Two New Glycerides from Red Sea Ascidian Didemnum Species Sabrin R.M

This article was downloaded by: [Gamal Mohamed] On: 20 June 2014, At: 08:21 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20 Didemnacerides A and B: two new glycerides from Red Sea ascidian Didemnum species Sabrin R.M. Ibrahimab, Gamal A. Mohamedcd, Lamiaa A. Shaalaef, Diaa T.A. Youssefcg & Ali A. Gab-Allah a Department of Pharmacognosy and Medicinal Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 41477, Saudi Arabia b Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt c Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia d Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt e Natural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia f Suez Canal University Hospital, Suez Canal University, Ismailia 41522, Egypt g Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt h Biological Sciences Department, Faculty of Science, Um Al-Qura University, Makkah, Saudi Arabia Published online: 18 Jun 2014.

To cite this article: Sabrin R.M. Ibrahim, Gamal A. Mohamed, Lamiaa A. Shaala, Diaa T.A. Youssef & Ali A. Gab-Alla (2014): Didemnacerides A and B: two new glycerides from Red Sea ascidian Didemnum species, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.927874 To link to this article: http://dx.doi.org/10.1080/14786419.2014.927874

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms- and-conditions Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.927874

Didemnacerides A and B: two new glycerides from Red Sea ascidian Didemnum species Sabrin R.M. Ibrahimab, Gamal A. Mohamedcd, Lamiaa A. Shaalaef, Diaa T.A. Youssefcg* and Ali A. Gab-Allah

aDepartment of Pharmacognosy and Medicinal Chemistry, Faculty of Pharmacy, Taibah University, Al Madinah Al Munawwarah 41477, Saudi Arabia; bDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt; cDepartment of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; dDepartment of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt; eNatural Products Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; fSuez Canal University Hospital, Suez Canal University, Ismailia 41522, Egypt; gDepartment of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt; hBiological Sciences Department, Faculty of Science, Um Al-Qura University, Makkah, Saudi Arabia (Received 13 April 2014; ﬁnal version received 20 May 2014)

Two new glycerides, didemnacerides A (1) and B (2), together with three known sterols, 24-ethyl-25-hydroxycholesterol (3), cholest-6-en-3,5,8-triol (4) and cholestane-3b,5a,6b-26-tetrol (5), were isolated from the Red Sea ascidian Didemnum sp. Their structures were elucidated by using extensive 1D (1H, 13C) and 2D (1H–1H COSY, HSQC and HMBC) NMR studies and mass spectroscopic data (GC-MS and HR-MS) as well as alkaline hydrolysis followed by GC–MS and NMR spectral analyses of the fatty acid methyl esters. This is the ﬁrst report of compounds 3–5 from the Red Sea ascidian Didemnum species. Keywords: marine ascidian; Didemnum species; glycerides; sterols

1. Introduction Recently, marine ascidians have been widely recognised as a rich source of bioactive natural products. Members of the genus Didemnum have yielded a variety of interesting secondary Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 metabolites. Examples of these secondary metabolites include alkaloids (Davis et al. 1999; Oku et al. 2000), lipids (Mitchell et al. 2000), peptides (Rudi et al. 2003; Toshiaki et al. 2008) and macrolides (Potts & Faulkner 1991; Pika & Faulkner 1995). Many of the isolated compounds were found to possess signiﬁcant biological activities including antiplasmodial (Wright et al. 2002), antibacterial (Kumaran et al. 2011), antiviral (Davis et al. 1999), cytotoxic (Segraves et al. 2003) and antileukaemic (Takeara et al. 2008). Previous investigation of the Red Sea Didemnum species led to the isolation of two new spiroketals, didemnaketals D and E (Mohamed et al. 2014). In continuation of our investigation of the same species, we describe here the isolation and structural elucidation of two new glycerides named didemnacerides A (1) and B (2), along with three known sterols, 24-ethyl-25-hydroxycholesterol (3) (Siddiqui et al. 1989), cholest-6-en-3,5,8-triol (4) (Youssef et al. 2010) and cholestane-3b,5a,6b-26-tetrol (5) (Liyanage & Schmitz 1996). Compounds 3–5 are reported here for the ﬁrst time from the Red Sea ascidian Didemnum species (Figure 1).

*Corresponding author. Email: [email protected]

q 2014 Taylor & Francis 2 S.R.M. Ibrahim et al.

Figure 1. Structures of the isolated compounds 1–5.

