Two New Jaspamide Derivatives from the Marine Sponge Jaspis splendens

Two new jaspamide derivatives 2 and 3, together with the parent compound jaspamide (1) have been isolated from the marine sponge Jaspis splendens collected in Kalimantan (Indonesia). The structures of the new compounds were unambiguously elucidated based on 1D and 2D NMR spectral data, mass spectrometry and comparison with jaspamide (1). The new derivatives inhibited the growth of mouse lymphoma (L5178Y) cell line in vitro with IC50 values of <0.1 μg/mL.

Jaspamide Q (2) was obtained as a white amorphous solid, and the ESIMS spectrum showed a pseudomolecular ion peak at m/z 631.3 [M+H] + , which was 79 amu smaller than that of jaspamide (1), the parent compound. This difference was assigned to the absence of the bromine atom at C-26 in jaspamide (1). The molecular formula of jaspamide Q (2) was C 36 H 46 N 4 O 6 , based on HRFTMS (m/z 631.3491 [M+H] + , ∆ + 1.0 ppm), therefore jaspamide Q (2) was identified as the debromo analogue of 1. 1 H-NMR spectral data (Table 1) revealed that the resonances of 2 were superimposable with those of 1 with only one additional proton resonance at δ H 6.87 (1H, br s) that was ascribed to H-26. The complete structure of jaspamide Q (2) was unambiguously elucidated and assigned on the basis of 1 H-1 H COSY, TOCSY, ROESY, and HMBC spectra.
In particular, the similarity of 1 H-, and 13 C-NMR resonances between jaspamide Q (2) and jaspamide (1) implied that the chiral centers of alanine, abrine (N-methyltryptophan), ß-tyrosine, and of the polypropionate fragment had the same relative configurations in both molecules. Therefore, the stereochemistry depicted in Figure 1 was tentatively assigned by analogy with the parent compound together with ROESY spectra that revealed a clear correlation between Me- 16 [20]. These differences in chemical shifts for the latter congeners were proven to be caused by D-Ala or L-Ala residues, respectively. Anaylsis of the absolute configurations of the amino acids of 2 could not be performed due to the small amount of compound isolated (0.7 mg).
A partial 13 C-NMR assignment of jaspamide Q (2) was achieved through HMBC spectra (  This difference was explained by the 1 H-NMR spectral data (Table 1), which revealed close similarity between jaspamide R (3) and jaspamide (1) (1) supports the notion that the chiral centers of alanine, 2,4-dibromoabrine, ß-tyrosine, and the polypropionate fragment have the same relative configurations in both molecules ( Figure 1). Again, due to the lack of material isolated of 3 (0.5 mg), analysis of the absolute configurations of the amino acids, e.g. by Marfey's method, could not be performed.
Jaspamide Q (2) and R (3) together with the parent jaspamide (1) differ in the bromination pattern of the abrine (N-methyltryptophan) moiety. Since these modifications were claimed as essential for the observed biological activity [44], compounds (2 and 3) together with jaspamide (1) were subjected to a cytotoxicity (MTT) assay against mouse lymphoma (L5178Y) cell lines. They exhibited potent activities with IC 50 values in the ng/mL range (<0.1 µg/mL, <0.16 µM), compared to kahalalide F (IC 50 = 6.3 µg/mL, 4.3 µM) which was used as a positive control. Further studies aimed at determining the effect of bromination pattern of the abrine residue on cytototoxic activity are in progress.

General experimental procedures
Column chromatography was carried out on Sephadex LH-20 using methanol as an eluent. For analytical HPLC analysis, samples were injected into a HPLC system equipped with a photodiode array detector (Dionex, Munich, Germany). Routine detection was at 235, 254, 280, and 340 nm. The separation column (125 × 4 mm ID) was prefilled with C-18 Eurosphere, 5 µm (Knauer, Berlin, Germany). Separation was achieved by applying a linear gradient from 90% H 2 O (pH 2.0) to 100% MeOH over 40 min. TLC analysis was carried out using aluminium sheet precoated with silica gel 60 F 254 (Merck, Darmstadt, Germany).
Preparative HPLC separations were performed on a LaChrom-Merck Hitachi HPLC system, pump L-7100, UV detector L-7400 using a C-18 column (Knauer, 300 × 8 mm ID, prefilled with C-18 Eurosphere, flow rate 5 mL/min, UV detection at 280 nm), and the solvent system consisted of a linear gradient of MeOH and nanopure H 2 O.
Optical rotations were measured on a Perkin-Elmer-241 MC polarimeter. ESIMS were obtained on a ThermoFinnigan LCQ DECA mass spectrometer coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. HRFTMS was recorded on a LTQ FT-MS-Orbitrap (ThermoFinnigan, Bremen, Germany). 1D and 2D NMR spectra were recorded at 300 ºK on a Bruker ARX-500. Samples were dissolved in deuterochloroform.

Biological material
In August 2008, specimens of J. splendens were collected on three neighboring Islands from East Kalimantan (Indonesia), namely Samama, Panjang, and Shoal Islands, at 10 meter depths. Numbers of voucher specimens are RMNH Por. 4234, 4266 and 4299, respectively. They were taxonomically identified as Jaspis splendens (order Astrophorida, family Ancorinidae) at the National Museum of Natural History, Leiden, Netherlands. HPLC and LCMS analyses of the three samples revealed that they were identical with regard to their peptide derivatives. Hence, the material was combined in order to obtain sufficient amounts of compounds for subsequent structure elucidation.

Extraction and isolation
The animal was freeze-dried, and the material (500 g) was extracted with methanol (3 × 2 L) and filtered. The extract was then combined, evaporated to dryness, and partitioned as follows. The methanolic extract (80 g) was dissolved in water and partitioned against n-hexane, ethyl acetate, and then n-butanol. The bioactive ethyl acetate soluble fraction (2 g) was chromatographed by CC using Sephadex LH20 as stationary phase and eluted with methanol followed by reversed-phase (C18 Eurosphere 100) HPLC using gradient elution of MeOH:H 2 O to yield 105 mg of jaspamide (1), 0.7 mg of jaspamide Q (2), and 0.5 mg of jaspamide R (3).

Cell proliferation assay
Cytotoxicity was tested against L5178Y mouse lymphoma cells using the microculture tetrazolium (MTT) assay as described earlier [11,45]. All experiments were carried out in triplicate and repeated three times. As controls, media with 0.1% EGMME/DMSO were included in the experiments.