagement of ERCC3_C342 by spironolactone (SP) or triptolide in 22Rv1 cells as measured by cysteine-directed ABPP. Right, waterfall plot showing the global cysteine reactivity profile for SP-treated cells. Cysteine-directed ABPP experiments were performed with 22Rv1 cells treated with SP (10 µM) or triptolide (1 µM) either with or without co-treatment with MLN4924 (10 µM) for 3 h. Data are average values ± SD for two independent experiments. (E) Western blots comparing effects of SP (10 µM, 3 h) and ZL- 12A or ZL-12B (50 µM, 3 h) on abundance of WT- versus C342T-ERCC3 in 22Rv1 cells. (F) Western blots showing blockade of ZL-12A (50 µM, 3 h) or SP (10 µM, 3 h) -dependent degradation of ERCC3 in 22Rv1 cells genetically disrupted for FBXL18 (sgFBXL18), but not in control cells (sgControl). (G) Left, line chart showing the relative cell proliferation at 12, 24, 48, and 72 h for 22Rv1 cells treated with ZL-12A (20 µM), ZL-12B (20 µM), SP (10 µM), or triptolide (50 nM) as determined by CellTiter-Glo. Right, quantification of relative cell proliferation at 72 h with the indicated compounds. Data are average values ± SD, n = 6. (H) Western blots showing time-dependent changes in the abundance of TFIIH complex proteins (ERCC3, GTF2H1, CDK7) and POLR2A in 22Rv1 cells treated with ZL-12A or ZL-12B (20 µM). For A, C, E, F, and H, data are from a single experiment representative of three independent experiments.

[0141] Figure 7. Cysteine-directed ABPP of spirocycle acrylamide stereoprobes. (A) Workflow for cysteine-directed ABPP experiments where stereoprobe reactivity with cysteines is determined using the broadly cysteine-reactive IA-DTB probe and multiplexed (tandem mass tagging, TMT10plex) MS-based proteomics, as described previously1. (B)-(D), Heat map showing stereoprobe-liganded cysteines for three different sets of spirocycle acrylamides (ZL-7A / B / / 10A / B (B), ZL-8A / B / / 11A / B (C), and ZL-9A / B / / 12A / B (D); 50 µM, 3 h). Data are from two independent cysteine-directed ABPP experiments each with two technical replicates.

[0142] Figure 8. Cysteine-directed ABPP data for spirocycle acrylamide-liganded proteins. (A) Bar graph showing cysteines (CRBN_C287 and GSTO1_C32) stereoselectively liganded by spirocycle acrylamides (50 µM, 3 h) in Ramos cells. Also shown are data for these cysteines from Ramos cells treated with azetidine acrylamides MY-1A / B / / 3A / B (20 µM, 3 h). (B), Structure of a representative compound from a recent patent2describing covalent inhibitors of PIKC3A-KRAS that appear to act through engaging PIKC3A_C242.

[0143] Figure 9. Evolutionary conservation of NEK9_C623 and RAB30_C168. (A), (B) Sequence alignment of NEK9 with orthologs from mouse, rat, dog, pig, zebrafish, andTSRI 2229.1PC chicken (A) and NEK family paralogs in humans (B). C623 is highlighted in blue and amino acid substitutions at this site in green. (C), (D) Sequence alignment of RAB30 with orthologs from mouse, rat, dog, pig, zebrafish, and chicken (C) and RAB family paralogs in humans (D). C168 is highlighted in blue and amino acid substitutions at this site in green.

[0144] Figure 10. (S)-(spiro)pyrrolidine acrylamide ZL-12A stereoselectively degrades ERCC3. (A) Workflow for protein-directed ABPP experiments where the stereoselective enrichment of proteins by alkyne stereoprobes is determined by multiplexed (tandem mass tagging, TMT16plex) MS-based proteomics3,4. (B) Left, western blots showing concentration-dependent, stereoselective effects of ZL-12A on ERCC3 abundance in Ramos (3 h). Right, quantification of ERCC3 signals from which an IC50value of 8.0 µM (95% confidence interval of 5.4-11.9 µM) was calculated for ZL-12A-induced decreases in ERCC3. Data are average values ± SD from three independent experiments. (C) Left, western blots showing concentration-dependent, stereoselective effects of ZL-12A on ERCC3 abundance in 22Rv1 cells (12 h). Right, quantification of ERCC3 signals from which an IC50value of 2.7 µM (95% confidence interval of 2.5-3.0 µM) was calculated for ZL-12A-induced decreases in ERCC3. Data are average values ± SD from three independent experiments. (D), (E) Bar graphs showing quantification of ERCC3 abundance in the indicated groups as measured by MS-based proteomics.22Rv1 cells were treated with ZL-12A or ZL-12B (50 µM, 3 h) either with or without co-treatment with MLN4924 or MG132 (10 µM). Data are average values ± SD from two independent experiments.

[0145] Figure 11. Distinct functional effects of covalent ligands engaging ERCC3_C342. (A) Heat map showing triptolide (1 µM, 3 h)-induced degradation of RNA polymerase II proteins and blockade of this degradation by pre-treatment with ZL-12A, but not ZL-12B (50 µM, 3 h) as measured by MS-based proteomics.22Rv1 cells were pre-treated with ZL-12A or ZL-12B (50 µM, 3 h) followed by the treatment with triptolide (1 µM, 3 h) or DMSO. Data are average values form 2-3 independent experiments. (B) Volcano plot of MS-based proteomic data comparing protein abundance measurements of 22Rv1 cells treated with DMSO or triptolide (1 µM, 3 h). RNA polymerase II proteins POLR2A, POLR2B, POLR2C, POLR2D, and POLR2G are highlighted in red. Data are average values from two independent experiments. (C) Quantification of representative RNA polymerase II proteins by MS-based proteomics from 22Rv1 cells treated with triptolide (1 µM, 3 h) with or without co-treatment with MLN4924 (10 µM). Data from average values ± SD for two independent experiments. (D) Top, western blots showing effects of MLN4924 on triptolide- dependentTSRI 2229.1PC degradation of POLR2A.22Rv1 cells were treated with triptolide (1 µM) with or without MLN4924 (10 µM) for 3 h. Bottom, quantification of relative POLR2A abundance in the indicated treatment groups. Data are average values ± SD for three independent experiments.

