New Lobane and Cembrane Diterpenes from Two Comorian Soft Corals

Preliminary biological investigation of a collection of Comorian soft corals resulted in the selection of two specimens, one of Sarcophyton and the other of Lobophytum, on the basis of their toxicity on larvae of the brine shrimp (Artemia salina) and inhibition of acetylcholinesterase, respectively. Bioassay-guided fractionations provided a known antitumor promoter cembrane diterpenoid, (+)-sarcophytol-A (1), along with a new lobane diterpenoid, carbomethoxyfuscol (2), from Sarcophyton sp., and a new cembranoid, crassumolide E (3), from Lobophytum sp. The structures of compounds 1–3 were determined by spectroscopic analysis and by comparison of the spectral data with previously reported values. The cembranoid 3 was found to exhibit a moderate inhibitory effect on acetylcholinesterase.


Introduction
Alcyonarians are rich sources of sesquiterpenes and diterpenes with unique structural diversity and pronounced biological activities [1]. Among these, lobane and cembrane diterpenoids are typical of coelenterates of the orders Alcyonacea and Gorgonacea, which have been recognized as the most prolific sources of these classes of diterpenes. Lobanes, which have been isolated from the Gorgonian species Eunicea fusca [2] and from soft corals of the genera Lobophytum [3][4][5][6] and Sinularia [7][8][9], are known for their antibacterial, fungicidal and cytotoxic properties. Cembranes, found in several genera of soft corals, in particular Lobophytum [10], Sinularia [11] and Sarcophyton [12], are believed to play a role in the protection of soft corals from predators [13]. They have been reported to have antitumor, antimicrobial, anti-inflammatory, calcium antagonistic effects and neuroprotective activities.
In our search for bioactive substances from marine organisms, a biological screen of a collection of Comorian soft corals was initiated on toxic activity using brine shrimp Artemia salina assay [14] and on acetylcholinesterase inhibition using a TLC assay [15]. These two biological assays were chosen specifically because they are simple and rapid. The TLC bioassay gives quick access to the activity and its localization in complex matrices. The latter was also chosen because of the implication of acetylcholinesterase (AChE) inhibitors in the therapeutic approach to the fight against Alzheimer's disease (AD).
AD is a progressive neurodegenerative disorder of the central nervous system that is characterized by an alteration of the cholinergic system and other neurotransmitter systems. One of the current therapeutic approaches to treating AD is aimed at restoring the native levels of acetylcholine in the central nervous system by using AChE inhibitors. Based on this strategy, AChE inhibitors are the most widely developed class of drugs approved for the symptomatic treatment of the disease. These include synthetic donepezil (Aticept®), rivastigmine (Exelon®) and the naturally occurring alkaloid galanthamine (Razadyne®) [16,17]. Some AChE inhibitors are known to have short half-time or strong side-effects. Even treatments with galanthamine, which is a potent long-acting drug with low toxicity, have shown a decline in clinical efficacy with time. The need for new potent selective and low side-effects AChE inhibitors is still enormous. After exploration of the terrestrial domain, marine organisms are being investigated.
Two specimens of soft corals were selected for their significant bioactivity against larvae of Artemia salina and acetylcholinesterase. Bioassays-guided separation resulted in the isolation of a known antitumor cembrane, (+)-sarcophytol-A (1), along with a new lobane, carbomethoxyfuscol (2) from a Sarcophyton sp. (family Alcyoniidae), and the isolation of a new cembranoid, crassumolide E (3), inhibitor of the acetylcholinesterase, from a Lobophytum sp. (family Alcyoniidae) ( Figure 1).

Results and Discussion
Specimens of the soft corals Lobophytum sp. and Sarcophyton sp. were collected by scuba diving at the lagoon of Southern Mayotte, Comoros Island, northwest of Madagascar. All specimens were treated following the same procedure: the freeze-dried biological material was extracted with a mixture of methanol-dichloromethane 1:1 to give a crude extract. The latter was partitioned between ethyl acetate (EtOAc) and water, and the organic phase was subjected to a silica gel column eluted sequentially with hexane, dichloromethane, ethyl acetate and methanol. The EtOAc fractions of both soft corals were selected on the basis of their bioactivity, i.e., the Sarcophyton EtOAc fraction showed toxicity against larvae of Artemia salina, and the Lobophytum EtOAc extract inhibited acetylcholinesterase. Fractions exhibiting activities were further chromatographied on silica gel column using different pentane-EtOAc mixtures. Final purifications were achieved by normal-phase HPLC to afford diterpenes 1-2 from Sarcophyton and 3 from Lobophytum.

