A novel aryl-hydrazide from the marine lichen Lichina pygmaea: Isolation, synthesis of derivatives, and cytotoxicity assays Catherine Roullier, Marylène Chollet-Krugler * , Pierre van de Weghe, Françoise Lohézic-Le Devehat, Joël Boustie EA 4090 ‘Substances licheniques et photoprotection’, Faculté des Sciences pharmaceutiques et Biologiques, Université Européenne de Bretagne, Université de Rennes 1, 2 Av. du Pr. Léon Bernard, 35043 Rennes Cedex, France article info Article history: Received 26 April 2010 Revised 1 June 2010 Accepted 2 June 2010 Available online 8 June 2010 Keywords: Lichen Pygmeine Aryl-hydrazide Cytotoxic activity Lichina pygmaea abstract A new aryl-hydrazide L-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of L-glutamic acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human mela- noma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC 50 = 1.6 lM) on B16 cells. Ó 2010 Elsevier Ltd. All rights reserved. As a continuation of our ongoing work 1 on bioactive secondary metabolites isolated from lichens, we focused on Lichina pygmaea, which is a shrubby black marine lichen, found on the rocky shore at the seaside, in the intertidal region. This lichen results from the symbiosis of a fungus and a Calothrix cyanobacterium. Forming dark cushions of some millimeters thick, it suffers from harmful UV radiation, exposure to sun and reverberation, a salty environment, immersion phases, and the violence of waves. So, it is assumed that this lichen may possess protective secondary metabolites, and antioxidant properties are claimed for a mycosporine compound. 2,3 Two amino acid derivatives had previously been isolated, namely mycosporine-serinol (1), which has a good UVB filter profile, and the L-glutamic acid 5-[(2,4-dimethoxyphenyl)-hydrazide] (2). 4 This last compound was found to have antioxidant activities, 5 and is claimed to be a myeloperoxidase inhibitor as isolated from Penicil- lium cultures. 6 A third compound (3) has additionally been isolated from a crude methanolic extract of L. pygmaea, by Centrifugal Par- tition Chromatography (CPC) (Fig. 1). Low quantities obtained and ambiguous spectroscopic data for structure identification led us to synthesize analogs which were also tested for cytotoxicity in a pre- liminary assay on melanoma cell lines. Extraction by three successive solvents was performed on 270 g of the dried lichen, 7 namely ethyl acetate (1.5 L  3) at room temperature for 2 h, then methanol (1.5 L  7) at 45 °C for 2 h, and methanol 50% in water (1.5 L  3) at 45 °C for 2 h. A first frac- tionation was conducted on the filtrate of the methanolic extract of L. pygmaea by a multiple dual-mode centrifugal partition chroma- tography method (MDM CPC) previously described, 4 using BuOH/ AcOH/H 2 O (4:1:5) as the biphasic solvent system. Fraction Lp6 re- sulted in the isolation of compound 2 (121 mg) and fraction Lp8 in the isolation of compound 1 (25 mg). Fraction Lp7 was evaporated to dryness (1.065 g), and the residue was further partitioned between CH 2 Cl 2 and H 2 O. The aqueous layer was concentrated and subjected to chromatography over a reversed-phase silica gel 0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2010.06.013 * Corresponding author. Tel.: +33 2 232 348 96; fax: +33 2 232 347 04. E-mail address: marylene.chollet@univ-rennes1.fr (M. Chollet-Krugler). NH 2 O H N N H O OH 5 4 3 2 (S) 1 NH 2 O H N N H 1' 2' 3' 4' 5' 6' H 3 CO O OH OCH 3 (S) O OCH 3 HO HOH 2 C NH Mycosporine serinol 1 L-glutamic acid 5-[(2,4-dimethoxy phenyl)-hydrazide] 2 OH OH HO Unknown 3 (S) NH 2 O H N N H O OH Agaritine HO Figure 1. Structures of compounds isolated from L. pygmaea, and the similar agaritine. Bioorganic & Medicinal Chemistry Letters 20 (2010) 4582–4586 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl