Renieramide, a Cyclic Tripeptide from the Vanuatu Sponge Reniera n. sp. Linda Ciasullo, † Agostino Casapullo, † Adele Cutignano, † Giuseppe Bifulco, † Ce ´cile Debitus, ‡ John Hooper, § Luigi Gomez-Paloma, † and Raffaele Riccio* ,† Dipartimento di Scienze Farmaceutiche, Universita ` di Salerno, 84084 Fisciano (SA), Italy, IRD, Centre de Noume ´ a, BP A5, 98848 Noume ´ a, New Caledonia, and Queensland Centre for Biodiversity, Queensland Museum, P.O. Box 3300, South Brisbane, Qld, 4101, Australia Received July 27, 2001 The polar extract of the Vanuatu sponge Reniera n. sp., which showed immunomodulating activity in preliminary tests, was found to contain a cyclic tripeptide, which we named renieramide (1). This metabolite is identical to a synthetic derivative mentioned in a patent concerning the preparation of cyclic peptides of the OF4949 family of anticancer agents. We describe here the first isolation of this metabolite from natural sources and its complete characterization by spectroscopic and chemical approaches. Renieramide (1) possesses a 17-membered cyclic side-chain-linked biphenyl ether skeleton, typical of the class that includes the natural products OF4949 I-IV, K13, and eurypamides. A tridimensional model of 1, obtained by NMR restrained molecular mechanics and dynamics, is also presented. Natural and synthetic cyclic peptides containing an isodityrosine moiety are reported in the literature as antimicrobial, antiviral, potential antitumor, and antihy- pertensive agents. 1-6 A few glycopeptidic antibiotic agents belonging to the vancomycin group, such as teicoplanin, avoparcin, and ristocetin, contain an aryl ether bond between two phenyl moieties of amino acids. 1 The K-13 molecule, produced by Micromonospora halophytica subsp. exilisia K-13, is a dityrosine cyclic tripeptide exhibiting a potent noncompetitive inhibition of the angiotensin con- verting enzyme (ACE). 2 In 1986, Sano et al. isolated from cultures of Penicillium rugulosum OF4949 four compounds named OF4949 I-IV, a series of tricyclic compounds that inhibited aminopeptidase B in a competitive way, thus showing an antitumor activity due to a strong stimulation of the immune system. 3 Euripamide A from Microciona eurypa 4 and bastadin-5 from Ianthella basta 5 are further examples of compounds with the isodityrosine feature from marine sponges. Recently, the diphenyl ether scaffold has also been taken in consideration for generating new leads for HIV protease inibition. 6 Under the auspices of MAST III project “Bioactive Marine Natural Products in the Field of Antitumoral, Antiviral and Immunomodulatory Activity” 7 we investi- gated the crude extracts of a Vanuatu sponge, Reniera n. sp. (Haplosclerida), that exhibited immunomodulating activity in a preliminary pharmacological screening, and this led to the isolation of renieramide (1), a cyclic tripep- tide belonging to the class of OF4949 and euripamide A. Renieramide (1) has not been reported previously as natural product, although it was mentioned as a synthetic compound in a patent concerning the preparation of a cyclic peptide of the OF 4949 family of anticancer agents. 8 Besides describing its isolation from a marine source, we also report for the first time its complete spectroscopic characterization. The freeze-dried organism (430 g) was preliminarily extracted with MeOH, and after solvent evaporation, the oily residue was partitioned according to a modified Kupchan method, 9 giving n-hexane, CCl 4 , CHCl 3 , and n-butanol extracts. The butanol-soluble material, which was more interesting by preliminary chromatographic and spectroscopic analysis, was desalted and sequentially fractionated by Sephadex LH20 chromatography and RP- HPLC, affording renieramide (1) as a pure compound. Complete structural elucidation of renieramide (1) was performed by 1D and 2D NMR, ESIMS, and MS/MS data, along with chemical analysis. The ESIMS spectrum of compound 1 showed an intense pseudomolecular ion at 456 m/z [M + H] + corresponding to the molecular formula C 24 H 29 N 3 O 6 , as deducted by HRESIMS data (see Experimental Section). The IR spectrum of 1 contained broad bands centered at 3430 cm -1 attributable to OH, NH, and COOH groups and at 1692 cm -1 compatible with an amide functionality. These data, together with signals contained in the 1 H NMR spectrum (DMSO-d 6 ), were suggestive of a peptide. In fact, the 1 H NMR spectrum of 1 showed two signals typical of amide protons at δ 7.59 (d, J ) 9.8 Hz) and 7.75 (d, J ) 10.0 Hz) and three R-methine protons of amino acid residues at δ 4.36 (dt, J ) 9.8, 3.8 Hz), 4.28 (m), and 3.50 (m). The 24 signals contained in the 13 C NMR spectrum were assigned, on the basis of the HSQC data, to 12 aromatic (of which seven are methines) and nine aliphatic carbons (four methines, three methylenes, and two methyls) and three acylic carbonyls. A complete analysis of homonuclear * To whom correspondence should be addressed. Tel: +39 089 962818. Fax: +39 089 962828. E-mail: riccio@unisa.it. † Universita ` di Salerno. ‡ IRD, Centre de Noume ´a. § Queensland Museum. 407 J. Nat. Prod. 2002, 65, 407-410 10.1021/np010383u CCC: $22.00 © 2002 American Chemical Society and American Society of Pharmacognosy Published on Web 02/14/2002