Callyaerins A–F and H, new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa Sabrin R. M. Ibrahim a,  , Cho Cho Min a , Franka Teuscher a,à , Rainer Ebel b , Christel Kakoschke c , Wenhan Lin d , Victor Wray c, * , RuAngelie Edrada-Ebel e, * , Peter Proksch a, * a Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität, Geb. 26.23, Universitätsstrasse 1, D-40225 Düsseldorf, Germany b Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Building, Meston Walk, Old Aberdeen, AB24 3UE, Scotland, UK c Department of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany d State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Xueyuan Road, 38, Beijing 100083, China e Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, Scotland, UK article info Article history: Received 22 March 2010 Revised 26 May 2010 Accepted 4 June 2010 Available online 11 June 2010 Dedicated to the memory of Cho Cho Min Keywords: Callyspongia aerizusa Callyaerins Proline-rich cyclic peptides Cytotoxicity Antibacterial Antifungal abstract Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A–F (1–6) and H (8). Their structures were determined using exten- sive 1D ( 1 H, 13 C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the L form. Callyaerins A–F (1–6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED 50 values of 0.39 and 0.48 lM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Marine sponges are a rich source of bioactive cyclic peptides and depsipeptides with unique structures involving a wide variety of unusual amino acids and other building blocks. 1–4 Structure elu- cidation of such compounds can be quite challenging insofar as they defy traditional amino acid sequencing strategies. Many cyclic peptides isolated from marine sponges exhibit interesting biologi- cal properties including a reverse of multi-drug resistance in tumour cells, 5 cytotoxicity, 6 HIV inhibition, 7,8 and nematocidal activity. 9 The genus Callyspongia includes 182 species, of which around 15 species only have been investigated chemically. Sponges belonging to the genus have been known to yield biologically active polyacetylenic 10 and nitrotetradecenyl pyridine 11 com- pounds with different chain lengths and unsaturation positions. Nematocidal depsipeptides, phoriospongin A and B were isolated from the Australian marine sponge Callyspongia bilamellata. 9 To date only our previous work describing a preliminary investigation of the metabolic diversity of the Indonesian sponge Callyspongia aerizusa has provided an insight into the chemical potential of this sponge species. We were able to isolate a new cytotoxic cyclic pep- tide callyaerin G (7) 12 and found indications of a considerable number of other cyclic peptides present in the extract. These pep- tides were related analogues of callynormine A, a cyclic peptide isolated from the southern Kenyan sponge Callyspongia abnormis. 13 As a continuation of this work we have now completed a detailed investigation of the bioactive ethyl acetate fraction of this sponge. In addition to the callyaerin G (7) previously described by us, 12 here we describe the isolation, structure elucidation, and biological activities of a further seven callyaerin congeners of variable ring size and side-chain length. Most of the isolated compounds dis- played strong activity against the tumour cell line L5178Y, and varying degrees of antibacterial and antifungal properties. 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.06.012 * Corresponding authors. Tel.: +49 211 81 14163; fax: +49 211 81 11923 (P.P.); tel.: +49 531 6181 7200; fax: +49 531 6181 7099 (V.W.); tel.: +44 (0)141 548 3924; fax: +44 (0)141 552 2562 (R.E.-E.). E-mail addresses: victor.wray@helmholtz-hzi.de (V. Wray), ruangelie.edrada- ebel@strath.ac.uk (RuAngelie Edrada-Ebel), proksch@uni-duesseldorf.de (P. Proksch).   Permanent address: Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt. à Present address: Malaria Drug Resistance and Chemotherapy Laboratory, The Queensland Institute of Medical Research, 300 Herston Rd., Herston, Brisbane, Queensland 4006, Australia. Bioorganic & Medicinal Chemistry 18 (2010) 4947–4956 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc