Discovery and Characterization of a Chemical Probe Targeting the Zinc-Finger Ubiquitin-Binding Domain of HDAC6

Abstract

Histone deacetylase 6 (HDAC6) inhibition is an attractive strategy for treating numerous cancers, and HDAC6 catalytic inhibitors are currently in clinical trials. The HDAC6 zinc-finger ubiquitin-binding domain (UBD) binds free C-terminal diglycine motifs of unanchored ubiquitin polymer chains and protein aggregates, playing an important role in autophagy and aggresome assembly. However, targeting this domain with small molecule antagonists remains an underdeveloped avenue of HDAC6-focused drug discovery. We report SGC-UBD253 (25), a chemical probe potently targeting HDAC6-UBD in vitro with selectivity over nine other UBDs, except for weak USP16 binding. In cells, 25 is an effective antagonist of HDAC6-UBD at 1 μM, with marked proteome-wide selectivity. We identified SGC-UBD253N (32), a methylated derivative of 25 that is 300-fold less active, serving as a negative control. Together, 25 and 32 could enable further exploration of the biological function of the HDAC6-UBD and investigation of the therapeutic potential of targeting this domain.

Introduction

There are 18 human histone deacetylases (HDACs) that regulate a plethora of important cellular functions by catalyzing the removal of acetyl groups from lysine residues of both histone and non-histone proteins.¹ HDAC6 is a structurally distinct microtubule-associated cytosolic deacetylase, harboring two tandem catalytic deacetylase domains^2,3 and a zinc-finger ubiquitin-binding domain (UBD).⁴⁻⁶ The catalytic domains interact with dynein to transport aggregated proteins via microtubules to the aggresome for degradation,⁷⁻⁹ whereas the UBD binds free C-terminal diglycine motifs of polyubiquitin or polyISG chains associated with protein aggregates or other cargo.^10,11

Inhibiting HDAC6 function is postulated to have therapeutic benefits in a number of different cancers, neurodegenerative diseases, and other pathologies.¹²⁻¹⁴ To date, HDAC6 drug discovery has focused on inhibitors targeting the catalytic activity of this protein and is currently being tested in the clinic, in some cases in combination with proteasome inhibitors.¹⁵⁻¹⁸ HDAC6 catalytic inhibitors prevent the deacetylation of microtubules, which disrupts dynein-mediated transport of protein cargoes to the aggresome. However, current selective catalytic hydroxamate HDAC6 inhibitors have demonstrated selectivity and toxicity liabilities.¹⁹ An alternative approach to inhibit or modulate HDAC6 function is to target protein aggregate recognition by the UBD with small molecule antagonists, both in isolation or in combination with catalytic inhibition, as has been demonstrated by HDAC6 knockdown or HDAC6-degraders in the context of inflammation,^20,21 infection,²²⁻²⁴ or proteinopathy pathologies.²⁵

We previously identified and characterized the first small molecule binders of HDAC6-UBD, which can displace the native C-terminal ubiquitin RLRGG peptide from a narrow and deep pocket within this domain.²⁶ These compounds have a carboxylate that mimics the C-terminus of the ubiquitin substrate and an extended aromatic structure that forms π-stacking interactions with W1182 and R1155. Together with our subsequent structure–activity relationship analysis of this chemical series,²⁷ we found that it was possible to induce a conformational remodeling of the pocket to open up an additional side pocket, which we postulated could be exploited to increase potency and selectivity.

We describe the discovery and characterization of SGC-UBD253 (25), a potent antagonist HDAC6-UBD chemical probe, which is active in cells at 1 μM and selective against other UBDs, albeit with weak activity for USP16. We present the optimization of our previous lead ligand (1)²⁷ to improve potency using a fluorescence polarization (FP) C-terminal ubiquitin peptide displacement assay. This led to the identification of our probe candidate (25) and a negative control compound (32), which is structurally similar to 25 with an additional methyl group that drastically decreases activity. We validated the binding parameters of 25 and 32 with multiple orthogonal biophysical assays and showed that the chemical probe (25) exhibits significant activity in cells at 1 μM, while the negative control (32) is inactive at all concentrations tested. We determined, with chemoproteomic approaches, that 25 has marked proteome-wide selectivity and has significant cellular activity while limiting off-target activity for USP16 at a working concentration of 3 μM. These tool compounds will enable the biological discovery of the functional roles of the UBD of HDAC6 and serve as a foundation for further optimization to develop drugs that target HDAC6-UBD.

Results

Structure–Activity Relationship

We previously reported 1, a fragment hit, that binds to HDAC6-UBD, which we characterized with established FP (K_disp ∼ 2.3 μM) and surface plasmon resonance (SPR) assays (K_D ∼1.5 μM).^26,27 In the co-crystal structure of 1 with the HDAC6 ubiquitin-binding domain (PDB ID: 6CED), the quinazolinone ring is sandwiched between R1155 and W1182 and the carboxylic acid group makes a hydrogen bond between G1154 and Y1184, as well as with R1155. The co-crystal structure also reveals that the N-Methyl group of 1 could be extended into the adjacent pocket to make additional interactions with the residues lining this site (Figure 1).

Figure 1.

Structure and co-crystal structure of 1 in complex with HDAC6-UBD, PDB ID 6CED. (a) The structure of compound 1 and associated binding parameters from FP and SPR assays. (b) The co-crystal structure of 1 with HDAC6-UBD showing key hydrogen bond interactions. (c) Space-filled diagram showing an adjacent pocket.

To test this hypothesis, we first explored simple N-alkyl groups such as cyclopropylmethyl and benzyl groups, which could potentially extend into the side pocket, but they led to a significant loss of activity (data not shown). Therefore, we explored extended linker groups, starting from the corresponding methylacetamide derivative (9), which was synthesized according to Scheme 1. Anthranilic acid (2a) was converted into amide (3a), which was subsequently treated with succinic anhydride to give 4a. Cyclization of 4a in the presence of sodium hydroxide followed by esterification with ethanol gave the ethyl ester intermediate (6a). Alkylation of 6a with N-methylbromoacetamide (7a) followed by hydrolysis of the ester gave the methylacetamide derivative (9).

Scheme 1. Synthesis of Compounds 9–32.

Reagent and conditions: (a) 1.5 equiv of CDI, DCM; rt, 0.5 h; (b) 10 equiv of NH₄OH, 12 h; (c) 1.1 equiv of succinic anhydride, PhMe, reflux, 4–48 h; (d) 2 M aq NaOH, reflux, 2 h; (e) cat. H₂SO₄, EtOH, reflux, 16 h; (f) 1.2 equiv of K₂CO₃, DMF, rt, 16 h; (g) 4 equiv of LiOH·H₂O, 3:1:1 THF/EtOH/H₂O, rt, 4–16 h.

Compared to 1, the corresponding methylacetamide derivative (9) showed a ∼4-fold loss of binding activity. However, we were able to obtain a 1.55 Å resolution (PDB: 8G43) co-crystal structure of 9 with HDAC6-UBD, which revealed the potential to improve activity further (Figure 2 and Table S2). In the co-crystal structure, R1155 moves to open the side pocket. The carbonyl and NH groups of the amide of 9 make additional hydrogen bonds with R1155 and Y1189, respectively, compared to 1, while maintaining the π-stacking of the quinazolinone core with R1155 and W1182 and hydrogen bond of the carboxylate with G1154 and Y1184. The co-crystal structure with 9 indicated that other larger amide groups could be extended into the side pocket to further improve activity.

Figure 2.

Structure and co-crystal structure of 9 with HDAC6-UBD, PDB ID 8G43. (a) The structure of compound 9. (b) Space-filled diagram of the co-crystal structure of 9 with HDAC6-UBD showing the adjacent pocket. (c) Key hydrogen bond interactions formed in this complex. (d) Overlay of the HDAC6-UBD co-crystal structure with 9 (in gray) and with 1 (in green) (for clarity, the structure of ligand 1 is not shown), highlighting the movement of R1155 by an arrow (in black).

To identify the optimal substitutions occupying the adjacent pocket and thus improve activity, we systematically explored an array of amides. These amide derivatives 10–22 were synthesized following Scheme 1 by using the corresponding amide derivatives of 7b–n, which were synthesized by simple amide coupling of commercially available amines with bromoacetyl bromide. The structure–activity relationship of these amides is summarized in Table 1. While simple amides such as ethyl (10), cyclopropylmethyl (11), and tertiary butyl (12) amides are tolerated, they did not improve activity significantly. However, the larger lipophilic adamantyl (13), cyclohexylmethyl (14), and benzyl groups (15) improve the binding activity 3–20 fold compared to methyl (9), from K_disp = 14 μM (9) to K_disp = 5.1, 2.0, and 0.62 μM respectively.

Table 1. Structure and Activity of Amide Substitutions (9–22)^a.

^aK_disp determination experiments were performed by FP with N ≥ 3, and the values are presented as mean ± SD, reported to 2 significant figures.

To confirm that the phenyl ring of 15 extends into the side pocket, we solved the crystal structure of HDAC6-UBD in complex with 15 to 1.55 Å resolution (PDB: 8G44) (Figure 3 and Table S2). As expected, the phenyl group of 15 occupies the side pocket and maintains the same interaction as 9 while making additional hydrophobic interactions with side pocket residues E1141 and I1177. The co-crystal structure with 15 also revealed that a hydrogen bond donor at the ortho position of the phenyl ring could pick up an additional interaction with Y1189, and indeed, the ortho-OMe group (16) improved binding activity 3-fold compared to the phenyl analogue (15), while a Cl group at this position (17) led to 3-fold loss of activity. A CF₃ group at the para position (18) was detrimental to binding. 3-Pyridyl (19) and para-OMe (20) analogues were designed to make additional interactions with D1178 or E1141 and S1175, respectively, but led to a significant loss of activity compared to 15. Extending the linker length of the amides (21, 22) was not beneficial.

Figure 3.