2. Results and discussion Compound 1 was isolated as colourless oil. Its HR-ESI-MS revealed a pseudo-molecular ion þ peak at m/z 1011.9239 [M þ H] , which is consistent with a molecular formula C66H122O6, requiring six degrees of unsaturation. Its IR spectrum revealed absorption bands at 2925 and 2850 (alkyl chain), 1655 (double bond) and 1732 (ester carbonyl) cm21 (Silverstein & Webster 1998; Ibrahim et al. 2009). The presence of a strong absorption band at 1732 cm21 in the IR 0 000 00 13 spectrum along with signals at dC 172.8 (C-1 ,1 ) and 173.3 (C-1 )in C NMR spectrum suggested the presence of three ester carbonyls in 1 (Shiao & Shiao 1989; Vlahov 1999; Swaroop et al. 2005; Vlahov 2009; Wang et al. 2010) (Supplementary Figure S2). The appearance of only two signals for three ester carbonyls suggested the symmetric nature of 1 (Swaroop et al. 2005; Ibrahim 2014). The signals at dH 4.29 and 4.15/dC 62.0 (H-1/C-1), 5.27/68.4 (H-2/C-2), and 4.31 and 4.14/62.1 (H-3/C-3) along with the fragment ion peaks at m/z þ þ 704.6235 [M þ H–C21H39O] and 397.3242 [M þ H–C42H78O2] confirmed that 1 is a triglyceride with C21 fatty acids, which was further confirmed by the observed COSY correlations (Supplementary Figures S1, S3, and S11). The 1H NMR spectrum exhibited signals 0 00 000 for olefinic protons at dH 5.36 (3H, each t, J ¼ 6.0 Hz, H-6 ,6,6 ) and 5.34 (3H, each t,

Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 0 00 000 0 000 00 J ¼ 6.0 Hz, H-7 ,7 ,7 ) correlated with carbons at dH 129.9 (C-6 ,6 ), 130.0 (C-6 ), 129.8 (C- 700) and 129.7 (C-70,7000) in the HSQC spectrum, also indicating the presence of three olefinic double bonds in 1. The geometry of the double bonds was deduced to be Z based on the coupling constant values (J60,70 ¼ J600,700 ¼ J6000,7000 ¼ 6.0 Hz) (de Carvalho et al. 2000; Ibrahim 2014). The 1H NMR spectrum revealed characteristic signals for the terminal methyl protons of the fatty 00 0 000 acids at dH 0.87 (t, J ¼ 6.5 Hz, H-21 ) and 0.85 (t, J ¼ 6.5 Hz, H-21 ,21 ), broad signals at dH 00 0 1.23–1.38 for the methylene protons, two methylene triplets at dH 2.32 (H-2 ) and 2.31 (H-2 , 000 00 0 000 00 2 ), and two multiplets at dH 1.63 (H-3 ) and 1.61 (H-3 ,3 ). In the COSY spectrum, H-2 and H-20,2000 correlated with H-300 and H-30,3000, which further correlated to H-400 and H-40,4000, respectively. The coupling observed in the COSY spectrum between the proton signals at dH 2.01 (H-50,5000,80,8000) and 2.02 (H-500,800) and the olefinic protons indicated its connection to the double bond carbon. In HMBC spectrum (Supplementary Figures S5 and S11), the carbonyl 00 0 000 carbon signals at dC 173.3 (C-1 ) and 172.8 (C-1 ,1 ) showed correlations with proton signals at 00 0 000 00 dH 2.32 (2H, t, J ¼ 7.5 Hz, H-2 ), 2.31 (4H, t, J ¼ 7.5 Hz, H-2 , H-2 ), 1.63 (2H, m, H-3 ) and 1.61 (4H, t, H-30,3000), also confirming that 1 is a triglyceride with long chain fatty acid esters. Furthermore, the HMBC correlations of H-1 to C-10, H-2 to C-100, and H-3 to C-1000 indicated the Natural Product Research 3

acyls of fatty acid moieties attached to C-1, C-2 and C-3. The proton signals at dH 2.01 (8H, m, 0 0 000 000 00 00 H-5 ,8,5 ,8 ) and 2.02 (4H, m, H-5 ,8 ) correlated with carbon signals at dC 129.7–130.0 (C- 0 0 00 00 000 000 6 ,7,6 ,7 ,6 and 7 ) along with the fragment peak at m/z 308.3004 (C21H40O) in the HR-ESI- MS confirmed the presence of (6Z)-henicos-6-enoic acid moiety in 1. Alkaline hydrolysis of 1 resulted in the fatty acid methyl ester (FAME). The FAME was subjected to 1D, 2D NMR and GC–MS analyses to afford (6Z)-henicos-6-enoic acid methyl ester (m/z 338 [M]þ). Accordingly, 1 wasassignedas1,2,3-tri-(6Z)-henicos-6-enoyl glycerol and named didemnaceride A. Compound 2 was isolated as colourless oil, with a molecular formula C45H84O7 as determined by HR-ESI-MS and NMR spectra. Its IR spectrum revealed absorption bands for OH at 3495 cm21 and an ester carbonyl at 1735 cm21 (Silverstein & Webster 1998; Ibrahim et al. þ 2009). The fragment ion peaks at m/z 720.6268 [M þ H–H2O] and 704.6325 [M þ H– þ 2H2O] suggested the presence of two hydroxyl functionalities in 2 and confirmed by signals for 0 00 two oxymethines at dH 4.18 (1H, t, J ¼ 6.2 Hz, H-2 ) and 3.43 (1H, quin, J ¼ 7.0 Hz, H-4 ) observed in the 1H NMR spectrum (Supplementary Figure S6). These protons correlated with 0 00 the carbon signals at dC 68.2 (C-2 ) and 71.7 (C-4 ) in the HSQC spectrum (Supplementary Figure S9). With the assistance of 2D NMR studies, including 1H–1H COSY, HSQC and HMBC experiments, the assignments of 1H and 13C NMR signals of 2 were established. The 1H and 13C NMR spectra (Supplementary Figures S6 and S7) of 2 revealed signals at dH 4.32 and 4.15 (each, dt, J ¼ 6.0 and 12.5 Hz, H-1)/dC 62.8 (C-1), dH 5.19 (H-2)/dC 70.1 (C-2) and dH 3.53 (H-3)/dC þ 68.9 (C-3), along with the fragment ion peaks at m/z 413.3625 [M þ H-(2H2O þ C20H35O)] þ and 323.3311 [M þ H-(2H2O þ C23H41O4)] characteristic for 2,3-diacyl glycerol moiety. The 0 00 two carbon signals at dC 174.6 (C-1 ) and 174.4 (C-1 ) suggested the presence of two ester carbonyls, confirming the diglyceride moiety of 2 (Momin et al. 2000; Vlahov et al. 2002; Ibrahim 2014). This was further secured by the HMBC correlations between H-1 and H-3 and C- 0 2, and H-2 and C-1 and C-3. Moreover, the signals at dH 5.35 (2H, dt, J ¼ 6.2 and 5.8 Hz, H-6 , 90), 5.36 (1H, dt, J ¼ 6.2 and 5.8 Hz, H-70) and 5.34 (1H, dt, J ¼ 6.2 and 5.8 Hz, H-100) 0 0 0 0 correlated with the carbon signals at dC 129.8 (C-6 ,9), 129.9 (C-7 ) and 128.8 (C-10 ) indicating the presence of two olefinic double bonds. The geometry of the double bonds was deduced to be Z based on the coupling constant values (J60,70 and J90,100 ¼ 5.8 Hz) (de Carvalho et al. 2000; Mohamed et al. 2013). The signals for methylene group connected to a double bond 0 0 carbon were observed at dH 2.81 (1H, t, J ¼ 6.2 Hz, H-8 )/dC 25.6 (C-8 ) and confirmed by its cross-peaks to H-70 and H-90 in COSY spectrum and with C-60 and C-100 in the HMBC spectrum. The positions of the hydroxy groups and the double bonds were confirmed by the COSY cross- Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 peaks of H-20 to H-30, H-60 to H-50 and H-70, H-100 to H-90 and H-110, and H-400 to H-300, H-500 and further secured by the HMBC correlations of H-20 to C-10 and C-40,H-40 with C-60, H-60 to C-80, H-70 to C-90, H-80 to C-60 and C-100, and H-9 to C-110 (Supplementary Figure S11). The connectivity of the fatty acid moieties to glycerol was confirmed by the HMBC correlations of H-2 to C-10 and H-3 with C-100. Alkaline hydrolysis of 2 followed by GC–MS, NMR and optical rotations analyses of the FAMEs indicated the presence of 4(S)-hydroxydocosanoic acid methyl ester (m/z 370 [M]þ) and (6Z,9Z)-2(S)-hydroxyicosa-6,9-dienoic acid methyl ester (m/z 338 [M]þ) moieties in 2. It was reported that a-hydroxy FAMEs (R-isomer) had negative optical rotations (Li et al. 1995; Asai et al. 2000; Qu et al. 2004). The FAMEs of 2 ([a]D þ 3.38 and þ5.48, respectively) had optical rotation signs that were different from those of analogous 2-(R)- hydroxy fatty acids reported earlier (Li et al. 1995; Asai et al. 2000; Qu et al. 2004), indicating that 2 had b-hydroxy fatty acids (S-isomer). Thus, the stereochemistry of 2 was assigned by comparing the 1H and 13C NMR chemical shifts, coupling constant values and optical rotation with the reported data for glyceride analogue (Dharma et al. 1985; Ibrahim 2014). On the basis of these findings, 2 was assigned as 2-(6Z,9Z)-2S-hydroxyicosa-6,9-dienoyl-3-(4S)-hydro- xydocosanoylglycerol and named didemnaceride B. 4 S.R.M. Ibrahim et al.

