Spiroisoxazoline Inhibitors of Acetylcholinesterase from Pseudoceratina verrucosa . Quantitative Chiroptical Analysis of Configurational Heterogeneity, and Total Synthesis of (±)-Methyl Purpuroceratate C.

Examination of the MeOH extract of the sponge, Pseudoceratina cf. verrucosa , Berquist 1995 collected near Ningaloo Reef, Western Australia for selective acetylcholinesterase (AChE) inhibitors, yielded five new bromotyrosine alkaloids, methyl purpuroceratates A and B ( 1b and 2b ), purpuroceratic acid C ( 3a ), and ningalamides A and B ( 4 and 5 ). The structures of 1 - 4 share the dibromo-spirocyclohexadienyl-isoxazoline (SIO) ring system found in purealidin-R, while ketoxime 5 is analogous to ianthelline and purpurealidin I. The planar structures of all five compounds were obtained from analysis of MS, 1D and 2D NMR data, and the absolute configuration of the spiroisoxazoline (SIO) unit was assigned by electronic circular dichroism (ECD) and comparison with standards prepared by total synthesis of methyl purpuroceratate C, (±)- 3b . Compound 4 is the most complex SIO described, to date. The configuration of the homoserine module ( C ) in 4 was ascertained, after acid hydrolysis, by derivatization of an l-tryptophanamide derivative based on Marfey's reagent. Chiral-phase HPLC, with comparison to synthetic standards, revealed that most SIOs isolated from P. cf. verrucosa were configurationally heterogeneous; some, essentially racemic. Chiral-phase HPLC, with UV-ECD detection, is demonstrated as a superlative method for configurational assignment and quantitation of the enantiomeric composition of SIOs. Two SIOs─aerophobin-1 and aplysinamisine II─emerged as selective inhibitors of AChE over butyrylcholinesterase (BuChE, IC 50 ratio >10), while aplysamine-2 moderately inhibited both cholinesterases (ChEs, IC 50 , (AChE) 0.46 μM; IC 50 , (BuChE) 1.03 μM). SIO alkaloids represent a potential new structural manifold for lead-discovery of new therapeutics for treatment of Alzheimer's disease.