Two New Oxodolastane Diterpenes from the Jamaican Macroalga Canistrocarpus cervicornis

The chemical investigation of the organic extract of Canistrocarpus cervicornis, collected at Drunken Man’s Cay at Port Royal, Jamaica, has led to the isolation of two new dolastane diterpenes 4R-acetoxy-8S,9S-epoxy-14S-hydroxy-7-oxodolastane (1) and 4R-hydroxy-8S,9S-epoxy-14S-hydroxy-7-oxodolastane (2) and the previously isolated dolastane (4R,9S,14S)-4,9,14-trihydroxydolast-1(15),7-diene (3) as a major diterpene constituent. The structures of the new compounds were elucidated by extensive spectroscopic analyses. Compounds 1–3 were evaluated for their cytotoxicity against human tumor cell lines PC3 and HT29. The results revealed that the dolastane diterpenes (1–3) displayed moderate, concentration dependent, cytotoxicity.


Introduction
Plant-derived drugs remain an important resource, especially in developing countries, and marine organisms provide a rich source of nutraceuticals and are potential candidates for the treatment of several human diseases [1].Cancer is a leading cause of deaths worldwide, accounting for 8.8 million deaths in 2015 [2].The most common causes of cancer deaths are cancers of the lungs, liver, colon, stomach, and breast.
Over the years, brown algae have caught the attention of many scientists in search of diverse and novel chemical structures.Marine algae of the order Dictyotales, (Phaeophyta) have been found to be distributed in tropical and sub-tropical waters.Chemical investigations have shown the order to be a rich source of monocyclic, bicyclic and tricyclic terpenes such as dolabellane, meroditerpenoid, dolastane and seco-dolastane skeleton types [3][4][5][6][7][8][9][10][11][12][13][14].Quite a number of diterpenes belonging to the Dictyotaceae family, inhabitants of the Atlantic and Indian Oceans, belong to the dolastane and seco-dolastane types.The species Canistrocarpus cervicornis (formerly classified as Dictyota cervicornis) has been shown to grow luxuriantly in Caribbean waters and are commonly found [3].
The chemical investigation of this macroalga has led to the isolation of a variety of dolastanes and seco-dolastanes with marked biological/ecological activity [4][5][6][7]9].They display a wide variety of pharmacological activities including antifouling, antifeedant, antibacterial, antiviral, and cytotoxic properties [4][5][6][7]9].In the course of our ongoing efforts to discover new biologically active secondary metabolites from brown algae, Canistrocarpus cervicornis, collected from off the coast of Port Royal, Drunken Man's Cay, Jamaica, was investigated.We report herein that the CH 2 Cl 2 extract of the dried brown alga resulted in the isolation and structure elucidation of two new oxodolastanes (1 and 2) and one known dolastane (3).The structure of the known dolastane (3) was identified by comparison of its physical and spectroscopic data with those reported in the literature (Figure 1).In addition, the cytotoxicity of the isolated compounds against human tumor cell lines PC3 and HT29 is also described.cytotoxicity of the isolated compounds against human tumor cell lines PC3 and HT29 is also described.

Results and Discussion
A series of chromatographic separations of the CH2Cl2 extracts of the brown alga Canistrocarpus cervicornis, collected at Drunken Man's Cay in Port Royal, Jamaica, resulted in the isolation of the new dolastanes 1 and 2 (Figure 1) and one previously reported dolastane, which was identified as (4R,9S,14S)-4,9,14-trihydroxydolast-1(15),7-diene (3) by comparison of its spectroscopic and physical characteristics with those reported in the literature [8].

Structural Elucidation
Compound 1 was isolated as a colorless gum.HRESIMS in positive ion mode showed a [M + Na] + peak at m/z 399.2029 implying the molecular formula of C22H32O5 (seven degrees of unsaturation) for compound 1.One olefinic carbon-carbon double bond and the two carbonyl groups accounted for three of the seven degrees of unsaturation, therefore the molecular structure of 1 was determined to be tetracyclic.The 13 C-NMR spectral data revealed the presence of 22 carbon resonances, which were assigned by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectra to eight non-protonated carbons, seven methylenes, five methyls, and two methines.A detailed analysis of 1D and 2D NMR spectra was necessary to determine the gross structure of 1.The evidence of an acetate residue could be easily deduced from the ester carbonyl signals at δC 169.0 along with the methyl group at δH 2.15.Therefore, the diterpene structure of 1 was inferred from the remaining 20 carbons.NMR signals also revealed the presence of an oxygenated methine at δC 81.7 which was attached to the proton signal at δH 4.81 (1H, br s) which was assigned to an acetoxy-bearing methine.
An indication of the existence of an exocyclic double bond was ascertained from the methylene carbon NMR signals observed at δC 110.7 with olefinic methylene protons at δH 5.01 (1H, br s) and 4.98 (1H, br s), along with an olefinic quaternary carbon at δC 149.6.Furthermore, the existence of a ketone functionality was inferred from the carbon signal at δC 207.7.The quaternary signals at δC 82.0, 78.2, and 72.0 were characteristic of oxygen-bearing quaternary carbons, two of which were deduced to be oxirane carbon atoms at 82.0 ppm (C-9) and 72.0 ppm (C-8).

