Discovery of New OTUB1 Covalent Ligands via Structure-activity Relationship Studies for Targeted Protein Stabilization

Abstract

Deubiquitinase-targeting chimera (DUBTAC) has recently emerged as a promising technology for inducing targeted protein stabilization (TPS). DUBTACs are heterobifunctional molecules that recruit deubiquitinases (DUBs) to induce deubiquitination and stabilization of target proteins. However, DUBTAC development has been hindered by the scarcity of DUB ligands. In this study, we report the discovery of novel covalent ligands of the OTUB1 DUB through structure-activity relationship (SAR) studies of the previously reported OTUB1 ligand EN523. Our lead compound 34 (MS8572), which features a new heterocyclic core, covalently modified OTUB1 faster and more effectively, while also displaying enhanced stability and aqueous solubility compared to EN523. Furthermore, 34 was selective for OTUB1 over several cysteine-containing proteins and did not inhibit the OTUB1 deubiquitinase activity. Lastly, by utilizing 34, we developed an effective CFTR DUBTAC. Overall, we developed new and improved OTUB1 covalent ligands, expanding the limited number of DUB ligands that can be harnessed for TPS.

Graphical Abstract

Introduction

Deubiquitinase-targeting chimeras (DUBTACs) represent a new class of heterobifunctional small molecules, which recruit deubiquitinases (DUBs) to proteins of interest (POI), thereby inducing targeted deubiquitination and subsequent stabilization. Targeted protein stabilization (TPS) of tumor suppressors has emerged as a promising anti-cancer therapeutic strategy compared with targeted protein degradation (TPD) of oncoproteins.^{1, 2} Since the first DUBTAC was reported in 2022³, significant progress has been made in this field. To date, a number of proteins have been successfully stabilized using the DUBTAC technology, including CFTR, Wee1, FOXO3A, p53, IRF3, AMPK, cGAS, PPARγ, UTX, PRCP, VHL and KEAP1.^3–10 Additionally, the repertoire of DUBs that can be harnessed for DUBTAC development has expanded from OTUB1 to USP7, USP28 and USP1.^{3–8, 10}

DUBs play a critical role in DUBTAC-induced TPS. To date, approximately 100 DUBs have been discovered in humans, classified into seven families: ubiquitin-specific protease (USP), ovarian tumor (OTU), JAB1/MPN/Mov34 metalloenzymes (JAMM), Josephin/Machado-Josephin domain (MJD), ubiquitin C-terminal hydrolase (UCH), zinc finger with UFM1-specific peptidase domain (ZUFSP) and motif interacting with Ub-containing novel DUB family (MINDY). Six of these families (USP, OTU, Josephin/MJD, UCH, ZUFSP and MINDY) are cysteine (Cys) proteases, relying on a catalytic cysteine residue for their deubiquitinase function.^{11, 12} In contrast, DUBs in the JAMM family are zinc-dependent metalloproteinases.¹² The OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) DUB is a member of the OTU family. It is widely expressed across human tissues and specifically cleaves K48-linked polyubiquitin chains, which are critical for proteasome-mediated protein degradation.^13–15 Given its broad expression and ubiquitin signal-agnostic substrate recognition property, OTUB1 is a particularly attractive DUB for DUBTACs development. Notably, Nomura and colleagues discovered EN523, an acrylamide-based allosteric binder that covalently modifies the non-catalytic cysteine 23 of OTUB1.³ This allosteric ligand, which does not inhibit the deubiquitinase activity of OTUB1, has been successfully employed in the development of DUBTACs that stabilize various protein targets, including CFTR,³ Wee1,³ FOXO3A,⁴ p53,⁴ and IRF3⁴. Recently, we developed an improved OTUB1 ligand, MS5105, based on EN523.⁶ MS5105 displayed enhanced OTUB1 covalent modification efficiency while preserving the enzymatic function of OTUB1. Utilizing this improved OTUB1 ligand, we developed effective CFTR DUBTACs and first-in-class cGAS DUBTACs.⁶ Despite these progresses, structure-activity relationship (SAR) studies for optimizing DUB ligands for TPS are very limited.

In this study, we present our comprehensive SAR exploration to optimize OTUB1 covalent ligands, based on the previously reported OTUB1 ligand EN523. By design, synthesis and evaluation of approximately 40 new compounds, we discovered several lead compounds including MS5105 (compound 13) and compound 34 (MS8572), a new and improved lead, which contains a novel heterocyclic core. Compound 34 covalently modified OTUB1 in a concentration- and timedependent manner and was more effective than EN523 and MS5105. Moreover, compound 34 showed improved stability and aqueous solubility, and did not inhibit the enzymatic function of OTUB1. Lastly, utilizing compound 34, we developed a CFTR DUBTAC (compound 43), which effectively stabilized mutant CFTR in ΔF508-CFTR cells. Overall, our extensive SAR exploration led to the discovery of a new and improved OTUB1 ligand. This study expands the limited number of DUB ligands that can be leveraged for targeted protein stabilization.

Results and Discussion

SAR Studies Led to Discovery of Compound 34: A New and Improved Covalent Ligand for OTUB1

Starting from EN523, we first designed and synthesized several analogs focusing on the left-hand side (LHS) moiety.³ To assess covalent modification of OTUB1 by these compounds, we incubated these compounds with purified OTUB1 protein and evaluated the ability of these ligands forming covalent adducts with OTUB1 using a mass spectrometry (MS)-based assay. EN523 (compound 1) was included as a positive control. As shown in Figure 1, EN523 showed around 44% covalent modification of OTUB1, which is consistent with our previous results.⁶ Compound 2, featuring a 5-methyl thiophene ring at the LHS, exhibited a modest improvement in modification efficiency (54%). Compound 3, bearing a 5-methylthiazol substitution, displayed modification efficiency comparable to that of EN523. Interestingly, altering the nitrogen position within the thiazole ring reduced modification efficiency as compound 4 modified OTUB1 by 20%. Replacing the methylthiazole ring in compound 4 with the 1,2-dimethyl-1H-imidazole ring in compound 5 resulted in little or no OTUB1 modification (<3%). Given the larger atomic radius of the sulfur atom, the thiophene ring could be considered to resemble a phenyl ring in terms of its overall size.¹⁶ Therefore, we designed and synthesized several compounds with a six-membered ring system such as phenyl, pyridinyl and pyrimidinyl at the LHS (compounds 6 – 10). However, most of these compounds showed minimal OTUB1 modification. Only compound 9 with a 2-methyl pyridinyl ring showed approximately 25% covalent modification of OTUB1.

Figure 1. SAR results of the left-hand side aromatic moiety.

A, Chemical structures of compound 1 (EN523) and its nine analogs, along with a summary of their effects on covalent modification of OTUB1. The formation of the OTUB1-ligand adduct was assessed using mass spectrometry (MS). Each compound was incubated with OTUB1 (10 μM) for 1 hour at a 250:1 ligand-to-protein molar ratio. The percentage of OTUB1-ligand adduct formation was calculated as follows: (%) = (OTUB1-ligand adduct / (OTUB1 + OTUB1-ligand adduct)) × 100. Data are presented as the range from two biological repeats. B, Representative mass spectra of OTUB1 wild-type protein incubated with or without compound 1 or 2.

Inspired by the initial SAR results, we preserved the 5-methylthiophene group as the LHS moiety and next explored the impact of various covalent warheads¹⁷ at the right-hand side (RHS) on OTUB1 covalent modification efficiency (Figure 2). First, we replaced the acrylamide warhead with but-2-ynamide moiety. Unfortunately, the resulting compound 11 completely lost the ability to modify OTUB1 in this experimental setting. We also tried 2-fluoro-acrylamide moiety (compound 12), however, the MS result indicated that this change was not favored either. Next, we explored compound 13 (MS5105)⁶ bearing a dimethylamino-2-enamide group, which has been successfully applied to several U.S. Food and Drug Administration (FDA)-approved drugs (such as Afatinib).¹⁸ This compound achieved 72% OTUB1 modification, which is around 25% higher than EN523. Encouraged by this result, we explored several other substituents at the terminal position of the double bond. Pyrrolidinyl (14) and piperidinyl (15) substitutions led to slight decrease in modification efficiency compared to compound 13 (14: 55%; 15: 58%). Interestingly, the morpholinyl substitution (16) drastically attenuated the modification efficiency (<10%). Additionally, two reversible covalent warheads were investigated: (E)-2-cyano-4,4-dimethylpent-2-enamide in compound 17 and (E)-2-cyano-4-methylpent-2-enamide in compound 18. However, both compounds showed poor OTUB1 modification (<15%). Moreover, we repositioned the carbonyl group adjacent to the double bond, generating compounds 19 and 20. Unfortunately, both compounds exhibited negligible OTUB1 modification (<3%). Finally, two α-haloacetyl warheads (α-chloroacetyl in compound 21 and α-fluoroacetyl in compound 22) as well as two α-sulfamate-acetyl warheads¹⁹ (in compounds 23 and 24) were explored. Among them, only compound 21 displayed appreciable OTUB1 modification (27%), while the others did not show significant covalent modification (<10%).

Figure 2. SAR results of the right-hand side covalent warheads.

A, Chemical structures of fourteen analogs, along with a summary of their effects on covalent modification of OTUB1. The formation of the OTUB1-ligand adduct was evaluated using MS. Each compound was incubated with OTUB1 (10 μM) for 1 hour at a 250:1 ligand-to-protein molar ratio. The percentage of OTUB1-ligand adduct formation was calculated as follows: (%) = (OTUB1-ligand adduct / (OTUB1 + OTUB1-ligand adduct)) × 100. Data are presented as the range from two biological repeats. B, Representative mass spectrum of compound 13.