The known compounds were identiﬁed by the interpretation of their spectral data (1D, 2D NMR and MS) and by comparing their data with those in the literature. The compounds were identiﬁed as 24-ethyl-25-hydroxycholesterol (3) (Siddiqui et al. 1989), cholest-6-en-3b,5a,8a- triol (4) (Youssef et al. 2010) and cholestane-3b,5a,6b-26-tetrol (5) (Liyanage & Schmitz 1996).

3. Experimental 3.1. General experimental procedures Melting point apparatus Electrothermal 9100 Digital Melting Point (Electrothermal Engineering, Southend-on-Sea, Essex, UK) was used to record the melting points. Optical rotations were measured on a Perkin-Elmer Model 341 LC polarimeter (Perkin-Elmer, Waltham, MA, USA). UV spectra were recorded in absolute MeOH on a Shimadzu 1601 UV/VIS spectrophotometer (Shimadzu, Kyoto, Japan). HR-ESI-MS data were determined on a Micromass Q-Tof mass spectrometer (ThermoFinnigan, Bremen, Germany). The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Shimadzu). 1D and 2D NMR spectra (chemical shifts in ppm and coupling constants in Hz) were recorded on Bruker Avance DRX 400 MHz spectrometers (Bruker BioSpin, Billerica, MA, USA) using CDCl3 as solvent, with TMS as the internal reference. A GC–MS was performed on Clarus 500 GC–MS (Perkin- Elmer). The software controller/integrator was Turbo Mass, version 4.5.0.007 (Perkin-Elmer). An Elite 5MS GC capillary column (30 mm £ 0.25 mm £ 0.5 mm, Perkin-Elmer) was used. The carrier gas was helium (purity 99.9999%) at a ﬂow rate of 2 mL/min (32 psi, ﬂow initial 55.8 cm/ s, split; 1:40). Temperature conditions were as follows: inlet line temperature, 2008C; source temperature, 1508C; trap emission, 1008C and electron energy, 70 eV. The column temperature program was as follows: 508C for 5 min, increased to 2208C (rate, 208C/min) and held for 5 min. The injector temperature was 2208C. MS scan was from 50 to 650 m/z. Column chromatographic separations were performed on silica gel 60 (0.04–0.063 mm, Merck, Darmstadt, Germany) and RP-18 (0.04–0.063 mm, Merck). The solvent systems used for TLC investigation were CHCl3– MeOH (97:3, S1) and CHCl3–MeOH (95:5, S2). Spots were visualised by UV absorption at lmax 255 and 366 nm followed by spraying with p-anisaldehyde/H2SO4.

3.2. Animal materials The marine ascidian Didemnum species was collected in the Mangrove located in Nabq/Sharm Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 El-Sheikh on the Egyptian Red Sea coast at depths (,1 to 2 m) in 2009. The ascidian forms thin (2–3 mm) deep-orange coloured sheets around the pneumatophores of the mangrove plant Avicennia marina. The colour of the ascidian fades on preservation in formalin. Didemnum species shares many characteristics that are common among colonial tunicate species. Characteristics such as colony shape and colour, where the colony grows, zooid structure and spicule shape have been used previously to separate and identify different Didemnum species. Didemnum species are characterised by many small zooids 1–2 mm in length, embedded in a sheet-like, gelatinous matrix called a tunic or test (Lambert 2002). White, calcareous, stellate spicules are embedded within the tunic’s surface among zooids that contain individual oral siphons while atrial siphons discharge into a common cloacal aperture maintained in deep crevices within the colony (Lambert 2002, 2005). Spicules are 40 mm in diameter on average, but can reach 100 mm. This combination of spicules and zooids give the tunic’s surface an overall appearance described as ‘small, white dots and pinhole-sized pores’. Descriptions of Didemnum species colony shape include long, ropey or beard-like colonies; low, undulating mats with short appendages that encrust or drape; sponge-like colonies. However, it should be noted that the shape of the Didemnum species colony changes with age. Colonies of young Natural Product Research 5

Didemnum species usually present as thin mats but as the colony matures, irregular lobes are formed, thereby greatly increasing the surface complexity. A voucher specimen (registration number 2009DY31) measuring 2.5 cm £ 3.0 cm has been deposited in the Red Sea Invertebrates collection of the Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University.

