Metallo‐β‐lactamases (MBLs) cause resistance of Gram‐negative bacteria to β‐lactam antibiotics and are of serious concern, because they can inactivate the last‐resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4‐amino‐2,4‐dihydro‐5‐(2‐methylphenyl)‐3H‐1,2,4‐triazole‐3‐thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non‐amino compound IIIB (1,2‐dihydro‐5‐(2‐methylphenyl)‐3H‐1,2,4‐triazole‐3‐thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole–thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC₅₀ values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM‐4, VIM‐2, NDM‐1, and IMP‐1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM‐1, VIM‐2, and IMP‐1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc‐dependent hydrolases belonging to the MBL‐fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC₅₀ values toward plasmodial glyoxalase II were in the 10 μm range.

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with K_i values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.