Based on compound 13, we next investigated the effect of different middle heterocyclic rings on OTUB1 modification efficiency. As shown in Figure 3A, we first relocated the amino group out of the ring system to get compound 25 and found that this change completely abolished OTUB1 covalent modification. Interestingly, compound 26, which has a larger ring size (1,4-diazepan-2-one) compared to compound 13, achieved about 29% OTUB1 modification. We also explored compounds without a ring system such as compounds 27 (with a glycine amide) and 28 (with an N,N’-dimethylglycine amide). However, neither compound showed appreciable modification of OTUB1 (27: 6%; 28: <3%). Next, we investigated compound 29, which contains a fused ring system (3,8-diazabicyclo[3.2.1]octan-2-one). This compound achieved near-complete modification of OTUB1 (96%). However, we also observed that this ligand overmodified OTUB1, forming a covalent adduct which contains one OTUB1 molecule and two small-molecule ligands (in about 10%), in the same assay conditions utilized to test all compounds (Figure 3B). Furthermore, compounds with piperazine (30), piperidine (31), and 3,8-diazabicyclo[3.2.1]octane (32) moieties in the middle region were also explored. Interestingly, these compounds, which lack the carbonyl group, displayed drastically attenuated efficiency in modifying OTUB1, suggesting that the carbonyl group in the middle heterocyclic ring of compounds 13 and 29 likely plays a critical role in facilitating covalent modification of OTUB1.

Figure 3. SAR results of the middle heterocyclic moiety.

A, Chemical structures of eight analogs, along with a summary of their effects on covalent modification of OTUB1. The formation of the OTUB1-ligand adduct was evaluated using MS. B, Representative mass spectrum of compound 29. C, Chemical structures of ten analogs, along with a summary of their effects on covalent modification of OTUB1. The formation of the OTUB1-ligand adduct was evaluated using MS. D, Representative mass spectrum of compound 34. For panels A and C, each compound was incubated with OTUB1 (10 μM) for 1 hour at a 250:1 ligand-to-protein molar ratio. The percentage of OTUB1-ligand adduct formation was calculated as follows: (%) = (OTUB1-ligand adduct / (OTUB1 + OTUB1-ligand adduct)) × 100. Data are presented as the range from two biological repeats.

Given the importance of this carbonyl group, we also investigated a series of compounds (compounds 33 – 42), in which the carbonyl group is moved out of the middle ring and directly linked to the LHS thiophene moiety (Figure 3C). Compound 33 with piperazine in the middle region modified OTUB1 with modest efficiency (24%). Notably, compound 34 bearing a 3,6-diazabicyclo[3.1.1]heptane ring system achieved very high OTUB1 modification efficiency (81%), which is better than that of EN523 and compound 13. In contrast, compounds with other bridged-ring systems such as a reversed diazabicyclo[3.1.1]heptane ring system in compound 35, 2,5-diazabicyclo[2.2.2]octane in compound 36, 3,8-diazabicyclo[3.2.1]octan-2-one ring systems in compounds 37 and 38, and 2,5-diazabicyclo[2.2.1]heptane ring systems in compounds 39 and 40 displayed much lower modification efficiency (11 – 24%). Furthermore, other middle region moieties such as 4-amino-piperidine in compound 41 and azetidin-2-ylmethanamine in compound 42 were investigated. However, while compound 42 modestly modified OTUB1 (18%), compound 41 did not modify OTUB1 in this experimental setting (<3%).

Overall, through these SAR studies, we discovered lead compounds 29 and 34, both of which remarkably modified OTUB1 and are more effective in modifying OTUB1 than EN523 and our previous lead compound MS5105. While compound 29 demonstrated higher modification effectiveness than compound 34, it also exhibited some over-modification of OTUB1, suggesting that it may have potential off-target effects in part due to its high reactivity. In contrast, compound 34 showed almost no over-modification. Therefore, we selected compound 34 as the lead compound for further characterization.

Further Characterization of Compound 34

We next compared compound 34 with the previously reported OTUB1 ligand EN523 in several assays.³ As illustrated in Figure 4A, compound 34 modified OTUB1 in a concentration-dependent manner, achieving significantly higher modification efficiency than EN523. Compound 34 can modify approximate 50% of OTUB1 protein at protein-to-ligand ratio of 1:100, while EN523 can modify only 20% of protein at the same ratio. Moreover, compound 34 was also slightly more effective than our recently discovered OTUB1 ligand MS5105⁶ (Figure S1). Furthermore, compound 34 modified OTUB1 more rapidly than EN523, with over 50% modification observed after 2 hours of incubation, compared to only about 20% for EN523 at the same time window. Compound 34 also exhibited superior stability in weakly acidic conditions (0.01 M HCl in methanol), with no decomposition observed after 48 hours, whereas over 90% of EN523 decomposed within 3 hours (Figure 4C). Additionally, compound 34 demonstrated significantly higher aqueous solubility (41.4 mg/mL) compared to EN523 (<0.6 mg/mL) (Figure 4D). Finally, an in vitro deubiquitination assay²⁰ confirmed that compound 34, similar to EN523,³ did not inhibit the catalytic activity of OTUB1 in cleaving a tetra-ubiquitin substrate (Figure 4E and S2). For comparison, the reversible OTUB1 inhibitor OTUB1/USP8-IN-1²¹ was also evaluated and, under identical conditions, it significantly inhibited OTUB1 deubiquitination activity (Figure S3).

Figure 4. Characterization of compound 34.

A, Bar graph of the OTUB1-ligand adduct formed after compound 34 (blue) or EN523 (red) was incubated with WT OTUB1 (10 μM) at the indicated ligand:OTUB1 ratio for 1 h. Data shown are the range from two biological repeats. B, Bar graph of the OTUB1-ligand adduct formed after compound 34 or EN523 (red) was incubated with WT OTUB1 (10 μM) for the indicated time at the 50:1 (ligand:OTUB1) ratio. Data shown are the range from two biological repeats. C, Representative HPLC spectra of EN523 (top) or compound 34 (bottom) at 1 M concentration after incubation for the indicated time in 0.01 M HCl MeOH solution from two independent experiments. D, Aqueous solubility of EN523 and compound 34. E, Representative western blot results of the K48-linked tetra-ubiquitin (3.6 μM) substrate incubated with OTUB1 (1.5 μM) in the presence or absence of compound 34 or EN523 (ligand:protein = 500:1) for the indicated time (0, 5, 15, 30 min), from two biological repeats.

We next assessed the selectivity of compound 34. We first evaluated which cysteine residue in OTUB1 was covalently modified by compound 34. There are 4 cysteine residues in human wild type OTUB1, cysteine 23 (C23), cysteine 91 (C91), cysteine 204 (C204) and cysteine 212 (C212).²² Among them, C91 is the catalytic cysteine, and C23 was reported as the residue modified by EN523.³ Therefore, we expressed and purified OTUB1 C23S mutant protein, and incubated it with compound 34, using EN523 as a control. As illustrated in Figure 5A, no covalent adduct was formed with the OTUB1 C23S mutant, indicating that C23 is the sole covalent modification site of OTUB1 by compound 34, which is the same as EN523. This result is consistent with our other results shown above: compound 34 neither over-modified OTUB1 (Figure 3D) nor inhibited the deubiquitinase activity of OTUB1 (Figure 4E). While our enzymatic assay suggested that modification on C23 did not affect the enzymatic activity of OTUB1 (Figure 4E and S2), evaluating the activity of the C23S mutant would provide additional support for this conclusion. This experiment is warranted in future studies. We also assessed the selectivity of compound 34 against two cysteine-containing proteins, the lysine methyltransferase GLP²³ and the CBP bromodomain²⁴, using the MS assays. As shown in Figure 5B, compound 34 did not significantly modify these two proteins. Taken together, these results suggest that our lead compound 34, which contains a novel heterocyclic core, is a highly effective allosteric OTUB1 covalent ligand, which specifically modifies the C23 residue of OTUB1 and does not inhibit the deubiquitinase activity of OTUB1. Moreover, compound 34 also possesses enhanced stability and aqueous solubility compared to EN523.

Figure 5. Selectivity assessment for compound 34.

A, MS results of compound 34 with the OTUB1 C23S mutant (C23SMu). EN523 (compound 1) was used as a control. B, MS results of compound 34 with different cysteine-containing proteins. For panels A – B, the formation of the protein-ligand adduct was detected by MS. Each compound was incubated with protein (10 μM) for 1 hour at a 50:1 ligand-to-protein molar ratio. The percentage of protein-ligand adduct formation was calculated as follows: (%) = (protein-ligand adduct / (protein + protein-ligand adduct)) × 100. ND: no protein-ligand adduct was detected. Data are presented as the mean ± SD from two biological repeats.

Compound 34 can be utilized to develop DUBTACs

Next, we generated a CFTR DUBTAC that utilizes compound 34 to recruit OTUB1 to demonstrate that compound 34 is a useful DUB ligand for developing DUBTACs. Using a similar linker strategy as the EN523-based CFTR DUBTAC reported by Nomura and colleagues,³ we extended the 5-methylthiophene ring of compound 34 with a propionic acidic handle to attach with the CFTR binder Lumacaftor via a 6-carbon linker, resulting in compound 43 (Figure 6A). We further evaluated the effect of this compound on stabilizing CFTR by treating CFBE41o-4.7 ΔF508-CFTR cells with this compound for 24 hours at multiple concentrations. As shown in Figure 6B, compound 43 effectively increased the CFTR protein level at 10 μM. The diffuse, smear-like appearance of CFTR bands on immunoblots likely reflects its extensive glycosylation and complex maturation as a multi-pass membrane protein, resulting in heterogeneous molecular weight species²⁵. Next, we evaluated the kinetics of OTUB1 stabilization induced by compound 43. As shown in Figure 6C, compound 43 increased the CFTR protein level in a time-dependent manner. Notably, the CFTR protein level increased as early as 6 hours, with the maximal stabilization observed at 18 hours. We then performed mechanism-of-action (MOA) studies to characterize CFTR stabilization by compound 43. First, treatment with either the CFTR ligand Lumacaftor or the OTUB1 ligand compound 34 alone did not alter CFTR protein levels (Figure 6D). In competition experiments, co-treatment of compound 43 with either Lumacaftor or compound 34 substantially abolished its stabilizing effect on CFTR (Figure 6E). Consistently, depletion of OTUB1 markedly attenuated the stabilization of CFTR induced by compound 43 (Figure 6F). Together, these results indicate that the compound 43-mediated CFTR stabilization depends on the engagement of both CFTR and OTUB1. Finally, CFTR mRNA levels remained unchanged after 24 h of treatment with compound 43 (Figure 6G), indicating that the observed increase in CFTR occurs through a post-transcriptional mechanism. Overall, our results demonstrate that our lead OTUB1 covalent ligand, compound 34, can be utilized to generate an effective DUBTAC, highlighting the potential of this new OTUB1 covalent ligand in developing effective DUBTACs.