3.3. Extraction and isolation

The fresh ascidian (350 g wet weight) was crushed into small pieces and extracted with CH2Cl2– MeOH (1:1). The crude extract was defatted with petroleum ether. The defatted extract was partitioned between CH2Cl2 and H2O. The CH2Cl2 extract (9.4 g) was subjected to vacuum liquid chromatography using n-hexane–EtOAc and EtOAc–MeOH gradients to afford 10 fractions: D-1 (210 mg, n-hexane 100%), D-2 (405 mg, n-hexane–EtOAc 90:10), D-3 (540 mg, n-hexane–EtOAc 75:25), D-4 (690 mg, n-hexane–EtOAc 50:50), D-5 (640 mg, n-hexane– EtOAc 25:75), D-6 (890 mg, EtOAc 100%), D-7 (970 mg, EtOAc–MeOH 75:25), D-8 (1.25 g, EtOAc–MeOH 50:50), D-9 (1.1 g, EtOAc–MeOH 25:75) and D-10 (1.9 g, MeOH 100%). Fraction D-2 (405 mg) was chromatographed over silica gel column (0.04–0.063 mm; 50 g £ 50 cm £ 2 cm) using n-hexane–EtOAc gradient elution to obtain two major subfractions: D- 2A (208 mg) and D-2B (155 mg). Subfraction D-2B was subjected to repeated silica gel column chromatography (0.04–0.063 mm; 30 g £ 50 £ 2 cm) using n-hexane–EtOAc gradient elution to afford 1 (9.2 mg, colourless oil). Repeated silica gel column chromatography (0.04– 0.063 mm; 60 g £ 50 cm £ 2 cm) using n-hexane–EtOAc gradient elution for fraction D-3 (540 mg) yielded 2 (10.7 mg, colourless oil). Fraction D-4 (690 mg) was chromatographed on silica gel column chromatography (70 g £ 50 cm £ 2 cm) and eluted with n-hexane–EtOAc gradient. Fractions (25 mL each) were collected and monitored with TLC. The fractions eluted with n-hexane–EtOAc (85:15) after repeated puriﬁcation steps through small columns yielded compounds 3 (6.5 mg, white crystals) and 4 (9.2 mg, white crystals). Fraction D-5 (640 mg) was subjected to silica gel column chromatography (70 g £ 50 cm £ 2 cm) to yield impure 5, which was further puriﬁed by RP-18 column chromatography (0.04–0.063 mm; 20 g £ 50 cm £ 2 cm) using MeOH–H2O (80:20 to 90:10) to yield 5 (11 mg, white crystals).

3.4. Alkaline treatment of compounds 1 and 2 Separate solutions of compounds 1 and 2 (5 mg each) in 3% NaOMe/MeOH (2 mL) were stirred at 408C for 2 h. The reaction mixture was neutralised with 2 N HCl in MeOH and partitioned Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 between MeOH and n-hexane. The n-hexane layer was concentrated under reduced pressure to yield FAMEs, which were analysed by GC–MS and NMR spectroscopy (Sayed et al. 2007).

