two new glycerides from Red Sea ascidian Dide

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Didemnacerides A and B: two new glycerides from Red Sea ascidian Didemnum species

Sabrin R.M. Ibrahim^ab, Gamal A. Mohamed^cd, Lamiaa A. Shaala^ef, Diaa T.A. Youssef^cg & Ali A. Gab-Alla^h

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

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Didemnacerides A and B: two new glycerides from Red Sea ascidian Didemnum species

Sabrin R.M. Ibrahim^ab, Gamal A. Mohamed^cd, Lamiaa A. Shaala^ef, Diaa T.A. Youssef^cg*and Ali A. Gab-Alla^h

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 choles- tane-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 compounds3–5from the Red Sea ascidianDidemnumspecies.

Keywords:marine ascidian;Didemnumspecies; 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 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 & Faulkner1991; Pika & Faulkner1995). 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 & Schmitz1996). Compounds3–5are reported here for the first time from the Red Sea ascidianDidemnumspecies (Figure 1).

q2014 Taylor & Francis

*Corresponding author. Email:dyoussef@kau.edu.sa Natural Product Research, 2014

http://dx.doi.org/10.1080/14786419.2014.927874

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2. Results and discussion

Compound1was isolated as colourless oil. Its HR-ESI-MS revealed a pseudo-molecular ion peak atm/z 1011.9239 [MþH]^þ, 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 cm²¹in the IR spectrum along with signals atdC 172.8 (C-1⁰, 1⁰⁰⁰) and 173.3 (C-1⁰⁰) in ¹³C 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 of1 (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 atm/z 704.6235 [MþH – C₂₁H₃₉O]^þ and 397.3242 [MþH – C₄₂H₇₈O₂]^þ 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). The¹H NMR spectrum exhibited signals for olefinic protons at dH 5.36 (3H, each t, J¼6.0 Hz, H-6⁰, 6⁰⁰, 6⁰⁰⁰) and 5.34 (3H, each t, J¼6.0 Hz, H-7⁰, 7⁰⁰, 7⁰⁰⁰) correlated with carbons atdH129.9 (C-6⁰, 6⁰⁰⁰), 130.0 (C-6⁰⁰), 129.8 (C- 7⁰⁰) and 129.7 (C-7⁰, 7⁰⁰⁰) in the HSQC spectrum, also indicating the presence of three olefinic double bonds in1. The geometry of the double bonds was deduced to beZbased on the coupling constant values (J₆⁰_,7⁰¼J₆⁰⁰_,7⁰⁰¼J₆⁰⁰⁰_,7⁰⁰⁰¼6.0 Hz) (de Carvalho et al.2000; Ibrahim2014). The

1H NMR spectrum revealed characteristic signals for the terminal methyl protons of the fatty acids atdH0.87 (t,J¼6.5 Hz, H-21⁰⁰) and 0.85 (t,J¼6.5 Hz, H-21⁰, 21⁰⁰⁰), broad signals atdH

1.23 – 1.38 for the methylene protons, two methylene triplets atdH2.32 (H-2⁰⁰) and 2.31 (H-2⁰, 2⁰⁰⁰), and two multiplets atdH1.63 (H-3⁰⁰) and 1.61 (H-3⁰, 3⁰⁰⁰). In the COSY spectrum, H-2⁰⁰and 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 atdH

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 carbon signals atdC173.3 (C-1⁰⁰) and 172.8 (C-1⁰, 1⁰⁰⁰) showed correlations with proton signals at dH2.32 (2H, t,J¼7.5 Hz, H-2⁰⁰), 2.31 (4H, t,J¼7.5 Hz, H-2⁰, H-2⁰⁰⁰), 1.63 (2H, m, H-3⁰⁰) and 1.61 (4H, t, H-3⁰, 3⁰⁰⁰), also confirming that1is 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-1⁰⁰⁰indicated the Figure 1. Structures of the isolated compounds1 – 5.

2 S.R.M. Ibrahimet al.

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acyls of fatty acid moieties attached to C-1, C-2 and C-3. The proton signals atdH2.01 (8H, m, H-5⁰, 8⁰, 5⁰⁰⁰, 8⁰⁰⁰) and 2.02 (4H, m, H-5⁰⁰, 8⁰⁰) correlated with carbon signals atdC129.7 – 130.0 (C- 6⁰, 7⁰, 6⁰⁰, 7⁰⁰, 6⁰⁰⁰and 7⁰⁰⁰) along with the fragment peak atm/z308.3004 (C₂₁H₄₀O) in the HR-ESI- MS confirmed the presence of (6Z)-henicos-6-enoic acid moiety in1. Alkaline hydrolysis of1 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 was assigned as 1,2,3-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 cm²¹and an ester carbonyl at 1735 cm²¹(Silverstein & Webster1998; Ibrahim et al.

