Two New Cytotoxic Candidaspongiolides from an Indonesian Sponge

Marine sponges have been recognized as potentially rich sources of various bioactive molecules. In our continuing search for new secondary metabolites from Indonesian marine invertebrates, we collected a sponge, whose extract showed cytotoxicity against cultured cells at 0.1 μg/mL. Purification of the extract yielded two new macrolides 2 and 3 along with known candidaspongiolide (1). The structures for compounds 2 and 3 were elucidated by spectral analysis (1H, 13C, COSY, HMQC, HMBC) and by comparison of their NMR data with those of 1. Compounds 2 and 3 exhibited a little more potent cytotoxicity (IC50 4.7 and 19 ng/mL) than that (IC50 37 ng/mL) of candidaspongiolide (1) against NBT-T2 cells.


Introduction
Sponges, a group of sedentary organisms, cannot move and escape from predators. Most sponges are filter feeders pumping water to its body to obtain foods and oxygen and to expel wastes and may be threatened by microorganisms during filtering seawater rich in bacteria and fungi [1,2]. In order to defend themselves against predators, pathogens and competitors, sponges may have developed to produce or accumulate secondary metabolites during their long evolution, such as feeding deterrent, antimicrobial, antifungal, and antifouling molecules. Interestingly, some of the compounds have also shown remarkable potency as drug candidates against various human diseases as discussed elsewhere [3][4][5][6][7][8][9].
In 1984, Schmitz and coworkers isolated tedanolide from the Caribbean marine sponge Tedania ignis [10]. Tedanolide is a unique 18-membered macrolide where lactonization occurs at a primary hydroxyl group instead of a common secondary one, and this class of macrolide has been reported to exhibit strong cytotoxicity at pico to nanomolar range [10,11]. The unique structure in combination with promising biological activity leads tedanolide as an intriguing target for formal and total syntheses [12][13][14]. More recently, Meragelman and coworkers reported a macrolide named candidaspongiolide (1) related to tedanolide with modification at C-11 to C-15 from the marine sponge Candidaspongia sp. Candidaspongiolide exhibited potent cytotoxicity in NCI 60 cells panel with GI 50 of 14 ng/mL [15], protein synthesis inhibition, and apoptosis induction [16].
In our continuing search for potential drug leads from Indonesian marine invertebrates [17,18], we obtained a sponge whose extract showed cytotoxicity at 0.1 μg/mL against NBT-T2 cells in a screening process. Purification of the extract provided candidaspongiolide (1) along with two new analogs 2 and 3, which are the subject of this paper. column. Optical rotations were measured on a Jasco P-1010 polarimeter using a cell with 3.5 mm aperture. IR spectra were recorded on a Jasco FT/IR-6100 instrument, whereas HRESIMS was measured on a Jeol JMS-T100LP spectrometer using reserpine or sodium trifluoroacetate as an internal standard. Most of 1 H and 13 C NMR spectra were measured in CDCl 3, while those of compound 3 were measured in CD 3 OD with TMS as an internal standard on a Jeol A500 and/or a Bruker AVANCE III-500 in CDCl 3 . The 1 H and 13 C chemical shifts were given in ppm, while coupling constants were in Hz.

Sponge.
Specimens of the sponge tagged K09-02 was collected by hand using SCUBA at 15-25 m depth at Kupang, West Timor, East Nusa Tenggara, Indonesia on August 2009. By comparing underwater images of our specimen with that of the specimen of NCI group [15], it is likely to be the same sponge. The specimen was kept frozen until extraction. The sponge K09-02 may be an endemic species to this region. The colonies are grey in color and stand.

2.7.
Acetylation. Compound 1 (0.2 mg) was dissolved in pyridine (50 μL) and acetic anhydride (50 μL). The mixture was stirred for three days under a nitrogen atmosphere at room temperature. After removal of excess reagents with nitrogen flow and vacuum, the reaction product 4 was checked with 1 H NMR and ESIMS. Compound 2 was similarly treated to give 4.

Results and Discussion
As an EtOAc soluble portion of a methanolic extract of the sponge K09-02 showed potent cytotoxicity against cultured NBT-T2 cells, the portion was separated repetitively on a silica gel column followed by Si-60 HPLC affording three compounds 1, 2, and 3 as shown in Figure 1. By inspecting 1 H and 13 C NMR spectra of compound 1 together with database search (Tables 1 and 2   at doi:10.5402/2011/852619)), we could readily identify that it is a member of candidaspongiolide, a series of 18membered cytotoxic macrolide retaining one of fatty acid moieties from C 14 to C 18 at C-28 [15]. HR-ESIMS of our material exhibited molecular-related ions at m/z 945.55514, 959.57079, 973.58884 [M+Na] + indicating that compound 1 is candidaspongiolide esterified with the homologs of three saturated fatty acids (palmitic, margaric, and stearic acids).
Compound 2 was obtained as a colorless glass with [α] 25 D +72. After elucidation of its 1 H and 13 C NMR spectra, compound 2 was found to be an analog of 1. However, the 13 C NMR spectrum showed two carbonyl carbons at δ C 171.3 q (C-1) and 173.1 (C-35) instead of three in 1 (Table 1, Figure S3). As the signals for an acetoxy group (δ H 2.04 s, δ C 21.5 q) in 1 are missing in 2, it was suggested that 2 is a deacetyl derivative of 1. and H-28 (δ H 3.75) indicated that 3 is devoid of acyl groups. Close similarity of 1 H and 13 C NMR data of 3 to 1 ( Table 2) indicated that the macrolide core structure of compound 3 is identical to compound 1.
All of natural compounds 1-3 exhibited potent cytotoxicity, IC 50 37, 4.7, and 19 ng/mL, against NBT-T2 cells. The result is not in good agreement with those reported by Meragelman and coworkers, that is, candidaspongiolide (1) showed stronger growth inhibition (GI 50 14 ng/mL) than the core compound (42 ng/mL) [15]. Additionally Paul et al. paperd the importance of a linear carbon chain on the cytotoxicity in the case of amphidinol [22]. The difference may be explained either by the number of cell lines or by different sensitivity of NBT-T2 cells.