New Antimalarial and Antimicrobial Tryptamine Derivatives from the Marine Sponge Fascaplysinopsis reticulata

The CH₂Cl₂-MeOH extract of the lyophilized sponge Fascaplysinopsis reticulata was first subjected to a reverse-phase silica gel column chromatography to yield fractions. The fractions were subjected to repetitive reverse-phase semi-preparative and analytical HPLC to yield eight compounds (1–7) (Figure 1). Three were new: one 6,6′-bis-(debromo)-gelliusine F (1) and two aplysinopsin derivatives 2 and 3, described below. In addition to the new compounds, four other known members were identified as 8-oxo-tryptamine (4), tryptamine (5) and a mixture of (E)-6-bromo-2′-demethyl-3′-N-methylaplysinopsin (6) and (Z)-6-bromo-2′-demethyl-3′-N-methylaplysinopsin (7) by comparison with published spectroscopic data.

6,6′-bis-(debromo)-gelliusine F (1) was obtained as a brown oil. The molecular formula, C₂₀H₂₃N₄, was established from HRESIMS molecular ion peak at m/z 319.2013 [M + H]⁺. Analysis of the 1D and 2D ¹H, and ¹³C NMR data for 1 (CD₃OD, Table 1) revealed resonances and correlations (Figure 2) consistent with those of a bis-tryptamine structure linked by the carbons C-2 and C-8′, like gelliusine F [10,11]. Analysis of the HSQC correlations and the comparison with latter compounds pointed the fragment C-8, C-9, C-9′ (δH 3.23, 3.00, 3.83–3.69; δC 23.7, 41.4, 44.3), one aliphatic methine C-8′ (δH 5.10; δC 34.3), nine aromatic methines C-4, C-5, C-6, C-7, C-2′, C-4′, C-5′, C-6′, C-7′ (δH 7.54, 7.38, 7.12, 7.06, 7.27, 7.58, 7.41, 7.14, 7.06; δC 118.9, 112.6, 123.1, 120.7, 124.0, 119.3, 112.9, 123.3, 120.6) and seven nonprotonated aromatic carbons C-2, C-3, C-3a, C-7a, C-3′, C-3a’, C-7a’ (δC 124.0, 113.8, 127.5, 135.3, 113.7, 129.2, 138.0). Compound 1 was different from gelliusine F by the presence of the two aromatic methines C-6 and C-6′ instead of two nonprotonated aromatic carbons substituted by bromine. Analysis of the COSY correlations revealed the presence of the spin systems C-4−C-5−C-6−C-7 and C-4′−C-5′−C-6′−C-7′ and confirmed this difference. These COSY correlations, in addition to the HMBC correlations between H-4 and C-7a, between H-5 and C-3a, between H-6 and C-7a, between H-2′, C-3′, C-3a’ and C-7a’, between H-4′ and C-7a’, between H-5′ and C-3a’, between H-6′ and C-7a’ and between H-7′ and C-3a’, confirmed the presence of two indole cores, the first one substituted in C-2 and C-3 and the second one substituted in C-3′. The COSY correlation between H-8 and H-9 and the HMBC correlation between H-9 and C-3 and between H-8, C-2, C-3 and C-3a indicated the substitution of the first indole core by an ethylamine chain in C-3. The COSY correlation between H-8′ and H-9′ and the HMBC correlations between H-9′ and C-3′ and between H-8′, C-2′, C-3′ and C-3a’ indicated the substitution of the second indole core by an ethylamine chain in C-3′. The two tryptamine patterns were linked between C-2 and C-8′ like gelliusine F. Compound 1 was named 6,6′-bis-(debromo)-gelliusine F according to gelliusine F, reported in 1995 [11].

