Topsensterols A–C, Cytotoxic Polyhydroxylated Sterol Derivatives from a Marine Sponge Topsentia sp.

Three new polyhydroxylated sterol derivatives topsensterols A–C (1–3) have been isolated from a marine sponge Topsentia sp. collected from the South China Sea. Their structures were elucidated by detailed analysis of the spectroscopic data, especially the NOESY spectra. Topsensterols A–C (l–3) possess novel 2β,3α,4β,6α-tetrahydroxy-14α-methyl Δ9(11) steroidal nuclei with unusual side chains. Compound 2 exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC50 value of 8.0 μM. Compound 3 displayed cytotoxicity against human erythroleukemia cell line K562 with an IC50 value of 6.0 μM.


Introduction
Most marine invertebrates are soft bodied and have a sedentary life style, resulting in the formation of distinctive chemical protection strategy [1,2]. The chemical means of defense necessitate the production of toxic compounds that can deter predators and paralyze their prey [2]. Undoubtedly, marine invertebrates represent a rich source of structurally novel and mechanistically unique secondary metabolites for the discovery of new drug leads [3]. Especially, sponges (Porifera) including the genus of Topsentia have been recognized as outstanding producers of sterols possessing novel side chains with unique alkylation and dealkylation patterns and displaying a variety of biological activities [4][5][6][7].
In our continuing efforts to discover new bioactive substances from marine invertebrates, a chemical investigation of a sponge of Topsentia sp. collected from the South China Sea was carried out. As a result, three new polyhydroxylated steroids, topsensterols A-C (1-3) (Figure 1), were isolated from its n-butanol extract. Compounds 2 and 3 showed significant cytotoxicity against human gastric carcinoma cell line SGC-7901 and human erythroleukemia cell line K562, respectively. Herein, we report the isolation, structural elucidation, plausible biogenetic pathway and biological evaluation of these steroids.

Results and Discussion
The frozen sample of sponge Topsentia sp. (5.1 kg, wet weight) was exhaustively extracted with 95% EtOH (8 L × 5 times) at room temperature, and the EtOH solution was evaporated under reduced pressure to give the crude extract. This extract was further partitioned between H2O and EtOAc. After evaporation of the organic solvents, the remaining aqueous suspension was extracted with n-butanol. Then the organic layer was concentrated to offer the n-butanol extract (27.1 g). This extract was subjected to a silica gel column chromatograph (CC) and the fractions were purified by octadecylsilyl silica gel CC and semi-preparative HPLC to obtain compounds 1-3.

Results and Discussion
The frozen sample of sponge Topsentia sp. (5.1 kg, wet weight) was exhaustively extracted with 95% EtOH (8 Lˆ5 times) at room temperature, and the EtOH solution was evaporated under reduced pressure to give the crude extract. This extract was further partitioned between H 2 O and EtOAc. After evaporation of the organic solvents, the remaining aqueous suspension was extracted with n-butanol. Then the organic layer was concentrated to offer the n-butanol extract (27.1 g). This extract was subjected to a silica gel column chromatograph (CC) and the fractions were purified by octadecylsilyl silica gel CC and semi-preparative HPLC to obtain compounds 1-3.
The stereochemistry of the steroid nucleus was established on the basis of coupling constants, COSY and NOESY data. Small coupling constants between H-2 and both H 2 -1 suggested that H-2 adopted an equatorial orientation. A large coupling constant between H-5 and H-6 (J = 10.8 Hz) and a small coupling constant between H-5 and H-4 (J = 2.4 Hz), implied that both H-5 and H-6 were axial, and H-4 was equatorial. In the COSY spectrum, a conspicuous W-type long-range crosspeaks between H-2 and H-4 further confirmed both H-2 and H-4 were equatorial. While W-type long-range crosspeaks between H-lβ and H-3 indicated that H-3 occupied an equatorial orientation. Therefore, the hydroxyl groups 2-OH, 3-OH, 4-OH, and 6-OH occupied the axial, axial, axial, and equatorial orientation, respectively. In the NOESY spectrum (  The stereochemistry of the steroid nucleus was established on the basis of coupling constants, COSY and NOESY data. Small coupling constants between H-2 and both H2-1 suggested that H-2 adopted an equatorial orientation. A large coupling constant between H-5 and H-6 (J = 10.8 Hz) and a small coupling constant between H-5 and H-4 (J = 2.4 Hz), implied that both H-5 and H-6 were axial, and H-4 was equatorial. In the COSY spectrum, a conspicuous W-type long-range crosspeaks   Careful inspection of the NMR spectra of 1-3 (Tables 1 and 2) showed that the common and highly conserved signals of the steroidal nuclei for 1-3 are strikingly similar, including the signals of three angular methyl groups, a C-9(11)-double bond, four oxygenated methines, and four hydroxyl groups. Combined with their NOESY data and comparison with the configurations of the known similar metabolites such as topsentiasterol sulfates A-F, chlorotopsentiasterol sulfate D, and Careful inspection of the NMR spectra of 1-3 (Tables 1 and 2) showed that the common and highly conserved signals of the steroidal nuclei for 1-3 are strikingly similar, including the signals of three angular methyl groups, a C-9(11)-double bond, four oxygenated methines, and four hydroxyl groups. Combined with their NOESY data and comparison with the configurations of the known similar metabolites such as topsentiasterol sulfates A-F, chlorotopsentiasterol sulfate D, and iodotopsentiasterol sulfate D also isolated from the marine sponge Topsentia sp. [7,8], compounds 1-3 were suggested to possess the same 2β,3α,4β,6α-tetrahydroxy-14α-methyl ∆ 9(11) sterol framework. The differences between these compounds were restricted to the respective aliphatic side chains.
Topsensterol C (3), with molecular formula of C 31 H 48 O 7 , was also isolated as a white amorphous powder. The 1 H and 13 C NMR signals (Tables 1 and 2, Supplementary Materials, Figures S17 and S18) of the side chain were very similar to those of 2. The only significant difference was that a methoxy (δ H 3.58, δ C 57.0) and an oxygenated methine (δ H 5.87, δ C 104.0) in 3 replaced the oxymethylene (δ H 4.87, δ C 71.7) in 2. Interpretation of HMBC data (Supplementary Materials, Figure S21) of 3 indicated that a 4-methoxy-2-alkyl butenolide moiety connected at the terminal of the side chain for 3.
Polyhydroxylated sterols with various novel side chains are common secondary metabolites from marine sponges. In present study, topsensterol A (1) possesses a unique side chain terminated with a 2-substituted-dimethyl maleate unit. To the best of our knowledge, it is the first report of polyhydroxylated sterol with a 2-substituted-dimethyl maleate side chain. The plausible biosynthesis mechanism to form the side chains of 1-3 was proposed in Scheme 1. According to the literature [6,7], the side chains present in 1-3 are likely formed via methylations by S-adenosylmethionine (SAM) to the side chain present in parkeol, along with oxidation and methoxylation. Scheme 1 illustrates that: (i) the addition of a methyl from SAM to C-24 and a loss of a proton to generate the 25,27-double bond; (ii) SAM adds a methyl to C-27 to form the C-25 cation; (iii) the C-25 cation followed by oxidation and tautomerization to form topsensterol B (2) (pathway a); and (iv) the C-25 cation followed by oxidation and methoxylation to form topsensterol C (3) and topsensterol A (1) (pathway b). Marine sponges of the genus Topsentia were reported to produce various structurally unique steroids including polyhydroxylated sterols [9] and polysulfated steroids [10][11][12]. In the present study, all of the isolated polar steroids were polyhydroxylated sterols, as the desulfated derivatives of similar sterol sulfates [8]. It seems that our isolated compounds maybe a group of desulfated artifacts. However, the desulfation of polysulfated sterols should be not so easy to occur during the Scheme 1. Plausible biosynthesis mechanism to form the side chains of 1-3.

