ABSTRACT
USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and allosterically inhibits the catalytic activity of the enzyme. Systematic exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 µM. 64 is selective over the structurally similar ZnF-UBD domain of HDAC6 and inhibits USP5 catalytic activity in vitro with an IC50 of 26 µM. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells.
INTRODUCTION
Ubiquitin specific proteases (USP) are the largest subfamily of deubiquitinating enzymes (DUB), consisting of more than 50 proteases with distinct roles in ubiquitin (Ub) biology. USP5 (also known as isopeptidase T, IsoT) cleaves unanchored ubiquitin chains, leading to the regeneration of monoUb1, 2 and removes Ub chains to stabilize post-translationally modified proteins in cells3–12. Deletion of UBP14, the gene encoding a yeast USP5 orthologue, leads to the accumulation of free polyUb chains and proteasome inhibition13. In addition to the proper functioning of the proteasome, USP5 has been shown to be important in neuropathic pain4, 14–17 and cancer3, 7, 12, 18–24. A genome-scale CRISPR-Cas9 knockout screen across 804 cells lines and 28 cancer types from the BROAD Institute reveals that, with USP36, USP5 is the most essential USP25, 26 (Figure 1). Interestingly, independent studies show that knocking-down USP5 is toxic in pancreatic cancer cell lines but has no effect in non-cancerous HEK293 cells19 and is well tolerated in mice4, suggesting that USP5 may be a valid therapeutic target in oncology.
USP5 has multiple Ub binding modules: an N-terminal zinc finger ubiquitin-binding domain (ZnF-UBD) which recognizes the proximal free C-terminus of a broad subset of polyUb substrates2, 27–29, a USP domain that forms the active site, and two ubiquitin-binding associated domains (UBA-1, UBA-2)30 (Figure 2a). The mechanism of polyUb processing by USP5 is not understood for all polyUb chain variants; site-directed mutagenesis experiments show USP5 processes Met1 and Lys48-linked polyUb chains sequentially from the proximal end of Ub chains2, 31. The USP domain is highly conserved making it difficult to develop selective catalytic inhibitors; to date, out of 56 human USPs, selective inhibitors have been reported for USP1, USP7, USP9x, USP14 and USP3032–38. The ZnF-UBD domain is present in a limited number of proteins in the human genome: USP3, USP5, USP13, USP16, USP20, USP22, USP33, USP39, USP44, USP45, USP49, USP51, HDAC6, a histone deacetylase protein, and BRAP, a BRCA1-associated protein39–41. The role of the USP5 ZnF-UBD is not well understood. Hydrolysis of the artificial substrate, ubiquitin amidomethyl coumarin (Ub-AMC) by USP5 is activated when free monoUb occupies the ZnF-UBD, suggesting that this domain participates in the allosteric modulation of USP542. However, in a separate study, deletion of the ZnF-UBD did not significantly affect hydrolysis of Ub-AMC30. It is therefore unclear whether targeting the ZnF-UBD is a valid strategy to antagonize the enzymatic activity of USP5.
We previously reported fragment-like molecules that bind to the USP5 ZnF-UBD with high micromolar affinity43. Here, we describe the progression of one of these compounds into a low micromolar USP5 inhibitor. We show that our ligand can displace Ub from the ZnF-UBD in the context of full-length (FL) USP5, inhibits the catalytic function of USP5 with a Lys48-linked di-ubiquitin (Ub2K48) substrate in vitro and is selective over HDAC6 ZnF-UBD. The accompanying crystal structures and structure activity relationship (SAR) provide a robust framework for the development of a chemical probe to interrogate the function of USP5 in cells and in vivo.
RESULTS & DISCUSSION
Hit Expansion
We focused on exploring the SAR around UBTR008295a, a 4-phenyl-4-oxobutanoic acid USP5 ZnF-UBD ligand previously reported (KD= 80 µM)43. A substructure search of the chemical scaffold from the Enamine REAL database resulted in 285 analogues, which were docked to the USP5 ZnF-UBD crystal structure (PDB: 6DXH)43 using Glide44 (Schrodinger, NY), with three hydrogen bond constraints at the side chain NH of Arg221, the NH backbone of Arg221 and the side chain OH of Tyr261. Ten compounds were ordered and tested using a fluorescence competition assay, in which displacement of a N-terminal fluorescently labeled Ub substrate from the ZnF-UBD was measured. Nine compounds displaced Ub from the ZnF-UBD at micromolar concentration and were tested using an orthogonal surface plasmon resonance (SPR) assay (Table S2). Compound 1, with an extended sulfonamide group, improved potency 7-fold against the USP5 ZnF-UBD (KD=12 µM) compared to UBTR008295a (KD=80 µM)43 (Table 1).
ZnF-UBDs share similar sequence and structural folds and the ZnF-UBD of USP3, USP5, USP16 and HDAC6 bind Ub31, 40, 45–47. We have previously shown that the ZnF-UBD of HDAC6 can be targeted by small molecules inhibitors that, as compound 1, include a carboxylic tail mimicking the C-terminal di-glycine motif of Ub48, 49. We therefore decided to use HDAC6 as an anti-target to monitor the selectivity of our compounds. Ultimately, an extended selectively panel should be applied to characterize the specificity of more advanced compounds. We found that 1 bound with equipotency to ZnF-UBDs of USP5 and HDAC6 (USP5 & HDAC6 KD=12 µM).
To confirm the binding pose of 1 to USP5 ZnF-UBD, the crystal structure of USP5 ZnF-UBD in complex with 1 was solved to 1.98 Å resolution (PDB: 7MS5). Compound 1 was well resolved in the electron density and modeled for two of the USP5 ZnF-UBD molecules in the crystallographic asymmetric unit.
The electron density of 1 was unambiguous in chain A and was reasonably modeled in chain B. The ligand pose in both molecules differs, with a RMSD of 3.11, due to, the fluorinated ring of 1 stacking on a Phe224 residue from an adjacent molecule in the lattice of the asymmetric unit in chain B (Figure S1). As expected, the carboxylate group of 1 recapitulates hydrogen bond interactions observed with the Ub C-terminal glycine carboxylic acid (Figure 2b). The backbone amide of Arg221 and side chain OH of Tyr261 are engaged in direct hydrogen bonds with the buried carboxylic acid of the ligand which also makes water-mediated hydrogen bonds with surrounding residues (Arg221, Val234). Additionally, the central aromatic ring is engaged in π-stacking interactions with Tyr259. The angle afforded by the sulfonyl favorably positions the 4-phenylpiperidine group for additional interactions with binding pocket residues. A shift in backbone conformation of a loop that is forming part of the binding pocket (residues 221-230) leads to repositioning of Try223 to optimally accommodate the 4-phenylpiperidine appendage of 1 (Figure 2b, Figure S2). In particular, Tyr223 engages in an edge-to-face π-stacking interaction with the terminal phenyl group of 1, which may account for the improved potency of the ligand.
To identify molecules structurally related to 1, a similarity search was performed against the Enamine REAL database and SciFinder. 26 compounds were docked to USP5 ZnF-UBD (chain A) in complex with 1 (PDB: 7MS5) with Glide44 with the three hydrogen bond constraints described previously. Sixteen compounds were ordered and tested by SPR (Table S3). Compound 3, which lacks fluorine substitutions at positions C3 and C4 of the terminal phenyl, and 5 which has a terminal pyrrolidine had marginally improved potency compared with 1 but were not selective (USP5 KD= 8 and 9 µM respectively; HDAC6 KD= 7 and 15 µM respectively). Interestingly, the introduction of an amide to the carboxylate chain in compound 2 resulted in a 9-fold gain in selectivity versus HDAC6 (USP5 KD=31 µM; HDAC6 KD=270 µM). The amide likely stabilizes the co-planarity of the carboxylate tail with the aromatic ring, which may be unfavorable for HDAC6 binding. We synthesized 4 and 6, the amide analogs of 3 and 5 respectively, to confirm that the amide confers selectivity for USP5 over HDAC6 (Scheme 1). Both compounds exhibited comparable affinity for USP5 (KD= 34, 33 µM respectively) and selectivity over HDAC6 (11 and 16-fold respectively). Compound 4 was identified as a more favorable scaffold for developing potent and selective ligands and we next focused on further exploring the SAR around 4.
Structure Activity Relationship
Our initial SAR studies focused on varying substitutions to the carboxylic acid to determine if less acidic moieties could interact with Arg221 and Tyr261 (Table S4). Substituting the carboxylic acid to an ester (S25) or tetrazole (S26, S27) resulted in a complete loss of binding against USP5 indicating that the conserved network of direct and water-mediated interactions between the carboxylate and Arg221, Tyr261 and Val234 is essential for binding, as was previously observed with the corresponding binding pocket of HDAC649. The addition of a (S)-methyl or dimethyl at the C2 position of the carboxylic chain and mono-N-methylation of the secondary amide were not tolerated (S28, S30, S31). A (R)-methyl at the C2 position of the carboxylic chain (S29) had weaker affinity for USP5 and improved affinity for HDAC6. Lengthening and shortening of the carboxylic chain was also not tolerated (S32, S24).
Molecules 9-38 were synthesized following a well-stablished synthetic procedure (Scheme 1). (4-(chlorosulfonyl)benzoyl)glycine was treated with various commercially available or synthesized 4-substituted-piperidylamines or related analogs under basic conditions to obtain the N-substituted-sulfonamides. Investigation of the SAR around the piperidine (Table 2, 9-27), revealed the 6-membered piperidine ring of 4 was preferred over 4, 5, and 7-membered rings (11-13). The phenylpiperidine of compound 4 had at least 3-fold improved affinity for USP5 ZnF-UBD over the bicyclic ring systems of compounds 9 and 10 which had isoindoline and dihydroisoquinoline groups, respectively. Finally, a piperidine was preferred over a piperazine (14), 1, 2, 3, 6-tetrahydropyridine (15), 2- and 3-methylpiperadine (16, 17), or a bridged-bicyclic ring (18). Substitutions of the piperidine ring (compound 19-25) showed that a phenyl ring (4) or a N-methyl-piperazine (25) were preferred at this position. The piperidine substituted with a phenyl (4) is preferred over other groups for efficient positioning of the terminal phenyl for π-stacking interactions with Tyr223, as seen with the complex structure of 1 (PDB: 7MS5). A spiro group (27) was found to be slightly more potent and selective than 4, but chemical tractability considerations led us to further explore substitutions on the phenyl ring of 4.
Substitutions at the terminal phenyl ring of 4 with methyl or halogen groups in ortho improved affinity by about 2-fold (28, 30-32), while a methoxy group decreased potency and selectivity (29) (Table 3). Meta-substitution with a methyl (34) markedly improved affinity (KD= 18 µM) while other groups at the same position were not favorable (33, 35-38).
To examine the role of the central aromatic core we synthesized and surveyed different substituted aromatic and heteroaromatic scaffolds (Table 4) following a previously described methodology (Scheme 2), with slight modifications, as shown in Scheme 3. For compounds, 44-57, 4-phenylpiperidine was reacted with the sulfonyl chloride of the core, to form the corresponding N-substituted sulfonamide. The condensation reaction between the carboxylic acid and tert-butyl glycinate was achieved through application of a coupling agent, HATU. The final step involved an ester deprotection under acid conditions to obtain the desired molecule. Compound 51 required a saponification step before the carboxamide formation.
