Toxicity reduction of imidazolium-based ionic liquids by the oxygenation of the alkyl substituent

Abstract

In this work, five different salicylate based ionic liquids were prepared in order to study their toxicity: 1-butyl-3-methylimidazolium salicylate, [bmim][Sal], 1-(4-hydroxy-2-oxybutyl)-3-methylimidazolium salicylate, [OHC₂OC₂mim][Sal], 1-(3-hydroxypropyl)-3-methylimidazolium salicylate, [OHC₃mim][Sal], 1-ethoxyethyl-3-methylimidazolium salicylate, [C₂OC₂mim][Sal] and imidazolium salicylate [Im][Sal]; aquatic organisms (Artemia salina) and a human non-tumor cell line (normal fetal lung fibroblasts, MRC-5) were also used in the investigation. The introduction of polar groups (in the form of hydroxide and/or ether group) into the alkyl side chain of the imidazolium cation, and their influence on the reduction of the ionic liquid's toxicity, were also demonstrated. The results indicate that the toxicity against A. salina and cytotoxicity against the healthy cell line of lipophobic ionic liquids were significantly lower than for the non-functionalized analogues, and are the same order of magnitude as the reference standard sodium salicylate. These facts open up the possibility of designing new non-toxic ionic liquids that can be used as active pharmaceutical ingredients in liquid form, adjusting only the lipophilicity of the cations by introducing polar oxygen groups into the side alkyl chain of the cation.

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1. Introduction

Pharmaceutical companies currently rely on crystalline solid forms for the delivery of active pharmaceutical ingredients (APIs) because of their high purity, thermal stability, synthetic methods and easy handling.^1–3 Nowadays, many therapeutics, as well as pharmaceutical formulations in the preclinical phase of development, are used in the form of salts, due to their better physical properties, better solubility, bioavailability, permeability and drug delivery potential.^2–6 Pharmacokinetic features of the APIs in the form of the salts directly depend on their absorption mechanism and lipophilicity. The main problem when salts are applied as APIs is their polymorphism or pseudopolymorphism, since each form shows different solubility and bioavailability.^1,2,5,6 To overcome these problems, the use of the liquid forms of the drugs has been proposed. Thus, ionic liquids (ILs), salts with melting temperatures below 100 °C, with biologically active components (cation and/or anion), were recently studied as potential APIs.^6–13

The ionic liquids are known as salts with tunable properties and high thermal stability, which give new perspective in the pharmaceutical industry. There are several strategies as to how ionic liquids can be used as APIs:^8,13–18

(1) API-ILs as pharmaceuticals prepared as single or dual-active liquid salts of APIs, by liquefaction using oligomeric ions or by the formation of liquid co-crystals.

(2) Synthesis of new API-ILs starting from existing API-ILs.

(3) ILs for solubilization of poorly soluble drugs, including (i) APIs solubilized within micelles acting as reservoirs for controlled release, (ii) IL-assisted, non-aqueous microemulsions stabilized by the addition of external surfactant, or (iii) ILs designed as hydrophilic–lipophilic balanced (HLB) hydrotropes that keep the APIs dissolved once added to water.

Application of the ionic liquids as potential drugs is still restricted, due to the lack of data concerning their toxicity and biodegradability.^8,19,20 It has been shown that many commercial ILs are toxic. Thus, the synthesis of the new ILs with environmentally friendly cations and biologically active anions, such as aminoacids is proposed. For this purpose, many choline-based ILs were synthesized, but most of them show thermal, hydrolytic and electrochemical instability.^21–24 On the other hand, commercially available imidazolium ILs are more stable, but more toxic, which greatly reduce their applications. One of the strategies in preparing low-toxic ILs is reducing the toxicity of the imidazolium ion by fine tuning the essential properties by the variation of the alkyl substituents at position N1 or N3 of the imidazolium ring.²⁵ Introducing oxygen atoms into the side alkyl chain decreases lipophilicity, due to the presence of the polar groups, while simultaneously decreasing the toxicity of the ILs.^25–27 Also, the presence of the ether and/or hydroxide functions increases the solubility of the ILs in water and the solubility of the salts in ILs, promoting them as excellent candidates for API-ILs synthesis.^26,28

The second strategy is to obtain biologically active ILs by selection of the adequate anion. One of the best studied anions is salicylate, which, in the form of acetylsalicylic acid (aspirin), is one of the most commonly used medicaments in the world.^5,7,29,30 It is well known that the aspirin is only partially soluble in water (0.33 g in 100 mL of water) and in the acidic stomach environment, which results in undissolved particles adhering to the gastrointestinal mucosa causing irritation and gastric distress.^29–31 It is obvious that better drug delivery can be achieved by applying the salicylate in the liquid form. Salicylic acid is also known as a cytotoxin with a prominent anticancer effect. Also, some salicylate based ionic liquids show luminescence properties,³² which facilitate their tracking and marking in the body.

