Sphaeroane and Neodolabellane Diterpenes from the Red Alga Sphaerococcus coronopifolius

Investigation of minor metabolites in the extracts of the red alga Sphaerococcus coronopifolius collected from the rocky coasts of Corfu Island in the Ionian Sea yielded two new diterpene alcohols, sphaerollanes I, and II (1, 2) featuring neodolabellane skeletons, and the new sphaeroane diterpene alcohol 16-hydroxy-9S*-acetoxy-8-epi-isosphaerodiene-2 (3), along with two previously reported metabolites 4, 5. The structures of the new natural products, as well as their relative stereochemistry, were elucidated on the basis of extensive spectral analysis, including 2D-NMR experiments.


Introduction
All organisms biosynthesize secondary metabolites for certain essential physiological functions and exhibit complex chemical profiles, frequently including terpene metabolites. The numerous ways that the basic C 5 units can be joined together and the different ecological pressures under which organisms have evolved justifies the enormous number and the diversity of the elaborated terpenoid structures [1].

OPEN ACCESS
Sphaerococcus coronopifolius has been the focus of previous chemical investigations, which showed the presence of interesting diterpenes with two, three and four ring carbon skeletons, most of which contain one or two bromine atoms [2,3].
In the course of our ongoing investigations toward the isolation of bioactive metabolites from marine organisms of the Greek seas [4,5], we recently studied the chemical composition of the red alga S. coronopifolius, collected from the west coast of Corfu Island. In this report we describe the isolation and structure elucidation of two new diterpenes 1, 2 with neodolabellane carbon skeletons, and one new sphaeroane diterpene 3, along with the already described metabolites presphaerol (4) [6][7][8] and isosphaerodiene-1 (5) [8,9] (Figure 1). The structures of the new metabolites were elucidated by extensive spectroscopic analyses and their relative stereochemistry was established by NOESY experiments. Moreover, detailed analyses of the 1D-and 2D-NMR spectra allowed full assignment of the 13 C-and 1 H-data which had not been previously reported for 4 and 5.