2009). The fragment ion peaks at m/z 720.6268 [MþH – H₂O]^þ and 704.6325 [MþH – 2H₂O]^þsuggested the presence of two hydroxyl functionalities in2and confirmed by signals for two oxymethines at dH 4.18 (1H, t, J¼6.2 Hz, H-2⁰) and 3.43 (1H, quin,J¼7.0 Hz, H-4⁰⁰) observed in the¹H NMR spectrum (Supplementary Figure S6). These protons correlated with the carbon signals atdC 68.2 (C-2⁰) and 71.7 (C-4⁰⁰) in the HSQC spectrum (Supplementary Figure S9). With the assistance of 2D NMR studies, including¹H –¹H COSY, HSQC and HMBC experiments, the assignments of¹H and¹³C NMR signals of2were established. The¹H and¹³C NMR spectra (Supplementary Figures S6 and S7) of2revealed signals atdH4.32 and 4.15 (each, dt,J¼6.0 and 12.5 Hz, H-1)/dC62.8 (C-1),dH5.19 (H-2)/dC70.1 (C-2) anddH3.53 (H-3)/dC

68.9 (C-3), along with the fragment ion peaks atm/z413.3625 [MþH-(2H₂OþC₂₀H₃₅O)]^þ and 323.3311 [MþH-(2H₂OþC₂₃H₄₁O₄)]^þcharacteristic for 2,3-diacyl glycerol moiety. The two carbon signals at dC 174.6 (C-1⁰) and 174.4 (C-1⁰⁰) suggested the presence of two ester carbonyls, confirming the diglyceride moiety of 2 (Momin et al. 2000; Vlahov et al. 2002;

Ibrahim2014). This was further secured by the HMBC correlations between H-1 and H-3 and C- 2, and H-2 and C-1 and C-3. Moreover, the signals atdH5.35 (2H, dt,J¼6.2 and 5.8 Hz, H-6⁰, 9⁰), 5.36 (1H, dt, J¼6.2 and 5.8 Hz, H-7⁰) and 5.34 (1H, dt, J¼6.2 and 5.8 Hz, H-10⁰) correlated with the carbon signals at dC 129.8 (C-6⁰, 9⁰), 129.9 (C-7⁰) and 128.8 (C-10⁰) indicating the presence of two olefinic double bonds. The geometry of the double bonds was deduced to beZbased on the coupling constant values (J₆0,7⁰andJ₉0,10⁰¼5.8 Hz) (de Carvalho et al.2000; Mohamed et al.2013). The signals for methylene group connected to a double bond carbon were observed atdH2.81 (1H, t,J¼6.2 Hz, H-8⁰)/dC25.6 (C-8⁰) and confirmed by its cross-peaks to H-7⁰and H-9⁰in COSY spectrum and with C-6⁰and C-10⁰in the HMBC spectrum.

The positions of the hydroxy groups and the double bonds were confirmed by the COSY cross- peaks of H-2⁰to H-3⁰, H-6⁰to H-5⁰and H-7⁰, H-10⁰to H-9⁰and H-11⁰, and H-4⁰⁰to H-3⁰⁰, H-5⁰⁰and further secured by the HMBC correlations of H-2⁰to C-1⁰and C-4⁰, H-4⁰with C-6⁰, H-6⁰to 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-1⁰and H-3 with C-1⁰⁰. Alkaline hydrolysis of2followed 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/z338 [M]^þ) moieties in2. It was reported thata-hydroxy FAMEs (R-isomer) had negative optical rotations (Li et al.1995; Asai et al.2000; Qu et al.2004). The FAMEs of2([a]_Dþ3.38and 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 that2 hadb-hydroxy fatty acids (S-isomer). Thus, the stereochemistry of2 was assigned by comparing the¹H and¹³C NMR chemical shifts, coupling constant values and optical rotation with the reported data for glyceride analogue (Dharma et al.1985; Ibrahim2014). 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.

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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 CDCl₃as 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£0.5mm, 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 650m/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 atlmax

255 and 366 nm followed by spraying withp-anisaldehyde/H2SO4.

3.2. Animal materials

The marine ascidianDidemnumspecies was collected in the Mangrove located in Nabq/Sharm 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 differentDidemnumspecies.

Didemnumspecies are characterised by many small zooids 1 – 2 mm in length, embedded in a sheet-like, gelatinous matrix called a tunic or test (Lambert2002). 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 (Lambert2002,2005). Spicules are 40mm in diameter on average, but can reach 100mm. 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 Didemnumspecies 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 theDidemnum species colony changes with age. Colonies of young 4 S.R.M. Ibrahimet al.

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Didemnumspecies 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 CH₂Cl₂and H₂O. The CH₂Cl₂extract (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) usingn-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) usingn-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) yielded2 (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 withn-hexane – EtOAc (85:15) after repeated purification steps through small columns yielded compounds3(6.5 mg, white crystals) and4(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 impure5, 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 yield5(11 mg, white crystals).