Comparing 4 vs. 44 indicated that transversal branching afforded by -1,4 substitution was favored over the -1,3 trajectory. Addition of a methyl or halogen in the C3 of the phenyl ring (45-47) improved neither potency nor selectivity; however, a fluorine in C2 resulted in a four-fold increase in potency 48: KD= 9 ± 2 µM, while bulkier groups such as chlorine or methyl (49, 50) were not tolerated. We solved the crystal structure of 48 (PDB: 7MS6) in complex with USP5 ZnF-UBD to a resolution of 1.55 Å with unambiguous electron density for the bound ligand (Figure S3). The fluorine group of 48 makes hydrophobic interactions with Trp209, which may account for the 4-fold increase in potency for USP5 ZnF-UBD in comparison to 4 (Figure 3). However, while the added fluorine increases affinity for USP5, it also results in a four-fold decrease in selectivity against HDAC6.
The effect of replacing the core phenyl ring with other ring systems was also explored (Table 4, compounds 51-57). Importantly, introducing a pyridine ring (compound 51) improved potency for USP5 five-fold (KD= 7 ± 2 µM) and selectivity over HDAC6 (KD= 200 ± 57 µM). Core hopping with 5-membered heterocycles led to no significant improvement in potency or selectivity. (52-57).
To build upon the improvement afforded by the fluoro-phenyl of 48 and the pyridine of 51, four derivatives of these compounds were synthesized and tested (Compounds 61-64; Scheme 3; Table 5). While no apparent benefit was observed in replacing the terminal phenyl ring of 48, we were pleased to observe further improvement in potency and selectivity when chlorinating this ring in 51 (compound 64: USP5 KD= 2.8 µM: HDAC6 KD=110 µM). We solved the crystal structure of 64 in complex with USP5 ZnF-UBD to 1.45 Å resolution (PDB: 7MS7). The USP5-64 complex is modeled with high confidence in the context of chain A; however, the electron density of 64 was not fully resolved in chain B and has ambiguous density for ligand positioning due to the adjacent molecule in the lattice partially blocking the binding site for the crystallographic asymmetric unit which is likely reducing occupancy of this ligand pocket (Figure S4). 64 makes similar interactions with binding pockets residues as 1 (PDB: 7MS5) and 48 (PDB: 7MS6), including hydrogen bond interactions with the backbone and side chains of Arg221, side chain of Tyr261, and π-stacking interaction with Tyr259 and Tyr223 (Figure 4). The pyridine nitrogen of 64 can engage in an intramolecular hydrogen bond with the amide nitrogen of the aliphatic tail, which reinforces π-stacking interactions with Tyr259, Tyr223 and Trp209 through electronic effects and contributes to the gain in potency. We observe significant variation in the backbone and side-chain conformation of residues 223-225, at the extremity of the loop lining the ligand binding pocket, confirming our previously reported observation that this loop can rearrange structurally to accommodate different ligands43 (Figure S5).
Characterization of Compound 64
We used SPR to show that lead compound 64 binds to the full-length (FL) USP5 with a KD of 8 ± 2 µM, 2.5-fold weaker in binding affinity compared to the isolated ZnF-UBD (KD = 2.8 ± 0.5 µM) (Figure 5ab). 64 shows no detectable binding to a FL R221A USP5 mutant (Figure S6), indicating that 64 is binding only at the ZnF-UBD in the context of the FL enzyme. In vitro displacement of Ub was tested using a fluorescence competition assay measuring the displacement of a N-terminal FITC-tagged Ub from both the ZnF-UBD and the FL USP5 (Figure 5c). We found that compound 64 decreased the interaction of Ub from the isolated ZnF-UBD and the FL USP5 in a dose-dependent manner with an IC50 of 7 ± 2 µM and 46 ± 15 µM, respectively. 64 displaces Ub approximately seven-fold less efficiently from FL USP5 than from the isolated ZnF-UBD; suggesting that in the context of the FL protein, the Ub molecule occupying the ZnF-UBD is also making stabilizing interactions with other domains of USP5; this is in agreement with the five-fold difference in binding affinity of Ub to the ZnF-UBD (KD= 1.4 µM) versus FL USP5 (KD=0.3 µM)43 (Figure S7). Importantly, we observed dose-dependent inhibition of USP5 catalytic activity by 64 in an internally quenched fluorophore (IQF) assay with a native K48-linked di-ubiquitin (Ub2K48) substrate, with an IC50 of 26 ± 9 µM in vitro (Figure 5d). Taken together, these data suggest that targeting the ZnF-UBD is a valid strategy for the selective inhibition of USP5, a putative cancer target. The potency of the compounds presented in this work needs to be further improved before inhibitors can be tested in cellular systems or animal models. To evaluate the development potential of this chemical series and support further optimization efforts, we verified that our most advanced compound was stable in mouse and human liver microsome stability assays (>90% compound remaining after 60 min, Table S5).
CONCLUSION
In summary, we optimized a preliminary chemical scaffold through structural analysis, docking and SAR studies to identify a small molecule antagonist targeting the USP5 ZnF-UBD with low micromolar affinity that is selective over a structurally similar ZnF-UBD in HDAC6. We show that compound 64 occupies the USP5 ZnF-UBD, can displace Ub in the context of FL USP5 and inhibits USP5 deubiquitinase activity.
The ZnF-UBD has been hypothesized to recognize and position substrates or allosterically modulate USP5 catalytic activity30, 31. Our antagonists inhibit USP5-mediated cleavage of a Ub2K48 substrate showing that when the ZnF-UBD is blocked, Ub2K48 cannot be properly positioned for catalytic cleavage. USP5 was reported to cleave a diverse array of differently branched polyubiquitin substrates, and it remains to be seen whether the inhibitory activity of ZnF-UBD ligands is substrate-specific. The chemical inhibitors and accompanying crystal structures reported here will enable further optimization into a potent and selective chemical probe to investigate the cellular function of USP5.
EXPERIMENTAL SECTION
Hit Expansion & Docking
Substructure search was run against ∼1.2 billion compounds from the Enamine REAL database (Enamine Ltd., 2019) and SciFinder. Ligprep (Schrodinger, New York) was used to prepare the ligands using default settings. The X-ray structures of USP5 ZnF-UBD (PDB ID: 6DXH43, 7MS5) were prepared with PrepWizard50 (Schrodinger, New York) for the assignment of bond order, assessment of correct protonation states and optimization of hydrogen-bond assignment at pH 7.3, and restrained minimization using the OPLS3e force field. Receptor grids were calculated at the centroid of the co-crystallized ligand for each respective complex. The library was docked to the USP5 ZnF-UBD structure for each respective complex using Glide SP44 (Schrodinger, New York) with hydrogen-bond constraints with the NH of Arg221 backbone, NH of Arg221 side chain and OH of Tyr261. Docked compounds were clustered in ICM51 and compounds were selected to be purchased based on docking pose, score, visual inspection, and cost.
Cloning, Protein Expression and Purification
cDNAs encoding Ub1–76, USP5171–290, USP51–835, USP51–835 R221A, HDAC61109–1215 were cloned as previously described43, 48, 49. Proteins were purified as before43, 48, 49; briefly, all proteins were expressed in Escherichia coli and purified by metal affinity chromatography, tag cleavage, gel filtration and ion exchange chromatography. The final concentration of purified proteins was 2-45 mg/mL by UV absorbance at 280 nm. Protein identity was confirmed by mass spectrometry and purity was assessed by SDS-PAGE, showing all samples to be >90% pure.
Fluorescence Competition Assay
All experiments were performed in 384-well black flat-bottomed streptavidin plates (Greiner). 20 µL of 1 µM biotinylated-USP5171–290, USP51–835 prepared with buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.005% Tween-20 (v/v), 1% DMSO (v/v)) was incubated in wells for 1 hour at 4⁰C. Wells were washed with 3x50 µL buffer to remove excess unbound protein. 20 µL of two-fold dilution series of ligand and 0.2 µM N-terminally FITC-labeled Ub (Boston Biochem) prepared in buffer were incubated in wells for 1 hour at 4⁰C. Wells were washed with 7x50 µL buffer to remove excess unbound FITC-UBQ. Fluorescence was measured using a Biotek Synergy H1 microplate reader (Biotek) at emission and excitation of 528 nm and 485 nm, respectively. The data were analyzed with GraphPad Prism 8.4.2.
Surface Plasmon Resonance Assay
Studies were performed with a Biacore T200 (GE Health Sciences) at 20⁰C. Approximately 3000-6000 response units (RU) of biotinylated USP5171-290, HDAC61109–1215, USP51–835, USP51–835 R221A were captured to flow cells of a streptavidin-conjugated SA chip per manufacturer’s protocol, and an empty flow cell used for reference subtraction. Serial dilutions were prepared in 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.005% Tween-20 (v/v), 1% DMSO (v/v). KD determination experiments were performed using multi-cycle kinetics with 60-120 s contact time, and 60-120 s dissociation time at a flow rate of 30 µL/min. KD values were calculated using steady state affinity fitting with the Biacore T200 evaluation software (GE Health Sciences).
Crystallization
12 mg/mL of tag-free USP5171–29 and 1.1% (v/v) of 200 mM DMSO-solubilized stock of compounds were prepared in 50 mM Tris pH 8, 150 mM NaCl, 1 mM TCEP. Sparse-matrix crystallization experiments yielded crystals in different conditions for each USP5 ZnF-UBD-compound complex. 1 was co-crystallized with 1:1 protein/compound: mother liquor (1 M sodium/potassium phosphate pH 6.9). 48 was co-crystallized with 1:1 protein/compound: mother liquor (2 M ammonium sulfate, 0.2 M sodium acetate, 0.1 M HEPES pH 7.5, 5% MPD). 64 was co-crystallized with 1:1 protein/compound: mother liquor (20% PEG3350, 0.2 M magnesium acetate). Co-crystals were soaked in a cryoprotectant consisting of mother liquor supplemented with 25% ethylene glycol (v/v) for 1 and 48 and 20% glycerol (v/v) for 64 prior to mounting and cryo-cooling.
Data Collection, Structure Determination and Refinement
X-ray diffraction data for USP5171–290 co-crystals with 1, 48 were collected at 100 K on RIGAKU FR-E X-ray generator with a RIGAKU Saturn A200 CCD detector. Diffraction data for 64 was collected at APS beamline 24-ID-E. Images were processed with HKL300052 or Xia253 and scaled with AIMLESS54. Graphics program COOT55 was used for model building and visualization, REFMAC56 for restrained refinement and MOLPROBITY57 for analysis of model geometry.
Internally Quenched Fluorophore Assay
Experiments were performed in a total volume of 60 µL in 384-well black polypropylene microplates (Greiner). Fluorescence was measured using a Biotek Synergy H1 microplate reader (Biotek) at excitation and emission wavelengths of 540 and 580 nm, respectively. Ligands were prepared in 20 mM Tris pH 7.5, 125 mM NaCl, 5 mM DTT, 0.01% TritonX-100 (v/v), 1% DMSO (v/v) for a two-fold dilution titration series. 125 nM USP51–835 was added and the plate was incubated at room temperature for 1 hour before the addition of 200 nM IQF Ub2K48 (LifeSensors, DU4802). Following a 1-minute centrifugation at 250 g, fluorescence readings were immediately taken for ten minutes. The data were analyzed with GraphPad Prism 8.4.2.
