Covalent Inhibition of Thioredoxin Reductase by Michael Acceptors: Rational Design, Synthesis, and Biological Evaluation of Oxazol-5(4H)-one Derivatives

Thioredoxin reductase 1 (TrxR1) is a key enzyme involved in maintaining redox homeostasis in cancer cells and is considered a promising target for anticancer drug development. This study aimed to rationally design, synthesize, and evaluate a novel series of oxazol-5(4H)-one derivatives as potent and selective inhibitors of TrxR1.

The design process was initiated by screening a commercial library of oxazolones, which informed the construction of a QSAR model. Guided by molecular docking studies against the TrxR1 catalytic site (PDB: 3EAN), a focused library of 18 target 4-benzylidene-oxazol-5(4H)-ones was synthesized via a multicomponent reaction using substituted hippuric acids and aromatic aldehydes. The inhibitory activity against TrxR1 and the off-target enzyme glutathione reductase (GR) was determined using a DTNB assay on A549 cell lysates. Cytotoxicity was evaluated against a panel of human cancer cell lines (A549, SHSY5Y, U251, and HELA) and normal HEK293T cells using an MTT assay. The mechanism of action was further investigated through immunoblotting analysis.

Several compounds demonstrated potent and selective TrxR1 inhibition. Lead compounds 1i, 1o, and 1s exhibited significant TrxR1 inhibition (over 50% at 25 µM) with low activity against GR. The IC₅₀ values for TrxR1 inhibition were in the nanomolar range (0.25 nM for 1i, 19.7 nM for 1o, and 4.2 nM for 1s). These compounds also showed pronounced cytotoxicity against cancer cells, with compound 1i being the most active against U251 glioblastoma cells (IC₅₀ = 12.58 µM) and demonstrating selectivity over normal cells. Immunoblotting analysis confirmed that inhibitors containing dual Michael acceptor motifs (e.g., 1q–s) induced the formation of covalent TrxR1 dimers, indicative of a unique mechanism of action.

The results validate our rational design strategy, highlighting the critical role of the Michael acceptor warhead and the oxazolone core in effective TrxR1 binding and inhibition. The high selectivity for TrxR1 over GR and the observed cytotoxic selectivity toward cancer cells underscore the therapeutic potential of this compound class. The discovery of TrxR1 dimerization provides a novel mechanism for irreversible enzyme inhibition.

A new series of oxazol-5(4H)-one derivatives was successfully developed as highly potent and selective TrxR1 inhibitors. The lead compounds 1i, 1o, and 1s showed promising anticancer activity in vitro, coupled with a favorable selectivity profile. This work not only establishes a robust structure–activity relationship but also introduces a rational strategy for designing novel TrxR1-targeted antitumor agents, supported by the discovery of a dimerization-based inhibition mechanism.