Based on our previous success in using cyclobutanone derivatives as enzyme inhibitors, we have designed and prepared a 37-member library of α-aminocyclobutanone amides and sulfonamides, screened for inhibition of the bacterial enzyme diaminopimelate desuccinylase (DapE), which is a promising antibiotic target, and identified several inhibitors with micromolar inhibitory potency. Molecular docking suggests binding of the deprotonated hydrate of the strained cyclobutanone, and thermal shift analysis with the most potent inhibitor (3y, IC₅₀ = 23.1 µM) enabled determination of a K_i value of 10.2 +/− 0.26 µM and observed two separate T_m values for H. influenzae DapE (HiDapE).

Based on the inhibitory potencies from earlier reported tetrazole thioether analogs, we now describe the synthesis and inhibition of pyrazole-based inhibitors of N-succinyl-l,l-2,6-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae (HiDapE). The most potent pyrazole analog 7d bears an aminopyridine amide with an IC₅₀ of 17.9 ± 8.0 μM, and the single enantiomer of ɑ-methyl analog 7q has an IC₅₀ of 18.8 µM, with potency residing in the (R)-enantiomer. Thermal shift revealed strong stabilization upon binding inhibitor (R)-7q with T_m = 50.2 °C and a K_i of 17.3 ± 2.8 μM. Enzyme kinetic experiments confirm competitive inhibition, and docking reveals key active site interactions.