Structure and co-crystal structure of 15 with HDAC6-UBD, PDB ID 8G44. (a) The structure of compound 15. (b) The co-crystal structure of 15 with HDAC6-UBD showing key hydrogen bond interactions. (c) The space-filled diagram showing a phenyl group occupying the adjacent pocket.

Based on the co-crystal structure of 15, we envisioned that a simple alkyl or halo group at the 8-position of the quinazolinone ring could make a hydrophobic interaction with L1162 and thus improve activity further. Hydrophobic substitutions could also improve cell permeability, so we also explored simple hydrophobic substitutions at the 6-position of the quinazolinone ring, which could point toward the adjacent pocket. To test this hypothesis, we synthesized analogues of 16 with substitutions at the 6- and/or 8-positions of the quinazolinone ring (23–26) according to Scheme 1, starting from appropriately substituted anthranilic acids (2b–2e). The structure–activity relationship of substituted quinazolinone analogues is summarized in Table 2. While fluoro (23) and methyl (24) groups maintain activity, a chloro (25) group at the 8-position improves activity 2-fold compared to the unsubstituted quinazolinone analogue (16), from K_disp= 0.18 μM (16) to K_disp = 0.095 μM. An additional iodo group at the 6-position (26) was also tolerated without significant loss of activity.

Table 2. Structure and Activity of Substituted Quinazolinones 16, 23–26.

compound	R1	R2	HDAC6 FP Kdisp [μM]a
16	H	H	0.18 ± 0.026
23	F	H	0.23 ± 0.12
24	Me	H	0.32 ± 0.27
25	Cl	H	0.095 ± 0.018
26	Cl	I	0.55 ± 0.26

^aK_disp determination experiments were performed by FP with N ≥ 3, and the values are presented as mean ± SD, reported to 2 significant figures.

To identify opportunities to further improve the activity, we solved the crystal structure of HDAC6-UBD in complex with 25 to 1.55 Å resolution (PDB: 8G45) (Figure 4 and Table S2). As expected, the quinazolinone ring is sandwiched between R1155 and W1182 and maintains the same hydrogen bond interactions as 15 (Figure 4). However, the quinazolinone ring of 25 moves slightly away from L1162 to accommodate the chloro group without significant movement of the rest of the molecule or the sidechains of the binding site.

Figure 4.

Structure and co-crystal structure of 25 with HDAC6-UBD, PDB ID 8G45. (a) Structure of compound 25 (SGC-UBD253) and negative control 32 (SGC-UBD253N), (b) Omit map (σ2) of compound 25, (c) co-crystal structure of 25 with HDAC6-UBD showing key hydrogen bond interactions, (d) space-filled diagram showing phenyl group occupying the adjacent pocket.

With the optimal substitutions identified at the quinazolinone core, we wanted to identify the best amide group for binding activity and cellular activity. As amides with both hydrogen bond donors and with lipophilic substitutions were tolerated, we explored additional amide groups incorporating these features, as summarized in Table 3.

Table 3. Structure and Activity of Amide Substitutions (27–32)^a.

^aK_disp determination experiments were performed by FP with N ≥ 3, and the values are presented as mean ± SD, reported to 2 significant figures.

To identify a close analogue that may serve as a negative control, we also synthesized 32 (SGC-UBD253N) by methylating the amide linker, which acts as a key hydrogen bond donor. As expected, 32 lost almost all binding activity compared to the close analogue 25 and is an excellent negative control for cellular experiments.

Furthermore, lipophilic substitutions at the ortho position, CH₃ (27), F (28), and Cl (29) groups, showed comparable activity to the ortho-OMe analogue (25). We explored tetrahydropyran (30) and tetrahydrofuran (31) analogues, as both have oxygen atoms that could make hydrogen bond interactions with Y1189. While the tetrahydropyran (30) analogue showed a modest decrease in activity, the tetrahydrofuran (31) analogue showed a significant drop in activity (10-fold), probably because the tetrahydrofuran group may not occupy the pocket efficiently.

In Vitro Characterization of 25 and 32

25 and 32 were selected as candidate probe and negative control compounds, respectively, both warranting further characterization by orthogonal biophysical assays (Figures 4 and S2, and Table 4). Both were characterized by SPR and isothermal calorimetry (ITC). 25 binds potently to HDAC6-UBD with K_D values of 0.084 and 0.080 μM as measured by SPR and ITC, respectively. In contrast, 32 was shown to bind weakly with a K_D of 32 μM as determined by SPR. Binding parameters of 25 were also determined for full-length HDAC6 using FP and SPR, yielding K_disp and K_D values of 0.44 and 0.26 μM for these assays, respectively. As we determined with the C-terminal ubiquitin peptide substrate (Table S1), 25 binds more potently to the isolated HDAC6-UBD than the full-length protein.

Table 4. Summary of Biophysical Characterization of 25 and 32^a,b.

^aK_disp determination experiments were performed with N ≥ 3, and the values are presented as mean ± SD, reported to 2 significant figures.

^bK_D determination experiments were performed with N ≥ 3, and the values are presented as mean ± SD. NT = not tested.

Next, we tested the selectivity of 25 and 32 for HDAC6-UBD using a panel of UBD proteins. 25 was at least ∼50-fold selective for HDAC6 over other UBD proteins tested, with the exception of USP16, for which 25 is 15-fold selective as measured by SPR (Table 5). USP16 has the highest degree of conservation of binding pocket residues compared to HDAC6, with 53.3% sequence identity for residues within 5 Å of the bound ligand in the pocket (PDB ID: 8G45). Extensive chemistry efforts were undertaken to further improve 25, generating large arrays of compounds with alternative substitutions on both the quinazolinone and methoxyphenyl moieties. Unfortunately, no compound was identified with improved selectivity for HDAC6 over USP16 while maintaining significant HDAC6 activity.

Table 5. Selectivity of 25 and 32 against a Panel of UBDs by SPR.

UBD	KD (μM)a25	KD (μM)a32	fold selectivity of 25 [UBD KD /HDAC6 KD]
USP3	11 ± 1.5	47 ± 4.4	130
USP5	4.5 ± 1.9	6.3 ± 2.1	53
USP13	NB	NB	NC
USP16	1.3 ± 0.04	4.9 ± 0.26	15
USP20	NB	NB	NC
USP33	NB	NB	NC
USP39	NB	NB	NC
USP49	NB	NB	NC
USP51	NB	NB	NC
BRAP	NB	92 ± 10	NC
HDAC6	0.084 ± 0.020	32 ± 7.3

^aK_D determination experiments were performed with N ≥ 3, and the values are presented as mean ± SD, reported to 2 significant figures. NB = K_D greater than the highest concentration of the compound tested. NC = not calculated as no K_D determined due to NB.

25 was tested to assess possible inhibition of the deacetylase activity of HDAC6 using a Boc-Lys(TFA)-AMC substrate assay, but no inhibition was observed (Figure S3). Similarly, inhibition of the ubiquitin peptidase activity of full-length USP proteins by 25 was assessed for USP3, USP5, USP16, and USP33 using a ubiquitin rhodamine substrate assay, but no inhibition was observed, except for USP5. However, the IC₅₀ in this in vitro assay of 25 for USP5 is 8.0 ± 1.1 μM, so this is unlikely to have significant consequences for the downstream use of this compound. We also tested HDAC6 catalytic activity in cells, monitoring α-tubulin acetylation, and determined that treatment with 25 did not inhibit the catalytic activity of HDAC6 (Figure S4).

Characterization of 25 and 32 in Cells

It has been demonstrated that designed ankyrin repeat proteins (DARPins) that antagonize the HDAC6-UBD can impair HDAC6 cellular function, so we sought to investigate 25 in cell-based assays.²⁴ To directly assess if 25 antagonizes HDAC6-UBD in live cells, we developed a nano-bioluminescence resonance energy transfer (NanoBRET) protein–protein interaction assay (Figure 5a). This assay detects protein interactions, and their disruption with antagonists, by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor.²⁸ ISG15 is a small ubiquitin-like modifier (SUMO) that is covalently attached to target proteins in a manner similar to ubiquitin. Importantly, both ISG15 and ubiquitin share the same C-terminal ‘LRLRGG’ motif,²⁹ allowing ISG15 to also be recognized by HDAC6-UBD. For cellular NanoBRET assays, ISG15 was selected as the acceptor protein for the HDAC6 donor instead of ubiquitin because of the latter’s high cellular abundance and, thus, high background signal in the assay. Using the HDAC6/ISG15 assay format, 25 was shown to significantly decrease the interaction between full-length HDAC6 and ISG15 compared to 32, with EC₅₀ values of 1.9 ± 0.61 and >30 μM, respectively, in HEK293T cells (Figure 5b). To validate the assay specificity, we identified a mutant HDAC6^{R1155A, Y1184A}, which removes key hydrogen bond interactions with the terminal glycine of ubiquitin substrates, resulting in decreased interaction with ISG15 in vitro (Table S1). Therefore, a donor construct bearing these mutations was used to define the baseline (binding-deficient HDAC6) BRET signal in the assay (Figure 5b). Compound 25 was shown to disrupt the wildtype HDAC6/ISG15 BRET signal in a dose-dependent manner to the same level as HDAC6^{R1155A, Y1184A}. To assess the selectivity of 25 in cells, a NanoBRET assay was designed to assess USP16-ISG15 interaction. Similar to the HDAC6 NanoBRET assay, an ISG15 binding-deficient mutant of USP16 (R84A/Y117A) was used to define the baseline of the assay. An EC₅₀ of 20 ± 2.7 μM was determined using this assay in HEK293T cells, indicating approximately 10-fold selectivity of 25 for HDAC6 over USP16 under these conditions. The negative control candidate, 32, was inactive. Analysis of the dose-dependent antagonism of both HDAC6 and USP16 suggests that using 25 at a concentration of 3 μM would limit the off-target effects of USP16 while achieving significant antagonism of HDAC6.

Figure 5.

