Enhancing Macrocyclic Diterpenes as Multidrug-Resistance Reversers: Structure-Activity Studies on Jolkinol D Derivatives Mariana Reis, † Ricardo J. Ferreira, † Maria M. M. Santos, † Daniel J. V. A. dos Santos, † Joseph Molna ́ r, ‡ and Maria-Jose ́ U. Ferreira* ,† † Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farma ́ cia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-019, Lisboa, Portugal ‡ Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dó m té r 10, H-6720 Szeged, Hungary * S Supporting Information ABSTRACT: The phytochemical study of Euphorbia pisca- toria yielded jolkinol D (1) in a large amount, whose derivatization gave rise to 12 ester derivatives (2-13) and hydrolysis to compound 14. The in vitro modulation of P-gp of compounds 1-14 was evaluated through a combination of transport and chemosensitivity assays, using the L5178 mouse T lymphoma cell line transfected with the human MDR1 gene. Apart from jolkinol D, all derivatives (2-14) showed potential as MDR reversal agents. In this small library of novel bioactive macrocyclic lathyrane diterpene derivatives, designed to evaluate structure-activity relationships essential in over- coming multidrug resistance (MDR), some correlations between MDR reversal and molecular weight, accessible solvent areas, and octanol/water partition coefficient were identified that can contribute to the development of new selective P-gp reversal agents. 1. INTRODUCTION Multidrug resistance (MDR) designates a phenomenon where resistance to one drug is accompanied by resistance to drugs that are structurally and functionally unrelated. The develop- ment of MDR and subsequent failure of chemotherapy are widespread problems in many types of tumors. One of the most significant mechanisms of MDR results from overexpression of membrane efflux pumps belonging to the evolutionarily conserved family of ATP binding cassette (ABC) proteins, which transports anticancer drugs out of the cells, preventing them from reaching their cellular targets. A very important member of this superfamily is P-glycoprotein (P-gp). 1-3 This protein, with 170 kDa, is the product of the human multidrug resistance (MDR1) gene. It is classified as a pseudosymmetrical heterodimer, where each monomer is composed of six membrane spanning segments (transmembrane domain, TMD) and one nucleotide-binding domain (NBD). The TMDs mediate the recognition and transport of substrates, and the NBDs are responsible for the ATP-binding and hydrolysis and consequently for the generation of conforma- tional changes on the protein. 4-7 One of the most accepted strategies to overcome MDR mediated by P-gp is based on the development of reversal agents that when coadministered with an anticancer drug will circumvent its efflux and consequently will avoid chemotherapy failure. 8,9 In this manner, a considerable number of natural and synthetic compounds have been described in the literature as P- gp reversal agents. Nevertheless, despite encouraging results in in vitro assays, to date, there are no reversal agents clinically available. 10-12 For instance, first-generation drugs like verapamil 13 and cyclosporine A, 14 besides revealing low affinity for P-gp, had distinct pharmacological actions on the cardiovascular (calcium channel blocker) and immune (immunosuppressant) systems. As the high serum concen- trations required for P-gp inhibition severely increased their adverse effects, analogues of these drugs (without intrinsic pharmacological activity) were designed as a way to reduce their side effects (second-generation modulators); however, they also showed increased toxicity 15 and alterations in the pharmacokinetics of cytotoxic drugs. 16 Third generation MDR modulators, like tariquidar (XR9576) 17 and zosuquidar (LY335979), 18 inhibit P-gp at the nanomolar level and were until recently under development but have not been approved for clinical use so far. The hampering of progress in this area can be attributable to the lack of knowledge concerning drug- transporter interactions to a certain extent because of the absence of detailed structural information of human P-gp. Meanwhile, in 2009 the structure of the first mammalian ABC transporter (murine, with 87% sequence identity to human P- gp) was published, showing for the first time P-gp complexed with a ligand. 19 Despite its low resolution (3.8 Å), it represents Received: July 12, 2012 Published: January 21, 2013 Article pubs.acs.org/jmc © 2013 American Chemical Society 748 dx.doi.org/10.1021/jm301441w | J. Med. Chem. 2013, 56, 748-760