Fragment-Sized Thiazoles in Fragment-Based Drug Discovery Campaigns: Friend or Foe?

Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.


S4
. Plots from the TNB 2reactivity assay. Compound background absorbance and baseline drift were subtracted from each measurement. Vertical dashed line denotes time of TCEP addition to determine the reversibility of the reaction. Orange lines denote baseline and blue lines denote compounds. 2-Chloro-N-(3-chlorophenyl)acetamide was used as a control compound. S5 Figure S4. Relationship between selected descriptors derived from quantum-chemical calculation and reactivity parameters, i.e., the number of flags (left column) and thiol reactivity (right column). S6

Experimental procedures
No unexpected or unusually high safety hazards were encountered.

ChEMBL substructure search
The bioactive compounds were downloaded in sdf format from the ChEMBL database, which contained 2,331,700 compounds. The database was accessed on 2022-10-21, and a substructure search was performed using the KNIME analysis platform. First, structures were converted to RDKit format using the RDKit From Molecule node. Second, a substructure search was performed using the RDKit Substructure Filter. The SMARTS patterns used for the substructure search are listed in Table S1. Table S1. SMARTS patterns used for the substructure search.

MurA assay
A recombinant E. coli MurA enzyme (UDP-N-acetylglucosamine enolpyruvyl transferase) was expressed in E. coli. 1 In a colorimetric assay using malachite green, inhibition of the enzyme was monitored by following the orthophosphate formed during the enzymatic reaction. Briefly, compounds were pre-incubated at a concentration of 500 µM with MurA and the substrate UDP-

MetAP1a assay
The enzymatic activity of M. tuberculosis MetAP1a, was measured by a kinetic assay using a fluorogenic substrate L-methionine 7-amido-4-methylcoumarin as described previously. 2

Aqueous stability assay
The aqueous stability of all compounds was determined spectrophotometrically by following the changes in the absorption spectra of the compounds, as described previously. 3

Redox activity assays
Assays were performed according to previously optimized procedures. 4 Experiments were performed in 96-well microplates in assay buffer (50 mM HEPES, 50 mM NaCl, pH 7.5).
Threshold values for activity flags were above 10-fold standard deviation compared to DMSO blanks. All reagent solutions were freshly prepared before performing the experiments. 3-Methyltoxoflavin was used as a control compound.

Thiol reactivity assays
Assays were performed according to previously optimized procedures. 4  Compound background absorbance and baseline drift were subtracted from each measurement. Additionally, electron density, average local ionization energy (ALIE), noncovalent interactions, and electrostatic potential were calculated and plotted on a grid.

General chemistry
Compounds from the in-house chemical library and commercially available compounds were

General procedure for bromination (general procedure 1)
Thiazole-2-amine (1.0 equiv.) was dissolved in MeCN (30 mL) on an ice bath. To a stirred solution, tert-butyl nitrite (1.2 equiv.) and CuBr2 (1.2 equiv.) were added, and the mixture was stirred on an ice bath overnight. The reaction mixture was extracted with ethyl acetate (2 × 50 mL) and water (2 × 50 mL). The combined organic phases were washed with brine, dried over S14 anhydrous sodium sulfate, filtered, and volatile components evaporated in vacuo to afford the crude product which was purified with flash column chromatography.

General procedure for esterification (general procedure 2)
2-Bromothiazole (1.0 equiv.) was suspended in anhydrous MeOH (20 mL) and cooled on an ice bath. SOCl2 (2.0 equiv.) was added dropwise and the mixture was refluxed overnight. The solvent was evaporated, the residue dissolved in water (2 mL) and neutralized with K2CO3. The aqueous phase was extracted with ethyl acetate (2 × 25 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and volatile components evaporated in vacuo to afford the crude product that was purified with flash column chromatography.

General procedure for demethylation (general procedure 3)
Methyl 2-oxoacetate (1.0 equiv.) was suspended in THF (20 mL) and cooled on an ice bath. LiOH (0.1 M, 5.0 equiv.) was added, and the mixture was stirred on an ice bath for 2 hours. The reaction mixture was neutralized with NaOH (1 M, 1-5 mL) and the impurities were extracted with ethyl acetate (2 × 20 mL). The aqueous phase was acidified to pH 1 with HCl (1 M) and the product precipitated from the solution.

General procedure for acylation (general procedure 4)
2-Aminothiazole (1.0 equiv.) was dissolved in DCM (20 mL) under argon. Next, Et3N (2.2-4.4 equiv.) was added and the reaction mixture was cooled on an ice bath. Methyl oxalyl chloride or methyl malonyl chloride (1.1-2.2 equiv.) was added dropwise and the reaction mixture was stirred under argon at room temperature overnight and extracted with DCM (50 mL), which was washed with saturated brine (2 × 15 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and volatile components evaporated in vacuo to afford the crude product.

2-(2-Bromothiazol-4-yl)acetic acid (29)
Methyl 2-(2-bromothiazol-4-yl)acetate (0.54 g, 2.3 mmol, 1.0 equiv.) was dissolved in MeOH (2 mL), on an ice bath. NaOH (2 M, 2.0 equiv.) was added to the solution and stirred for 2 h at room temperature. The mixture was neutralized with HCl (1 M, 1 mL) and MeOH was evaporated. The reaction mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with brine (100 mL), dried over anhydrous sodium sulfate, filtered, and volatile components evaporated in vacuo to afford the crude product which was purified with flash column chromatography to afford 29 as a white crystalline solid in 76% of yield (388 mg