Assessment of cell-based target engagement of 25 and 32. (a) Schematic of NanoBRET assay, which measures the BRET from the interaction between HDAC6 or USP16 tagged with the NanoLuc donor and ISG15 tagged with acceptor HaloTag (HT) in the presence of the HaloTag NanoBRET 618 Ligand (HL). (b) 25 inhibited interaction of HDAC6 and ISG15 (EC₅₀ = 1.9 ± 0.61 μM) and USP16 and ISG15 (EC₅₀ = 20 ± 2.7 μM) in HEK293T cells, while 32 was inactive. 3 μM is the recommended concentration (highlighted in green) to achieve selectivity for HDAC6 over USP16 for 25 in cells. Experimental triplicates of mean corrected NanoBRET ratios normalized to DMSO control are presented. (c) (i) The structure of compound 33 (the biotin derivative of 25). (ii) Western blot analysis of proteins pulled down from lysates by 33 after pretreatment with DMSO, of 10 μM of 32, or 25. Replicate experiments show that pretreatment with 25, but not 32, inhibits HDAC6 pull-down with 33. (d) Selectivity profiling by label-free chemical proteomics in the cytoplasmic fraction. The volcano plot is annotated with significantly enriched proteins where log 2(Fold Change) <−2 and p-value ≤0.05 (n = 3 independent replicates). Data analysis was completed using the Bioconductor packages DEP.³¹ Parallel analysis using proDA³² gave the output in Supporting Data Files.

To further characterize the selectivity of 25, we synthesized 33 (Figure 5c(i)), a biotin-labeled derivative of 25, for use as an affinity reagent for proteome-wide cellular target engagement profiling. The affinity of 33 for HDAC6-UBD was measured by FP and was found to be comparable to 25. Two enrichment-based experiments were performed. First, we completed a competition assay in which HDAC6 was pulled down from HEK293T cell lysates treated with DMSO, 32, or 25 (10 μM each). Treated lysates were incubated with 33 bound to streptavidin beads. After washing, the bound fraction was analyzed by western blot (Figure 5c(ii)), revealing that pretreatment with 25, but not DMSO or 32, inhibited the pull-down of HDAC6 from cells with 33. Next, we performed a chemical proteomics analysis of material pulled down from the cytoplasmic fraction of HEK293T cells by 33-conjugated streptavidin beads. Preincubation with 25 prevented enrichment of HDAC6 by 33, whereas preincubation with 32 did not affect the enrichment profile (Figure 5d). USP16 was not observed in the analysis from either pretreatment condition. Since USP16 and HDAC6 are both localized to the cytoplasm,^2,30 this demonstrates that 25 is selective for endogenous HDAC6 over endogenous USP16.

Conclusions

We report the successful identification of an HDAC6-UBD chemical probe (25) (SGC-UBD253) and its negative control (32) (SGC-UBD253N), which have been thoroughly characterized with in vitro biophysical assays, crystal structures, and functional cellular target engagement assays. 25 is a potent antagonist of HDAC6-UBD, is active in cells, and shows good selectivity against other UBD, albeit with slight activity against the UBD of USP16 under some conditions. Additionally, we generated a biotin derivative of 25, which we validated as a useful tool for chemoproteomic experiments and demonstrated strong proteome-wide selectivity of 25 for HDAC6. We believe that the tools presented here will enable biological investigation of HDAC6-UBD and serve as a robust foundation for future applications such as targeted degradation agents and/or other proximity-inducing agents.

Experimental Section

Compound Synthesis

General Considerations

Unless otherwise stated, all reactions were carried out under an inert atmosphere of dry argon or nitrogen utilizing glassware that was either oven (120 °C) or flame-dried. Workups and isolation of the products were conducted on the benchtop using standard techniques. Reactions were monitored using thin-layer chromatography (TLC) on SiliaPlate Silica Gel 60 F254 plates. Visualization of the plates was performed under ultraviolet (UV) light (254 nm) or using KMnO₄ stains. Toluene was distilled over calcium hydride, and anhydrous N,N-dimethylformamide (DMF) was purchased from Fisher Sciences and used as received. Silica gel flash column chromatography was performed on Silicycle 230–400 mesh silica gel. Mono- and multidimensional NMR characterization data were collected at 298 K on a Varian Mercury 300, Varian Mercury 400, Bruker Avance II, Agilent 500, or a Varian 600. ¹H NMR spectra were internally referenced to the residual solvent peak (CDCl₃ = 7.26 ppm, DMSO-d₆ = 2.50 ppm). ¹³C{¹H} NMR spectra were internally referenced to the solvent peak (CDCl₃ = 77.16 ppm, DMSO-d₆ = 39.52 ppm). ¹⁹F NMR chemical shifts are reported in ppm with absolute reference to ¹H NMR data are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant (Hz), and integration. Coupling constants have been rounded to the nearest 0.05 Hz. The NMR spectra were recorded at the NMR facility of the Department of Chemistry at the University of Toronto. Infrared spectra were recorded on a Perkin-Elmer Spectrum 100 instrument equipped with a single-bounce diamond/ZnSe ATR accessory and are reported in wavenumber (cm^–1) units. High-resolution mass spectra (HRMS) were obtained on a micromass 70S-250 spectrometer (EI), an ABI/Sciex QStar Mass Spectrometer (ESI), or a JEOL AccuTOF model JMS-T1000LC mass spectrometer equipped with an IONICS direct analysis in real time (DART) ion source at the Advanced Instrumentation for Molecular Structure (AIMS) facility of the Department of Chemistry at the University of Toronto. Analytical HPLC analyses were carried out on an Agilent 1100 series instrument equipped with a Phenomenex KINETEX column (2.6 μm, C18, 50 mm × 4.6 mm). A linear gradient starting from 5% acetonitrile and 95% water (0.1% formic acid) to 95% acetonitrile and 5% water (0.1% formic acid) over 4 min followed by 5 min of elution at 95% acetonitrile and 5% water (0.1% formic acid) was employed. The flow rate was 1 mL/min, and UV detection was set to 254 and 214 nm. HPLC analyses were conducted at room temperature. All compounds submitted for testing were at ≥95% purity (by HPLC, by UV detection at 254 nm) unless otherwise stated. Purities of all compounds were estimated to be >95%, as no significant impurities were detected in the chromatogram.

General Procedure 1 (Synthesis of α-Bromoamide Derivatives 7b–7t)

The aldehyde derivative (1 equiv) was added dropwise to a suspension of the hydroxylamine·HCl salt in absolute ethanol (1.5 M) and stirred at room temperature for 3–16 h (depending on the ketone or aldehyde). Upon consumption of the starting material, concentrated HCl (4 equiv) was added to the reaction. The solution was then cooled to 0 °C, and zinc powder was added portion-wise. Following the complete addition of the zinc powder, the reaction was warmed to room temperature and stirred for 10 min. A 2:1 solution of 6 M NaOH and NH₄OH (1.07 M, relative to starting material) was added, resulting in the formation of a white precipitate. The suspension was filtered through a pad of Celite. The white solids were washed three times with DCM. The filtrate was collected and extracted three times with DCM, dried over MgSO₄, filtered, and concentrated in vacuo. The amine was used in the next step without further purification. Bromoacetyl bromide (1.2 equiv) was added dropwise at 0 °C to a solution of the amine (1 equiv) and K₂CO₃ (1.2 equiv) in DCM (0.4 M). The solution was warmed to room temperature and stirred for 4–12 h. The reaction was carefully quenched by dropwise addition of water. The aqueous layer was extracted three times with DCM. The organic layers were combined, dried over MgSO₄, filtered, and concentrated in vacuo. The resultant α-bromoamides were used without further purification.

General Procedure 2 (Synthesis of Quinazolinone Derivatives: 9–32)

3a–3e: The anthranilic acid derivative (1 equiv) and CDI (1.5 equiv) were added to a round-bottomed flask connected to an oil bubbler. The reagents were dissolved in DCM (0.4 M) and stirred at room temperature for 30 min. The round-bottom flask was fitted with an addition funnel, and NH₄OH (10 equiv) was added dropwise. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc, washed twice with 1 M HCl, once with an aqueous solution of NaHCO₃, once with water, and once with brine. The organic layer was dried over MgSO₄, filtered, and concentrated in vacuo. The resulting anthranilamide derivatives were used in the next step without further purification.

4a–4e: In a flask equipped with a reflux condenser, succinic anhydride (1.1 equiv) and the anthranilamide (3a–e) (1 equiv) were dissolved in toluene and stirred at reflux for 4–48 h. The reaction mixture was cooled to room temperature and filtered. The white precipitate was washed with water, Et₂O, and a small amount of absolute ethanol. The product was dried and used without further purification in the next step.

5a–5e: In a flask equipped with a reflux condenser, the intermediate bis-amide (4a–e) was dissolved in 2 M NaOH (0.2 M). The resulting solution was stirred at reflux and then cooled to room temperature or treated with 2 M K₂CO₃ at room temperature for 2 h. The solution was carefully acidified to pH 4–6 using concentrated HCl while stirring. The white precipitate was filtered and washed with water and dried. The resulting quinazolinone was used in the next step without further purification.

6a–6e: The acid (5a–e) was dissolved in EtOH (0.1 M) in a flask equipped with a reflux condenser. A catalytic amount of H₂SO₄ (1 drop/mmol) was added, and the solution was stirred at reflux for 16 h. The reaction was cooled to room temperature and diluted with water and then cooled to −40 °C in a freezer. The resulting white solid was filtered, washed with a saturated solution of NaHCO₃ and then water, and dried. The product was used in the next step without further purification.

8a–8t: NaH (2 equiv) was added to a solution of the secondary amide (6a–e) (2 equiv) in THF (0.4 M) at 0 °C in a flask. The solution was stirred at 0 °C until bubbling had ceased. α-Bromoamide derivatives (7a–s) (2 equiv) were added dropwise at 0 °C. The solution was warmed to room temperature and stirred for 12 h. The reaction was quenched with water and diluted with EtOAc. The aqueous layer was extracted three times with EtOAc. The organics were combined, dried over MgSO₄, filtered, and concentrated in vacuo. The resulting tertiary amide was used in the next step without further purification.

9–32: In a flask containing the ester (8a–t) (1 equiv) and LiOH·H₂O (4 equiv) was added a solution of 3:1:1 THF/H₂O/EtOH (0.05 M). Upon complete consumption of the starting material, the solution was neutralized to pH 6–7 using 1 M HCl. The organics were removed in vacuo, and the resulting solid was filtered, and washed with water, acetone, and then Et₂O. In some cases, the material was further purified by flash column chromatography (1:1 acetone/PhMe with 0.1% acetic acid) or by other means (specified for each compound).

3-(3-(2-(Methylamino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (9)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.18 (s, 1H), 8.23 (q, J = 4.6 Hz, 1H), 8.09 (dd, J = 7.9, 1.6 Hz, 1H), 7.81 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.58 (dd, J = 8.2, 1.1 Hz, 1H), 7.50 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 2.97 (t, J = 6.8 Hz, 2H), 4.78 (s, 2H), 2.75 (t, J = 6.8 Hz, 2H), 2.64 (d, J = 4.6 Hz, 3H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.7, 166.9, 161.2, 156.3, 146.7, 134.4, 126.8, 126.4, 126.2, 119.7, 45.2, 30.0, 28.7, 25.7. IR (neat): 3298, 2936, 1675, 1655, 1597, 1588, 1391, 1236, 1185, 974, 905, 765, 705. HRMS: ESI⁺, calcd for C₁₄H₁₆N₃O₄ 290.11408 [M + H]⁺, found 290.11398.

3-(3-(2-(Ethylamino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (10)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.17 (s, 1H), 8.31 (t, J = 5.5 Hz, 1H), 8.09 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.58 (dd, J = 8.2, 1.0 Hz, 1H), 7.50 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 4.78 (s, 2H), 3.12 (qd, J = 7.2, 5.4 Hz, 2H), 2.97 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H), 1.05 (t, J = 7.2 Hz, 3H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 174.1, 166.6, 161.6, 156.8, 147.1, 134.9, 127.2, 126.9, 126.7, 120.2, 45.6, 34.1, 30.5, 29.2, 15.0. IR (neat): 3300, 2927, 1653, 1597, 1555, 1393, 1184, 942, 766, 708, 690. HRMS: ESI⁺, calcd for C₁₅H₁₈N₃O₄ 304.12973 [M + H]⁺, found 304.12995.

3-(3-(2-(Cyclopropylamino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (11)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.18 (s, 1H), 8.41 (d, J = 4.1 Hz, 1H), 8.09 (ddd, J = 7.9, 1.6, 0.6 Hz, 1H), 7.81 (ddd, J = 8.6, 7.1, 1.6 Hz, 1H), 7.58 (dt, J = 8.0, 0.9 Hz, 1H), 7.50 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 4.74 (s, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H), 2.62–2.71 (m, 1H), 0.61–0.67 (m, 2H), 0.42–0.47 (m, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.6, 167.5, 161.1, 156.3, 146.6, 134.4, 126.8, 126.4, 126.2, 119.7, 45.1, 30.0, 28.7, 22.4, 5.6. IR (neat): 3283, 3083, 2960, 1648, 1654, 1598, 1557, 1397, 1272, 1182, 775, 708. HRMS: ESI⁺, calcd for C₁₆H₁₈N₃O₄ 316.12973 [M + H]⁺, found 316.13027.

3-(3-(2-(tert-Butylamino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (12)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.16 (s, 1H), 8.09 (ddd, J = 7.9, 1.6, 0.6 Hz, 1H), 8.00 (s, 1H), 7.80 (ddd, J = 8.2, 7.1, 1.6 Hz, 1H), 7.58 (ddd, J = 8.2, 1.1, 0.5 Hz, 1H), 7.50 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 4.76 (s, 2H), 2.94 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H), 1.27 (s, 9H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.6, 165.6, 161.1, 156.4, 146.7, 134.4, 126.8, 126.4, 126.2, 119.6, 50.5, 45.2, 30.0, 28.7, 28.4. IR (neat): 3473, 3298, 3097, 2981, 2927, 1722, 1636, 1592, 1567, 1394, 1364, 1222, 1183, 780. HRMS: ESI⁺, calcd for C₁₇H₂₂N₃O₄ 332.16103 [M + H]⁺, found 332.16063.

3-(3-(2-((Adamantan-1-yl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (13)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.14 (s, 1H), 8.09 (dd, J = 8.0, 1.5 Hz, 1H), 7.88 (s, 1H), 1.61 (s, 6H), 7.80 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.45–7.53 (m, 1H), 4.76 (s, 2H), 2.93 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H), 2.01 (s, 3H), 1.94 (s, 6H). ¹³C{¹H}-NMR (DMSO-d₆, 125 MHz): δ 173.6, 165.3, 161.1, 156.4, 146.6, 134.4, 126.7, 126.4, 126.2, 119.6, 51.2, 45.2, 40.9, 30.0, 28.8, 28.7. IR (neat): 3309, 2910, 2853, 2663, 1600, 1547, 1343, 1278, 1245, 1182, 780. HRMS: ESI⁺, calcd for C₂₃H₂₈N₃O₄ 410.20798 [M + H]⁺, found 410.20843.

3-(3-(2-((Cyclohexylmethyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (14)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.18 (s, 1H), 8.28 (t, J = 5.9 Hz, 1H), 8.09 (dd, J = 8.0, 1.5 Hz, 1H), 7.80 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.58 (dd, J = 8.2, 0.9 Hz, 1H), 7.49 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 4.80 (s, 2H), 3.04–2.89 (m, 2H), 2.75 (t, J = 6.9 Hz, 2H), 1.74–1.64 (m, 4H), 1.63–1.57 (m, 1H), 1.49–1.31 (m, 1H), 1.26–1.03 (m, 3H), 0.95–0.79 (m, 2H). ¹³C{¹H}H NMR (DMSO-d₆, 125 MHz): δ 173.6, 166.4, 161.2, 156.3, 146.7, 134.4, 126.8, 126.4, 126.2, 119.7, 45.1, 45.1, 37.5, 30.4, 30.0, 28.8, 26.0, 25.4. HRMS: ESI⁺, calcd for C₂₀H₂₆N₃O₄ 372.19233 [M + H]⁺, found 372.19233.

3-(3-(2-(Benzylamino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (15)

¹H NMR (DMSO-d₆, 500 MHz): δ 8.90 (t, J = 5.8 Hz, 1H), 8.11 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.60 (dd, J = 8.2, 1.0 Hz, 1H), 7.51 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 4.89 (s, 2H), 7.21–7.40 (m, 5H), 4.34 (d, J = 5.9 Hz, 2H), 3.01 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). The signal of the COOH group is extremely broad. ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.6, 166.6, 161.2, 156.4, 146.5, 139.0, 134.5, 128.3, 127.2, 126.9, 126.7, 126.5, 126.2, 119.7, 45.3, 42.3, 30.1, 28.8. IR (neat): 3285, 3065, 2940, 1652, 1597, 1552, 1391, 1248, 1179, 971, 774, 744, 695. HRMS: ESI⁺, calcd for C₂₀H₂₀N₃O₄ 366.14538 [M + H]⁺, found 366.14496.

3-(3-(2-((2-Methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (16)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.20 (s, 1H), 8.67 (t, J = 5.8 Hz, 1H), 8.11 (dd, J = 8.0, 1.6 Hz, 1H), 7.81 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.65–7.57 (m, 1H), 7.53–7.45 (m, 1H), 7.34–7.18 (m, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.93 (t, J = 7.3 Hz, 1H), 4.89 (s, 2H), 4.29 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H), 3.01 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.7, 166.6, 161.2, 156.7, 156.3, 146.7, 134.5, 128.2, 127.9, 126.8, 126.5, 126.3, 126.2, 119.7, 110.5, 55.3, 45.2, 37.5, 30.1, 28.8. HRMS: ESI⁺, calcd for C₂₁H₂₂N₃O₅ 396.15595 [M + H]⁺, found 396.15592.

3-(3-(2-((2-Chlorobenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (17)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.22 (s, 1H), 8.88 (t, J = 5.8 Hz, 1H), 8.11 (dd, J = 7.9, 1.5 Hz, 1H), 7.81 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.59 (dd, J = 8.3, 1.0 Hz, 1H), 7.50 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.43 (ddd, J = 12.0, 7.6, 1.7 Hz, 2H), 7.38–7.25 (m, 2H), 4.92 (s, 2H), 4.40 (d, J = 5.7 Hz, 2H), 3.02 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.7, 166.9, 161.2, 156.2, 146.7, 135.9, 134.5, 132.1, 129.2, 128.9, 128.8, 127.2, 126.8, 126.5, 126.2, 119.7, 45.3, 40.3, 30.1, 28.8. HRMS: ESI⁺, calcd for C₂₀H₁₉ClN₃O₄ 400.10641 [M + H]⁺, found 400.10649.

3-(4-Oxo-3-(2-oxo-2-((4-(Trifluoromethyl)benzyl)amino)ethyl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (18)

¹H NMR (DMSO-d₆, 400 MHz): δ 9.19 (t, J = 6.0 Hz, 1H), 8.10 (dd, J = 8.1, 1.1 Hz, 1H), 7.80 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.60 (dt, J = 8.2, 1.0 Hz, 1H), 7.56–7.45 (m, 3H), 4.43 (d, J = 5.8 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 2.60 (t, J = 7.1 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 100 MHz,): δ 174.3, 167.2, 161.4, 157.8, 147.0, 144.1, 134.3, 127.8, 127.6, 127.3, 126.7, 126.2, 125.2 (1, J = 3.6 Hz), 119.7, 45.7, 41.9, 33.1, 30.4. ¹⁹F NMR (DMSO-d₆, 377 MHz) δ −60.8. IR (neat): 3281, 2930, 1657, 1597, 1548, 1421, 1387, 1327, 1251, 1166, 1122, 1069, 778, 703, 648. HRMS: DART, calcd for C₂₁H₁₉F₃N₃O₄ 434.13277 [M + H]⁺, found 434.13209.

