Alendronic Acid as Ionic Liquid: New Perspective on Osteosarcoma

Herein the quantitative synthesis of eight new mono- and dianionic Organic Salts and Ionic Liquids (OSILs) from alendronic acid (ALN) is reported by following two distinct sustainable and straightforward methodologies, according to the type of cation. The prepared ALN-OSILs were characterized by spectroscopic techniques and their solubility in water and biological fluids was determined. An evaluation of the toxicity towards human healthy cells and also human breast, lung and bone (osteosarcoma) cell lines was performed. Globally, it was observed that the monoanionic OSILs showed lower toxicity than the corresponding dianionic structures to all cell types. The highest cytotoxic effect was observed in OSILs containing a [C2OHMIM] cation, in particular [C2OHMIM][ALN]. The latter showed an improvement in IC50 values of ca. three orders of magnitude for the lung and bone cancer cell lines as well as fibroblasts in comparison with ALN. The development of OSILs with high cytotoxicity effect towards the tested cancer cell types, and containing an anti-resorbing molecule such as ALN may represent a promising strategy for the development of new pharmacological tools to be used in those pathological conditions.


Introduction
Alendronic acid is an aminobisphosphonate derivative that has shown efficacy in postmenopausal osteoporosis, malignant hypercalcemia and Paget's disease [1]. Alendronate localizes preferentially at active sites of bone resorption, which has been inhibited at doses that have no effect on bone mineralization [2].
Bisphosphonates bind at the bone mineral surface, where they potently inhibit osteoclast-mediated bone resorption and subsequently embed in the bone, being released only during subsequent resorption [3]. In contrast to other antiresorptive agents, bisphosphonates with the greatest binding affinity to bone (zoledronic acid > alendronate > ibandronate > risedronate) may persist in bone, and patients continue to be exposed to the pharmacologic effects of these drugs several years after discontinuation.
All bisphosphonates rapidly reduce bone resorption, which leads to decreased bone formation because resorption and formation are coupled. Within three to six months, equilibrium is reached at a lower rate of bone turnover [4].
It is described that alendronate in rats exhibited 200 to 1000 times more potency than etidronate and approximately 100 times more in comparison with clodronate or tiludronate [5]. It is recognized that the presence of amino group side-chain from alendronate chemical structure contributes to greater potency and specificity [3][4][5].
The introduction of specific functional groups in the BPs structure can lead to modifications in their physicochemical, biological, therapeutic and toxicological properties. In recent literature studies, alendronic acid is reported as an FDA drug already approved for the prevention and treatment of osteoporosis in men and women, either postmenopausal or glucocorticoid-induced [6]. However, BPs present low bioavailability when administered orally and frequently require parenteral administration, which is not the most convenient route in case of continuous treatment.
Thus, considering the pharmaceutical importance of bisphosphonate drugs as antiresorptive and potential antitumoral agents, it is of paramount importance that novel formulations of such drugs, which render higher bioavailability and lower systemic toxicity than the latter are developed. Hence, in this paper, we report our latest results on our research line regarding bisphosphonate-based OSILs. In particular, we describe the synthesis of eight new Ionic Liquids and Organic Salts from alendronic acid (ALN-OSILs) as mono-and dianion by combination with one or two units of biocompatible cations, respectively. The desired compounds were prepared in quantitative yields by two distinct sustainable and straightforward methodologies, according to the type of cation. All prepared ALN-OSILs were characterized by spectroscopic techniques and their solubility in water and biological fluids was determined. Finally, evaluation of the toxicity towards human healthy cells and lung, breast and bone (osteosarcoma) cell lines was performed. Figure 1 depicts the structures of ALN and the selected cations, which were combined in 1:1 or 1:2 stoichiometric ratios, thereby deprotonating one or two phosphonate groups, respectively.
On the other hand, a combination of ALN with one and two equivalents of [Ch] and [C2OHMIM] cations yielded four more ALN-based OSILs (compounds 5-8) by following another methodology already described by our group [15,22]. In this case, quaternary ammonium hydroxide cations are previously prepared by the corresponding halide exchange with hydroxide exchange resins (e.g., Amberlyst A26-OH) in methanolic solution. The very basic solutions were then neutralized by addition to an aqueous solution of bisphosphonate yielding the desired salts in quantitative yields. From the four synthesized compounds, two are RTILs while the other two are solids (see below).
All products were characterized by NMR ( 1 H and 13 C) and FTIR spectroscopic techniques, as well as elemental analysis. The thermal properties were evaluated by DSC and the solubility in water and saline solution was determined for all compounds.
The NMR spectra of the ALN-OSILs were acquired in D2O taking advantage of their high solubility in water (see below). In all cases, the 1 H NMR spectra showed that the cation/anion The preparation of compounds 1-4 consisted of the addition of the diluted superbases to a dispersion of ALN (pathway A). These particular compounds, termed Superionic Liquids of APIs, were prepared according to a previous methodology reported by us for zoledronic acid [22] as well as other APIs [16]. While both monoanionic ALN-OSILs were obtained as solids, the two dianionic were isolated as pastes.
