Oxygenated Cembrene Diterpenes from Sarcophyton convolutum: Cytotoxic Sarcoconvolutum A–E

The soft coral genus Sarcophyton contains the enzymatic machinery to synthesize a multitude of cembrene-type diterpenes. Herein, highly oxygenated cembrenoids, sarcoconvolutum A–E (1–5) were purified and characterized from an ethyl acetate extract of the red sea soft coral, Sarcophyton convolutum. Compounds were assemblies according to spectroscopic methods including FTIR, 1D- and 2D-NMR as well as HRMS. Metabolite cytotoxicity was tested against lung adenocarcinoma, cervical cancer, and oral-cavity carcinoma (A549, HeLa and HSC-2, respectively). The most cytotoxic compound, (4) was observed to be active against cell lines A549 and HSC-2 with IC50 values of 49.70 and 53.17 μM, respectively.

In a pursuit of novel metabolites with biological activity, polyoxygenated cembrenetype diterpenoids (Figure 1) have been isolated from the Red Sea soft coral S. convolutum. Metabolite antiproliferative activity was assayed against a non-small-cell lung adenocarcinoma, a cervical cancer, and an oral-cavity squamous-cell carcinoma, A549, HeLa, and HSC-2, respectively.

Results and Discussion
Sarcoconvolutum A (1) was isolated as yellow oil with a positive optical rotation (+10.6) in a MeOH solvent. The HRCIMS molecular ion peak at m/z 367.2122 [M + H] + was assigned predicting a molecular formula of C20H30O6 (calcd. 367.2121, [M + H] + ) with six degrees of unsaturation suggesting a bicyclic skeleton. FTIR spectrum exhibited bands at 3465 cm −1 and 1765 cm −1 corresponding to hydroxyls and carbonyl groups. From 1 H NMR spectrum (Table 1) Twenty carbon resonances were predicted from 13 C NMR (Table 1) and categorized into 6 quaternary (including carbonyl at δC 175.3; three olefinics at δC 123.1, 143.9 and 162.3; alongside of two oxygenated carbons at δC 74.5 and 84.1), five methines (comprising two oxygenated carbons at δC 71.6 and 79.4; as well as three olefinic at δC 121.1, 127.8, and 134.8), five methylenes, four methyls (δC 8.8, 16.5, 20.3, and 24.2) depending upon DEPT and HSQC experiments. The above analyses of 1D NMR of 1 revealed the cembrene-based diterpenoid [6,7,24]. The 1D NMR of 1 was similar to 12-hydroperoxylsarcoph-10-ene isolated previously from S. glaucum [24]. Observed signal differences for the newly isolated compound included a down field shift of H-7 and C-7 by 0.73 and 12.1 ppm, respectively; a down field shift of 15.3 ppm for C-8; and a down field shift of 6 ppm for Me-19 consistent with the substitution of a hydroxyl group at C-7 instead of an epoxide ring coupling C-7 and C-8. The proposed structure was confirmed by 1

Results and Discussion
Sarcoconvolutum A (1) was isolated as yellow oil with a positive optical rotation (+10.6) in a MeOH solvent. The HRCIMS molecular ion peak at m/z 367.2122 [ (Table 1) and categorized into 6 quaternary (including carbonyl at δ C 175.3; three olefinics at δ C 123.1, 143.9 and 162.3; alongside of two oxygenated carbons at δ C 74.5 and 84.1), five methines (comprising two oxygenated carbons at δ C 71.6 and 79.4; as well as three olefinic at δ C 121.1, 127.8, and 134.8), five methylenes, four methyls (δ C 8.8, 16.5, 20.3, and 24.2) depending upon DEPT and HSQC experiments. The above analyses of 1D NMR of 1 revealed the cembrene-based diterpenoid [6,7,24]. The 1D NMR of 1 was similar to 12-hydroperoxylsarcoph-10-ene isolated previously from S. glaucum [24]. Observed signal differences for the newly isolated compound included a down field shift of H-7 and C-7 by 0.73 and 12.1 ppm, respectively; a down field shift of 15.3 ppm for C-8; and a down field shift of 6 ppm for Me-19 consistent with the substitution of a hydroxyl group at C-7 instead of an epoxide ring coupling C-7 and C-8. The   (Table 1), 6 quartenary (including carbonyl at δC 174.8; three olefinics at δC 123.5, 143.5 and 161.9; alongside of two oxygenated carbons at δC 74.5 and 84.0), 5 methenes (comprising two oxygenated carbons at δC 78.7 and 71.1, as well as three olefinic signals at δC 121.6, 126.9, 134.6), 5 methylenes, 4 methyls (δC 8.9, 16.1, 23.6, and 24.1) were identified based on DEPT and HSQC signals. A down field shift of δC 84.0 by ca. 7 ppm suggested the presence of a hydroperoxyl unit at C-12 [24] which was consistent with mass spectral data. A 13 C NMR comparison of the two isolated compounds indicated a down field shift of Me-20 for 2. The 2D NMR comparisons  (Table 1), 6 quartenary (including carbonyl at δ C 174.8; three olefinics at δ C 123.5, 143.5 and 161.9; alongside of two oxygenated carbons at δ C 74.5 and 84.0), 5 methenes (comprising two oxygenated carbons at δ C 78.7 and 71.1, as well as three olefinic signals at δ C 121.6, 126.9, 134.6), 5 methylenes, 4 methyls (δ C 8.9, 16.1, 23.6, and 24.1) were identified based on DEPT and HSQC signals. A down field shift of δ C 84.0 by ca. 7 ppm suggested the presence of a hydroperoxyl unit at C-12 [24] which was consistent with mass spectral data. A 13 C NMR comparison of the two isolated compounds indicated a down field shift of Me-20 for 2. The 2D NMR comparisons showed otherwise similar 1 H 1 H COSY and HMBC signals ( Figure 2). A 13 C NMR down field shift of 3.3 ppm for Me-20 indicated the opposite stereochemistry at C-12.
Sarcoconvolutum E (5) was obtained as yellow oil exhibiting a positive optical rotation (+61.7, c 0.003, CH 3 OH). The mass spectroscopy exhibited a HREIMS molecular ion peak at m/z 367.2130 [M + H] + that was identified as C 20 H 30 O 6 (calcd. 367.2042, [M + H] + ) with a bicylic skeleton and six degrees of unsaturation. The FTIR broad bands corresponding to hydroxyls and carbonyl groups were detected at 3464 cm −1 and 1759 cm −1 . 1 H NMR (Table 1) (20) and the oxygenation of C-11 ( Figure 2). Consistent with the mass spectral data and the observed up-field shift of C-11, the oxygenation of C-11 was identified as a hydroperoxide group.

Coral Material
The animal soft coral Sarcophyton convolutum was collected from the Egyptian Red Sea (the coast of Hurghada) in March 2017 and was recognized by M Al-Hammady with a voucher specimen (08RS1071). It was deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

Coral Material
The animal soft coral Sarcophyton convolutum was collected from the Egyptian Red Sea (the coast of Hurghada) in March 2017 and was recognized by M Al-Hammady with a voucher specimen (08RS1071). It was deposited in the National Institute of Oceanography and Fisheries, marine biological station, Hurghada, Egypt.

Extraction and Separation
The frozen soft coral (2.5 kg, total wet weight) was chopped into small pieces and extracted with ethyl acetate at room temperature (3 L × 5 times). The collective extracts were concentrated in vacuo to a brown gum. The dried material (95 g) was subjected to a silica gel column (6 × 120 cm) eluting with n-hexane (2000 mL), followed by a gradient of