3.5 Spectral data

Didemnaceride A (1): colourless oil; Rf 0.84, Si 60 F254 (Figure S1); [a]D þ 39.28 (c ¼ 1.5, 21 CHCl3); UV lmax (MeOH): 207 nm; IR (KBr) nmax 2925, 2850, 1732, 1655, 1296, 1050 cm ; 1 H NMR (CDCl3, 400 MHz): dH 4.29 (1H, dd, J ¼ 12.0 and 6.0 Hz, H-1A), 4.15 (1H, dd, J ¼ 12.0 and 6.0 Hz, H-1B), 5.27 (1H, m, H-2), 4.31 (1H, dd, J ¼ 12.0 and 6.0 Hz, H-3A), 4.14 0 000 (1H, dd, J ¼ 12.0 and 6.0 Hz, H-3B), 2.31 (2 £ CH2, each t, J ¼ 7.5 Hz, H-2 ,2 ), 1.61 (2 0 000 0 000 0 000 £ CH2, each m, H-3 ,3 ), 1.32 (2 £ CH2, each m, H-4 ,4 ), 2.01 (2 £ CH2, each m, H-5 ,5 ), 5.36 (2 £ CH, each t, J ¼ 6.0 Hz, H-60,6000), 5.34 (2 £ CH, each t, J ¼ 6.0 Hz, H-70,7000), 2.01 (2 0 000 0 0 000 000 £ CH2, each m, H-8 ,8 ), 1.23–1.38 (20 £ CH2 groups, each m, H-9 -18 and H-9 -18 ), 1.25 0 000 0 000 (2 £ CH2, each m, H-19 ,19 ), 1.50 (2 £ CH2, each m, H-20 ,20 ), 0.85 (2 £ CH3, each t, J ¼ 6.5 Hz, H-210,21000), 2.32 (2H, t, J ¼ 7.5 Hz, H-200), 1.63 (2H, m, H-300), 1.32 (2H, m, H-400), 2.02 (2H, m, H-500), 5.36 (1H, t, J ¼ 6.0 Hz, H-600), 5.34 (1H, t, J ¼ 6.0 Hz, H-700), 2.02 (2H, m, 6 S.R.M. Ibrahim et al.

00 00 00 00 00 H-8 ), (20 £ CH2, each m, H-9 -18 ), 1.25 (2H, m, H-19 ), 1.50 (2H, m, H-20 ), 0.87 (3H, t, 00 13 J ¼ 6.5 Hz, H-21 ); C NMR (CDCl3, 100 MHz): dC 62.0 (C-1), 68.4 (C-2), 62.1 (C-3), 172.8 (C-10,1000), 34.0 (C-20,2000), 24.8 (C-30,3000), 28.9 (C-40,4000), 27.4 (C-50,5000), 129.9 (C-60,6000), 129.7 (C-70,7000), 27.2 (C-80,8000), 29.1–29.9 (C-90-180, C-9000-18000), 31.7 (C-190,19000), 22.6 (C- 200,20000), 14.1 (C-210,21000), 173.3 (C-100), 34.2 (C-200), 28.8 (C-300), 29.0 (C-400), 27.9 (C-500), 130.0 (C-600), 129.8 (C-700), 27.1 (C-800), 29.1-29.9 (C-900-1800), 31.9 (C-1900), 22.7 (C-2000), 14.1 00 þ (C-21 ); HR-ESI-MS m/z 1011.9239 (calcd for C66H123O6,[Mþ H] , 1011.9241).

Didemnaceride B (2): colourless oil; Rf 0.76, Si 60 F254 (Figure S1); [a]D þ 61.38 (c ¼ 1.5, 21 1 CHCl3); UV lmax (MeOH): 224 nm; IR (KBr) nmax 3495, 1735, 1291, 1056 cm ; HNMR (CDCl3, 400 MHz): dH 4.32 (1H, dt, J ¼ 12.5 and 6.0 Hz, H-1A), 4.15 (1H, dt, J ¼ 12.5 and 6.0 Hz, H-1B), 5.19 (1H, m, H-2), 3.53 (2H, m, H-3), 4.18 (1H, t, J ¼ 6.2 Hz, H-20), 1.77 (2H, m, H-30), 1.42 (2H, m, 40), 2.10 (2H, m, H-50), 5.35 (1H, dt, J ¼ 6.2 and 5.8 Hz, H-60), 5.36 (1H, dt, J ¼ 6.2 and 5.8 Hz, H-70), 2.81 (2H, t, J ¼ 6.2 Hz, H-80), 5.35 (1H, dt, J ¼ 6.2 and 5.8 Hz, H-90), 0 0 0 5.34 (1H, dt, J ¼ 5.8 Hz, H-10 ), 2.10 (2H, m, H-11 ), 1.38 (2H, m, H-12 ), 1.21–1.35 (6 £ CH2, each m, H-130-H-180), 1.40 (2H, m, H-190), 0.89 (3H, t, J ¼ 7.0 Hz, H-200), 2.33 (2H, m, H-200), 1.55 (2H, m, H-300), 3.43 (1H, quin, J ¼ 7.0 Hz, H-400), 2.02 (2H, m, H-500), 1.21–1.35 (15 00 00 00 00 13 £ CH2, each m, H-6 -H-20 ), 1.15 (2H, m, H-21 ), 0.87 (3H, t, J ¼ 6.8 Hz, H-22 ); CNMR 0 0 0 (CDCl3, 100 MHz): dC 62.8 (C-1), 70.1 (C-2), 68.9 (C-3), 174.6 (C-1 ), 68.2 (C-2 ), 34.2 (C-3 ), 31.9 (C-40), 27.9 (C-50), 129.8 (C-60), 129.9 (C-70), 25.6 (C-80), 129.8 (C-90), 128.8 (C-100), 27.9 (C-110), 31.8 (C-120), 29.8–29.9 (C-130-C-180), 22.7 (C-190), 14.4 (C-200), 174.4 (C-100), 34.1 (C- 200), 29.6 (C-300), 71.7 (C-400), 31.9 (C-500), 29.8–29.9 (C-600-C-2000), 22.6 (C-2100), 14.4 (C-2200); þ HR-ESI-MS m/z 737.6241 (calcd for C45H85O7,[Mþ H] , 737.6217).

