The voltage-gated sodium ion channel inhibitory activities of a new tetrodotoxin analogue, 4,4a-anhydrotetrodotoxin, and three other analogues evaluated by colorimetric cell-based assay

• Nav inhibitory activities of four TTX analogues were evaluated by cytotoxicity protection assay.
• EC50 of 4,4a-anhydroTTX was 750 fold larger than that of TTX, supporting the implication of 4-OH.
• The high activity of 11-oxoTTX as same as TTX was confirmed by this assay.
• The activity of 11-norTTX-6,6-diol by this assay was much less than that previously reported.
• Modification of C-6 in 11-norTTX-6,6-diol by introducing a maleimide moiety reduced activity.

The voltage-gated sodium ion channel inhibitory activities of four tetrodotoxin analogues were evaluated for their ability to reduce the cytotoxicity of ouabain and veratridine in mouse neuroblastoma Neuro-2a cells. EC50 of the novel analogue, 4,4a-anhydrotetrodotoxin purified from pufferfish, was 750 fold larger than that of tetrodotoxin, supporting the implication of 4-OH in activity. The high activity of 11-oxotetrodotoxin was confirmed. Modification of C-6 of 11-nortetrodotoxin-6,6-diol to form an oxime derivative decreased the activity to 1/22.