Figure 6. Compound 43, a CFTR DUBTAC based on compound 34, stabilizes the ΔF508-CFTR mutant protein.

A, Chemical structure of compound 43. B, WB analysis of compound 43’s effect on stabilizing the CFTR protein level in ΔF508-CFTR cells treated with compound 43 for 24 h at indicated concentrations. C, WB analysis of compound 43’s effect on stabilizing the CFTR protein level in ΔF508-CFTR cells treated with compound 43 at 10 μM for the indicated times. D, WB analysis of CFTR protein levels in ΔF508-CFTR cells treated with indicated compounds for 24 h. E, WB analysis of CFTR protein levels in ΔF508-CFTR cells treated with DMSO, Lumacaftor, compound 34, or compound 43, either alone or in combination as indicated for 24h. F, WB analysis of CFTR protein levels in ΔF508-CFTR cells infected with the lentiviral sgControl or sgOTUB1, followed by treatment with DMSO or compound 43 for 24 h. G, Quantification of CFTR mRNA levels in ΔF508-CFTR cells treated with DMSO or compound 43 (10 μM)for 24 h, as determined by RT-qPCR. Results in B-F are representative of two independent experiments. Error bars in G indicate the standard deviation (SD) from at least two independent experiments. Data are presented as mean ± SD and were analyzed using unpaired two-tailed Student’s t-tests in GraphPad Prism. ns, not significant.

Chemical Synthesis

Compounds 2 – 10 were synthesized following similar procedures for preparing EN523 (Scheme 1–I).⁶ Starting from commercially available I-1 and different aryl bromides, through copper-catalyzed Ullmann reaction followed by removal of the Boc protecting group under acid condition, intermediates I-2 – I-10 were obtained. These intermediates were then conjugated with acryloyl chloride to yield compounds 2 – 10 in moderate yields. Starting from intermediates I-2 (obtained in Scheme 1–I), compounds 11 – 18, 21, and 22 were obtained in moderate to good yields through a one-step condensation reaction with various covalent warheads (Scheme 1–II). Similarly, intermediate I-2 was subjected to a substitution reaction with 2-(bromomethyl)acrylic acid or (E)-4-bromobut-2-enoic acid followed by condensation reaction with dimethylamine to obtain compounds 19 and 20 (Scheme 1–III). Compounds 23 and 24 were synthesized from intermediate I-2 via a condensation reaction with 2-hydroxyacetic acid, followed by a substitution reaction with either methylsulfamoyl chloride or benzylsulfamoyl chloride (Scheme 1–IV).

Scheme 1.

Synthesis of compounds 1 – 24^a

^aReaction and conditions: (a) aryl bromides, CuI, K₂CO₃, N,N’-dimethylethylenediamine, 1,4-dioxane, 100 °C; (b) TFA, DCM, rt, yield 18%-43%; (c) acryloyl chloride, Et3N, DCM, rt, yield 55%; (d) acids, HATU, DIEA, DMF, rt, yield 14%-57%; (e) 2-(bromomethyl)acrylic acid or (E)-4-bromobut-2-enoic acid, K2CO3, KI, DMF, rt; (f) dimethylamine, HATU, DIEA, DMF, rt, yield 19%-23%; (g) 2-hydroxyacetic acid, HATU, DIEA, DMF, rt; (h) methylsulfamoyl chloride or benzylsulfamoyl chloride, Et3N, DCM, rt, yield 15%-29%.

The synthetic routes for compounds 25 – 32 are shown in Scheme 2. Intermediate I-11 was first coupled with various amides followed by deprotection of the Boc group with the treatment of trifluoroacetic acid (TFA) to yield intermediates I-12 – I-16. These intermediates were then conjugated with (E)-4-(dimethylamino)but-2-enoic acid to yield compounds 25 – 29 (Scheme 2–I). Coupling of intermediate I-11 with tert-butyl piperazine-1-carboxylate or tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate, followed by deprotection, yielded intermediates I-17 and I-18. Installing (E)-4-(dimethylamino)but-2-enoic acid into these two intermediates resulted in compounds 30 and 32 (Scheme 2–II). Coupling of I-11 with I-19, followed by hydrogenation and TFA-mediated deprotection, generated intermediate I-20. This intermediate was further conjugated with (E)-4-(dimethylamino)but-2-enoic acid to yield compound 31 (Scheme 2–III).

Scheme 2.

Synthesis of compounds 25 – 32^a

^aReation and conditions: (a) amides, CuI, K₂CO₃, N,N’-dimethylethylenediamine, 1,4-dioxane, 100 °C; (b) TFA, DCM, rt, yield 19%-44%; (c) (E)-4-(dimethylamino)but-2-enoic acid, HATU, DIEA, DMF, rt, yield 17%-47%; (d) tert-butyl piperazine-1-carboxylate derivatives, Pd₂(dba)₃, Cs₂CO₃, Xantphos, 1,4-dioxane, 100 °C; (e) TFA, DCM, rt; (f) (E)-4-(dimethylamino)but-2-enoic acid, HATU, DIEA, DMF, rt; (g) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (I-19), Pd(PPh₃)Cl₂, K₃PO₄, Xantphos, 1,4-dioxane, H₂O, 100 °C; (h) Pd/C, H₂, MeOH, rt, yield 42%.

The synthetic routes for compounds 33 – 42 are illustrated in Scheme 3. Starting from commercially available intermediate I-21, conjugation with various Boc-protected amines followed by TFA-mediated deprotection and subsequent installation of the covalent warheads in the final step afforded the desired compounds.

Scheme 3.

Synthesis of compounds 33 – 42^a

^aReaction and conditions: (a) various Boc-protected amines, HATU, DIEA, DMF, rt; (b) TFA, DCM, rt, yield 23%-61%; (c) (E)-4-(dimethylamino)but-2-enoic acid, HATU, DIEA, DMF, rt yield 18%-59%.

The synthetic route for compound 43 is illustrated in Scheme 4. Starting from commercially available intermediate I-32, coupling with tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate, followed by TFA-mediated deprotection and subsequent hydrolysis, afforded I-33. I-33 was further converted to I-34 by reacting with (E)-4-(dimethylamino)but-2-enoic acid. I-34 was then reacted with the previously reported intermediate I-35⁶ via a regular coupling reaction to get the final DUBTAC 43.

Scheme 4.

Synthesis of compound 43^a

^aReaction and conditions: (a) tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate, HATU, DIEA, DCM, rt, then TFA; (b) LiOH, THF/H₂O, rt, yield 52%; (c) (E)-4-(dimethylamino)but-2-enoic acid, HATU, DIEA, DMF, rt, yield 47%; (d) I-35, HATU, DIEA, DMF, rt, yield 39%.

Conclusions

While the DUBTAC technology is very promising, one major limitation in the targeted protein stabilization field is the scarcity of the DUB ligands that can be utilized for developing DUBTACs. In particular, SAR studies to deliberately generate new DUB ligands for TPS have barely been pursued by the research community. In this study, we performed comprehensive SAR exploration on the scaffold of EN523, a previously reported covalent ligand of OTUB1, which is a widely expressed DUB across human tissues. We investigated multiple structural motifs of this scaffold, including the left-hand side heteroaromatic ring, middle heterocyclic ring, and right-hand side covalent warhead. By design, synthesis and evaluation of approximately 40 new compounds, we discovered several lead compounds including compound 34, a new and improved OTUB1 allosteric covalent ligand. Compound 34, which contains a new heterocyclic middle core, covalently modified OTUB1 faster and more effectively and possesses improved stability and aqueous solubility than EN523. In addition, compound 34 specifically targets C23 among several cysteine residues including the catalytic cysteine residue C91 of OTUB1. Furthermore, we determined that compound 34 did not inhibit the deubiquitinase activity of OTUB1. Lastly, compound 34 was successfully utilized to develop a CFTR DUBTAC, which effectively stabilized mutant CFTR protein, highlighting its potential as a DUB ligand in developing effective DUBTACs.

Although compound 34 shows improved modification efficiency and faster kinetics, it remains a relatively weak covalent ligand compared with previously reported covalent drugs. This limitation is likely attributable to the targeted C23 residue residing within the intrinsically disordered region (IDR) of OTUB1, which lacks a stable, well-defined binding pocket to enable efficient ligand positioning for covalent modification. Future structural studies will be essential to guide further optimization and improve the potency of this compound.

In this SAR study, intact-MS was employed to assess compounds’ potency. While MS is a widely adopted biophysical method to characterize and identify covalent binders, it has inherent limitations. Therefore, complementary assays, such as cysteine-reactive fluorophore competition and MS-based activity-based protein profiling (ABPP) assays, would help further validate these results. Furthermore, more in-depth MOA studies – such as co-treatment with a proteasome inhibitor or PNGase F, as well as cell cycle analysis and assessment of the total ubiquitin pool – will help further elucidate the underlying mechanism of the CFTR DUBTAC compound 43.

Overall, our comprehensive SAR studies resulted in the discovery of new and improved OTUB1 covalent ligands including compound 34. This work expands the limited number of DUB ligands that can be utilized for DUBTAC development, thus advancing the TPS field.

Experimental Section

Expression and Purification of Proteins

Human OTUB1 wild-type (NM_017670.3) and C23S mutation were cloned, expressed and purified according to the previously described protocol³. Catalytic domains of human GLP (982-1266) were cloned, expressed and purified according to the previously described protocol.²⁶ Bromodomain of CBP was custom-designed by GenScript, the expression sequence is listed below:

MHHHHHHSSGVDLGTENLYFQSMAPGQSKKKIFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLG

Mass Spectrometry-Based Analysis of Protein-Covalent Ligand Adducts

To test covalent ligands, recombinant OTUB1 protein (10 μM) was respectively incubated with DMSO, compound 1 (EN523) or 41 new compounds (2500 μM) in reaction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.5 mM TCEP pH 7.5) for 1 h at room temperature.