Taking all the above mentioned facts into account, we synthesized a series of ionic liquids with imidazolium cations functionalized with ether and/or hydroxide groups and salicylate anions, in order to study their toxicity and compare the obtained results with the unsubstituted analogues of 1-butyl-3-methylimidazolium salicylate, as reported herein. In this way, the influence of the oxygen functions in the substituent on the imidazole ring on the ILs toxicity can be discussed.

2. Experimental section

All chemicals for ILs synthesis were used as purchased from the manufacturer, without further purification: 1-methylimidazole (Sigma Aldrich, CAS number: 616-47-7, ω ≥ 0.99), 2-chloroethyl ether (Sigma Aldrich, CAS number: 628-34-2, ω ≥ 0.99), 3-chloro-1-propanol (Sigma Aldrich, CAS number: 627-30-5, ω ≥ 0.98), 2-(2-chloroethoxy)ethanol (Sigma Aldrich, CAS number: 628-89-7, ω ≥ 0.99), ethyl acetate (Sigma Aldrich, CAS number: 141-78-6, ω ≥ 0.998), sodium salicylate (Reanal, CAS number: 54-21-7; ω ≥ 0.995), imidazole (Sigma Aldrich, CAS number: 288-32-4, ω ≥ 0.99), N-methylimidazole (Sigma Aldrich, CAS number: 616-47-7, ω ≥ 0.99), hydrochloric acid (Sigma Aldrich, CAS number: 7647-01-0), acetone (Lachner, CAS number: 67-64-1).

Five different salicylate based ionic liquids were synthesized. The synthesis of 1-butyl-3-methylimidazolium salicylate ionic liquid, [bmim][Sal], is described in our previous paper.³³ Other ILs, namely: [OHC₂OC₂mim][Sal], [OHC₃mim][Sal], [C₂OC₂mim][Sal] and [Im][Sal] were prepared, starting from the corresponding chloride salts: [OHC₂OC₂mim][Cl], [C₃OHmim][Cl], [C₂OC₂mim][Cl] and [Im][Cl], according to the synthetic path presented in Fig. 1.

Fig. 1 Synthetic paths for [OHC₂OC₂mim][Sal], [OHC₃mim][Sal], [C₂OC₂mim][Sal] and [Im][Sal].

3-Chloro-1-propanol (or 2-(2-chloroethoxy)ethanol or 2-chloroethyl ether) and 1-methylimidazole were added to a round-bottom flask. Ethyl acetate was used as a solvent, and 3-chloro-1-propanol (or 2-(2-chloroethoxy)ethanol or 2-chloroethyl ether) was added in 10% excess. The mixture was kept under reflux for 48 h at 70 °C with stirring, until two phases were formed. The top phase, containing unreacted starting material was removed. The bottom phase was washed four times with new portions of ethyl acetate. The products were obtained in the liquid state, and stored under vacuum with P₂O₅ for the next 72 h.

Non-substituted imidazolium chloride, [Im][Cl], was synthesized by potentiometric acid–base titration. The reaction was conducted by the slow addition of HCl (0.1053 mol dm⁻³) to an aqueous solution of imidazole, with constant stirring until an adequate pH was achieved. Additionally, the IL was dried under vacuum for the next 24 h in order to remove water, and the obtained solid [Im][Cl] was stored under P₂O₅.

The obtained chloride ionic liquids were transformed into salicylates by the addition of equimolar amounts of sodium salicylate, using acetone as a solvent. The resulting solution was stirred and refluxed for 12 h. After that, the white precipitate (NaCl) was removed and the acetone solution of the ionic liquid was obtained. Acetone was removed by evaporation under vacuum at 70 °C for 1 h, achieving constant mass. The water content was determined by Karl-Fischer titration, and the chloride content by ion chromatography. It was found that the water content was less than 200 ppm and the chloride content was less than 8.3 ppm in all synthesized ionic liquids.