[0146] Figure 12. Evidence that spironolactone degrades ERCC3 by covalent modification of C342. (A) Volcano plot of MS-based proteomic data comparing the protein abundance measurements of 22Rv1 cells treated with DMSO or spironolactone (SP) (10 µM, 3 h). ERCC3 is highlighted in red. Data are from a single experiment representative of two independent experiments each with two technical replicates. (B) Western blots showing effects of proteasome (Carfilzomib) and Neddylation (MLN4924) inhibitors on SP-dependent degradation of ERCC3.22Rv1 cells were treated with spironolactone (10 µM) either with or without Carfilzomib (2 µM, 1 h pre-treatment) or MLN4924 (10 µM) for 3 h. (C) Western blots showing the blockade of ZL-12A or SP-induced ERCC3 degradation by triptolide. 22Rv1 cells were pre-treated with triptolide (1 µM, 1 h) followed a PBS wash and treatment with ZL-12A (50 µM), ZL-12B, or SP (10 µM) for 3 h. (D) Western blots showing similar triptolide-dependent degradation of POLR2A in sgControl and sgFBXL1822Rv1 cells. Cells were treated with triptolide (1 µM) for 3 h. For B-D, Data are from a single experiment representative of three independent experiments.

[0147] Figure 13. Cancer Dependency Map profiles for TFIIH complex and POLR2 members. (A) to (H), Cancer cell line dependency profiles for indicated genes in the Cancer Dependency Map (https: / / depmap.org / portal / ). Representative compounds of the invention:TSRI 2229.1PCTSRI 2229.1PC EXAMPLES Abbreviations

[0148] 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), 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 prefixesTSRI 2229.1PC 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 Cell culture

[0149] All human cancer cell lines were from ATCC. Ramos and 22Rv1 cell lines were maintained in RPMI-1640 (Gibco) and HEK293T cell line was maintained in DMEM (Gibco) at 37˚C with 5% CO2. All media were supplemented with 10% FBS (Omega Scientific), 2 mM GlutaMax, 100 U / mL penicillin, and 10 µg / mL streptomycin. Reagents

[0150] Additional reagents, source and catalog numbers are found in Supplementary Table 2. Cloning and mutagenesis

[0151] NEK9 and RAB30 plasmids were obtained from OriGene and the mutagenesis was performed with Q5®Site-Directed Mutagenesis Kit (New England BioLabs, E0554S), using primers shown in Supplementary Table 2. Generation of sgERCC322Rv1 cells stably expressing WT-ERCC3-HA or C342T- ERCC3-HA

[0152] Lentiviral expression plasmid pLV416_WT-ERCC3-HA (codon optimized, Supplementary Table 2) was cloned using the gateway recombinase cloning strategy (Invitrogen, USA) from commercially synthesized gene blocks (IDT, USA). Lentiviral expression plasmid pLV416_C342T-ERCC3-HA was cloned by mutagenesis from pDONR221_WT-ERCC3-HA with Q5® Site-Directed Mutagenesis Kit followed by gateway recombinase cloning strategy.

[0153] Lentivirus was produced using FuGENE® 6 (Promega, E2691) transfection protocol. Lenti-X 293T cells (3 million, Takara, 632180) were seeded in 10 cm plate with 8 mL DMEM 24 h before transfection.33 µL FuGENE® 6 was dissolved in 572 µL serum-free DMEM and incubated for 5 min followed by the addition of Lentiviral expression plasmid (5 µg), pSPAX2 (5 µg), and pM2D.G (1 µg). The mixture was added gently to the plate after 15TSRI 2229.1PC min incubation at room temperature. The next day, replaced the media with 8 mL fresh pre- warmed complete DMEM and harvested viral supernatant after 48 h.

[0154] For transduction, 1 million 22Rv1 cells were mixed with 300 µL of freshly harvested virus in a total of 3 mL full RPMI supplemented with 8 µg / mL polybrene in a 6- well plate. Cells were spin-infected at 800 g and 30 ˚C for 1 h and incubated for 24 h at 37 ˚C. The selection was initiated with 800 µg / mL G418 (Gibco, 10131035) for 3 days then increased the concentration to 1 mg / mL for another 2 weeks, when the no-virus control cells were completely killed. Selected pools were characterized by Western blotting.

[0155] Stable knockout cell lines were generated by transduction of 22Rv1_WT-ERCC3- HA or 22Rv1_C342T-ERCC3-HA with lentiCRISPR v2 vector (sgRNAs cloned into the vector, Addgene #52961) using standard CRISPR / Cas9 and lentivirus protocols (Supplementary Table 3)3. Lentivirus was produced as described above. For transduction, 1 million 22Rv1_WT-ERCC3-HA cells or 22Rv1_C342T-ERCC3-HA cells were infected with 300 µL lentivirus in 3 mL RPMI supplemented with 8 µg / mL polybrene in a 6-well plate. Cells were spin-infected at 800 g and 30˚C for 1 h and incubated for 24 h at 37 ˚C. Selection was initiated with 2 µg / mL puromycin for 1 week. Selected pools were characterized by gDNA PCR. Generation of sgFBXL18 and sgControl 22Rv1 cells

[0156] Stable knockout cell lines were generated by transduction of 22Rv1 cells with lentiCRISPR v2-Blast and lentiGuide-puro virus (sgRNAs cloned into vectors, Adgene #83480 or Adgene #52963) using standard CRISPR / Cas9 and lentivirus protocols (Supplementary Table 2). Subsequent process was described above. Selection was initiated with 10 µg / mL blasticidin for 1 week followed by 10 µg / mL blasticidin and 2 µg / mL puromycin for one additional week. Selected pools were characterized by Western blotting. Gel-ABPP for proteome-wide reactivity

[0157] Ramos cells (5 mL of 3 million cells / mL) were treated with alkyne probes for 1 h and harvested. Cell pellets were resuspended in 250 µL of cold PBS and lysed by probe sonication (3×8 pulses).50 µL of 1 mg / mL of normalized whole protein samples were prepared after quantification with Pierce™ BCA Protein Assay Kit. Subsequent click reactions were performed ('click' mixture: 3 µL of 1.7 mM TBTA in t-BuOH / DMSO (4:1), 1 µL of 50 mM CuSO4 in H2O, 1 µL of 1.25 mM rhodamine-PEG-Azide in DMSO, 1 µL ofTSRI 2229.1PC freshly prepared 50 mM TCEP in PBS) for 1 h followed by the addition of 4X SDS gel loading buffer for SDS-PAGE and imaging by in-gel fluorescent scanning. Gel-ABPP with recombinant proteins