Results and Discussion
A series of chromatographic separations of the CH 2 Cl 2 extracts of the brown alga Canistrocarpus cervicornis, collected at Drunken Man's Cay in Port Royal, Jamaica, resulted in the isolation of the new dolastanes 1 and 2 (Figure 1) and one previously reported dolastane, which was identified as (4R,9S,14S)-4,9,14-trihydroxydolast-1(15),7-diene (3) by comparison of its spectroscopic and physical characteristics with those reported in the literature [8].

Structural Elucidation
Compound 1 was isolated as a colorless gum.HRESIMS in positive ion mode showed a [M + Na] + peak at m/z 399.2029 implying the molecular formula of C 22 H 32 O 5 (seven degrees of unsaturation) for compound 1.One olefinic carbon-carbon double bond and the two carbonyl groups accounted for three of the seven degrees of unsaturation, therefore the molecular structure of 1 was determined to be tetracyclic.The 13 C-NMR spectral data revealed the presence of 22 carbon resonances, which were assigned by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectra to eight non-protonated carbons, seven methylenes, five methyls, and two methines.A detailed analysis of 1D and 2D NMR spectra was necessary to determine the gross structure of 1.The evidence of an acetate residue could be easily deduced from the ester carbonyl signals at δ C 169.0 along with the methyl group at δ H 2.15.Therefore, the diterpene structure of 1 was inferred from the remaining 20 carbons.NMR signals also revealed the presence of an oxygenated methine at δ C 81.7 which was attached to the proton signal at δ H 4.81 (1H, br s) which was assigned to an acetoxy-bearing methine.
An indication of the existence of an exocyclic double bond was ascertained from the methylene carbon NMR signals observed at δ C 110.7 with olefinic methylene protons at δ H 5.01 (1H, br s) and 4.98 (1H, br s), along with an olefinic quaternary carbon at δ C 149.6.Furthermore, the existence of a ketone functionality was inferred from the carbon signal at δ C 207.7.The quaternary signals at δ C 82.0, 78.2, and 72.0 were characteristic of oxygen-bearing quaternary carbons, two of which were deduced to be oxirane carbon atoms at 82.0 ppm (C-9) and 72.0 ppm (C-8).
Analysis of the presented NMR data revealed a close alignment to the tricyclic dolastane skeleton, which appears to be a skeletal framework characteristic to this alga species [1][2][3][4][5][6][7][8][9][10][11].However, differences in the chemical shifts because of the oxygenation pattern suggested that compound 1 was a new natural product. 1 H-1 H correlation spectroscopy (COSY) experiments established the fragments outlined in Figure 2 after assignments of the direct 1 H- 13 C correlations via HSQC analysis. 1H-  2).2).Further structural information for compound 1 was obtained by 2D analysis; heteronuclear multiple bond correlation (HMBC) spectra (Figure 2).The location of the acetate group at C-4 was established through the HMBC correlations between δ H 4.81 proton (H-4) and the carbonyl signal at δ C 169.0.HMBC correlations of C-1, C-14, and C-15 to H-2, located the exocyclic double bond as a part of the six-membered ring and placed the hydroxyl group at C-14.Also, HMBC correlations of C-5, Mar.Drugs 2017, 15, 150 4 of 8 C-7, C-14, to H-6 and C-8, C-12, C-14, with H-13 established the position of the ketone functionality and allowed for the assignment of the seven-membered ring.Similarly, the epoxy C-atom, C-9, was HMBC correlated to H-17, H-18, and H-19 confirming the ether bridge and the isopropyl fragment on the five-membered ring.Based on the above evidence, the planar structure of dolastane 1 could be established as 4-acetoxy-8,9-epoxy-14-hydroxy-7-oxodolastane.
The relative configuration of compound 1 was established by extensive analysis of nuclear Overhauser effect spectroscopy (NOESY) spectrum.The assignment of the stereocenter was based on careful analysis of the NOESY spectrum as well as comparison with the reference [8].Additionally, OH-Me 1 H NMR shifts effect helped with resolving the relative configuration.It is of note that, when an H or Me group is coplanar to a nearby OH group, it is deshielded.The usual mean position of a methyl substituent in a 1,3 relationship with a hydroxyl group is 0.72 ppm.Consequently, the 14S-hydroxy stereocenter was deduced from the 1 H-NMR signal of Me-20 at 1.37 ppm, deshielded by 0.65 ppm from the mean, thus co-facial with H 3 -20 [15,16].In addition, strong NOE interaction between H-4 and H 3 -16 provided evidence that they were co-facial (Figure 3).This inferred trans-fusion of the six and seven membered ring.The absence of nuclear Overhauser effect (NOE) between H 3 -16 and H 3 -18 suggested that they were on opposite sides, while the NOE enhancement of H 3 -20 ad H 3 -18 (Figure 3) provided evidence for the trans-fusion of the seven and five membered rings and determining the relative configuration of the chiral centers C-4, C-5, C-8, C-9, C-12, C-14, as 4R*,5R*,8S*,9S*,12R*, and 14S*.