For additional characterization, the IR and NMR spectra (Fig. S1 and S2 in ESI†) of the newly synthesized ionic liquids were recorded. NMR spectra were recorded in D₂O at 25 °C on a Bruker Advance III 400 MHz spectrometer. Tetramethylsilane was used as the accepted internal standard for calibrating the chemical shift for ¹H and ¹³C. The ¹H homodecoupling and the 2D COSY method were used routinely for the assigning of the obtained NMR spectra. ¹³C spectra were assigned by a selective decoupling technique.

2.1. [OHC₂OC₂mim][Sal]

¹H NMR (D₂O): 3.45 (m, 2H, NCH₂CH₂OCH₂CH₂OH); 3.58 (m, 2H, NCH₂CH₂OCH₂CH₂OH); 3.67 (s, 3H, CH₃); 3.67 (t, 2H, NCH₂CH₂OCH₂CH₂OH); 4.13 (t, 2H, J = 4.7, NCH₂CH₂OCH₂CH₂OH); 6.77 (d, 1H, J_3′,4′ = 8.2 Hz, H-3′); 6.10 (t, 1H, J_3′,4′ = 7.5 Hz, H-5′); 7.17 and 7.25 (2xs, 2H, H-4 and H-5); 7.29 (m, 1H, H-4′); 7.67 (dd, 1H, J_4′,6′ = 1.3 Hz, J_5′,6′ = 7.8 Hz, H-6′); 8.44 (s, 1H, H-2).

¹³C NMR (D₂O): 38.63 (NCH₃); 51.77 (NCH₂CH₂OCH₂CH₂OH); 63.08 (NCH₂CH₂OCH₂CH₂OH); 70.99 (NCH₂CH₂OCH₂CH₂OH); 74.51 (NCH₂CH₂OCH₂CH₂OH); 118.97 (C-3′); 120.75 (C-1′); 122.03 (C-5′); 125.17 and 126.07 (C-5 and C-4); 133.18 (C-6′); 136.66 (C-4′); 138.83 (C-2); 162.47 (C-2′); 177.80 (C=O).

2.2. [C₂OC₂mim][Sal]

¹H NMR (D₂O): 1.05 (t, 3H, J = 7.0 NCH₂CH₂OCH₂CH₃); 3.43 (q, 2H, J = 7.1, NCH₂CH₂OCH₂CH₃); 3.66 (t, 2H, NCH₂CH₂OCH₂CH₃); 3.73 (s, 3H, NCH₃); 4.14 (t, 2H, NCH₂CH₂OCH₂CH₃); 6.80 (d, 1H, J_3′,4′ = 8.3 Hz, H-3′); 6.84 (t, 1H, J_3′,4′ = 7.6 Hz, H-5′); 7.23 and 7.27 (2xs, 2H, H-4 and H-5); 7.31 (m, 1H, H-4′); 7.72 (bd, 1H, J_5′,6′ = 7.8 Hz, H-6′); 8.47 (s, 1H, H-2).

¹³C NMR (D₂O): 16.83 (NCH₂CH₂OCH₂CH₃); 38.39 (NCH₃); 51.76 (NCH₂CH₂OCH₂CH₃); 69.39 (NCH₂CH₂OCH₂CH₃); 70.38 (NCH₂CH₂OCH₂CH₃); 74.51 (NCH₂CH₂OCH₂CH₃); 118.94 (C-3′); 120.88 (C-1′); 121.99 (C-5′); 125.12 and 126.19 (C-5 and C-4); 133.19 (C-6′); 136.59 (C-4′); 138.70 (C-2); 162.54 (C-2′); 177.71 (C=O).

2.3. [OHC₃mim][Sal]

¹H NMR (D₂O): 2.00 (m, 2H, NCH₂CH₂CH₂OH); 3.55 (t, 2H, J = 6.1, NCH₂CH₂CH₂OH); 3.78 (s, 3H, NCH₃); 4.15 (t, 2H, J = 7.2, NCH₂CH₂CH₂OH); 6.84–6.94 (m, 2H, H-3′ and H-5′); 7.29 and 7.34 (2xs, 2H, H-4 and H-5); 7.40 (m, 1H, H-4′); 7.75 (d, 1H, J_5′,6′ = 8.1 Hz, H-6′); 8.54 (s, 1H, H-2).