[0158] HEK293T cells (0.3 million) were seeded in a 6-well plate for 12 h before transfection with 1-2 µg plasmids (PEI:DNA = 3:1) for 48 h. Treatment was performed with alkyne probe only for 1 h or 3 h for competitor followed by the incubation with alkyne probe for 1 h. Subsequent process as described above. Gel-ABPP (IP)

[0159] 22Rv1 cells (10 million), 22Rv1 (WT-ERCC3-HA / ∆ERCC3) cells, or 22Rv1 (C342T-ERCC3-HA / ∆ERCC3) were seeded in a 10 cm plate for 24 h before treatment with ZL-14A or ZL-14B (20 µM) only for 1 h or 3 h for ZL-12A or ZL-12B (20 µM) followed by 1 h for ZL-14A or ZL-14B (20 µM) and harvested. Cell pellets were resuspended in NP-40 lysis buffer (25mM Tris-HCl pH 7.4, 150mM NaCl, 10% glycerol, 1% Nonidet P-40 and Roche cOmplete protease inhibitor cocktail) and lysed by probe sonication (2×8 pulses). Centrifugation (16,000 g, 15 min) was performed after rotating in the cold room for 10 min and the supernatant was collected for quantification with Pierce™ BCA Protein Assay Kit. 200 µL of 2 mg / mL normalized samples were subsequently incubated with anti-HA magnetic beads (30 µL slurry per sample) in the cold room for 2 h followed by washing with immunoprecipitation washing buffer (0.2% NP-40, 25 mM Tris-HCl pH 7.4 and 150 mM NaCl, 4 times). PBS (30 µL) was added to each sample followed by 3 µL 'click' mixture as describes above. The affinity gel was heated at 95 °C for 10 min in 2X SDS gel loading buffer. The supernatant was collected for SDS-PAGE and imaging by in-gel fluorescent scanning as described above. Cell lysis and Western blotting

[0160] Cell pellets were lysed with RIPA buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Sodium Deoxycholate, 0.1% SDS) supplemented with Pierce™ Universal Nuclease (Life Tech, 88701) and Protease / Phosphatase Inhibitor Cocktail (Cell Signaling Tech, 5872). All samples were normalized to 2 - 3 mg / mL as described above, run, and transferred for subsequent chemiluminescent detection by ChemiDoc MP imaging system (Bio-Rad). Antibodies used were FLAG (1:2,000, Sigma, A8592), RAB30 (1:500, ProteinTech, 16328-1-AP), ERCC3 (1:1,000, R&D Systems Inc, AF6349-SP), ERCC3TSRI 2229.1PC (1:1,000, Bethyl Laboratories, A301-337A), HA (1:2,000, Sigma, 12013819001), FBXL18 (1:250, Santa Cruz Biotechnology, sc-100738), GTF2H1 (1:300, Santa Cruz Biotechnology, sc-25329 HRP), CDK7 (1:1,000, Cell Signaling Tech, 2916S), POLR2A (1:1,000, Cell Signaling Tech, 2629S), and Actin (1:5,000, Santa Cruz Biotechnology, sc-47778 HRP). Secondary antibodies used were mouse anti-goat (1:5,000, Santa Cruz Biotechnology, sc- 47778 HRP), goat anti-mouse (1:5,000, Invitrogen, 32430), and goat anti-rabbit (1:5,000, Invitrogen, 31460). Cell proliferation assays

[0161] 22Rv1 cells were seeded in a 96-well plate (5,000 cells per well in 50 µL media) for 24 h before treatment. The next day, 50 µL of media containing compounds or DMSO were added to each well. A reference plate was assayed by CellTiter-Glo (Promega, G9242) reagent (30 µL) to determine the cell population density at time 0 (t0). Remaining plates were assayed at 12 h, 24 h, 48 h, or 72 h by CellTiter-Glo. Raw values were analyzed using GraphPad PRISM software version 9.0.0. Binding-site mapping of RAB30

[0162] Binding-site detection was performed on a crystallographic structure of RAB30 (PDB: 2EW1), following removal of the ligand GTP, using SiteMap on Schrödinger Maestro (version 13.2.128, MMshare Version 5.8.128, Release 2022-2, Platform Darwin-x86_64). The RAB30 GTP binding site scored similarly to a site, located on the opposite side of the protein, containing RAB30_C168: GTP site SiteScore 0.944, Dscore 0.759; C168 site SiteScore 0.820, Dscore 0.829. Glutathione (GSH) reactivity assay

[0163] This assay was performed as described previously4. Briefly, 100 µL GSH (50 µM in 0.1 M Tris (pH = 8.8) and 30% acetonitrile) was aliquoted in a clear 384-well plate (Greiner 781101).5 µL stereoprobes (10 mM) were employed 2 h or 6 h incubation at room temperature followed by the absorbance measurement at 440 nm. The concentration of GSH remaining was derived from a standard curve and observed rate (kobs / [I]) was calculated assuming pseudo first-order reaction kinetics from the following equations: d[GSH] / dt = −k∗[GSH], [GSH]t = [GSH]t0∗e−kt. Cysteine-directed ABPPTSRI 2229.1PC

[0164] Ramos cells (10 mL of 3 million / mL in 25 mL flask, seeded 30 min before treatment) or 22Rv1 cells (20 mL of 1 million / mL in 15 cm plate, seeded 24 h before treatment) were treated with DMSO or stereoprobes for 3 h and harvested. Cell pellets were lysed by probe sonication and normalized to 500 µL of 2 mg / mL as described above. Subsequent sample processes including IA-DTB labeling, alkylation, trypsin digestion, streptavidin enrichment, TMT labeling, and HPLC fractionation were conducted as reported previously1, 5.