6-bromo-8,1′-dihydro-isoplysin A (2) was obtained as a yellow oil. Its molecular formula, C₁₄H₁₆BrN₄O (9 degree of unsaturation), was established from HRESIMS pseudo-molecular ion peak at m/z 337.0483 (see Supplementary Materials) indicating the presence of one bromine atom in the molecule. Analysis of the 1D and 2D ¹H, and ¹³C NMR data for 2 (CD₃OD, Table 2) revealed resonances and correlations (Figure 2) consistent with those of a 1′,8-dihydroaplysinopsin structure: the HSQC correlations revealed the presence of one methylene C-8 (δH 3.35; δC 28.1), one aliphatic methine C-1′ (δH 4.62; δC 61.8), four aromatic methines C-2, C-4, C-5, C-7 (δH 7.11, 7.51, 7.14, 7.50; δC 126.4, 121.1, 123.3, 115.5), four nonprotonated aromatic carbons C-3, C-3a, C-6, C-7a (δC 109.0, 127.6, 116.6, 138.1), one guanidine-like carbon C-3′ (δC 159.2) and one amide carbonyl C-5′ (δC 174.9). The structure of the indole core was determined by the analysis of COSY correlations between H-4 and H-5, the ⁴J coupling constant between H-5 and H-7 (J = 1.8 Hz) and HMBC correlations between H-2, C-3, C-3a, and C-7a, between H-4, C-6 and C-7a and between H-5, C-3a and C-7. The HMBC correlation between H-2 and C-8 indicated the substitution of the non-protonated carbon C-3 by the methylene C-8. The COSY correlation between H-8 and H-1′ indicated link between the heterocycle core and C-8. The structure of the heterocycle core was determined by the HMBC correlations between H-1′ and C-5′, between CH₃-6′ and C-3′ and between CH₃-7′, C-3′ and C-5′.

5,6-dibromo-8,1′-dihydro-isoplysin A (3) was obtained as a yellow oil. Its molecular formula C₁₄H₁₅Br₂N₄O (9 degrees of unsaturation), was established from HRESIMS pseudo-molecular ion peak at m/z 414.9630 (see Supplementary Materials) indicating the presence of two bromine atom in the molecule. Analysis of the ¹H and ¹³C NMR data for 3 and comparison with the ¹H and ¹³C NMR data for 2 (CD₃OD, Table 3) revealed a 1′,8-dihydroaplysinopsin structure close to the above-described 6-bromo-8,1′-dihydro-isoplysin A (2), where one hydrogen was replaced by a bromine atom. The spectra showed two N-methyles C-6′, C-7′ (δH 2.86, 2.94; δC 25.4, 28.9), one methylene C-8 (δH 3.73; δC 28.2), one aliphatic methine C-1′ (δH 4.60; δC 61.4), three aromatic methines C-2, C-4, C-7 (δH 7.16, 7.96, 7.69; δC 126.6, 123.6, 117.4), five nonprotonated aromatic carbons C-3, C-3a, C-5, C-6, C-7a (δC 109.0, 129.8, 116.9, 115.9, 137.5), one guanidine-like carbon C-3′ (δC 157.9) and one amide carbonyl C-5′ (δC 175.8). 5,6-dibromo-8,1′-dihydro-isoplysin A (3) differed from 6-bromo-8,1′-dihydro-isoplysin A (2) by the presence of one more aromatic nonprotonated aromatic carbon and one less aromatic methine. The chemical shifts and the multiplicity of C-4 and C-7 also differed between compound 3 (two singlets) and compound 2 (two doublets). For compound 3, the multiplicity of C-4 and C-7 indicated that H-4 was para to H-7. These spectroscopic features, as well as the molecular formula, supported that the position of the proton H-5 of 2 was substituted by a bromine in compound 3.

The capacity of compounds to interfere with microfouling was assessed by screening the pure compounds against five bacterial strains that are involved in the initial formation of the fouling biofilm: Shewanella putrefaciens, Roseobacter litoralis, Vibrio carchariae, Vibrio natrigens and Vibrio proteolyticus. The effects on both adhesion and growth (A and G) were studied, and the results expressed as the minimal inhibitory concentration (MIC) are summarized in Table 4. The two new 6-bromo-8,1′-dihydro-isoplysin A (2) and 5,6-dibromo-8,1′-dihydro-isoplysin A (3) showed promising antifouling activity against Vibrio natrigens, with MIC values of 0.01 and 1.00 µg/mL, respectively, towards growth inhibition. Vibrio natrigens is a major component of biofilms due to its fast generation doubling time, its biofilm producing ability and steel corrosion behavior. Thus, it has considerable negative economic impacts on man-made immersed surfaces [12,13].