¹³C NMR (D₂O): 34.35 (NCH₂CH₂CH₂OH); 38.39 (NCH₃); 49.19 (NCH₂CH₂CH₂OH); 60.64 (NCH₂CH₂CH₂OH); 119.04 (C-3′); 120.74 (C-1′); 122.14 (C-5′); 124.99 and 126.33 (C-5 and C-4); 133.24 (C-6′); 136.77 (C-4′); 138.63 (C-2); 162.44 (C-2′); 178.08 (C=O).

2.4. [Im][Sal]

¹H NMR (D₂O): 6.35–6.45 (m, 2H, H-3′ and H-5′); 6.83–6.93 (m, 3H, H-4, H-5 and H-4′); 7.34 (dd, 1H, J_5′,6′ = 7.8 Hz, J_4′,6′ = 0.9 Hz, H-6′); 8.10 (s, 1H, H-2).

¹³C NMR (D₂O): 118.62 (C-3′); 120.34 (C-1′); 121.21 (C-5′); 121.62 (C-5 and C-4); 132.84 (C-6′); 135.52 (C-2); 136.26 (C-4′); 162.14 (C-2′); 177.81 (C=O).

Infrared spectra were recorded on neat samples from (4000–650) cm⁻¹ on a Thermo-Nicolet Nexus 670 spectrometer fitted with a Universal ATR Sampling Accessory.

2.5. [OHC₂OC₂mim][Sal]

3500–3300 (H-bond OH); 3145 (sym. stretching ν HC((4)C(5)H)); 3094 (sym. stretching ν HC(2)); 2867 (sym. stretching ν CH₃); 1584 (in-plane vibrations of imidazolium ring); 1484 (stretching ν CC); 1380 (stretching ν C–O); 1323 (in-plane bending mode δ O–H); 1125 and 1067 (stretching ν C–O from ether group); 807 (in-plane bending mode δ CC).

2.6. [C₂OC₂mim][Sal]

3142 (sym. stretching ν HC((4)C(5)H)); 3077 (sym. stretching ν HC(2)); 2974 (asym. stretching ν CH₃); 2871 (sym. stretching ν CH₃) 1585 (in-plane vibrations of imidazolium ring); 1483 and 1284 (stretching ν CC); 1376 (stretching ν C–O); 1166 (stretching ν C–O); 1144 and 1026 (stretching ν C–O from ether group); 806 (in-plane bending mode δ CC);

2.7. [OHC₃mim][Sal]

3260 (stretching OH); 2954 (asym. stretching ν CH₃); 2880 (sym. stretching ν CH₃); 3081 (sym. stretching ν HC(2)); 3144 (sym. stretching ν HC((4)C(5)H)); 1585 (in-plane vibrations of imidazolium ring); 1483 and 1284 (stretching ν CC); 1376 (stretching ν C–O); 1166 (stretching ν C–O); 806 (in-plane bending mode δ CC); 702 (out-of-plane deformation γ CC).

2.8. [Im][Sal]

3159 (sym. stretching ν HC((4)C(5)H)); 3047 (sym. stretching ν HC(2)); 2978 (asym. stretching ν HC(2)); 1602 (in-plane vibrations of imidazolium ring); 1488 (stretching ν CC) 1383 (stretching ν C–O); 1463 and 1346 (stretching ν C–N); 809 (in-plane bending mode δ CC); 667 (out-of-plane deformation γ CC).

2.9. Toxicity assays

2.9.1. Toxicity on aquatic microcrustacean A. salina. Mean lethal concentration (LC₅₀) was determined by modification of the in vitro test described by Parra et al.³⁴ Cultures of A. salina (Great Salt Lake Artemia Cysts, Ogden UT, USA) were incubated in artificial sea water (ASW) at a temperature of 30 °C under constant illumination and aeration for 30 h. After incubation, 10–20 larvae of A. salina were transferred to each test tube and another 200 μL of ASW and 20 μL of the proper ionic liquids. Tests were carried out in five replicates for each concentration of each IL, with the negative control of 220 μL of ASW without the addition of ionic liquids. The control solvent was distilled water (20 μL). Microplates were incubated at 30 °C under constant illumination and the mortality of the larvae was monitored after 24 and 48 h.