OTUB1 C23SMu (10 μM) was incubated with DMSO, compound 1 (EN523), or compound 34 (500 μM) in reaction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.5 mM TCEP pH 7.5) for 1 h at room temperature.

Recombinant OTUB1 WT, GLP, or CBP BD protein (10 μM) was respectively incubated with DMSO, compound 1, or compound 34 (500 μM) in reaction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.5 mM TCEP pH 7.5) for 1 h at room temperature.

For quantitative analysis of the effect of different protein:ligand ratios, recombinant OTUB1 protein (10 μM) was incubated with compound 34 (100 μM, 200 μM, 500 μM, 1000 μM, 2500 μM) in reaction buffer for 1 h at room temperature.

To quantitatively assess the time course of the OTUB1 covalent modification by the ligands, recombinant OTUB1 protein (10 μM) was incubated with compound 34 (500 μM) in reaction buffer for 0.5 h, 1 h, 2 h, 4 h, and 8 h at room temperature.

Then, the reaction solution was exchanged and concentrated to 50 mM ammonium bicarbonate with a 10 kDa MWCO centrifugal filter unit (UFC901024, Millipore) and diluted in MS buffer (30% ACN, 0.2% FA) to a final concentration of 10 μM.

Mass spectrometry detection was performed by an Agilent LC/MSD Time-Of-Flight (TOF) mass spectrometer (Agilent Technologies) equipped with an electrospray ion source or an Acquity H-Class UPLC combined with Xevo G2 XS Q-TOF mass spectrometer in the positive electrospray ionization mode located in Columbia University chemistry department Mass Spectrometry Core Facility. The data was analyzed by TOF Protein Confirmation Software (Agilent BioConfirm 2.0 or 11.0). All experiments were performed in duplicates.

In vitro Deubiquitinase Assay

Recombinant OTUB1 protein (10 μM) was preincubated with DMSO, Compound 1 (EN523, 5 mM) or Compound 34 (5 mM) in reaction buffer for 1 h at room temperature. Then, the pretreated protein was purified with a 10 kDa MWCO centrifugal filter unit (UFC901024, Millipore). To initiate the assay, untreated or pretreated OTUB1 (1.5 μM) was incubated with K48-Linked Tetra-Ubiquitin (3.6 μM, SI-4804-0025, LifeSensors) in reaction buffer for 0, 0.5, 1, 2.5, 5, 15, and 30 min at 37 °C.

OTUB1 (1.5 μM) was incubated with K48-Linked Tetra-Ubiquitin (3.6 μM, SI-4804-0025, LifeSensors) and OTUB1/USP8-IN-1 (500 μM, HY-151563, MedChemExpress) in reaction buffer for 0, 5, 15, and 30 min at 37 °C.

The reaction was terminated by adding 4x Laemmli Sample Buffer (1610747, BIO-RAD) with 100 mM TCEP pH 7.5 and heating at 95 °C for 6 min. The appearance of tetra-ubiquitin and monoubiquitin was monitored with western blotting. The primary antibody was Ubiquitin (P4D1) Mouse mAb (3936S, Cell Signaling Technology). The secondary antibody was IRDye 680RD Donkey anti-Mouse IgG (926-68072, LI-COR). Protein signals were detected by OdysseyCLx imaging system (LI-COR) and then analyzed by Image Studio Lite software (LI-COR).

Stability Assay

46.85 mg of EN523 and 66.69 mg of compound 34 were weighed and prepared into 1 M solution with 0.01 M HCl in MeOH respectively. The two solutions were incubated at room temperature for 0 h, 3 h, 24 h, and 48 h, respectively, and the purity was determined by HPLC after the specified incubation time.

Solubility Assay

In sample bottle, 0.5 mL of deionized water was added followed by 0.1 mg of EN523 or compound 34. Ultrasonic shaking was applied to facilitate dissolution. Upon complete dissolution of the compound, the weighing and dissolution procedures were sequentially repeated until the solution reached saturation.

Cell Culture

The human cystic fibrosis bronchial epithelial CFBE41o-4.7 ΔF508-CFTR cells were obtained from Millipore Sigma (SCC159). The cell line was generated from parental CFBE41o- cells by stably introducing the ΔF508-CFTR construct. CFBE41o- 4.7 ΔF508-CFTR cells were cultured in α-MEM medium (Sigma, #M2279) supplemented with 10% FBS, 2 mM L-Glutamine, 300 μg/mL Hygromycin B, and penicillin-streptomycin (10,000 units/mL Penicillin and 10,000 μg/mL Streptomycin). For treatment, cells were plated in 6-well plates pre-coated with a matrix mixture containing 10 μg/mL Fibronectin (Sigma, #F2006), 30 μg/mL PureCol Collagen (Sigma, #5006), and 100 μg/mL BSA (Sigma, #126575). Cells were treated with the indicated compounds once they reached approximately 60-70% confluency.

Western Blot Assay

Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease (Pierce) and phosphatase inhibitors (Calbiochem, cocktail set I and II). Protein concentration was determined using the previously described standard method ²⁷. The lysates (60 μg protein) were separated by SDS-PAGE, 6% gels (130 V for 120 min) for CFTR or 10% gels (130 V for 80 min) for other proteins, and then transferred to PVDF membranes, which were incubated with the indicated antibodies at 4°C overnight. After washing four times with Tris-buffered saline containing 0.1% Tween-20 (TBST), membranes were incubated with the HRP-conjugated secondary antibody for 1 hour at room temperature, and washed four times with TBST. The CFTR antibody (#78335, 1:1,000) was obtained from Cell Signaling Technology. The Vinculin antibody (V-4505, 1:50,000), HRP-conjugated anti-mouse (A-4416, 1:3,000), and HRP-conjugated anti-rabbit (A-4914, 1:3,000) secondary antibodies were purchased from Sigma.

sgRNA and Lentiviral Transduction

Lentiviral particles were generated by co-transfecting HEK293T cells with a CRISPR–Cas9 lentiviral vector encoding sgRNAs targeting OTUB1 (sgOTUB1-1, 5′-TATCAACAGAAGATCAAGGT-3′; sgOTUB1-2, 5′-CACCGGACCTCTGTCGCCGACCTGC-3′), together with the packaging plasmids psPAX2 and pMD2.G. Viral supernatants were collected at 48 and 72 h post-transfection, filtered, and used for subsequent infection. CFBE41o– 4.7 ΔF508-CFTR cells were infected with lentivirus expressing sgOTUB1 or a non-targeting control sgRNA (sgControl) in the presence of polybrene (4 μg/mL). Following infection, cells were cultured for 6 days to allow for efficient gene depletion. Cells were then treated with DMSO or compound 43 for 24 h prior to downstream analyses.

RNA Isolation and Quantitative Reverse-transcription PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using iScript^™ Reverse Transcription Supermix (#1708841, Bio-Rad). Quantitative PCR (qPCR) was performed using SYBR^™ Select Master Mix (#4472908, Thermo Fisher Scientific), and amplification was monitored on a CFX96^™ Real-Time PCR Detection System (Bio-Rad). Primer sequences used in this study are list in Table 1.

Table 1.

Primer sequences.

Human CFTR-F: AAAAGGCCAGCGTTGTCTCC;

Human CFTR-R: AACATCGCCGAAGGGCATTA;

Human ACTIN-F: GCTCGTCGTCGACAACGGCTC;

Human ACTIN-R: CAAACATGATCTGGGTCATCTTCTC;

Chemistry General Procedures

All commercial chemical reagents and solvents were used for the reactions without further purification. Flash column chromatography was performed on Teledyne ISCO CombiFlash Rf+ instrument equipped with a 220/254/280 nm wavelength UV detector and a fraction collector. Normal phase column chromatography was conducted on silica gel columns with either hexane/ethyl acetate or dichloromethane/methanol as eluent. Reverse phase column chromatography was conducted on HP C18 RediSep Rf columns, and the gradient was set to 10% of acetonitrile in H₂O containing 0.1% TFA progressing to 100% of acetonitrile. All final compounds were purified with preparative high-performance liquid chromatography (HPLC) on an Agilent Prep 1290 infinity II series with the UV detector set to 220/254/280 nm at a flow rate of 40 mL/min. Samples were injected onto a Phenomenex Luna 750 X 30 mm, 5 μm C18 column, and the gradient was set to 10% of acetonitrile in H₂O containing 0.1% TFA progressing to 100% of acetonitrile. LCMS was performed by an Agilent 1200 series system with DAD detector and a 2.1 mm x 150 mm Zorbax 300SB-C18 5 μm column for chromatography and high-resolution mass spectra (HRMS) that were acquired in positive ion mode using an Agilent G6230BA Accurate Mass TOF with an electrospray ionization (ESI) source. Samples (0.8 μL) were injected onto a C18 column at room temperature, and the flow rate was set to 0.6 mL/min with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX 400 MHz for proton (¹H NMR) and 101 MHz for carbon (¹³C NMR). Chemical shifts for all compounds are reported in parts per million (ppm, δ). The format of chemical shift was reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (J values in Hz), and integration. All final compounds had > 95% purity using the HPLC methods described above.

1-(5-methylthiophen-2-yl)piperazin-2-one (I-2)

To a solution of 2-bromo-5-methylthiophene (200.0 mg, 1.12 mmol) dissolved in dioxane (1 mL), N,N’-dimethylethylenediamine ( 0.59 mL, 3.4 mmol, 0.3 eq), K₂CO₃ ( 0.47 g, 3.4 mmol, 3.0 eq), CuI ( 21.4 mg, 0.112 mmol, 0.1eq ) followed by tert-butyl 3-oxopiperazine-1-carboxylate (0.32 g, 1.68 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield crude product. Then the crude product was dissolved in DCM/TFA (1:1, 1 mL). The reaction mixture was stirred at rt 1 h. After excess TFA was removed, resulting crude mixtures were purified via ISCO to yield intermediate I-2 as a white powder (107.8 mg, 49%).¹H NMR (400 MHz, Methanol-d₄) δ 6.65 (d, J = 3.8 Hz, 1H), 6.59 (d, J = 3.5 Hz, 1H), 4.08 – 3.98 (m, 4H), 3.71 – 3.61 (m, 2H), 2.38 (s, 3H).