3-(4-Oxo-3-(2-oxo-2-((Pyridin-3-ylmethyl)amino)ethyl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (19)

¹H NMR (DMSO-d₆, 400 MHz): δ 12.19 (s, 1H), 8.90 (t, J = 5.9 Hz, 1H), 8.52 (s, 1H), 8.47 (d, J = 4.7 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.50 (td, J = 7.6, 7.1, 1.2 Hz, 1H), 7.36 (dd, J = 7.8, 4.7 Hz, 1H), 4.88 (s, 2H), 4.37 (d, J = 5.8 Hz, 2H), 3.00 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 101 MHz): δ 173.7, 166.9, 161.2, 156.2, 148.7, 148.2, 146.7, 135.0, 134.5, 134.5, 126.8, 126.5, 126.2, 123.5, 119.7, 45.4, 40.0, 30.0, 28.8. HRMS: DART, calcd for C₂₀H₁₈ClFN₃O₄ 418.09644 [M + H]⁺, found 418.09634.

3-(3-(2-((4-Methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (20)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.21 (s, 1H), 8.77 (t, J = 5.9 Hz, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 4.86 (s, 2H), 4.26 (d, J = 5.9 Hz, 2H), 3.73 (s, 3H), 3.00 (t, J = 6.9 Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.7, 166.5, 161.2, 158.3, 156.3, 146.7, 134.5, 130.9, 128.61 126.8, 126.5, 126.3, 119.8, 113.8, 55.1, 45.3, 41.9, 30.1, 28.8. HRMS: ESI⁺, calcd for C₂₁H₂₂N₃O₅ 396.15595 [M + H]⁺, found 396.15549.

3-(4-Oxo-3-(2-oxo-2-(Phenethylamino)ethyl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (21)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.20 (s, 1H), 8.41 (t, J = 5.6 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.58 (dd, J = 8.2, 1.0 Hz, 1H), 7.50 (ddd, J = 8.1, 7.2, 1.2 Hz, 1H), 7.36–7.27 (m, 2H), 7.25–7.15 (m, 3H), 4.77 (s, 2H), 3.40–3.25 (m, 3H), 2.91 (t, J = 6.8 Hz, 2H), 2.74 (td, J = 7.1, 3.2 Hz, 4H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.6, 166.4, 161.1, 156.2, 146.6, 139.3, 134.4, 128.7, 128.3, 126.8, 126.4, 126.2, 126.1, 119.7, 45.1, 40.4, 35.0, 30.0, 28.7. HRMS: ESI⁺, calcd for C₂₁H₂₂N₃O₄ 380.16103 [M + H]⁺, found 380.16087.

3-(3-(2-((2-Methoxyphenethyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (22)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.19 (s, 1H), 8.38 (t, J = 5.7 Hz, 1H), 8.09 (dd, J = 8.0, 1.5 Hz, 1H), 7.81 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.64–7.55 (m, 1H), 7.50 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.20 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.13 (dd, J = 7.4, 1.8 Hz, 1H), 6.95 (dd, J = 8.3, 1.0 Hz, 1H), 6.87 (td, J = 7.4, 1.1 Hz, 1H), 4.77 (s, 2H), 3.78 (s, 3H), 3.32–3.26 (m, 2H), 2.92 (t, J = 6.8 Hz, 2H), 2.74 (dt, J = 10.3, 6.8 Hz, 4H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.7, 166.3, 161.1, 157.2, 156.2, 146.7, 134.4, 130.1, 127.6, 126.9, 126.8, 126.4, 126.2, 120.3, 119.7, 110.7, 55.3, 45.1, 38.9, 30.0, 29.9, 28.7. HRMS: DART, calcd for C₂₂H₂₄N₃O₅ 410.17160 [M + H]⁺, found 410.17235.

3-(8-Fluoro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (23)

¹H NMR (DMSO-d₆, 500 MHz): δ 8.68 (t, J = 5.8 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 9.2 Hz, 1H), 7.48 (td, J = 8.0, 4.5 Hz, 1H), 7.25 (ddd, J = 10.2, 7.4, 2.4 Hz, 2H), 6.98 (d, J = 8.1 Hz, 1H), 6.92 (t, J = 7.4 Hz, 1H), 4.90 (s, 2H), 4.30 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H), 3.03 (t, J = 6.9 Hz, 2H), 2.77 (t, J = 6.8 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.6, 166.4, 160.4 (d, J = 3.2 Hz), 157.2, 157.0, 156.7, 155.0, 136.0 (d, J = 11.6 Hz), 128.3, 127.9, 126.8 (d, J = 7.5 Hz), 126.2, 121.8, 120.2, 110.5, 55.4, 55.3, 45.5, 37.6, 30.0, 29.1. ¹⁹F NMR (DMSO-d₆, 377 MHz): δ -126.28. HRMS: DART, calcd for C₂₁H₂₁FN₃O₅ 414.14352 [M + H]⁺, found 414.14687.

3-(3-(2-((2-Methoxybenzyl)amino)-2-oxoethyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (24)

¹H NMR (DMSO-d₆, 500 MHz): δ 8.69 (t, J = 5.8 Hz, 1H), 7.92 (dd, J = 8.0, 1.6 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.25 (td, J = 7.3, 6.8, 1.9 Hz, 2H), 6.97 (d, J = 8.1 Hz, 1H), 6.93 (t, J = 7.4 Hz, 1H), 4.89 (s, 2H), 4.29 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H), 2.99 (t, J = 6.6 Hz, 2H), 2.73 (t, J = 6.6 Hz, 2H), 2.52 (s, 3H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 174.1, 166.7, 161.5, 156.7, 155.4, 145.1, 135.0, 134.6, 128.2, 127.9, 126.3, 125.9, 120.2, 119.6, 110.5, 55.3, 55.3, 45.2, 37.5, 30.8, 29.4, 16.7. HRMS: DART, calcd for C₂₂H₂₄N₃O₅ 410.17160 [M + H]⁺, found 410.17144.

3-(8-Chloro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (25)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.20 (br s, 1H), 8.68 (t, J = 5.8 Hz, 1H), 8.06 (dd, J = 8.0, 1.4 Hz, 1H), 7.97 (dd, J = 7.7, 1.4 Hz, 1H), 7.48 (t, J = 7.9 Hz, 1H), 7.17–7.32 (m, 2H), 6.98 (d, J = 7.7 Hz, 1H), 6.92 (td, J = 7.4, 1.0 Hz, 1H), 4.89 (s, 2H), 4.29 (d, J = 5.7 Hz, 2H), 3.80 (s, 3H), 3.04 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.7 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz): δ 173.5, 166.3, 160.7, 157.3, 156.7, 143.1, 134.5, 130.4, 128.3, 127.9, 126.9, 126.2, 125.4, 121.4, 120.2, 110.5, 55.3, 45.5, 37.5, 29.8, 29.1. IR (neat): 3294, 2977, 1746, 1722, 1690, 1658, 1610, 1434, 1401, 1373, 1233, 1261, 756. HRMS: ESI⁺, calcd for C₂₁H₂₁ClN₃O₅ 430.11697 [M + H]⁺, found 430.11749.

3-(8-Chloro-6-iodo-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (26)

¹H NMR (DMSO-d₆, 500 MHz): δ 1H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.69 (t, J = 5.8 Hz, 1H), 8.34 (d, J = 1.9 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H), 7.30–7.25 (m, 1H), 7.25–7.21 (m, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.97–6.90 (m, 1H), 4.89 (s, 2H), 4.30 (d, J = 5.7 Hz, 2H), 3.81 (s, 3H), 3.04 (t, J = 6.7 Hz, 2H), 2.79 (t, J = 6.7 Hz, 2H). HRMS: ESI calcd for C₂₁H₁₉ClIN₃O₅ 556.01307 [M + H]⁺, found 556.01.

3-(8-Chloro-3-(2-((2-methylbenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (27)

¹H NMR (CD₃OD, 500 MHz): δ 8.09 (dd, J = 7.9, 1.4 Hz, 1H), 7.85 (dd, J = 7.7, 1.4 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.34–7.23 (m, 1H), 7.20–7.05 (m, 3H), 4.98 (s, 2H), 4.42 (s, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H), 2.33 (s, 3H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz,): δ 179.1, 169.0, 163.5, 158.9, 145.3, 137.4, 137.0, 135.7, 132.9, 131.3, 129.3, 128.5, 127.6, 127.1, 126.3, 122.9, 47.0, 42.6, 33.7, 31.9, 19.1. IR (neat): 3359, 3283, 3080, 2957, 1687, 1645, 1580, 1422, 1386, 1327, 1246, 1225, 1170, 1003, 994, 981, 770, 761, 683, 451. HRMS: ESI⁺, calcd for C₂₁H₂₁ClN₃O₄ 414.12206 [M + H]⁺, found 414.12249.

3-(8-Chloro-3-(2-((2-fluorobenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (28)

¹H NMR (DMSO-d₆, 500 MHz): δ 8.87 (t, J = 5.8 Hz, 1H), 8.05 (dd, J = 8.0, 1.4 Hz, 1H), 7.95 (dd, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.38 (td, J = 7.8, 1.9 Hz, 1H), 7.32 (tdd, J = 7.5, 5.3, 1.8 Hz, 1H), 7.21–7.12 (m, 2H), 4.89 (s, 2H), 4.38 (d, J = 5.8 Hz, 2H), 3.03 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.7 Hz, 2H). HRMS: DART, calcd for C₂₀H₁₈ClFN₃O₄ 418.09644 [M + H]⁺, found 418.09634.

3-(8-Chloro-3-(2-((2-chlorobenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (29)

¹H NMR (DMSO-d₆, 500 MHz): δ 8.98 (t, J = 5.9 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.44 (dt, J = 15.3, 7.7 Hz, 3H), 7.31 (dt, J = 25.1, 7.4 Hz, 2H), 4.94 (s, 2H), 4.40 (d, J = 5.7 Hz, 2H), 3.02 (t, J = 7.0 Hz, 2H), 2.75 (t, J = 6.9 Hz, 2H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz,): δ 174.1, 166.8, 160.8, 157.9, 143.2, 135.9, 134.5, 132.0, 130.4, 129.1, 128.9, 128.7, 127.3, 126.8, 125.4, 121.4, 45.7, 31.2, 29.8. HRMS: DART, calcd for C₂₀H₁₈Cl₂N₃O₄ 434.06744 [M + H]⁺, found 434.06703.

