Discovery and Optimization of a Covalent AKR1C3 Inhibitor.

We tested whether cycloalkyl substituents on the triazole leaving group of SuTEx compounds affects reactivity and selectivity across the proteome. Our rationale is based on previous studies showing sensitivity of the SuTEx electrophile to cycloalkyl modifications resulting in abolishment of reactivity or promotion of specificity for target proteins 38 . We synthesized 2 series of SuTEx probes for evaluation: (i) para - and (ii) meta -analogs of the broad-spectrum SuTEx probe HHS-475 39 bearing a cycloalkyl-1,2,4-triazole leaving group and a propargylamide or N -methylpropargylamide substitution ( Figure 1A ). Synthesis of substituted 1,2,4-triazoles and subsequent coupling to sulfonyl-chlorides to form the SuTEx probes was carried out as previously reported 40 and described in the Supporting Methods . To evaluate binding activity, we treated Colo205 cells with respective SuTEx probes (100 μM, 4 h, 37 °C) followed by cell lysis and isolation of soluble proteome, CuAAC conjugation of desthiobiotin-azide, trypsin digest, avidin chromatography and tandem liquid chromatography-mass spectrometry (LC-MS/MS) detection of probe-modified peptides (See Supporting Methods for quality control criteria used in our label-free LC-MS/MS chemoproteomic analyses). Cycloalkyl-triazole modifications to the HHS-475 scaffold improved chemoselectivity for tyrosine (Y) versus lysine (K) modification with comparable proteome-wide coverage as compared to HHS-475 (Y/K ratios >6 compared with ~3.8, respectively ( Figure 1B ). Modification of the 1,2,4-triazole with a cyclopropyl group or replacement of the propargylamide with a N -methylpropargylamide on the HHS-475 scaffold resulted in a comparable number of probe-modified sites and tyrosine chemoselectivity (RJG-2302 or RJG-2300 vs HHS-475, respectively, Figure 1B ). Moving the propargylamide position from the para to the meta position retained high chemoselectivity for tyrosine (Y/K ratio >10) without a concomitant loss in proteome-wide coverage (RJG-2260 vs HHS-475, Figure 1B ). Different combinations of modifications (cycloalkyl-triazole, N -methylpropargylamide, para -/ meta -substitution) resulted in varying effects on proteome-wide reactivity and chemoselectivity with no obvious synergy ( Figure 1B ). The HHS-475 analogs tested also modified distinct sites and inferred proteins compared with the parent probe in proteomes from treated cells ( Figure S1 ). Next, we asked whether probe-modified sites were enriched for particular proteins, which would help prioritize protein classes based on binding for further evaluation. We examined the collective set of probe-modified peptides by comparing peptide quality (Byonic score) as a function of relative abundance (spectral count, i.e. , peptide spectral matches or PSMs). Interestingly, a cluster of SuTEx probe-modified peptides belonging to AKR1C3 was identified, suggesting that either AKR1C3 is overexpressed in Colo205 cells and/or SuTEx electrophiles are potentially privileged for binding this family of proteins ( Figure 1C ). We further analyzed SuTEx-labeled peptides and discovered Y24- and Y55-containing peptides of AKR1C3 were highly enriched by several RJG probes, e.g. , RJG-2043, RJG-2045, RJG-2300, and RJG-2260 ( Figure 1D ). We also found that RJG-3014 and RJG-2302 were selective for Y55 in AKR1C1 and AKR1C2, respectively ( Figure 1D ). The enrichment of RJG probes for the AKR1Cs prompted further medicinal chemistry efforts to develop AKR1C-tailored probes and selective ligand(s) for functional studies. We selected RJG-2261 as a starting point for developing a AKR1C3-tailored probe because of this scaffold’s high tyrosine chemoselectivity and tempered proteome-wide binding activity ( Figure 1A , B and D ). The adduct group of RJG-2261 was modified with a piperazine-phenol moiety previously demonstrated to bind reductase enzymes (PTGR2 41 ) to provide a site for introducing an alkyne reporter tag through functionalization of the hydroxyl group (RJG-2121, Figure 2A ). Treatment of Colo205 cells with RJG-2121 resulted in prominent and specific labeling of a ~36 kDa protein band as measured by in-gel fluorescence detection of proteomes from treated cells subjected to CuAAC conjugation with rhodamine-azide ( Figure 2B ). Insensitivity of the RJG-2121-labeled protein band to competition with a covalent PTGR2 inhibitor RJG-2096 41 , which should block probe labeling and reduce fluorescent protein signal, suggested another potential enzyme target ( Figure 2B ). To identify the labeled protein target, soluble proteomes from RJG-2121-treated Colo205 cells were subject to label-free LC-MS/MS chemoproteomic analyses. The most enriched target based on spectral counting was Y24 in AKR1C3 (520 PSMs) followed by a handful of additionally modified proteins, which included PTGR1-Y265 (50 PSMs), BIP-Y635 (54 PSMs), CYTB-Y97 (111 PSMs), and GSTP1-Y8 (45 PSMs). RJG-2121 probe labeling of AKR1C3 was substantially enriched for Y24 compared with Y55 as well as other probe-modified sites detected (>500 vs <75 spectral counts for Y24 compared with Y55 and other bound sites, respectively; Figure 2C ). We detected low level labeling of other AKR1C members with RJG-2121 treatments except for moderate binding to AKR1C1 Y24 site although the magnitude was >10-fold less than AKR1C3 Y24 modification ( Figure 2D ). Evaluation of MS2 spectra showed confident identification of major y- and b-ions as well as fragment ions belonging to tyrosine covalently bound to the desthiobiotin-clicked RJG-2121 probe ( Figure 2E ). Collectively, these data support RJG-2121 as a tailored covalent probe enriched for binding a non-catalytic tyrosine (Y24) on AKR1C3 and minimal binding activity towards other AKR proteins detected ( Figure 2D ). Moreover, the proximity of Y24 to catalytic tyrosine Y55, its location in the active site of AKR1C3, and the fact that Y24 is not conserved amongst AKR family members supports targeting Y24 as a promising strategy to selectively inhibit AKR1Cs. Inspired by the ability to target a non-catalytic tyrosine 24 (Y24) in cells, we synthesized a series of RJG-2121-based compounds for developing a covalent SuTEx inhibitor of AKR1C3. To identify covalent AKR1C3 inhibitors, we screened compounds in live cells and evaluated the following parameters: (i) covalency via a RJG-2121-mediated competitive activity-based protein profiling (ABPP) target engagement assay (gel-based readout, Figure S2 - 6 ), and (ii) biochemical activity via turnover of coumberone to coumberol through AKR1C-mediated reduction. 42 - 43 We designed a series of compounds expecting the likelihood of reactivity between the SuTEx electrophile and nucleophilic tyrosine would be enhanced by installing an electron-withdrawing group (EWG) at the 4-position relative to the sulfonyl. While the 3-cylopropyl-1,2,4-triazole leaving group in RJG-2121 likely assisted in selectivity for AKR1C3, we began our structure-activity relationship (SAR) studies with an unsubstituted 1,2,4-triazole leaving group to increase reactivity given results from proteome-wide profiling with a previously published PTGR2 inhibitor HHS-0703 40 ( Table 1 ). We synthesized compound 1 and observed poor inhibition of probe labeling and modest inhibition of biochemical activity. Moving the methoxy substitution from the 2- to the 3- or 4-positions as in compounds 2 and 3 , respectively, improved inhibition of probe labeling and biochemical activity. Similarly, the large, inductively withdrawing 4-iodo ( 4 ) substitution improved inhibition of probe labeling and biochemical activity, whereas the stronger electron-withdrawing 2-pyridyl ( 5 ) substitution was less effective. Next, we explored how substitutions on the 1,2,4-triazole affected inhibitory activity. Installing a 3-phenyl-1,2,4-triazole leaving group generated compound 6 and improved inhibition of probe labeling compared to 1 while maintaining inhibition of biochemical activity. Increasing steric bulk with a 3-[1,1'-biphenyl]-4-yl-1,2,4-triazole leaving group ( 7 ) reduced competition with RJG-2121 and inhibition of biochemical activity. Less bulky substitutions such as 4-bromo ( 8 ) and 4-methoxyphenyl ( 9 ) were potent inhibitors of probe labeling and biochemical activity. We synthesized 2- ( 10 ), 3- ( 11 ), and 4-fluorophenyl ( 12 ) analogs to assess effects of inductive withdrawing fluorine substitution and observed improved inhibition of RJG-2121 labeling and biochemical activity with 11 and 12 . We installed inductively withdrawing 4-trifluoromethyl ( 13 ) and 4-trifluoromethoxy ( 14 ) substituents and, as expected given results with compound 12 , we observed enhanced inhibition of probe labeling with 13 compared to 14 , and comparable inhibition of biochemical activity, thus hinting that increased hydrophobicity on triazole substitutions could improve inhibition of AKR1C3. We synthesized heterocycle-substituted 1,2,4-triazoles with hydrophilic and