4-Acryloyl-1-(5-methylthiophen-2-yl)piperazin-2-one (2)

To a solution of I-2 (19.7 mg, 0.1 mmol, 1.0 eq) in DCM Et₃N (13 μL, 0.089 mmol, 2.0 eq) was added. At 0 °C the acryloyl chloride (4.8 μL, 0.053 mmol, 1.2 eq.) was added, the reaction mixture stirred at 0 °C for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield (2) as white solid (13.8 mg, yield 55%). ¹H NMR (400 MHz, Methanol-d₄) δ 6.92 – 6.68 (m, 1H), 6.68 – 6.54 (m, 2H), 6.31 (d, J = 16.7 Hz, 1H), 5.90 – 5.76 (m, 1H), 4.48 (d, J = 28.0 Hz, 2H), 4.15 – 3.81 (m, 4H), 2.42 (d, J = 2.2 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₂H₁₅N₂O₂S, 251.0849; found: 251.0857.

Compounds 3 – 10 were synthesized following the same procedure for preparing compound 2.

4-Acryloyl-1-(5-methylthiazol-2-yl)piperazin-2-one (3)

White solid, 6.5 mg, 26% yield. ¹H NMR (400 MHz, Chloroform-d) δ 8.19 (s, 1H), 6.70 – 6.49 (m, 1H), 6.48 – 6.33 (m, 1H), 5.98 – 5.73 (m, 1H), 4.73 – 4.45 (m, 2H), 4.30 (s, 2H), 4.14 – 3.91 (m, 2H), 2.44 (d, J = 2.3 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₁H₁₄N₃O₂S, 252.0801; found: 252.0796.

4-Acryloyl-1-(2-methylthiazol-5-yl)piperazin-2-one (4)

White solid, 9.8 mg, 39% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.92 – 7.44 (m, 1H), 6.94 – 6.65 (m, 1H), 6.32 (d, J = 17.9 Hz, 1H), 5.85 (d, J = 9.8 Hz, 1H), 4.64 – 4.47 (m, 2H), 4.22 – 4.07 (m, 2H), 4.04 – 3.93 (m, 3H), 2.65 (d, J = 11.5 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₁H₁₄N₃O₂S, 252.0801; found: 252.0821.

4-Acryloyl-1-(1,2-dimethyl-1H-imidazol-5-yl)piperazin-2-one (5)

White solid, 4.7 mg, 19% yield. ¹H NMR (400 MHz, Chloroform-d) δ 7.38 (d, J = 20.7 Hz, 1H), 6.65 – 6.41 (m, 1H), 6.34 (d, J = 16.6 Hz, 1H), 5.79 (d, J = 10.4 Hz, 1H), 4.41 (s, 2H), 3.98 (d, J = 19.4 Hz, 4H), 3.69 (s, 3H), 2.58 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₂H₁₇N₄O₂, 249.1346; found: 249.1320.

4-Acryloyl-1-(m-tolyl)piperazin-2-one (6)

White solid, 13.9 mg, 57% yield. ¹H NMR (400 MHz, Chloroform-d) δ 7.34 – 7.26 (m, 1H), 7.14 – 7.03 (m, 3H), 6.64 – 6.50 (m, 1H), 6.43 (d, J = 16.7 Hz, 1H), 5.84 (d, J = 9.9 Hz, 1H), 4.51 – 4.37 (m, 2H), 4.05 – 3.94 (m, 2H), 3.80 – 3.75 (m, 2H), 2.37 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₄H₁₇N₂O₂, 245.1285; found: 245.1277.

4-Acryloyl-1-(p-tolyl)piperazin-2-one (7)

White solid, 7.8 mg, 32% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.31 – 7.20 (m, 4H), 6.92 – 6.69 (m, 1H), 6.33 (dt, J = 16.7, 2.4 Hz, 1H), 5.85 (d, J = 10.6 Hz, 1H), 4.54 – 4.30 (m, 2H), 4.12 – 3.97 (m, 2H), 3.88 – 3.74 (m, 2H), 2.37 (d, J = 2.2 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₄H₁₇N₂O₂, 245.1285; found: 245.1265.

3-(3-(4-Acryloyl-2-oxopiperazin-1-yl)phenyl)propanoic acid (8)

White solid, 9.1 mg, 30% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.35 (t, J = 6.3 Hz, 1H), 7.24 – 7.16 (m, 3H), 6.90 – 6.66 (m, 1H), 6.32 (dd, J = 16.9, 3.7 Hz, 1H), 5.84 (d, J = 10.5 Hz, 1H), 4.43 (d, J = 25.3 Hz, 2H), 4.04 (t, J = 5.9 Hz, 2H), 3.90 – 3.77 (m, 2H), 2.95 (q, J = 7.1, 6.5 Hz, 2H), 2.63 (q, J = 7.5, 5.6 Hz, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₁₉N₂O₄, 303.1339; found: 303.1363.

4-Acryloyl-1-(6-methylpyridin-2-yl)piperazin-2-one (9).

White solid, 13.0 mg, 53% yield. ¹H NMR (400 MHz, Chloroform-d) δ 7.82 – 7.65 (m, 1H), 7.60 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 6.63 – 6.46 (m, 1H), 6.40 (dd, J = 16.7, 1.9 Hz, 1H), 5.89 – 5.69 (m, 1H), 4.44 (d, J = 27.3 Hz, 2H), 4.20 (d, J = 29.6 Hz, 2H), 3.94 (d, J = 25.4 Hz, 2H), 2.49 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₃H₁₆N₃O₂, 246.1237; found: 246.1262.

4-Acryloyl-1-(2-methylpyrimidin-4-yl)piperazin-2-one (10).

White solid, 11.3 mg, 46% yield. ¹H NMR (400 MHz, Chloroform-d) δ 8.68 (d, J = 6.3 Hz, 1H), 8.35 (s, 1H), 6.54 (t, J = 13.2 Hz, 1H), 6.44 (d, J = 16.4 Hz, 1H), 5.85 (dd, J = 10.0, 2.6 Hz, 1H), 4.64 – 4.45 (m, 2H), 4.44 – 4.26 (m, 2H), 4.05 – 3.87 (m, 2H), 2.77 (d, J = 2.2 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₂H₁₅N₄O₂, 247.1190; found: 247.1187.

4-(But-2-ynoyl)-1-(5-methylthiophen-2-yl)piperazin-2-one (11)

To a solution of I-2 (19.7 mg, 0.1 mmol, 1.0 eq) in DMF (1 mL), HATU (41.8 mg, 0.11 mmol, 1.1 eq), DIEA (35 μL, 0.2 mmol, 2.0 eq) was added. At rt but-2-ynoic acid (8.4 mg, 0.1 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield (11) as colorless oil (3.8 mg, 14% yield). ¹H NMR (400 MHz, Chloroform-d) δ 6.55 (q, J = 3.6, 3.2 Hz, 1H), 6.47 (dt, J = 8.8, 2.8 Hz, 1H), 4.59 (d, J = 2.1 Hz, 1H), 4.43 (d, J = 2.0 Hz, 1H), 4.18 – 4.07 (m, 1H), 4.04 – 3.93 (m, 1H), 3.91 – 3.84 (m, 1H), 3.84 – 3.75 (m, 1H), 2.42 (d, J = 2.4 Hz, 3H), 2.04 (dd, J = 5.0, 2.1 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₃H₁₅N₂O₂S, 263.0849; found: 263.0825.

Compounds 12 – 18 were synthesized following the same procedure for preparing compound 11.

4-(2-Fluoroacryloyl)-1-(5-methylthiophen-2-yl)piperazin-2-one (12)

White solid, 10.5 mg, 39% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.67 – 6.62 (m, 1H), 6.60 (d, J = 4.0 Hz, 1H), 5.50 – 5.35 (m, 1H), 5.35 – 5.29 (m, 1H), 4.44 (s, 2H), 4.05 (t, J = 5.5 Hz, 2H), 3.93 (t, J = 5.5 Hz, 2H), 2.41(s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₂H₁₄FN₂O₂S, 269.0755; found: 269.0773.

(E)-4-(4-(dimethylamino)but-2-enoyl)-1-(5-methylthiophen-2-yl)piperazin-2-one (13)

White solid, 17.2 mg, 56% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.92 (dd, J = 35.8, 15.2 Hz, 1H), 6.75 (dt, J = 14.9, 7.2 Hz, 1H), 6.62 – 6.48 (m, 2H), 4.43 (d, J = 29.9 Hz, 2H), 4.09 – 3.97 (m, 2H), 3.99 – 3.77 (m, 4H), 2.89 (d, J = 2.1 Hz, 6H), 2.36 (d, J = 2.1 Hz, 3H). ¹³C NMR (101 MHz, Methanol-d₄) δ 167.75, 166.51, 146.94, 142.85, 132.26, 127.19, 123.33, 61.26, 60.90, 58.82, 49.43, 45.33, 29.74, 15.14. HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₅H₂₂N₃O₂S, 308.1427; found: 308.1437.

(E)-1-(5-methylthiophen-2-yl)-4-(4-(pyrrolidin-1-yl)but-2-enoyl)piperazin-2-one (14)

White solid, 19.3 mg, 58% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.90 (dd, J = 36.2, 15.2 Hz, 1H), 6.81 – 6.68 (m, 1H), 6.65 – 6.44 (m, 2H), 4.43 (d, J = 30.7 Hz, 2H), 4.00 (d, J = 7.1 Hz, 4H), 3.94 – 3.78 (m, 2H), 3.62 (s, 2H), 3.12 (s, 2H), 2.37 (d, J = 2.3 Hz, 3H), 2.08 (d, J = 48.5 Hz, 4H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₆N₃O₂S, 334.1584; found: 334.1611.