[0165] For in vitro treatment, 22Rv1 cell pellets were lysed and normalized to 500 µL of 2 mg / mL as described above.5 µL of stereoprobes or DMSO (100X of final concentration) were added and incubated at room temperature for 3 h. Subsequent process was as described above. Protein-directed ABPP

[0166] Ramos cells (10 mL of 3 million / mL in 25 mL flask, seeded 30 min before treatment) or 22Rv1 cells (20 mL of 1 million / mL in 15 cm plate, seeded 24 h before treatment) were treated with alkyne stereoprobes for 1 h or 3 h and harvested. Cell pellets were lysed by probe sonication and normalized to 500 µL of 2 mg / mL as described above. Subsequent sample processing including conjugation to biotin-azide, streptavidin enrichment, trypsin digestion, TMT labeling, and HPLC fractionation was conducted as reported previously4. Whole proteome proteomics

[0167] 22Rv1 cells (20 mL of 1 million / mL in 15 cm plate, seeded 24 h before treatment) were treated with DMSO or stereoprobes for 3 h (another treatment was performed after PBS wash if needed) and harvested. Cell pellets were lysed by probe sonication in PBS supplemented with Roche cOmplete protease inhibitor cocktail and normalized to afford 200 µg proteome as described above. Subsequent sample processing including alkylation after reduction, trypsin digestion, TMT labeling, and HPLC fractionation was conducted as reported previously3, 6. TMT liquid chromatography-mass-spectrometry (LC-MS) analysis

[0168] All LC-MS analysis and data analysis were performed as described previously1, 4,5. Briefly, samples were run on an Oribtrap Fusion mass spectrometer (Thermo Scientific) coupled to an UltiMate 3000 Series Rapid Separation LC system and autosampler (ThermoTSRI 2229.1PC Scientific Dionex). Raw files were analyzed by Integrated Proteomics Pipeline (IP2, http: / / ip2.scripps.edu / ip2 / mainMenu.html). Data and Code availability

[0169] The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE7partner repository with the dataset identifier PXD046356. Synthetic Chemistry General information

[0170] Unless otherwise noted, all materials were used as received from commercial sources without further purification. Chemicals and solvents were purchased from Aldrich, Alfa Aesar, Oakwood, TCI, or Combi-Blocks. NMR spectra were recorded on a Bruker AV- 600 instrument. Spectra were internally referenced to SiMe4 or residual solvent signals. The following abbreviations (or combinations thereof) were used to explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, sx = sextet, m = multiplet, br = broad signal. High-resolution mass spectra (HRMS) for new compounds were recorded on an Agilent LC / MSD TOF mass spectrometer. Reverse-phase preparative liquid chromatography was performed on a Waters Autopurification LC with a Waters BEH C18 column (19 × 160 mm, 5 μm) using a 0.1% aqueous formic acid:acetonitrile gradient (30 mL / min, main segment of gradient at 65–95% acetonitrile over 8 min) at ambient temperature. Fractionation was triggered by a Waters QDa single quadrupole mass spec in ESI+mode (cone voltage 15V). Enantiomeric excess (ee) was determined on a Waters UPC2 system using commercially available chiral-phase columns. Exemplary procedures for the preparation of spirocycle stereoprobes

[0171] Spirocyclic core scaffold ZL-6A (Scheme 1) and its enantiomer ZL-6B were prepared by adapting previously described procedures8. The absolute stereochemical configuration of ZL-6A was confirmed by X-ray crystallography.TSRI 2229.1PC

[0172] Scheme 1. Synthesis of spirocycle acrylamide stereoprobes derived from ZL- 6A. Identical procedures were employed to access the corresponding enantiomers, starting with the enantiomeric substrate ZL-6B. Preparation of common intermediate 2a

[0173] To a suspension of ZL-6A (47.3 mg, 0.1 mmol) and K2CO3(27.6 mg, 2 equiv.) in DMF (0.5 mL) under inert atmosphere was added PhSH (12.2 µL, 1.2 equiv.). The resulting suspension was stirred for 30 minutes and purified by silica gel column chromatography directly (elution with Hexane, Hexane / EtOAc = 2:1, and DCM / MeOH = 10:1 with 1% Et3N subsequently) to give crude product 2a (22.5 mg, 78% yield) which was used in subsequent steps without further characterization. Preparation of common intermediate 4a-1

[0174] To a precooled (0 ˚C) solution of 2a (22.5 mg, 0.078 mmol) and DIPEA (34.0 µL, 2.5 equiv.) in DCM (1 mL) was added acryloyl chloride (12.6 µL, 2 equiv.) under inert atmosphere. The reaction mixture was allowed to warm to room temperature and stirred for another 3 h. The resulting mixture was then concentrated under vacuum and purified by silica gel column chromatography (Hexane / EtOAc = 1:1) to afford product 3a-1 (19.2 mg, 72% yield).

[0175] To a solution of 3a-1 (19.2 mg, 0.056 mmol) in DCM (1.5 mL) was added trifluoroacetic acid (0.5 mL) slowly at room temperature. The resulting mixture was stirred for 2 h and dried by blowdown evaporation with N2to afford the crude product 4a-1 (12.3 mg, 91%) which was used for the next step without further characterization. General procedure A (Scheme 1a)TSRI 2229.1PC

[0176] To a solution of 4a-1, RCO2H (2.0 equiv.), and HATU (2.0 equiv.) in DCM (0.2 M) was added DIPEA (2.5 equiv.). The resulting mixture was stirred for 12 h and subsequently concentrated under vacuum. The crude product was purified by silica gel column chromatography to afford final product, which was further purified by preparative HPLC to afford the final product for assays. General procedure B (Scheme 1b)

[0177] To a solution of 2a, RCO2H (2.0 equiv.), and HATU (2.0 equiv.) in DCM (0.2 M) was added DIPEA (2.5 equiv.). The resulting mixture was stirred for 12 h and concentrated under vacuum. The crude product was purified by silica gel column chromatography to afford product 3a-2 which was characterized by LC-MS.

[0178] To a solution of 3a-2 in DCM (0.1 M) was added trifluoroacetic acid (DCM / TFA = 3:1) slowly at room temperature. The resulting mixture was stirred for 2 h and dried by blowdown evaporation with N2 subsequently to afford the crude product 4a-2 which was used for the next step without further characterization.