Results and Discussion
S. coronopifolius was collected in Palaiokastritsa Bay on the west side of Corfu Island and the CH 2 Cl 2 /MeOH extract of the freeze-dried alga was subjected to a series of column chromatography fractionations on silica gel, as well as normal, reversed and chiral phase high performance liquid chromatography (HPLC) separations to yield compounds 1 -5 in pure form.  1 H-1 H TOCSY, HSQC, and HMBC data allowed the remaining two degrees of unsaturation to be assigned to a neodolabellane carbon skeleton. The H 3 -16 signal at δ H 1.24 (s) showed HMBC correlations to C-10 (δ C 38.2), C-11 (δ C 73.4), and C-12 (δ C 137.3), which placed the methyl on the quaternary oxygenated carbon adjacent to the 1,2-disubstituted double bond. Cross peaks in the COSY spectrum revealed couplings between H-10α (δ H 1.66) and H-9b (δ H 1.42), H-10β (δ H 1.48) and H-9a (δ H 1.89), and from both H 2 -9 to H-8 (δ H 5.41). Furthermore, the H-8 methine signal showed HMBC correlations to C-9 (δ C 28.3), C-10 (δ C 38.2), C-7 (δ C 149.8), C-17 (δ C 107.0), and to the acetate signal at δ C 170.8, confirming the position of methylene carbons C-9 and C-10 between the acetoxy-bearing carbon C-8 and the oxygenated carbon C-11. The correlations from H-5β (δ H 1.28) and both H 2 -17 (δ H 4.74 and 4.65) to C-6 (δ C 28.4) required the presence of the exomethylene at C-7. The H 3 -15 signal at δ H 0.77 (s) correlated with the C-3 (δ C 55.4), C-4 (δ C 47.1), C-5 (δ C 37.0), and C-14 (δ C 59.4) signals, allowing it to be placed at the C-4 bridge-head position. Homonuclear coupling between H-13 (δ H 5.66) and H-14 (δ H 2.18), and HMBC correlations of both H-1β (δ H 1.47) and H-12 (δ H 5.52) to C-14 (δ C 59.4) completed the 11-membered ring by positioning the disubstituted double bond next to the ring junction. The correlations from both methyl signs H 3 -19 and H 3 -20 at δ H 0.83 (d) and 0.95 (d), respectively, to C-3 (δ C 55.4), C-18 (δ C 30.3) clearly positioned the isopropyl group at C-3. The signal of H-1α at δ H 1.36 (m), belonging to one of the remaining intercoupling methylene groups, showed HMBC correlation with carbon C-13 (δ C 127.5). Thus, C-1 had to be connected to C-14, and the remaining C-2 methylene had to be connected to C-3, forming a cyclopentane ring.  The relative stereochemistry shown for 1 ( Figure 2) was assigned by interpretation of 1 H-NMR coupling constants and NOESY data. The E-geometry of the 12,13-olefin was assigned on the basis of a 15.3 Hz coupling constant. The NOE correlations observed between H-1β and H-12 with H-14, as well as the correlations between H-1α, H-13 and H 3 -15, required a trans ring fusion and indicated that protons H-1β, H-12 and H-14 were on the same (upper) side of the compound, while H-1α, H-13 and H-15 were on the opposite. The stereochemistry at C-3 was deduced by the NOE correlation between proton H-3 and methyl protons H 3 -15. The H-8 signal showed a correlation to the H-10β signal, which in turn showed a NOE to the H-12 signal, thereby establishing the stereochemistry at C-8. The correlation between H-12 and H 3 -16 indicated that methyl H 3 -16 is also on the upper side of the ring, resulting in the (3R*,4S*,8S*,11R*,12E,14R*)-geometry proposed for sphaerollane I (1). Compound 2 proved to have the same molecular formula as 1, C 22 H 36 O 3 , which was derived from HRMS and NMR data (Table 1), and is an isomer of 1 that differs only in the location of one of the double bonds. The structural type and substitution pattern of 2 was elucidated by means of 1D-and 2D-NMR correlated spectroscopy including HSQC, HMBC and 1 H-1 H COSY. Sphaerollane II (2) contained a Δ 6 trisubstituted double bond instead of the Δ 7,17 olefinic methylene in 1. In the 1 H-NMR spectrum (Table 1), both H 2 -5 signals at δ H 2.52 (dd) and 2.28 (ddq) were coupled to the H-6 olefinic signal at δ H 5.25 (ddq) that along with methylene proton H-5α (δ H 2.28) showed long-range couplings to the H 3 -17 methyl sign at δ H 1.61 (t). The Z geometry of Δ 6 olefinic bond was assigned on the basis of NOE correlations between H-6 and H 3 -17, and between H-5β and H-8. Further analysis of the NOESY spectrum revealed that the relative stereochemistry at C-3, C-4, C-8, C-11 and C-14 was the same as in 1 (Table 1, Figure 3). (3R*,4S*,8R*,9S*,13R*,14R*)-16-Hydroxy-9-acetoxy-8-epi-isosphaerodiene-2 (3) was isolated as a colorless oil. The high-resolution mass measurement established the molecular formula as C 22 H 34 O 3 , requiring six unsaturation equivalents. The IR spectrum showed bands that were assigned to ester (v max 1,733, 1,240, 1,024 cm -1 ) and hydroxyl (v max 3,367 cm -1 ) functionalities, respectively These assignments were  (Table 2), being for one carbon-oxygen and two carbon-carbon double bonds, indicated compound 3 to be tricyclic. The 1 H-and 13 C-NMR spectra (  1 t), and a primary alcohol (δ H 3.99 br. s, 2H; δ C 66.8 t). Extensive analyses of the 2D-NMR data of 3 including COSY, HSQC and HMBC spectra led to the unambiguous assignment of all protons and carbons on the sphaeroane skeleton. The