Cytotoxic Sesterterpenoids from a Sponge Hippospongia sp.

One new pentacyclic sesterterpene, hippospongide A (1), and one new scalarane sesterterpenoid, hippospongide B (2), along with six previously reported known scalarane–type sesterterpenes (3–8), were isolated from a sponge Hippospongia sp. The structures of these compounds were elucidated on the basis of their spectroscopic data and comparison of the NMR data with those of known analogues. These metabolites are the first pentacyclic sesterterpene and scalarane-type sesterterpenes to be reported from this genus. Compounds 3–5 exhibited significant cytotoxicity against DLD-1, HCT-116, T-47D and K562 cancer cell lines.


Results and Discussion
The EtOAc extract of the freeze-dried specimen was fractionated by silica gel column chromatography and the eluted fractions were further separated utilizing normal phase HPLC to yield metabolites 1-8 (Chart 1).   (Table 1): five methyls, seven sp 3 methylenes, four sp 3 methines (including one oxygenated carbon at δ 75.9), two sp 2 methines, and four sp 3 quaternary carbons. The remaining three signals appearing in the downfield region of the spectrum are due to the quaternary carbons of two olefinic carbons (δ 122.9 and 159.0) and one ketone carbonyl (δ 196.8). From the 1 H NMR (Table 1) spectrum of 1, the 1 H NMR data revealed the presence of two olefinic methine protons (δ 7.33 Hz; d, J = 1.5 Hz; 6.76 Hz; d, J = 1.5 Hz). Furthermore, one oxygenated methine (δ 4.58, s) was also designated from the 1 H NMR signal. Careful analysis of the 1 H-1 H COSY correlations observed for 1 led to the establishment of five partial structures, as shown in Figure 2. The molecular framework of 1 was further established by a HMBC experiment ( Figure 2). The five rings and their connectivities were elucidated on the basis of the following key HMBC correlations: both methyls H 3 -19 and H 3 -20 to C-3, C-4 and C-5, H 3 -21 to C-7, C-8, C-9 and C-13, H 3 -22 to C-1, C-5, C-9 and C-10, H 3 -23 to C-11, C-12, C-13 and C-18, H-13 to C-15, H-14 to C-15 and C-16, H-18 to C-17 and C-16, and both olefinic methines H-24 and H-25 to C-16 and C-17. Thus, 1 was found to possess two double bonds at C-16/C-17 and C-24/C-25, one hydroxy group at C-18, and one ketone group at C-15. Linking all the above functional groups to the sesterterpene skeleton thus yielded the gross structure of 1.
The relative configuration of 1, elucidated mainly from the NOESY spectrum, was corroborated by MM2 force field calculations, which suggested the most stable conformation to be that shown in Figure 2. In the NOESY spectrum, H-9 showed NOEs with H-5 and H-13 but not with three methyls H 3 -21, H 3 -22 and H 3 -23. Thus, assuming an α-orientation of H-5, both H-9 and H-13 must also be on the α face whilst the three methyls H 3 -21, H 3 -22 and H 3 -23 must be located on the β face. Moreover, the NOE correlations of H 3 -23 with H-18 indicated the β-orientation of H-18. On the basis of the above findings and other detailed NOE correlations (Figure 3), the relative structure of 1 was determined. After determining the structure of 1, we discovered that its molecular framework has been obtained as known sesterterpenoids salmahyrtisol A and similan A, which were isolated previously from sponges Hyrtios erecta [8] and Hyrtios gumminae [9], respectively.   (Table 1). Moreover, it was found that the NMR data of the tricyclic skeleton (C-1 to C-14) of 2 were quite similar to those of 3 and 8, indicating the same substitution and stereochemistry at C-5, C-8, C-9, C-10, C-12, C-13 and C-14. Furthermore, analysis of the 1 H-1 H COSY and HMBC correlations established the remaining structure, including another two rings from C-13 to C-18 ( Figure 2). Finally, the relative stereochemistries at C-17 and C-18 were resolved by careful interpretation of the NOE correlations ( Figure 4). Key NOE correlations for 2 showed interactions between H-18 to H-12 and H-14. Thus, H-18 should be located on the α face. NOE correlations were also detected between H-17 and H 3 -23, revealing the β-orientation of H-17, as suggested by a molecular model of 2. After structural determination of 2, we found that this compound had been obtained previously by hydrogenation of the natural product hydroxylactone IV [10]. In the original report, the authors gave a planar structure. However, our study led to the isolation of 2 for the first time from natural sources. In addition, we successfully elucidated the full structure of 2. Moreover, our work also provides full assignment for the 1 H and 13 C NMR spectral data of 2.  The cytotoxicities of compounds 1-8 against DLD-1, HCT-116, T-47D and K562 cancer cells are shown in Table 2. The results showed that compounds 3-5 were found to exhibit cytotoxicity against all or part of the above carcinoma cell lines, while compound 3 (IC 50 values 0.001, 0.001, 0.001 and 0.001 μM against the above carcinoma cell lines, respectively) was the most potent.

General Experimental Procedures
Optical rotation values were measured with a Jasco P-1010 digital polarimeter. IR spectra were recorded on a Varian Digilab FTS 1000 Fourier transform infrared spectrophotometer. The NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR instrument at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR, respectively, in CDCl 3 . ESIMS data were obtained with a Finnigan LCQ ion-trap mass spectrometer. HRESIMS data were recorded on a LTQ Orbitrap XL mass spectrometer. Gravity column chromatography was performed on silica gel (230-400 mesh, Merck). TLC was carried out on pre-coated Kieselgel 60 F254 (0.2 mm, Merck) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. High-performance liquid chromatography was performed using a system comprised of a Hitachi L-7100 pump and a Rheodyne 7725 injection port. A preparative normal phase column (250 × 21.2 mm, 5 μm) was used for HPLC.

Animal Material
The specimen of Hippospongia sp. was collected by scuba diving at a depth of 20 m from coral reefs off the coast of Tai-tung, Taiwan. Voucher specimen was deposited in the National Museum of Marine Biology and Aquarium, Taiwan (specimen No. 2011SP-1). This genus is often confused with Hyattella (Lendenfeld, 1888), whereas Hippospongia is more elastic and compressible with fewer primary fibers ( Figure 5). Taxonomic identification was performed by Li-Lian Liu of the National Sun Yat-sen University, Kaohsiung, Taiwan.

Molecular Mechanics Calculations
Implementation of the MM2 force filed in Chem3D Pro software [13] was used to calculate the molecular models.

Conclusions
Previous chemical investigations of sponges of the genus Hippospongia have led to the isolation and identification of various metabolites . Some of these have been found to possess several kinds of biological activities, such as isocitrate lyase (ICL) inhibitory [14], RCE protease inhibitory [15] and cytotoxic [16][17][18][19][20][21] activities. In the present study, two new sesterterpenoids, hippospongides A and B (1 and 2), together with six known scalarane sesterterpenoids were isolated from the sponge Hippospongia sp. Compounds 3-5 showed significant cytoxicities against DLD-1, HCT-116, T-47D and K562 cell lines. However, the new compounds 1 and 2 and the other known compounds had no significant activity. Furthermore, it is worth mentioning that these compounds are the first pentacyclic sesterterpene and scalarane-type sesterterpenes to be reported from this genus. However, this genus is often confused with Hyattella and the sesterterpenoids are not likely to assist in chemical differentiation of the species.