New cytotoxic halogenated sesquiterpenes from the Egyptian sea hare, Aplysia oculifera Mohamed-Elamir F. Hegazy a,⇑ , Alaa Y. Moustfa b , Abou El-Hamd H. Mohamed c , Montaser A. Alhammady d , Serag Eldin I. Elbehairi e , Shinji Ohta f , Paul W. Paré g a Chemistry of Medicinal Plants Department, and Center of Excellence for Advanced Sciences, National Research Centre, El-Tahrir Street, Dokki, Giza 12622, Egypt b Zoology Department, Faculty of Science, Sohag University, Egypt c Chemistry Department, Faculty of Science, Aswan University, Aswan, Egypt d National Institute of Oceanography and Fisheries, Red Sea Branch, Hurghada, Egypt e Egyptian Organization for Biological Products and Vaccines, Agouza, Giza, Egypt f Graduate School of Biosphere Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan g Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA article info Article history: Received 27 August 2013 Revised 11 January 2014 Accepted 22 January 2014 Available online 31 January 2014 Keywords: Aplysia oculifera Opisthobranch Halogenated sesquiterpenes Cytotoxicity abstract Two new sesquiterpenes, oculiferane (1) and epi-obtusane (2) have been isolated and identified from an acetone extract of the digestive gland of the sea hare, Aplysia oculifera. The structures were elucidated by spectroscopic analysis including HREIMS, 1 H, 13 C, DEPT, 1 H– 1 H COSY, HMQC, and HMBC NMR; the relative configuration was confirmed by X-ray analysis. Compounds 1 and 2 exhibited cytotoxic activity in vitro against several human cancer cell lines with IC 50 values in the low lg/ml range. Ó 2014 Elsevier Ltd. All rights reserved. Sea hares (Opisthobranch: Anaspidea) are herbivorous gastro- pod molluscs that feed on a diverse array of marine algae. 1,2 Based on a chemical defense strategy, sea hares store and concentrate food in digestive glands, 1,3,4 and as such, these glands are a rich source of toxic metabolites including polyketides, alkaloids, peptides, and terpenes. 5 In addition to these digestive glands being a diverse source of natural products, previous studies have estab- lished that such metabolites are also a rich source of medically active components ranging from general cytotoxic agents to antineoplastic agents that target specific cancers. Such biological activity has made sea hares a promising source of new natural products with high drug-lead potential. 6–9 Most chemical studies that focus on sea hare digestive glands from the genus Aplysia have led to the isolation of an abundance of halogenated mono-, di- and sesqui-terpenes that are thought to be derived from two red algae sources: Laurenica spp. and Plocamium spp. 9–12 Although sea hares have a worldwide distribution and have been studied chemically for over five decades, 11 this is the first report of Aplysia oculifera terpenes being isolated from the Red Sea. 13 The two isolated sesquiterpenes, oculiferane (1) and epi-obtusane (2), exhibited cytotoxic activity against several different cancer cell lines. An acetone extract of the digestive glands of the sea hare A. oculifera, was subjected to normal and reverse phase chromatogra- phy to afford two new halogenated sesquiterpenes, named oculife- rane (1) and epi-obtusane (2)(Fig. 1). Compound 1 was obtained as colorless crystals, ½a 25 D +46.0 (c 0.05, MeOH), and its low resolution mass spectrum showed a molecular ion peak [M] + at m/z 414. The HREIMS exhibited the molecular ion peak [M] + at m/z 414.9901 (calcd for C 15 H 25 Br 2 ClO: 414.9912). The molecular formula of C 15 H 25 Br 2 ClO was confirmed by 13 C NMR and DEPT data (Table 1), which showed 15 carbon signals representing: four quaternary carbons (47.7, 50.3, 67.5, and 78.7 ppm), two methines (63.9, and 67.3 ppm), five methyl- enes (29.6, 30.7, 30.9, 40.1, and 41.2 ppm), and four methyl groups (22.2, 22.6, 23.2, and 23.4 ppm). The carbon chemical shifts indicated the presence of one hydroxylated carbon at d C 78.7 (C- 6), as well as three halogenated carbons at d C 67.5 (C-3), d C 67.3 (C-4), and d C 63.9 (C-10). 1 H NMR analysis established the methyl groups appearing as four singlets at d H 1.01, 1.03, 1.17, and 1.78. The signals at d H 1.03 (H 3 -13) and d H 1.17 (H 3 -12) correlated in the HMBC indicating that both were located on the same quater- nary carbon at d C 47.7 (C-11). A doublet of doublets at d H 4.28 http://dx.doi.org/10.1016/j.tetlet.2014.01.096 0040-4039/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +20 1220073557; fax: +20 233370931. E-mail address: elamir77@live.com (M.-E.F. Hegazy). Tetrahedron Letters 55 (2014) 1711–1714 Contents lists available at ScienceDirect Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet