The β-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with β-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-β-lactam moiety displayed highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. LC-MS experiments revealed that tripeptide-β-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the β-lactam ring with the catalytic serine, followed by release of the inhibitor with opened β-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.

The enantioseparation of syn- and anti-3,5-disubstituted hydantoins 5a–i was investigated on three immobilized polysaccharide-based columns (CHIRAL ART Amylose-SA, CHIRAL ART Cellulose-SB, CHIRAL ART Cellulose-SC) by high performance liquid chromatography (HPLC) using n-hexane/2-PrOH (90/10, v/v) or 100% dimethyl carbonate (DMC) as mobile phases, respectively, and by supercritical fluid chromatography (SFC) using CO₂/alcohol (MeOH, EtOH, 2-PrOH; 80/20, v/v) as a mobile phase. The chromatographic parameters, such as separation and resolution factors, have indicated that Amylose-SA is more suitable for enantioseparation of the most analyzed syn- and anti-3,5-disubstituted hydantoins than Celullose-SB and Cellulose-SC in both HPLC and SFC modalities. All three tested columns showed better enantiorecognition ability toward anti-hydantoins compared to syn-hydantoins, both in HPLC and SFC modes. We have demonstrated that environmentally friendly solvent DMC can be efficiently used as the mobile phase in HPLC mode for enantioseparation of hydantoins on the immobilized polysaccharide-based chiral stationary phases.

Hydantoins, a class of five-membered heterocyclic compounds, exhibit diverse biological activities. The aim of this study was to synthesize and characterize a series of novel 3,5-disubstituted hydantoins and to investigate their antiproliferative activity against human cancer cell lines. The new hydantoin derivatives 5a–i were prepared as racemic mixtures of syn- and anti-isomers via a base-assisted intramolecular amidolysis of C-3 functionalized β-lactams. The enantiomers of syn-5a and anti-hydantoins 5b were separated by preparative high-performance liquid chromatography (HPLC) using n-hexane/2-propanol (90/10, v/v) as the mobile phase. The absolute configuration of the four allyl hydantoin enantiomers 5a was assigned based on a comparison of the experimental electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra with those calculated using density functional theory (DFT). The antiproliferative activity evaluated in vitro against three different human cancer cell lines: HepG2 (liver hepatocellular carcinoma), A2780 (ovarian carcinoma), and MCF7 (breast adenocarcinoma), and on the non-tumor cell line HFF1 (normal human foreskin fibroblasts) using the MTT cell proliferation assay. In silico drug-like properties and ADMET profiles were estimated using the ADMET Predictor ver. 9.5 and the online server admetSAR. Eighteen new 3,5-disubstituted hydantoins were synthesized and characterized. The compound anti-5c showed potent cytotoxic activity against the human tumor cell line MCF7 (IC₅₀ = 4.5 µmol/L) and the non-tumor cell line HFF1 (IC₅₀ = 12.0 µmol/L). In silico analyzes revealed that the compounds exhibited moderate water solubility and membrane permeability and are likely substrates for CYP3A4 and P-glycoprotein and have a high probability of antiarthritic activity.