Structure-activity relationship and nsp15-dependent mechanism of spirothiazolidinone derivatives with pan-coronavirus activity.

The ubiquitous presence of pathogenic coronaviruses (CoVs) in humans and animals underscores the need for small-molecule antivirals with pan-CoV efficacy. Here, we report the design and synthesis of a novel series of spirothiazolidinone derivatives (2a-2p), prepared via an efficient one-pot cyclocondensation reaction. The compounds were initially evaluated for antiviral activity against human CoVs HCoV-229E and SARS-CoV-2. Analysis of the structure-activity relationship revealed that antiviral potency and selectivity were strongly influenced by the substituent at C-8 of the spirocyclic core, with bulky hydrophobic or aromatic groups yielding the most favourable profiles. The resulting lead compound, 2p, was evaluated against a broader panel of human and animal CoVs, including HCoV-NL63, HCoV-OC43, murine hepatitis virus (MHV-A59) and porcine transmissible gastroenteritis virus (TGEV). 2p demonstrated pan-CoV activity, with EC90 values ranging from 2.2 to 30 μM, and without cytotoxicity at 100 μM. Virus selected for 2p resistance carried mutations in nsp15, a CoV non-structural protein with endoribonuclease (EndoU) functionality. Biochemical EndoU assays showed no inhibition by 2p, while surface plasmon resonance demonstrated direct binding to nsp15, indicating non-catalytic target engagement. Time-of-compound-addition experiments showed that 2p exerts its inhibitory effect during the post-entry phase of the CoV life cycle. 2p displayed additive antiviral effect when combined with the CoV polymerase inhibitor GS-441524, indicating mechanistic complementarity and potential for combination therapy. Collectively, these findings position our spirothiazolidinones as promising leads for further optimization toward pan-CoV drug development.