H 2 -16 oxygenated methylene protons at δ H 3.99 (br. s) showed heteronuclear long range couplings to C-10 (δ C 32.7), C-11 (δ C 133.5), and C-12 (δ C 127.4), which confirmed the presence of the primary allylic alcohol at C-11. Correlations from H-8 methine (δ H 2.74) and both H 2 -10 (δ H 2.59 and 2.04) to C-9 (δ C 68.7), as well as from H-9 (δ H 5.35) to the carbonyl signal at δ C 170.8 required the acetyl group to be positioned at C-9. The olefinic proton H-12 (δ H 5.68) showed HMBC correlations to C-8 (δ C 51.3) and C-13 (δ C 41.6), thus positioning both the double bond and the acetate group next to the 6-membered ring junctions. The H 2 -17 olefinic methylene protons at δ H 4.79 (br. s) correlated with C-6 (δ C 30.8), C-7 (δ C 149.4), and C-8 (δ C 51.3), had to be placed on C-7. Long range heteronuclear couplings from H 2 -6 (δ H 2.23 and 2.22) to both C-4 (δ C 46.4) and C-5 (δ C 38.9), and from H 2 -5 (δ H 1.87 and 1.48) to C-7 (δ C 149.4) were observed in the HMBC spectrum. The H 3 -15 methyl signal at δ H 0.76 showed correlations to C-3 (δ C 58.0), C-4 (δ C 46.4), C-5 (δ C 38.9), and C-14 (δ C 53.7), and was placed on the quaternary carbon C-4. The cycloheptane ring was completed by the HMBC correlation observed between H-8 (δ H 2.74) and C-14 (δ C 53.7). The correlation of both H 3 -19 and H 3 -20 (δ H 0.82 and 0.92) with C-3 (δ C 58.0) and C-18 (δ C 29.8), required the presence of the isopropyl group at C-3. The correlations observed between H-1α (δ H 1.25) and C-13 (δ C 41.6), and between H-2b (δ H 1.28) and C-18 (δ C 29.8), confirmed the place of methylene carbons C-1 and C-2 in the 5-membered ring.  Stereochemical assignment of the chiral centers was facilitated by interpretation of 1 H-NMR and NOESY spectra. NOE correlations between H 3 -15 and H-13 and H-1α required trans fusion between the 7-membered and the cyclopentane ring. Furthermore, the correlation between H 3 -15 and H-3 positioned both H-3 and H-13 co-facial to methyl H 3 -15. The strong NOEs observed between the pairs of protons H-9, H-14 and H-8, H-13 suggested the unusual for a sphaeroane skeleton cis-fusion of the 7-membered and the cyclohexene rings. Both C-8 and C-13 were assigned as R*, based on the NOE correlations of H 3 -15 with H 2 -17, of H 2 -17 with H-8, of the latter with H-10α, and of H 3 -15 with H-13, H-5α and H-6α. Moreover, the NOEs observed between H-14 and H-9, and between H-9 and H-5β, H-6β and H-10β, secured the relative stereochemistry at chiral centers C-9 and C-14, thereby defining the structure as (3R*,4S*,8R*,9S*,13R*,14R*)-16-hydroxy-9-acetoxy-8-epi-isosphaerodiene-2 (3) (Figure 4). Along with the above described metabolites two previously reported compounds, presphaerol (4) [6][7][8] and isosphaerodiene-1 (5) [8,9], were isolated and spectroscopically characterized. Complete 1 Hand 13 C-NMR assignments, based on extensive analyses of their 2D-NMR spectra, are now provided for the first time for 4 and 5. The co-occurrence of compounds 1, 2 and 3 with the previously described 4 and 5 in the same organism indicated the possibility that they all derive from a common precursor neodolabellane cation [10], as illustrated in the proposed biogenetic pathway (Scheme 1). Thus, successive nucleophilic substitution of a H 2 O molecule, oxidation of the trisubstituted olefinic bond [11], and acetylation can yield the neodolabellanes 1 and 2. Alternatively, nucleophilic attack of a H 2 O molecule on the trisubstituted Δ 7 double bond can produce presphaerol (4) [10], which through water elimination generates isosphaerodiene-1 (5). Metabolite 3 can be derived from 5 by oxidation of Δ 7 double bond and successive eliminations, additions of H 2 O molecules, and a final acetylation.
Diterpenes of the neodolabellane class have previously been isolated only from soft corals of the genera Cespitularia, Clavularia, and Lobophytum of the Indian and Pacific Ocean [12][13][14][15][16], while diterpenes of the sphaeroane skeleton have been isolated, only from the red alga S. coronopifolius and the macromycete Mycena tintinnabulum [17].

Scheme 1. Proposed biogenetic origin of 1-5.
algorithm: Polak-Ribiere). High-Resolution mass spectral data were provided by the University of Notre Dame, Department of Chemistry and Biochemistry, Indiana, USA. Low-Resolution Electron Impact or Chemical Ionisation MS data were recorded on a Thermo DSQ Mass Detector using Direct Exposure Probe (DEP) and methane as the CI gas. Vacuum Liquid Chromatography (VLC) separation was performed with Kieselgel 60 (Merck), gravity column chromatography (GCC) was performed with Kieselgel 60H (Merck), thin layer chromatography (TLC) was performed with Kieselgel 60 F 254 aluminum support plates (Merck) and spots were detected after spraying with 15% H 2 SO 4 in MeOH and charring. HPLC separations were conducted using an Agilent 1100 system equipped with refractive index detector and a SupelcoSil 5μ (250×10 mm) HPLC normal phase column or a Kromasil 100 C 18 5μ (250×8 mm) HPLC reversed phase column or a Chiralcel OD 5μ (250×4.6 mm) analytical chiral column.