3-(8-Chloro-4-oxo-3-(2-oxo-2-(((tetrahydro-2H-pyran-2-yl)methyl)amino)ethyl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (30)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.17 (s, 1H), 8.43 (t, J = 5.8 Hz, 1H), 8.04 (dd, J = 8.0, 1.4 Hz, 1H), 7.96 (dd, J = 7.8, 1.4 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 4.83 (s, 2H), 3.91–3.84 (m, 1H), 3.38–3.27 (m, 2H), 3.17 (ddd, J = 13.6, 6.0, 4.5 Hz, 1H), 3.08 (ddd, J = 13.6, 7.0, 5.6 Hz, 1H), 3.00 (t, J = 6.8 Hz, 2H), 2.79 (t, J = 6.7 Hz, 2H), 1.79–1.73 (m, 1H), 1.58–1.51 (m, 1H), 1.49–1.38 (m, 3H), 1.21–1.09 (m, 1H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz,): δ 173.6, 166.3, 160.7, 157.4, 143.1, 134.5, 130.4, 126.9, 125.4, 121.4, 75.8, 67.3, 45.3, 44.0, 29.8, 29.0, 28.9, 25.6, 22.6. IR (neat): 3445, 3285, 2946, 1726, 1654, 1597, 1573, 1447, 1201, 1165, 1096, 1047, 993, 925, 906, 768, 686. HRMS: ESI⁺, calcd for C₁₉H₂₃ClN₃O₅ [408.13207] [M + H]⁺, found [408.13234].

3-(8-Chloro-4-oxo-3-(2-oxo-2-(((tetrahydrofuran-2-yl)methyl)amino)ethyl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (31)

¹H NMR (DMSO-d₆, 500 MHz): δ 12.18–12.15 (m, 1H), 8.45 (t, J = 5.8 Hz, 1H), 8.04 (dd, J = 8.0, 1.4 Hz, 1H), 7.95 (dd, J = 7.8, 1.4 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 4.83 (s, 2H), 3.85 (qd, J = 6.6, 5.0 Hz, 1H), 3.77 (ddd, J = 8.2, 7.0, 6.1 Hz, 1H), 3.66–3.58 (m, 1H), 3.25–3.12 (m, 2H), 3.00 (t, J = 6.7 Hz, 2H), 2.79 (t, J = 6.7 Hz, 2H), 1.93–1.72 (m, 3H), 1.50 (ddt, J = 12.5, 9.0, 7.3 Hz, 1H). ¹³C{¹H} NMR (DMSO-d₆, 125 MHz,): δ 173.6, 166.4, 160.7, 157.4, 143.1, 134.5, 130.4, 126.8, 125.4, 121.4, 77.1, 67.2, 45.3, 42.9, 29.8, 29.0, 28.5, 25.2. IR (neat): 3289, 3087, 2938, 1705, 1680, 1652, 1596, 1556, 1441, 1328, 1169, 1138, 1069, 984, 796, 764. HRMS: ESI⁺, calcd for C₁₈H₂₁ClN₃O₅ [394.11642] [M + H]⁺, found [394.11661].

3-(8-Chloro-3-(2-((2-methoxybenzyl)(methyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic Acid (32)

¹H NMR (DMSO-d₆, 500 MHz) (Hindered rotation): δ 8.04 (dd, 1H), 7.95 (d, 1H), 7.46 (t, 1H), 7.33 (t, 0.5H), 7.24 (t, 1H), 7.07 (t, 1H), 7.01 (t, 1H), 6.92 (t, 0.5H), 5.19 (d, 2H), 4.63–4.50 (2xs, 2H), 3.88–3.79 (2xs, 3H), 3.10–2.95 (s,t,t 3.5H), 2.77–2.48 (2xt, 3.5H). HRMS: ESI⁺, calcd for C₂₂H₂₃ClN₃O₅ 444.13262 [M + H]⁺, found 444.13319.

3-(8-Chloro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-6-(1-(13-oxo-17-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl)-1H-1,2,3-triazol-4-yl)-3,4-dihydroquinazolin-2-yl)propanoic Acid (33)

Ethyl 3-(8-chloro-6-iodo-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoate (prepared according to the general scheme) (805.2 mg, 1.38 mmol, 1 equiv), Pd(PPh₃)₂Cl₂ (9.7 mg, 0.014 mmol, 1 mol %), and CuI (13.1 mg, 0.069 mmol, 5 mol %) were added to a 2-dram vial. DMF (1.4 mL) and then NEt₃ (0.58 mL) were added sequentially, followed by TMS-acetylene (0.23 mL, 1.66 mmol, 1.2 equiv), and the solution was stirred at room temperature for 30 h. The mixture was filtered through a short plug of silica gel, eluting with EtOAc. The filtrate was washed twice with H₂O, three times with brine, and dried over MgSO₄. The mixture was filtered through a second plug of silica gel, eluting with EtOAc. The material was purified via flash column chromatography (40% EtOAc/pent, dry loaded with silica gel) to afford ethyl 3-(8-chloro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-6-((trimethylsilyl)ethynyl)-3,4-dihydroquinazolin-2-yl)propanoate as an off-white solid (412.7 mg, 66% yield).

Ethyl 3-(8-chloro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-6-((trimethylsilyl)ethynyl)-3,4-dihydroquinazolin-2-yl)propanoate (412.7 mg, 0.74 mmol, 1 equiv) was added to a 2-dram vial and dissolved in THF (1.5 mL). A 1.0 M solution of TBAF in THF (0.89 mL, 0.89 mmol, 1.2 equiv) was added dropwise at room temperature, and the solution was stirred for 2 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified via flash column chromatography (20% EtOAc/DCM) to afford ethyl 3-(8-chloro-6-ethynyl-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoate as an off-white solid (194.9 mg, 54% yield).

Ethyl 3-(8-chloro-6-ethynyl-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoate (38.0 mg, 0.079 mmol, 1 equiv) and LiOH·H₂O (13.2 mg, 0.315 mmol, 4 equiv) were added to a 2-dram vial, followed by THF (0.95 mL), H₂O (0.32 mL), and EtOH (0.32 mL). The mixture was stirred at room temperature for 16 h. The solution was then neutralized with 1 M HCl (pH paper), and the organics were concentrated in vacuo. The crude material was purified via flash column chromatography (1:1 PhMe/acetone w/0.1% v/v AcOH, dry loaded with silica gel) to afford 3-(8-chloro-6-ethynyl-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid as a white solid (27.1 mg, 76% yield).

An oven-dried 2-dram vial was equipped with a magnetic stir bar and cooled to room temperature under a positive flow of argon gas. 3-(8-Chloro-6-ethynyl-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid (11.3 mg, 0.025 mmol, 1 equiv), CuI (1 mg, 0.005 mmol, 20 mol %), and TBTA (2.7 mg, 0.005 mmol, 20 mol %) were added sequentially to the vial. Then, 0.13 mL of DMF followed by 0.13 mL of H₂O was added to the solids, followed by the addition of biotin azide (250 μL of a 100 mM DMSO stock solution, 0.025 mmol, 1 equiv). The reaction mixture was placed in an oil bath preheated to 85 °C and stirred for 20 h. The vial was removed from the oil bath and allowed to cool to room temperature. The crude material was filtered through a pad of Celite eluting with EtOAc. The material was purified via reverse-phase column chromatography to afford 3-(8-chloro-3-(2-((2-methoxybenzyl)amino)-2-oxoethyl)-4-oxo-6-(1-(13-oxo-17-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-3,6,9-trioxa-12-azaheptadecyl)-1H-1,2,3-triazol-4-yl)-3,4-dihydroquinazolin-2-yl)propanoic acid. MS (ESI⁺): m/z = 898.54 [M + H]⁺.

Protein Purification

HDAC6^1109–1213, with an N-terminal His-tag, and a thrombin protease site, ISG15^81–157, with an N-terminal His-tag and a TEV protease site, were expressed in Escherichia coli BL21 (DE3) codon plus cells from a pET28a-LIC and pET28-MHL vector, respectively. HDAC6^1109–1215, HDAC6^{1108-1215 R1155A_Y1184A}, USP3^1–131, USP5^171–290, USP16^25–185, USP20^1–141, USP33^29–134, USP39^84–194, USP49^1–115, USP51^176–305, and BRAP^304–390 encoding an N-terminal AviTag for biotinylation and a C-terminal His6 tag were expressed in BirA cells from p28BIOH-LIC vectors, while USP13^183–307 with an N-terminal His-tag and TEV protease cleavage site, and a C-terminal biotinylation sequence was expressed in BirA cells from a pNICBIO2 vector. All cells, apart from cells expressing ISG15^81–157 and USP5^1–835, were grown in M9 minimal media in the presence of 50 μM ZnSO₄, 50 μg/mL of kanamycin, and 30 μg/mL of chloramphenicol to an OD₆₀₀ of 0.8 and induced by isopropyl-1-thio-d-galactopyranoside (IPTG) for a final concentration of 0.5 mM; cultures were incubated overnight for 18 h at 15 °C. ISG15^81–157 and USP5^1–835 were grown in Terrific broth (TB) media (Sigma-Aldrich) with the same conditions. All p28BIOH-LIC or pNICBIO2 expressing cells were also supplemented with 1× of 100× biotin stock (10 mM; 2.4 mg/mL).

HDAC6^1–1215, USP3^1–520, USP16^1–823, and USP33^36–825 were overexpressed in Sf9 cells where cultures were grown in HyQ SFX Insect Serum Free Medium (Fisher Scientific) to a density of 4 × 10⁶ cells/mL and infected with 10 mL of P3 viral stock per 1 L of cell culture. Cell culture medium was collected after 4 days of incubation in a shaker at 27 °C.