electron-withdrawing character such as 2-furanyl ( 15 ), 3-pyridyl ( 16 ), and 4-pyridyl ( 17 ). We observed improved inhibition of probe labeling compared to 1 but only modest biochemical inhibition (>4 μM). We synthesized compounds 18 – 21 with 3-cycloalkyl-substituted 1,2,4-triazoles leaving groups spanning 3-6 membered rings, respectively, and observed a trend in strong inhibition of probe labeling and biochemical activity as the cycloalkyl ring increased in size. Next, we compared meta -substituted analogs of 1 with different 1,2,4-triazole leaving groups as well as another series with a 2° p -amide ( Table 2 ). Compounds 22 - 25 , the meta -substituted analogs of compounds 1 , 18 , 21 , and 16 , respectively, were weak inhibitors of RJG-2121 probe labeling and AKR biochemical activity, a surprising result given their structural similarity to RJG-2121. Similarly, anilino substitutions on the amide in compounds 26 - 28 resulted in reduced blockade of probe labeling and biochemical activity. Finally, we synthesized 29 to test effects of a piperidine and secondary anilino substitution but only observed modest inhibition. Indomethacin and PTUPB have been described as inhibitors of AKR1C3 in the literature; we tested these compounds in the biochemical assay and observed weak inhibition of AKRs ( Table 1 ). We attribute the discrepancy between our findings here and published results 24 , 35 using indomethacin and PTUPB to differences from compound activity in vitro versus in cells. SAR evaluation identified compounds 18 - 21 ( RJG-2048-2051) as the most promising lead AKR1C3 inhibitors based on potency observed in the biochemical assay and the degree of covalent target engagement by gel-based competitive ABPP ( Table 1 ). To prioritize further, we evaluated potency of these compounds as well as a structurally analogous negative control compound 22 (RJG-2036) for covalent binding to AKR1C3 using the RJG-2121-based competitive ABPP assay ( Figure 3A , B and S7A ). As expected, 22 was a poor inhibitor of AKR probe labeling, whereas potency of compounds 18-21 improved as the cycloalkyl ring size increased from cyclopropyl to cyclohexyl ( Figure 3B , S7B and C ). From these studies, compound 21 (RJG-2051) was determined to be the most potent inhibitor (IC 50 = 35 nM, Figure 3B and S7C ). Since RJG-2051 displayed optimal potency in biochemical and chemoproteomic assays, we proceeded with time-dependence evaluation to further support covalent mechanism of this inhibitor. AKR1C1-, 2-, 3-, or 4-expressing HEK293T cells were treated with vehicle, RJG-2036 ( 22 ), or RJG-2051 ( 21 ) for 5 – 120 minutes followed by gel-based competitive ABPP analysis. AKR1C1 and 2 were efficiently labeled by RJG-2121, but no inhibition of probe labeling was observed ( Figure 3C ). Recombinant AKR1C4 probe labeling was weak, which made it difficult to assess ligand competition. In contrast, recombinant AKR1C3 was readily detected by RJG-2121 and, moreover, RJG-2051 pretreatment clearly blocked probe labeling in a time-dependent manner ( Figure 3C , D ). Next, RJG-2051 ( 21 ) but not the negative control compound 22 (RJG-2036) was confirmed to be a significantly more potent inhibitor of AKR biochemical activity compared with the existing AKR1C3 inhibitors PTUPB and indomethacin in live cells ( Figure 3E ). Finally, we performed a live cell coumberone reduction assay in HEK293T cells overexpressing AKR1C1, 2, 3, or 4 to directly compare selectivity of RJG-2051 ( 21 ) against related AKR protein members. We observed potent (IC 50 = 13 nM) inhibition of AKR1C3 with clear selectivity against AKR1C1 (2350-fold), AKR1C2 (1790-fold), and AKR1C4 (429-fold; Figure 3F , G ). Collectively, we provide data in support of RJG-2051 as a potent and AKR1C-selective covalent inhibitor of AKR1C3. Next, we utilized our reported tandem mass tag (TMT) chemical proteomics workflow, TMT-SuTEx 41 , and a broadly reactive SuTEx probe RJG-2043 to determine whether the lead AKR1C3 inhibitors exhibit proteome-wide selectivity. We used gel-based screening conditions resulting in near maximal blockade of probe labeling and biochemical activity for TMT-SuTEx evaluation of AKR1C3 inhibitors (RJG-2048-2051, 10 μM, 2 h). TMT-SuTEx is a quantitative LC-MS/MS chemoproteomic method for assessing ligandability of tyrosine and lysine sites 41 . An effective liganding event at a protein site will block probe labeling and reduce detection signals of the respective probe-modified peptide signals that can be quantified and multiplexed across different ligand treatment conditions. For these studies, cells were lysed after compound pretreatment, proteomes labeled with RJG-2043 followed by CuAAC conjugation of desthiobiotin-azide, trypsin digestion, avidin chromatography enrichment of probe modified peptides, TMT labeling and LC-MS/MS quantification (see Supporting Methods for additional details). Liganded sites were identified from probe-modified peptides exhibiting ≥75% blockade of probe labeling in SuTEx compound- compared with DMSO vehicle-treated cells (competition ratio or CR ≤ 0.25, Figure 4A , B ). Using these criteria, the lead AKR1C3 inhibitors significantly liganded AKR1C3 Y24 ( Figure 4B ). We did not observe inhibitory activity of AKR1C3 inhibitors against probe-modified sites on additional AKR proteins quantified (CR of ~1 for AKR1B10 (Y49), AKR1C1 (Y24 or Y196), AKR1C3 (Y272); Figure 4B ). While the AKR1C3 inhibitors showed comparable activity against AKR proteins, RJG-2051 exhibited the highest degree of proteome-wide selectivity. Across >1800 probe-modified sites quantified, AKR1C3 Y24 was the most significantly liganded site with reduced binding activity against only a handful of other proteins detected in proteomes from RJG-2051-treated cells ( Figure 4A ). In summary, TMT-SuTEx identified RJG-2051 as a targeted covalent inhibitor of AKR1C3 that engages a non-catalytic tyrosine site (Y24) with proteome-wide selectivity in cells.

Sulfonyl-azoles (SufAz) are electrophilic compounds that enable development of covalent ligands targeting tyrosine or lysine-containing protein pockets 39 , 41 . The tunable nature of this electrophile is best exemplified by sulfonyl-triazoles (SuTEx) that can be synthesized as either global activity-based probes or fragment compounds for ligandability assessment 39 . Adjustments to proteome-wide reactivity and selectivity can be accomplished through sulfone and/or azole leaving group modifications that produce varying impacts on electrophile activity 39 , 41 , 44 . Whether SuTEx or this electrophilic class in general can be optimized from fragment ligand binders to targeted covalent inhibitors remains largely underexplored. Here, we disclose the development of a potent, selective and cell-active AKR1C3 inhibitor that targets a non-catalytic tyrosine in the active site using SuTEx chemistry. The current study identified cycloalkyl triazole modifications as a means to achieve higher tyrosine preference and while tempering proteome reactivity of SuTEx probes ( Figure 1 ). We leveraged these features to develop a highly tailored covalent probe for AKR1C3 (RJG-2121) that unexpectedly modified the non-catalytic (Y24) as opposed to the catalytic – and presumably more reactive – tyrosine site (Y55, Figure 2 ). The ability to target the less conserved, non-catalytic tyrosine provided the rationale for SAR exploration of RJG-2121 analogs as ligand counterparts for selective AKR1C3 inactivation ( Tables 1 and 2 ). The combination of chemoproteomics and a cellular coumberone biochemical assay enabled rapid optimization of potent and cell-active AKR inhibitors that were subsequently confirmed to be AKR1C3 Y24-selective inhibitors by TMT-SuTEx. Importantly, we show the optimized AKR1C3 inhibitor RJG-2051 exhibits selectivity not only among AKR family members but also across the broader proteome ( Figures 3 and 4 ). Thus, RJG-2051 exhibits the proper balance of potency and proteome-wide selectivity to serve as a targeted covalent inhibitor of AKR1C3. An advantage of using covalent inhibitors is the ability to expedite cell biological studies via rapid confirmation of target engagement and off-target activity in situ . For AKR1C3 studies, this feature is particularly enabling given humans express 14 known AKR proteins 45 - 46 . The high selectivity of RJG-2051 for AKR1C3 is likely due to covalent modification of a non-conserved, non-catalytic tyrosine that to the best of our knowledge has not been explored as an inhibitory mechanism. Future studies will focus on deploying RJG-2051 in relevant tumor cell models to understand the impact of AKR1C3 inactivation on steroid and lipid metabolism 11 , 35 , 37 . In summary, we report the development of a targeted covalent inhibitor of AKR1C3 that achieves potency and proteome-wide selectivity in cells by liganding a non-catalytic tyrosine to disrupt biochemical function and can serve as a tool compound for investigating AKR1C3 biology and therapeutic potential.