3.4. Alkaline treatment of compounds 1 and 2

Separate solutions of compounds1and2(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 between MeOH andn-hexane. Then-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; R_f0.84, Si 60 F₂₅₄(Figure S1); [a]_Dþ39.28 (c¼1.5, CHCl3); UVlmax(MeOH): 207 nm; IR (KBr)nmax2925, 2850, 1732, 1655, 1296, 1050 cm²¹;

1H NMR (CDCl₃, 400 MHz): dH 4.29 (1H, dd, J¼12.0 and 6.0 Hz, H-1A), 4.15 (1H, dd, J¼12.0 and 6.0 Hz, H-1B), 5.27 (1H, m, H-2), 4.31 (1H, dd,J¼12.0 and 6.0 Hz, H-3A), 4.14 (1H, dd, J¼12.0 and 6.0 Hz, H-3B), 2.31 (2£CH₂, each t, J¼7.5 Hz, H-2⁰, 2⁰⁰⁰), 1.61 (2

£CH₂, each m, H-3⁰, 3⁰⁰⁰), 1.32 (2£CH₂, each m, H-4⁰, 4⁰⁰⁰), 2.01 (2£CH₂, each m, H-5⁰, 5⁰⁰⁰), 5.36 (2£CH, each t,J¼6.0 Hz, H-6⁰, 6⁰⁰⁰), 5.34 (2£CH, each t,J¼6.0 Hz, H-7⁰, 7⁰⁰⁰), 2.01 (2

£CH₂, each m, H-8⁰, 8⁰⁰⁰), 1.23 – 1.38 (20£CH₂groups, each m, H-9⁰-18⁰and H-9⁰⁰⁰-18⁰⁰⁰), 1.25 (2£CH₂, each m, H-19⁰, 19⁰⁰⁰), 1.50 (2£CH₂, each m, H-20⁰, 20⁰⁰⁰), 0.85 (2£CH₃, each t, J¼6.5 Hz, H-21⁰, 21⁰⁰⁰), 2.32 (2H, t,J¼7.5 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,J¼6.0 Hz, H-6⁰⁰), 5.34 (1H, t,J¼6.0 Hz, H-7⁰⁰), 2.02 (2H, m, Natural Product Research 5

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H-8⁰⁰), (20£CH₂, each m, H-9⁰⁰-18⁰⁰), 1.25 (2H, m, H-19⁰⁰), 1.50 (2H, m, H-20⁰⁰), 0.87 (3H, t, J¼6.5 Hz, H-21⁰⁰);¹³C NMR (CDCl₃, 100 MHz):dC62.0 (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 (C-21⁰⁰); HR-ESI-MSm/z1011.9239 (calcd for C₆₆H₁₂₃O₆, [MþH]^þ, 1011.9241).

Didemnaceride B (2): colourless oil; R_f0.76, Si 60 F₂₅₄ (Figure S1); [a]_Dþ61.38 (c¼1.5, CHCl3); UV lmax (MeOH): 224 nm; IR (KBr) nmax 3495, 1735, 1291, 1056 cm²¹; ¹H NMR (CDCl3, 400 MHz):dH4.32 (1H, dt, J¼12.5 and 6.0 Hz, H-1A), 4.15 (1H, dt,J¼12.5 and 6.0 Hz, H-1B), 5.19 (1H, m, H-2), 3.53 (2H, m, H-3), 4.18 (1H, t,J¼6.2 Hz, H-2⁰), 1.77 (2H, m, H-3⁰), 1.42 (2H, m, 4⁰), 2.10 (2H, m, H-5⁰), 5.35 (1H, dt,J¼6.2 and 5.8 Hz, H-6⁰), 5.36 (1H, dt, J¼6.2 and 5.8 Hz, H-7⁰), 2.81 (2H, t,J¼6.2 Hz, H-8⁰), 5.35 (1H, dt,J¼6.2 and 5.8 Hz, H-9⁰), 5.34 (1H, dt,J¼5.8 Hz, H-10⁰), 2.10 (2H, m, H-11⁰), 1.38 (2H, m, H-12⁰), 1.21 – 1.35 (6£CH₂, each m, H-13⁰-H-18⁰), 1.40 (2H, m, H-19⁰), 0.89 (3H, t,J¼7.0 Hz, H-20⁰), 2.33 (2H, m, H-2⁰⁰), 1.55 (2H, m, H-3⁰⁰), 3.43 (1H, quin, J¼7.0 Hz, H-4⁰⁰), 2.02 (2H, m, H-5⁰⁰), 1.21 – 1.35 (15

£CH₂, each m, H-6⁰⁰-H-20⁰⁰), 1.15 (2H, m, H-21⁰⁰), 0.87 (3H, t,J¼6.8 Hz, H-22⁰⁰);¹³C NMR (CDCl₃, 100 MHz):dC62.8 (C-1), 70.1 (C-2), 68.9 (C-3), 174.6 (C-1⁰), 68.2 (C-2⁰), 34.2 (C-3⁰), 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);

HR-ESI-MSm/z737.6241 (calcd for C₄₅H₈₅O₇, [MþH]^þ, 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 ascidianDidemnumspecies.

Supplementary material

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.

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