On the other hand, a combination of ALN with one and two equivalents of [Ch] and [C2OHMIM] cations yielded four more ALN-based OSILs (compounds 5-8) by following another methodology already described by our group [15,22]. In this case, quaternary ammonium hydroxide cations are previously prepared by the corresponding halide exchange with hydroxide exchange resins (e.g., Amberlyst A26-OH) in methanolic solution. The very basic solutions were then neutralized by addition to an aqueous solution of bisphosphonate yielding the desired salts in quantitative yields. From the four synthesized compounds, two are RTILs while the other two are solids (see below).
All products were characterized by NMR ( 1 H and 13 C) and FTIR spectroscopic techniques, as well as elemental analysis. The thermal properties were evaluated by DSC and the solubility in water and saline solution was determined for all compounds.
The NMR spectra of the ALN-OSILs were acquired in D2O taking advantage of their high solubility in water (see below). In all cases, the 1 H NMR spectra showed that the cation/anion The preparation of compounds 1-4 consisted of the addition of the diluted superbases to a dispersion of ALN (pathway A). These particular compounds, termed Superionic Liquids of APIs, were prepared according to a previous methodology reported by us for zoledronic acid [22] as well as other APIs [16]. While both monoanionic ALN-OSILs were obtained as solids, the two dianionic were isolated as pastes.
On the other hand, a combination of ALN with one and two equivalents of [Ch] and [C 2 OHMIM] cations yielded four more ALN-based OSILs (compounds 5-8) by following another methodology already described by our group [15,22]. In this case, quaternary ammonium hydroxide cations are previously prepared by the corresponding halide exchange with hydroxide exchange resins (e.g., Amberlyst A26-OH) in methanolic solution. The very basic solutions were then neutralized by addition to an aqueous solution of bisphosphonate yielding the desired salts in quantitative yields. From the four synthesized compounds, two are RTILs while the other two are solids (see below).
All products were characterized by NMR ( 1 H and 13 C) and FTIR spectroscopic techniques, as well as elemental analysis. The thermal properties were evaluated by DSC and the solubility in water and saline solution was determined for all compounds.
The NMR spectra of the ALN-OSILs were acquired in D 2 O taking advantage of their high solubility in water (see below). In all cases, the 1 H NMR spectra showed that the cation/anion proportion is strictly 1.0:1.0 or 2.0:1.0, in agreement with the intended stoichiometry (see Figures S1-S16). In addition, only one set of signals was observed, meaning that the reactions were complete and only one product was formed. No comparison with alendronic acid is achievable because of its lack of solubility in the same solvents as the ALN-OSILs. In the 13 C NMR spectra, the resonance of the quaternary carbon atom of ALN appears at ca. 74 ppm, with no particular difference if only one or two neighboring phosphonate groups are deprotonated. Similarly, the 1 H NMR data is also irreflective of the ionic state of the bisphosphonates and also of the cations. In contrast, the collected FTIR spectra (Figures S17-S25) show pristine variations between the neutral bisphosphonates and the ALN-OSILs, as well as between mono-and dianions. The FTIR spectrum of ALN shows two characteristic regions, namely a weak and undefined structure at 2400-2000 cm −1 with maximum intensity at 2256 cm −1 , and also very intense multiple peaks at 1250-900 cm −1 (see Figure 2A). While the first one accounts for O-H stretches from the O=P-OH groups, the second region contains peaks assignable to the stretch of both P=O and P-OH bonds [24]. In the spectra of the synthesized ALN-OSILs (see Figure 2 for [TMGH]-based ALN-OSILs), the peaks in the first region become sharper and much weaker, and two other sets of peaks appear in the vicinity (at ca. 2400-2300 cm −1 and 2200-2100 cm −1 ) which are more intense for the dianionic ALN-OSILs than for the monoanionic. This is in complete agreement with changes in the vibrational modes of the OH groups from the phosphonate moieties that are particularly enhanced when both groups are deprotonated. This corollary is also sustained by the changes observed in the second region of peaks, which in general displays two very intense broad peaks at ca. 1160 cm −1 and 1060 cm −1 , and one or two of medium intensity between 960 and 900 cm −1 .
Pharmaceutics 2020, 12, x FOR PEER REVIEW 4 of 10 proportion is strictly 1.0:1.0 or 2.0:1.0, in agreement with the intended stoichiometry (see Figures S1-S16). In addition, only one set of signals was observed, meaning that the reactions were complete and only one product was formed. No comparison with alendronic acid is achievable because of its lack of solubility in the same solvents as the ALN-OSILs. In the 13 C NMR spectra, the resonance of the quaternary carbon atom of ALN appears at ca. 74 ppm, with no particular difference if only one or two neighboring phosphonate groups are deprotonated. Similarly, the 1 H NMR data is also irreflective of the ionic state of the bisphosphonates and also of the cations. In contrast, the collected FTIR spectra (Figures S17-S25) show pristine variations between the neutral bisphosphonates and the ALN-OSILs, as well as between mono-and dianions. The FTIR spectrum of ALN shows two characteristic regions, namely a weak and undefined structure at 2400-2000 cm −1 with maximum intensity at 2256 cm −1 , and also very intense multiple peaks at 1250-900 cm −1 (see Figure 2A). While the first one accounts for O-H stretches from the O=P-OH groups, the second region contains peaks assignable to the stretch of both P=O and P-OH bonds [24]. In the spectra of the synthesized ALN-OSILs (see Figure 2 for [TMGH]-based ALN-OSILs), the peaks in the first region become sharper and much weaker, and two other sets of peaks appear in the vicinity (at ca. 2400-2300 cm −1 and 2200-2100 cm −1 ) which are more intense for the dianionic ALN-OSILs than for the monoanionic. This is in complete agreement with changes in the vibrational modes of the OH groups from the phosphonate moieties that are particularly enhanced when both groups are deprotonated. This corollary is also sustained by the changes observed in the second region of peaks, which in general displays two very intense broad peaks at ca. 1160 cm −1 and 1060 cm −1 , and one or two of medium intensity between 960 and 900 cm −1 .

