New cytotoxic cyclic peroxide acids from Plakortis sp. marine sponge

Bioassay-guided fractionation of the extract of Jamaican marine sponge Plakortis sp. followed by preparative TLC and HPLC yielded several known methyl ester cyclic peroxides ( 1a, 2a, 3a, 4, 5 ), known plakortides ( 6,7 ), known bicyclic lactone ( 8 ) and new cyclic peroxide acids ( 1b, 2b, 3b ). The chemical structures were elucidated by extensive interpretation of their spectroscopic data. These natural products showed remarkable in vitro cytotoxicity against several cancer cell lines.


Introduction
Sponges of the genus Plakortis are well known among marine chemists as prolific producers of biologically active secondary metabolites.2][3][4] The first of these compounds to be reported was plakortin, a six membered ring cycloperoxide found in 1978 by Faulkner's group in a Plakortis halicondrioides. 5ubsequently, a series of related bioactive metabolites have been isolated.They include plakinic acids the strongly antifungal peroxyketals named peroxyplakoric acids and the recently reported activators of cardiac SR-Ca 2+ ATPase plakortones A-D.In addition, [6][7][8][9][10] an unusual furano unsaturated ester (6,7) has also been described. 4,11,12 a part of systematic endeavors to isolate bioactive compounds from marine organisms, we have investigated constituents of the sponge Plakortis sp., collected at depths of -10 to -20 m in the Discovery Bay, Jamaica.These efforts resulted in the isolation and characterization of known methyl ester cyclic peroxides (1a, 2a, 3a, 4, 5), known plakortides (6, 7), known bicyclic lactone (8) and new acid peroxides (1b, 2b, 3b) (Figure 1).

Results and Discussion
The chloroform/methanol extract of the Plakortis sponge, was fractionated on silica gel using nhexane-ethyl acetate as gradient solvent.The fractions were monitored by cytotoxicity bioassays using proliferating mouse cell lines: NIH3T3, KAI3T and Vero cells to afford, in order of elution, eleven compounds (1-8).The structures of known compounds 4, 5 5, 6 4 , 7, 12 8, 9 and 1a, 2a, 3a 10 were established by comparing their physical and spectral data with those in the literature.
The new cyclic peroxide acid, plakortide 1b was isolated as an oil, the molecular formula C 21 H 40 NO 4 was deduced by HRCIMS of 370.2960 for [M + NH 4 ] + and 13 C NMR which required four degrees of unsaturation.The 1 H NMR spectrum of 1b contained signals that were typical of cyclic peroxides having the plakortin ring system and a side chain. 10It exhibited three olefinic multiplets at 5.13,5.09 and a double doublet of doublets at 4.46.In addition there were a number of methylene multiplets between and four methyl triplets at0.95,0.90, 0.84 and 0.83 and methyl singlet at 1.70 brs in the upfield region.The proton signals were divided into a number of separate spin systems by interpretation of 1 H 1 H COESY experiments and the resulting assignments were verified by HMQC and HMBC data.In the side chain of the molecule, the two olefinic multiplets at 5.09 (H-11) and 5.37 (H-12) shared a 15.5 Hz trans coupling.The 1 H 1 H COESY NMR spectrum of 1b showed a correlation between H-12 and the methylene H-13 at (1.99) of an ethyl group.The H-11 was coupled to the H-10 methine multiplet at 2.04 which also shared additional correlations with the methylene signals of an ethyl group H-20 at (1.40, 1.17) and a vinylic methylene H-9 at (2.10, 1.93).Decoupling of the brood H-7 olefinic multiplet at  5.13 resulted in the sharpening of the signals due to the H-9 methylene group and the H-19 methyl group at  1.70.Therefore there must be ethyl group attached to C-10 and methyl group attached to C-8.The remaining two ethyl groups were located in the cyclic peroxide of 1b which also incorporated a methane multiplet at 4.46 (H-3) which further coupled to a non-equivalent methylene pair (H-2).The DEPT NMR spectrum displayed five methyl, seven methylene, six methine and three quaternary carbon signals .The structure of the cyclic peroxide of the plakortide 1b was established by evidence based on HMBC data.The H-7 multiplet at  5.13 showed correlations to C-8, C-9, and Me-19 of the side chain of 1b and oxygenated quaternary C-6 at  84.3 and C-5 methylene at  34.4 in the cyclic peroxide moiety.
