An Acetylenic Alkaloid from the Calcareous Sponge Leucetta sp.

A new acetylenic alkaloid was isolated from the sponge Leucetta sp. The structure was established by analyzing spectroscopic data. The alkaloid showed cytotoxicity IC50 2.5 μg/mL against NBT-T2 cells.


Introduction
More than 8000 species of sponges have been recorded from intertidal to deep oceanic floors, from tropical to polar regions, and from marine to freshwater environments [1]. The majority of sponges prefer to grow in shallow warm waters such as coral reefs. Since coral reef sponges have been major subjects from an early stage of marine natural products study, they are recognized as the most prolific sources of diverse bioactive secondary metabolites among macrobenthos [2]. However, dereplication has become an increasingly major issue in natural product chemistry as it is common to encounter known molecules probably reflecting the fact that many overlapping specimens have been examined. To overcome this problem and to increase the diversity of source organisms, we collected sponges in a coral reef twilight zone (50-100 m), where the depth is too deep for conventional scuba diving and the fauna there have not been well examined. A few examples from the zone include seragamides [3] and a unique fatty acid [4]. In this short note, we describe a structure of a new cytotoxic acetylenic alkaloid from a sponge collected in the zone.
A number of polyacetylenic molecules have been reported from marine sources [4][5][6][7], however, compound 1 is the first example of polyacetylene with an alkaloidal functionality from a marine sponge.

General Procedures
FTIR spectrum was taken on a Varian FTS-3000 instrument. 1 H, 13 C and 2D (COSY, HSQC, HMBC) NMR spectra were obtained on a Bruker Avance III 500 spectrometer in CDCl 3 with reference to an internal standard of TMS. Chemical shifts and coupling constants were given as  and Hz. ESIMS was measured on a Jeol JMS-T100LP instrument.

Animal Material
The sponge, an undescribed Leucetta sp. (Figure 2, Leucettidae, Clathrinida, Calcarea), was collected at 50 m depth off Kume Island in Okinawa on September, 2009 and kept frozen until extraction. The sponge was identified by one of us (NJdV) and deposited at NCB Naturalis under the code RMNH POR 3927. The sponge is small, pink, pyriform and has one prominent osculum. The skeleton consists of regular triactines and tetractines densely and irregularly scattered throughout the ectosome and choanosome. The rays of the spicules range from 5-110 μm, of which the smaller ones are juvenile forms.

Cytotoxicity Testing
NBT-T2 cells (BRC-1370, purchased from Riken BioResource Center) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and antimicrobials under standard protocol and seeded in 200 L wells. After preincubation (37 °C, 24 h), cells were exposed to graded concentrations of compound 1 in duplicate (37 °C, 48 h). Then, the cells were treated with MTT solution (15 L, 5 mg/mL in PBS) after removal of the medium and incubated for 3 h. The residual formozan was dissolved in DMSO (100 L) and the absorbance was measured with a Tecan sunrise microplate reader at 560 nm. The IC 50 values were found by plotting the absorbance values against concentrations. The alkaloid showed cytotoxicity IC 50 2.5 g/mL against NBT-T2 cells.

Conclusions
A new acetylenic alkaloid 1 was characterized with spectroscopic methods. Together with our previous work [4], Calcareous sponges are still promising sources of unique bioactive molecules.