Cyano-containing Daphniphyllum alkaloids with antienteroviral activity from the leaves and stems of Daphniphyllum calycinum Benth.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease (HFMD), is often associated with severe clinical outcomes. In this study, three rare cyano-containing alkaloids, cyanodaphcalycines A-C (1-3), featuring an unusual 5/6/6/7/5/5-hexacyclic scaffold, and an undescribed daphmanidin A-type alkaloid, daphmanidin G (4), were isolated from the leaves and stems of the medicinal plant Daphniphyllum calycinum Benth., along with three known Daphniphyllum alkaloids (5-7). The previously undescribed structures and their absolute configurations were determined using a combination of spectroscopic analysis, a single crystal X-ray diffraction analysis, and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. In the biological evaluation, the antiviral activities of compounds 1-4 were tested against EV71 in human malignant embryonal rhabdomyosarcoma (RD) cells. Compounds 3 and 4 exhibited potent antiviral effects against human EV71 in RD cells, with EC50 values of 3.78 ± 0.23 and 6.87 ± 0.30 μg/mL, respectively, demonstrating superior efficacy to ribavirin (EC50 65.77 ± 0.11 μg/mL). This is the first report of Daphniphyllum alkaloids as potent inhibitors of EV71.