2.9.2. In vitro antiproliferative assay.
Cell lines and cell culture. Human non-tumor cell line (normal fetal lung fibroblasts MRC-5, ATCC CCL 171) was used in this study. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 4.5% of glucose. Media were supplemented with 10% of fetal calf serum (FCS, Sigma) and antibiotics: 105 IU mL⁻¹ of penicillin and 100 μg mL⁻¹ of streptomycin (ICN Galenika). The cell line was cultured in a flask (Costar, 25 cm²) at 37 °C in a 100% humidity atmosphere containing 5% of CO₂. Only viable cells were used in the assays. Cell viability was determined by the trypan blue dye exclusion assay.
Antiproliferative activity. Antiproliferative activity of the tested salicylate derivatives was evaluated by tetrazolium colorimetric MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay.³⁵ Cells were exposed to the test compounds over 72 h, in concentrations ranging from 10⁻⁸ to 10⁻⁴ mol L⁻¹. The reference compound used in the MTT assay was sodium salicylate. Exponentially growing cells were harvested, counted by trypan blue dye exclusion test, seeded onto 96-well plates at a density of 5000 cells/well and allowed to stand overnight. Solutions of the tested compounds in medium (10 μmol per L per well) were added and therefore, the final concentrations ranged from 10⁻⁸ to 10⁻⁴ mol L⁻¹. After the 72 h treatment period, cell viability was determined by the addition of 10 μL of sterile MTT solution (5 mg mL⁻¹). The precipitated formazan crystals were solubilized with acidified 2-propanol (100 μL of 0.04 mol L⁻¹ HCl in 2-propanol) and the absorbance was recorded (Multiscan MCC340, Labsystems) at 540 and 690 nm after a few minutes incubation at room temperature. Wells containing cells without tested compounds were used as controls. Wells without cells, containing only complete medium and MTT were used as the blank. Cytotoxicity (CI) was calculated according to the following formula:
Data analysis. Two independent experiments were conducted in quadruplicate for each concentration of tested compound. Mean values and standard deviations (σ) were calculated for each concentration. Antiproliferative activity was expressed as IC₅₀ value, defined as the dose of compound that inhibits cell growth by 50%. The IC₅₀ of each tested compound was determined by median effect analysis.³⁶

3. Results and discussion

Described tests on aquatic organisms (Artemia salina) and on the human non-tumor cell line (normal fetal lung fibroblasts, MRC-5) were performed in order to investigate the influence of the alkyl chain substituent on the ILs toxicity. The assay based on toxicity against A. salina is considered rapid, convenient, low cost and one of the most reliable methods for the preliminary detection of mycotoxins, heavy metals and pesticide toxicity.^34,37,38 Also, this test is often used for the general toxicity testing of compounds before their commercial use.^37,39 In this work, it was used for the lethal concentration (LC₅₀) determination.

In order to investigate toxicity against healthy human cells, the MRC-5 cell line was used, which is commonly applied in vaccine development⁴⁰ as a transfection host in virology research,⁴¹ and for in vitro cytotoxicity testing.⁴² In order to evaluate the cytotoxicity of the synthesized salicylate ionic liquids, obtained IC₅₀ values were compared with the results obtained for non-functionalized ionic liquid [bmim][Sal] and for the sodium salicylate. The first reference compound is highly toxic, while the second one is commonly used in medicine as an analgetic, antipyretic, and non-steroidal anti-inflammatory drug that has antitumor potential, due to inducing necrosis and/or apoptosis in cancer cells.^43–46

The obtained toxicity results are presented in Table 1 and in Fig. 2 and 3.

Table 1 Toxicity of the studied salicylate salts^a

	Salt	LC50 (A. salina)/μM	IC50 (MRC-5)/μM
a * very high toxicity at low concentrations, LC50 could not be determined.** IC50 is not detected in the investigated concentration range, meaning that these compounds can be considered as non-toxic.
1	Sodium salicylate	8.87	**
2	[bmim][Sal]	*	27.54
3	[OHC2OC2mim][Sal]	8.41	**
4	[C2OC2mim][Sal]	8.18	**
5	[OHC3mim][Sal]	10.18	**
6	[Im][Sal]	*	**

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Fig. 2 A. salina mortality as a function of concentration of (a) [OHC₂OC₂mim][Sal], (b) [Im][Sal], (c) [OHC₃mim][Sal], (d) [C₄Omim][Sal], (e) [bmim][Sal], (f) Na-Sal (■, after 24 hours; ○, after 48 hours).

