A new cembranoid from the Red Sea soft coral Sarcophyton acutum

Abstract The Red Sea soft coral Sarcophyton acutum ethyl acetate extract has afforded one new cembranoid; sarcacutumolid A (1), along with six known metabolites have been isolated from S. acutum for the first time (2-7). Chemical structures were elucidated by employing several spectroscopic analyses. The cytotoxic potential of the isolated compounds was assessed against four human cancer cell lines; hepatocellular (HepG2), cervical (HeLa), breast (MCF-7) and colorectal cancer (Colo-205). Sarcacutumolid A (1) and gorgosterol (7) inhibited colorectal cancer cell proliferation in a concentration-dependent manner with IC50 values of 35.5 and 44.0 μM, respectively.


Introduction
Marine ecosystems possess a broad habitat covering nearly 70% of the earth's surface and are supposed to comprise more than 80% of the world's animal and plant species (McCarthy and Pomponi 2004).Adaptation to diverse and complex marine ecosystems boosted marine creatures' metabolic and physiologic potential (Abraham et al. 2012;Sawadogo et al. 2015;Farag et al. 2016).About one-third to two-thirds of marine organisms haven't been described yet (Appeltans et al. 2012;Hegazy et al. 2015).
Consequently, marine life is deemed to be a prospective provider for a myriad of new biologically active metabolites (Hamed et al. 2015;Farag et al. 2016;Farag et al. 2017).
The Red Sea ecosystem is considered the world's warmest and most saline habitat (Edwards and Head1987;Hegazy et al. 2015).It is a one-of-a-kind marine spot with a high endemic biome, covering the farthest northern tropical reefs with both soft and stony corals (Farag et al. 2016).Despite the fact that the Red Sea is home to about 40% of all globally known soft corals, merely a few chemical investigations of the Red Sea marine invertebrates have been conducted in recent decades (Hegazy et al. 2015), so intensive examinations of the Red Sea marine lifeforms have developed during the last ten years (Hegazy et al. 2012;Elkhateeb et al. 2014;Farag et al. 2016;Hegazy et al. 2017).
Cancer is a complicated chronic disease characterized by aberrant processes of signaling that results in abnormal growth of cells, leading to untimely death (Cragg et al. 2012., Newman andCragg 2016).On a global scale, the World Health Organization (WHO) ranked malignant neoplasms as the second main cause of death (Hegazy et al. 2017).
Marine flora and fauna provide an enormous prospect for the discovery of new anticancer leads (Wali et al. 2019), as numerous compounds of marine origin proved to have a beneficial effect in combating cancer by either suppressing the proliferation of malignant cells or enhancing apoptosis in human cancerous cell lines (Wali et al. 2019).
Up-to-date, five drugs isolated from marine invertebrates have been licensed as pharmaceuticals for the treatment of cancer; cytarabine (Cytosar-UV R ), eribulin mesylate (Halaven V R ), trabectedin (Yondelis V R ), ziconotide (Prilat V R ) and brentuximab vedotin (Adcetris V R ) (Mudit and El Sayed 2016).Additionally, many other marine natural compounds have revealed promising anticancer effects in preclinical trials and are being promoted to clinical phases (Mudit and El Sayed 2016).
Compound 1 was obtained as colorless crystals with a negative optical rotation in methanol ½a 25 D À1.0 (C 0.1, MeOH).The HR-TOF-ESI-MS exhibited a (m/z of 405.1889 and six degrees of unsaturation were deduced the tricyclic skeleton.The IR spectrum showed the characteristic bands at 3430 cm À1 (OH) and 1750 (CO) (Figure S14).
The saturation of the lactone ring, besides the appearance of resonances typical of oxygenated quaternary carbons at d c 86.1 and d c 79.5, suggested the presence of oxygen atoms at C-1 and C-15, respectively.The previously-mentioned functionalities elucidate five of the six degrees of unsaturation.The remaining one proposed a tricyclic structure with peroxide linkage to fulfill the mass and molecular formula of the compound.The presence of an oxygenated bridge was determined from the six degrees of unsaturation that were deduced the tricyclic skeleton.Furthermore, by the deshielding of C-1 and C-12 the bridge was identified as a peroxide bridge compared to hydroxy group-bearing carbons (Thao et al. 2014).
The signal at d H 4.77 (d, J ¼ 9.9 Hz) correlated with the proton signal at d H 5.18 (d, J ¼ 9.9 Hz) and quaternary olefinic carbons at d C 143.3 and d C 86.1 in 1 H-1 H COSY and HMBC (Figure S1), respectively, allowing for the assignments of H-2, H-3, C-4, and C-1, respectively.The HMBC spectrum's correlations revealed a number of informative linkages; H-3 at d H 5.18 (d, J ¼ 9.9 Hz) to carbon signals at d C 15.9 (q, olefinic) and d C 36.1(t) allowed for the assignment of methyl group at d H 1.67 (s, H-18), and H-5 at d H 2.20 (ddd, J ¼ 13.1, 3.7, 3.7) and 2.42 (ddd, J ¼ 13.7, 13.1, 3.7 Hz), respectively.Also, the methyl signal at d  A methyl signal at d H 1.21 (s, H 3 -20) was observed proximal to C-12 determined from HMBC correlations between C-12 (d C 81.9) with proton signals at d H 1.21 (s) and 5.67 (ddd, J ¼ 16.4, 10.1, 4.5 Hz) allowing for the assignments of H-20, H-10, respectively and C-13 (d C 26.2) and a methyl signal at d H 1.21 (s, H 3 -20).Additionally, a correlation was detected between H-9 (d H 2.54, dd, J ¼ 13.5, 10.1 Hz; d H 2.13, dd, J ¼ 13.5, 3.6 Hz) and olefinic methine carbon signals at d c 127.6 (C-10) and 135.1 (C-11) revealed the presence of oxygenated quaternary carbon at C-12 and a double bond at C-10/C-11.HMBC correlations (Figure S1) were also observed between oxygenated methine signal at H-7 d H 3.60 (dd, J ¼ 11.0, 5.0 Hz) and C-5 (d C 36.1), C-6 (d C 25.0), C-8 (d C 73.9), and C-19 (d C 22.7) as well as methyl signal at d H 1.22 (H 3 -19) exhibited HMBC correlations with C-7 (d C 69.5), C-8 (d C 73.9) and C-9 (d C 43.2) indicating the location of the hydroxyl groups at C-7 and C-8.The relative configuration of 1 was assigned based on the coupling constants along with the correlations of NOESY (Figure S2).The vicinal coupling between H-2 at d H 4.77 (d, J ¼ 9.9 Hz) and H-3 at d H 5.18 (d, J ¼ 9.9 Hz) along with the NOESY correlations between H-2 and methyl protons (H 3 -18) verified the trans linkage between the c-lactone (H-2) and the olefinic proton (H-3).Also, H-2 showed NOESY correlations with (H 3 -17) and H-14 (d H 1.85) as well as NOESY correlation between H 3 -18 with H-7 and H-5 (d H 2.20) showed that H-2, H-7, H 3 -17, H 3 -19 and H 3 -20 in the a-orientation.Supporting CD data for 1 (Figure S3), CD spectral comparison between 1 and sarcophine (2) indicated the reverse absolute configuration for the two compounds at C-2.The positive effect at 245 nm followed by the negative Cotton effect at 225 nm and for sarcacutumolid A (1) indicated a left hand (M) helix configuration for the five-membered a,b-unsaturatedc-lactone ring (Nii et al. 2008), in comparison with the negative Cotton effect at 247 nm followed by the positive Cotton effects at 223 nm for sarcophine (2) (Figure S3) (Hegazy et al. 2011;Hegazy et al. 2012).From the above spectral data, 1 was established as sarcacutumolid A.