(E)-1-(5-methylthiophen-2-yl)-4-(4-(piperidin-1-yl)but-2-enoyl)piperazin-2-one (15)

White solid, 19.1 mg, 55% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.91 (dd, J = 34.6, 15.2 Hz, 1H), 6.82 – 6.67 (m, 1H), 6.57 (dt, J = 11.4, 3.5 Hz, 2H), 4.43 (d, J = 29.1 Hz, 2H), 4.02 (q, J = 8.4, 6.8 Hz, 2H), 3.88 (dd, J = 25.4, 6.4 Hz, 4H), 3.51 (d, J = 12.4 Hz, 2H), 2.94 (t, J = 12.5 Hz, 2H), 2.37 (d, J = 2.3 Hz, 3H), 1.95 (d, J = 14.4 Hz, 2H), 1.88 – 1.63 (m, 3H), 1.59 – 1.40 (m, 1H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₆N₃O₂S, 348.1740; found: 348.1764.

(E)-1-(5-methylthiophen-2-yl)-4-(4-morpholinobut-2-enoyl)piperazin-2-one (16)

White solid, 16.8 mg, 48% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.95 (dd, J = 34.0, 15.1 Hz, 1H), 6.84 – 6.68 (m, 1H), 6.65 – 6.45 (m, 2H), 4.44 (d, J = 30.6 Hz, 2H), 4.13 – 3.63 (m, 10H), 3.44 (s, 2H), 3.20 (s, 2H), 2.37 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₃S, 350.1533; found: 350.1558.

(E)-4,4-dimethyl-2-(4-(5-methylthiophen-2-yl)-3-oxopiperazine-1-carbonyl)pent-2-enenitrile (17)

White solid, 15.5 mg, 47% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.12 – 6.97 (m, 1H), 6.67 – 6.43 (m, 2H), 4.38 (s, 2H), 4.08 – 3.76 (m, 4H), 2.39 (s, 3H), 1.32 (s, 9H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₂N₃O₂S, 332.1427; found: 332.1450.

(E)-4-methyl-2-(4-(5-methylthiophen-2-yl)-3-oxopiperazine-1-carbonyl)pent-2-enenitrile (18)

White solid, 18.0 mg, 57% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.03 (d, J = 10.1 Hz, 1H), 6.68 – 6.59 (m, 2H), 4.43 (s, 2H), 4.08 – 3.91 (m, 5H), 3.06 – 2.84 (m, 1H), 2.42 (s, 3H), 1.22 – 1.16 (m, 6H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₀N₃O₂S, 318.1271; found: 318.1249.

N,N-dimethyl-2-((4-(5-methylthiophen-2-yl)-3-oxopiperazin-1-yl)methyl)acrylamide (19)

To a solution of I-2 (19.7 mg, 0.1 mmol, 1.0 eq) in DMF (1 mL), 2-(bromomethyl)acrylic acid (16.5 mg, 0.1 mmol, 1. eq), KI (1.6 mg, 0.1 mmol, 1.0 eq) and K₂CO₃ (3.64, 0.2 mmol, 2.0 eq) were added. The reaction mixture was stirred at rt for 6 h. Resulting crude mixtures were purified via prep-HPLC to yield crude product. To a solution of the crude product in DMF (1 mL) were added dimethylamine(1 M in THF) (0.1 mL, 0.1 mmol, 1.0 eq), HATU (41.8 mg, 0.11 mmol, 1.1 eq), DIEA (35 ∝L, 0.2 mmol, 2.0 eq) was added. The reaction mixture was stirred at rt for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield titled compound as white solid (7.1 mg, 23% yield). ¹H NMR (400 MHz, Methanol-d₄) δ 6.64 (d, J = 3.9 Hz, 1H), 6.60 – 6.53 (m, 1H), 5.94 (s, 1H), 5.77 (s, 1H), 4.14 – 3.89 (m, 6H), 3.64 (t, J = 5.7 Hz, 2H), 3.12 (s, 3H), 2.98 (s, 3H), 2.37 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₅H₂₂N₃O₂S, 308.1427; found: 308.1430.

(E)-N,N-dimethyl-4-(4-(5-methylthiophen-2-yl)-3-oxopiperazin-1-yl)but-2-enamide (20)

Compound was synthesized following the same procedure for preparing compound 19. White solid, 5.8 mg, 19% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.98 (d, J = 15.0 Hz, 1H), 6.74 – 6.59 (m, 3H), 4.16 – 4.02 (m, 6H), 3.72 (t, J = 5.7 Hz, 2H), 3.17 (s, 3H), 3.03 (s, 3H), 2.41 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₅H₂₂N₃O₂S, 308.1427; found: 308.1452.

Compounds 21 and 22 was synthesized following the same procedure for preparing compound 11.

4-(2-Chloroacetyl)-1-(5-methylthiophen-2-yl)piperazin-2-one (21)

White solid, 8.7 mg, 32% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.64 (d, J = 3.9 Hz, 1H), 6.60 (d, J = 3.9 Hz, 1H), 4.48 – 4.30 (m, 4H), 4.08 – 3.84 (m, 4H), 2.41 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₁H₁₄ClN₂O₂S, 273.0459; found: 273.0469.

4-(2-Fluoroacetyl)-1-(5-methylthiophen-2-yl)piperazin-2-one (22)

White solid, 10.2 mg, 40% yield. ¹H NMR (400 MHz, Chloroform-d) δ 6.56 (d, J = 3.7 Hz, 1H), 6.49 (d, J = 5.5 Hz, 1H), 5.10 (d, J = 10.6 Hz, 1H), 4.99 (d, J = 10.2 Hz, 1H), 4.40 (d, J = 34.5 Hz, 2H), 4.09 – 3.77 (m, 4H), 2.42 (d, J = 2.1 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₁H₁₄FN₂O₂S, 257.0755; found: 257.0774.

(2-(4-(5-Methylthiophen-2-yl)-3-oxopiperazin-1-yl)-2-oxoethyl methylsulfamate (23).

To a solution of I-2 (20 mg, 0.102 mmol, 1.0 eq) dissolved in DMF (1 mL), and the HATU ( 42.7 mg, 0.112 mmol, 1.1 eq), DIPEA(0.1 mL), 2-hydroxyacetic acid (9.4 mg, 0.102 mmol, 1.0 eq) were added. The reaction mixture was stirred at rt for 1 h, the crude mixtures were purified by prep-HPLC yield the crude product. The crude product was re-dissovled in DCM (1 mL), Et₃N (0.1 mL, 2.0 eq) and methylsulfamoyl chloride (9.3 mg, 1.2 eq) were added. The reaction mixture was stirred at rt for 5 h. Resulting crude mixture was purified by prep-HPLC to yield the titled compound. White solid, 10.1 mg, 29% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.75 – 6.51 (m, 2H), 4.91 (s, 2H), 4.38 (s, 2H), 3.95 (m, 4H), 2.75 (s, 3H), 2.41 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₂H₁₈N₃O₅S₂, 348.0682; found: 348.0705.

2-(4-(5-Methylthiophen-2-yl)-3-oxopiperazin-1-yl)-2-oxoethyl benzylsulfamate(24)

Compound 24 was synthesized following the same procedure for preparing compound 23. White solid, 6.3 mg, 15% yield ¹H NMR (400 MHz, Methanol-d₄) δ 7.56 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.23 – 7.14 (m, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.83 – 6.64 (m, 2H), 4.24 – 4.01 (m, 2H), 3.99 – 3.86 (m, 5H), 3.79 – 3.68 (m, 2H), 3.65 – 3.53 (m, 1H), 2.95 – 2.84 (m, 6H), 2.38 – 2.15 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₂N₃O₅S₂, 424.0995; found: 424.1023.

Compounds 25 – 29 were synthesized following the same procedure for preparing compound 2.

(E)-4-(dimethylamino)-N-(1-(5-methylthiophen-2-yl)-2-oxopiperidin-4-yl)but-2-enamide (25)

White solid, 8.7 mg, 27% yield ¹H NMR (400 MHz, Methanol-d₄) δ 6.81 – 6.66 (m, 1H), 6.63 – 6.51 (m, 2H), 6.37 (d, J = 15.3 Hz, 1H), 4.37 – 4.24 (m, 1H), 3.96 – 3.76 (m, 4H), 2.89 (s, 7H), 2.58 – 2.46 (m, 1H), 2.38 (s, 3H), 2.32 – 2.21 (m, 1H), 2.11 – 2.00 (m, 1H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₄N₃O₂S, 322.1584; found: 322.1605.

(E)-4-(4-(Dimethylamino)but-2-enoyl)-1-(5-methylthiophen-2-yl)-1,4-diazepan-2-one (26)

White solid, 15.1 mg, 47% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.04 – 6.88 (m, 1H), 6.83 – 6.64 (m, 1H), 6.63 – 6.50 (m, 2H), 4.62 – 4.47 (m, 2H), 4.18 – 4.01 (m, 2H), 4.00 – 3.81 (m, 4H), 2.89 (s, 6H), 2.38 (s, 3H), 2.12 – 1.87 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₄N₃O₂S, 322.1584; found: 322.1599.

(E)-4-(Dimethylamino)-N-(2-((5-methylthiophen-2-yl)amino)-2-oxoethyl)but-2-enamide (27)

White solid, 5.6 mg, 20% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.74 – 6.56 (m, 1H), 6.46 – 6.32 (m, 3H), 4.04 – 3.95 (m, 2H), 3.90 – 3.81 (m, 2H), 2.81 (s, 6H), 2.28 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₃H₂₀N₃O₂S, 282.1271; found: 282.1294.

(E)-4-(Dimethylamino)-N-methyl-N-(2-(methyl(5-methylthiophen-2-yl)amino)-2-oxoethyl)but-2-enamide (28)

White solid, 8.1 mg, 26% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.93 – 6.75 (m, 2H), 6.69 (s, 1H), 6.51 (d, J = 15.2 Hz, 1H), 4.14 (d, J = 7.3 Hz, 2H), 4.03 (s, 2H), 3.26 (s, 3H), 3.16 (s, 9H), 2.48 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₅H₂₄N₃O₂S, 310.1584; found: 310.1604.

(E)-8-(4-(dimethylamino)but-2-enoyl)-3-(5-methylthiophen-2-yl)-3,8-diazabicyclo[3.2.1]octan-2-one (29)

White solid, 12.9 mg, 39% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.05 – 6.73 (m, 2H), 6.55 (d, J = 8.4 Hz, 2H), 5.07 (d, J = 37.4 Hz, 2H), 4.21 – 3.90 (m, 3H), 3.84 – 3.59 (m, 1H), 2.93 (s, 6H), 2.53 – 1.88 (m, 7H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₂S, 334.1584; found: 334.1606.