Further structural information for compound 1 was obtained by 2D analysis; heteronuclear multiple bond correlation (HMBC) spectra (Figure 2).The location of the acetate group at C-4 was established through the HMBC correlations between δH 4.81 proton (H-4) and the carbonyl signal at δC 169.0.HMBC correlations of C-1, C-14, and C-15 to H-2, located the exocyclic double bond as a part of the six-membered ring and placed the hydroxyl group at C-14.Also, HMBC correlations of C-5, C-7, C-14, to H-6 and C-8, C-12, C-14, with H-13 established the position of the ketone functionality and allowed for the assignment of the seven-membered ring.Similarly, the epoxy C-atom, C-9, was HMBC correlated to H-17, H-18, and H-19 confirming the ether bridge and the isopropyl fragment on the five-membered ring.Based on the above evidence, the planar structure of dolastane 1 could be established as 4-acetoxy-8,9-epoxy-14-hydroxy-7-oxodolastane.
The relative configuration of compound 1 was established by extensive analysis of nuclear Overhauser effect spectroscopy (NOESY) spectrum.The assignment of the stereocenter was based on careful analysis of the NOESY spectrum as well as comparison with the reference [8].Additionally, OH-Me 1 H NMR shifts effect helped with resolving the relative configuration.It is of note that, when an H or Me group is coplanar to a nearby OH group, it is deshielded.The usual mean position of a methyl substituent in a 1,3 relationship with a hydroxyl group is 0.72 ppm.Consequently, the 14Shydroxy stereocenter was deduced from the 1 H-NMR signal of Me-20 at 1.37 ppm, deshielded by 0.65 ppm from the mean, thus co-facial with H3-20 [15,16].In addition, strong NOE interaction between H-4 and H3-16 provided evidence that they were co-facial (Figure 3).This inferred trans-fusion of the six and seven membered ring.The absence of nuclear Overhauser effect (NOE) between H3-16 and H3-18 suggested that they were on opposite sides, while the NOE enhancement of H3-20 ad H3-18 (Figure 3) provided evidence for the trans-fusion of the seven and five membered rings and determining the relative configuration of the chiral centers C-4, C-5, C-8, C-9, C-12, C-14, as 4R*,5R*,8S*,9S*,12R*, and14S*.Compound 2, obtained as a white solid, gave the molecular formula C20H30O4, as calculated from the [M + Na] + pseudo molecular ion at m/z 357.1923 and NMR data.The spectroscopic characteristics of 2 were comparable to those of compound 1.Thus, the gross structure of 2 was determined by comparison of the spectroscopic data with that of compound 1.By contrast, the differences between 1 and 2 lie in the NMR signal at δH 3.53 connected to δC 78.7, assigned as a hydroxy methine at C-4 due to its upfield shift with respect to δH 4.81 connected to carbon at δC 81.7, in compound 1 and the absence of the chemical shifts that correspond to the acetate group in compound 1.Furthermore, HMBC correlations established the hydroxyl group at C-4 methine as H-4 correlated with C-1, C-2, C-5, C-14, and C-16 in the HMBC spectrum.Based on the above evidence, the planar structure of dolastane 2 could be established as 4-hydroxy-8,9-epoxy-14-hydroxy-7-oxodolastane.
It should be noted that the occurrence of the epoxide in the dolastanes remains uncommon with only one other such isolate being identified.Spectroscopic data and X-ray diffraction analysis provided evidence for this isolate to be identified as 10β-acetoxy-8α,9α-epoxy-14β-hydroxy-7oxodolastane [1].Compound 2, obtained as a white solid, gave the molecular formula C 20 H 30 O 4 , as calculated from the [M + Na] + pseudo molecular ion at m/z 357.1923 and NMR data.The spectroscopic characteristics of 2 were comparable to those of compound 1.Thus, the gross structure of 2 was determined by comparison of the spectroscopic data with that of compound 1.By contrast, the differences between 1 and 2 lie in the NMR signal at δ H 3.53 connected to δ C 78.7, assigned as a hydroxy methine at C-4 due to its upfield shift with respect to δ H 4.81 connected to carbon at δ C 81.7, in compound 1 and the absence of the chemical shifts that correspond to the acetate group in compound 1.Furthermore, HMBC correlations established the hydroxyl group at C-4 methine as H-4 correlated with C-1, C-2, C-5, C-14, and C-16 in the HMBC spectrum.Based on the above evidence, the planar structure of dolastane 2 could be established as 4-hydroxy-8,9-epoxy-14-hydroxy-7-oxodolastane.
It should be noted that the occurrence of the epoxide in the dolastanes remains uncommon with only one other such isolate being identified.Spectroscopic data and X-ray diffraction analysis provided evidence for this isolate to be identified as 10β-acetoxy-8α,9α-epoxy-14β-hydroxy-7-oxodolastane [1].