All tested compounds were confirmed to be ≥95.0% purity by HPLC, and the representative HPLC traces for the compounds profiled are included in the Supporting Information . Colo205 cells were cultured at 37 °C with 5% CO 2 in RPMI with 10% fetal bovine serum and 1% L-glutamine in 6 or 10 cm 2 tissue culture dishes. Cells were cultured to 80-90% confluency for experimental use or to passage. Colo205 cells were grown to 80-90% confluency. Media was aspirated and cells were washed twice with warm 1X PBS. For probe treatments, cells were treated with 100 μM probe in serum-free media and incubated at 37 °C with 5% CO 2 for 2 hours. For compound treatments, cells were treated with either DMSO or inhibitors from a 1,000X stock in serum-free media (final [DMSO] = 0.1%) and subsequently incubated at 37 °C with 5% CO2 for 2 hours, followed by treatment with RJG-2121 at a final concentration of 1 μM from a 1,000X stock in serum-free media. Cells were harvested and pelleted at 500 × g for 3 min and the supernatant was aspirated. Cells were re-suspended in cold PBS and centrifuged at 500 × g for 3 min and the supernatant was aspirated once more. The PBS wash was repeated for a second time before cells were snap frozen and stored at −80 °C for future experiments. HEK293T cells were cultured at 37°C under 5% CO2 in DMEM medium containing 10% FBS (US Source, Omega Scientific), 1% L-glutamine (Fisher Scientific). Recombinant protein production via transient transfection of HEK293T cells was performed according to previous work. 44 In summary, HEK293T cells were allowed to grow to 50-60% confluency and then were transfected for 48 h with 2,600ng of PTGR2 plasmid and PEI 20μg. After the transfection period cells were lysed in DPBS and processed as in previous work to then be used in both proteomic and in substrate assay experiments. Human AKR plasmids were purchased from GenScript. The cell-based AKR activity assay described in this manuscript was adapted from a previously published protocol. 43 Confluent Colo205 cells were detached from a plate with 0.25% trypsin. Cells were pelleted at 500 x g for 3 min and the supernatant aspirated. Cells were resuspended in serum- and phenol-red free RPMI and pelleted at 500 x g for 3 min and the supernatant aspirated once more. Cells were resuspended in 1 mL serum- and phenol-red free RPMI and counted with a hemocytometer. Cells were plated at 20,000 cells/well in 50 μL serum- and phenol-red free RPMI. Cells were treated with either DMSO or 2X inhibitors from a 1,000X stock in serum-free media (final [DMSO] = 0.1%) and subsequently incubated at 37 °C with 5% CO2 for 2 hours. Cells were treated with 100 μL of either DMSO, 2X coumberone, or 2X coumberol standard curve from a 1,000X stock in serum-free media (final [DMSO] = 0.1%) and subsequently incubated at 37 °C with 5% CO2 for 3 hours. Coumberol formation was assessed by using an excitation λ = 385 nm, emission λ = 510 nm, gain = 800, and focal height = 6-7 mm. Data were plotted as a percent of DMSO control and IC 50 value calculated by non-linear regression in GraphPad Prism 7. Cell pellets were lysed in 1X PBS buffer by sonicating 3 seconds × 20% amplitude. The lysate was fractionated by centrifuging at 100,000 × g for 45 min at 4 °C, separating membrane and soluble fractions. The soluble fractions were used for assays. Protein concentrations were measured using the Bio-Rad DC protein assay, and fractions were diluted to a concentration of 1 mg/mL in 1X PBS for SDS-PAGE experiments, respectively. For lysate SDS-PAGE experiments, 48 μL of lysate was treated with 1 μL 50x compound stock in DMSO for respective time then 1 μL probe for respective time, followed by CuAAC as described in the following sentence. For live cell SDS-PAGE experiments, 50 μL aliquots of proteome was aliquoted and treated with CuAAC reagents in the following manner: 1 μl of 1.25 mM stock of rhodamine-azide in DMSO (25 μM final), 1 μl of freshly prepared 50 mM TCEP stock in water (1 mM final), 3 μl of a 1.7 mM TCEP stock in 4:1 t -butanol/DMSO (100 μM final), and 1 μl of a 50 mM CuSO 4 stock (1 mM final concentration). Samples were immediately and gently flicked, and the reaction proceeded for 1 hr at room temperature. Reactions were quenched with 17 μL of 4X SDSPAGE loading buffer and β-mercaptoethanol. Samples (30 μL) were analyzed by SDS-PAGE followed by in-gel fluorescence scanning using a rhodamine azide tag conjugated by CuAAC. Coomassie staining or Western blot, e.g. , using anti-AKR1C3 primary antibodies produced in rabbit followed by secondary antibody treatment with a conjugated DyLight 650 tag, were used to assess equivalent protein loading and presence of AKR1C3 across lanes. Probe modified proteome was prepared as previously described. 40 , 47 Label-free or TMT-tagged SuTEx modified peptides were analyzed on a ThermoFisher Q-Exactive Plus coupled to an Ultimate 3000 RSLC nanoSystem using previously described acquisition parameters. 40 , 47 Peptide identification and quantification was accomplished with the Byonic ™ (Protein Metrics Inc.) or Proteome Discoverer ™ (ThermoFisher) software packages using protocols and quality control metrics previously described. 48 Aliquots of Colo 205 proteome (1 mg) were incubated with 100 μM RJG-2043 (1 hr, RT). Desthiobiotin-azide was conjugated to the probe modified proteome using CuAAC and chloroform methanol extractions were used to remove click reagents as previously described 48 . Following resuspension in 6M Urea/ 25 mM AmBic, proteins were reduced by dithiothreitol and alkylated with iodoacetamide as previously described 48 . Denaturing reagents were removed via chloroform methanol extraction. Proteins were digested overnight with Tryp-Lys-C in 25mM AmBic (7.5 μg, 37 °C). The resulting peptides were desalted for TMT labeling using Pierce ™ Peptide Desalting Spin Columns. Peptide concentrations were normalized to 5 μg/ μL in EPPS (pH 8.5) using the Pierce ™ Quantitative Colorimetric Peptide Assay. TMTsixplex ™ was used to isotopically label 50 μg of peptide per channel at a 2:1 w/w ratio of label to peptide (1 hr, RT, 500 rpm shaking). The TMT reaction was quenched with 6% hydroxylamine (15 min, RT) and channels were mixed 1:1. Samples were dried using a speed vac and reconstituted in 500 μL DPBS. SuTEx modified peptides were enriched with avidin agarose and eluted using 150 μL of 50% ACN + 0.1% formic acid (3X) as previously described. 48 Enriched peptides were dried, resuspended in 25 μL 0.1% formic acid, and stored at −80 °C until analysis. TMT-tagged SuTEx modified peptides were analyzed on a Thermo Fisher Q-Exactive Plus coupled to an Ultimate 3000 RSLC nanoSystem using previously described acquisition parameters. 