4. Conclusion Two new glycerides: didemnacerides A (1) and B (2) together with the previously reported sterols, 24-ethyl-25-hydroxycholesterol (3), cholest-6-en-3,5,8-triol (4) and cholestane- 3b,5a,6b-26-tetrol (5) were isolated from the Red Sea ascidian Didemnum species. Their structures were determined using spectroscopic studies including 1D and 2D NMR data and high-resolution spectral determination. The sterols are reported here for the ﬁrst time from the ascidian Didemnum species.

Supplementary material Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 Supplementary material relating to this article is available online, alongside Figures S1–S11.

Acknowledgements We thank Mr Volker Brecht (Nuclear Magnetics Resonance, Institute fu¨r Pharmazeutische Wissenschaften, Albert-Ludwigs-Universita¨t Freiburg, Germany) for NMR and MS spectral measurements.

References Asai N, Fusetani N, Matsunaga S, Sasaki J. 2000. Sex pheromones of the hair crab Erimacrus isenbeckii. Part 1: Isolation and structures of novel ceramides. Tetrahedron. 56:9895–9899. Davis RA, Carroll AR, Pierens GK, Quinn RJ. 1999. New lamellarin alkaloids from the Australian ascidian Didemnum chartaceum. J Nat Prod. 62:419–424. de Carvalho MG, de Oliveira MCC, Werle AA. 2000. Chemical constituents from Luxemburgia nobilis (EICHL). J Braz Chem Soc. 11:232–236. Dharma RK, Trevor GR, Donald MS. 1985. Synthesis and polymorphism of 3-acyl-sn-glycerols. Biochemistry. 24:519–525. Ibrahim SRM. 2014. New chromone and triglyceride from Cucumis melo seeds. Nat Prod Commun. 9:205–208. Natural Product Research 7