Chemistry
All reagents were purchased from commercial vendors and used without further purification. Volatiles were removed under reduced pressure by rotary evaporation or by using the V-10 solvent evaporator system by Biotage®. Very high boiling point (6000 rpm, 0 mbar, 56 °C), mixed volatile (7000 rpm, 30 mbar, 36 °C) and volatile (6000 rpm, 30 mbar, 36 °C) methods were used to evaporate solvents. The yields given refer to chromatographically purified and spectroscopically pure compounds. Compounds were purified using a Biotage Isolera One system by normal phase chromatography using Biotage® SNAP KP- Sil or Sfär Silica D columns (Part No.: FSKO-1107/FSRD-0445) or by reverse-phase chromatography using Biotage® SNAP KP-C18-HS or Sfär C18 D (Part No.: FSLO-1118/FSUD-040). If additional purification was required, compounds were purified by solid phase extraction (SPE) using Biotage Isolute Flash SCX-2 cation exchange cartridges (Part No.: 532-0050-C and 456-0200-D). Products were washed with 2 cartridge volumes of MeOH and eluted with a solution of MeOH and NH4OH (9:1 v/v). Final compounds were dried using the LabconcoTM Benchtop FreeZoneTM Freeze-Dry System (4.5 L Model). 1H and proton-decoupled 19F NMRs were recorded on a Bruker Avance-III 500 MHz spectrometer at ambient temperature. An Agilent D500 was used for 13C NMRs data collection. Residual protons of CDCl3, DMSO-d6 and CD3OD solvents were used as internal references. Spectral data are reported as follows: chemical shift (δ in ppm), multiplicity (br = broad, s = singlet, d = doublet, dd = doublet of doublets, m = multiplet), coupling constants (J in Hz) and proton integration. Compound purity was determined by UV absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (LCMS) using a Waters Acquity separations module. All compounds have ≥95% purity, with exceptions for compounds 5 (90%), and S22 (93%) as determined using this method. Low resolution mass spectrometry (LRMS) was conducted in positive/negative ion mode using a Waters Acquity SQD mass spectrometer (electrospray ionization source) fitted with a PDA detector. Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B consisted of 0.1% formic acid in acetonitrile. One of three types of columns were used: Method 1: Acquity UPLC CSH C18 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186005296), Method 2: Acquity UPLC BEH C8 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186002877) or Method 3: Acquity UPLC HSS T3 (2.1 x 50 mm, 100 Å, 1.8 µm. Part No. 186003538). All were used with column temperature maintained at 25 °C. High resolution mass spectrometry (HRMS) was conducted using a Waters Synapt G2-S quadrupole-time-of-flight (QTOF) hybrid mass spectrometer system coupled with an Acquity ultra-performance liquid chromatography (UPLC) system (Table S6). Chromatographic separations were carried out on an Acquity UPLC CSH C18 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186005296, Method 1), Acquity UPLC BEH C8 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186002877) or Acquity UPLC HSS T3 (2.1 x 50 mm, 100 Å, 1.8 µm. Part No. 186003538). The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Leucine Enkephalin was used as lock mass. MassLynx 4.1 was used for data analysis.
(4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (4)
A solution of 2-{[4-(chlorosulfonyl)phenyl]formamido}acetic acid (7, 54 mg, 0.194 mmol), 4-phenyl-piperidine (8, 47 mg, 0.292 mmol) and DIPEA (68 µL, 0.389 mmol) in DCM (2 mL) was stirred for 30 min at room temperature (rt), after which time LC analysis indicated total consumption of the starting material and only one major peak. The volatiles were removed by evaporation to yield a yellow solid to which DCM (10 mL) and aqueous HCl 10% (10 mL) were added. Upon combination of organic and aqueous solutions, an insoluble precipitate appears, which floats between the two layers. The organic phase alongside the insoluble precipitate was collected and washed with (2x 10% HCl). The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). After evaporation of the desired fractions the product was suspended in water and freeze dried for 2 days. White powder; yield 61% (48 mg, 0.119 mmol). 1H NMR (500 MHz, DMSO) δ 9.12 (t, J = 5.8 Hz, 1H), 8.12 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 7.30 – 7.24 (m, 2H), 7.21 – 7.15 (m, 3H), 3.96 (d, J = 5.9 Hz, 2H), 3.80 (d, J = 11.7 Hz, 2H), 2.37 – 2.31 (m, 2H), 1.81 (d, J = 11.2 Hz, 2H), 1.66 (qd, J = 12.7, 3.9 Hz, 2H). LC: Method 1, tR = 1.68 min. MS (ESI): m/z = 401.56 [M - 1]-; HRMS (ESI) for C20H23N2O5S [M + H]+: m/z = calcd, 403.1328; found, 403.1326.
(4-((4-(pyrrolidin-1-yl)piperidin-1-yl)sulfonyl)benzoyl)glycine (6)
A solution of 2-{[4-(chlorosulfonyl)phenyl]formamido}acetic acid (52 mg, 0.188 mmol), 4-(1-pyrrolidinyl)piperidine (44 mg, 0.282 mmol)) and DIPEA (66 µL, 0.377 mmol) in DCM (2 mL) was stirred for 30 min at rt, after which time LC analysis indicated total consumption of the starting material and only one major peak. The volatiles were removed by evaporation to yield a yellow solid. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). After evaporation of the desired fractions the product was suspended in water and freeze dried for 2 days. White powder; yield 39% (29 mg, 0.073 mmol). Obtained as a formic acid salt. 1H NMR (500 MHz, DMSO) δ 9.01 (t, J = 5.7 Hz, 1H), 8.08 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 3.90 (d, J = 5.8 Hz, 2H), 3.49 (d, J = 12.0 Hz, 4H), 2.46 (s, 4H), 2.05 (t, J = 9.6 Hz, 1H), 1.85 (d, J = 10.3 Hz, 2H), 1.63 (s, 4H), 1.47 – 1.39 (m, 2H). LC: Method 1, tR = 0.94 min. MS (ESI): m/z = 394.56 [M - 1]-; HRMS (ESI) for C18H26N3O5S [M + H]+: m/z = calcd, 396.1593; found, 396.1584.
(4-(isoindolin-2-ylsulfonyl)benzoyl)glycine (9)
Compound 9 was prepared as described for 4, using 2,3-dihydro-1H-isoindole hydrochloride (56 mg, 0.360 mmol, 2 equiv) as amine. Off-white powder; yield 54% (35 mg, 0.096 mmol). 1H NMR (500 MHz, DMSO) δ 12.63 (s, 1H), 9.05 (t, J = 5.9 Hz, 1H), 8.04 (d, J = 8.6 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 1.0 Hz, 4H), 4.61 (s, 4H), 3.92 (d, J = 5.9 Hz, 2H). LC: Method 1, tR = 1.47 min. MS (ESI): m/z = 359.27 [M - H]-; HRMS (ESI) for C17H17N2O5S [M+ H]+: m/z = calcd, 361.0858; found, 361.0860.
(4-((3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)benzoyl)glycine (10)
Compound 10 was prepared as described for 4, using 1,2,3,4-tetrahydroisoquinoline (52 mg, 0.388 mmol, 2 equiv) as amine. White powder; yield 63% (44 mg, 0.117 mmol).1H NMR (500 MHz, DMSO) δ 12.66 (s, 1H), 9.08 (t, J = 5.9 Hz, 1H), 8.07 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 7.14 (dd, J = 5.7, 3.7 Hz, 3H), 7.11 (dt, J = 7.9, 3.9 Hz, 1H), 4.24 (s, 2H), 3.94 (d, J = 5.9 Hz, 2H), 3.34 (t, J = 6.0 Hz, 2H), 2.85 (t, J = 5.9 Hz, 2H); LC: Method 1, tR = 1.54 min. MS (ESI): m/z = 373.26 [M - H]-; HRMS (ESI) for C18H19N2O5S [M + H]+: m/z = calcd, 375.1015; found, 375.1021.
(4-((3-phenylazetidin-1-yl)sulfonyl)benzoyl)glycine (11)
Compound 11 was prepared as described for 4, using 7 (100 mg, 0.360 mmol) and 3-phenylazetidine, HCl (92 mg, 0.540 mmol, 2 equiv) as amine. White powder; yield 36% (48 mg, 0.129 mmol). 1H NMR (500 MHz, DMSO) δ 9.18 (t, J = 5.8 Hz, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.24 – 7.15 (m, 3H), 6.89 – 6.84 (m, 2H), 4.16 (dd, J = 10.6, 5.0 Hz, 2H), 4.00 – 3.97 (m, 2H), 3.75 – 3.65 (m, 3H); LC: Method 1, tR= 1.63 min. MS (ESI): m/z=375.48 [M + H]+; HRMS (ESI) for C18H19N2O5S [M + H]+: m/z = calcd, 375.1015; found, 375.1008.
(4-((3-phenylpyrrolidin-1-yl)sulfonyl)benzoyl)glycine (12)
Compound 12 was prepared as described for 4, using 7 (100 mg, 0.360 mmol) and 3-phenylpyrrolidine (0.105 mL, 0.720 mmol, 2 equiv) as amine. White powder; yield 56% (79 mg, 0.202 mmol). 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.7 Hz, 1H), 8.10 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.4 Hz, 2H), 7.26 (t, J = 7.3 Hz, 2H), 7.22 – 7.17 (m, 1H), 7.14 (d, J = 7.2 Hz, 2H), 3.95 (d, J = 5.7 Hz, 2H), 3.72 (dd, J = 9.6, 7.4 Hz, 1H), 3.46 (ddd, J = 10.4, 8.4, 3.0 Hz, 1H), 3.30 – 3.24 (m, 2H), 3.17 (dt, J = 16.6, 8.2 Hz, 1H), 3.09 (t, J = 9.5 Hz, 1H), 2.15 (dtd, J = 9.7, 6.8, 3.0 Hz, 1H), 1.78 (td, J = 18.5, 9.1 Hz, 1H); LC: Method 1, tR= 1.70 min. MS (ESI): m/z= 389.27 [M + H]+; HRMS (ESI) for C19H21N2O5S [M + H]+: m/z = calcd, 389.1171; found, 389.1171.
(4-((4-phenylazepan-1-yl)sulfonyl)benzoyl)glycine (13)
Compound 13 was prepared as described for 4, using 4-phenylazepane, HCl (57 mg, 0.270 mmol) as amine. Off-white powder; yield 46% (35 mg, 0.083 mmol).1H NMR (500 MHz, DMSO) δ 9.08 (t, J = 5.9 Hz, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 7.18 – 7.10 (m, 3H), 3.95 (d, J = 5.9 Hz, 2H), 3.55 – 3.50 (m, 2H), 3.22 – 3.17 (m, 2H), 2.67 – 2.61 (m, 1H), 1.94 – 1.87 (m, 2H), 1.79 – 1.59 (m, 4H); LC: Method 1, tR = 1.75 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1484.