40 , 47 Peptides were separated an 85 min gradient by reverse phase LC using 3 μm C18 (20 cm) as follows: (A: 0.1% formic acid/H 2 O; B: 80% ACN, 0.1% formic acid in H 2 O, 300 nL/min): 0–7 min 1% B; 7–14 min 13% B; 14–114 min 32% B; 114–154 46% B; 154–156 min 95% B; 156–165 min 95% B; 165–166 min 1% B; 166–180 min 1% B. Data was acquired using a top 10 ddMS2 method. MS1 spectra were acquired at 70K resolution with a maximum injection time of 60 ms and MS2 spectra were taken with an AGC of 10K, quadrupole isolation width of 0.7 m/z , and max IT of 200 ms. Peptide identification and quantification was accomplished with Proteome Discoverer 3.0 and a PMI-Byonic node. MS2 spectra were searched against the human protein database (Uniprot, download date 01/17/2024) using the following parameters: ≤2 missed cleavages, 10 ppm precursor mass tolerance, 20 ppm fragment mass tolerance, and 1% protein false discovery rate. Modifications considered included SuTEx (+635.2737, Y, K, variable), methionine oxidation (+15.9949, M, variable), cysteine carbamidomethylation (+57.021464, C, fixed), and TMT modification (+229.1629, N-term, K, variable). Peptides used for quantification met the following quality control criteria: PMI-Byonic Score ≥300, delta ppm err. 5, co-isolation interference threshold ≤ 50%, reporter ion S/N ≥10, and were present in n=2 biological replicates. TMT channels were normalized to peptides from 14 housekeeping proteins commonly and non-specifically enriched. Volcano plots were generated by grouping PSMs using the peptide isoform node with cutoffs of Log 2 FC≥0.5, and p-value ≤0.05. For sites of interest, a median ratio was calculated from all isoforms adducted at the same position. All chemicals used were reagent grade and used as supplied, except where noted. Dichloromethane (CH 2 Cl 2 ) and tetrahydrofuran (THF) were used without any further purification steps. Analytical thin layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Flash column chromatography was carried out using forced flow of the indicated solvent on Silica Gel 60 (230-400 mesh) purchased from Fisher Scientific. Compounds were visualized by UV-irradiation and iodine chamber. 1 H and 13 C NMR spectra were recorded on a Varian Inova 500 (500 MHz), 600 (600 MHz), or Bruker Avance III 800 (800 MHz) spectrometers in CDCl 3 , Acetone-d 6 , or DMSO-d 6 with chemical shifts referenced to internal standards (CDCl 3 : 7.26 ppm 1H, 77.16 ppm 13C; (CD 3 ) 2 CO: 2.05 ppm 1H, 29.84 and 206.26 ppm 13C; (CD 3 )2SO: 2.50ppm 1H, 40.00 ppm 13C) unless stated otherwise. Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet for 1 H-NMR data. NMR chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in Hz. High resolution mass spectral (HRMS) data were obtained by an Agilent 6545B LC/Q-TOF (Agilent Technologies, Santa Clara, CA, USA). High-performance liquid chromatography (HPLC) data was obtained by a Shimadzu 1100 Series spectrometer with UV detection at 254 nm using a reverse-phase column with a 10-min acidified water/acetonitrile gradient as previously described. 47 Samples were injected on a Shimadzu 1100 Series spectrometer with UV detection at 254nm using a reverse-phase Kinetex C18 column (50 x 4.6 mm, 2.6 μm, 100 Å) using a 10-minute 15-85% gradient of solvent B in solvent A. Solvent A: HPLC-grade water with 0.1% acetic acid; Solvent B: HPLC-grade acetonitrile with 0.1% acetic acid. 4-(prop-2-yn-1-ylcarbamoyl)benzenesulfonyl chloride was synthesized as previously described. 41 A 6-dram vial fixed with a stir bar was charged with sulfonyl chloride (0.24 mmol) and purged with N 2 via Schlenk technique. Dry THF (48 mL) was added and the reaction stirred. DIEA (0.26 mmol) was added followed by triazole (0.26 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and purified by flash chromatography (10-40% gradient ethyl acetate in hexanes). White solid (403 mg, 61%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.55 (s, 1H), 8.15 – 8.11 (m, 2H), 8.00 – 7.95 (m, 2H), *7.94 (d, J = 8.4 Hz, 2H), *7.85 (d, J = 8.4 Hz, 2H), *7.73 (s, 1H), 6.39 (s, 1H), 4.29 – 4.25 (m, 2H), *2.84 – 2.77 (m, 1H), 2.31 (dd, J = 2.9, 2.1 Hz, 1H), 2.02 (ddt, J = 10.3, 7.9, 3.9 Hz, 1H), *1.25 – 1.19 (m, 2H), *1.19 – 1.15 (m, 2H), 1.01 – 0.95 (m, 4H). 13 C NMR (201 MHz, CDCl 3 ) δ 170.30, 165.07, *162.66, *152.12, 145.24, 140.03, 139.05, 129.08, *128.87, 128.46, *128.42, *128.17, *127.95, 78.77, 72.69, 30.25, *10.91, 9.17, 8.56. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 15 H 15 N 4 O 3 S + , 331.0859, Found, 331.0823. * indicates rotamer peaks White solid (315 mg, 49%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.62 (s, 1H), 8.16 – 8.11 (m, 2H), 8.00 – 7.94 (m, 2H), *7.80 (s, 1H), 6.41 (s, 1H), 4.26 (dd, J = 5.2, 2.6 Hz, 2H), *3.86 (p, J = 7.9 Hz, 1H), 3.16 (p, J = 7.8 Hz, 1H), 2.30 (t, J = 2.6 Hz, 1H), *2.19 – 2.10 (m, 2H), 2.03 – 1.94 (m, 2H), *1.93 – 1.81 (m, 2H), 1.81 – 1.68 (m, 2H), 1.62 (dq, J = 9.3, 3.6, 3.0 Hz, 2H). 13 C NMR (201 MHz, CDCl 3 ) δ 172.34, 165.09, *164.68, *151.93, 145.35, 140.01, *139.90, 139.08, 129.08, *129.01, *128.61, 128.45, *128.38, *127.56, 78.78, 72.66, 38.80, *37.31, *32.89, 32.12, *30.28, 30.23, *25.89, 25.74. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 17 H 19 N 4 O 3 S + , 359.1172, Found, 359.1171. * indicates rotamer peaks White solid (320 mg, 45%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.62 (s, 1H), 8.15 – 8.11 (m, 2H), 7.99 – 7.95 (m, 2H), *7.81 (s, 1H), 6.45 (s, 1H), 4.25 (dd, J = 5.2, 2.6 Hz, 2H), *3.48 (tt, J = 11.8, 3.5 Hz, 1H), 2.72 (tt, J = 11.6, 3.6 Hz, 1H), 2.30 (t, J = 2.6 Hz, 1H), 1.99 – 1.90 (m, 2H), *1.87 (ddd, J = 13.3, 5.0, 2.0 Hz, 2H), 1.76 (dq, J = 12.7, 3.5 Hz, 2H), 1.68 (dddd, J = 12.6, 5.0, 3.2, 1.4 Hz, 1H), *1.62 (qd, J = 12.8, 3.4 Hz, 2H), 1.49 (qd, J = 12.3, 3.5 Hz, 2H), *1.45 – 1.41 (m, 2H), 1.32 (qt, J = 12.4, 3.3 Hz, 2H), 1.27 – 1.19 (m, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 172.43, 165.09, *164.56, 152.07, 145.16, 139.99, 139.11, 129.05, *129.02, 128.42, *128.40, 78.79, 72.69, 37.74, *36.80, 32.07, 31.25, 30.24, 26.02, 25.88, *25.70. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 18 H 21 N 4 O 3 S + , 373.1329, Found, 373.1329. * indicates rotamer peaks Light brown semisolid (411 mg, 73%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.15 (d, J = 8.4 Hz, 2H), 8.05 (s, 1H), 7.69 (dd, J = 45.4, 8.0 Hz, 2H), 4.37 (s, 1H), 3.90 (s, 1H), 3.17 (s, 1.5H), 3.00 (s, 1.5H), 2.40 (s, 0.5H), 2.31 (s, 0.5H). 13 C NMR (201 MHz, CDCl 3 ) δ 168.8, 154.7, 144.9, 142.7, 129.2, 128.5, 128.3, 74.2, 73.0, 41.5, 36.7. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 13 H 13 N 4 O 3 S + , 305.0703, Found 305.0703. Light brown semisolid (439 mg, 55%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.54 (d, J = 0.7 Hz, 1H), 8.12 (d, J = 8.3 Hz, 2H), 7.68 (dd, J = 45.2, 7.9 Hz, 2H), 4.38 (s, 1H), 3.92 (s, 1H), 3.18 (s, 1.5H), 3.02 (s, 1.5H), 2.40 (s, 0.5H), 2.31 (s, 0.5H), 2.04 (q, J = 6.7 Hz, 1H), 1.26 – 1.15 (m, 1H), 1.03 – 0.96 (m, 4H). 13 C NMR (201 MHz, CDCl 3 ) δ 170.2, 162.6, 152.1, 145.2, 142.3, 137.5, 129.0, 128.4, 128.2, 74.1, 73.0, 41.5, 36.7, 33.1, 10.9, 9.2, 8.6, 7.9. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 16 H 16 N 4 O 3 S(Na + ), 367.0835, Found, 367.0831. White solid (144 mg, 64%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.78 (s, 1H), 8.46 (td, J = 1.8, 0.5 Hz, 1H), 8.23 – 8.17 (m, 2H), 8.04 (s, 1H), 7.72 (td, J = 7.9, 0.5 Hz, 1H), 6.61 (s, 1H), 4.28 (dd, J = 5.2, 2.6 Hz, 2H), 2.32 (t, J = 2.6 Hz, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 164.4, 154.8, 144.9, 136.6, 135.8, 134.5, 131.7, 130.6, 127.1, 78.8, 72.8, 30.3. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 12 H 10 N 4 O 3 S(Na + ), 313.0366, Found, 313.0364. White solid (195 mg, 76%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.56 (d, J = 0.6 Hz, 1H), 8.42 (td, J = 1.8, 0.5 Hz, 1H), 8.19 (dddd, J = 10.6, 7.8, 1.8, 1.1 Hz, 2H), 7.73 – 7.68 (m, 1H), 6.48 (s, 1H), 4.28 (dt, J = 4.9, 2.4 Hz, 3H), 2.33 (td, J = 2.5, 1.2 Hz, 1H), 2.07 – 1.97 (m, 1H), 1.01 – 0.94 (m, 4H). 13 C NMR (201 MHz, CDCl 3 ) δ 170.3, 164.6, 152.1, 145.2, 137.0, 135.6, 134.3, 134.0, 131.5, 131.2, 130.5, 130.4, 126.9, 78.9, 72.7, 30.2, 11.0, 9.2, 8.6. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 15 H 14 N 4 O 3 S(Na + ), 353.0679, Found, 353.0682. Light brown semisolid (301 mg, 54%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.17 (s, 2H), 8.04 (s, 1H), 7.86 (d, J = 26.4 Hz, 1H), 7.69 (t, J = 7.9 Hz, 1H), 4.38 (s, 1H), 3.91 (s, 1H), 3.18 (s, 1.5H), 3.05 (s, 1.5H), 2.43 (s, 0.5H), 2.32 (s, 0.5H). 13 C NMR (126 MHz, CDCl 3 ) δ 168.32, 154.57, 144.76, 142.18, 137.28, 136.28, 134.38, 134.00, 130.24, 130.00, 127.46, 127.19, 124.99, 74.09, 72.88, 41.54, 36.79, 33.29. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 13 H 12 N 4 O 3 S(Na + ), 327.0522, Found, 327.0533. White solid (336 mg, 53%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.54 (s, 1H), 8.18 (d, J = 40.5 Hz, 2H), 7.90 – 7.78 (m, 1H), 7.67 (t, J = 7.9 Hz, 1H), 4.38 (s, 1H), 3.94 (s, 1H), 3.18 (s, 1.5H), 3.05 (s, 1.5H), 2.37 (d, J = 55.2 Hz, 1H), 2.02 (p, J = 6.7 Hz, 1H), 0.98 (d, J = 7.8 Hz, 4H). 13 C NMR (201 MHz, CDCl 3 ) δ 170.2, 152.0, 145.2, 137.3, 134.2, 133.8, 130.3, 127.4, 127.1, 74.2, 72.9, 41.7, 38.1, 36.9, 10.9, 9.2, 8.5. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 16 H 16 N 4 O 3 S(Na + ), 367.0835, Found, 367.0837. A flame-dried 6-dram vial fixed with a stir bar was charged with sulfonyl chloride (0.24 mmol) and purged with N 2 via Schlenk technique. Dry THF (48 mL) was added and the reaction stirred. DIEA (0.26 mmol) was added followed by triazole (0.26 mmol). The reaction was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and purified by flash chromatography (10-40% gradient ethyl acetate in hexanes). A flame-dried 6-dram vial fixed with a stir bar was charged with triazole (0.41 mmol) and purged with N 2 via Schlenk technique. Dry THF (0.40 mL) was added and the reaction stirred and cooled to 0 °C with an ice/water bath. After cooling for 10 minutes, sodium hydride (0.41 mmol, 60% in paraffin oil) was added. The reaction was stirred at 0 °C for 30 minutes. The sulfonyl chloride (0.37 mmol) was dissolved in dry THF (0.40 mL). The sulfonyl chloride solution was added to the triazolide solution and the reaction slowly warmed to room temperature overnight. The reaction mixture was concentrated in vacuo and purified by flash chromatography (10-40% gradient ethyl acetate in hexanes). Sulfonyl fluoride (0.27 mmol), triazole (0.30 mmol), and potassium carbonate (0.30 mmol) were added to a 2-dram vial. Water (0.54 mL) was added and the reaction stirred overnight. About 2 mL of water was added to the reaction. The aqueous layer was extracted three times with ethyl acetate (5 mL each time). The combined organic layers was dried over sodium sulfate and concentrated in vacuo . The crude residue was purified by flash chromatography (10-40% gradient ethyl acetate in hexanes). Light brown semisolid (144 mg, 45%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.54 (s, 1H), 8.13 (t, J = 1.8 Hz, 1H), 7.81 (dt, J = 7.7, 1.4 Hz, 1H), 7.71 – 7.65 (m, 1H), 7.08 – 6.91 (m, 4H), 4.77 (d, J = 2.4 Hz, 2H), 3.99 (s, 2H), 3.57 (s, 2H), 3.19 (s, 2H), 3.06 (s, 2H), 2.52 (d, J = 2.4 Hz, 1H), 2.02 (p, J = 6.7 Hz, 1H), 0.99 (d, J = 1.6 Hz, 2H), 0.97 (s, 2H). 13 C NMR (MHz, CDCl 3 ) δ 170.21, 167.65, 150.38, 145.17, 137.57, 136.77, 134.18, 130.35, 129.66, 129.18, 128.37, 127.29, 122.65, 114.39, 56.44, 51.35, 50.61, 48.28, 42.82, 9.20, 8.56. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 25 H 26 N 5 O 4 S + , 492.1700, Found, 492.1697. White solid (155 mg, 71%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.18 – 8.13 (m, 2H), 8.05 (s, 1H), 7.67 – 7.61 (m, 2H), 7.07 – 7.03 (m, 1H), 6.93 (td, J = 7.6, 1.3 Hz, 1H), 6.89 (ddd, J = 8.0, 3.6, 1.6 Hz, 2H), 3.97 (t, J = 5.0 Hz, 2H), 3.86 (s, 3H), 3.51 (t, J = 4.9 Hz, 2H), 3.14 (t, J = 5.1 Hz, 2H), 2.99 (t, J = 4.9 Hz, 2H). 13 C NMR (126 MHz, CDCl 3 ) δ 167.68, 154.66, 152.33, 144.87, 140.34, 136.77, 129.25, 128.44, 124.00, 121.21, 118.61, 111.48, 60.53, 55.56, 51.19, 48.03, 42.59, 32.01, 22.82, 21.19, 14.33, 14.25. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 20 H 22 N 5 O 4 S + , 428.1387, Found, 428.1378. Off-white solid (14.1 mg, 46%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.19 – 8.14 (m, 2H), 8.06 (s, 1H), 7.68 – 7.62 (m, 2H), 7.22 (t, J = 8.1 Hz, 1H), 6.63 – 6.49 (m, 3H), 3.99 (s, 2H), 3.80 (s, 3H), 3.54 (s, 3H), 3.30 (s, 2H), 3.13 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.79, 165.58, 152.36, 145.64, 137.21, 130.83, 129.19, 129.12, 128.85, 128.42, 127.28, 121.29, 111.62, 55.60, 51.27, 50.68, 48.04, 42.45. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 20 H 22 N 5 O 4 S + , 428.1387, Found, 428.1378. White solid (11.0 mg, 26%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.78 (s, 1H), 8.18 – 8.13 (m, 2H), 8.06 (s, 1H), 7.67 – 7.63 (m, 2H), 7.00 – 6.89 (m, 2H), 6.86 (d, J = 8.3 Hz, 2H), 3.96 (s, 2H), 3.78 (s, 3H), 3.50 (s, 2H), 3.17 (s, 2H), 3.01 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.71, 154.71, 144.97, 142.91, 136.95, 129.29, 128.46, 119.38, 114.77, 55.69, 51.68, 50.94, 47.69, 42.39. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 20 H 22 N 5 O 4 S + , 428.1387, Found, 428.1382. White solid (40.6 mg, 41%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.20 (ddd, J = 4.9, 2.0, 0.8 Hz, 1H), 8.19 – 8.15 (m, 2H), 8.06 (s, 1H), 7.69 – 7.62 (m, 2H), 7.52 (ddd, J = 8.5, 7.1, 2.0 Hz, 1H), 6.70 (ddd, J = 7.2, 4.9, 0.8 Hz, 1H), 6.66 (dt, J = 8.5, 0.9 Hz, 1H), 3.90 (s, 2H), 3.64 (s, 2H), 3.54 (s, 2H), 3.45 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.88, 159.03, 154.71, 148.24, 144.90, 142.90, 137.94, 137.01, 129.32, 128.48, 114.53, 107.56, 47.40, 45.66, 45.39, 42.08. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 18 H 19 N 6 O 3 S + , 399.1234, Found, 399.1239. White solid (17.9 mg, 42%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.20 – 8.17 (m, 2H), 8.14 – 8.06 (m, 3H), 7.66 – 7.62 (m, 2H), 7.45 – 7.41 (m, 4H), 7.03 (d, J = 8.2 Hz, 1H), 6.94 – 6.89 (m, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 4H), 3.50 (s, 3H), 3.14 (s, 3H), 2.98 (s, 3H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.72, 160.81, 154.69, 144.91, 142.82, 136.98, 130.21, 129.30, 128.46, 109.69, 103.67, 55.39, 50.12, 49.67, 47.47, 42.22. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 26 N 5 O 4 S + , 504.1700, Found, 504.1703. Off-white solid (97.4 mg, 45%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.78 (s, 1H), 8.24 – 8.17 (m, 4H), 7.65 (s, 0H), 7.65 – 7.59 (m, 2H), 7.46 (t, J = 7.7 Hz, 2H), 7.39 – 7.34 (m, 1H), 7.04 (td, J = 7.7, 1.8 Hz, 1H), 6.94 – 6.86 (m, 3H), 3.97 (s, 2H), 3.85 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.79, 165.39, 152.35, 145.69, 143.58, 142.84, 140.36, 137.23, 129.20, 129.03, 128.45, 127.99, 127.73, 127.53, 127.25, 121.31, 111.62, 55.61, 51.29, 50.70, 47.89, 42.46. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 32 H 30 N 5 O 4 S + , 580.2013, Found, 580.2021. White solid (20.4 mg, 39%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.21 – 8.16 (m, 2H), 8.00 – 7.95 (m, 2H), 7.67 – 7.63 (m, 2H), 7.60 – 7.54 (m, 2H), 7.08 – 7.02 (m, 1H), 6.95 – 6.86 (m, 3H), 3.97 (s, 2H), 3.85 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.97, 164.98, 152.60, 145.96, 143.28, 137.25, 132.34, 129.47, 129.03, 128.71, 128.33, 125.56, 121.50, 111.81, 55.83, 51.48, 50.86, 48.26, 42.82. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 25 BrN 5 O 4 S + , 582.0805, Found, 582.0797. White solid (93.4 mg, 74%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.19 – 8.14 (m, 2H), 8.06 (s, 1H), 7.68 – 7.61 (m, 2H), 7.57 – 7.51 (m, 2H), 6.70 – 6.65 (m, 2H), 3.93 (s, 2H), 3.48 (s, 2H), 3.24 (s, 2H), 3.08 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.74, 154.71, 150.41, 144.91, 142.73, 138.22, 137.07, 129.32, 128.47, 118.95, 49.76, 49.21, 47.42, 41.85. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 19 H 19 IN 5 O 3 S + , 524.0248, Found, 524.0226. Light yellow solid (437 mg, 64%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.72 (s, 1H), 8.20 – 8.16 (m, 2H), 8.07 – 8.02 (m, 2H), 7.66 – 7.62 (m, 2H), 7.04 (td, J = 7.7, 1.8 Hz, 1H), 6.97 – 6.93 (m, 2H), 6.92 (dd, J = 7.4, 1.3 Hz, 1H), 6.88 (ddd, J = 8.2, 5.0, 1.5 Hz, 2H), 3.97 (s, 2H), 3.85 (s, 3H), 3.85 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 13 C NMR (201 MHz, CDCl 3 ) δ 167.76, 165.41, 161.70, 152.31, 145.55, 142.81, 140.37, 137.21, 129.08, 128.82, 128.42, 128.35, 127.56, 123.91, 121.68, 121.18, 118.58, 114.19, 111.47, 55.52, 55.47, 51.17, 50.56, 48.01, 42.56. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 27 H 28 N 5 O 5 S + , 534.1806, Found, 534.1802. White solid (25.4 mg, 19%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.80 (s, 1H), 8.24 – 8.20 (m, 2H), 8.04 (td, J = 7.6, 1.9 Hz, 1H), 7.67 – 7.63 (m, 2H), 7.46 – 7.41 (m, 1H), 7.23 (td, J = 7.6, 1.1 Hz, 1H), 7.18 (ddd, J = 10.7, 8.3, 1.1 Hz, 1H), 7.04 (ddd, J = 8.1, 7.3, 1.8 Hz, 1H), 6.93 (td, J = 7.6, 1.4 Hz, 1H), 6.89 (ddd, J = 8.2, 5.5, 1.5 Hz, 3H), 3.97 (s, 2H), 3.86 (s, 2H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 19 F NMR (564 MHz, CDCl 3 ) δ −173.28. 13 C NMR (151 MHz, CDCl 3 ) δ 188.39, 167.77, 162.08, 160.71 (d, J = 257.0 Hz), 152.37, 145.22, 143.03, 140.41, 137.02, 132.32 (d, J = 8.6 Hz), 130.64 (d, J = 2.2 Hz), 129.28, 128.41, 124.38 (d, J = 3.5 Hz), 123.96, 121.23, 118.63, 116.86 (d, J = 21.4 Hz), 111.52, 55.56, 51.21, 50.61, 48.05, 42.61. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 25 FN 5 O 4 S + , 522.1606, Found, 522.1607. White solid (44.4 mg, 30%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.21 – 8.16 (m, 2H), 7.89 (ddd, J = 7.8, 1.5, 1.0 Hz, 1H), 7.80 (ddd, J = 9.6, 2.7, 1.5 Hz, 1H), 7.66 – 7.63 (m, 2H), 7.40 (td, J = 8.0, 5.7 Hz, 1H), 7.13 (tdd, J = 8.4, 2.7, 1.0 Hz, 1H), 7.03 (ddd, J = 8.0, 7.3, 1.7 Hz, 1H), 6.91 (td, J = 7.6, 1.3 Hz, 1H), 6.87 (ddd, J = 8.1, 4.8, 1.5 Hz, 3H), 3.96 (s, 2H), 3.84 (s, 3H), 3.50 (s, 2H), 3.13 (s, 2H), 2.98 (s, 2H). 19 F NMR (564 MHz, CDCl 3 ) δ −173.91. 13 C NMR (151 MHz, CDCl 3 ) δ 167.72, 164.55, 163.03 (d, J = 246.3 Hz), 152.36, 145.70, 140.39, 136.97, 131.25 (d, J = 7.7 Hz), 130.52 (d, J = 8.1 Hz), 129.24, 128.47, 122.98 (d, J = 3.0 Hz), 121.23, 118.64, 117.76 (d, J = 21.4 Hz), 114.26 (d, J = 23.7 Hz), 111.53, 55.57, 51.22, 50.61, 48.06, 42.61. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 25 FN 5 O 4 S + , 522.1606, Found, 522.1608. White solid (26.6 mg, 59%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.74 (s, 1H), 8.22 – 8.16 (m, 2H), 8.14 – 8.07 (m, 2H), 7.68 – 7.63 (m, 2H), 7.16 – 7.08 (m, 2H), 7.08 – 7.03 (m, 1H), 6.95 – 6.86 (m, 3H), 3.97 (s, 2H), 3.85 (s, 3H), 3.52 (s, 2H), 3.20 – 3.12 (m, 2H), 3.00 (s, 2H). 19 F NMR (564 MHz, CDCl 3 ) δ −109.43 (tt, J = 8.5, 5.4 Hz). 13 C NMR (151 MHz, CDCl 3 ) δ 167.74, 164.74, 164.46 (d, J = 256.4 Hz), 152.35, 145.68, 142.96, 137.08, 129.36 (d, J = 8.6 Hz), 129.19, 128.43, 125.37, 121.25, 115.96 (d, J = 22.0 Hz), 111.57, 55.57, 51.23, 50.62, 42.52. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 25 FN 5 O 4 S + , 522.1606, Found, 522.1598. White solid (26.8 mg, 54%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.79 (s, 1H), 8.26 – 8.20 (m, 2H), 8.22 – 8.19 (m, 2H), 7.72 – 7.68 (m, 2H), 7.68 – 7.64 (m, 2H), 7.05 (td, J = 7.6, 7.2, 1.8 Hz, 1H), 6.95 – 6.87 (m, 3H), 3.97 (s, 2H), 3.85 (s, 3H), 3.51 (s, 2H), 3.15 (s, 2H), 2.99 (s, 2H). 19 F NMR (564 MHz, CDCl 3 ) δ −62.91. 13 C NMR (151 MHz, CDCl 3 ) δ 188.28, 167.69, 164.29, 152.35, 145.83, 143.13, 136.89, 132.48 (t, J = 16.4 Hz), 129.27, 128.49, 127.58, 125.90 – 125.66 (m), 123.98 (q, J = 272.3 Hz), 121.26, 111.58, 55.58, 51.23, 50.61, 47.92, 42.56. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 27 H 25 F 3 N 5 O 4 S + , 572.1574, Found, 572.1562. White solid (161 mg, 81%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.22 – 8.18 (m, 2H), 8.18 – 8.13 (m, 2H), 7.68 – 7.63 (m, 2H), 7.30 – 7.27 (m, 2H), 7.07 – 7.01 (m, 1H), 6.95 – 6.86 (m, 3H), 3.97 (s, 2H), 3.86 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 19 F NMR (564 MHz, CDCl 3 ) δ −57.75. 13 C NMR (151 MHz, CDCl 3 ) δ 188.52, 167.73, 164.44, 152.38, 151.07, 145.77, 143.10, 140.38, 137.01, 129.23, 128.97, 128.47, 127.78, 124.02, 121.25, 121.12, 118.64, 111.54, 55.58, 51.24, 50.61, 48.08, 42.63. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 27 H 25 F 3 N 5 O 5 S + , 588.1523, Found, 588.1537. White solid (57.0 mg, 43%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.73 (s, 1H), 8.22 – 8.16 (m, 2H), 7.67 – 7.62 (m, 2H), 7.55 (dd, J = 1.8, 0.8 Hz, 1H), 7.09 (dd, J = 3.4, 0.8 Hz, 1H), 7.04 (ddd, J = 8.0, 7.3, 1.8 Hz, 1H), 6.93 (td, J = 7.6, 1.3 Hz, 1H), 6.89 (ddd, J = 8.1, 5.3, 1.5 Hz, 2H), 6.52 (dd, J = 3.4, 1.8 Hz, 1H), 3.96 (s, 2H), 3.86 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.73, 158.26, 152.37, 145.69, 144.78, 144.58, 143.06, 136.89, 129.26, 128.48, 123.98, 121.23, 118.64, 112.73, 111.95, 111.52, 55.57, 51.21, 50.61, 48.04, 42.60. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 24 H 24 N 5 O 5 S + , 494.1493, Found, 494.1492. White solid (52.3 mg, 52%). 