Ibrahim SRM, Mohamed GA, Fouad MA, El-Khayat ES, Proksch P. 2009. Iotrochotamides I and II new ceramides from the Indonesian sponge Iotrochota purpurea. Nat Prod Res. 23:86–92. Kumaran NS, Bragadeeswaran S, Meenakshi VK. 2011. Evaluation of antibacterial activity of crude extracts of ascidian Didemnum psammathodes Sluiter, against isolated human and fish pathogens. Asian Pac J Trop Biomed. 1: S90–S99. Lambert G. 2002. Non indigenous ascidians in tropical waters. Pac Sci. 56:291–298. Lambert G. 2005. Ecology and natural history of the protochordates. Can J Zool. 83:34–50. Li H, Matsunaga S, Fusetani N. 1995. Halicylindrosides, antitfungal and cytotoxic cerebrosides from the marine sponge Halchonria cylindrata. Tetrahedron. 51:2273–2280. Liyanage GK, Schmitz FJ. 1996. Cytotoxic amides from the octocoral Telesto riisei. J Nat Prod. 59:148–151. Mitchell SS, Rhodes D, Bushman FD, Faulkner D. 2000. Cyclodidemniserinol trisulfate, a sulfated serinolipid from the Palauan ascidian Didemnum guttatum that inhibits HIV-1 integrase. Org Lett. 2:1605–1607. Mohamed GA, Ibrahim SRM, Badr JM, Youssef DTA. 2014. Didemnaketals D and E, bioactive terpenoids from a Red Sea ascidian Didemnum species. Tetrahedron. 70:35–40. Mohamed GA, Ibrahim SRM, Ross SA. 2013. New ceramides and isoflavone from the Egyptian Iris germanica L. rhizomes. Phytochem Lett. 6:340–344. Momin RA, Ramsewak RS, Nair MG. 2000. Bioactive compounds and 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9,12- octadecadienoyl]glycerol from Apium graveolens L. seeds. J Agric Food Chem. 48:3785–3788. Oku N, Matsunaga S, Fusetani N. 2000. Shishijimicins A-C, novel enediyne antitumor antibiotics from the ascidian Didemnum proliferum. J Am Chem Soc. 125:2044–2045. Pika J, Faulkner DJ. 1995. A reinvestigation of the didemnaketals from the palauan ascidian Didemnum sp. Nat Prod Lett. 7:291–296. Potts BCM, Faulkner DJ. 1991. Didemnaketals A and B, HIV-1 protease inhibitors from the ascidian Didemnum sp. J Am Chem Soc. 113:6321–6322. Qu Y, Zhang H, Liu J. 2004. Isolation and structure of a new ceramide from the basidiomycete Hygrophorus eburnesus.Z Naturforsch. 59B:241–244. Rudi A, Chill L, Aknin M, Kashman Y. 2003. Didmolamide A and B, two new cyclic hexapeptides from the marine ascidian Didemnum molle. J Nat Prod. 66:575–577. Sayed HM, Mohamed MH, Farag SF, Mohamed GA, Proksch P. 2007. A new steroid glycoside and furochromones from Cyperus rotundus L. Nat Prod Res. 21:343–350. Segraves NL, Lopez S, Johnson TA, Said SA, Fu X, Schmitz FJ, Pietraszkiewicz H, Crews P. 2003. Structures and cytotoxicities of fascaplysin and related alkaloids from two marine phyla-Fascaplysinopsis sponges and Didemnum tunicates. Tetrahedron Lett. 44:3471–3475. Shiao T, Shiao M. 1989. Determination of fatty acid composition of triacylglycerols by high resolution NMR spectroscopy. Bot Bull Acad Sin. 30:191–199. Siddiqui S, Faizi S, Siddiqui BS, Sultana N. 1989. Triterpenoids and phenanthrenes from leaves of Bryophyllum pinnatum. Phytochemistry. 28:2433–2438. Silverstein RM, Webster FX. 1998. Spectrometric identification of organic compounds. 6th ed. New York, NY: Wiley. Swaroop A, Sinha AK, Chawla R, Arora R, Sharma RK, Kumar JK. 2005. Isolation and characterization of 1,3- dicapryloyl-2-linoleoylglycerol: a novel triglyceride from berries of Hippophae rhamnoides. Chem Pharm Bull. 53:1021–1024. Downloaded by [Gamal Mohamed] at 08:21 20 June 2014 Takeara R, Jimenez PC, Wilke DV, Odorico de Moraes M, Pessoa C, Peporine Lopes N, Lopes JL, Monteiro da Cruz Lotufo T, Costa-Lotufo LV. 2008. Antileukemic effects of Didemnum psammatodes (Tunicata: Ascidiacea) constituents. Comp Biochem Physiol A: Mol Integr Physiol. 151:363–369. Toshiaki T, Hiroaki S, Kiyotake S. 2008. Hexamollamide, a hexapeptide from an Okinawan ascidian Didemnum molle. Tetrahedron Lett. 49:5297–5299. Vlahov G. 1999. Application of NMR to the study of olive oils. Prog Nucl Magn Reson Spectrosc. 35:341–357. Vlahov G. 2009. 13C nuclear magnetic resonance spectroscopy to determine fatty acid distribution in triacylglycerols of vegetable oils with ‘high – low oleic acid’ and ‘high linolenic acid’. Open Magn Reson J. 2:8–19. Vlahov G, Chepkwony PK, Ndalut PK. 2002. 13C NMR characterization of triacylglycerols of Moringa oleifera seed oil: an oleic-vaccenic acid oil. J Agric Food Chem. 50:970–975. Wang W, Tian S, Stark RE. 2010. Isolation and identification of triglycerides and ester oligomers from potential degradation of Potato suberin. J Agric Food Chem. 58:1040–1045. Wright AD, Goclik E, Ko¨nig GM, Kaminsky R. 2002. Lepadins D-F: antiplasmodial and antitrypanosomal decahydroquinoline derivatives from the tropical marine tunicate Didemnum sp. J Med Chem. 45:3067–3072. Youssef DTA, Ibrahim AK, Khalifa SI, Mesbah MK, Mayer AMS, van Soest RWM. 2010. New anti-inflammatory sterols from the Red Sea sponges Scalarispongia aqabaensis and Callyspongia siphonella. Nat Prod Commun. 5:27–31.