Screening of the antiproliferative activity
The isolated compounds 1-7 were initially screened for their antiproliferative action against three human cancer cell lines derived from the liver (HepG-2), cervix (HeLa) and breast tissue (MCF-7) at concentrations of 100 and 10 mM utilizing the Sulpho-Rhodamine-B (SRB) cytotoxicity assay.Among the tested compounds, only gorgosterol (7) inhibited the proliferation of HeLa cells by 58.3% at a concentration of 100 lM compared to DMSO as a solvent control, while the anti-proliferative effect of the other compounds on HeLa cells was less than 50%.None of the isolated compounds exhibited cytotoxic activity against either human liver (HepG2) or breast cells (MCF-7) at the tested concentrations.

Determination of the minimum inhibitory concentration
The minimum inhibitory concentration of gorgosterol ( 7) and the new cembranoid, sarcacutumolid A (1), were further tested against colorectal cancer (Colo-205) using different concentrations within the range of 1000 to 0.1 lM.The IC 50 (concentration causing 50% loss of cell viability) was calculated from the dose response curve using a non-linear regression analysis model on GraphPad Prism V8.0 (San Diego, USA).Gorgosterol and sarcacutumolid A inhibited cell proliferation in a concentrationdependent manner compared to DMSO as the solvent control, with IC 50 values of 35.5 and 44.0 lM, respectively (FigureS4).

General experimental procedure
The spectra of 1 H and 13 C NMR were recorded in deuterated chloroform (CDCl 3 ) using Varian Unity INOVA 500 ( 1 H 500 MHz, 13 C 125 MHz).All NMR chemical shifts (d) were illustrated in ppm units relative to the internal standard TMS and the coupling constants (J) were given in Hz.
High performance liquid chromatography (HPLC) was accomplished on an Agilent pump 1260 Infinity connected to a refractive index detector RID10A and a semi-preparative reverse-phase column (Cosmosil, 5 C 18 -MS-II, 10 ID x 250 mm).
Normal phase silica gel 60 (Merck, 230-400 mesh) was utilized for column chromatography.While, for TLC analyses, the pre-coated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) were applied.Visualization of spots was achieved by heating the plates after spraying with vanillin/H 2 SO 4 .

Coral material
The soft coral Sarcophyton acutum was gathered from the Egyptian Red Sea coast in Hurghada in March 2017.Dr. Montaser Al-Hammady was identified the soft coral and a voucher specimen (08RS1069) placed in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

Extraction and isolation
The frozen soft coral specimens (3 kg, total wet weight) were cut up into small pieces and extracted with ethyl acetate (4 L Â 3 times) at room temperature.The combined ethyl acetate extract was evaporated to dryness to yield a brown gum.The dried ethyl acetate extract (60 g) was chromatographed using low pressure pump column chromatography (6 Â 120 cm) with gradient elution of n-hexane-EtOAc followed by EtOAc-MeOH.Fractions were collected, concentrated, and monitored based on their TLC profile.Fractions with similar profiles were combined together to give ten main fractions.Fraction 4 (17.7 g), eluted with n-hexane-EtOAc (8:1), was further purified using silica gel column chromatography (4 Â 80 cm) to afford 2 (25 mg) and 7 (20 mg).The remaining samples of this fraction were collected based on the TLC profile and rechromatographed on another silica gel column (4 Â 80 cm) using n-hexane-EtOAc (gradient elution) followed by purification on reversed phase HPLC using MeOH: H 2 O (60:40), 2 mL/min, to produce 1 (16 mg), 3 (12 mg), 4 (16 mg), 5 (19 mg) and 6 (13 mg).).The IR spectrum showed the distinctive bands at 3430 cm À1 (OH) and 1750 (CO). 1 H and 13 C NMR data (CDCl 3 , 500 Hz), see Table S1.
HeLa and Colo-205 cell lines were maintained in Roswell Park Memorial Institute medium (RPMI), while HepG2 and MCF-7 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM).All media were supplemented with 100 units/mL of penicillin, 100 mg/mL of streptomycin and 10% of heat-inactivated fetal bovine serum in a humidified 5% (v/v) CO 2 atmosphere at 37 C.

Sulpho-Rhodamine-B (SRB) cytotoxicity assay
The anti-proliferative effect of the compounds on the viability of various cell lines was investigated using the SRB assay (Skehan et al. 1990;Allam et al. 2018).Aliquots of 100 lL cell suspension (5 Â 10 3 cells) were incubated in complete media in 96-well plates for 24 hrs to allow adhesion of cells to the wells.Cells were treated with another aliquot of 100 lL media including the isolated compounds at tested concentrations for 72 hrs.In parallel, Doxorobucin HCl (Dox) was used as a reference cytotoxic drug.Then the cells were fixed by substituting media with 150 lL of 10% tricarboxylic acid (TCA) and incubated at 4 C for 1 hr.followed by removal of the TCA solution and washing cells with distilled water 5 times.Aliquots of 70 lL SRB solution at a concentration of (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min.Plates were washed 3 times with 1% acetic acid and air-dried overnight.Thereafter, 150 lL of TRIS (10 mM) was applied for dissolving protein-bound SRB stain and the absorbance at 540 nm was measured using a BMG LABTECHV R -FLUOstar Omega microplate reader (Ortenberg, Germany).

Conclusions
One new (1) and five known cembranolides (2-6) along with one steroid (7) were isolated and chemically characterized from the Red Sea soft coral Sarcophyton acutum.The anti-proliferative potential of the isolated compounds was evaluated against four human cancer cell lines, which resulted in sarcacutumolid A and gorgosterol being active against the Colo-205 cancer cell line.
H 1.51 (s) exhibited HMBC correlations with d C 176.9 (C ¼ O), d C 79.5 and d c 86.1 attributed to H-17, C-16, C-15, and C-1 respectively, as well as supporting the location of the C-1/C-2 lactone ring.

Figure 1 .
Figure 1.Structures of the isolated compounds from Sarcophyton acutum.