Enhancing thiazolide efficacy: C-4 aryl modifications drive selective SARS-CoV-2 inhibition.

Background: COVID-19 highlighted urgent need for broad-spectrum antivirals. Nitazoxanide (NTZ), a broad-spectrum antiviral with an established safety profile, shows promise against SARS-CoV-2; however, its moderate potency and pharmacokinetic limitations necessitate the development of improved analogues.

Methods: We synthesized 24 thiazolide analogues, including novel molecules bearing 4´- or 5´-aryl substitutions, dual 4´,5´-modifications, or fused benzothiazole cores, and evaluated their in vitro efficacy against SARS-CoV-2 (Vero E6 cells). Antiviral potency (IC₅₀), cytotoxicity (CC₅₀), and selectivity indices (SI = CC₅₀/IC₅₀) were determined by qRT-PCR and MTT assays. In silico ADMET profiling predicted drug-likeness, absorption, distribution, metabolism, excretion, and toxicity.

Results: Among the analogues, 4´-aryl thiazolides (6d-6 g) and one dual-substituted thiazolide (7b) exhibited outstanding selectivity index (SI > 30), significantly surpassing NTZ (SI ≈ 14). The analogue 6e (3-OCF₃-phenyl) demonstrated the highest SI of ≈ 51 (IC₅₀ ≈0.21 µM; CC₅₀ ≈10.8 µM). Benzothiazole analogue 8a (OCF₃ at 4´-position) also showed favorable SI (≈11). ADMET predictions confirmed acceptable oral bioavailability, minimal cytochrome P450 (CYP450) inhibition, and low cardiotoxicity risk.

Conclusions: Lipophilic, electron-withdrawing substituents at C-4 of the thiazole core markedly enhance antiviral potency and therapeutic potential. In particular, 4´-(PhOCF₃) substitutions emerge as lead scaffolds for further preclinical development. These insights provide a way forward for optimizing thiazolides against SARS-CoV-2 and other emerging viruses.