Cells were harvested by centrifugation at 4000–6000 RPM at 10 °C, and cell pellets were lysed by sonification in lysis buffer 50 mM Tris (pH 8), 150 mM NaCl, 1 mM TCEP (for proteins: HDAC6^1109–1213, ISG15^81–157, HDAC6^1109–1215, HDAC6^{1108-1215 R1155A_Y1184A}, USP3^1–131, USP5^171–290, USP13^183–307, USP16^25–185, USP20^1–141, USP33^29–134, USP39^84–194, USP49^1–115, USP51^176–305 and BRAP^304–390, USP16^1–823), 50 mM Tris (pH 8), 500 mM NaCl, 2.5% glycerol (v/v), 1 mM TCEP (for proteins: USP3^1–520, USP33^36–825), 50 mM Tris (pH 8), 150 mM NaCl, 1 mM TCEP, 5% glycerol (v/v) (for protein: USP5^1–835) supplemented with 50 μL of benzonase, 1 mM protease inhibitor (PMSF, benzamidine), and 0.5% NP-40 (only for sf9 expression). The crude extract was centrifuged at 14,000 RPM for 1 h at 10 °C. The clarified lysate was incubated with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) for 1 h with agitation. The protein-bound resin was washed in wash buffer 1 (lysis buffer with no additives), then wash buffer 2 (wash buffer 1 supplemented with 15 mM imidazole), and finally, bound proteins were eluted with elution buffer (wash buffer 1 supplemented with 300 mM imidazole) and monitored with Bradford reagent.

HDAC6^1109–1213 was dialyzed overnight in 2 L of wash buffer 1 with 100 U of thrombin. The dialyzed sample was supplemented with 2 mM CaCl₂ and treated daily with 100 U of thrombin until protein cleavage was complete, as assessed by SDS-PAGE (days 5–7). The cleaved protein was rocked with 5 mL of equilibrated Ni-NTA resin at 4 °C for 30 min, poured through an open column, and washed with wash buffers 1 and 2, followed by elution buffer. All other proteins were dialyzed in their respective wash buffer 1, with no tag cleavage.

HDAC6^1–1215 and USP16^1-823were diluted 3-fold with Buffer A (20 mM Tris (pH 8), 1 mM TCEP), and then concentrated to 5 mL and loaded onto a MonoQ 5/50 column (Buffer A: 20 mM Tris (pH 8), 1 mM TCEP; Buffer B: 20 mM Tris (pH 8), 1 M NaCl, 1 mM TCEP). Peak fractions were analyzed by SDS-PAGE, and protein fractions were pooled and then loaded onto an S200 16/60 gel filtration column for further purification (gel filtration buffer: 50 mM Tris (pH 8), 150 mM NaCl, 1 mM TCEP). USP3^1–520 and USP33^36–825 were concentrated to 5 mL and loaded onto a S200 16/60 gel filtration column. All other dialyzed or cleaved proteins were concentrated to 5 mL and loaded onto an S75 16/60 gel filtration column. Protein peak fractions were analyzed by SDS-PAGE, pooled, and concentrated. Protein concentration was measured by UV absorbance at 280 nm, and protein identity was confirmed by mass spectrometry.

Fluorescence Polarization Assay

Experiments were performed in 384-well black polypropylene PCR plates (Axygen) in 10 μL volume. In each well, 9 μL of compound solutions in buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, 0.005% Tween-20 (v/v), 1% DMSO (v/v)) were diluted, followed by the addition of 1 μL of 30 μM HDAC6^1109–1213 or 10 μM HDAC6^1–1215 and 500 nM N-terminally FITC-labeled RLRGG or 1 μL of 2 μM HDAC6^1109–1213 and 500 nM N-terminally FITC-labeled LRLRGG were added to each well. The LRLRGG substrate was used for compounds with K_D <∼1 μM. Following 1 min centrifugation at 1000 RPM, the plate was incubated for 10 min before FP measurements with a BioTek Synergy 4 (BioTek) at excitation and emission wavelengths of 485 and 528 nm, respectively. The data was processed in GraphPad Prism using Sigmoidal, 4PL, X is log(concentration) fit.

Isothermal Titration Calorimetry

HDAC6^1109–1215 was diluted to 10 μM and dialyzed for 24 h at 4 °C in assay buffer (PBS, 0.005% Tween-20 (v/v), 0.25% DMSO (v/v)). HDAC6^1109–1215 concentration was assessed after dialysis using UV absorbance at 280 nm. 25 was initially diluted from the solubilized DMSO stock to 0.25% DMSO (v/v) in the assay buffer (no DMSO) for a final concentration of 100 μM. The pH of the protein and compound solution were measured and assessed to be within 0.1 pH units of each other. ITC measurements were performed at 25 °C on a Nano ITC (TA Instruments), with 300 μL of 10 μM HDAC6 in the sample cell and 50 μL of 100 μM 25 in the injection syringe. A total of 24 × 2 μL titrations with an initial 0.5 μL injection that was omitted from the fitting analysis were delivered into 0.167 mL of sample cells at a 180 s interval. The data was analyzed using Nano Analyze software and fitted with an independent-binding site model.

Surface Plasmon Resonance

Studies were performed using a Biacore T200 (GE Health Sciences). A SA chip was primed with 3 × 60 s injection with 50 mM NaOH, followed by approx. 500–6000 response units (RU) of biotinylated protein (HDAC6^1109–1215, HDAC6^1108–1215 R1155A_Y1184A HDAC6^1–1215 USP3^1–131, USP5^171–290, USP13^183–307, USP16^25–185, USP20^1–141, USP33^29–134, USP39^84–194, USP49^1–115, USP51^176–305, and BRAP^304–390) diluted in assay buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, 0.005% Tween-20 (v/v), and 1% DMSO (v/v)) coupling to flow channels, with an empty flow channel used for reference subtraction. Following protein capture, the assay buffer was flowed over the chip until a stable baseline was achieved. Compound dilutions were prepared in assay buffer, and experiments were performed using multicycle kinetics with 60–400 s contact time, 60–400 s dissociation time, and 20–30 μL/minute flow rate at 20 °C. K_D values were calculated using steady-state affinity fitting with the Biacore T200 evaluation software.

X-ray Crystallography

Apo 3.5 mg/mL HDAC6^1109–1213 was crystallized in mother liquor: 2 M Na formate, 0.1 M Na acetate, pH 4.6, 5% ethylene glycol. 1 μL of apo HDAC6^1109–1213 crystals were diluted 1:1000 with mother liquor and vortexed vigorously to make a seed mix. Crystallization solutions of 3.5 mg/mL tag-free HDAC6^1109–1213 and 1% (v/v) of a 200 mM DMSO-solubilized stock of 2, 5, 9, and 18 were prepared in buffer (50 mM Tris (pH 8), 150 mM NaCl, 1 mM TCEP) and incubated on ice for at least 1 h. One microliter of HDAC6^1109–1213: compound solution and 1 μL of the mother liquor were set up in a 96-well Intelli-plate using a PHOENIX liquid dispensing robot followed by the addition of 200 nL of 1:2-8-fold dilution of seed mix using a MOSQUITO instrument. X-ray diffraction data for HDAC6^1109–1213 co-crystals were collected at 100 K at a Rigaku FR-E SuperBright at a wavelength of 1.54178 Å for compounds 9 and 15. X-ray diffraction data for HDAC6^1109–1213 co-crystal with 25 were collected at 100 K at APS 24-ID-E at a wavelength of 0.9792 Å. Diffraction data were processed with Xia2 and Aimless.³³ Models were refined with cycles of Coot³⁴ for model building and visualization, with REFMAC³⁵ for restrained refinement, and validated with MOLPROBITY.³⁶

HDAC6 Catalytic Activity Assay

The final concentration of 200 nM HDAC6^1–1215 was incubated with compound dilutions prepared with 20 mM HEPES (pH 8.0), 1 mM MgCl₂, 137 mM NaCl, 2.7 mM KCl, 0.05% BSA (v/v), and 2% DMSO (v/v) in a 384-well microplate (Greiner). A final concentration of 50 μM Boc-Lys(TFA)-AMC was added and incubated for 1 hour at room temperature, followed by the addition of developer solution containing a final concentration of 50 μM TSA. Following a 1 min centrifugation at 1000 RPM, fluorescence intensity was read using BioTek Synergy 4 (BioTek) with excitation and emission of 360 and 460 nm. The data were analyzed with GraphPad Prism 8.2.0.

Ubiquitin Rhodamine Dub Assay

Experiments were performed in a total volume of 60 μL in 384-well black polypropylene microplates (Grenier). Fluorescence was measured using a BioTek Synergy H1 microplate reader (BioTek) at excitation and emission wavelengths of 485 and 528 nm, respectively. Compound dilutions were prepared in assay buffer (USP3^1–520: 20 mM Tris (pH 7.5), 125 mM NaCl, 1 mM DTT, 0.01% TX-100 (v/v), 1% DMSO (v/v); USP5^1–835: 20 mM Tris (pH 7.5), 30 mM NaCl, 1 mM DTT, 0.01% Triton-X (v/v), and 1% DMSO (v/v); USP16^1–823: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.002% TX-100 (v/v), 1% DMSO (v/v); USP33^36–825: 20 mM Tris (pH 7.5), 30 mM NaCl, 5 mM DTT, 0.01% TX-100 (v/v), 1% DMSO (v/v)). 500 nM USP3^1–520, 1 nM USP5¹⁻⁸³⁵, 16 nM USP16^1–823, 8 nM USP33^36–825 and ubiquitin-rhodamine110 (UBPBio) at 200 nM for USP3^1–520 and USP5¹⁻⁸³⁵, or 500 nM for USP16^1–823, USP33^36–825 was added to each well. Following 1 min centrifugation at 1000 RPM, fluorescence readings were immediately taken for 10 min. The data were analyzed with GraphPad Prism 8.2.0.