(4-((4-phenylpiperazin-1-yl)sulfonyl)benzoyl)glycine (14)
Compound 14 was prepared as described for 4, using 1-phenylpiperazine (63 mg, 0.388 mmol) as amine. White powder; yield 74% (55 mg, 0.137 mmol); 1H NMR (500 MHz, DMSO) δ 9.09 (t, J = 5.8 Hz, 1H), 8.12 – 8.08 (m, 2H), 7.92 – 7.87 (m, 2H), 7.22 – 7.17 (m, 2H), 6.90 (dd, J = 8.7, 0.8 Hz, 2H), 6.80 (t, J = 7.3 Hz, 1H), 3.94 (d, J = 5.9 Hz, 2H), 3.22 – 3.18 (m, 4H), 3.08 – 3.03 (m, 4H); LC: Method 1, tR = 1.56 min. MS (ESI): m/z = 402.23 [M - H]-; HRMS (ESI) for C19H22N3O5S [M + H]+: m/z = calcd, 404.1280; found, 404.1274.
(4-((4-phenyl-3,6-dihydropyridin-1(2H)-yl)sulfonyl)benzoyl)glycine (15)
Compound 15 was prepared as described for 4, using 7 (100 mg, 0.360 mmol) and 4-phenyl-1,2,3,6-tetrahydropyridine, HCl (106 mg, 0.540 mmol) as amine. White powder; yield 35% (50 mg, 0.125 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 7.1 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.27 – 7.23 (m, 1H), 6.09 (t, J = 3.4 Hz, 1H), 3.95 (d, J = 4.4 Hz, 1H), 3.94 (s, 1H), 3.72 (d, J = 2.8 Hz, 2H), 3.27 (t, J = 5.7 Hz, 2H), 2.54 (d, J = 1.5 Hz, 2H); LC: Method 1, tR= 1.78 minzz. MS (ESI): m/z= 401.5 [M + H]+; HRMS (ESI) for C20H21N2O5S [M + H]+: m/z = calcd, 401.1171; found, 401.1166.
(4-((3-methyl-4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (16)
Compound 16 was prepared as described for 4, using 3-methyl-4-phenylpiperidine, mixture of diastereomers (47 mg, 0.270 mmol) as amine. Off-white powder; yield 69% (55 mg, 0.124 mmol); 1H NMR (500 MHz, DMSO) δ 9.09 (q, J = 6.1 Hz, 1H), 8.06 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 7.27 – 7.16 (m, 3H), 7.11 – 7.05 (m, 2H), 3.95 (d, J = 5.9 Hz, 2H), 3.50 (dd, J = 10.0, 7.4 Hz, 1H), 3.20 – 3.16 (m, 1H), 2.85 (dd, J = 10.0, 8.4 Hz, 1H), 2.76 (dd, J = 9.9, 8.6 Hz, 1H), 2.67 (dd, J = 13.7, 5.4 Hz, 1H), 1.90 – 1.81 (m, 1H), 1.74 – 1.66 (m, 1H), 0.82 (d, J = 6.6 Hz, 3H) major rotamer reported. LC: Method 1, tR = 1.74 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1486.
(4-((2-methyl-4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (17)
Compound 17 was prepared as described for 4, using 2-methyl-4-phenylpiperidine, mixture of diastereomers (68.0 mg, 0.388 mmol) as amine. Light yellow powder; yield 37% (29 mg, 0.069 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.7 Hz, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.28 – 7.23 (m, 2H), 7.18 – 7.14 (m, 1H), 7.09 – 7.05 (m, 2H), 4.35 – 4.27 (m, 1H), 3.95 (d, J = 5.9 Hz, 2H), 3.82 (dq, J = 13.7, 2.1 Hz, 1H), 3.19 – 3.13 (m, 1H), 2.95 – 2.84 (m, 1H), 1.69 (dd, J = 13.0, 2.6 Hz, 1H), 1.57 (dd, J = 8.9, 4.1 Hz, 2H), 1.40 – 1.31 (m, 1H), 1.12 (d, J = 6.9 Hz, 3H); LC: Method 1, tR = 1.70 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1488.
Rac-(4-(((1R,3R,5S)-3-phenyl-8-azabicyclo[3.2.1]octan-8-yl)sulfonyl)benzoyl)glycine (18)
Compound 18 was prepared as described for 4, using 7 (100 mg, 0.360 mmol) and rac-(1R,3R,5S)-3-phenyl-8-azabicyclo[3.2.1]octane, HCl (120 mg, 0.540 mmol) as amine. Yellow powder; yield 15% (24 mg, 0.055 mmol); 1H NMR (500 MHz, DMSO) δ 9.06 (t, J = 5.8 Hz, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 2H), 7.29 – 7.25 (m, 4H), 7.19 – 7.14 (m, 1H), 4.29 (s, 2H), 3.93 (d, J = 5.6 Hz, 2H), 2.83 (p, J = 9.3 Hz, 1H), 2.40 – 2.34 (m, 2H), 1.61 (dd, J = 13.1, 10.0 Hz, 2H), 1.49 (d, J = 7.4 Hz, 2H), 1.35 (dd, J = 8.4, 4.2 Hz, 2H); LC: Method 1, tR= 1.83 min. MS (ESI): m/z= 429.32 [M + H]+; HRMS (ESI) for C22H25N2O5S [M + H]+: m/z = calcd, 429.1484; found, 429.1487.
(4-((4-phenoxypiperidin-1-yl)sulfonyl)benzoyl)glycine (19)
Compound 19 was prepared as described for 4, using 4-phenoxy-piperidine (49 mg, 0.277 mmol) as amine. White powder; yield 79% (61 mg, 0.146 mmol); 1H NMR (500 MHz, DMSO) δ 9.12 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.22 (dd, J = 8.5, 7.5 Hz, 2H), 6.90 – 6.85 (m, 3H), 4.47 – 4.41 (m, 1H), 3.96 (d, J = 5.9 Hz, 2H), 2.90 – 2.82 (m, 2H), 2.54 (dd, J = 3.8, 1.9 Hz, 2H), 2.02 – 1.93 (m, 2H), 1.70 – 1.61 (m, 2H); LC: Method 1, tR = 1.67 min. MS (ESI): m/z = 417.28 [M - H]-; HRMS (ESI) for C20H23N2O6S [M + H]+: m/z = calcd, 419.1277; found, 419.1278.
(4-((4-(2-oxoindolin-1-yl)piperidin-1-yl)sulfonyl)benzoyl)glycine (20)
Compound 20 was prepared as described for 4, using 1-piperidin-4-yl-1,3-dihydro-2H-indol-2-one (58.4 mg, 0.270 mmol) as amine. Off-white powder; yield 70% (59 mg, 0.126 mmol); 1H NMR (500 MHz, DMSO) δ 12.69 (s, 1H), 9.14 (t, J = 5.9 Hz, 1H), 8.17 – 8.13 (m, 2H), 7.96 – 7.92 (m, 2H), 7.22 (dd, J = 7.3, 0.7 Hz, 1H), 7.15 (td, J = 7.8, 0.7 Hz, 1H), 6.95 (td, J = 7.8, 0.7 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.10 (tt, J = 12.0, 3.9 Hz, 1H), 3.97 (d, J = 5.9 Hz, 2H), 3.86 (d, J = 12.2 Hz, 2H), 3.50 (s, 2H), 2.56 (td, J = 12.5, 1.8 Hz, 2H), 2.32 (qd, J = 12.9, 4.3 Hz, 2H), 1.65 (d, J = 10.1 Hz, 2H); LC: Method 1, tR = 1.47 min. MS (ESI): m/z = 456.41 [M - H]-; HRMS (ESI) for C22H24N3O6S [M + H]+: m/z = calcd, 458.1386; found, 458.1384.
(4-((3H-spiro[isobenzofuran-1,4’-piperidin]-1’-yl)sulfonyl)benzoyl)glycine (21)
Compound 21 was prepared as described for 4, using 3H-spiro[2-benzofuran-1,4’-piperidine] (86 mg, 0.382 mmol) as amine.
White powder; yield 53% (43 mg, 0.101 mmol);1H NMR (500 MHz, DMSO) δ 9.13 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 8.4 Hz, 2H), 7.29 – 7.23 (m, 4H), 4.87 (s, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.68 (d, J = 11.1 Hz, 2H), 2.02 (td, J = 13.3, 4.6 Hz, 2H), 1.67 (d, J = 12.6 Hz, 2H); LC: Method 1, tR = 1.61 min. MS (ESI): m/z = 429.47 [M - H]-; HRMS (ESI) for C21H23N2O6S [M + H]+: m/z = calcd, 431.1277; found, 431.1264.
(4-((4-(dimethylamino)piperidin-1-yl)sulfonyl)benzoyl)glycine (22)
Compound 22 was prepared as described for 6, using 4-(dimethylamino)piperidine (36 mg, 0.277 mmol) as amine. Off-white powder; yield 47% (36 mg, 0.082 mmol); obtained as a formic acid salt; 1H NMR (500 MHz, DMSO) δ 9.09 (t, J = 5.9 Hz, 1H), 8.09 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.6 Hz, 2H), 3.95 (d, J = 5.9 Hz, 2H), 3.67 (d, J = 12.0 Hz, 2H), 2.52 (d, J = 1.8 Hz, 2H), 2.33 (s, 6H), 1.85 (d, J = 11.5 Hz, 2H), 1.50 (qd, J = 12.3, 3.9 Hz, 2H); LC: Method 1, tR = 0.92 min. MS (ESI): m/z = 368.30 [M - H]-; HRMS (ESI) for C16H24N3O5S [M + H]+: m/z = calcd, 370.1437; found, 370.1434.
(4-((4-cyclopentylpiperidin-1-yl)sulfonyl)benzoyl)glycine (23)
Compound 23 was prepared as described for 4, 4-cyclopentylpiperidine hydrochloride (50 mg, 0.264 mmol) as amine. White powder; yield 53% (37 mg, 0.093 mmol); 1H NMR (500 MHz, DMSO) δ 9.07 (t, J = 5.8 Hz, 1H), 8.08 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.4 Hz, 2H), 3.94 (d, J = 5.7 Hz, 2H), 3.65 (d, J = 11.7 Hz, 2H), 2.18 (td, J = 11.7, 1.8 Hz, 2H), 1.71 (d, J = 11.5 Hz, 2H), 1.68 – 1.61 (m, 2H), 1.56 – 1.47 (m, 2H), 1.47 – 1.38 (m, 3H), 1.15 (qd, J = 12.5, 3.9 Hz, 2H), 1.05 – 0.93 (m, 3H); LC: Method 1, tR= 1.97 min. MS (ESI): m/z= 395.54 [M + H]+; HRMS ESI) for C19H27N2O5S [M + H]+: m/z = calcd, 395.1641; found, 395.1638.
(4-((4-morpholinopiperidin-1-yl)sulfonyl)benzoyl)glycine (24)
Compound 24 was prepared as described for 6, using 4-(piperidin-4-yl)-morpholine (46 mg, 0.270 mmol) as amine. Off-white powder; yield 62% (51 mg, 0.112 mmol); obtained as a formic acid salt; 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.9 Hz, 1H), 8.08 (d, J = 8.5 Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 3.95 (d, J = 5.9 Hz, 2H), 3.66 (d, J = 12.0 Hz, 2H), 3.52 – 3.50 (m, 4H), 2.40 – 2.37 (m, 4H), 2.29 (td, J = 11.9, 9.9 Hz, 2H), 2.14 (tt, J = 10.9 Hz, 1H), 1.79 (d, J = 11.2 Hz, 2H), 1.41 (qd, J = 12.4, 3.9 Hz, 2H); LC: Method 1, tR = 0.92 min. MS (ESI): m/z = 410.36 [M - H]-; HRMS (ESI) for C18H26N3O6S [M + H]+: m/z = calcd, 412.1542; found, 412.1550.
