Sarcoconvolutums F and G: Polyoxygenated Cembrane-Type Diterpenoids from Sarcophyton convolutum, a Red Sea Soft Coral

Natural products and chemical analogues are widely used in drug discovery, notably in cancer and infectious disease pharmacotherapy. Sarcophyton convolutum (Alcyoniidae) a Red Sea–derived soft coral has been shown to be a rich source of macrocyclic diterpenes and cyclized derivatives. Two previously undescribed polyoxygenated cembrane-type diterpenoids, sarcoconvolutums F (1) and G (2), as well as four identified analogues (3–6) together with a furan derivate (7) were isolated from a solvent extract. Compounds were identified by spectroscopic techniques, including NMR, HREIMS, and CD, together with close spectral comparisons of previously published data. Sarcoconvolutum F (1) contains a rare 1-peroxid-15-hydroxy-10-ene functionality. Isolated metabolites (1–7) were screened against lung adenocarcinoma (A549), cervical cancer (HeLa) and oral cavity carcinoma (HSC-2) lines. Compound 4 exhibited an IC50 56 µM and 55 µM against A549 and HSC-2 cells, respectively.


Introduction
Drug development relies heavily on natural products and structural analogues, particularly for cancer and infectious disease pharmacotherapy's [1]. While terrestrial plants and soil microorganisms are traditionally a rich source of biologically-active natural products, marine organisms also produce a broad assortment of chemical defenses with significant chemical diversity and activity [2]. The Red Sea with both tropical and subtropical ecosystems supports robust and diverse marine life including rich native biota. Indeed, while over 180 soft coral species have been discovered around the world, a significant percentage reside and are native to the Red Sea [3]. Soft coral are traditionally catagorized based on a sclerite classification system; these sclerites are small aggregates of calcium carbonate embedded in the soft coral tissue that provide colony support. Having eight tentacles, they are classified in the class Octocorallia. Alcyonacean, an order of Octocorallia are massive sessile invertebrates with an unique stalk and a mushroom-shaped capitulum [4,5]. The genus Sarcophyton, in the order Alcyonacea includes 35 species, six of which were recently recognized as new [6][7][8][9][10][11]. The chemistry and pharmacology of Sarcophyton metabolites have been investigated extensively. Sesquiterpenes, diterpenes, steroids and fatty acids have been proposed to be a major source of pharmacological activity [12][13][14]. Indeed, macrocyclic cembranes and their derivatives, have been confirmed to be potent anticancer, antibacterial, anti-inflammatory, anti-osteoporotic, antimetastatic, antiangiogenic, and neuroprotective natural bioactive agents [15]. Diterpenoids from the cembrane family serve in reef chemical defense against predators or competitors as they are often highly cytotoxic [13,15,16]. The genus includes 39 biscembranoids, 323 diterpenes, 11 sesquiterpenes, 53 polyoxygenated sterols, and 55 miscellaneous metabolites, many of which have documented pharmacological efficacies [17].
Cembrane-type diterpenoid biosynthesis utilizes a geranylgeraniol precursor that is cyclized between carbons 1 and 14, generating a 14-membered cembrane or thumbergane skeleton [18]. An E double bond geometry from the geranylgeraniol substrate generates a (+)-cembrene, that was first isolated from pine oleoresin [19]. Kobayashi and colleagues later isolated the cembranoid diterpene, sarcophytol A, a potent antitumor agent from Okinawan soft coral S. glaucum [20]. Subsequently, hundreds of cembranoids have been isolated and identified from insects, and marine organisms alike [15]. Cembranoids generally include a cyclic ether and a lactone, or furane moiety usually present between C-1, C-2, C-15 and C-16 (type I) or C-2, C-3, C-4 and C-18 (type II). At C-17, the α-position of an α,β-unsaturated butenolactone moiety, is frequently substituted with a Me group or an exo-CH 2 = group in type-I cembranoids. A CH 2 OH group can exist as a linkage intermediate between a Me and a terminal C=C bond. Several cembranoids have been reported as type II. Sarcophyocrassolide A, isolated from a CHCl 3 extract of S. crassocaule, was the first cembranolide skeleton bearing an 8-hydroperoxy group in the α-methylidene-γ-lactone skeleton [21].
