Discovery of thymoquinone analogues with high anthelmintic activity against monogenean infections in goldfish (Carassius auratus).

Monogenean parasites are harmful pathogens in aquaculture systems. Current treatment strategies for monogenean infections are unsatisfactory, making the discovery of new drugs urgent. Thymoquinone (TQ), a natural monoterpene isolated from Nigella sativa L., has demonstrated its potential as lead structure against the monogenean parasites. In the present study, the anthelmintic activity of 26 selected TQ analogues was investigated against the monogenean parasite Gyrodactylus kobayashii in the goldfish (Carassius auratus). The results indicated that 18 TQ analogues displayed the EC₅₀ value below 1.0 mg/L, 14 of them showed more potent anthelmintic activity than TQ (EC₅₀=0.303 mg/L). Particularly, compounds 9 and 23 exhibited the best efficacies with the EC₅₀ values of 0.058 and 0.034 mg/L, outperforming the standard drug praziquantel as well. Structure-activity relationship analysis revealed that large electron-donating substituents at the 2-position of the quinone ring contribute to enhanced in vivo anthelmintic activity. Additionally, four analogues (9, 12, 23 and 25) displayed low fish toxicity, with the therapeutic index (TI, LC₅₀/EC₅₀) above 9.23, and were selected for further in silico drug‑likeness assessment. The results demonstrated an excellent drug-likeness profile and an adherence to major pharmaceutical companies' filters for compounds 9 and 23. Besides, TQ analogue 9 was chosen for an in vivo study in large-scale trial based on its potency, low fish toxicity, and excellent drug-likeness profiles. Treatment of G. kobayashii infected goldfish for 24 h at 0.1 and 0.2 mg/L was effective, reducing the infection prevalence from 100 % to 0 % post-treatment. The experimental fish exhibited normal behavior, despite minor modifications were displayed in the gills and liver according to histopathological results. Collectively, this study suggested that TQ analogues are excellent scaffolds in the discovery of novel anthelmintic agents against monogenean infections.