(E)-4-(dimethylamino)-1-(4-(5-methylthiophen-2-yl)piperazin-1-yl)but-2-en-1-one (30)

To a solution of 2-bromo-5-methylthiophene (177.1 mg, 1 mmol, 1.0 eq) dissolved in dioxane (3 mL), Xantphos (173 mg, 0.3 mmol, 0.3 eq), Cs₂CO₃ (655.6 mg, 2.0 eq), Pd₂(dba)₃ (91.6 mg, 0.1 mmol, 0.1 eq) were added, followed by tert-butyl piperazine-1-carboxylate (279.6 mg, 1.5 mmol, 1.5 eq). The reaction mixture was stirred at 100 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate I-17 as a white solid, 34.6 mg, 19% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.38 (t, J = 3.0 Hz, 1H), 6.07 (t, J = 3.0 Hz, 1H), 3.25 (q, J = 2.8, 2.3 Hz, 4H), 3.21 (q, J = 2.6 Hz, 4H), 2.31 (d, J = 2.2 Hz, 3H).

To a solution of I-17 dissolved in DCM (1 mL), TFA( 1 mL) was added. The reaction mixture was stirred at rt for 1 h. After excess TFA was removed, resulting crude product was re-dissolved in DMF (1 mL), followed by DIEA (380 μL, 2.68 mmol, 3.0 eq) and (E)-4-(dimethylamino)but-2-enoic acid (129.1 mg, 1.00 mmol, 1.0 eq.). The reaction mixture was stirred at rt for 30 mins.

Resulting crude mixtures were purified via prep-HPLC to yield titled compound. White solid, 49.8 mg, 17% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.00 – 6.72 (m, 2H), 6.73 – 6.30 (m, 2H), 4.12 – 3.84 (m, 8H), 3.76 – 3.69 (m, 2H), 2.89 – 2.85 (m, 9H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₅H₂₄N₃OS, 294.1635; found: 294.1634.

4-(5-methylthiophen-2-yl)piperidine (I-20)

To a solution of the 2-bromo-5-methylthiophene (177.1 mg, 1 mmol, 1 eq) dissolved in dioxane/H₂O (4:1, 3 mL), K₃PO₄ (424.6 mg, 2 mmol, 2.0 eq), Pd (PPh₃)₂Cl₂ (70.2 mg, 0.1 mmol, 0.1 eq), followed by tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (618.4 mg, 2.0 mmol, 2.0 eq). The reaction mixture was stirred at 90 °C under nitrogen atmosphere overnight. After cooling down to rt, resulting crude mixtures were purified via silica gel column chromatography to yield intermediate. To a solution of intermediate (200 mg, 0.72 mmol) dissolved in MeOH (3 mL), Pd/C( 20 mg) was added. The reaction mixture was stirred at hydrogen atmosphere (balloon). After stirred at rt for 6 h, MeOH was removed. Resulting crude product was re-dissolved in DCM (1 mL), TFA (1 mL) was added. The reaction mixture was stirred at rt for 1 h, resulting crude mixtures were purified via ISCO to yield intermediate I-20 as a white powder (55.1 mg, 42%).¹H NMR (400 MHz, Methanol-d₄) δ 6.57 (d, J = 3.2 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 3.35 (d, J = 12.8 Hz, 2H), 3.06 – 2.93 (m, 3H), 2.31 (s, 3H), 2.08 (d, J = 13.8 Hz, 2H), 1.86 – 1.69 (m, 2H). [M+H]⁺182.11

(E)-4-(dimethylamino)-1-(4-(5-methylthiophen-2-yl)piperidin-1-yl)but-2-en-1-one (31)

To a solution of the I-20 (18.1 mg, 0.1 mmol, 1.0 eq) in DMF (1 mL), HATU (41.8 mg, 0.11 mmol, 1.1 eq), DIEA (35 μL, 0.2 mmol, 2.0 eq) was added. At rt the but-2-ynoic acid (8.4 mg, 0.1 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 30 mins. Resulting crude mixtures were purified via prep-HPLC to yield (31) as a white solid, 12.6 mg, 43% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.95 (d, J = 15.1 Hz, 1H), 6.72 – 6.58 (m, 2H), 6.55 (d, J = 2.8 Hz, 1H), 4.59 (d, J = 13.4 Hz, 1H), 4.13 (d, J = 13.9 Hz, 1H), 3.92 (dd, J = 7.3, 2.2 Hz, 2H), 3.24 (d, J = 13.1 Hz, 1H), 3.14 – 3.00 (m, 1H), 2.95 – 2.76 (m, 7H), 2.38 (d, J = 2.3 Hz, 3H), 2.13 – 1.96 (m, 2H), 1.74 – 1.42 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₅N₂OS, 293.1682; found: 293.1701.

(E)-4-(dimethylamino)-1-(3-(5-methylthiophen-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)but-2-en-1-one (32)

Compound 31 was synthesized following the same procedure for preparing compound 30. White solid, 6.1 mg, 19% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 6.94 – 6.74 (m, 3H), 6.36 (s, 1H), 4.78 – 4.40 (m, 3H), 3.99–3.95 (m, 4H), 2.92 (m, 8H), 2.31 (s, 3H), 2.09 – 2.03 (m, 2H), 1.98 (td, J = 14.5, 12.6, 8.6 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₆N₃OS, 320.1791; found: 320.1816.

(E)-4-(dimethylamino)-1-(4-(5-methylthiophene-2-carbonyl)piperazin-1-yl)but-2-en-1-one (33)

To a solution of 5-methylthiophene-2-carboxylic acid (75 mg, 0.5 mmol, 1.0 eq) in DMF (1 mL), HATU (209 mg, 0.55 mmol, 1.1 eq) and DIPEA (0.18 mL, 2.0 eq) were added. tert-Butyl piperazine-1-carboxylate (93.3 mg, 0.5 mmol, 1.0 eq.) was added, the reaction mixture was stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to get intermediate. Then the intermediate was re-dissolved in DCM (0.5 mL), and the TFA( 0.5 mL) was added. The reaction mixture stirred at rt 1 h. After excess TFA was removed, resulting crude product was purified via prep-HPLC to yield intermediate I-22 as a white solid, 60.4 mg, 58% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.23 (d, J = 3.7 Hz, 1H), 6.82 – 6.63 (m, 1H), 4.02 – 3.83 (m, 4H), 3.31 – 3.15 (m, 4H), 2.46 (s, 3H).

To a solution of I-22 (20 mg, 0.095 mmol) in DMF (1 mL), HATU (39.8 mg, 0.1 mmol, 1.1 eq) and DIPEA (0.1 mL, 2.0 eq) were added. Then (E)-4-(dimethylamino)but-2-enoic acid (15.8 mg, 0.095 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 2 h. Resulting crude mixtures were purified via prep-HPLC to yield titled compound White solid, 28.9 mg, 18% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.28 – 7.18 (m, 1H), 7.01 – 6.86 (m, 1H), 6.78 (d, J = 4.5 Hz, 1H), 6.74 – 6.60 (m, 1H), 4.02 – 3.86 (m, 2H), 3.86 – 3.75 (m, 4H), 3.75 – 3.62 (m, 4H), 2.87 (s, 6H), 2.48 (s, 3H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₄N₃O₂S, 322.1584; found: 322.1604.

Compounds 34 – 42 were synthesized following the same procedure for preparing compound 33.

(E)-4-(dimethylamino)-1-(3-(5-methylthiophene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)but-2-en-1-one (34).

White solid, 17.8 mg, 54% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.43 (d, J = 3.4 Hz, 1H), 6.83 (d, J = 3.0 Hz, 1H), 6.77 (dt, J = 14.4, 7.1 Hz, 1H), 6.58 (d, J = 15.2 Hz, 1H), 4.83 – 4.76 (m, 1H), 4.59 – 4.54 (m, 1H), 4.44 – 4.06 (m, 2H), 4.04 – 3.87 (m, 4H), 2.89 (s, 6H), 2.87 – 2.78 (m, 1H), 2.50 (s, 3H), 1.72 (d, J = 9.2 Hz, 1H). ¹³C NMR (101 MHz, Methanol-d₄) δ 165.27, 164.77, 145.71, 133.96, 131.55, 130.53, 128.07, 125.83, 60.22, 57.73, 57.32, 41.89, 28.52, 13.71. HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₂S, 334.1584; found: 334.1610.

(E)-4-(dimethylamino)-1-(6-(5-methylthiophene-2-carbonyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)but-2-en-1-one (35)

White solid, 18.8 mg, 57% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.48 – 7.31 (m, 1H), 6.88 – 6.73 (m, 2H), 6.73 – 6.60 (m, 1H), 5.12 – 4.89 (m, 1H), 4.71 – 4.42 (m, 1H), 4.35 – 4.00 (m, 1H), 3.96 – 3.75 (m, 4H), 3.73 – 3.60 (m, 1H), 2.88 – 2.76 (m, 7H), 2.46 (s, 3H), 1.69 – 1.57 (m, 1H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₂S, 334.1584; found: 334.1608.

(E)-4-(dimethylamino)-1-(5-(5-methylthiophene-2-carbonyl)-2,5-diazabicyclo[2.2.2]octan-2-yl)but-2-en-1-one (36)

White solid, 14.2 mg, 41% yield ¹H NMR (400 MHz, Methanol-d₄) δ 7.24 (d, J = 75.4 Hz, 1H), 6.91 – 6.54 (m, 3H), 4.73 (d, J = 24.1 Hz, 1H), 4.62 – 4.33 (m, 1H), 4.11 – 3.71 (m, 4H), 3.69 – 3.49 (m, 2H), 2.81 (s, 6H), 2.41 (s, 3H), 2.16 – 1.70 (m, 4H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₆N₃O₂S, 348.1740; found: 348.1767.

