Isolation and Structure Elucidation of Three New Dolastanes from the Brown Alga Dilophus spiralis

Three new dolastane diterpenes (1–3) and five previously reported perhydroazulenes were isolated from the organic extracts of the brown alga Dilophus spiralis. The structure elucidation and the assignment of the relative configurations of the isolated natural products were based on extensive analyses of their spectroscopic data, whereas the absolute configuration of metabolite 2 was determined through its chemical conversion to a previously isolated compound of known configuration.


Introduction
The family Dictyotaceae comprises cosmopolitan species of brown algae which are considered a prolific source of secondary metabolites. Representatives of the family have been the subject of numerous chemical studies over the last 50 years yielding approximately 500 new natural products. Many of these metabolites have been evaluated for and proven to possess different levels of antibacterial, antiviral, cytotoxic, antifeedant, ichthyotoxic, algicidal, and/or antifouling activities. Among these, species of the genera Dictyota and Dilophus produce mainly sesquiterpenes and diterpenes of normal biosynthesis featuring a wide range of carbon skeletons [1,2].
In the course of our ongoing research focusing on the isolation of bioactive secondary metabolites from marine organisms found along the coastlines of Greece, we initiated a thorough investigation of the OPEN ACCESS chemical composition of Dilophus spiralis (Montagne) Hamel (syn. ligulatus). Previously, we described the isolation and structural characterization of five new dolastanes, one new 2,6-cyclo-xenicane, twenty new dolabellanes, two diterpenes featuring novel carbon skeletons, and several known compounds [3][4][5][6][7]. Herein, we report the isolation and structure elucidation of three new dolastanes (1)(2)(3) and five known perhydroazulene diterpenes.

