Indole alkaloid from the Red Sea sponge Hyrtios erectus

Indole-3-carbaldehyde and 5-deoxyhyrtiosine A are reporterd for the first time in the genus Hertios, in addition to other indole alkaloids and three scalarane sesterterpenes, from the Red Sea sponge Hertios erectus . The isolated compounds were tested for their cytotoxic and antimicrobial activities.


Introduction
The secondary metabolites from the genus Hyrtios particularly Hertios erectus (synonym H. erecta) have been investigated extensively [1][2][3][4][5] .Previous chemical investigations of different Hyrtios sp. and their associated microorganisms revealed the presence of numerous structurally unique natural products including scalarane sesterterpenes, acyclic triterpenes, indole alkaloids, and macrolides in addition to steroids.Many of these compounds possess interesting biological activities.Spongistatins, the most important metabolites of the genus Hyrtios discovered to date, showed powerful anticancer activity 6,7 , which encouraged an exhaustive recollection of the H. erecta sponge (600 kg wet wt.) for further chemical investigations, which afforded the antineoplastic agents sesterstatins [8][9][10] .

Results and Discussion
The CH 2 Cl 2 -soluble residue of the alcoholic extract of the red sea sponge H. erectus was subjected to vacuum-liquid chromatography eluting with CH 2 Cl 2 -MeOH followed by silica gel column chromatography to afford four compounds (6-9).While the MeOH-soluble residue was subjected to vacuum-liquid chromatography eluting with gradient CH 2 Cl 2 -MeOH followed by silica gel column chromatography and semipreparative HPLC to afford five compounds (1-5).
Compound 2. The EIMS showed a molecular ion peak at m/z 146 [M+H] + .UV λ max (MeOH): 206, 243, 255, and 298 nm.The 1 H NMR (DMSO-d 6 , Table 1), as in 1 showed four protons resonances for the aromatic ABCD system at δ H 8.07 (1H, br.d, J = 7.4 Hz ; H-4), 7.10 (1H, dt, J = 1.2, 7.4 Hz; H-5), 7.30 (1H, dt, J = 1.3, 7.4 Hz; H-6) and 7.60 (1H, br.d, J = 7.4 Hz; H-7), in addition to three other proton signals at δ H 8.30 (1H, s ; H-2), an aldehydic proton at δ H 9.95 (1H, s; H-8) and the most downfield broad singlet at δ H 12.13 (1H, s ; H-1).The HMBC (Figure 2), showed correlations which identified an indole-3-carbaldehyde structure.The NMR data were identical to those reported in literature 14 , but this is the first isolation of 2 from genus Hyrtios.Compound 3. Isolated as a translucent needle crystals.EIMS showed molecular ion peak of m/z 191 [M] + , the HREIMS revealed its formula to be C 10 H 9 NO 3 .The 1 H and 13 C NMR data were consistent with those of 5-hydroxy-1H-indole-3-carboxylic acid methyl ester; this compound was previously isolated from the same sponge. 15ompound 4. Isolated as a brownish translucent amorphous powder with a characteristic odour and had a molecular formula of C 9 H 7 NO 2 based on the results of FABMS, 1 H and 13 C NMR data.The 1 H and 13 C NMR spectral data of 4 were consistent with those of 5-hydroxy-1Hindol-3-carbaldehyde which was previously isolated from H. erectus 11 and from Dragmacidon sp. 16ompound 5. Isolated as a brownish translucent amorphous powder, with slightly offensive characteristic odour and had a molecular formula of C 10 H 9 NO 3 based on the results of FABMS, 1 H and 13 C NMR data.The 1 H and 13 C NMR spectral data of 5 were consistent with those of hyrtiosine A that was previously isolated from the same sponge 11 and from Dragmacidon sp. 16ompound 6. Isolated as a colourless amorphous solid and had a molecular formula of C 25 H 38 O 4 as established from its FABMS and 13 C NMR data.The structural assignments of 6 were based on an accurate study of the 1D and 2D (COSY, HMQC, HMBC) NMR spectra.The 1 H and 13 C NMR spectral data of 6 were consistent with the data for 16-hydroxyscalarolide, which was previously reported from the Okinawan sponge H. erectus 2 .
Compound 7. Isolated as a brownish white amorphous solid and had a molecular formula of C 25 H 38 O 3 , as established from its FABMS and 13 C NMR data.The structural assignments of 7 were based on an accurate study of the 1D and 2D (COSY, HMQC, HMBC) NMR spectra.The 1 H and 13 C NMR spectral data of 7 were consistent with the data for scalarolide, which was previously reported from the sponge Spongia idia 17 .
Compound 8. Isolated as a white amorphous solid and had a molecular formula of C 25 H 38 O 4 as established from its FABMS and 13 C NMR data.The structural assignments of 8 were based on an accurate study of the 1D and 2D (COSY, HMQC, HMBC) NMR spectra.The 1 H and 13 C NMR spectral data of 8 were consistent with the data for 12-episcalarin 18,19 and suggested that 8 was a deacetyl derivative of 12-episcalarin and identical with the previously published 12-Odeacetyl-12-epi-scalarin which isolated from Spongia sp. 18ompound 9. Isolated as white needle crystals and had a molecular formula of C 27 H 44 O 3 as established from its FABMS and 13 C NMR data.The structural assignments of 9 were based on an accurate study of the 1D and 2D (COSY, HMQC, HMBC) NMR spectra.The 1 H and 13 C NMR spectral data of 5α,8α-epidioxy-cholesta-6-en-3β-ol. 20n testing the cytotoxic activities of the isolated compounds, it was found that compound 6 (16 hydroxyscalarolide) showed 40.5 and 55.3% growth inhibition activity against L5178Y cells at concentrations 3.0 µg and 10.0 µg respectively, compound 3 (5-hydroxy-1H-indole-3carboxylic acid methyl ester) showed 36 % cytotoxic activity at concentration 10.0 µg/ml, while compound 2 (indole-3-carbaldehyde) showed 53% cytotoxic activity at concentration 10.0 µg/ml.On testing the antimicrobial activity it was found that compounds 1 (deoxyhyrtiosine), 5 ARKAT USA, Inc.

Experimental Section
General Procedures. 1 H (1D, 2D COSY) and 13 C (1D, 2D HMBC) NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer.Mass spectra recorded on Finningan MAT TSQ-7000 mass spectrometer, while HREIMS were obtained on a Finningan MAT 900 mass spectrometer.UV spectra were measured in methanol on a Perkin-Elmer UV/Vis lambda spectrophotometer.Solvents were distilled prior to use, and spectral grade solvents were used for spectroscopic measurements.TLC was performed on TLC plates pre-coated with silica gel F 254 (Merck, Darmstadt, Germany).Semipreparative HPLC was performed on HPLC system (Merck, Darmstadt, Germany) coupled with UV detector L7400 (UV detection at 280 nm), the separation column (8 × 250 mm) pre-packed with Eurosphere C 18 (Knauer, Berlin, Germany).The compounds were eluted with solvent system of MeOH/H 2 O containing 1% TFA for improved separation, at a flow rate of 5 ml/min.Animal material.The H. erectus was collected at a depth of 30 feet 5 km to the north of Hurghada, Egypt in March 2003 and was kept in ethanol immediately after collection.A voucher specimen has been deposited in the Zoological Museum Amsterdam under registration number ZMA POR18348.

Figure 1 .
Figure 1.Structure of the isolated compounds.