(4-((4-(4-methylpiperazin-1-yl)piperidin-1-yl)sulfonyl)benzoyl)glycine (25)
Compound 25 was prepared as described for 6, using 1-methyl-4-(piperidin-4-yl)piperazine (49.5 mg, 0.270 mmol) as amine. Off-white powder; yield 59% (55 mg, 0.106 mmol); obtained as a 2X formic acid salt; 1H NMR (500 MHz, DMSO) δ 9.03 (t, J = 5.8 Hz, 1H), 8.08 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 3.92 (d, J = 5.8 Hz, 2H), 3.65 (d, J = 11.9 Hz, 4H), 2.41 (s, 4H), 2.34 (s, 2H), 2.28 (td, J = 11.9, 10.0 Hz, 2H), 2.16 (s, 3H), 1.75 (d, J = 11.0 Hz, 2H), 1.41 (qd, J = 12.4, 3.9 Hz, 2H); LC: Method 1, tR = 0.88 min. MS (ESI): m/z = 423.38 [M - H]-; HRMS (ESI) for C19H29N4O5S [M + H]+: m/z = calcd, 425.1859; found, 425.1852.
(4-(N-(1-methylpiperidin-4-yl)sulfamoyl)benzoyl)glycine (26)
Compound 26 was prepared as described for 6, using 4-amino-1-methylpiperidine (32 mg, 0.277 mmol) as amine. White powder; yield 22% (16 mg, 0.040 mmol); obtained as a formic acid salt; 1H NMR (500 MHz, DMSO) δ 9.06 (t, J = 5.9 Hz, 1H), 8.04 (d, J = 8.6 Hz, 2H), 8.00 (d, J = 6.6 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 3.95 (d, J = 5.9 Hz, 2H), 3.01 (s, 2H), 2.54 (dd, J = 3.8, 2.0 Hz, 2H), 1.67 (d, J = 11.0 Hz, 2H), 1.50 (d, J = 10.4 Hz, 2H); LC: Method 1, tR = 0.88 min. MS (ESI): m/z = 354.23 [M - H]-; HRMS (ESI) for C15H22N3O5S [M + H]+: m/z = calcd, 356.1280; found, 356.1282.
(4-((9-methyl-3,9-diazaspiro[5.5]undecan-3-yl)sulfonyl)benzoyl)glycine (27)
Compound 27 was prepared as described for 6, using 3-methyl-3,9-diazaspiro[5.5]undecane (46 mg, 0.270 mmol) as amine. Off-white powder; yield 67% (55 mg, 0.120 mmol); obtained as a formic acid salt; 1H NMR (500 MHz, DMSO) δ 8.93 (t, J = 5.6 Hz, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 3.87 (d, J = 5.7 Hz, 2H), 2.94 – 2.90 (m, 4H), 2.37 (s, 4H), 2.22 (s, 3H), 1.49 – 1.44 (m, 4H), 1.34 – 1.30 (m, 4H); LC: Method 1, tR = 1.00 min. MS (ESI): m/z = 408.25 [M - H]-; HRMS (ESI) for C19H28N3O5S [M + H]+: m/z = calcd, 410.1750; found, 410.1748.
(4-((4-(p-tolyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (28)
Compound 28 was prepared as described for 4, using 4-(4-methylphenyl)piperidine, HCl (57 mg, 0.270 mmol) as amine. Off-white powder; yield 75% (60 mg, 0.135 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.5 Hz, 2H), 7.09 – 7.03 (m, 4H), 3.96 (d, J = 5.9 Hz, 2H), 3.78 (d, J = 11.7 Hz, 2H), 2.43 (tt, J = 11.9, 3.5 Hz, 1H), 2.32 (td, J = 12.0, 2.2 Hz, 2H), 2.23 (s, 3H), 1.78 (d, J = 11.0 Hz, 2H), 1.62 (qd, J = 12.7, 4.0 Hz, 2H); LC: Method 1, tR = 1.79 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1475.
(4-((4-(4-methoxyphenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (29)
Compound 29 was prepared as described for 4, using 4-(4-methoxyphenyl)piperidine (52 mg, 0.270 mmol) as amine. Off-white powder; yield 75% (59 mg, 0.135 mmol); 1H NMR (500 MHz, DMSO) δ 9.11 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.78 (d, J = 11.7 Hz, 2H), 3.70 (s, 3H), 2.42 (tt, J = 11.9, 3.4 Hz, 1H), 2.33 (td, J = 11.8, 1.9 Hz, 2H), 1.77 (d, J = 11.0 Hz, 2H), 1.61 (qd, J = 12.7, 4.0 Hz, 2H); LC: Method 1, tR = 1.67 min. MS (ESI): m/z = 431.43 [M - H]-; HRMS (ESI) for C21H25N2O6S [M + H]+: m/z = calcd, 433.1433; found, 433.1430.
(4-((4-(4-fluorophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (30)
Compound 30 was prepared as described for 4, using 4-(4-fluorophenyl)-piperidine (32 mg, 0.180 mmol) as amine. Off-white powder; yield 77% (59 mg, 0.138 mmol); 1H NMR (500 MHz, DMSO) δ 9.12 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.24 – 7.20 (m, 2H), 7.11 – 7.06 (m, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.7 Hz, 2H), 2.33 (td, J = 12.0, 2.2 Hz, 2H), 1.79 (d, J = 11.0 Hz, 2H), 1.64 (qd, J = 12.7, 3.9 Hz, 2H); 19F NMR (471 MHz, DMSO) δ -116.85 (s); LC: Method 1, tR = 1.72 min. MS (ESI): m/z = 419.39 [M - H]-; HRMS (ESI) for C20H22FN2O5S [M + H]+: m/z = calcd, 421.1233; found, 421.1226.
(4-((4-(4-chlorophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (31)
Compound 31 was prepared as described for 4, using 4-(4-chlorophenyl)piperidine, HCl (63 mg, 0.270 mmol) as amine. Off-white powder; yield 71% (58 mg, 0.128 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.7 Hz, 2H), 2.33 (td, J = 12.0, 2.1 Hz, 2H), 1.79 (d, J = 11.1 Hz, 2H), 1.64 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.82 min. MS (ESI): m/z = 435.42 [M - H]-; HRMS (ESI) for C20H22ClN2O5S [M + H]+: m/z = calcd, 437.0938; found, 437.0928.
(4-((4-(4-bromophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (32)
Compound 32 was prepared as described for 4, using 4-(4-bromophenyl)piperidine (93 mg, 0.389 mmol) as amine. White powder; yield 56% (56 mg, 0.108 mmol); 1H NMR (500 MHz, CD3CN) δ 8.01 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 7.52 (s, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 4.07 (d, J = 5.9 Hz, 2H), 3.86 (dd, J = 9.7, 2.1 Hz, 2H), 2.47 (tt, J = 12.2, 3.6 Hz, 1H), 2.38 (td, J = 12.1, 2.5 Hz, 2H), 1.84 (d, J = 11.8 Hz, 2H), 1.70 (qd, J = 12.7, 4.0 Hz, 2H); LC: Method 1, tR = 1.82 min. MS (ESI): m/z = 479.36 [M - 1]-, 481.46 [M - 1]- +2; HRMS (ESI) for C20H22BrN2O5S [M + H]+: m/z = calcd, 481.0433; found, 481.0434.
(4-((4-(3-(trifluoromethyl)phenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (33)
Compound 33 was prepared as described for 4, using 4-(3-trifluoromethylphenyl)piperidine, HCl (103 mg, 0.389 mmol) as amine. White powder; yield 76% (66 mg, 0.141 mmol); 1H NMR (500 MHz, DMSO) δ 8.53 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.01 – 6.94 (m, 4H), 3.38 (d, J = 5.8 Hz, 2H), 3.25 (d, J = 11.6 Hz, 2H), 2.12 – 2.04 (m, 1H), 1.77 (td, J = 10.3, 2.0 Hz, 2H), 1.28 (d, J = 10.9 Hz, 2H), 1.17 (qd, J = 12.6, 3.8 Hz, 2H); 19F NMR (471 MHz, CD3CN) δ -63.04 (s); LC: Method 1, tR = 1.81 min. MS (ESI): m/z = 469.35 [M - H]-; HRMS (ESI) for C21H22F3N2O5S [M + H]+: m/z = calcd, 471.1202; found, 471.1192.
(4-((4-(m-tolyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (34)
Compound 34 was prepared as described for 4, using 4-(3-methylphenyl)piperidine, HCl (57 mg, 0.270 mmol) as amine. Off-white powder; yield 42% (33 mg, 0.076mmol); 1H NMR (500 MHz, DMSO) δ 9.11 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.15 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 9.1 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.7 Hz, 2H), 2.43 (tt, J = 12.1, 3.4 Hz, 1H), 2.33 (td, J = 12.0, 2.2 Hz, 2H), 2.25 (s, 3H), 1.78 (d, J = 11.0 Hz, 2H), 1.64 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.79 min. MS (ESI): m/z = 415.33 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1480.
(4-((4-(3-methoxyphenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (35)
Compound 35 was prepared as described for 4, using 4-(3-methoxyphenyl)piperidine (52 mg, 0.270 mmol) as amine. Off-white powder; yield 80% (63 mg, 0.145 mmol); 1H NMR (500 MHz, DMSO) δ 9.11 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.18 (t, J = 7.8 Hz, 1H), 6.74 (dd, J = 7.9, 2.0 Hz, 2H), 6.72 (d, J = 1.6 Hz, 1H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.6 Hz, 2H), 3.70 (s, 3H), 2.46 (tt, J = 12.1, 3.5 Hz, 1H), 2.34 (td, J = 11.6, 1.8 Hz, 2H), 1.79 (d, J = 11.0 Hz, 2H), 1.66 (qd, J = 12.6, 3.9 Hz, 2H); LC: Method 1, tR = 1.68 min. MS (ESI): m/z = 431.28 [M - H]-; HRMS (ESI) for C21H25N2O6S [M + H]+: m/z = calcd, 433.1433; found, 433.1444.
(4-((4-(3-fluorophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (36)
Compound 36 was prepared as described for 4, using 4-(3-fluorophenyl)piperidine (48 mg, 0.270 mmol) as amine. Off-white powder; yield 74% (56 mg, 0.134 mmol); 1H NMR (500 MHz, DMSO) δ 9.12 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.6 Hz, 2H), 7.31 (td, J = 8.1, 6.4 Hz, 1H), 7.06 – 6.97 (m, 3H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.6 Hz, 2H), 2.32 (td, J = 11.9, 2.1 Hz, 2H), 1.82 (d, J = 11.1 Hz, 2H), 1.67 (qd, J = 12.6, 3.9 Hz, 2H); 19F NMR (471 MHz, DMSO) δ -113.27 (s); LC: Method 1, tR = 1.72 min. MS (ESI): m/z = 419.39 [M - H]-; HRMS (ESI) for C20H22FN2O5S [M + H]+: m/z = calcd, 421.1233; found, 421.1227.