Results and Discussion
A series of chromatographic separations of the organic extracts of the brown alga D. spiralis, collected in Elafonissos island, Greece, resulted in the isolation of the new dolastanes 1-3 ( Figure 1) and five previously reported perhydroazulenes, which were identified as dictytriene B [8], dictyoxide [9], pachydictyol A [10], isopachydictyol A [11], and dictyol E [12] by comparison of their spectroscopic and physical characteristics with those reported in the literature.   (Table 2) revealed 20 carbon signals, which corresponded to four quaternary carbon atoms, four methines, eight methylenes, and four methyls, as determined from DEPT experiments. Among them, two olefinic (δ C 112.8 and 147.4) and two oxygenated (δ C 84.6 and 85.5) carbons were evident. Since the carbon-carbon double bond accounted for one of the five degrees of unsaturation, the molecular structure of 1 was determined as tetracyclic. Due to the presence of two oxygenated carbons but only one oxygen atom in the molecule, in combination with the absence of an absorption band at either 1670-1750 or 3300-3500 cm −1 in the IR spectrum, the oxygen atom was assigned to an ether function. Comparison of these spectroscopic characteristics to those previously reported for dolastane diterpenes [3] and analysis of the correlations observed in the HMBC and COSY spectra (    The relative configurations of the stereocenters of metabolite 1 were established by analysis of the key correlations displayed in the NOESY spectrum ( Figure 3). The NOE enhancements of H-8/H-9, H-8/H 3 -20, and H-14/H 3 -16 provided evidence that H-14 and H 3 -16 were cofacial, whereas H-8, H-9, and H 3 -20 were on the opposite side of the molecule, thus suggesting the trans fusion of the six-and seven-membered rings, as well as of the seven-and five-membered rings and determining the relative configurations of the chiral centers C-5, C-8, C-9, C-12, and C-14 as 5S*,8S*,9S*,12R*,14S*, in accordance with previously reported dolastane derivatives isolated from the same algal specimens [3]. Taking into account that the ether bridge formation between C-1 and C-4 required the cis orientation of the substituents at the α and α′ positions to the ether linkage, namely H-4 and H 3 -15, in conjunction with the interactions of H-4 with Η 2 -3, Η 2 -6, and Η 3 -20, as well as of Η-3α with Η-6α observed in the NOESY spectrum, the relative configurations at C-1 and C-4 were determined as 1R*,4S*. The absence of a COSY correlation between H-3α and H-4, indicating that the dihedral angle Η-3α-C-3-C-4-H-4 was approaching 90°, further supported the proposed conformation.
Compound 2, obtained as a yellow oil, had the molecular formula C 20 H 32 O 3 , as calculated from the HRFABMS measurements and NMR data. The spectroscopic characteristics of 2 were rather similar to those of metabolite 1. Specifically, the 1 H NMR spectrum (Table 1), as in the case of 1, included signals for four singlet methyls (δ H 0.88, 1.03, 1.07, and 1.78), one exomethylene group (δ H 4.68 and 4.96), and an oxygenated methine (δ H 3.19). The 13 C NMR spectrum (Table 2)   Compound 3 was isolated as a colorless oil. The structural elements displayed in the 1 H and 13 C NMR spectra of 3 closely resembled those of metabolite 2. The 1 H NMR spectrum (Table 1) included signals for two aliphatic and two vinylic singlet methyls (δ H 0.84, 1.07, 1.63, and 1.82), one exomethylene group (δ H 4.71 and 4.99), an olefinic methine (δ H 5.32), and an oxygenated proton (δ H 3.42), whereas the 13 C NMR spectrum (Table 2) revealed 20 carbon signals, among which a carbonyl (δ C 224.1), one oxygenated (δ C 74.9), and four olefinic (δ C 113.4, 120.4, 135.6, and 147.2) carbons were apparent. In agreement with the molecular formula C 20 H 30 O 2 , as deduced from the HRFABMS data, it was obvious that the difference between 2 and 3 was the absence of one hydroxy group and the formation of a second carbon-carbon double bond. The trisubstituted double bond was placed between C-1 and C-2 as indicated by the HMBC correlations of both C-4 and C-15 with H-2. The relative configurations of the stereogenic centers of 3 were established by analysis of the key NOE enhancements observed, in accordance with those of 2, as 4S*,5S*,8S*,9S*,12R*,14R*.
Reduction of metabolite 2 according to Molander et al. [13] yielded the 11-deoxo derivative of 2, identical in all respects to (1S,4S,8S,14S)-1,4-dihydroxy-17-dolastene [3]. Since the semisynthetic compound exhibited the same sign of optical rotation as the natural product previously isolated from the same algal collection [3], for which the absolute configuration was determined by application of Mosher's method, the absolute configuration of 2 was established as depicted. The absolute configurations of metabolites 1 and 3 were not determined due to the limited available amounts, but on the basis of biogenetic considerations they are expected to be the same.
Among the new dolastanes isolated in the present study, metabolite 2, which was obtained in adequate quantity, was evaluated for its cytotoxic activity against four human apoptosis-resistant (U373, A549, SKMEL28, OE21) and two human apoptosis-sensitive (PC3, LoVo) cancer cell lines, since previously isolated dolastanes had shown moderate cytotoxicity [3]. Furthermore, compound 2 was tested for its inhibitory effect on the hypoxia-inducible factor-1 (HIF-1). However, in both cases metabolite 2 exhibited no activity.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer. IR spectra were obtained on a Paragon 500 Perkin-Elmer spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on a δ (ppm) scale using TMS as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. High resolution FAB mass spectral data were provided by the University of Notre Dame, Department of Chemistry and Biochemistry, Notre Dame, IN, USA. Low resolution EI mass spectra were measured on a Hewlett Packard 5973 mass spectrometer. Column chromatography separations were performed with Kieselgel 60 (Merck). HPLC separations were conducted using a CECIL 1100 Series liquid chromatography pump equipped with a GBC LC-1240 refractive index detector, using the following columns: (i) Econoshpere Silica 10u (Alltech, 25 cm × 10 mm) and (ii) Chiralcel OD 10 μm (Daicel Chemical Industries Ltd., Osaka, Japan, 25 cm × 10 mm). TLC were performed with Kieselgel 60 F 254 (Merck aluminum support plates) and spots were detected after spraying with 15% H 2 SO 4 in MeOH reagent and heating at 100 °C for 1 min. The lyophilization was carried out in a Freezone 4.5 freeze dry system (Labconco).

Plant Material
Specimens of Dilophus spiralis were collected by hand in Elafonissos island, south of Peloponnese, Greece, at a depth of 0.1-1 m, in April of 2004. A voucher specimen of the alga has been deposited at the Herbarium of the Department of Pharmacognosy and Chemistry of Natural Products, University of Athens (ATPH/MO/159).

Extraction and Isolation
Specimens of the freeze-dried alga (272 g) were exhaustively extracted with CH 2 Cl 2 and subsequently with MeOH at room temperature. Evaporation of the solvents in vacuo afforded two dark green oily residues. The CH 2 Cl 2 residue (9.2 g) was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield fifteen fractions (A1-A15). Fraction A3 (20% EtOAc in cyclohexane, 1.17 g) was further fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford twenty-one fractions (A3a-A3u). Fraction A3b (1% EtOAc in cyclohexane, 355.7 mg) was subjected to gravity column chromatography on silica gel, using cyclohexane with increasing amounts of CH 2 Cl 2 , followed by CH 2 Cl 2 with increasing amounts of EtOAc as the mobile phase, to yield eleven fractions (A3b1-A3b11).