Mar. Drugs
Marine sponges of the genus Topsentia were reported to produce various structurally unique steroids including polyhydroxylated sterols [9] and polysulfated steroids [10][11][12]. In the present study, all of the isolated polar steroids were polyhydroxylated sterols, as the desulfated derivatives of similar sterol sulfates [8]. It seems that our isolated compounds maybe a group of desulfated artifacts. However, the desulfation of polysulfated sterols should be not so easy to occur during the ordinary extraction and separation processes. To date many polysulfated sterols have been reported to be obtained from marine sponges via ordinary separation processes, indicating that polysulfated sterols are stable enough. More importantly, on the basis of biogenetic considerations, the 2β,3α,4β,6α-tetrahydroxy-14α-methyl ∆ 9(11) steroidal nuclei pattern could be biosynthesized directly from parkeol in marine sponges [6,13,14]. Given the above, compounds 1-3 were more likely to be produced by ecological conditions, but the probability of desulfated artifacts could not be ruled out.
Compounds 1-3 were assessed for their cytotoxic activity against human gastric carcinoma SGC-7901 and human erythroleukemia K562 cell lines by MTT method [15]. Compound 2 exhibited cytotoxicity against SGC-7901 and K562 cell lines with IC 50 values of 8.0 and 20 µM, respectively. Compound 3 exhibited cytotoxicity against SGC-7901 and K562 cell lines with IC 50 values of 28 and 6.0 µM, respectively. However, compound 1 showed no cytotoxicity against the two cell lines. The above results revealed that the terminal butenolide moiety in the side chain may play a key role in the cytotoxicity.
Compounds 1-3 were also tested for their antimicrobial activity against human pathogenic bacteria including Staphylococcus aureus (ATCC 51650), Methicillin-resistant Staphylococcus aureus (ATCC 9551), and Candida albicans (ATCC 10231) using the method developed by Fromtling et al. [16]. However, no compound was found to be active against these bacteria.

Animal Material
The marine sponge Topsentia sp. was collected from Xuwen, Guangdong Province, China, in April 2006, and was identified by Nicole J. de Voogd, National Museum of Natural History, The Netherlands. A voucher specimen (GD-XW-20060007) was deposited at the Key Laboratory of Marine Drugs, Ministry of Education, Ocean University of China, Qingdao, China.

Conclusions
In summary, three new polyhydroxylated steroid derivatives topsensterols A-C (l-3) possessing unusual side chains were successfully isolated from the marine sponge Topsentia sp. The plausible biosynthesis mechanism to form the uncommon side chains of 1-3 was proposed. Compounds 2 and 3 exhibited significant cytotoxicities, demonstrating that the terminal butenolide moiety in the side chain may act an important part in the cytotoxicity.