(4-((4-(3-chlorophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (37)
Compound 37 was prepared as described for 4, using 4-(3-chlorophenyl)piperidine, HCl (63 mg, 0.270 mmol) as amine. Off-white powder; yield 65% (51 mg, 0.116 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.8 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.31 (dd, J = 8.5, 7.7 Hz, 1H), 7.25 – 7.23 (m, 2H), 7.16 (d, J = 7.7 Hz, 1H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.7 Hz, 2H), 2.31 (td, J = 11.9, 2.0 Hz, 2H), 1.81 (d, J = 11.1 Hz, 2H), 1.67 (qd, J = 12.6, 3.9 Hz, 2H); LC: Method 1, tR = 1.80 min. MS (ESI): m/z = 435.27 [M- H]-; HRMS (ESI) for C20H22ClN2O5S [M + H]+: m/z = calcd, 437.0938; found, 437.0937.
(4-((4-(3-bromophenyl)piperidin-1-yl)sulfonyl)benzoyl)glycine (38)
Compound 38 was prepared as described for 4, using 4-(3-bromophenyl)piperidine, HCl (75 mg, 0.270 mmol) as amine. Off-white powder; yield 55% (48 mg, 0.099 mmol); 1H NMR (500 MHz, DMSO) δ 9.10 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.40 – 7.36 (m, 2H), 7.26 – 7.22 (m, 1H), 7.20 (dt, J = 7.8, 1.2 Hz, 1H), 3.96 (d, J = 5.9 Hz, 2H), 3.79 (d, J = 11.6 Hz, 2H), 2.31 (td, J = 11.8, 2.0 Hz, 2H), 1.81 (d, J = 11.1 Hz, 2H), 1.66 (qd, J = 12.6, 3.9 Hz, 2H); LC: Method 1, tR = 1.83 min. MS (ESI): m/z = 479.28 [M- 1]-, 481.09 [M - 1]- +2; HRMS (ESI) for C20H22BrN2O5S [M + H]+: m/z = calcd, 481.0433; found, 481.0436.
(3-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (44)
Step 1, A solution of 3-(chlorosulfonyl)benzoic acid (100 mg, 0.453 mmol), 4-phenyl-piperidine (8, 110 mg, 0.680 mmol) and DIPEA (0.158 mL, 0.906 mmol) in DCM (4.5 mL) was stirred for 30 min at rt, after which time LC analysis indicated total consumption of the starting material and only one major peak. The volatiles were removed by evaporation to yield a white solid to which DCM (10 mL) and aqueous HCl 10% (10 mL) were added. Upon combination of organic and aqueous solutions, an insoluble precipitate appears, which floats between the two layers. The organic phase alongside the insoluble precipitate was collected and washed with (2x 10% HCl). The crude product was suspended in water and freeze dried for 2 days to obtain 3-((4-phenylpiperidin-1-yl)sulfonyl)benzoic acid (41), that was used in the next step without further purification. White powder; yield 71% (130 mg, 0.324 mmol). LC: Method 1, tR = 1.84 min. MS (ESI): m/z = 344.37 [M - 1]-. Step 2, To a solution of 3-((4-phenylpiperidin-1-yl)sulfonyl)benzoic acid (50 mg, 0.145 mmol), DIPEA (0.101 mL, 0.579 mmol) and HATU (83 mg, 0.217 mmol) in N,N- Dimethylformamide (DMF) (2 mL) was added tert-butyl 2-aminoacetate, HCl (42, 36.4 mg, 0.217 mmol). The orange solution was stirred at rt for 24 h under N2 atmosphere, after which time water (1 mL) was added to the suspension. The volatiles were removed by evaporation to yield a white solid to which DCM (10 mL) and aqueous HCl 10% (10 mL) were added. The organic phase was collected and washed with (2x 10% HCl). The remaining DCM was evaporated to obtain a yellow solid. The protecting group was removed by dissolving the sample in DCM (2 to 3 mL) and adding TFA (0.750 mL), the solution was stirred at rt for 1 h. The volatiles were evaporated. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). After evaporation of the desired fractions the product was suspended in water and freeze dried for 2 days. Off-white powder; yield 82% (50 mg, 0.118 mmol); 1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 9.20 (t, J = 5.8 Hz, 1H), 8.25 (t, J = 1.6 Hz, 1H), 8.23 – 8.21 (m, 1H), 7.95 (ddd, J = 7.8, 1.6, 1.1 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.27 (dd, J = 8.0, 7.1 Hz, 2H), 7.19 (d, J = 7.3 Hz, 3H), 3.97 (d, J = 5.9 Hz, 2H), 3.81 (d, J = 11.6 Hz, 2H), 2.34 (td, J = 12.0, 2.0 Hz, 2H), 1.82 (d, J = 11.1 Hz, 2H), 1.67 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.67 min. MS (ESI): m/z = 401.41 [M - H]-; HRMS (ESI) for C20H23N2O5S [M + H]+: m/z = calcd, 403.1328; found, 403.1317.
(3-fluoro-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (45)
Compound 45 was prepared as described for 44, using 4-(chlorosulfonyl)-3-fluorobenzoic acid (100 mg, 0.419 mmol) as a core. Off-white powder; yield 73% (44 mg, 0.100 mmol); 1H NMR (500 MHz, DMSO) δ 12.71 (s, 1H), 9.18 (t, J = 5.9 Hz, 1H), 7.97 – 7.90 (m, 3H), 7.31 – 7.26 (m, 2H), 7.21 – 7.16 (m, 3H), 3.97 (d, J = 5.9 Hz, 2H), 3.83 (d, J = 11.9 Hz, 2H), 2.64 (t, J = 12.2 Hz, 2H), 2.58 (tt, J = 12.0, 3.3 Hz, 1H), 1.84 (d, J = 11.2 Hz, 2H), 1.65 (qd, J = 12.7, 4.0 Hz, 2H); 19F NMR (471 MHz, DMSO) δ -107.65; LC: Method 1, tR = 1.72 min. MS (ESI): m/z = 419.39 [M - H]-; HRMS (ESI) for C20H22FN2O5S [M + H]+: m/z = calcd, 421.1233; found, 421.1231.
(3-chloro-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (46)
Compound 46 was prepared as described for 44, using 3-chloro-4-(chlorosulfonyl)benzoic acid (100 mg, 0.392 mmol) as a core. Off-white powder; yield 92% (53 mg, 0.121 mmol); 1H NMR (500 MHz, DMSO) δ 12.72 (s, 1H), 9.21 (t, J = 5.9 Hz, 1H), 8.14 (d, J = 1.7 Hz, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.01 (dd, J = 8.2, 1.7 Hz, 1H), 7.29 (dd, J = 8.0, 7.1 Hz, 2H), 7.20 (d, J = 7.2 Hz, 3H), 3.96 (d, J = 5.9 Hz, 2H), 3.86 (d, J = 12.4 Hz, 2H), 2.84 (td, J = 12.4, 2.1 Hz, 2H), 2.65 (tt, J = 6.0, 3.5 Hz, 1H), 1.83 (d, J = 11.0 Hz, 2H), 1.61 (qd, J = 12.7, 4.1 Hz, 2H); LC: Method 1, tR = 1.76 min. MS (ESI): m/z = 435.42 [M - H]-; HRMS (ESI) for C20H22ClN2O5S [M + H]+: m/z = calcd, 437.0938; found, 437.0925.
(3-methyl-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (47)
Compound 47 was prepared as described for 44, using 4-(chlorosulfonyl)-3-methylbenzoic acid (100 mg, 0.426 mmol) as a core. Off-white powder; yield 79% (48 mg, 0.110 mmol); 1H NMR (500 MHz, DMSO) δ 12.66 (s, 1H), 9.03 (t, J = 5.7 Hz, 1H), 7.94 – 7.91 (m, 2H), 7.87 (dd, J = 8.3, 1.4 Hz, 1H), 7.31 – 7.26 (m, 2H), 7.22 – 7.18 (m, 3H), 3.94 (d, J = 5.9 Hz, 2H), 3.76 (d, J = 12.0 Hz, 2H), 2.70 (td, J = 12.2, 2.1 Hz, 2H), 2.65 (s, 3H), 2.60 (tt, J = 12.4, 3.6 Hz, 1H), 1.83 (d, J = 11.0 Hz, 2H), 1.62 (qd, J = 12.6, 4.0 Hz, 2H); LC: Method 1, tR = 1.7 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1482.
(2-fluoro-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (48)
Compound 48 was prepared as described for 44, using 4-(chlorosulfonyl)-2-fluorobenzoic acid (100 mg, 0.419 mmol) as a core. Off-white powder; yield 84% (50 mg, 0.116 mmol); 1H NMR (500 MHz, DMSO) δ 13.14 (s, 1H), 9.27 (dd, J = 5.8, 4.3 Hz, 1H), 8.32 (t, J = 7.3 Hz, 1H), 8.16 – 8.11 (m, 2H), 7.70 (dd, J = 8.0, 7.1 Hz, 2H), 7.61 (d, J = 7.2 Hz, 3H), 4.39 (d, J = 5.9 Hz, 2H), 4.23 (d, J = 11.6 Hz, 2H), 2.97 – 2.94 (m, 1H), 2.83 (td, J = 12.0, 2.1 Hz, 2H), 2.25 (d, J = 11.0 Hz, 2H), 2.10 (qd, J = 12.7, 4.0 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 171.14, 163.31, 159.33 (d, 1J C-F = 254.7 Hz), 145.53, 139.47 (d, 3J C-F = 6.8 Hz), 131.95, 128.84, 127.98 (d, 2J C-F = 15.0 Hz), 127.12, 126.74, 124.07 (d, 3J C-F = 3.5 Hz), 115.98 (d, 2J C-F = 25.6 Hz), 46.96, 41.77, 40.83, 32.40; 19F NMR (471 MHz, DMSO) δ -110.90 (s); LC: Method 1, tR = 1.73 min. MS (ESI): m/z = 419.54 [M - H]-; HRMS (ESI) for C20H22FN2O5S [M + H]+: m/z = calcd, 421.1233; found, 421.1241.
(2-chloro-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (49)
Compound 49 was prepared as described for 44, using 2-chloro-4-(chlorosulfonyl)benzoic acid (100 mg, 0.392 mmol) as a core. Off-white powder; yield 94% (55 mg, 0.124 mmol); 1H NMR (500 MHz, DMSO) δ 12.73 (s, 1H), 8.98 (t, J = 5.4 Hz, 1H), 7.84 (d, J = 1.6 Hz, 1H), 7.82 (dd, J = 7.9, 1.7 Hz, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.28 (dd, J = 10.0, 5.0 Hz, 2H), 7.20 (dd, J = 8.0, 1.7 Hz, 3H), 3.95 (d, J = 5.9 Hz, 2H), 3.81 (d, J = 11.6 Hz, 2H), 2.39 (td, J = 11.6, 1.7 Hz, 2H), 1.84 (d, J = 11.2 Hz, 2H), 1.68 (qd, J = 12.6, 3.9 Hz, 2H); LC: Method 1, tR = 1.74 min. MS (ESI): m/z = 435.34 [M - H]-; HRMS (ESI) for C20H22ClN2O5S [M + H]+: m/z = calcd, 437.0938; found, 437.0934.