Sarcophyton
Compound 1 was identified as (+)-sarcophytol-A by comparison of its spectral data with previously reported values [18]. The toxic activity against Artemia salina was mainly provided by this compound. Sarcophytol A has been reported to show antitumor activity [19] as well as potent inhibitory activities against several kinds of tumor promoters [20].
The 1 H and 13 C NMR data were consistent with a lobane derivative (Table 1). In particular, the 1 H spectrum revealed the presence of vinyl and isoprenyl systems of a lobane skeleton. In addition, three olefinic protons were observed in the downfield region at δ Η 6.  19 and H-20) connected to one oxygenated carbon (δ C 71.0, C-18) suggested that a hydroxyl group is located at C-18. Confirmation of this site was achieved by an HMBC experiment that unambiguously established the atom connectivities in 2 (Figure 2), i.e., long-range correlations were found between the olefinic proton at δ Η 6.05 (H-17) and the quaternary carbon at δ C 71.0 (C-18), and the methyl carbons at δ C 29.6 (C-19 and C-20). Besides, long-range correlations were found between the methyl protons at δ Η 1.36 and C-17 and C-18. The sp 2 quaternary carbon C-13 could be connected to a carbomethoxy group (δ C 167.  (4), a lobane congener isolated from the gorgonian species Eunicea fusca [21] and from the soft coral Lobophytum microlobulatum [22], which present a methyl group instead of the carboxylate function.  The relative configuration of the various assymetric centers was deduced from the comparison with NMR data of diverse lobane congeners (the lobanes have all been described with the same stereochemistry as 1R, 2R, 4S); the similarity in the 13 C chemical shifts of C-1 to C-12 in all compounds indicated the same relative configuration of the cyclohexane system. Thus, 2 was deduced to be methyl (1R*, 2R*, 4S*)-18-hydroxyloba-8,10,13(Z),16(E)-tetraen-13-carboxylate.

Lobophytum
After assignment of each direct C-H bonding based on HSQC data, the partial structures a, b, and c ( Figure 3) were established by 1 H-1 H COSY analysis and supported by long-range HMBC correlations. The partial structures were reasonably connected to each other following HMBC correlations (Figure 4). The link between segments a and b via C-4 and C-5 was deduced from longrange correlations between H-5 to C-18. The connection between b and c via C-8 and C-10 was revealed by correlations between H-7, H-19, H-10a and H-10b to C-9. Finally, the connection between c and a via C-12 and C-13 was determined by correlations between H-13a and H-13b to C-11, C-12, C-20.    The E-configuration of the two methyl-substituted olefins was deduced from the 13 C NMR data [δ C 15.5 (q, C-18), 17.0 (q, C-19)] [28]. The E-configuration of the carboxylic-substituted olefin was suggested by the large downfield shifts of the vinylic proton (H-11) and carbon C-11 [δ H 7.03 (H-11) and δ C 151.5 (C-11)] [29]. In order to deduce the stereochemistry of the lactone ring junction, NOESY experiments have been recorded in different solvent conditions but no dipolar coupling could be observed between H-1 and H-14. According to literature [30][31][32][33], the coupling constant between the lactone methine protons ( 3 J 1,14 ) did not permit univocal stereochemical assignments due to the flexibility of the cembrane ring. However, previous studies tended to show that small coupling constants (ranging from 2.9 to 7.5 Hz) indicate a trans-fused γ-lactone ring while large coupling constants (from 6.5 to 9.3 Hz) suggest a cis-fused γ-lactone ring [33][34][35][36]. These propositions were done in agreement with several X-ray studies [29,33,37]. The 3 J 1,14 coupling constant of the lactone methine protons for 3 being 2.3 Hz, this suggests a trans-fused γ-lactone as in crassumolide A (5) [38], a cembrane isolated from Lobophytum crassum, whose structure is very similar. Thus, 3 was proposed as (1R*,14S*)-cembra-3(E),7(E),11(Z),15(17)-tetraen-14,16-olide-12-carboxylic acid.
Moreover, this compound was found to exhibit inhibitory effects on AChE. A TLC bioautographic method for detection of acetylcholinesterase inhibitors was used for the screening of soft coral extracts. The active fraction of Lobophytum sp. was directly detected on the TLC. After purification on HPLC, the active fraction afforded compound 3, that was further tested in order to establish the detection limit for its activity ( Figure 5). 3 showed weak activity (1 μg at least) as AChE inhibitor, compared to galanthamine, an alkaloid isolated from plants of the Amaryllidaceae family (the smallest amount required 0.01 μg, cf. lit. [15]). Only few AChE inhibitors have been isolated from marine organisms. One other terpene from the Alcyonacea family has been found to exhibit a modest AChE inhibition activity: cladidiol isolated from a Cladiella species [40]. Other examples are: a series of natural pigments with tetrazacyclopentazulene skeleton found in the zoanthid coral Parazoanthus axinellae (pseudozoanthoxanthins and zoanthoxanthins) [41], an irreversible inhibitor from the mollusk Onchidella binneyi (onchial) [42], a lipoidal metabolite isolated from the dinoflagellate Gymnodinium breve (BTX-B) [43], a nemertine alkaloid toxin (anabaseine) [44], a large 3-alkylpyridinium polymer isolated from the sponge Reniera sarai (poly-APS) [45,46], a bromotyrosine-derived metabolite of an unidentified verongid sponge (aplysamine-4) [47] and spirocompounds isolated from the mediterranean sponge Aplysina cavernicola (aerothionin, homoaerothionin, 11,19-dideoxyfistularin) [48], a diiodotyramine derivative from the Japanese gastropod Turbo marmorata (turbotoxin A) [49] and a pyridoacridine alkaloid from a Thai marine sponge Petrosia n. sp. (petrosamine) [50].
The biological interest of the newly isolated cembrane lies not only in its inhibitory effect on AChE, but also in the possibility of using this compound as a tool in the regulation of acetylcholine receptors. Over recent years, new results have emerged demonstrating that AChE inhibitors have additional pharmacological properties, i.e., protection against neurotoxic agents, such as glutamate, and up-regulation of nicotinic acetylcholine receptors (nAChRs). Neuroprotection against toxic insults and use of nAChR modulators appears to be a promising new strategy against neurodegenerative diseases. Several tobacco and marine cembranoids have been found to interact with nAChRs in complex ways: as irreversible inhibitors at the agonist sites, as non competitive inhibitors, or as positive modulators. Studies of neuroprotection by a tobacco cembranoid and by the marine cembranoid sarcophytolide, isolated from the soft coral Sarcophyton glaucum, revealed apparent similarities between the two compounds, and allowed one to conclude that neuroprotection is based on a nicotinic mechanism [51]. It remains to be determined whether the AChR-mediated neuroprotection by those two cembranoids is the exception or a general property of most cembranoids. The isolation of new marine cembranoids with acetylcholinesterase inhibitory effects will give the opportunity to explore the specific action of cembranoids on the nicotinic acetylcholine receptors.

General Experimental Procedures
TLC were performed on pre-coated TLC plates with Sigel 60 F 254 (Marchey-Nagel, Düren, Germany). HPLC was conducted with a MerckLiChrospher Si 60 (250 x 4 mm, 5 μm) column (Merck, Darmstadt, Germany), using different cyclohexane-EtOAc mixtures as eluent, and a refractometric detection. Optical rotations were measured at 20 °C on a Jasco P-1010 polarimeter. Ultraviolet (UV) spectra were recorded in methanol on a Perkin Elmer 551 spectrometer and Infrared (IR) spectra were obtained in chloroform on a Perkin Elmer 1600 FTIR spectrometer. 1 H and 13 C spectra, and 2D-NMR experiments were recorded on Brücker Avance DPX-250 NMR and Jeol EX 400 NMR spectrometers. Chemical shifts are given on a δ (ppm) scale with CHCl 3 ( 1 H, 7.26 ppm; 13 C, 77.0 ppm) as internal standard. EI and HRESI mass spectra were acquired on an ATI UNICAM and a micromass LCT (ES) mass spectrometer, respectively.

Biological Material
Specimens of the soft corals Lobophytum sp. and Sarcophyton sp. were collected at the lagoon of Southern Mayotte, Comoros Islands, northwest of Madagascar, by scuba diving at a depth of 10 meters. Samples were identified by G. Williams (California Academy of Sciences). A positive identification could not be done, but the Lobophytum specimen showed similarities to Lobophytum crassum (Marenzeller, 1886), and the Sarcophyton specimen appeared close to Sarcophyton infundibuliforme (Tixier-Durivault, 1956). Voucher fragments are maintained at the California Academy of Sciences.

Brine Shrimp Lethality Test
Eggs of Artemia salina (Dohse, Aquaristik GmbH, Bonn, Germany) were hatched in a small tank filled with natural sea water at 25 °C under continuous light regime. After 48 h, the phototropic nauplii were collected; 10 shrimps were transferred to each sample vial using a pipette, and seawater was added to a final volume of 180 μL. 20 μL of each sample (solution of 1 mg.mL -1 in water and 10% EtOH) were added in the 96-well plate. The toxicity was determined after 24 and 48 h of exposure. The experiments were done twice in duplicate.

Acetylcholinesterase Inhibition Test
Acetylcholinesterase (Sigma, St Louis, USA; 1000 U) was dissolved in 150 mL of 0.05 M Tris-hydrochloric acid buffer at pH 7.8, with 150 mg of bovine serum albumin (Merck, Darmstadt, Germany). After deposition of the samples on TLC (100 μg) and migration in a suitable solvent, the TLC plate was dried to remove the solvent, sprayed with the enzyme solution and dried again. The plate was placed in an incubator at 90% humidity and 37 °C for 20 min. For detection of the enzyme, 10 mL of a 1-naphtylacetate solution (250 mg in 100 mL of ethanol) and 40 mL of a Fast Blue Salt solution (400 mg in 160 mL of distilled water) were mixed and sprayed onto the plate to give a purple coloration after 1-2 min. Inhibitors of acetylcholinesterase appear as white spots.