Labdane diterpenes with a new oxidation pattern from the marine pulmonate Trimusculus peruvianus Ana R. Dı ´az-Marrero, a Enrique Dorta, a Mercedes Cueto, a Juana Rovirosa, b Aurelio San-Martı ´n, b Alberto Loyola c and Jose ´ Darias a, * a Instituto de Productos Naturales y Agrobiologı ´a del CSIC, Avda. Astrofı ´sico F. Sa ´nchez 3, Apdo 195, 38206 La Laguna, Tenerife, Spain b Departamento de Quı ´mica, Facultad de Ciencias, Universidad de Chile, Santiago de Chile, Chile c Departamento de Quı ´mica, Facultad de Ciencias, Universidad de Antofagasta, Antofagasta, Chile Received 18 February 2003; revised 31 March 2003; accepted 30 April 2003 Abstract—Four new labdane diterpenes, 1-4, with an unusual oxidation pattern have been isolated from the marine pulmonate Trimusculus peruvianus. The structure and stereochemistry of these compounds were determined by spectroscopic data and chemical transformations. The absolute stereochemistry of 4 was assigned by application of the modified Mosher method. These compounds exhibit in vitro moderate cytotoxic activity against human colon carcinoma cell lines. q 2003 Elsevier Science Ltd. All rights reserved. 1. Introduction Whereas shell-less marine opisthobranch and nudibranch molluscs in particular, have the ability to sequester a wide variety of natural compounds from dietary source, 1 as well as to produce some de novo metabolites 2–4 that are presumably involved in their defence against predators, shell-bearing molluscs such as intertidal pulmonate limpets of the family Trimusculidae seem only to produce de novo secondary metabolites. 5 These sessile limpets, that live with unusual constraints, produce diterpenes which appear to be repellent to predatory fish, 6 and also toxic to larvae settling around them, 7 in order to avoid the presence of other invertebrates. At the present time, four species of Trimusculus have been studied and all of them have a common feature: they produce diterpenes belonging to a unique class of labdane skeleton revealing preferred oxidation sites along the framework. All naturally occurring diterpenes from T. reticulatus, 5,6 T. peruvianus, 8,9 T. conica 6 and T. costatus, 7 are oxidized at carbons C-6, C-7 and C-15, except one from T. peruvianus 8 which lacked oxidation at C-7. Only two metabolites, one isolated from T. reticulatus 5 and the other from T. costatus, are additionally oxidized at C-2. The labdane diterpenes from Trimusculus differ in the degree and type of esterification with acetoxy and isovaleroxy ester predominantly. In this work we report on the structure elucidation of four new diterpenes 1-4 featuring a novel oxidation pattern at C-3 and C-19, unlike other metabolites from this genus. These compounds exhibit in vitro moderate cytotoxic activity against human colon carcinoma cell lines. 2. Results and discussion From the crude ethyl acetate extract of T. peruvianus, collected near Antofagasta coast (Chile), compounds 1-4 were obtained after flash chromatography followed by gel filtration and successive HPLC. 0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-4020(03)00731-2 Tetrahedron 59 (2003) 4805–4809 * Corresponding author. Tel.: þ34-922-25-21-44; fax: þ34-922-26-01-35; e-mail: jdarias@ipna.csic.es Keywords: labdane diterpenes; pulmonate; carcinoma.