In this study, the soft coral S. convolutum, native to and collected from the Red Sea was solvent extracted and chemically characterized. A series of cembrene diterpenoids were identified ( Figure 1) and metabolites were assayed for cytotoxicity against a series of tumor cell lines. tentacles, they are classified in the class Octocorallia. Alcyonacean, an order of Octocorallia are massive sessile invertebrates with an unique stalk and a mushroom-shaped capitulum [4,5]. The genus Sarcophyton, in the order Alcyonacea includes 35 species, six of which were recently recognized as new [6][7][8][9][10][11]. The chemistry and pharmacology of Sarcophyton metabolites have been investigated extensively. Sesquiterpenes, diterpenes, steroids and fatty acids have been proposed to be a major source of pharmacological activity [12][13][14]. Indeed, macrocyclic cembranes and their derivatives, have been confirmed to be potent anticancer, antibacterial, anti-inflammatory, anti-osteoporotic, antimetastatic, antiangiogenic, and neuroprotective natural bioactive agents [15]. Diterpenoids from the cembrane family serve in reef chemical defense against predators or competitors as they are often highly cytotoxic [13,15,16]. The genus includes 39 biscembranoids, 323 diterpenes, 11 sesquiterpenes, 53 polyoxygenated sterols, and 55 miscellaneous metabolites, many of which have documented pharmacological efficacies [17].
Cembrane-type diterpenoid biosynthesis utilizes a geranylgeraniol precursor that is cyclized between carbons 1 and 14, generating a 14-membered cembrane or thumbergane skeleton [18]. An E double bond geometry from the geranylgeraniol substrate generates a (+)-cembrene, that was first isolated from pine oleoresin [19]. Kobayashi and colleagues later isolated the cembranoid diterpene, sarcophytol A, a potent antitumor agent from Okinawan soft coral S. glaucum [20]. Subsequently, hundreds of cembranoids have been isolated and identified from insects, and marine organisms alike [15]. Cembranoids generally include a cyclic ether and a lactone, or furane moiety usually present between C-1, C-2, C-15 and C-16 (type I) or C-2, C-3, C-4 and C-18 (type II). At C-17, the α-position of an α,β-unsaturated butenolactone moiety, is frequently substituted with a Me group or an exo-CH2= group in type-I cembranoids. A CH2OH group can exist as a linkage intermediate between a Me and a terminal C=C bond. Several cembranoids have been reported as type II. Sarcophyocrassolide A, isolated from a CHCl3 extract of S. crassocaule, was the first cembranolide skeleton bearing an 8-hydroperoxy group in the α-methylidene-γ-lactone skeleton [21].
In this study, the soft coral S. convolutum, native to and collected from the Red Sea was solvent extracted and chemically characterized. A series of cembrene diterpenoids were identified ( Figure 1) and metabolites were assayed for cytotoxicity against a series of tumor cell lines.
Compound 1 was generated as a colorless oil with a positive optical rotation in methanol [α] 25 D + 41.0 in MeOH. Based on HREIMS data that exhibited an [M] + ion at m/z 400.2094 (calcd m/z 400.2098), the chemical formula was estimated to be C 20 H 32 O 8 , reflecting a five degrees of unsaturation. Two double bonds and one carbonyl from NMR data are responsible for three of the five elements of unsaturation, giving rise in a bicyclic molecule. An absorption for a hydroxyl (υ max 3450 cm −1 ), a carbonyl (υ max 1750 cm −1 ) and an olefin (υ max 1669 cm −1 ) were confirmed by IR analysis. The 1 H NMR spectrum (Table 1) (Table 1), which were further differentiated by DEPT to four methyls (three oxygenated at δ C 22.7, 22.9, 27.3 and one olefinic at δ C 15.9), five methylenes (19.0, 24.9, 26.7, 36.1, and 43.3), five methines (two oxygenated at δ C 69.7 and 80.9, three olefinic at δ C 117.5, 126.6 and 136.3), and six quaternary carbons (four oxygenated at δ C 73.9, 77.5, 79.9 and 86.9, one olefinic at δ C 143.5 and one keto at δ C 175.2). The cembrene-based diterpenoid was generated using the 1D NMR analysis described above of 1 [14,23,[27][28][29][30]. Combining spectral data of compound 1 indicated to a cembranoid diterpene molecular framework containing a rare 1-peroxid-15-hydroxy-10-ene with a close similarity to sarcoroseolide B previously isolated from S. roseum [31], with the exception that 1 was missing the oxygen bridge which was found in sarcoroseolide B expected to be replaced by the peroxy group at C-1. Moreover, compound 1 possessed an 34 amu in its molecular weight increases than sarcoroseolide B, which predict that 1 had a peroxy group. Besides, functionalities with a molecular formula of sarcoroseolide B suggesting a tricyclic structure compared with 1 validated by HREIMS to be a bicyclic frame skelton. Spectral study of 1 H-1 H COSY and HMBC validated the predicted structure. The signal at δ H 5.04 (d, J = 10.4 Hz, 1H) correlated with a proton signal at δ H 5.22 (d, J = 10.4 Hz, 1H) and quaternary olephnic carbon at δ C 143.5 (Figure 2), respectively, allowed for the assignments of H-2, H-3, C-4 [14,27,29]. The long-range HMBC correlations ( Figure 2) between H 3 -17 and carbon signals at δ C 86.9 (C-1), δ C 77.5 (C-15) and keto group at δ C 175.2 (C-16) indicated the missing double bond resulted in a saturated lactone ring and consequently led the oxygenated tertiary carbons (δ C 86.9 and 77.5) at C-1 and C-15 [32].  (Figure 4), [14,23]. From the above spectral data, 1 was established as sarcoconvolutum F. sarcoroseolide B previously isolated from S. roseum [31], with the exception th missing the oxygen bridge which was found in sarcoroseolide B expected to be by the peroxy group at C-1. Moreover, compound 1 possessed an 34 amu in its m weight increases than sarcoroseolide B, which predict that 1 had a peroxy group. functionalities with a molecular formula of sarcoroseolide B suggesting a tricycl ture compared with 1 validated by HREIMS to be a bicyclic frame skelton. Spectr of 1 H-1 H COSY and HMBC validated the predicted structure. The signal at δH 5. 10.4 Hz, 1H) correlated with a proton signal at δH 5.22 (d, J = 10.4 Hz, 1H) and qu olephnic carbon at δC 143.5 (Figure 2), respectively, allowed for the assignments H-3, C-4 [14,27,29]. The long-range HMBC correlations (Figure 2) between H3-17 bon signals at δC 86.9 (C-1), δC 77.5 (C-15) and keto group at δC 175.2 (C-16) indic missing double bond resulted in a saturated lactone ring and consequently led genated tertiary carbons (δC 86.9 and 77.5) at C-1 and C-15 [32].  (Figure 4), [14,23]. From the above spectral data, 1 was est as sarcoconvolutum F.   (Table 1), which were further differentiated by DEPT to three methyls (one oxygenated at δ C 19.8 and two olephenic at δ C 8.8, 20.7), seven methylenes (one exomethelene at δ C 111.1 and six aliphatic at δ C 26.8, 27.2, 30.8, 32.9, 33.4 and 36.1), four methines (three oxygenated at δ C 74.6, 79.5 and 83.1, one olefinic at δ C 120.4), and six quaternary carbons (one oxygenated at δ C 85.1, one keto at δ C 174.8, four olefinic at δ C 125.0, 145.9,148.6 and 162.0). The presence of an ether linkage was suggested by a high downfield carbon signals at δ C 83.1 and 85.1, which were functionally validated by HREIMS [24]. Seven degrees of unsaturation were deduced, suggesting a tricyclic cembrane frame skeleton.  A combination of the intensity of the 1 H and 13 C NMR signals for 2, together w [M] + ion at m/z = 332.1991 (calcd. 332.2222) in the HREI mass spectrum, supported ical formula of C20H28O4 corresponding to seven double bond equivalents. Comp was produced as a colorless oil with a negative optical rotation of [α] − 3.4 in me The IR spectra revealed two distinct bands at 3450 cm −1 (OH) and 1750 cm −1 (CO). NMR spectrum displayed twenty carbon signals (Table 1) 16 (H-19) with oxygenated methine at δ C 74.6 (C-7), oxygenated quaternary carbon at δ C 85.1 (C-8) and aliphatic methylene at δ C 36.1 (C-9) and the data comparison with those of crassumol G [24], revealed that the sole difference between 2 and crassumol G was in the stereochemistry. On the basis of coupling constants and NOESY experiments, the relative configuration of 2 was modeled. A cis configuration between the γ-lactone (H-2) and the olefinic proton (H-3) was established using a vicinal coupling constant of 9.8 Hz between H-2 (δ H 5.47) and H-3 (δ H 5.09) as well as a NOESY correlation of α-orientation of H-2 with H 3 -18 (δ H 1.96).
The value of vicinal coupling constant which equally to 10.1 Hz between H-7 (δ H 3.22) and H-6 (δ H 1.58), as well as 13.7 Hz vicinal coupling between H-6 (δ H 1.99) and H-5 (δ H 2.08) with the aid of NOESY spectrum supported the orientation of H-7 in α-orientation and differentiate between C-6 protons chemical shifts together with the comparison with those of crassumol G [24].  (3); compound (2) indicated the same absolute configuration for 1 and reverse absolute configuration for sarcophine 3 at C-2 [14,23]. From the above spectral data, 2 was established as sarcoconvolutum G.
Despite the fact that many cembrane-type diterpenoids have been identified from soft corals in the genus Sarcophyton, the solvent extract of the soft coral S. convolutum resulted in the isolation of two novel cembrane diterpenoids (1-2), one of which, sarcoconvolutum F (1), has a rare 1-peroxid-15-hydroxy-10-ene cembrane skeleton. Putative biosynthetic pathways are postulated as depicted in Figure 5, to explain the biogenetic origin of the secondary metabolites (2-6), along with 1 which is outlined separately in Figure 6. An alternative biosynthetic route would be that the enolate is oxidized directly to form an epoxide, leading to an alpha-hydroxy ketone via an epoxide intermediate (not shown). Besides the previously described phytochemistry from the same species (Sarconvolutum A, B, D & E) [1], the metabolites may all be traced back to a common precursor, sarcophine (3) which is a typical cembrane diterpenoids discovered in many soft coral belonging to the Sarcophyton genus and accumulating from various locations [25]. Sarcophine is a common compound for this soft coral species and can be used as a chemotaxonomic tool. We propose a biogenetic pathway to 1 that begins with the nucleophilic attack of α,β-unsaturation-γlactone moeity to form tetra-substituted epoxide, followed by the production of a hemiketal intermediate to convert the double bond to 1-peroxy-2-hydroxy, a rare constituent of sarcoconvolutum F (1).

General Procedures
A JASCO P-2300 polarimeter was used to measureoptical rotation (Jasco Corpora tion, Tokyo, Japan). A Shimazi FTIR-8400S was used to measure IR spectra (Columbia

General Procedures
A JASCO P-2300 polarimeter was used to measureoptical rotation (Jasco Corporation, Tokyo, Japan). A Shimazi FTIR-8400S was used to measure IR spectra (Columbia, MD 21046, USA). A Bruker 600 or 500 Hz NMR spectrometer was used to record 1D and 2D NMR spectra (MA, USA). Chemical shifts were expressed in parts per million (ppm), while coupling constants were expressed in hertz (Hz). On a JEOL JMS-700 instrument, HR-MS spectra were collected (Tokyo, Japan). A JASCO 810 polarimeter was used to measure electronic circular dichroism (ECD). For column chromatography, Merck's Silica Gel 60 (230-400 mesh, Merck, Darmstadt, Germany) was employed. TLC examination was performed on precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm, Merck, Darmstadt, Germany). A Jasco PU-980 pump clever HPLC pump was used to perform high-performance liquid chromatography (HPLC).

Animal Material
In March 2017, the soft coral S. convolutum was collected from the Red Sea coast in Hurghada, Egypt. A voucher specimen (08RS1071) from the National Institute of Oceanography and Fisheries' marine biological station in Hurghada, Egypt, was the basis for coral identification (by M Al-Hammady).

Cell Culture and Treatment Conditions
Squamous cell carcinoma of the oral cavity (HSC-2), non-small cell lung adenocarcinoma (A549), and human cervical cancer cell (HeLa) (ATCC ® ) were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 4 mM l-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin sulphate Monolayers at 70-90 percent confluence were passed using a trypsin-EDTA solution. In a humidified CO 2 incubator with 5% CO 2 , cell were held at 37 • C. All materials and reagents for the cell cultures were purchased from Lonza (Verviers, Belgium).

Cytotoxicity Assay
A modified MTT (3-[4,5,[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) test based on a previously published technique [33] was used to assess forcytotoxicity. A96-well plate was seeded with appropriate cell densities of exponentially growing A549, HeLa, and HSC2 cells (5000-10000 cells/well). Stock test compounds (1-5) dissolved in dimethyl sulfoxide (DMSO) were screened at concentrations of 100, 10 and 1 µM after a 24 h incubation period at 37 • C with 5 percent CO 2 , followed by varying concentrations for the generation of concentration-dependent curves of the most cytotoxic compounds with culture medium (final DMSO concentration in medium = 0.1 percent, by volume). MTT solution in PBS (5 mg/mL) was added to each well after 48 h of incubation, and the incubation was continued for another 90 min. A phase contrast microscopic analysis verified the production of intracellular formazan crystals (mitochondrial reduction product of MTT). The medium was withdrawn at the conclusion of the incubation time, and 100 µL of DMSO were added to each well with shacking for 10 min to dissolve the produced formazan crystals (200 rpm). The absorbance at 492 nm (OD) of dissolved crystals was measured on a microplate reader (SunriseTM microplate reader, Tecan Austria Gmbh, Grödig, Austria) and utilized as a marker of cell proliferation.

Anti-Proliferation Quantitative Analysis
IC 50 values were obtained using GraphPad Prism ® v6.0 software and the concentrationresponse curve fit to the non-linear regression model (GraphPad Software Inc., San Diego, CA, USA).

Conclusions
Although there are few chemical studies that focus specifically on the species S. convolutum, the biological activity of key Sarcophyton metabolites has drawn extensive chemical analyses of the genus. Herein a solvent extract of the soft coral afforded seven cembrane-type diterpenoids, including sarcoconvolutum F and G (1,2), previously undescribed. Compound 4 exhibited modest cytotoxic activity with an IC 50 of 56 µM and 55 µM against lung adenocarcinoma (A549) and oral cavity carcinoma (HSC-2) lines, respectively.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.