(2-methyl-4-((4-phenylpiperidin-1-yl)sulfonyl)benzoyl)glycine (50)
Compound 50 was prepared as described for 44, using 4-(chlorosulfonyl)-2-methylbenzoic acid (100 mg, 0.426 mmol) as a core. Off-white powder; yield 100% (65 mg, 0.139 mmol); 1H NMR (500 MHz, DMSO) δ 12.67 (s, 1H), 8.80 (t, J = 6.0 Hz, 1H), 7.65 (dd, J = 7.9, 1.2 Hz, 2H), 7.59 – 7.57 (m, 1H), 7.30 – 7.26 (m, 2H), 7.19 (dd, J = 8.0, 1.8 Hz, 3H), 3.93 (d, J = 6.0 Hz, 2H), 3.79 (d, J = 11.6 Hz, 2H), 2.46 (s, 3H), 2.33 (td, J = 11.9, 2.1 Hz, 2H), 1.82 (d, J = 11.1 Hz, 2H), 1.68 (qd, J = 12.7, 4.0 Hz, 2H); LC: Method 1, tR = 1.66 min. MS (ESI): m/z = 415.40 [M - H]-; HRMS (ESI) for C21H25N2O5S [M + H]+: m/z = calcd, 417.1484; found, 417.1472.
(5-((4-phenylpiperidin-1-yl)sulfonyl)picolinoyl)glycine (51)
Step 1, A solution of methyl 5-(chlorosulfonyl)pyridine-2-carboxylate (50 mg, 0.212 mmol), 4-phenyl-piperidine (8, 51 mg, 0.318 mmol) and DIPEA (0.074 mL, 0.424 mmol) in DCM (2 mL) was stirred for 30 min at rt, after which time LC analysis indicated total consumption of the starting material and only one major peak. The volatiles were removed by evaporation to yield a white solid to which DCM (10 mL) and aqueous HCl 10% (10 mL) were added. Upon combination of organic and aqueous solutions, an insoluble precipitate appears, which floats between the two layers. The organic phase alongside the insoluble precipitate was collected and washed with (2x 10% HCl). Saponification of the crude product was carried out by stiring the solution with NaOH (1 pellet) in THF, water, MeOH. The volatiles were removed by evaporation and the aqueous solution was neutralized using formic acid. The product was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)), the fractions containing the product were freeze dried for 2 days to obtain 5-((4-phenylpiperidin-1-yl)sulfonyl)picolinic acid. White powder; yield 34% (25 mg, 0.072 mmol). LC: Method 1, tR = 1.73 min. MS (ESI): m/z = 345.27 [M - 1]-. Step 2, To a solution of 5-((4-phenylpiperidin-1-yl)sulfonyl)picolinic acid (25 mg, 0.072 mmol), DIPEA (0.050 mL, 0.289 mmol) and HATU (41.2 mg, 0.108 mmol) in DMF (1 mL) was added tert-butyl 2-aminoacetate, HCl (42, 18.2 mg, 0.108 mmol). The orange solution was stirred at rt for 24 h under N2 atmosphere, after which time water (1 mL) was added to the suspension. The volatiles were removed by evaporation to yield a white solid to which DCM (10 mL) and aqueous HCl 10% (10 mL) were added. The organic phase was colected and washed with (2x 10% HCl). The remaining DCM was evaporated to obtain a yellow solid. The tert-butyloxycarbonyl protecting group was removed by dissolving the sample in DCM (2 to 3 mL) and adding TFA (∼0.750 mL), the solution was stirred at rt for 1 h. The volatiles were evaporated. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid). After evaporation of the desired fractions the product was suspended in water and freeze dried for 2 days. Off-white powder; yield 85% (25 mg, 0.061 mmol); 1H NMR (500 MHz, DMSO) δ 12.74 (s, 1H), 9.18 (t, J = 6.0 Hz, 1H), 9.00 (dd, J = 2.2, 0.7 Hz, 1H), 8.40 (dd, J = 8.2, 2.3 Hz, 1H), 8.29 (dd, J = 8.2, 0.7 Hz, 1H), 7.30 – 7.25 (m, 2H), 7.21 – 7.16 (m, 3H), 4.00 (d, J = 6.0 Hz, 2H), 3.84 (d, J = 11.6 Hz, 2H), 2.54 (tt, J = 12.3, 3.6 Hz, 1H), 2.45 (td, J = 12.0, 2.2 Hz, 2H), 1.83 (d, J = 11.0 Hz, 2H), 1.68 (qd, J = 12.6, 3.9 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 170.79, 162.84, 152.60, 146.82, 145.07, 137.53, 134.35, 128.40, 126.65, 126.30, 122.69, 46.31, 41.25, 40.22, 31.94; LC: Method 1, tR = 1.79 min. MS (ESI): m/z = 402.23 [M - H]-; HRMS (ESI) for C19H22N3O5S [M + H]+: m/z = calcd, 404.1280; found, 404.1274.
(5-((4-phenylpiperidin-1-yl)sulfonyl)thiophene-2-carbonyl)glycine (52)
Compound 52 was prepared as described for 44, using 5-(chlorosulfonyl)thiophene-2-carboxylic acid (100 mg, 0.441 mmol) as a core.
Off-white powder; yield 69% (43 mg, 0.099 mmol);1H NMR (500 MHz, DMSO) δ 12.75 (s, 1H), 9.23 (t, J = 5.9 Hz, 1H), 7.92 (d, J = 4.0 Hz, 1H), 7.69 (d, J = 4.0 Hz, 1H), 7.30 – 7.27 (m, 2H), 7.20 (dd, J = 9.2, 4.3 Hz, 3H), 3.94 (d, J = 5.9 Hz, 2H), 3.77 (d, J = 11.6 Hz, 2H), 2.58 (tt, J = 12.3, 3.5 Hz, 1H), 1.86 (d, J = 11.1 Hz, 2H), 1.71 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.71 min, MS (ESI): m/z = 407.28 [M- H]-; HRMS (ESI) for C18H21N2O5S2 [M + H]+: m/z = calcd, 409.0892; found, 409.0886.
(3-methyl-5-((4-phenylpiperidin-1-yl)sulfonyl)thiophene-2-carbonyl)glycine (53)
Compound 53 was prepared as described for 44, using 5-(chlorosulfonyl)-3-methylthiophene-2-carboxylic acid (50 mg, 1.208 mmol) as a core. Off-white powder; yield 71% (43 mg, 0.097 mmol); 1H NMR (500 MHz, DMSO) δ 12.69 (s, 1H), 8.63 (t, J = 5.9 Hz, 1H), 8.38 (s, 1H), 7.31 – 7.27 (m, 2H), 7.23 – 7.17 (m, 3H), 3.90 (d, J = 5.9 Hz, 2H), 3.77 (d, J = 11.8 Hz, 2H), 2.66 – 2.59 (m, 3H), 2.56 (s, 3H), 1.85 (d, J = 11.2 Hz, 2H), 1.65 (qd, J = 12.7, 4.0 Hz, 2H); LC: Method 1, tR = 1.77 min. MS (ESI): m/z = 421.20 [M - H]-; HRMS (ESI) for C19H23N2O5S2 [M + H]+: m/z = calcd, 423.1048; found, 423.1048.
(4-((4-phenylpiperidin-1-yl)sulfonyl)thiophene-2-carbonyl)glycine (54)
Compound 54 was prepared as described for 44, using 4-(chlorosulfonyl)thiophene-2-carboxylic acid (100 mg, 0.441 mmol) as a core. Off-white powder; yield 71% (43 mg, 0.101 mmol); 1H NMR (500 MHz, DMSO) δ 9.19 (t, J = 5.9 Hz, 1H), 8.45 (d, J = 1.4 Hz, 1H), 8.10 (d, J = 1.4 Hz, 1H), 7.30 – 7.27 (m, 2H), 7.23 – 7.18 (m, 3H), 3.93 (d, J = 5.9 Hz, 2H), 3.76 (d, J = 11.5 Hz, 2H), 2.57 (tt, J = 12.3, 3.7 Hz, 1H), 2.45 (td, J = 12.1, 2.2 Hz, 2H), 1.85 (d, J = 11.3 Hz, 2H), 1.70 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.67 min. MS (ESI): m/z = 407.43 [M - H]-; HRMS (ESI) for C18H21N2O5S2 [M + H]+: m/z = calcd, 409.0892; found, 409.0880.
(5-((4-phenylpiperidin-1-yl)sulfonyl)thiophene-3-carbonyl)glycine (55)
Compound 55 was prepared as described for 44, using 5-(chlorosulfonyl)thiophene-3-carboxylic acid (100 mg, 0.441 mmol) as a core. Off-white powder; yield 59% (37 mg, 0.084 mmol); 1H NMR (500 MHz, DMSO) δ 12.68 (s, 1H), 8.97 (t, J = 5.9 Hz, 1H), 8.58 (t, J = 1.7 Hz, 1H), 8.04 (d, J = 1.6 Hz, 1H), 7.31 – 7.27 (m, 2H), 7.24 – 7.18 (m, 3H), 3.92 (d, J = 5.9 Hz, 2H), 3.77 (d, J = 11.2 Hz, 2H), 2.57 (tt, J = 12.5, 3.6 Hz, 1H), 2.47 (td, J = 12.1, 2.4 Hz, 2H), 1.87 (d, J = 11.2 Hz, 2H), 1.72 (qd, J = 12.7, 3.9 Hz, 2H); LC: Method 1, tR = 1.69 min. MS (ESI): m/z = 407.35 [M - H]-; HRMS (ESI) for C18H21N2O5S2 [M + H]+: m/z = calcd, 409.0892; found, 409.0883.
(5-((4-phenylpiperidin-1-yl)sulfonyl)furan-2-carbonyl)glycine (56)
Compound 56 was prepared as described for 44, using 5-(chlorosulfonyl)furan-2-carboxylic acid (100 mg, 0.475 mmol) as a core. Off-white powder; yield 68% (40 mg, 0.101 mmol); 1H NMR (500 MHz, DMSO) δ 12.75 (s, 1H), 9.00 (t, J = 5.9 Hz, 1H), 7.36 (d, J = 3.7 Hz, 1H), 7.33 (d, J = 3.7 Hz, 1H), 7.30 – 7.26 (m, 2H), 7.19 (d, J = 7.3 Hz, 3H), 3.92 (d, J = 6.0 Hz, 2H), 3.83 (d, J = 12.3 Hz, 2H), 2.78 (td, J = 12.3, 2.2 Hz, 2H), 2.63 (tt, J = 12.1, 3.3 Hz, 1H), 1.82 (d, J = 11.0 Hz, 2H), 1.62 (qd, J = 12.8, 4.1 Hz, 2H); LC: Method 1, tR = 1.66 min. MS (ESI): m/z = 391.25 [M - H]-; HRMS (ESI) for C18H21N2O6S [M + H]+: m/z = calcd, 393.1120; found, 393.1121.