Thermal Analysis of ALN-OSILs
All prepared OSILs from alendronic acid were studied by Differential Scanning Calorimetry (DSC) techniques (see Figures S26-S33). Table 1 contains the obtained data, namely melting, crystallization and glass transition temperatures, as well as the physical state of the analyzed compounds.
In general, the monoanionic ALN-OSILs are foam-like solids while the dianionic ones were obtained as thick colorless pastes at room temperature, thus being considered Room Temperature Ionic Liquids (RTILs). The exceptions to this rule are the compounds with the [C2OHMIM] cation, where the mono-and dianionic are, respectively, a paste and a foam. This inversion of the trend is probably related to specific interactions of ALN with the electron-rich imidazolium ring [25].

Thermal Analysis of ALN-OSILs
All prepared OSILs from alendronic acid were studied by Differential Scanning Calorimetry (DSC) techniques (see Figures S26-S33). Table 1 contains the obtained data, namely melting, crystallization and glass transition temperatures, as well as the physical state of the analyzed compounds.
In general, the monoanionic ALN-OSILs are foam-like solids while the dianionic ones were obtained as thick colorless pastes at room temperature, thus being considered Room Temperature Ionic Liquids (RTILs). The exceptions to this rule are the compounds with the [C 2 OHMIM] cation, where the mono-and dianionic are, respectively, a paste and a foam. This inversion of the trend is probably related to specific interactions of ALN with the electron-rich imidazolium ring [25]. In comparison with sodium alendronate, which presents a melting temperature of 259.     Figure 3B) showed an endothermic signal at ca. 150 °C of the third cycle that could be assigned to a melting process. However, it is preceded by a  Figure 3B) showed an endothermic signal at ca. 150 • C of the third cycle that could be assigned to a melting process. However, it is preceded by a glass transition at ca. 40 • C in the same cycle, meaning that the compound is in an amorphous state. So, the referred endothermic signal is most likely due to evaporation, consistent with the irregular shape of the curve caused by the formation of bubbles in the thick pasty compound. A similar observation can also be observed in [C 2

OHMIM][ALN] (Figure 3C). From the set of eight compounds, [DBNH][ALN]
is the only one that possesses a polymorphic structure, given by the two melting temperatures at 130.3 and 133.2 • C recorded in the DSC thermogram ( Figure 3D) Figures S29 and S33, respectively).

Solubility Studies
As expected, all OSILs were more soluble in water and saline solution at 37 • C than alendronic acid as well as its sodium salt. Figure 4 contains the data obtained from the solubility studies.

Solubility Studies
As expected, all OSILs were more soluble in water and saline solution at 37 °C than alendronic acid as well as its sodium salt. Figure 4 contains the data obtained from the solubility studies.

Cytotoxicity on Human Cells
The cytotoxicity of the prepared ALN-OSILs was determined on human cells, by means of IC50 calculation. The analysis was performed on human gingival fibroblasts (GF) and on different cancer

Cytotoxicity on Human Cells
The cytotoxicity of the prepared ALN-OSILs was determined on human cells, by means of IC 50 calculation. The analysis was performed on human gingival fibroblasts (GF) and on different cancer cell lines, namely T47D (breast), A549 (lung) and MG63 (osteosarcoma). The results obtained with the starting materials of the synthesis, as well as the prepared ALN-OSILs upon incubation for 24 h are presented in Table 2. The cytotoxic effect of the ALN-OSILs on the cancer cell lines was also evaluated after 72h of exposure, and the data is presented in Table 3. A comparison with the standard anticancer drug paclitaxel is presented for both exposure times.  Globally, it was observed that the IC 50 values determined at 72 h of incubation were higher than those obtained with a culture period of 24 h. The IC 50 values obtained for paclitaxel in the tumor cell lines were the lowest ones, which is in line with its well-known cytotoxic potential. However, it showed no selectivity between healthy and cancer cells.
In the case of the ALN-OSILs, the obtained data clearly shows that the monoanionic OSILs elicited lower cytotoxicity than the corresponding dianionic versions for all cell types. Regarding  Breast and lung cancers are often associated with bone osteolytic metastases [27][28][29] and also osteosarcoma, which are usually caused by disturbances in bone metabolism and increases in bone turnover rates [6,30,31]. Thus, the development of OSILs with high cytotoxicity towards the tested cancer cell types and containing an anti-resorbing molecule (alendronate) may represent a promising strategy for the development of new pharmacological tools to be used in those pathological conditions.
Overall, [C 2 OHMIM][ALN] was found to be particularly active against lung cancer and osteosarcoma cell lines while retaining very low toxicity towards healthy cells. These enhanced biological properties, in addition to the absence of polymorphism in this monoanionic compound, suggest that this room temperature ionic liquid could be a very promising alendronic acid formulation.

General Procedure (A) for the Synthesis of ALN-OSILs with Organic Superbases as Cations
To a dispersion of alendronic acid (400 mg, 1.61 mmol) in MeOH/H 2 O (15 mL, 1:1) a methanolic solution of 1 or 2 molar equivalents of organic superbase (15 mg/mL) was added dropwise at room temperature under magnetic stirring. After reacting for 1 h, the solvent was evaporated and the desired product was dried under vacuo for 24 h.

General Procedure (B) for the Preparation of ALN-OSILs with Ammonium and Methylmidazolium Cations
The halide salts of the selected ammonium and methylimidazolium cations were dissolved in methanol and passed slowly through an anion-exchange column A-26(OH) (3 equivalents). The freshly formed methanolic solutions of the corresponding hydroxide salts (1 or 2 equivalents) were consequently added dropwise to alendronic acid (400 mg, 1.61 mmol) dispersed in H 2 O under magnetic stirring at room temperature. After 1 h, the solvent of the clear solution was evaporated, and the desired product was dried under vacuo for 24 h.
Toxicity studies are described as supporting information.