An ovel series of C12-keto-type saxitoxin (STX) derivatives bearinga nu nusualn onhydrated form of the ketone at C12 has been synthesized,a nd their Na V -inhibitory activity has been evaluated in ac ell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3a)s howedp otent inhibitory activity againstn euroblastoma Neuro2Ai nboth cell-baseda nd electrophysiological analyses,w ithE C 50 and IC 50 valueso f8 .5 and 30.7 nm,r espectively.I nterestingly,t he compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of Na V 1.5, with an IC 50 value of 94.1 nm.D erivatives 3a-d and 3f showedl ow recovery rates from Na V 1.2 subtype (ca4 5-79 %) comparedt on atural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-ketod erivatives with Na V in which the enonem oiety in the STX derivatives 3 worksa sM ichaela cceptorf or the carboxylateo fA sp 1717 .

Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 μM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 μM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 μM AA (1), while 50 μM DGLA (5), 50 μM EPA (6), and 10 μM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.