[0179] To a precooled (0 ˚C) solution of 4a-2 and DIPEA (2.5 equiv.) in DCM (0.1 M) under inert atmosphere was added acryloyl chloride (2 equiv.). The reaction mixture was allowed to warm to room temperature and stirred for another 3 h. The resulting mixture was then concentrated under vacuum and purified by silica gel column chromatography to afford final product, which was further purified by preparative HPLC to afford the final product for assays. Compound characterization

[0180] Note: All peaks in both1H NMR and13C NMR broad and / or split due to the presence of rotamerstert-Butyl (S)-2-((2-nitrophenyl)sulfonyl)-8-phenyl-2,6-diazaspiro[3.4]octane-6- carboxylate (ZL-6A). ee > 99%.1H NMR (600 MHz, CDCl3) δ 7.90 – 7.82 (m, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.67 – 7.59 (m, 2H), 7.34 – 7.19 (m, 3H), 7.12 (br m, 2H), 4.01 (br m, 2H), 3.91 – 3.86 (m, 1H), 3.75 – 3.56 (m, 5H), 3.36 – 3.24 (m, 1H), 1.49 (br m, 9H).13C NMR (150 MHz, CDCl3) δ 154.22, 148.30, 137.91, 137.53, 133.76, 131.83, 131.40, 131.27, 130.59,TSRI 2229.1PC 128.89, 128.85, 127.84, 127.79, 127.67, 124.19, 80.12, 80.07, 61.76, 61.04, 55.84, 55.66, 54.78, 54.05, 50.52, 50.11, 49.45, 42.91, 42.21, 28.45. HRMS (ESI-TOF) calculated for C23H28N3O6S+[M+H]+474.1699, found 474.1691.nitrophenyl)sulfonyl)-8-phenyl-2,6-diazaspiro[3.4]octane- 6-carboxylate (ZL-6B). ee > 99%.1H NMR (600 MHz, CDCl3) δ 7.90 – 7.82 (m, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.67 – 7.59 (m, 2H), 7.38 – 7.19 (m, 3H), 7.12 (br m, 2H), 4.01 (br m, 2H), 3.92 – 3.86 (m, 1H), 3.75 – 3.56 (m, 5H), 3.37 – 3.25 (m, 1H), 1.49 (br m, 9H). HRMS (ESI-TOF) calculated for C23H28N3O6S+[M+H]+474.1699, found 474.1680.(S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-7A). Prepared from ZL-6A following Scheme 1a. ee > 99%.1H NMR (600 MHz, CDCl3) δ 7.61 – 7.55 (m, 2H), 7.44 – 7.21 (m, 4H), 7.10 (br s, 1H), 6.98 – 6.87 (m, 2H), 6.36 – 5.93 (m, 2H), 5.70 – 5.55 (m, 1H), 4.33 – 3.29 (m, 12H). HRMS (ESI-TOF) calculated for C23H25N2O3+[M+H]+377.1865, found 377.1868.(R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-7B). Prepared from ZL-6B following Scheme 1a. ee > 99%.1H NMR (600 MHz, CDCl3) δ 7.60 – 7.54 (m, 2H), 7.44 – 7.21 (m, 4H), 7.10 (br s, 1H), 6.98 – 6.87 (m, 2H), 6.36 – 5.93 (m, 2H), 5.71 – 5.55 (m, 1H), 4.33 – 3.25 (m, 12H).13C NMR (150 MHz, CDCl3) δ 169.75, 165.60, 161.35, 137.63, 137.40, 129.40, 129.37, 129.08, 129.06, 127.92, 127.85, 127.73, 127.54, 125.44, 113.69, 60.27, 58.68, 58.46, 57.96, 56.93, 55.37, 54.61, 54.16, 53.62,TSRI 2229.1PC 52.42, 51.62, 51.46, 50.42, 50.18, 49.32, 49.12, 44.05, 42.63. HRMS (ESI-TOF) calculated for C23H25N2O3+[M+H]+377.1865, found 377.1868.(S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-8A). ee > 99%. Prepared from ZL-6A following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 9.06 – 8.92 (m, 1H), 8.25 – 8.13 (m, 1H), 7.94 – 7.84 (m, 1H), 7.83 – 7.72 (m, 1H), 7.66 – 7.21 (m, 7H), 6.36 – 5.88 (m, 2H), 5.71 – 5.51 (m, 1H), 4.43 – 3.75 (m, 5H), 3.70 – 3.17 (m, 4H). HRMS (ESI-TOF) calculated for C25H24N3O2+[M+H]+398.1869, found 398.1872.(R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-8B). ee > 99%. Prepared from ZL-6B following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 9.07 – 8.91 (m, 1H), 8.26 – 8.13 (m, 1H), 7.94 – 7.83 (m, 1H), 7.83 – 7.72 (m, 1H), 7.66 – 7.21 (m, 7H), 6.36 – 5.87 (m, 2H), 5.71 – 5.51 (m, 1H), 4.43 – 3.75 (m, 5H), 3.71 – 3.17 (m, 4H).13C NMR (150 MHz, CDCl3) δ 169.21, 169.13, 169.10, 165.71, 165.67, 165.53, 151.03, 151.01, 144.35, 144.29, 138.04, 137.73, 136.74, 136.71, 136.63, 136.43, 136.38, 136.30, 136.23, 130.00, 129.72, 129.42, 129.37, 129.25, 129.23, 128.98, 128.95, 128.93, 128.91, 128.31, 128.29, 128.12, 127.91, 127.77, 127.74, 127.71, 127.68, 127.61, 127.40, 126.55, 126.52, 126.41, 125.59, 125.57, 125.46, 125.40, 121.85, 121.82, 121.77, 61.12, 60.67, 59.00, 57.85, 56.22, 55.89, 54.41, 53.87, 52.08, 51.82, 51.80, 51.32, 51.19, 51.14, 50.00, 49.86, 49.71, 49.61, 43.95, 43.85, 42.98. HRMS (ESI-TOF) calculated for C25H24N3O2+[M+H]+398.1869, found 398.1869.TSRI 2229.1PC(S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-9A). ee > 99%. Prepared from ZL-6A following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 8.80 (br m, 1H), 7.90 (br m, 1H), 7.48 – 7.21 (m, 7H), 7.11 (br s, 1H), 6.60 (s, 1H), 6.36 – 5.91 (m, 2H), 5.74 – 5.53 (m, 1H), 4.34 – 3.29 (m, 9H).13C NMR (150 MHz, CDCl3) δ 171.45, 165.62, 137.82, 137.55, 137.35, 136.84, 129.99, 129.71, 129.05, 129.02, 127.90, 127.80, 127.71, 127.57, 127.27, 127.24, 125.69, 125.46, 121.47, 121.45, 120.59, 111.10, 60.41, 58.72, 58.17, 56.96, 55.49, 54.64, 54.17, 53.84, 53.77, 52.44, 51.58, 51.43, 50.41, 50.16, 49.44, 49.23, 44.09, 43.99, 42.71. HRMS (ESI-TOF) calculated for C24H24N3O2+[M+H]+386.1869, found 386.1866.