1 H NMR (600 MHz, CDCl 3 ) δ 9.32 (s, 1H), 8.78 (s, 1H), 8.68 (d, J = 4.8 Hz, 1H), 8.36 (dt, J = 8.0, 1.9 Hz, 1H), 8.22 – 8.16 (m, 2H), 7.71 – 7.60 (m, 2H), 7.38 (dd, J = 8.0, 4.8 Hz, 1H), 7.03 (td, J = 7.7, 1.7 Hz, 1H), 6.94 – 6.84 (m, 3H), 3.95 (s, 2H), 3.84 (s, 3H), 3.50 (s, 2H), 3.13 (s, 2H), 2.98 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.68, 163.33, 152.36, 151.50, 148.50, 145.83, 143.19, 140.35, 136.84, 134.60, 129.29, 128.50, 124.00, 123.70, 121.23, 118.64, 111.52, 55.57, 51.22, 50.59, 48.06, 42.61. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 25 H 25 N 6 O 4 S + , 505.1653, Found, 505.1647. White solid (64.6 mg, 38%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.81 (s, 1H), 8.78 – 8.71 (m, 2H), 8.24 – 8.19 (m, 2H), 8.03 – 7.96 (m, 2H), 7.69 – 7.64 (m, 2H), 7.05 (ddd, J = 8.1, 7.3, 1.8 Hz, 1H), 6.96 – 6.86 (m, 3H), 3.97 (s, 2H), 3.86 (s, 3H), 3.51 (s, 2H), 3.14 (s, 2H), 2.99 (s, 2H). 13 C NMR (151 MHz, CDCl 3 ) δ 167.78, 163.33, 150.45, 145.94, 143.42, 140.46, 136.73, 129.35, 128.56, 124.04, 121.25, 121.21, 118.64, 111.55, 55.58, 51.55, 50.95, 48.23, 42.67. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 25 H 25 N 6 O 4 S + , 505.1653, Found, 505.1663. Off-white solid (430 mg, 60%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.54 (s, 1H), 8.12 (d, J = 8.3 Hz, 2H), 7.65 – 7.62 (m, 2H), 7.05 (td, J = 7.2, 5.8, 2.5 Hz, 1H), 6.97 – 6.88 (m, 3H), 3.98 (s, 2H), 3.86 (s, 3H), 3.53 (s, 2H), 3.15 (s, 2H), 3.01 (s, 2H), 2.07 – 2.00 (m, 1H), 0.99 (d, J = 6.6 Hz, 4H). 13 C NMR (201 MHz, CDCl 3 ) δ 170.14, 167.86, *162.53, 152.34,* 152.02, 145.17, 142.70, *140.25, 137.24, 129.05, *128.85, 128.32, *128.27, *128.18, *127.80, 124.05, 121.22, 118.68, 111.52, 55.56, 51.20, 50.60, 48.00, 42.55, 9.15, 8.52. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 23 H 26 N 5 O 4 S + , 468.1700, Found, 468.1707. * indicates rotamer peaks White solid (405 mg, 54%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.62 (s, 1H), 8.13 – 8.10 (m, 1H), *8.10 – 8.07 (m, 1H), *7.83 (s, 1H), 7.64 – 7.58 (m, 2H), 7.06 – 7.00 (m, 1H), 6.94 – 6.84 (m, 3H), 3.96 (d, J = 6.7 Hz, 2H), *4.12 (m, 1H), 3.85 (s, 3H), 3.61 (p, J = 8.6 Hz, 1H), 3.50 (s, 2H), 3.13 (s, 2H), 2.98 (s, 2H), 2.49 – 2.42 (m, 1H), 2.38 – 2.27 (m, 3H), *2.14 – 2.06 (m, 1H), 2.06 – 1.99 (m, 1H), 1.98 – 1.89 (m, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 171.12, 167.81, *162.90, 152.33, *152.03, 145.32, 142.73, *142.59, *140.33, *137.93, 137.21, *129.08, 128.91, 128.30, *128.23, *128.16, *127.79, 123.97, 121.20, 118.62, 111.50, *60.48, 55.54, 51.17, 50.58, 47.99, 42.53, 33.47, *32.42, *27.86, 27.62, 18.77, *18.48. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 24 H 28 N 5 O 4 S + , 482.1857, Found, 482.1867. * indicates rotamer peaks White solid (337 mg, 49%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.62 (s, 1H), 8.15 – 8.10 (m, 2H), *7.81 (s, 1H), 7.66 – 7.60 (m, 2H), 7.05 (t, J = 8.3 Hz, 1H), 6.96 – 6.84 (m, 3H), 3.97 (s, 2H), 3.86 (s, 3H), 3.52 (s, 2H), 3.22 – 3.12 (m, 3H), 3.00 (s, 2H), *2.18 – 2.11 (m, 2H), 2.05 – 1.96 (m, 2H), *1.94 – 1.88 (m, 2H), *1.88 – 1.82 (m, 2H), 1.84 – 1.68 (m, 2H), 1.69 – 1.58 (m, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 172.25, 167.93, 152.40, 145.35, 142.77, 139.88, 137.34, 129.12, 128.35, 124.08, 121.29, 118.73, 111.60, 55.61, 51.25, 50.66, 48.04, 42.58, 38.81, 32.15, 25.76. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 25 H 30 N 5 O 4 S + , 496.2013, Found, 496.2026. * indicates rotamer peaks White solid (180 mg, 33%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.61 (s, 1H), 8.16 – 8.11 (m, 2H), *7.85 (d, J = 8.0 Hz, 2H), *7.83 (s, 1H), 7.65 – 7.60 (m, 2H), 7.05 (d, J = 8.2 Hz, 1H), 6.91 (dd, J = 21.9, 7.6 Hz, 3H), 3.98 (s, 2H), 3.87 (s, 3H), 3.53 (s, 2H), 3.16 (s, 2H), 3.01 (s, 2H), 2.78 – 2.72 (m, 1H), 2.01 – 1.91 (m, 2H), 1.88 (d, J = 13.3 Hz, 2H), 1.78 (dt, J = 13.1, 3.5 Hz, 2H), *1.69 (d, J = 12.6 Hz, 2H), *1.63 (dd, J = 12.2, 3.4 Hz, 2H), 1.62 – 1.40 (m, 4H), 1.39 – 1.29 (m, 2H), 1.29 – 1.21 (m, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 172.34, 167.89, *164.49, 152.38, 152.04, 145.11, *137.38, 129.09, *129.06, 128.32, *128.27, *128.23, *127.84, 121.29, 111.60, 55.62, 51.26, 50.68, 47.97, 37.75, 36.81, 32.11, 31.26, 26.05, 25.90, *25.72. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 32 N 5 O 4 S + , 510.2170, Found, 510.2175. * indicates rotamer peaks White solid (415 mg, 82%). 1 H NMR (497 MHz, CDCl 3 ) δ 8.75 (s, 1H), 8.16 (ddd, J = 6.1, 1.9, 1.2 Hz, 2H), 8.04 (s, 1H), 7.82 (dt, J = 7.7, 1.3 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.09 (s, 1H), 6.99 – 6.89 (m, 2H), 4.03 (d, J = 17.4 Hz, 2H), 3.89 (s, 3H), 3.58 (s, 2H), 3.20 (s, 2H), 3.08 (s, 2H). 13 C NMR (201 MHz, CDCl 3 ) δ 167.48, 154.71, 152.40, 144.90, 136.51, 134.41, 130.43, 129.91, 127.55, 121.43, 111.81, 55.71, 51.31, 50.79, 48.04, 42.26. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 20 H 22 N 5 O 4 S + , 428.1387, Found, 428.1387. White solid (143 mg, 60%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.54 (s, 1H), 8.13 (d, J = 1.4 Hz, 1H), 7.81 (dt, J = 7.8, 1.4 Hz, 1H), 7.71 – 7.66 (m, 1H), 7.06 (ddd, J = 8.6, 6.8, 2.3 Hz, 1H), 6.97 – 6.87 (m, 3H), 3.99 (s, 2H), 3.88 (s, 3H), 3.57 (s, 2H), 3.16 (s, 2H), 3.04 (s, 2H), 2.04 – 2.00 (m, 1H), 0.98 (d, J = 6.6 Hz, 4H). 13 C NMR (126 MHz, CDCl 3 ) δ 170.19, 167.59, 152.36, 145.17, 140.43, 137.61, 136.76, 134.19, 130.34, 129.65, 127.27, 123.98, 121.26, 118.68, 111.50, 55.58, 51.24, 50.85, 48.30, 42.80, 9.19, 8.55. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 23 H 26 N 5 O 4 S + , 468.1700, Found, 468.1691. White solid (156 mg, 61%). 1 H NMR (500 MHz, CDCl 3 ) δ 8.64 (s, 1H), 8.15 (d, J = 1.5 Hz, 1H), 7.83 (dt, J = 7.9, 1.4 Hz, 1H), 7.73 – 7.68 (m, 1H), 7.11 – 7.05 (m, 1H), 7.00 – 6.89 (m, 3H), 4.01 (s, 2H), 3.90 (s, 3H), 3.59 (s, 2H), 3.19 (s, 2H), 3.05 (s, 2H), 2.75 (dp, J = 11.6, 3.6 Hz, 1H), 1.96 (dd, J = 13.4, 3.1 Hz, 2H), 1.79 (dt, J = 13.1, 3.5 Hz, 2H), 1.74 – 1.61 (m, 1H), 1.51 (td, J = 12.5, 3.4 Hz, 2H), 1.41 – 1.19 (m, 3H). 13 C NMR (126 MHz, CDCl 3 ) δ 172.33, 167.62, 152.37, 145.08, 140.42, 137.58, 136.78, 134.18, 130.33, 129.68, 127.24, 123.99 (d, J = 2.2 Hz), 121.25, 118.68, 111.50, 55.60, 51.24, 50.62, 48.27, 42.82, 37.76, 31.27, 25.89. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 26 H 32 N 5 O 4 S + , 510.2170, Found, 510.2160. White solid (83%, 248 mg) 1 H NMR (497 MHz, CDCl 3 ) δ 9.31 (d, J = 2.2 Hz, 1H), 8.79 (d, J = 1.2 Hz, 1H), 8.66 (dd, J = 4.7, 1.6 Hz, 1H), 8.34 (dd, J = 7.8, 2.2 Hz, 1H), 8.21 (dd, J = 7.8, 1.3 Hz, 2H), 7.82 (dt, J = 7.7, 1.3 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.33 (dd, J = 8.0, 4.9 Hz, 1H), 7.08 – 7.03 (m, 1H), 6.98 – 6.87 (m, 4H), 3.98 (s, 2H), 3.86 (s, 3H), 3.55 (s, 2H), 3.16 (s, 2H), 3.00 (s, 2H). 13 C NMR (126 MHz, Acetone-d6) δ 167.7, 163.7, 153.5, 152.4, 148.7, 148.1, 142.0, 139.3, 137.2, 135.3, 134.8, 131.4, 130.2, 128.1, 126.2, 124.7, 124.0, 121.8, 119.3, 112.8, 55.8. