Original article Low toxicity and unprecedented anti-osteoclast activity of a simple sulfur-containing gem-bisphosphonate: A comparative study Donatella Granchi a, * , Alessandro Scarso b, 1 , Giulio Bianchini b, 1 , Andrea Chiminazzo b,1 , Alberto Minto b,1 , Paolo Sgarbossa c , Rino A. Michelin c , Gemma Di Pompo a , Sofia Avnet a , Giorgio Strukul b, * , 1 a Laboratorio di Fisiopatologia Ortopedica e Medicina Rigenerativa, Istituto Ortopedico Rizzoli, via di Barbiano 1/10, I-40136 Bologna, Italy b Dipartimento di Scienze Molecolari e Nanosistemi, Università Ca’ Foscari di Venezia, Dorsoduro 2137, I-30123 Venezia, Italy c Dipartimento di Processi Chimici dell’Ingegneria, Università degli Studi di Padova, via F. Marzolo 9, I-35131 Padova, Italy article info Article history: Received 22 January 2013 Received in revised form 8 April 2013 Accepted 11 April 2013 Available online 11 May 2013 Keywords: Bisphosphonates S-containing drugs Osteoclast Apoptosis Cytotoxicity abstract Bisphosphonates (BPs) are key drugs for the treatment of bone resorption diseases like osteoporosis, Paget’s disease and some forms of tumors. Recent findings underlined the importance of lipophilic N- containing BPs to ensure high biological activity. Herein we present some unprecedented results con- cerning the low toxicity and good anti-osteoclast activity of low molecular weight hydrophilic S-con- taining BPs. A series of S and N-containing BPs bearing aromatic and aliphatic substitution were prepared through Michael addition reaction between vinylidenebisphosphonate tetraethyl ester and the proper nucleophile under basic catalysis. S-containing BPs showed a generally low toxicity, determined with the neutral-red assay using the L929 cell line, and, in particular for an aliphatic one, a good biological activity assessed on primary cultures of human osteoclasts. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction Since 1970s, gem-bisphosphonates (BPs) 1 (Scheme 1) have been employed as drugs for the treatment of bone disorders, hypocalcaemia and osteoporosis [1] thanks to their structural similarity with pyrophosphate 2 a major constituent of hydroxy- apatite (HAP) present in the mineral portion of bones. This property ensures that bisphosphonate-based drugs are targeted only to bones (magic bullets) [2] and do not accumulate on other organs or tissues. The mineral portion of bone is a mix of inorganic HAP, carbonatoapatite (a carbonated form of the stoichiometric HAP), and proteins, mainly collagen. The continuous equilibrium action between osteoclasts, involved in resorption of the mineral portion of bones, and osteoblasts, involved in mineral formation, ensures health to the skeleton of mammals. Deregulation of osteoclast ac- tivity is associated with osteoporosis, metastasis-induced osteol- ysis, Paget’s disease, rheumatoid arthritis, and periodontal disease. In 2011 BPs celebrated 40 years of application in medicinal chemistry [3], but still the correlation between their chemical structure and biological activity is a highly debated topic [4,5]. Despite their successful use, a critical feature of BPs is their low oral bioavailability (1e2%) since these derivatives are highly hydrophilic and extensively charged and consequently poorly absorbed from the gastrointestinal tract after oral administration thus limiting transcellular permeation across the epithelial cells [6]. Two enzymes, both involved in the mevalonate pathway that provide lipids such as cholesterol and isoprenoids to the cell, are targets of BPs in the inhibition of osteoclasts, namely farnesyldi- phosphate synthase (FPPS) and geranylgeranyl diphosphate syn- thase (GGPPS) [7]. This feature allows also to achieve good inhibition properties toward a wide range of parasitic protozoan diseases like those caused by Plasmodium, Leishmania and Toxo- plasma species [8,9]. FPPS is an enzyme that converts dimethylallyldiphosphate in a longer farnesyldiphosphate containing a polyunsaturated C 10 fragment while GGPPS converts farnesyldiphosphate into a longer C 20 -diphosphate moiety. Inhibition of these enzymes turns out into osteoclast inactivation and apoptosis. Farnesyldiphosphate (FPP) biosynthesis proceeds via carbocationic transition state/reactive intermediates and this explains the high activity of the latest * Corresponding authors. E-mail addresses: donatella.granchi@ior.it (D. Granchi), strukul@unive.it (G. Strukul). 1 Tel.: þ39 041 2348569; fax: þ39 041 2438517. Contents lists available at SciVerse ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.04.032 European Journal of Medicinal Chemistry 65 (2013) 448e455