Biological Activities and 3D QSAR Studies of a Series of Delisea pulchra (cf. fimbriata) Derived Natural Products Anthony D. Wright,* ,† Rocky de Nys, ‡ Cindy K. Angerhofer, § John M. Pezzuto, | and Marion Gurrath ⊥ Australian Institute of Marine Science, PMB No. 3, TownsVille MC, Qld 4810, Australia, School of Marine Biology and Aquaculture, James Cook UniVersity, TownsVille, Qld 4811, Australia, AVeda Corporation, Minneapolis, Minnesota 55449, College of Pharmacy, Nursing, and Health Sciences, Purdue UniVersity, West Lafayette, Indiana 47907, and Institute for Pharmaceutical Chemistry, Heinrich-Heine UniVersity, UniVersita ¨tsstrasse 1, D-40225 Du ¨sseldorf, Germany ReceiVed December 6, 2005 Twenty-five natural products, mainly halogenated furanones, isolated from the temperate red algae Delisea pulchra were investigated for their cytotoxic, antimicrobial, and antiplasmodial effects, their inhibition of the activity of the enzymes HIV-1-RT (HIV-1-reverse transcriptase), PKC (protein kinase C), and TK (tyrosine kinase), and their inhibition of the biosynthesis of IL-1 (interleukin-1). All were found to mediate a positive response in one or more of these test systems. In particular, compounds 9, 11, 12, 14, 16, 17, 19, and 20 demonstrated cytotoxic activity in all of the assays they were tested in; compounds 11, 12, 17, 19, and 20 were also active in the majority of the anti-infective screens. In the antimalarial and tyrosine kinase assays, compounds 17, 19, and 20 were all active. Molecular modeling studies employing 3D QSAR with receptor modeling methodologies performed with 16 halogenated furanones generated a pharmacophore hypothesis consistent with the experimentally derived cytotoxicity data. This hypothesis is developed around an active molecule having a framework based on compound 11 with an OH function or OAc (assay dependent) at C-7 and bulky electron-rich groups at C-6, such as Cl and Br but not I. Delisea pulchra (cf. fimbriata) 1 (Greville) Montage (Bonnemai- sonales, Bonnemaisoniaceae) is a common subtidal red alga in South Eastern Australia. It has been the focus of early pharmacological studies based on its strong antimicrobial activity. 2 D. pulchra produces a series of halogenated furanones 3-5 that play an important role in the ecology of the plant. 6,7 Furanones have strong feeding deterrent properties against generalist herbivores 7 and are bioac- cumulated by more specialized predators, possibly as a chemical defense. 8 They are also natural antifouling agents located at the surface of the plant, 9 where they deter the settlement and growth of epiphytic bacteria 6 and macrofouling organisms. 10 Further studies based on the potent biological activity of furanones against epiphytic bacteria have led to the elucidation of the mechanism of action of these compounds in inhibiting the growth and biofilm development of Gram-negative bacteria. Furanones specifically interfere with the acylated homoserine lactone (AHL) genetic regulatory system that controls the expression of density-dependent phenotypes in many bacteria 11,12 and also affect the alternative autoinducer 2 (AI- 2) signaling systems in both Gram-negative and Gram-positive bacteria. 13,14 They have been demonstrated to inhibit AHL regulated phenotypes in a diversity of bacteria including clinically important pathogens. 15,16 While the natural products chemistry and function of furanones have been well studied, there is less information on the pharma- cological activity of these compounds. The present study was undertaken to establish the potential of furanones as chemical leads that could be useful in the development of new therapeutic agents. To do this, a 3D QSAR molecular modeling study was performed with biologically active molecules to develop a pharmacophore hypothesis for cytotoxicity. Results and Discussion D. pulchra was collected from Cape Banks, New South Wales, Australia, and yielded 25 natural products, the majority of which are halogenated furanones. 4,5 Biological testing of these compounds toward pharmacologically relevant targets was undertaken using in vitro bioassays. These studies targeted cytotoxic, antimicrobial, and antimalarial activities, enzyme inhibition for HIV-1-RT, PKC and TK, and the inhibition of IL-1 and protein biosynthesis. Compounds were limited in availability, and therefore not all compounds were used in all assays. Evaluation of Cytotoxic Potential with Cultured Cancer Cell Lines. To determine the antineoplastic potential of the natural products, cytotoxic effects were evaluated with a battery of cultured tumor cells (Table 1). Of the 19 compounds that were tested (Table 1), 13 demonstrated ED 50 values of e4 μg/mL in one or more human cancer cell test systems and were classified as active. 16 Compounds 11, 12, 14, 16, 17, and 20 demonstrated the broadest spectrum of activity. Five compounds (3, 8, 13, 21, and 25) had weak activity (20 g ED 50 g 4 μg/mL), while only one, an acyclic molecule (23), was completely inactive (ED 50 g 20 μg/mL). Of the 13 active compounds, 11, 14, and 16 reversed vinblastine resistance in KB-V1 cells to some extent, demonstrating greater activity toward “drug-resistant” KB+V1 cells treated with vinblas- tine, compared to the cells without vinblastine treatment. Some selective cytotoxicity was also observed for compounds 13 and 21. They were inactive toward the sensitive KB-V1 cells but mod- erately toxic toward human melanoma cells (Mel2). Antimicrobial Activity. Furanones have strong antimicrobial activity; however, testing has been limited to the four major metabolites of D. pulchra. 2 A larger array of furanones and other metabolites from D. pulchra were tested using bioautographic assays that are efficient at detecting antimicrobial compounds. 18 Localiza- tion of activities on developed TLC plates is useful for testing crude extracts and directing isolation procedures. The current study employed a direct bioautographic technique with microorganisms grown directly on TLC plates. 19 Twenty-four compounds were evaluated using the fungus Penicillium oxalicum, the Gram-positive bacteria Bacillus subtilis and Micrococcus luteus, and the Gram- negative bacterium Escherichia coli as test organisms. Furanones were significantly more active than the acyclic compounds (22-24) in all assays. There was a marked distinction between the antimicrobial effects of furanones against Gram- positive and Gram-negative bacteria, with more compounds having * To whom correspondence should be addressed. Tel: +61 7 4753 4204. E-mail: a.wright@aims.gov.au. Web site: www.aims.gov.au. † Australian Institute for Marine Science. ‡ James Cook University. § Aveda Corporation. | Purdue University. ⊥ Heinrich-Heine University. 1180 J. Nat. Prod. 2006, 69, 1180-1187 10.1021/np050510c CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 08/04/2006