The activity of these compounds was lower towards inhibition of adhesion (respectively 100 and >100 µg/mL for (2) and (3)). Based on MICs values and mode of action, 6-bromo-8,1′-dihydro-isoplysin A (2) is the most potent compound as it has the ability to reduce growth when used at very low concentration and can also affect adhesion at higher doses. Regarding the known compound, tryptamine (5) showed promising antimicrobial activity against Vibrio carchariae with MIC value of 1 µg/mL. This result is of high interest, as Vibrio carchariae is responsible for mass mortalities of fish [14] and invertebrates [15]. Thus, Vibrio carchariae is considered to be a major nuisance for the aquaculture sector [16], and new ways to stop its development are sought after.

All the isolated compounds were also tested against the protozoan parasite Plasmodium falciparum (3D7 strain). The 8-oxo-tryptamine (4) and the mixture of the known (E) and (Z)-6-bromo-2′-demethyl-3′-N-methylaplysinopsin (6, 7) exhibited antiplasmodial activity against Plasmodium falciparum with IC₅₀ values of 8.8 and 8.0 µg/mL respectively while 6,6′-bis-(debromo)-gelliusine F (1), 6-bromo-8,1′-dihydro-isoplysin A (2), 5,6-dibromo-8,1′-dihydro-isoplysin A (3) and tryptamine (5) did not show significant antimalarial activity. Hu et al. [17] have already reported the antiplasmodial activity of (E) and (Z)-6-bromo-2′-demethyl-3′-N-methylaplysinopsin (6, 7) together with the activity of two other aplysinopsins, isoplysin A and 6-bromoaplysinopsin isolated from the sponge Smenospongia aurea. Bialonska et Zjawiony [1] also reported, for 27 aplysinopsins, their biological activities, among which the antiplasmodial activity seems to be dependent on the skeleton: all the aplysinopsins that presented antiplasmodial activity had a double bond between C-8 and C-1′. The lack of antiplasmodial activity for compounds (2) and (3) confirms this study. These activities are moderate compared to control drugs, but these simple molecular scaffolds could be investigated for further pharmacomodulations in order to improve final bioactivity.

Optical rotations were measured on a MCP 300 polarimeter (Anton Paar, Les Ulis, France) at 25 °C (MeOH, c in g/100 mL). ¹H and ¹³C NMR data were acquired with a Brucker UltraShield Avance-300 and 600 MHz spectrometers (CNRS-ICSN, Brucker, Wissembourg, France). Chemical shifts were referenced using the corresponding solvent signals (δ_H 3.31 and δ_C 49.00 for CD₃OD). The spectra were processed using TopSpin software (TopSpin 3.5, Brucker, Wissembourg, France). HRESIMS spectra were recorded using a Waters Acquity BEH C18, 1.7 μm, 50 × 2.1 mm column on a Waters Micromass LCT-Premier TOF mass spectrometer (Waters, Guyancourt, France) with a Waters Acquity UPLC system.

The sponge was lyophilized with Cosmos −80 °C CRYOTEC and extracted with Dionex ASE 300. Reversed phase column chromatography separations were carried out on glass column (150 × 10 mm i.d.) packed with Acros Organics C18-RP, 23%C, silica gel (40−63 μm). Precoated TLC sheets of silica gel 60, Alugram SIL G/UV254 were used, and spots were visualized on the basis of the UV absorbance at 254 nm and by heating silica gel plates sprayed with formaldehyde−sulfuric acid or Dragendorff reagents. Analytical HPLC was carried out using a Waters Sunfire C₁₈ (150 × 4.6 mm i.d., 5 μm) column and was performed on an Agilent 1100 series system controller equipped with a photodiode array detector (Serie Agilent 1100 G1315B, Agilent Technologies, Wilmington, Germany) and a mass spectrometer detector (Serie Agilent 1100 G1956A, Agilent Technologies, Wilmington, Germany) with Chemstation software (Version B.04.03. Agilent Technologies, Wilmington, Germany). Preparative HPLC was carried out using a Waters Sunfire Prep RP₁₈ (150 × 19 mm i.d., 5 μm) column and was performed on a Waters 600 system controller equipped with a photodiode array detector (Waters 2996, Waters, Guyancourt, France). Semi-preparative HPLC was carried out using Waters Sunfire Prep RP₁₈ (250 × 10 mm i.d., 5 μm) column and was performed on a Waters 600 system controller (Waters, Guyancourt, France) equipped with photodiode array detectors (Waters 2996 and Waters 486). All solvents were analytical or HPLC grade and were used without further purification.

The sponge Fascaplysinopsis reticulata (phylum Porifera, class Demospongiae, order Dictyoceratida, family Thorectidae) was collected in May 2013 in Passe Bateau, Mayotte (12°58,653′ S, 44°58,949′ E at 15–17 m depth). One voucher specimen (RMNH POR 8466) was deposited in Naturalis, the Netherlands Centre for Biodiversity. Sponge samples were frozen immediately and kept at −20 °C until processed.

The frozen sponge (28 g, dry weight) was chopped into small pieces and extracted by ASE first with Water (×1) and then with MeOH/CH₂Cl₂ (1:1, v:v) (×2). After evaporating the solvents under reduced pressure, a brown, oily residue (2.91 g) was obtained. The extract (2.90 g) was then subjected to fractionation by C-18 SPE, eluted with a combination of Water, MeOH and CH₂Cl₂ of decreasing polarity and twelve fractions were obtained (F1–F12).

Fraction F3 (543 mg). Separation of only 100 mg of this fraction was performed by preparative HPLC (Waters Sunfire Prep C₁₈ Column, 5 µm, 150 × 19 mm i.d., 18 mL min⁻¹ gradient elution with 2% ACN-H₂O (+0.1% formic acid) over 5 min, then 10% ACN-H₂O (+0.1% formic acid) to 100% ACN over 30 min; UV 280 nm) to furnish pure compound 1 (6,6′-bis-(debromo)-gelliusine F, 0.6 mg).

Fraction F4 (355.4 mg). Only 200 mg was subjected to preparative HPLC (Waters Sunfire Prep C₁₈ Column, 5 µm, 150 × 19 mm i.d., 18 mL min⁻¹ gradient elution with 2% ACN-H₂O (+0.1% formic acid) over 5 min, then 2% ACN-H₂O (+0.1% formic acid) to 100% ACN (+0.1% formic acid) over 35 min; UV 280 nm) to give pure compounds 2 (6-bromo-8,1′-dihydro-isoplysin A, 4 mg), 3 (5,6-dibromo-8,1′-dihydro-isoplysin A, 4 mg) and 5 (tryptamine, 4.0 mg).

Fraction F5 (99.1 mg) was subjected to preparative HPLC (Waters Sunfire Prep C₁₈ Column, 5 µm, 150 × 19 mm i.d., 18 mL min⁻¹ gradient elution with 2% ACN-H₂O (+0.1% formic acid) over 5 min, then 2% ACN-H₂O (+0.1% formic acid) to 100% ACN (+0.1% formic acid) over 45 min; UV 280 nm) to give pure compound 1 (6,6′-bis-(debromo)-gelliusine F, 1.5 mg), 4 (8-oxo-tryptamine, 0.7 mg) and 5 (tryptamine, 3.0 mg).