NanoBRET Assay

HEK293T cells were plated in 6-well plates (8 × 10⁵/well) in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). After 4 h, cells were co-transfected with 0.02 μg of N-terminally NanoLuc-tagged HDAC6 or USP16 (wildtype or R1155A/R84A, Y1184A/Y117A mutant, respectively), 0.4 μg of N-terminally HaloTag-tagged ISG15, and 1.6 μg of empty vector. The following day cells were trypsinized and seeded in a 384-well white plate (20 μl/well) in DMEM F12 (no phenol red, 4% FBS) +/– HaloTag NanoBRET 618 Ligand (1 μL/mL, Promega) and +/– compounds (DMSO concentration in each sample was kept the same). Four hours later, 5 μL/well of NanoBRET Nano-Glo substrate (10 μL/mL in DMEM no phenol red, Promega) was added, and 460 nm donor and 618 nm acceptor signals were read within 10 min of substrate addition using a CLARIOstar microplate reader (Mandel). Mean corrected NanoBRET ratios (mBU) were determined by subtracting the mean of 618/460 signal from cells without NanoBRET 618 Ligand ×1000 from the mean of 618/460 signal from cells with NanoBRET 618 Ligand ×1000. The IC₅₀ values were determined using GraphPad Prism 7 software.

HDAC6 Pull-Down From Whole Cell Lysate and Western Blot

HEK-293 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in 15 cm plates. To isolate whole cell lysate, media was removed from the plate, and cells were washed once in 2 mL of phosphate-buffered saline (PBS). Cells were scraped off the plate using a cell scraper, transferred into a 15 mL tube, and centrifuged for 3 min at 230g. Cells were lysed in 1 mL of lysis buffer with salt (450 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, 1× protease inhibitors) and mixed by vigorous pipetting and vortexing. Cells were incubated on ice for 20 min before dilution with lysis buffer without salt (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Triton X-100, 1× protease inhibitors) to a final concentration of 150 mM NaCl. Lysates were centrifuged at 20,000g for 2 min at 4 °C, and the supernatant was transferred to a fresh tube. Protein concentration was determined using a Pierce BCA Protein Assay, and lysates were snap-frozen in liquid nitrogen and stored at −80 °C. Lysates were thawed, and a small volume was set aside as an input control and stored at −20 °C. To each 3 mg of thawed lysate, 25, 32, or DMSO control (same volume of DMSO) was added to achieve a final compound concentration of 10 μM. Tubes were incubated with rotation for 1 h at 4 °C. Then, 25 μL of Streptavidin magnetic Dynabeads (M270, Thermo-Fischer Scientific 65305) per sample were combined into a single tube and washed twice in 500 μL of low salt wash buffer (100 mM NaCl, 10 mM Tris-HCl (pH 7.9), 0.1% NP-40), with beads isolated from the buffer between each step using a magnetic rack. An aliquot of 25 μL of washed beads was removed and added to lysate without preincubation with any compounds or DMSO (beads alone control). The remaining washed beads were incubated for 1 h at 4 °C with rotation in 2 mL of low salt wash buffer containing 10 μM 33. The excess unbound compound was removed by washing the beads three times in 1 mL of low salt wash buffer before resuspending the beads in 100 μL of low salt wash buffer per 25 μL of beads. After 1 h preincubation of lysates with DMSO, 25, or 32, 100 μL of 33-bound beads were added and lysates were incubated for 1 h with rotation at 4 °C. Beads were washed three times in low salt wash buffer. After the final wash, beads were pelleted and resuspended directly in 15 μL of 1× SDS loading buffer and boiled at 95 °C for 1 min. Beads were pelleted, and the entire volume of supernatant was used for western blotting along with input lysate using the NuPAGE electrophoresis and transfer system (Invitrogen) and near-infrared detection for HDAC6 (CST #7558; 1:1000) using IRDye 800CW Secondary Antibody (1:5000). Immunoblots were imaged on a Li-Cor Odyssey CLx.

HDAC6 Tubulin Acetylation Assay

HEK293T cells were treated for 24 h with 10 or 30 μM 25 or 10 μM Tubacin or DMSO control. Cell lysates were analyzed by Western blot using the NuPAGE electrophoresis and transfer system (Invitrogen), and proteins detected with primary antibodies anti-α tubulin-K40ac (1:2000, Abcam, ab179484) and anti-α tubulin (1:2000, Abcam, ab7291) and secondary antibodies IRDye 800CW (1:5000, ThermoFisher, A32735) and IRDye 488CW (1:5000, ThermoFisher, A11029). Immunoblots were imaged on a Li-Cor Odyssey CLx.

HDAC6 Chemoproteomics in Cytoplasmic Fraction

To isolate the cytoplasmic fraction, cell pellets were collected as above and resuspended in 3 mL of hypotonic lysis buffer, Buffer A (10 mM HEPES (pH 7.4), 10 mM NaCl, 1.5 mM MgCl₂, 0.005% (v/v) Tween-20, 1× protease inhibitors), and kept on ice for 20 min, with vigorous vortexing for 5 s every 5 min. Cells were centrifuged for 5 min at 380g and 4 °C, and the supernatant was collected and transferred to 2 mL tubes before being centrifuged again for 1 min at 18,500g and 4 °C. The cleared supernatant was collected and transferred to 2 mL tubes. NaCl was added to adjust the final concentration to 150 mM. Protein concentration was determined using the Pierce BCA Protein Assay, and lysates were snap-frozen in liquid nitrogen and stored at −80 °C.

25 nanomoles of 33 were bound to 20 μL of Streptavidin Sepharose High-Performance (MilliporeSigma, GE17-5113-01) beads for 1 h at 4 °C in PBS. The beads were washed three times with Buffer A. Meanwhile, to each 1.5 mg of cytoplasmic fraction, a final compound concentration of 10 μM compound 25, 32, or DMSO control (same volume of DMSO) was added, and the samples were incubated for 1 h at 4 °C. Protein and beads were then mixed and rocked for a further 1 h at 4 °C. The supernatant was removed, and the beads were washed once with Buffer A and transferred to a new tube. The beads were then washed three times with 50 mM ammonium bicarbonate, and then 1 μg of chymotrypsin was added for 15 min at RT. To this solution, 1 μg of trypsin was added and incubated for 2.25 h at 37 °C. Disulfide bonds were reduced by adding DTT to a final concentration of 5 mM. After incubating for 30 min at 56 °C, the reduced cysteines were alkylated with 20 mM iodoacetamide in the dark for 45 min. An additional 1 μg of trypsin was added, and the solution was left overnight at 37 °C.

The digested peptides were analyzed using reversed-phase (Reprosil-Pur 120 C18-AQ, 1.9 μm), nano-HPLC (Vanquish Neo UHPLC) coupled to an Orbitrap Fusion Lumos Tribrid. Peptides were eluted from the column with an acetonitrile gradient starting from 3.2% acetonitrile with 0.1% formic acid to 35.2% acetonitrile with 0.1% formic acid using a linear gradient of 90 min. The MS1 scan had an accumulation time of 50 ms within a mass range of 400–1500 Da, using an orbitrap resolution of 120,000, 60% RF lens, AGC target of 125%, and 2400 V. This was followed by MS/MS scans with a total cycle time of 3 s. An accumulation time of 50 ms and 33% HCD collision energy was used for each MS/MS scan. Each candidate ion was required to have a charge state from 2–7 and an AGC target of 400%, isolated using an orbitrap resolution of 15,000. Previously analyzed candidate ions were dynamically excluded for 9 s. The RAW files were searched with FragPipe v18.0, using MSFragger v3.5 and Philosopher v4.4.0. The LFQ-MBR workflow was utilized using chymotrypsin/trypsin enzymatic digestion with human Uniprot ID UP000005640 (with decoys and contaminants appended). Differential protein expression was determined using R Package DEP,³¹ and independently validated with ProDA.

Glossary

Abbreviations Used

FP

fluorescence polarization

HDAC

histone deacetylase

K_D

dissociation constant

NanoBRET

nano-bioluminescence resonance energy transfer

SPR

surface plasmon resonance

UBD

ubiquitin-binding domain

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00314.

• Data collection and refinement statistics for compounds 9, 15, and 25; general practical considerations; synthetic procedures; characterization data; and purity analysis for compounds investigated in this study (PDF)

• coord_8g43_9 (CIF)

• sf_8g43_9 (CIF)

• coord_8g44_15 (CIF)

• sf_8g44_15 (CIF)

• coord_8g45_25 (CIF)

• sf_8g45_25 (CIF)

• Molecular formula strings and the associated biological data (CSV)

Accession Codes

PDB deposition: HDAC6-UBD in complex with 9—8G43, HDAC6-UBD in complex with 15—8G44, HDAC6-UBD in complex with 25—8G45. Authors will release the atomic coordinates and experimental data upon article publication. Mass spectrometry data deposition: Data are available via ProteomeXchange with identifier PXD039880. Processed mass spectrometry data files and other associated materials are available via Zenodo #7618095. Files in the deposition are as follows: combined_protein_Int.txt (output from database search); hdac6_dep_submitted.R (script used to process data (from DEP package)); hdac6_dep_submitted.R.RData (workspace environment from DEP package); hdac6probecontrol_dep.csv (exported list of p-value and fold-change from DEP); hdac6probecontrol_proda.csv (exported list of p-value and fold-change from proDA); hdac6proda_submitted.R (proDA script); hdac6proda_submitted.RData (proDA workspace environment).

Author Contributions

^∇ R.J.H., I.F., M.K.M., M.M.S., and B.M. contributed equally to this work. V.S. and M.S. designed compounds, R.J.H. wrote the manuscript, R.J.H. solved structures, R.J.H., M.K.M., M.S., S.A., and D.O. tested compounds, and I.F., B.M., A.S., K.J., R.S., R.B., and C.D. synthesized compounds. C.H.A. conceived the project and C.H.A., M.L., and P.J.B. advised throughout the project. All of the authors approved the final version of the manuscript.

Open Access is funded by the Austrian Science Fund (FWF).

The authors declare no competing financial interest.

Author Status

^# Deceased July 1, 2021