key: cord-0942160-mfipcln2
authors: Kashman, Y.; Hirsch, S.; Koehn, F.; Cross, S.
title: Reiswigins A and B, novel antiviral diterpenes from a deepwater sponge
date: 1987-12-31
journal: Tetrahedron Letters
DOI: 10.1016/s0040-4039(00)96754-6
sha: 5897bc7b92f1335a9bf85fb054342a5287d7dc3b
doc_id: 942160
cord_uid: mfipcln2

Abstract The structures of reiswigins A( 1 ) and B( 2 ), new reduced azulene diterpene enones have been determined by combined one and two dimensional NMR and mass spectral techniques.

beendetermined by canbined one andtwodimansionalNMR andmass spectral techniques.

As part Of our continuing program aimed at the isolation of novel antiviral metabolites from deepwater marine organisms we found the diterpenes 1 and 2 in a specimen of Epipolasis reiswiqi( Class LXmosponqiae, Order Halichondria)'collected by submersible at 330 meters.

Reiswigins A and B show potent in-vitro activity against Herpes simplex type 1 virus and mrine A59 hepatitis virus2, and are novel additions to the vast array of terpane metabolites isolated fran marine sponges.3 Initial in-vitro assay of a 3:l methanol/toluene extract of the frozen sponge showed strong antiviral activity against Herpes simplex virus type 1 (HSV-1). Accordingly, lyophilized sponge(55 gr. dry wt.) was extracted with diciiioranethane to give 1.3 grams of crude extract. Bioassay guided isolation began by flash chrcmatography on silica gel with successive elution by heptane, chloroform and finally 1:l methanol/chloroform. The antiviral fraction was subjected to repeated vacum flash chromatography (silica gel H) followed by HPLC (silica gel, 10,20% ethyl acetate/heptane) to give l(SOOmg,O.9% of lyophilized sponge) and 2(4Omg,O.O7%) as tan oils.

Reiswigin A was assigned molecular formnila C20H3202 by HREIM?. 4 The 13C NMR spectrum showed two singlet carbonyl resonances at 210.4 and 206.9 pp. These, considered along with infrared ab--1 sorptions at 1700 and 1630 an , and ultra violet absorbance bends at 223nm(z=23OO,heptane) and 205nm(z=5700) established the presence of ketone and enone functionalities. A DEPT experiment 2 showed the remaining carbon resonances to consist of 2 quaternary(one sp and one sp3), 5 methine(one sp2 and four sp3), 6 methylene and 5 methyl resonances. With 5 sites of unsaturation required by the apparent molecular formula, and 3 accounted for by the Cl3 data, 1 was determined to be a bicyclic diterpene.

The proton NMR data clearly established the presence of a terminal isopropyl group, observed at 1.0 and 0.98 ppn, a bridgehead methyl resonance at 1.20 ppm, vinyl methyl of an a,p unsaturated ketone observed at 1.98 ppn, along with the a proton resonance of the enone at 5.85

With the identity of several key functional elements estabished by the preceding inspec tion, and the relatively congested nature of the upfield region of the 'H NMR spectrum, we turned to 2 dimensional methods to establish the overall molecular connectivities. A standard 'H CZSY6 experiment along with a one bond HEIUX' experiment established several proton-proton and one bond proton-carbon connectivities. In addition, several 3 bond correlations were obtained by a long range HEX?X*experiment. All correlations observed in these experiments are presented in Table 1 . A determination of the canplete carbon connectivity network by this method was precluded by substantial overlap of the signals for protons on C-5,C-6, and C-13, and for protons bound to C-8, C-9, and C-10. We subsequently performed a 2D iNAD~TEg'loexperiment to establish the remaining carbon connectivities. A contour plot of the upfield region of the symmetrical INADEQUATE 2D matrix is presented in Figure 2 . Cross peaks delineating the canplete connectivity network for all sp3 carbons are evident. Additional one-bond correlations for C-2,C-3,C-4, and C-16 were observed in the downfield region (not shown).The resulting structure assignment is consistent with the major mass spectral fragmentations, shown in Figure 1 .

Relative stereochemical relationships between the C-11 methyl group, H-7,and H-8, were assigned by phase sensitive NOESY " ,NCE difference and spin decoupling experiments.

The HREIMS spectrum of reiswigin B(2) indicated a molecular formula of C20H3002.5

Intercanparison of 'H and 13C NMR data with those of (1) showed the presence of an additional double bond in (2), the location of which must be as an isopropenyl terminus, evidenced in the 1 HNMR spectrum by resonances for H-14 at 6.04 ppn and for terminal H-17 and H-16 methyl singlets at 2.13 and 1.88 ppn respectively. All 'H and l3 C NMR assignments for 2 were confirmed by 0=6Y and m experiments. 0 

Taxonanic identification of the sponge was made by

New Swies of Derrrsspcngiae(Porifera) fran the National Park "Archi~lago de 10s Rcques

Antiviral assays for herpes simplex virus type 1 (HSV-1) and vesicular stanatitis virus(VSV) were plaque reduction tests in infected CV-1 monkey kidney cells

Readings in these assays range from "-'I for no observable viral inhibition, to "3+", denoting canplete inhibition of virus by the test canpound. Reiswigin A(&) was nontoxic at 2pg and ccmpletely inhibited HSV-1 and VSV. A59 was partially inhibited at 2Opg with a 2+ reading

MS(HRE1) m/z 304,C20H3202 , 1.8%, 0.3~~ deviation, 1H NMR(360 MHz,CLC13): 6 5.85s(lH,H-5)

223nm(~=2300) 205nm(&=5700)

Me-20),1.88s(6H,Me-19,Me-12), 1.82m(lH,H-10) 1.74m(lH,H-8), 1.68m(3H,H-6,H-lo)

Standard m @se sequence with suitable delays to emphasize Ja= 1OHz and 7Hz

Sweep width in Fl and F2 18.5KH.z, 256 experiments (t, increments) at 256 scans per experiment were recorded. Zero filling in Fl to lK, no zero filling in F2 to give a total 2D matrix 2K X 2K, Effective resolution in both Fl and F2 9 Hz/point