(1-methyl-4-((4-phenylpiperidin-1-yl)sulfonyl)-1H-pyrrole-2-carbonyl)glycine (57)
Compound 57 was prepared as described for 44, using 4-(chlorosulfonyl)-1-methyl-1H-pyrrole-2-carboxylic acid (50 mg, 0.224 mmol) as a core. Off-white powder; yield 58% (32 mg, 0.080 mmol); 1H NMR (500 MHz, DMSO) δ 12.61 (s, 1H), 8.69 (t, J = 6.0 Hz, 1H), 7.59 (d, J = 1.8 Hz, 1H), 7.31 – 7.26 (m, 2H), 7.23 (t, J = 4.2 Hz, 2H), 7.21 – 7.17 (m, 1H), 7.16 (d, J = 1.9 Hz, 1H), 3.90 (s, 3H), 3.85 (d, J = 6.0 Hz, 2H), 3.66 (d, J = 11.4 Hz, 2H), 2.53 (tt, J = 12.3, 3.4 Hz, 1H), 2.32 (td, J = 11.9, 2.1 Hz, 2H), 1.84 (d, J = 10.9 Hz, 2H), 1.71 (qd, J = 12.6, 3.9 Hz, 2H); LC: Method 1, tR = 1.66 min. MS (ESI): m/z = 404.19 [M - H]-; HRMS (ESI) for C19H24N3O5S [M + H]+: m/z = calcd, 406.1437; found, 406.1432.
(4-((4-(4-chlorophenyl)piperidin-1-yl)sulfonyl)-2-fluorobenzoyl)glycine (61)
Compound 61 was prepared as described for 44, using 4-(chlorosulfonyl)-2-fluorobenzoic acid (50 mg, 0.210 mmol) as a core. Off-white powder; yield 78% (69 mg, 0.151 mmol); 1H NMR (500 MHz, DMSO) δ 12.71 (s, 1H), 8.85 (dd, J = 5.8, 4.3 Hz, 1H), 7.89 (dd, J = 7.9, 6.9 Hz, 1H), 7.71 (ddd, J = 9.6, 8.8, 1.6 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 3.97 (d, J = 5.9 Hz, 2H), 3.80 (d, J = 11.6 Hz, 2H), 2.55 (tt, J = 12.1, 3.3 Hz, 1H), 2.39 (td, J = 11.9, 2.0 Hz, 2H), 1.81 (d, J = 11.3 Hz, 2H), 1.66 (qd, J = 12.7, 3.9 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 170.68, 162.84, 158.88 (d, 1JC-F = 254.6 Hz), 144.07, 138.96 (d, 3JC- F = 6.8 Hz), 131.49 (d, 3JC-F = 3.0 Hz), 130.80, 128.62, 128.31, 127.54 (d, 2JC-F = 14.9 Hz), 123.63 (d, 4JC-F = 3.4 Hz), 115.53 (d, 2JC-F = 25.6 Hz), 46.41, 41.32, 31.78; 19F NMR (471 MHz, DMSO) δ -110.88 (s); LC: Method 1, tR = 1.85 min. MS (ESI): m/z = 453.25 [M - H]-; HRMS (ESI) for C20H21ClFN2O5S [M +H]+: m/z = calcd, 455.0844; found, 455.0844.
(4-((4-(4-bromophenyl)piperidin-1-yl)sulfonyl)-2-fluorobenzoyl)glycine (62)
Compound 62 was prepared as described for 44, using 4-(chlorosulfonyl)-2-fluorobenzoic acid (50 mg, 0.210 mmol) as a core. Off-white powder; yield 76% (66 mg, 0.131 mmol); 1H NMR (500 MHz, DMSO) δ 8.85 (td, J = 5.6, 1.4 Hz, 1H), 7.90 (t, J = 7.3 Hz, 1H), 7.74 – 7.68 (m, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 3.96 (d, J = 5.9 Hz, 2H), 3.80 (d, J = 11.7 Hz, 2H), 2.43 – 2.37 (m, 2H), 1.81 (d, J = 11.3 Hz, 2H), 1.65 (qd, J = 12.7, 3.9 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 170.69, 162.86, 158.88 (d, 1JC-F = 254.6 Hz), 144.49, 138.97 (d, 3J C-F = 6.9 Hz), 131.50 (d, 3J C-F = 2.9 Hz), 131.23, 129.03, 127.54 (d, 2J C-F = 14.9 Hz), 123.63 (d, 4J C-F = 3.5 Hz), 119.26, 115.53 (d, 2J C-F = 25.3 Hz), 46.40, 41.32, 39.78, 31.72; 19F NMR (471 MHz, DMSO) δ -110.88 (s); LC: Method 1, tR = 1.91 min. MS (ESI): m/z = 497.27 [M - H]-, 499.30 [M - H]- +2; HRMS (ESI) for C20H21BrFN2O5S [M + H]+: m/z = calcd, 499.0339; found, 499.0333.
(2-fluoro-4-((4-(pyrrolidin-1-yl)piperidin-1-yl)sulfonyl)benzoyl)glycine (63)
Compound 63 was prepared as described for 44, using 4-(chlorosulfonyl)-2-fluorobenzoic acid (50 mg, 0.210 mmol) as a core and 4-(1-pyrrolidinyl)piperidine (49 mg, 0.314 mmol) as amine. Off-white powder; yield 80% (66 mg, 0.143 mmol); obtained as a formic acid salt; 1H NMR (500 MHz, DMSO) δ 9.61 (s, 1H), 8.84 (dd, J = 5.8, 4.1 Hz, 1H), 7.93 – 7.88 (m, 1H), 7.75 (dd, J = 9.5, 1.5 Hz, 1H), 7.70 (dd, J = 8.0, 1.6 Hz, 1H), 3.96 (d, J = 5.9 Hz, 2H), 3.78 (d, J = 12.4 Hz, 2H), 3.49 (d, J = 4.9 Hz, 2H), 3.17 – 3.08 (m, 1H), 3.04 – 2.95 (m, 2H), 2.40 (t, J = 11.4 Hz, 2H), 2.10 (d, J = 11.4 Hz, 2H), 2.02 – 1.92 (m, 2H), 1.86 – 1.76 (m, 2H), 1.59 (qd, J = 12.3, 4.1 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 170.67, 162.78, 158.92 (d, 1JC-F = 255.0 Hz), 138.97 (d, 3JC-F = 6.9 Hz), 131.61 (d, 3JC-F = 3.0 Hz), 127.65 (d, 2JC-F = 15.0 Hz), 123.59 (d, 4JC-F = 3.8 Hz), 115.55 (d, 2JC-F = 25.2 Hz), 59.66, 51.00, 44.36, 41.30, 27.41, 22.48; 19F NMR (471 MHz, DMSO) δ -110.70 (s); LC: Method 1, tR = 0.99 min. MS (ESI): m/z = 414.35 [M - H]-; HRMS (ESI) for C18H25FN3O5S [M + H]+: m/z = calcd, 414.1499; found, 414.1501.
(5-((4-(4-chlorophenyl)piperidin-1-yl)sulfonyl)picolinoyl)glycine (64)
Compound 64 was prepared as described for 51, using methyl 5-(chlorosulfonyl)pyridine-2-carboxylate (50 mg, 0.212 mmol) as a core and 4-(4-chlorophenyl)piperidine hydrochloride (74 mg, 0.318 mmol) as amine. Off-white powder; yield 97% (30 mg, 0.066 mmol); 1H NMR (500 MHz, DMSO) δ 12.73 (s, 1H), 9.19 (t, J = 6.1 Hz, 1H), 9.00 (dd, J = 2.2, 0.7 Hz, 1H), 8.39 (dd, J = 8.2, 2.3 Hz, 1H), 8.29 (dd, J = 8.2, 0.7 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 4.01 (d, J = 6.1 Hz, 2H), 3.83 (d, J = 11.6 Hz, 2H), 2.57 (tt, J = 12.1, 3.4 Hz, 1H), 2.44 (td, J = 11.8, 2.0 Hz, 2H), 1.82 (d, J = 11.2 Hz, 2H), 1.66 (qd, J = 12.7, 4.0 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 170.79, 162.88, 152.59, 146.81, 144.05, 137.53, 134.29, 130.81, 128.60, 128.32, 122.70, 46.21, 41.17, 39.58, 31.77; LC: Method 1, tR = 1.83 min. MS (ESI): m/z = 436.09 [M - H]-; HRMS (ESI) for C19H21ClN3O5S [M + H]+: m/z = calcd, 438.0890; found, 438.0882.
ASSOCIATED CONTENT
Supporting Information
USP5-1 coordinates (CIF)
USP5-1 sf (CIF)
USP5-48 coordinates (CIF)
USP5-48 sf (CIF)
USP5-64 coordinates (CIF)
USP5-64 sf (CIF)
Supporting tables and figures, X-ray crystallographic statistics, methods, characterization data, 1H NMR, 13C NMR, 19F NMR, HLPC purity analysis for the compounds (PDF)
Molecular formula strings (CSV)
3D PDB file used for docking studies (PDB)
Hit Expansion 1 6DXH
Hit Expansion 2 7MS5
Accession Codes
7MS5, 7MS6, 7MS7
Authors will release atomic coordinates and experimental data upon article publication.
AUTHOR INFORMATION
Author Contributions
The manuscript was written by M.K.M, and M.S. and revised by R. J. H. All authors have given approval to the final version of the manuscript. M.K.M. completed hit expansion and docking, expressed and purified proteins, designed and optimized biophysical assays and tested compounds, and performed co-crystallization screens. A.D. solved the X-ray structure with compound 1. R. J. H. solved the X-ray crystal structures with 48 and 64. Analogs were synthesized and characterized by C.A.Z. and H.G.A. T.K. and A.A. performed liver microsomal stability studies and generated compound HRMS data. R.A., C.H.A, R.J.H. and M.S. advised throughout the project.
Funding Sources
M.S. gratefully acknowledges support from NSERC [Grant RGPIN-2019-04416]. R.J.H. is the recipient of the Huntington’s Disease Society of America Berman Topper Career Development Fellowship. H.G.A. acknowledges Mexican National Council on Science and Technology (CONACyT) for financial support of this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].We thank Albina Bolotokova for compound management, Darcy Burns and Jack Sheng for 13C NMR data collection, and Amir Jafari and Rick Briltz for their help in the procurement of the starting materials.
ABBREVIATIONS
- BRAP
- BRCA-1 associated protein
- DCM
- dichloromethane
- DMF
- dimethylformamide
- DMSO
- dimethylsulfoxide
- DIPEA
- N,N-diisopropylethylamine
- DUB
- deubiquitinase
- FL
- full-length
- HATU
- hexafluorophosphate azabenzotriazole tetramethyl uranium
- HDAC6
- histone deacetylase 6
- HRMS
- high resolution mass spectrometry
- IQF
- internally quenched fluorophore
- KD
- dissociation constant
- LCMS
- liquid chromatography mass spectrometry
- LRMS
- low resolution mass spectrometry
- QTOF
- quadrupole-time-of-flight
- SAR
- structure activity relationship
- SD
- standard deviation
- SPE
- solid phase extraction
- SPR
- surface plasmon resonance
- TFA
- trifluoroacetic acid
- Ub
- ubiquitin
- Ub2K48
- Lys48-linked di-ubiquitin
- UBA
- ubiquitin-binding associated domain
- Ub-AMC
- ubiquitin amidomethyl coumarin
- nUBP
- N-terminal ubiquitin-binding domain
- USP
- ubiquitin specific protease
- UPLC
- ultra-performance liquid chromatography
- WT
- wild-type
- ZnF-UBD
- zinc finger ubiquitin-binding domain
REFERENCES
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