The H-4 multiplet at  2.07 showed correlations to C-5 methylene at  34.4,C-6 at  84.3, C-15 at  25.1, C-16 at  11.0 and oxygenated metaine C-3 at  78.3 in the cyclic peroxide moiety.The H-3 signals showed a one bond correlation to the oxygenated C-3 at  78.3 and long range correlation to the C-2 at 31.5, C-4 at 35.7, C-15 at  25.1, and carboxlic C-1 at 177.9 The new cyclic peroxide acid, plakortide 2b was isolated as an oil, the molecular formula C 20 H 40 NO 4 was deduced by HRCIMS of 358.2946 for [M + NH 4 ] + and 13 C NMR which required three degrees of unsaturation.The 1 H NMR spectrum of 2b contained signals that were typical of a cyclic peroxide having the plakortin ring system 10 and a side chain.It exhibited two olefinic multiplets at 1H, dt, J 15.0, 6.5, H-12), 5.08 (1H, dd, J 15.0, 8.5, H-11) with trans coupling and a double doublet of doublets at 4.47.In addition there were a number of methylene multiplets between and and four methyl triplets at0.96,0.92, 0.85 and 0.82.The proton signals were divided into a number of separate spin systems by interpretation of 1 H 1 H COESY experiments and the resulting assignments were verified by HMQC and HMBC data.The DEPT 13 C NMR spectrum displayed four methyl, nine methylene, five methine and two quaternary carbon signals .The 1 H 1 H COESY NMR spectrum of 2b showed a correlation between H-12 and the methylene of an ethyl group H-13/14.The H-11 was coupled to the H-10 methine multiplet at 1.78 which also shared additional correlations with the methylene signals of an ethyl H-19/20 group at 1.38, 1.18 and methylene at H-9 (1.47,1.20).The H-9 protons further correlated to the H-8 and H-7 methylene protons.In the HMBC spectrum of 2b the nonequivalent H-7 protons at  1.32 and 1.20 correlated to C-8 and C-9 in the side chaine, to the C-6 quaternary carbon at the junction of the peroxide ring and the side chain, to the C-5 methylene ring carbon, and finally to C-17 of the isolated C-17/18 ethyl group which was attached to C-6.The Me-18 proton at  0.85, which are attached to the upfield methyl carbon at  7.1, correlated to C-17 and C-6, establishing that this was the ethyl group attached at the fully substituted C-6.
In the other extended spin system the H-3 methine multiplet at  4.47 correlated to the nonequivalent H-2 methylene doublet of doublets  3.04 ( dd, J 15.5, 9.5), 2.38 (dd, J 15.5, 3.0) and C-1 at 177.3 of the carboxylic group on one side and the C-4 and C-5 in the ring.The Me-16 triplet at  0.92 correlated to C-15 (24.2) and C-4 (34.4),confirming that it was part of the remaining ethyl substituent attached at C-4.
The relative stereochemistry about the peroxide ring was established by NOESY correlations, the results of which are summarized with arrows in Figure 2. On saturation of the H-2 methylene protons, correlations with H-3 signal and H-5ax were viewed, establishing that H-2 and H-5ax were on the same face of the peroxide ring.Both the H-3 and Me-16 hydrogens showed a correlation with the axial H-4 signal.The H-4 multiplet shared a 4.5 Hz coupling constant with H-3 and a 12.5 Hz axial/axial coupling constant with H-5ax.The H-4 showed a correlation with the H-5eq.The H-5eq also showed correlation with H-17 methylene protons.Thus, H-4, H-3 and H-17 must be in the opposite face of the peroxide ring.The new cyclic peroxide acid, plakortide 3b was isolated as an oil, the molecular formula C 20 H 40 NO 4 was deduced by HRCIMS of 358.2935 for [M + NH 4 ] + .The NMR spectrum was similar to that of 2b.The COESY spectrum of plakortide 3b indicated the presence of nearly identical spin systems in 2b and 3b.The DEPT 13 C NMR and HMQC spectrum of 3b exhibited 20 carbon signals as was the case for 2b, and most of the resonances had virtually the same chemical shifts except for two carbon signals.The C-7 resonance in 3b was observed at  32.1, 3.0 ppm further upfield than its  35.1 counterpart in 2b, and the C-17 resonance in 3b was observed at  29.5, 4.4 ppm further downfield than its  25.1 value in plakortide 2b.Since the NMR data indicated that 2b and 3b were very similar in structure, these differences suggested that plakortide 3b is a diastereomer of 2b with inverted stereochemistry at C-6.This conclusion was further supported by measuring coupling constants and NOESY experiments which are summarized in Figure 3. On saturation of the H-7` at  1.49, the methylene protons in the side chain correlated with the axial H-4 signals at  2.15, and H-4 showed also correlation with H-3 signal.Thus, H-4 must be on the same face of the peroxide ring as is H-3 and the side chain containing H-7`.Therefore, the C-6 sterochemistry of plakortide 2b is opposite of that of plakortide 3b.This conclusion was consistent with the downfield shift of C-7, which is closer to the peroxide ringe oxygens in plakortide 2b than in 3b, and the up field shift of C-17 which is further away from the ring oxygen in plakortide 2b than 3b.
Finally, cytotoxic assays of the individual compounds after purification (Table 1) were performed using proliferating mouse cell lines, NIH3T3, KA3IT and one Verocells. 13,14 mpounds 1a and 2a, 3a exhibited very low cytotoxicity and 6-8 exhibited moderate cytotoxicity on all cell lines, but compounds 1b and 2b, 3b exhibited high cytotoxicity on all cell lines.

Experimental Section
General. 1 H NMR spectra were recorded at 300 or 500 MHz and 13 C NMR at 75 MHz.Chemical shifts are given in  (ppm) relative to TMS as internal standard.Electron impact mass spectra were determined at 70 ev on a Kratos MS-25 instrument.Thin layer chromatography was performed on silica gel (kieselgel 60, F254) of 0.25 mm layer thickness.Preparative thin layer chromatography (PTLC) was performed on silica gel plates (20 cm x 20 cm) of 500 m thicknesses.The spray reagent used is ammonium molybdate, which was prepared from, a solution of molybdenum trioxide (8.64 g in 7 mL NH 4 OH, stirred for 30 min) was added to a solution of amm.Cr IV sulphate (4 g in 50 mL 50% H 2 SO 4 , stirred for 30 min) and completed to 250 mL by dist.water.The obtained solution was stirred overnight.The chromatoplate, after spraying, was heated at 60-70 o C until the spots attained maximum color intensity.
Extraction and isolation.The sponge specimens were collected at depths of -10 to -20 m in the Discovery Bay, Jamaica.Each specimen was freeze-dried (100 g) and then ground and extracted with methanol/chloroform (1:1).The extract was evaporated under vacuum to afford ~10 g of a brown oily residue.This oil was chromatographed on silica gel (200 g, 50 x 3 cm) eluting first with hexane followed by a gradient of hexane-ethyl acetate.Fractions of ~100 mL were collected.The fractions containing material of the same R f were combined and further purified by sequential preparative TLC, MPLC, HPLC to give pure compounds in the order described below.
Scheme 1. Isolation of the metabolite from Plakortis sp.