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 25 H 25 N 6 O 4 S + 505.1653, found 505.1652. White solid (194 mg, 90%). 1 H NMR (600 MHz, DMSO-d 6 ) δ 10.60 (s, 1H), 9.49 (s, 1H), 8.40 (s, 1H), 8.26 (d, J = 8.1 Hz, 2H), 8.20 (d, J = 8.2 Hz, 2H), 7.77 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.38 – 7.26 (m, 2H), 7.11 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 3.77 (s, 3H). 13 C NMR (201 MHz, DMSO-d 6 ) δ 163.94, 156.14, 154.89, 146.76, 141.67, 137.41, 137.30, 130.20, 129.54, 129.43, 129.30, 128.71, 128.49, 120.79, 119.90, 111.77, 55.50. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 22 H 18 N 4 O 4 S(Na + ) 457.0941, found 457.0942. White solid (168 mg, 78%). 1 H NMR (600 MHz, DMSO-d 6 ) δ 10.67 (s, 1H), 9.49 (s, 1H), 8.41 (s, 1H), 8.29 – 8.24 (m, 2H), 8.23 – 8.18 (m, 2H), 7.88 – 7.84 (m, 2H), 7.58 – 7.53 (m, 1H), 7.50 (td, J = 8.3, 7.3, 4.3 Hz, 5H). 19 F NMR (564 MHz, DMSO-d 6 ) δ −56.22. 13 C NMR (201 MHz, DMSO-d 6 ) δ 164.14, 154.90, 146.77, 145.28, 141.63, 138.37, 137.36, 134.30, 131.82, 131.60, 131.51, 129.45, 129.39, 129.21, 128.51, 128.04, 122.22, 121.73, 120.18, 120.00 (q, J = 257.2 Hz). HRMS (ESI-TOF) m/z: [M+H] + calculated for C 22 H 15 F 3 N 4 O 4 S(Na + ) 511.0658, found 511.0658. White solid (63.8 mg, 56%). 1 H NMR (600 MHz, DMSO-d 6 ) δ 10.68 (s, 1H), 9.49 (s, 1H), 8.91 (d, J = 2.4 Hz, 1H), 8.55 (dd, J = 4.8, 1.6 Hz, 1H), 8.41 (s, 1H), 8.27 (d, J = 8.6 Hz, 2H), 8.21 (d, J = 8.5 Hz, 2H), 8.08 (dt, J = 7.8, 2.0 Hz, 1H), 7.94 – 7.85 (m, 2H), 7.79 – 7.75 (m, 2H), 7.48 (dd, J = 8.0, 4.7 Hz, 1H). 13 C NMR (201 MHz, DMSO-d 6 ) δ 164.08, 154.89, 148.18, 147.34, 146.77, 141.55, 138.78, 137.37, 134.97, 133.71, 132.61, 129.46, 128.50, 127.19, 123.87, 120.75. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 20 H 16 N 5 O 3 S + 406.0968, found 406.0967. White solid (14 mg, 45%). 1 H NMR (600 MHz, CDCl 3 ) δ 8.76 (s, 1H), 8.17 – 8.11 (m, 2H), 8.05 (s, 1H), 7.65 – 7.59 (m, 2H), 6.85 (td, J = 7.7, 1.4 Hz, 1H), 6.79 (dd, J = 8.0, 1.4 Hz, 1H), 6.69 (t, J = 7.7 Hz, 1H), 6.62 (d, J = 7.9 Hz, 1H), 4.52 (s, 0H), 3.84 (s, 3H), 3.62 – 3.54 (m, 2H), 3.21 – 3.12 (m, 3H), 2.21 (s, 1H), 2.09 – 2.02 (m, 1H), 1.56 (s, 1H), 1.41 (t, J = 7.2 Hz, 0H), 1.30 – 1.23 (m, 1H). 13 C NMR (201 MHz, CDCl 3 ) δ 167.72, 154.67, 147.08, 144.88, 143.39, 136.75, 136.30, 129.27, 128.19, 121.36, 117.22, 110.57, 109.95, 55.56, 49.61, 46.49, 41.24, 32.99, 32.09, 29.84, 28.61. HRMS (ESI-TOF) m/z: [M+H] + calculated for C 21 H 24 N 5 O 4 S + , 442.1544, Found, 442.1543.

The aldo-keto reductases (AKRs) are a superfamily of enzymes that reduce aldehydes or ketones to their respective alcohols. 1 - 2 Of note, AKR family 1 member C3 (AKR1C3) reduces androgen, estrogen, and prostaglandin substrates to produce potent drivers of proliferation in hormone-dependent malignancies. 3 - 8 For example, AKR1C3 reduces 5α-androstanedione to dihydrotestosterone which binds androgen receptor and promotes prostate cancer. 9 - 12 First-line therapies for prostate cancer are androgen deprivation or treatment with androgen receptor signaling inhibitors (ARSIs, e.g., enzalutamide, abiraterone acetate, apalutamide, and darolutamide), both of which often result in castration-resistant prostate cancer (CRPC). 13 - 17 Resistance to ARSIs is usually derived from androgen receptor mutations producing the constitutively active AR-V7. 18 - 21 More recently, Liu et al . demonstrated that up-regulation of AKR1C3 and AR-V7 is associated with cross-resistance to ARSIs. 13 The interplay between AKR1C3 and AR-V7 provides a different route to target this oncogenic pathway. Small molecule inhibitors that perturb AKR1C3 enzymatic activity have been developed and utilized for investigating AKR1C3 function and therapeutic potential in cancer cell proliferation. 22 , 3 , 9 , 23 Many of these agents are existing non-steroidal anti-inflammatory drugs (NSAIDs) or natural products (steroids, flavones, jasmonates) that have been repurposed as AKR1C3 inhibitors. For example, the NSAIDs indomethacin and flufenamic acid are cyclooxygenase (COX) inhibitors but also reported to have inhibitory activity against AKR1C3 24 - 28 . Efforts to optimize NSAIDs activity towards AKR1C3 have produced inhibitors with improved selectivity for AKR1C3 over COX 24 - 25 , 29 - 34 . Additional inhibitor scaffolds have been explored with varying degrees of potency and selectivity against AKR1C3 compared with related AKR1C enzymes 9 . AKR1C3 inhibitors have been tested in cancer cells lines and in xenografts in vivo and shown promising activity as single agents or in combination with other chemotherapeutics 10 , 19 , 28 , 35 . An important consideration in AKR1C3 inhibitor development is the ability to achieve selectivity against related family members that exhibit high sequence homology. The AKR1C subfamily consists of four isoforms (AKR1C1-4) with 86% sequence homology and all enzyme members possess a conserved catalytic tetrad (Y55, D50, K84 and H117 numbering for AKR1C3, Figure 1E ) 3 . While the reported inhibitors exhibit good potency and selectivity between AKR1C members in biochemical assays using purified protein, the selectivity of these compounds against endogenous AKR1C enzymes and other potential off targets in the proteome is not known. The latter is particularly important considering evaluation of a clinical AKR1C3 inhibitor BAY1128688 in a phase 2 clinical trial for treatment of endometriosis was terminated for hepatoxicity from off-target inhibition of AKR1D1 36 . Newer modalities including targeted protein degradation using a AKR1C3 PROTAC have been pursued but exhibits cross-reactivity with AKR1C1 and AKR1C2 37 . Development of chemical tool compounds with demonstrated potency and selectivity in cellular systems are therefore needed to assess AKR1C3 function and therapeutic potential. Here, we report the development of RJG-2051 as a covalent inhibitor that blocks AKR1C3 activity through covalent targeting of a non-catalytic tyrosine (Y) in the active site. RJG-2051 was discovered from evaluation of a series of sulfonyl-triazole (SuTEx) probes bearing cycloalkyl-triazole leaving groups that demonstrate enhanced tyrosine chemoselectivity in cells. Development of a tailored SuTEx probe for cellular detection of endogenous AKR1C3 guided optimization of cell-active inhibitors of AKR1C3. Using multiplexed quantitative chemical proteomics, we show direct evidence that site specific engagement of the AKR1C3 Y24 site results in potent inactivation of biochemical activity in cells. Remarkably, RJG-2051 exhibited proteome-wide selectivity including negligible activity against detectable AKR1 enzymes in compound-treated cells.

Supporting figures, experimental methods, chemical synthesis and characterization, NMR spectra and full-length gels for gel-based ABPP screens. Supporting Tables: Table S1 - LC-MS data for SuTEx probes; Table S2: LC-MS data for TMT-SuTEx.