(R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one (ZL-9B). ee > 99%. Prepared from ZL-6B following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 8.49 (br m, 1H), 7.90 (br m, 1H), 7.49 – 7.20 (m, 7H), 7.11 (br s, 1H), 6.62 (s, 1H), 6.37 – 5.91 (m, 2H), 5.73 – 5.51 (m, 1H), 4.34 – 3.30 (m, 9H). HRMS (ESI-TOF) calculated for C24H23N3O2. HRMS (ESI-TOF) calculated for C24H24N3O2+[M+H]+386.1869, found 386.1871.(S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-10A). ee > 99%. Prepared from ZL-6A following Scheme 1b.1H NMR (600 MHz,TSRI 2229.1PC CDCl3) δ 7.50 (br m, 2H), 7.39 – 7.27 (m, 3H), 7.20 – 7.11 (m, 2H), 6.86 (br m, 2H), 6.50 – 6.42 (m, 2H), 5.80 – 5.72 (m, 1H), 4.37 – 3.64 (m, 11H), 3.46 (br m, 1H). HRMS (ESI-TOF) calculated for C23H25N2O3+[M+H]+377.1865, found 377.1865.(R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-10B). ee > 99%. Prepared from ZL-6B following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 7.51 (br m, 2H), 7.39 – 7.27 (m, 3H), 7.21 – 7.11 (m, 2H), 6.86 (br m, 2H), 6.50 – 6.42 (m, 2H), 5.80 – 5.72 (m, 1H), 4.37 – 3.67 (m, 11H), 3.45 (br m, 1H).13C NMR (150 MHz, CDCl3) δ 170.06, 164.64, 164.61, 161.90, 161.82, 137.54, 129.98, 129.77, 129.08, 129.00, 128.82, 128.71, 128.68, 127.90, 127.87, 127.79, 127.72, 127.64, 127.62, 124.95, 124.81, 113.61, 113.57, 64.25, 63.94, 59.05, 58.33, 57.80, 55.33, 55.11, 54.66, 52.85, 52.60, 51.21, 50.62, 49.73, 49.01, 44.67, 42.88. HRMS (ESI-TOF) calculated for C23H25N2O3+[M+H]+377.1865, found 377.1867.(S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-11A). ee > 99%. Prepared from ZL-6A following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 9.02 – 8.91 (m, 1H), 8.22 – 8.12 (m, 1H), 7.91 – 7.81 (m, 1H), 7.75 – 7.69 (m, 0.5H), 7.60 – 7.19 (m, 6.5H), 7.16 – 7.10 (m, 1H), 6.51 – 6.38 (m, 2H), 5.80 – 5.68 (m, 1H), 4.35 – 4.28 (m, 1H), 4.27 – 4.20 (m, 0.5H), 4.04 – 3.37 (m, 7.5H). HRMS (ESI-TOF) calculated for C25H24N3O2+[M+H]+398.1869, found 398.1871.TSRI 2229.1PC (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-11B). ee > 99%. Prepared from ZL-6B following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 9.02 – 8.91 (m, 1H), 8.22 – 8.12 (m, 1H), 7.92 – 7.81 (m, 1H), 7.75 – 7.69 (m, 0.5H), 7.60 – 7.19 (m, 6.5H), 7.16 – 7.10 (m, 1H), 6.51 – 6.38 (m, 2H), 5.80 – 5.68 (m, 1H), 4.35 – 4.28 (m, 1H), 4.27 – 4.20 (m, 0.5H), 4.04 – 3.37 (m, 7.5H).13C NMR (150 MHz, CDCl3) δ 170.04, 169.96, 164.61, 164.58, 151.07, 151.00, 150.98, 150.90, 144.62, 144.57, 144.52, 137.90, 137.88, 137.84, 137.50, 136.37, 136.30, 136.21, 136.17, 134.24, 134.16, 134.10, 134.05, 130.00, 129.86, 129.72, 129.70, 129.63, 129.08, 129.00, 128.92, 128.86, 128.84, 128.75, 128.71, 128.65, 128.63, 128.59, 128.23, 128.15, 128.11, 127.96, 127.91, 127.81, 127.76, 127.73, 127.70, 127.67, 127.63, 126.27, 126.24, 126.21, 126.17, 121.65, 121.61, 121.55, 61.83, 61.72, 58.84, 57.94, 55.38, 55.22, 55.06, 54.87, 54.51, 54.28, 52.46, 52.28, 51.12, 50.67, 50.62, 49.83, 49.56, 48.97, 48.78, 44.68, 44.56, 42.84, 42.78. HRMS (ESI-TOF) calculated for C25H24N3O2+[M+H]+398.1869, found 398.1870.(S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-12A). ee > 99%. Prepared from ZL-6A following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 8.93 (br s, 1H), 7.97 – 7.72 (m, 1H), 7.53 – 7.06 (m, 8H), 6.56 (br s, 1H), 6.51 – 6.42 (m, 2H), 5.81 – 5.71 (m, 1H), 4.39 – 4.07 (m, 3H), 4.05 – 3.72 (m, 5H), 3.44 (br m, 1H). HRMS (ESI-TOF) calculated for C24H24N3O2+[M+H]+386.1869, found 386.1870.(R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one (ZL-12B). ee > 99%. Prepared from ZL-6B following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 8.57 (br s, 1H), 7.96 – 7.72 (m, 1H), 7.53 – 7.08 (m, 8H), 6.58 (br s, 1H), 6.51 – 6.44 (m, 2H), 5.80 – 5.70 (m, 1H), 4.39 – 4.08 (m, 3H), 4.07 – 3.72 (m, 5H), 3.46 (br m, 1H).TSRI 2229.1PC13C NMR (150 MHz, CDCl3) δ 171.84, 164.68, 164.66, 137.68, 137.28, 137.24, 130.00, 129.72, 129.08, 129.00, 128.70, 128.67, 127.93, 127.86, 127.75, 127.70, 127.64, 127.22, 125.57, 125.49, 124.33, 124.19, 121.99, 121.95, 121.33, 121.29, 110.96, 110.93, 103.52, 103.50, 64.61, 64.33, 59.21, 58.52, 57.94, 55.58, 55.15, 54.73, 52.81, 52.57, 51.25, 50.72, 49.82, 49.04, 44.67, 42.87. HRMS (ESI-TOF) calculated for C24H24N3O2+[M+H]+386.1869, found 386.1869.(S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2- en-1-one (ZL-13A). ee > 99%. Prepared from ZL-6A following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 7.61 – 7.56 (m, 2H), 7.44 – 7.22 (m, 4H), 7.10 (br m, 1H), 7.06 – 6.95 (m, 2H), 6.35 – 5.92 (m, 2H), 5.71 – 5.55 (m, 1H), 4.73 (br m, 2H), 4.31 – 3.29 (m, 9H), 2.54 (br m, 1H). HRMS (ESI-TOF) calculated for C25H25N2O3+[M+H]+401.1865, found 401.1863.