Research paper Synthesis and anticancer activity of novel fluorinated asiatic acid derivatives Bruno M.F. Gonçalves a, b , Jorge A.R. Salvador a, b, * , Silvia Marín c, d , Marta Cascante c, d, ** a Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548, Coimbra, Portugal b Center for Neuroscience and Cell Biology, Coimbra, Portugal c Department of Biochemistry and Molecular Biology, Faculty of Biology, Institute of Biomedicine of University of Barcelona, Diagonal 643, 08028 Barcelona, Spain d Institute of Biomedicine of University of Barcelona (IBUB) and Associated Unit to CSIC, Barcelona, Spain article info Article history: Received 18 August 2015 Received in revised form 26 January 2016 Accepted 24 February 2016 Available online xxx Keywords: Triterpenoids Asiatic acid Fluorinated derivatives Anticancer activity Apoptosis abstract A series of novel fluorinated Asiatic Acid (AA) derivatives were successfully synthesized, tested for their antiproliferative activity against HeLa and HT-29 cell lines, and their structure activity relationships were evaluated. The great majority of fluorinated derivatives showed stronger antiproliferative activity than AA in a concentration dependent manner. The most active compounds have a pentameric A-ring con- taining an a,b-unsaturated carbonyl group. The compounds with better cytotoxic activity were then evaluated against MCF-7, Jurkat, PC-3, A375, MIA PaCa-2 and BJ cell lines. Derivative 14 proved to be the most active compound among all tested derivatives and its mechanism of action was further investigated in HeLa cell line. The results showed that compound 14 induced cell cycle arrest in G0/G1 stage as a consequence of up-regulation of p21 cip1/waf1 and p27 kip1 and down-regulation of cyclin D 3 and Cyclin E. Furthermore, compound 14 was found to induce caspase driven-apoptosis with activation of caspases-8 and caspase-3 and the cleavage of PARP. The cleavage of Bid into t-Bid, the up-regulation of Bax and the down-regulation of Bcl-2 were also observed after treatment of HeLa cells with compound 14. Taken together, these mechanistic studies revealed the involvement of extrinsic and intrinsic pathways in the apoptotic process induced by compound 14. Importantly, the antiproliferative activity of this compound on the non-tumor BJ human fibroblast cell line is weaker than in the tested cancer cell lines. The enhanced potency (between 45 and 90-fold more active than AA in a panel of cancer cell lines) and selectivity of this new AA derivative warrant further preclinical evaluation. © 2016 Elsevier Masson SAS. All rights reserved. 1. Introduction Natural products are an indispensable source of lead structures for development of new drugs [1e4]. Triterpenoids are one of the largest classes of natural products with over 20 000 known members, and they are synthesized in a wide spectrum of plants by cyclization of squalene [5]. Over the last decades, an unique range of pharmacological activities of pentacyclic triterpenoids have been reported, including their enormous chemopreventive and anti- neoplastic potential [5e9]. Asiatic acid (AA,2a,3b,23-trihydroxyurs-12-ene-28-oic acid, Fig. 1) is a member of the ursane family, extracted mainly from the tropical medicinal plant Centella Asiatica [10]. This compound ex- hibits several pharmacological effects including hepatoprotective [11,12], neuroprotective and anti-alzheimer [13,14], antidiabetic, antihyperlipidemic [15,16], anti-inflammatory [17] and antioxidant [18]. Moreover, AA induces apoptotic cell death in several cancer cell lines [19e23], inhibits tumor cell proliferation [24], induces cell cycle arrest [25], inhibits TPA-induced tumor promotion in a rat model [26], increases sensitivity of colon cancer cells to treatment with camptothecin (CPT-11) [27], and exerts anti-angiogenic ac- tivity [28]. In recent years, some studies showed that chemical modifica- tion of AA can improve its anticancer activities. Jian-Fei et al. * Corresponding author. Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548, Coimbra, Portugal. ** Corresponding author. Department of Biochemistry and Molecular Biology, Faculty of Biology, Institute of Biomedicine of University of Barcelona, Diagonal 643, 08028 Barcelona, Spain. E-mail addresses: salvador@ci.uc.pt (J.A.R. Salvador), martacascante@ub.edu (M. Cascante). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech http://dx.doi.org/10.1016/j.ejmech.2016.02.057 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved. European Journal of Medicinal Chemistry 114 (2016) 101e117