Bioactive Indole Derivatives from the South Pacific Marine Sponges Rhopaloeides odorabile and Hyrtios sp.

Indole derivatives including bromoindoles have been isolated from the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp. Their structures were established through analysis of mass spectra and 1D and 2D NMR spectroscopic data. Their potential inhibitory phospholipase A2 (PLA2), antioxidant and cytotoxic activities were evaluated. The new derivative 5,6-dibromo-l-hypaphorine (9) isolated from Hyrtios sp. revealed a weak bee venom PLA2 inhibition (IC50 0.2 mM) and a significant antioxidant activity with an Oxygen Radical Absorbance Capacity (ORAC) value of 0.22. The sesquiterpene aureol (4), also isolated from Hyrtios sp., showed the most potent antioxidant activity with an ORAC value of 0.29.


Introduction
A great variety of simple and substituted indole derivatives, including halogenated indoles, bisindoles and tryptamine derivatives, have been previously isolated from marine organisms [1]. Indole derivatives are known to display various bioactivities such as anticancer, antibiotic, and anti-inflammatory activities [2]. Antioxidant activities were also recently reported for some analogues such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengers, highlighting an additional bioactivity in the series [3].
In our ongoing search for bioactive compounds within the frame of the CRISP program (Coral Reef Initiative in the South Pacific), the crude extracts of two South Pacific marine sponges were investigated, based on their significant anti-PLA 2 activities. One specimen of Rhopaloeides odorabile was collected from the Solomon Islands and one specimen of Hyrtios sp. from the Fiji Islands. Fractionation of each of the crude extracts led to the isolation of a series of indole derivatives.
Three known monomeric indoles were isolated from the marine sponge R. odorabile and five dibromoindole derivatives, including the new derivative, 5,6-dibromo-L-hypaphorine (9), in addition to the sesquiterpene aureol (4) were obtained from the sponge Hyrtios sp.
The current report describes the isolation of alkaloids 1-9 and structural identification of the new analogue, 5,6-dibromo-L-hypaphorine (9). Anti-PLA 2 , antioxidant and cytotoxic activities of the series were evaluated and are presented.
Chromatographic fractionation of the CH 2 Cl 2 extract of Hyrtios sp. using silica gel afforded aureol (4), rapidly identified by comparison with literature data [6]. Chromatographic fractionation of the MeOH extract of Hyrtios sp. using C18 and LH 20 columns followed by successive ODS C18 HPLC revealed the presence of five dibromoalkaloids 5-9. The structures of the known compounds 5-8 were rapidly determined as 5,6-dibromotryptamine 5, N-methyl-5,6-dibromotryptamine (6) [7], N,N-dimethyl-5,6-dibromotryptamine (7) [8], and 5,6-dibromoabrine (8) [9] by comparison with literature data. The structure of the new metabolite, 5,6-dibromo-L-hypaphorine (9), was obtained through detailed examination of mass spectrometric data and extensive 1D and 2D NMR studies ( Figure 2). The main difference between 8 and 9 was the presence of a N + Me 3 cation, indicated by a nine-proton singlet in the 1 H NMR spectrum of 9 at   3.17 (9H, s). In addition, two broad singlet protons at  H 11.20 (1H, brs) and 8.45 (1H, brs) suggested the presence of an amine and hydroxyl function, respectively. Furthermore, COSY correlations between the methine proton at  H 3.67 with methylene protons at  H 3.21 and between the amine proton at  H 11.20 with proton at  H 7.27 indicated the presence of a CH 2 -CH group and a NH-CH group, respectively. Five non-protonated aromatic carbons at  C 135.7 (C-7a), 128.2 (C-3a), 114.8 (C-6), 112.6 (C-5) and 109.5 (C-3) suggested 5,6 dibromosubstitution of the indole nucleus, which was supported by the observed HMBC correlations as presented in Table 1 and by comparison with literature values for 8 [9]. Thus, the new alkaloid, was identified as 5,6-dibromo-L-hypaphorine (9), a new member of the hypaphorine family. Halogenation on the benzene ring of tryptophan derivatives does not affect the sign of optical rotation [10], therefore 9 was assigned as L-configuration (  Several halogenated indoles bearing a N,N,N-trimethyltryptophane betaine moiety including di-and tri-iodo as well as both chlorine and iodine atoms on the indole nucleus have been reported from the Caribbean sponge Plakortis simplex [10,13]. The monobromoderivatives D-6-and L-6bromohypaphorine were previously reported from the Okinawan marine sponge Aplysina sp. and from the sponge Pachymatisma johnstoni, respectively [11,12] and the dibromoderivative 5,7-dibromo-L-hypaphorine was previously obtained by synthesis [12]. This is the first report of 5,6-dibromo-L-hypaphorine (9) as a natural product.

Biological Activities of Compounds 1-9
Compounds 1-9 were evaluated for their inhibitory activity against bee venom PLA 2 and their antioxidant activity was estimated with the ORAC assay. The results from the in vitro assays are presented in Table 2. In addition, their cytotoxicity against the human pharyngeal carcinoma cell line was also determined.

General Experimental Procedures
Optical rotations were recorded on a Perkin Elmer 341 polarimeter. UV spectra were recorded on a UVIKON 930 spectrometer and IR spectra were recorded on a FT-IR Shimadzu 8400 S spectrometer. NMR spectra were obtained on a Bruker AVANCE 400 spectrometer. HSQC and HMBC experiments were acquired at 400.13 MHz using a 1 H-13 C Dual probehead. HMBC spectra were optimized for 7 Hz coupling. Mass spectra were recorded on an API Q-STAR PULSAR I of Applied Biosystem. HPLC were performed with an Alliance apparatus (model 2695, Waters) equipped with a photodiode array detector (model 2998, Waters), an evaporative light-scattering detector (model Sedex 80, Sedere) and the software Empower. HPLC solvents were purchased from Carlo-Erba.

Extraction and Isolation
Lyophilized sponge sample Rhopaloeides odorabile (20 g) was extracted with CH 2 Cl 2 (5 × 200 mL, sonicated each time for 15 min) at room temperature. The five extracts were filtered, combined and concentrated under reduced pressure to yield 4 g of CH 2 Cl 2 extract which was chromatographed on a silica gel (Merck) column using an initial gradient of cyclohexane/ethyl acetate from 80/20 to 60/40 followed by a second gradient of CH 2 Cl 2 /acetone from 80/20 to 60/40. The 80/20 CH 2 Cl 2 /acetone fraction (98 mg) exhibited anti-PLA 2 activity and was submitted to semi-preparative reversed-phase HPLC column chromatography (Interchim, Uptisphere C18 (100 mL of each). The anti-PLA 2 active fraction H 2 O/MeOH 1:2 (220 mg) was eluted from an Sephadex LH 20 column (GE Healthcare) with MeOH to give an anti-PLA 2 active yellow fraction (30 mg) which was further submitted to semi-preparative reversed-phase HPLC column chromatography (Interchim, Uptisphere C18 7.8 × 250 mm) with increasing amounts of CH 3 CN/0.1% formic acid in H 2 O/0.1% formic acid as eluent (flow rate: 3 mL/min, wavelength: 254 nm) through a linear gradient for 30 min. Five peaks between 12 and 22 min were obtained, and further purified through an analytical reversed-phase HPLC column (Interchim, Uptisphere C18 4.6 × 250 mm) with increasing amounts of CH 3 CN/0.1% formic acid in H 2 O/0.1% formic acid as eluent (flow rate: 1 mL/min, wavelength: 254 nm) through a linear gradient for 30 min and yielded pure compounds 5-9 (0.5 mg for 5, 1.4 mg for 6, 1.3 mg for 7, 1.7 mg for 8, 3.0 mg for 9). An aliquot of the crude CH 2 Cl 2 extract (0.4 g) was chromatographed on a silica gel column, using a linear gradient of acetone in CH 2 Cl 2 as eluent. The 50% acetone fraction afforded pure 4 (4 mg). ; spectroscopic data matched those previously published [7].

PLA 2 Inhibition Assay
Bioassay guided fractionation was based on a colorimetric bioassay [14]. Assays were performed in duplicate in 96 well plates and read on a CERES 900 spectrophotometer. Extracts (250 µg) or fractions (100 µg) dissolved in 10 µL of DMSO were incubated with 2 µL of a PLA 2 solution (1 mg/mL in DMSO) from Apis mellifera bee venom (Sigma) for 1 hr at 25 °C. Then 198 µL of the substrate solution L--lecithin (Sigma) 3.5 mM containing Triton X-100 (7 mM), NaCl (100 mM), CaCl 2 (10 mM) and red phenol (0.055 mM) as colorimetric indicator, at pH 7.6 were added and the absorbance at 550 nm read at time 0 and 5 min. Percent inhibition of the enzyme activity was determined by comparison with a control without drug. Manoalide (Aldrich) was used as a positive control.

Antioxidant Assays
Qualitative DPPH screening: The potential antioxidant activity of crude extracts and pure compounds were screening using the scavenging activity of the DPPH (Sigma) free radicals. Active extracts were visualized by spraying a purple DPPH solution (2 mg/mL in MeOH) on a Tlc plate (Merck, Silica gel 60 F 254 ), where compounds have been deposited. Immediate discoloration of DPPH around tested samples reveals their antioxidant activity.
Quantitative ORAC assay: The antioxidant activity of pure compounds was assessed with the ORAC assay. The ORAC assay is a kinetic assay measuring the decrease in fluorescence of fluorescein (FL) (Sigma) by adding the oxidative species AAPH (Aldrich, 2.2'-azobis(2amidinopropane dihydrochloride). Thus, antioxidant protection of compounds was evaluated over time. The antioxidant Trolox (Aldrich, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as a positive control delaying the loss of FL fluorescence in a dose dependent manner [15,16]. The antioxidant activity is normalized to equivalent Trolox units to quantify the antioxidant activity of each compound. The assay was performed with a spectrofluorimeter Berthold Mithras LB 940. Reaction mixtures containing 25 µL of different 2-fold dilutions of pure compounds (dissolved in phosphate buffer 75 mM, pH 7.4 containing 5% DMSO) or Trolox (200 µM-12.5 µM) and 150 µL of FL solution (10 nM in phosphate buffer) were distributed in 96 well microplates in triplicate and incubated at 37°C for 15 min. Fluorescence was measured (Ex. 485 nm, Em. 520 nm) every 90 sec to determine the background signal. After 3 cycles of measurement, 25 µL of an AAPH solution (240 mM in phosphate buffer) was added via an automated injector and 60 fluorescence measurements were taken over a 90 min time period. The final relative ORAC values for tested compounds were calculated by using a regression equation and were expressed as Trolox equivalents according to Ou et al. [16]. Trolox and ascorbic acid (Acros Organics) solutions were used as positive controls and FL solution with AAPH as blank.

Cytotoxicity Assay
The human KB cell line was obtained from ECACC (Salisbury, UK) and grown in D-MEM medium supplemented with 10% fetal calf serum (Invitrogen), in the presence of penicillin, streptomycin and fungizone in a 75 cm² flask under 5% CO 2 . Cells were plated in 96-well tissue culture microplates at a density of 650 cells/well in 200 µL medium and treated 24 hrs later with compounds dissolved in DMSO using a Biomek 3000 automate (Beckman-Coulter). Controls received the same volume of DMSO (1% final volume). After 72 hrs exposure MTS reagent (Celltiter 96 Aqueous One solution, Promega) was added and incubated for 3 hrs at 37°C: the absorbance was monitored at 490 nm and results expressed as the inhibition of cell proliferation calculated as the ratio [(1 − (OD 490 treated/OD 490 control)) × 100]. For IC 50 determinations (50% inhibition of cell proliferation) experiments were performed with compound concentrations ranging from 1 µM to 100 µM in duplicate.

Conclusions
In conclusion, our search for new inhibitors of PLA 2 and/or antioxidant natural products has led to the investigation of specimens of the South Pacific marine sponges Rhopaloeides odorabile and Hyrtios sp. Eight indole derivatives including the new 5,6-dibromo-L-hypaphorine (9), and the sesquiterpene aureol (4) were isolated and their chemical structures were resolved by spectroscopic analysis. Evaluation of anti-PLA 2 and antioxidant activities of the series led to the identification of both 4 and 9 as potential antioxidant compounds. In contrast to 4, the new derivative 9 did not show any cytotoxic activity towards the human KB cancer cell line. Consequently, 9 could be promising in cosmetics and/or in pharmaceutics due to its anti-inflammatory and antioxidant potentials.