(E)-4-(dimethylamino)-1-(3-(5-methylthiophene-2-carbonyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)but-2-en-1-one (37)

White solid, 12.5 mg, 36% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.30 – 7.17 (m, 1H), 7.01 – 6.88 (m, 1H), 6.88 – 6.72 (m, 2H), 4.76 (s, 1H), 4.61 (t, J = 4.8 Hz, 1H), 4.38 (d, J = 15.7 Hz, 2H), 3.99 (t, J = 5.1 Hz, 2H), 3.34 (s, 3H), 2.94 (s, 6H), 2.53 (s, 3H), 2.22 – 1.70 (m, 4H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₆N₃O₂S, 348.1740; found: 348.1763.

(E)-4-(dimethylamino)-1-(8-(5-methylthiophene-2-carbonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)but-2-en-1-one (38)

White solid, 17.0 mg, 49% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.38 – 7.29 (m, 1H), 6.94 – 6.82 (m, 1H), 6.80 (d, J = 4.6 Hz, 1H), 6.76 – 6.57 (m, 1H), 4.74 (s, 2H), 4.48 – 4.30 (m, 1H), 4.00 – 3.82 (m, 3H), 3.56 – 3.41 (m, 1H), 3.09 – 2.98 (m, 1H), 2.86 (s, 6H), 2.47 (s, 3H), 2.05 – 1.83 (m, 2H), 1.83 – 1.59 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₈H₂₆N₃O₂S, 348.1740; found: 348.1764.

(E)-4-(dimethylamino)-1-((1S,4S)-5-(5-methylthiophene-2-carbonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)but-2-en-1-one (39)

White solid, 13.9 mg, 42% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.31 – 7.14 (m, 1H), 6.80 – 6.34 (m, 3H), 4.86 – 4.78 (m, 2H), 3.80 (h, J = 18.8, 15.5 Hz, 3H), 3.67 – 3.31 (m, 3H), 2.73 (d, J = 7.4 Hz, 6H), 2.34 (s, 3H), 2.03 – 1.77 (m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₂S, 334.1584; found: 334.1605.

(E)-4-(dimethylamino)-1-((1R,4R)-5-(5-methylthiophene-2-carbonyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)but-2-en-1-one (40)

White solid, 19.5 mg, 59% yield.. ¹H NMR (400 MHz, Methanol-d₄) δ 7.35 – 7.18 (m, 1H), 6.90 – 6.40 (m, 3H), 4.87 – 4.80 (m, 2H), 3.98 – 3.71 (m, 3H), 3.72 – 3.32 (m, 3H), 2.82 – 2.70 (m, 6H), 2.36 (s, 3H), 2.03 – 1.78 (m, 2H).MS (ESI) HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₄N₃O₂S, 334.1584; found: 334.1603.

(E)-4-(dimethylamino)-N-(1-(5-methylthiophene-2-carbonyl)piperidin-4-yl)but-2-enamide (41)

White solid, 15.4 mg, 46% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.11 (t, J = 4.0 Hz, 1H), 6.72 (d, J = 4.8 Hz, 1H), 6.69 – 6.56 (m, 1H), 6.35 – 6.19 (m, 1H), 4.28 (d, J = 13.5 Hz, 2H), 4.06 – 3.89 (m, 1H), 3.84 (t, J = 5.5 Hz, 2H), 3.20 – 3.03 (m, 2H), 2.82 (s, 6H), 2.42 (s, 3H), 1.90 (d, J = 12.9 Hz, 2H), 1.54 – 1.35(m, 2H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₇H₂₆N₃O₂S, 336.1740; found: 336.1761.

(E)-N-((1-(4-(dimethylamino)but-2-enoyl)azetidin-2-yl)methyl)-5-methylthiophene-2-carboxamide (42)

White solid, 10.9 mg, 34% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 7.55 – 7.42 (m, 1H), 6.85 – 6.63 (m, 2H), 6.59 – 6.41 (m, 1H), 4.78 – 4.57 (m, 1H), 4.24 (t, J = 7.8 Hz, 1H), 4.02 – 3.89 (m, 2H), 3.88 – 3.73 (m, 2H), 3.69 – 3.50 (m, 1H), 2.88 (d, J = 11.0 Hz, 6H), 2.59 – 2.41 (m, 4H), 2.24 – 2.08 (m, 1H). HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₁₆H₂₄N₃O₂S, 322.1584; found: 322.1602.

3-(5-(3,6-Diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanoic acid (I-33)

To a solution of 5-(3-methoxy-3-oxopropyl)thiophene-2-carboxylic acid (I-32) (214.2 mg, 1 mmol, 1.0 eq) and tert-butyl 3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (198.3 mg, 1 mmol, 1.0 eq) in DMF (10 mL) were added HATU (418.2 mg, 1.1 mmol, 1.1 eq) and DIEA(258.4 mg, 1 mmol, 2.0 eq). The reaction mixture was stirred at rt for 1 h, resulting crude product was purified via revers-ISCO to yield crude product. To a solution of crude product in THF/H₂O (1:1, 2 mL), was added LiOH (95.8 mg, 4 mmol, 4.0 eq). The reaction mixture was stirred at rt for 1 h, resulting crude product were purified via revers-ISCO to yield titled compound as a brown oil (146.1 mg, 52%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.52 – 7.31 (m, 1H), 7.01 – 6.80 (m, 1H), 4.51 – 4.42 (m, 1H), 4.42 – 3.94 (m, 2H), 3.84 – 3.64 (m, 1H), 3.59 – 3.49 (m, 1H), 3.13 – 3.05 (m, 2H), 3.03 – 2.89 (m, 1H), 2.69 – 2.56 (m, 2H), 1.99 – 1.78 (m, 1H), 1.34 – 1.31 (m, 1H). [M+H]⁺ 281.09

(E)-3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanoic acid (I-34)

To a solution of I-33 (280.3 mg, 1 mmol, 1.0 eq) and (E)-4-(dimethylamino)but-2-enoic acid (129.2 mg, 1 mmol, 1.0 eq) in DMF (5 mL) were added HATU (418.2 mg, 1.1 mmol, 1.1 eq) and DIEA(258.4 mg, 1 mmol, 2.0 eq). The reaction mixture was stirred at rt for 1 h, resulting crude product was purified via revers-ISCO to yield titled compound as brown oil(184.1 mg, 47%). ¹H NMR (400 MHz, Methanol-d₄) δ 7.46 (d, J = 3.8 Hz, 1H), 6.92 (d, J = 3.7 Hz, 1H), 6.79 – 6.69 (m, 1H), 6.62 (d, J = 15.9 Hz, 1H), 4.62 – 4.47 (m, 2H), 4.01 – 3.91 (m, 4H), 3.17 – 3.11 (m, 4H), 2.74 – 2.65 (m, 8H), 1.74 – 1.68 (m, 1H), 1.23 – 1.18 (m, 1H). [M+H]⁺ 392.16

(E)-3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropane-1-carboxamido)-3-methylpyridin-2-yl)-N-(6-(3-(5-(6-(4-(dimethylamino)but-2-enoyl)-3,6-diazabicyclo[3.1.1]heptane-3-carbonyl)thiophen-2-yl)propanamido)hexyl)benzamide (43)

To a solution of I-34 (10.2 mg, 0.026 mmol, 1.0 eq) in DMF (1 mL), HATU (41.8 mg, 0.11 mmol, 1.1 eq), DIEA (35 μL, 0.2 mmol, 2.0 eq) were added. Then, I-35 (14.3 mg, 0.026 mmol, 1.0 eq.) was added, the reaction mixture stirred at rt for 30 mins. After that, resulting reaction mixtures were purified via prep-HPLC to yield titled compound as a white solid, 9.4 mg, 39% yield. ¹H NMR (400 MHz, Methanol-d₄) δ 8.07 (d, J = 8.5 Hz, 1H), 7.89 (dt, J = 6.1, 2.2 Hz, 2H), 7.86 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.63 – 7.52 (m, 2H), 7.44 (s, 1H), 7.38 (d, J = 1.5 Hz, 1H), 7.32 (dd, J = 8.3, 1.7 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 3.6 Hz, 1H), 6.76 (dt, J = 14.7, 7.1 Hz, 1H), 6.57 (d, J = 15.3 Hz, 1H), 4.80 – 4.72 (m, 1H), 4.59 – 4.50 (m, 1H), 4.44 – 4.00 (m, 1H), 4.00 – 3.87 (m, 3H), 3.36 (t, J = 7.1 Hz, 2H), 3.21 – 3.09 (m, 4H), 2.89 (s, 6H), 2.83 – 2.77 (m, 1H), 2.54 (t, J = 7.2 Hz, 2H), 1.72 – 1.66 (m, 3H), 1.65 – 1.54 (m, 2H), 1.53 – 1.43 (m, 2H), 1.42 – 1.22 (m, 6H). ¹³C NMR (101 MHz, Methanol-d₄) δ 172.88, 172.57, 168.00, 165.19, 154.42, 149.37, 148.55, 143.95, 143.47, 142.24, 138.90, 135.02, 134.89, 134.72, 131.71, 131.52, 131.42, 130.74, 129.21, 128.36, 128.08, 127.61, 127.29, 127.01, 126.97, 125.32, 113.23, 112.30, 109.84, 60.20, 57.71, 57.38, 41.93, 39.52, 38.81, 36.91, 30.92, 28.94, 28.85, 28.53, 26.20, 26.07, 25.47, 17.49, 16.49.

HRMS (ESI-TOF) m/z: [M+H]⁺ calcd for C₄₉H₅₆F₂N₇O₇S, 924.3925; found: 924.3949.

Supplementary Material

The Supporting Information is available free of charge at

Molecular formula strings for all compounds (CSV); Figure S1, OTUB1 modification by compound 34 and MS5105; Figure S2, Western blot analysis of OTUB1 activity via deubiquitination assay; Figure S3, OTUB1/USP8-IN-1 inhibits OTUB1 deubiquitinase activity; and ¹H NMR, ¹³C NMR, and LC-MS spectra of compounds 34 and 43 (PDF).

Abbreviations Used

ACN

acetonitrile

Boc

tert-butyloxycarbonyl

DCM

dichloromethane

DIEA

diisopropylethylamine

DMF

dimethylformamide

HATU

O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

RT

room temperature

TFA

trifluoroacetic acid

WB

western blot

WT

wild type