1
A New Synthe+c Spiroketal: Studies on An+tumor Ac+vity on Murine Melanoma Model In Vivo and Mechanism of Ac+on In Vitro Maria Pia Fugge+a *1 , Pietro Spanu *2 , Fausta Ulgheri 2 , Francesco Deligia 2 , Giovanni Loriga 2 , Paola Carta 2 , Alberto Mannu 2 , Veronica Tro+a 3 , Rosanna De Cicco 3 , Adriano Barra 3 , Enrica Zona 3 , Franco Morelli *3 1 Is$tuto di Farmacologia Traslazionale - Consiglio Nazionale delle Ricerche, Roma, Italy 2 Is$tuto di Chimica Biomolecolare - Consiglio Nazionale delle Ricerche, Sassari, Italy 3 Is$tuto di Gene$ca e Biofisica A. Buzza$ Traverso - Consiglio Nazionale delle Ricerche, Napoli, Italy • INTRODUCTION: Inspired by bioacHve natural spiroketals, spirocyclic rigidified structures have demonstrated to be privileged scaffolds for the design of new tool compounds endowed with diversified biological acHviHes. We synthesised the natural-like spiroketal, 2-hydroxy-8-methyl-1,7- dioxaspiro[5.5]undec-3-en-5-one (5) that showed a potent anHcancer acHvity against human cancer cells of different nature and histotype. 1 In order to confirm the therapeuHc potenHal of this molecule we verified in vitro and in vivo, in a syngenic murine melanoma model, that our product is able to induce cancer regression and growth inhibiHon. Modulator of tubulin cytoskeleton integrity Phosphatase inhibitors Cytotoxic against cancer cells microtuble assembly inhibitor Proapoptotic activity O O Me N 2 OMe MeO O O O O O HN O O O COOH O HO OH HO O HO N O O O Me 2 3 4 5 6 7 8 O O Me Me OTBS AcO O O OH HO O O OBn Me 1 Me Cytotoxic agents towards primary CLL cells Telomerase inhibitor proapototic activity antitumor activity SGLT2 inhibitor Modulator of the RXRα gene tanscription factor anti-Helicobacter pylori activity OH • SYNTHESIS OF THE SPIROKETAL 5: The synthesis of the spiroketalic stereoisomeric mixture 5 a,b was carried out by using a two steps procedure based on 2-furyl ketone oxidaHon-rearrangement method. Because, in our previous experiments, the inhibiHon acHvity of the enanHomers was comparable, the steroisomeric mixture 5 a,b was used as such for in vitro and in vivo experiments. • CONCLUSION: We have idenHfied the spiroketal 5 a,b as a new promising anHcancer agent with in vivo acHvity on a murine melanoma model. Compound 5 a,b showed potent dose-dependent anHcancer efficacy in syngenic murine model (C57Black mice) of melanoma, suppressing cancer growth by an average of 90% at a dose of 5 mg/kg by one intra-peritoneum administraHon at alternate days for 15 days. It also displayed high anHcancer acHvity in the B16 cells in vitro with nanomolar IC 50 value. In addiHon to the proapoptoHc and telomerase inhibiHon acHvity previously observed, the compound 5 a,b has also shown to inhibit cell migraHon and the deterioraHon of the acHn cytoskeleton. Moreover our spiroketal strongly reduces the HIF1α expression that is considered as regulator of mulHple cellular funcHons related to the progression from primary to metastaHc disease. Therefore, although the full mechanism of acHon has yet to be completely elucidated, we can conclude that spiroketal 5 a,b is a promising anHcancer drug candidate for the clinical treatment of melanoma. O O O + O OH O NBS THF/H 2 0 BuLi TMEDA, THF * * 9 10 11 O O O 5a O O O 5b O O O HO OH HO + 12 • IN VITRO ACTIVITY: In the first set of experiments, the effect of spiroketal 5 a,b was evaluated in vitro on B16 cell growth in order to confirm on this murine model, the previously reported efficacy on human cell lines. Moreover, to acquire addiHonal informaHon on the origin of the anHproliferaHve acHvity, we have also studied the role of cell cycle modificaHon, the apoptosis inducHon, the migraHon characterisHcs of B16 cultured cells, the gene expression of the hypoxia inducible factor 1α (HIF1α) in B16 murine melanoma cells and the modificaHon of the acHn structure and cytoskeleton conformaHon induced by 5 a,b . • IN VIVO ACTIVITY: The assessment of the in vivo acHvity of the spiroketal 5 a,b in a well-known B16/C57BL/6J syngenic model of murine melanoma was performed. The effect of this compound on cancer growth reported in the Figures, showed a tumor volume reducHon with respect to the control. Control 24 h 48h ac.n HIF1α PCR expression analysis of HIF1α in presence of spiroketal 5 a,b Cell migra*on analysis. Culture of untreated control cells (le6 plate) and treated with 10 μg/ mL of 5 a,b a6er 24 hours (right plate). Proapopto'c in Vitro Ac'vity 0 50 100 control 5ab 0.62 μg/ml 5ab 1.25μg/ml5ab 2.5 μg/ml Luminescence An#prolifera#ve effect of 5a,b on B16 tumor cell line Cell growth inhibi#on evaluated by MTT test Cell Prolifera.on Inhibi.on Assay 0 200 400 600 800 0h 24h 48h 72h live cell number x 1000 B16 CT 0,781 3,125 12,5 25 50 200 0 20 40 60 0,39 0,78 1,56 3,125 6,25 12,5 25 50 100 200 Percent of growth inhibi.on Reorganiza*on of the cytoskeleton in response to 5a,b (B16 Cells) Tumor Growth Inhibi/on in Spiroketal Treated Mice • Acknowledgements: This study was supported by Regione Autonoma della Sardegna - Italy (research grant Dr. Paola Carta). We thank Dr. Maria CrisHna Porcu and Mr. Salvatore Baiano for technical assistance and Mr. Antonio Guicciardi for animal experiments. • References: 1) Fugge+a, M. P.; De Mico, A.; Co+arelli, A.; Morelli, F.; Zonfrillo, M.; Ulgheri, F.; Peluso, P.; Mannu, A.; Deligia, F.; Marchei, M.; Roviello, G.; Reyes Romero, A.; Dömling, A.; Spanu, P. Synthesis and EnanHomeric SeparaHon of a Novel Spiroketal DerivaHve: A Potent Human Telomerase Inhibitor with High in Vitro AnHcancer AcHvity. J. Med. Chem. 2016, 59, 9140−9149.
![2016 [Advances in Virus Research] Coronaviruses Volume 96 __ The Nonstructural Proteins Directing Coronavirus RNA Synthe](https://static.documents.pub/doc/80x56/613ca6cf9cc893456e1e874e/2016-advances-in-virus-research-coronaviruses-volume-96-the-nonstructural-proteins.jpg)