(R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2- en-1-one (ZL-13B). ee > 99%. Prepared from ZL-6B following Scheme 1a.1H NMR (600 MHz, CDCl3) δ 7.61 – 7.56 (m, 2H), 7.44 – 7.23 (m, 4H), 7.10 (br m, 1H), 7.06 – 6.96 (m, 2H), 6.35 – 5.93 (m, 2H), 5.72 – 5.54 (m, 1H), 4.73 (br m, 2H), 4.31 – 3.30 (m, 9H), 2.54 (br m, 1H).13C NMR (150 MHz, CDCl3) δ 169.59, 165.71, 165.69, 165.68, 165.62, 165.62, 165.56, 159.21, 129.32, 129.10, 128.84, 127.89, 127.78, 127.53, 125.43, 114.66, 77.94, 76.02, 60.24, 58.72, 58.41, 57.94, 55.82, 54.14, 51.67, 50.21, 49.14, 44.18, 44.07. HRMS (ESI-TOF) calculated for C25H25N2O3+[M+H]+401.1865, found 401.1861.TSRI 2229.1PC(S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2- en-1-one (ZL-14A). ee > 99%. Prepared from ZL-6A following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 7.64 – 7.41 (m, 2H), 7.40 – 7.28 (m, 3H), 7.21 – 7.12 (m, 2H), 6.95 (br m, 2H), 6.50 – 6.42 (m, 2H), 5.81 – 5.72 (m, 1H), 4.71 (s, 2H), 4.33 – 3.70 (m, 8H), 3.54 – 3.37 (m, 1H), 2.53 (s, 1H). HRMS (ESI-TOF) calculated for C25H25N2O3+[M+H]+401.1865, found 401.1857.(R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2- en-1-one (ZL-14B). ee > 99%. Prepared from ZL-6B following Scheme 1b.1H NMR (600 MHz, CDCl3) δ 7.64 – 7.41 (m, 2H), 7.40 – 7.28 (m, 3H), 7.21 – 7.12 (m, 2H), 6.95 (br m, 2H), 6.51 – 6.42 (m, 2H), 5.80 – 5.72 (m, 1H), 4.71 (s, 2H), 4.33 – 3.70 (m, 8H), 3.54 – 3.37 (m, 1H), 2.53 (s, 1H).13C NMR (150 MHz, CDCl3) δ 169.88, 164.66, 164.63, 159.77, 159.69, 137.63, 137.44, 129.75, 129.12, 129.04, 128.76, 128.74, 127.95, 127.87, 127.83, 127.75, 127.64, 125.88, 125.72, 114.52, 77.90, 77.86, 76.03, 76.00, 64.23, 59.11, 57.69, 55.79, 54.73, 54.63, 51.24, 50.63, 49.85, 49.70, 49.61, 49.03, 44.70, 42.91. HRMS (ESI- TOF) calculated for C25H25N2O3+[M+H]+401.1865, found 401.1857. References 1. Vinogradova, E. V.; Cravatt, B. F., Multiplexed proteomic profiling of cysteine reactivity and ligandability in human T cells. STAR Protoc.2021, 2, 100458. 2. Xu, R.; Wang, B.; Wallace, E.; Wehn, P.; Eeltran, P.; Stice, J.; Sinkevicius, K.; Yang, Y.; Bisignano, P.; Lightstone, F.; Simanshu, D.; Turner, D. M.; Czyzyk, D. J.; Singh, S.; Yerabolu, J. R. Compounds having a T-structure formed by at least four cycles forTSRI 2229.1PC use in the treatment of cancer and other indications. WO 2023 / 154282 A1.17 Aug. 2023. 3. Lazear, M. R.; Remsberg, J. R.; Jaeger, M. G.; Rothamel, K.; Her, H. L.; DeMeester, K. E.; Njomen, E.; Hogg, S. J.; Rahman, J.; Whitby, L. R.; Won, S. J.; Schafroth, M. A.; Ogasawara, D.; Yokoyama, M.; Lindsey, G. L.; Li, H.; Germain, J.; Barbas, S.; Vaughan, J.; Hanigan, T. W.; Vartabedian, V. F.; Reinhardt, C. J.; Dix, M. M.; Koo, S. J.; Heo, I.; Teijaro, J. R.; Simon, G. M.; Ghosh, B.; Abdel-Wahab, O.; Ahn, K.; Saghatelian, A.; Melillo, B.; Schreiber, S. L.; Yeo, G. W.; Cravatt, B. F., Proteomic discovery of chemical probes that perturb protein complexes in human cells. Mol. Cell 2023, 83, 1725-1742. 4. Njomen, E.; Hayward, R.; DeMeester, K.; Ogasawara, D.; Dix, M.; Nguyen, T.; Ashby, P.; Simon, G.; Schreiber, S.; Melillo, B.; Cravatt, B. F., comprehensive mapping of electrophilic small molecule protein interactions in human cells. ChemRxiv 2023. 5. Vinogradova, E. V.; Zhang, X.; Remillard, D.; Lazar, D. C.; Suciu, R. M.; Wang, Y.; Bianco, G.; Yamashita, Y.; Crowley, V. M.; Schafroth, M. A.; Yokoyama, M.; Konrad, D. B.; Lum, K. M.; Simon, G. M.; Kemper, E. K.; Lazear, M. R.; Yin, S.; Blewett, M. M.; Dix, M. M.; Nguyen, N.; Shokhirev, M. N.; Chin, E. N.; Lairson, L. L.; Melillo, B.; Schreiber, S. L.; Forli, S.; Teijaro, J. R.; Cravatt, B. F., An Activity- Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells. Cell 2020, 182, 1009-1026. 6. Tao, Y.; Remillard, D.; Vinogradova, E. V.; Yokoyama, M.; Banchenko, S.; Schwefel, D.; Melillo, B.; Schreiber, S. L.; Zhang, X.; Cravatt, B. F., Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1. J. Am. Chem. Soc.2022, 144, 18688-18699. 7. Perez-Riverol, Y.; Bai, J.; Bandla, C.; Garcia-Seisdedos, D.; Hewapathirana, S.; Kamatchinathan, S.; Kundu, D. J.; Prakash, A.; Frericks-Zipper, A.; Eisenacher, M.; Walzer, M.; Wang, S.; Brazma, A.; Vizcaino, J. A., The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022, 50, D543-D552.TSRI 2229.1PC 8. Hudson, L.; Mason, J. W.; Westphal, M. V.; Richter, M. J. R.; Thielman, J. R.; Hua, B. K.; Gerry, C. J.; Xia, G.; Osswald, H. L.; Knapp, J. M.; Tan, Z. Y.; Kokkonda, P.; Tresco, B. I. C.; Liu, S.; Reidenbach, A. G.; Lim, K. S.; Poirier, J.; Capece, J.; Bonazzi, S.; Gampe, C. M.; Smith, N. J.; Bradner, J. E.; Coley, C. W.; Clemons, P. A.; Melillo, B.; Hon, C. S.; Ottl, J.; Dumelin, C. E.; Schaefer, J. V.; Faust, A. M. E.; Berst, F.; Schreiber, S. L.; Zecri, F. J.; Briner, K., Diversity-oriented synthesis encoded by deoxyoligonucleotides. Nat. Commun.2023, 14, 4930.

[0181] 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 appended 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.

[0182] 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 2229.1PC WHAT IS CLAIMED IS:

1. A method of covalently binding to cysteine-342 in ERCC3 which leads to subsequent degradation of ERCC3 using the following spirocycle acrylamides of Formula I or II:wherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaromatic ring system; including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

2. The method of Claim 1, wherein R is 1H-indole.

3. The method of Claim 1, wherein R is quinoline.

4. The method of Claim 1, wherein R is substituted phenyl.

5. The method of Claim 4, wherein R is Ph-OMe.

6. The method of Claim 1, wherein R is (prop-2-yn-1-yloxy)benzene.

7. The method of Claim 1, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one; andTSRI 2229.1PC (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one.

8. The method of Claim 1, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one.9.The method of Claim 1, wherein the compound of Formula I is (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4]octan-6-yl)prop-2-en-1-one.

10. A compound of Formula I selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one.

11. A compound of Formula II selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one;TSRI 2229.1PC (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one. (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one.

12. A compound selected from the group consisting of: tert-Butyl (S)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate; and tert-Butyl (R)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate.

13. A composition comprising the compound of any one of Claims 10-12, admixed with a pharmaceutically acceptable carrier, diluent, or excipient.

14. The composition of Claim 13, further comprising one or more therapeutic compounds or compositions.

15. The composition of Claim 14, wherein the one or more therapeutic compounds or compositions is a second anti-cancer compound or composition.

16. A method of preventing, ameliorating, or treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the compound of any one of Claims 10-12 or composition of any one of claims 13-15.

17. The method of Claim 16, further comprising combination with one or more therapeutic compounds or compositions.

18. The method of Claim 17, wherein the one or more therapeutic compounds or compositions is another anti-cancer compound or composition.

19. The method of Claim 18, wherein at least one of the one or more therapeutic compounds or compositions is another degrader of ERCC3.

20. An ERCC3 protein covalently bound to a compound, wherein the compound is covalently bound to cysteine residue 342 of the ERCC3 protein.

21. The ERCC3 protein of Claim 20, wherein the compound is covalently and stereoselectively bound to cysteine residue 342 of the ERCC3 protein.

22. The ERCC3 protein of Claim 20 or Claim 21, wherein the compound covalently bound to cysteine residue 342 of ERCC3 has the structure of the following spirocycle acrylamides of Formula I or II:TSRI 2229.1PCwherein: each R is independently a substituted or unsubstituted phenyl or a fused bicyclic heteroaromatic ring system; including stereoisomers, racemic and scalemic mixtures, and pharmaceutically acceptable salts thereof.

23. The ERCC3 protein of Claim 22, wherein R is 1H-indole.

24. The ERCC3 protein of Claim 22, wherein R is quinoline.

25. The ERCC3 protein of Claim 22, wherein R is substituted phenyl.

26. The ERCC3 protein of Claim 22, wherein R is Ph-OMe.

27. The ERCC3 protein of Claim 22, wherein R is (prop-2-yn-1-yloxy)benzene.

28. The ERCC3 protein of Claim 22, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en- 1-one.TSRI 2229.1PC 29. The ERCC3 protein of Claim 22, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy)benzoyl)-2,6-diazaspiro[3.4]octan-2-yl)prop-2-en- 1-one.

30. The ERCC3 protein of Claim 22, wherein the compound of Formula I is (S)-1-(2-(1H- Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro[3.4]octan-6-yl)prop-2-en-1-one.

31. The ERCC3 protein of Claim 22, wherein the compound of Formula I is selected from the group consisting of: (S)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-2-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (R)-1-(2-(1H-Indole-6-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-6-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-2-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-6-yl) prop-2- en-1-one.

32. The ERCC3 protein of Claim 22, wherein the compound of Formula II is selected from the group consisting of: (S)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(6-(4-Methoxybenzoyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (R)-1-(8-Phenyl-6-(quinoline-8-carbonyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(6-(1H-Indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one;TSRI 2229.1PC (R)-1-(6-(1H-indole-5-carbonyl)-8-phenyl-2,6-diazaspiro [3.4] octan-2-yl) prop-2-en-1-one; (S)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one; and (R)-1-(8-Phenyl-6-(4-(prop-2-yn-1-yloxy) benzoyl)-2,6-diazaspiro [3.4] octan-2-yl) prop-2- en-1-one.

33. The ERCC3 protein of Claim 22, wherein the compound is selected from the group consisting of: tert-Butyl (S)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate; and tert-Butyl (R)-2-((2-nitrophenyl) sulfonyl)-8-phenyl-2,6-diazaspiro [3.4] octane-6- carboxylate.

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