Fraction F6 (51.1 mg) was subjected to semi-preparative HPLC (Waters Sunfire Prep RP₁₈ Column, 5 μm, 250 × 10 mm i.d., 4.5 mL min⁻¹ gradient elution with 2% ACN-H₂O (+0.1% formic acid) over 5 min, then 2% ACN-H₂O (+0.1% formic acid) to 100% ACN (+0.1% formic acid) over 35 min; UV 280 nm) to give pure compounds 2 (6-bromo-8,1′-dihydro-isoplysin A, 1.2 mg), 4 (8-oxo-tryptamine, 0.4 mg) and 5 (tryptamine, 0.6 mg).

Fraction F7 (266.8 mg) was subjected to semi-preparative HPLC (Waters Sunfire Prep RP₁₈ Column, 5 μm, 250 × 10 mm i.d., 4.5 mL min⁻¹ gradient elution with 2% ACN-H₂O (+0.1% formic acid) over 5 min, then 2% ACN-H₂O (+0.1% formic acid) to 100% ACN (+0.1% formic acid) over 35 min; UV 280 nm) to give pure compounds 5 (tryptamine, 0.4 mg) and the mixture of 6 and 7 ((E) and (Z)-6-bromo-2′-demethyl-3′-N-methylaplysinopsin, 10 mg).

6,6′-bis-(debromo)-gelliusine F (1): brown oil, ¹H and ¹³C NMR, see Table 2; HRESIMS m/z 319.2015 [M + H]⁺ (calcd for C₂₀H₂₃N₄, 319.1923).

6-bromo-8,1′-dihydro-isoplysin A (2): yellow oil, αD20 0.0 (c 0.5, MeOH); ¹H and ¹³C NMR, see Table 2; HRESIMS m/z 337.0483 [M + H]⁺ (calcd for C₁₄H₁₆N₄O⁸¹Br, 337.0487).

5,6-dibromo-8,1′-dihydro-isoplysin A (3): yellow oil, αD20 0.0 (c 0.5, MeOH); ¹H and ¹³C NMR, see Table 3; HRESIMS m/z 414.9630 [M + H]⁺ (calcd for C₁₄H₁₅N₄O⁷⁹Br⁸¹Br, 414.9592).

Activity against Plasmodium falciparum chloroquine-sensitive 3D7 strains was assessed following the procedure already described in Frédérich et al. [18]. The parasites were obtained from MR4-BEI Resources (Manassas, VA, US). Each compound, fraction and extract was applied in a series of eight 2-fold dilutions (final concentrations ranging from 0.8 to 100 μg/mL for an extract and from 0.08 to 10 μg/mL for a pure substance) on two rows of a 96-well microplate and were tested in triplicate (n = 3). Parasite growth was estimated by determination of lactate dehydrogenase activity as described previously [19]. Artemisinin (98%, Sigma-Aldrich, Saint-Louis, MO, USA) was used as positive control with IC₅₀ of 0.006 ± 0.002 µg/mL.

All compounds were tested against five marine bacterial strains commonly found on biofilms, Roseobacter litoralis (ATCC 495666), Shewanella putrefaciens (ATCC 8071), Vibrio carchariae (ATCC 35084), Vibrio natrigens (ATCC 14048) and Vibrio proteolyticus (ATCC 15338). Bacterial adhesion and growth rates were determined according to the methods of Thabard et al. [20], Messina et al. [21] and Trepos et al. [22]. Bacterial suspensions (100 μ aliquots, 2 × 10⁸ colony forming units/mL) were aseptically added to the microplate wells containing compound (0.01–10 μg/mL), and the plates were incubated for 48 h at 26 °C prior to assessment of bioactivity. Media only (Marine Broth 2216, Difco) was used as a blank. Bacterial growth was monitored spectroscopically at 630 nm. The minimal inhibitory concentration (MIC) for bacterial growth was defined as the lowest concentration which results in a decrease in OD, compared to the blank. The microplates were then emptied, and the bacterial adhesion assay was performed using aqueous crystal staining method [22]. The MIC for bacterial adhesion was defined as the lowest concentration of compound that, after 48-h incubation, produced a decrease of the OD at 595 nm compared to the blank.