Original article
Low toxicity and unprecedented anti-osteoclast activity of a simple sulfur-containing gem-bisphosphonate: A comparative study

https://doi.org/10.1016/j.ejmech.2013.04.032Get rights and content

Highlights

  • We prepared five bisphosphonates (BPs) classified into four classes.

  • Toxicological screening of BPs was performed comparing to a commercially available BP.

  • Cytotoxicity of BPs was measured with neutral-red (NR) assay on the L929 cell line.

  • Biological activity of BPs was evaluated on primary cultures of osteoclasts.

  • S-containing BPs provides new contributions for more potent and better tolerated BPs.

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 concerning the low toxicity and good anti-osteoclast activity of low molecular weight hydrophilic S-containing 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.

Graphical abstract

A comparative study on several bisphosphonates showed that aliphatic S-containing species provide low toxicity and good anti-osteoclast activity assessed through primary culture of osteoclasts.

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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 hydroxyapatite (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 activity is associated with osteoporosis, metastasis-induced osteolysis, 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 (1–2%) 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 farnesyldiphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (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 Toxoplasma species [8], [9].

FPPS is an enzyme that converts dimethylallyldiphosphate in a longer farnesyldiphosphate containing a polyunsaturated C10 fragment while GGPPS converts farnesyldiphosphate into a longer C20-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 generation bisphosphonates bearing pyridinium residues. The active site of FDPS accommodates the BP moiety via coordination to a Mg(II) metal center and the most potent inhibitors, containing basic N atoms on C4 that are protonated at physiological pH, provide strong hydrogen bonding recognition with specific aminoacids (Lys200 and Thr201). GGPPS is inhibited by more hydrophobic BPs having long aliphatic or aromatic residues because in this case the active site of the enzyme is more hydrophobic and does not contain hydrogen bonding aminoacids. The hydrophobic character of BPs is a common aspect that greatly influences their activity. Very recently it has been demonstrated that such enzymes can be efficiently inhibited by more hydrophobic BPs lacking the gem-OH moiety and containing a positive N center and a long aliphatic chain [6](b), [10]. Similarly, hydrophobic BPs bearing rigid polycyclic bile acid moieties showed enhanced biological activity compared to commercial BPs drugs like neridronate [11].

Although BPs are the most potent of the currently approved anti-resorptive agents, their effectiveness is related strictly to drug adherence, both in terms of compliance (i.e., proportion of the total treatment time that is covered by the dispensed medication) and persistence (i.e., continuation of treatment over time). Epidemiologic studies have shown that independent predictors of poor adherence to BP therapy include more frequent dosing, adverse upper gastrointestinal effects, treatment cost, lack of disease symptoms, and practical difficulties with the administration regimen [12], [13]. BPs with R2 = OH and containing nitrogen derivatives in R1, like pamidronate 3a, alendronate 3b and zoledronate 4, are by far the most studied class of anti-resorption commercial drugs [14]. On the other hand, N-containing aromatic species are characterized by high toxicity that favors the non-adherence to treatment thus limiting the therapeutic benefit [15]. However, the BP moiety is intrinsically poorly toxic and the development of new BPs characterized by low toxicity and good anti-osteoclast activity is a very important issue.

We turned our attention to S-containing BPs that are an under-investigated class of BPs. One example is the commercially available tiludronate 5 (Scheme 1), known under the commercial name Skelid®, that contains a S atom in β-position. On the other hand in the more recent literature only a limited number of contributions deal with S-containing BPs. Some describe exclusively the synthesis of such class of BPs in the ester protected form [16], and only a few report the formation of the corresponding bisphosphonic acids [17] and their potential effects on osteoclast's activity [18]. Tiludronate 5, like other non-nitrogenous BPs can be incorporated into molecules of adenosine triphosphate (ATP), creating non hydrolysable analogs of ATP molecules called cytotoxic ATP. Nevertheless, tiludronate 5 has low cellular potency and low mineral affinity in comparison to the more potent N-containing BPs, and the clinical use is limited to the treatment of Paget's disease and in veterinary orthopedics [19]. Worth of mention is a very recent contribution where sulphonylamino derived BPs 6 bearing sulphone moiety in the α or β-position proved to be powerful bone resorption inhibitors as assessed by cell line J774 and HepG2 survival assay, thus suggesting that chemical structure of S-containing BPs could have a pivotal role in enhancing their therapeutic effectiveness [20].

Herein we present a comparative study on the toxicity and osteoclast inhibition activity between some known N-containing BPs and one new S-containing BPs (812, Scheme 2), either containing an aromatic portion or an aliphatic substituent on position R,1 in all cases lacking the presence of the gem-hydroxyl moiety R2 which has been demonstrated not to be crucial for the biological activity of this class of molecules [10]. The aim of the work is to address whether small S-containing BPs provide good anti-osteoclast activity while maintaining a low toxicity level which is an extremely important aspect for further drug development.

Section snippets

Chemistry: synthesis of BP derivatives bearing N and S substituents

We prepared five BPs reported in Scheme 2 that can be classified into four classes depending on the presence of heteroatoms in R1 like N (810) or S (1112) and on the presence of an aromatic ring (910 and 12) or an aliphatic moiety (8 and 11). The synthesis of the BPs was accomplished by reaction of the proper heteroatomic nucleophile with vinylidenebisphosphonate tetraethyl ester 7 [21] under triethylamine catalysis, with an improved procedure compared to those reported in the literature [22]

Conclusion

In this contribution a comparative study on the HAP affinity, toxicity and anti-osteoclast activity of a series of aromatic and aliphatic N- and S-containing BPs showed that the latter class of molecules provides new contributions for the development of more potent and better tolerated BPs. This observation well complements recent finding on other classes S-containing BPs that showed promising properties as antitumor agents for breast, cervix, liver and colon cancer diseases [18](b), [18](c).

Reagents and materials

General. 1H NMR, 31P{1H} NMR, spectra were run on a Bruker Avance 300 spectrometer operating at 300.15, (121.5), MHz, respectively, at 298 K, unless otherwise stated. δ values in ppm are relative to Si(CH3)4, 85% H3PO4. GC–MS analyses were performed on a GC Trace GC 2000 coupled with a quadrupole MS Thermo Finnigan Trace MS with Full Scan method. Experimental conditions are reported in the following Table 3.

Vinilydenebisphosphonate tetraethyl ester 7 was prepared as reported in the literature

Acknowledgments

The authors acknowledge MIUR for funding PRIN project 2008. L. Sperni is gratefully acknowledged for GC–MS analysis.

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