Cytotoxicity Evaluation against PC3 and HT29 Cancer Cell Lines
Evaluation of the impact of compound 1 on the viability of the human prostate adenocarcinoma cell line-PC3-and the human colon adenocarcinoma cell line-HT29-revealed moderate, concentration-dependent cytotoxicity.The EC 50 value, the concentration at which cell viability is decreased by 50%, was estimated to be above 100 µM for both PC3 and HT29.Sixty-eight percent (68%) and 59% cell viability was observed respectively for these two cell lines at 133 µM of compound 1.Such patterns are comparable with the chosen positive control drug, bicalcutamide for PC3 cells which yielded an EC 50 value of 103.9 µM.Bicalcutamide is known to have no effect on PC3 at low doses, but significant cytotoxicity against PC3 at high doses [17][18][19][20].More potent activity was observed for positive control drugs, doxorubicin and tamoxifen, against HT29 with EC 50 values of 11.4 and 39.0 µM, respectively.Doxorubicin is known for a broad spectrum of antitumor activity [21].
EC 50 values for compound 2 against both these cell lines were estimated to be above 50 µM, with cells observed to be fully viable at the tested concentration of 47.8 µM.Unfortunately, limited supply restricted investigations at higher concentrations.Like compound 1, the EC 50 values for compound 3 were above 100 µM for both PC3 and HT29, indicating moderate cytotoxicity by this compound against these two cell lines.
These initial, yet promising indications of cytotoxicity against two human carcinoma cell lines indicate the need for a full evaluation against a larger panel of cell lines, particularly for the two novel compounds.

General Experimental Procedures
Optical rotations were measured on an Anton Paar model MCP 300 polarimeter (Ostfildern scharnhausen, Germany) with a 0.5 dm cell.Nuclear magnetic resonance spectra were recorded on a Bruker Avance DRX-500 MHz spectrometer (Bruker BioSpin GmbH, Silberstreifen, Germany), equipped with a 5-mm broadband inverse probe and a 5-mm dual probe, employing deuterochloroform (CDCl 3 ) as a solvent and referenced to tetramethysilane (TMS) as internal standard. 13C spectral editing were obtained by DEPT experiments.Chemical shifts (δ) were expressed in ppm and coupling constants in Hz.HR-ESI-MS spectra were taken on Bruker micro TOF focus II mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany).Separation and purification were performed by column chromatography on silica gel 60 Å (230-400 mesh, SiliCycle Inc., Quebec City, QC, Canada).Compound detection in the thin-layer chromatography (TLC) plate was achieved by molybdenum spray detection.All the solvents were analytical grade.

Algal Sample Collection
Specimens of C. cervicornis (Dictyotaceae, Ochrophyta) were collected at Drunken Man's Cay, Port Royal, Kingston, Jamaica in November 2015 at a depth of 15-20 m.Immediately after collection, the algal material was cleaned and air-dried for two days.

Extraction, Isolation, and Structural Elucidation of Compounds
The air-dried algal material (82.50 g) was extracted exhaustively at room temperature successively with hexane, methylene chloride, ethyl acetate, and methanol for three days each.After removal of the solvent, the methylene chloride extract yield was 2 g of a dark green gum residue.The methylene chloride extract was subjected to flash silica gel column chromatography (CC) (6 × 30 cm), eluting with 100% CH 2 Cl 2 , increasing portions of EtOAc in CH 2 Cl 2 followed by increasing portions of methanol in EtOAc to produce 12 main fractions, which were monitored by thin layer chromatography (TLC).
Figure 2. Key COSY and HMBC correlations observed for 1.