Fig. 3 MRC-5 cytotoxicity as a function of concentration of (a) [OHC₂OC₂mim][Sal], (b) [Im][Sal], (c) [OHC₃mim][Sal], (d) [C₄Omim][Sal], (e) [bmim][Sal], (f) Na-Sal (symbol + lines – experimental values; dashed red lines – calculated values).

Ionic liquids [bmim][Sal] and [Im][Sal] showed high toxicity, even at the lowest ILs concentrations (3 μM) on A. salina larvae within the first 24 h; thus, the corresponding values of LC₅₀ were not detected.

The introduction of oxygen in the form of a hydroxy and/or ether group into the side alkyl chain of the imidazolium ion leads to a significant reduction of the ILs toxicity to A. salina, where the LC₅₀ values of these newly synthesized ILs after 48 h were found to be similar to the LC₅₀ obtained for the reference sodium salicylate. A significant reduction in toxicity can be observed by comparing the results obtained for [bmim][Sal] and ILs with the oxygen in the alkyl substituent (Table 1, Fig. 2). It can be seen that introduction of the oxygen in the form of the hydroxide group in [OHC₃mim][Sal], had a greater impact on reducing toxicity than the presence of oxygen in the form of the ether group in the side alkyl chain of [C₂OC₂mim][Sal]. This observation is in agreement with that reported by Stolte et al.^27,47 In the case of [OHC₂OC₂mim][Sal], the introduction of the OH group in the alkyl chain with the existing ether oxygen had a lower impact on reducing the toxicity, compared to the introduction of the OH group in the non-functionalized alkyl chain.

In the case of healthy lung cells, MRC-5, it was observed that only [bmim][Sal] expressed significant toxicity, its IC₅₀ was 27.54 μM (Table 1). Otherwise, oxygen-modified ILs were not toxic against the healthy cell line MRC-5. The cytotoxicity tests were performed in the ionic liquid concentration range from 0.01 to 100 μmol mL⁻¹ and the results are presented graphically in Fig. 3.

It is known that the structure of the cation has the greater impact on the ILs toxicity.^{25,27,47–49} From the results presented in this manuscript, it is obvious that the alkyl side chain is the primary factor that affects the toxicity of imidazolium based ionic liquids, i.e., a change in the polarity. This phenomenon can be explained by the assumption that the lipophilic cations are adsorbed or intercalated into the cell membrane, causing “perturbations” in the membrane (expansion or swelling, increase in fluidity, lowering of the phase transition temperature and alteration of the ion permeability of the membrane).^25,49–52 The presented results indicate that the presence of a lipophilic butyl group leads to proportional destabilization of the lipid double layer membrane, which results in a linear increase of the toxicity with increasing IL concentration (Fig. 3e). Introduction of oxygen into the alkyl substituent increases the polarity of the ion, while the lipophilicity decreases. This weakens the interactions between lipid cell membrane and the ionic liquid, which significantly reduces the toxicity (Fig. 3a, c and d). The lower toxicity of the ionic liquid containing the hydroxide group in the side chain, in relation to the corresponding ether group, leads to the conclusion that the toxicity is largely affected by the type of functional group of the cation. The terminal group of the alkyl chain is the most accessible for the interactions with the cellular membrane, and its reactivity, namely hydrophilicity/hydrophobicity, is mainly responsible for the toxicity of the whole ionic liquid. The [C₂OC₂mim][Sal] ionic liquid has a more hydrophobic methyl group at the end of the alkyl chain, compared to [OHC₃mim][Sal], where the side chain terminates with a distinctly polar OH group, thus explaining the higher toxicity of [C₂OC₂mim][Sal]. Also, it can be observed that additional oxygenation of the alkyl chain does not decrease the toxicity of the ionic liquids, which is in agreement with the results reported by Samori et al.⁵³

The ionic liquids examined in this work, contain the biologically active salicylate anion and cation as drug carriers. Due to the dissociation of the ionic compounds, the cation and anion can be delivered separately into the cells. Thus, by decreasing the lipophilicity of the cation, its toxicity and bioavailability can be reduced without affecting the bioavailability of the salicylate anion.

4. Conclusions

In this paper the toxicity of newly synthesized salicylate based ionic liquids has been investigated using aquatic organisms (A. salina) and human non-tumor cell line (normal fetal lung fibroblasts, MRC-5). Also, the influence of the oxygenation (in the form of hydroxy and/or ether groups) of the alkyl side chain on the toxicity reduction was studied. The results indicate that both toxicity against A. salina and cytotoxicity against healthy cell line of lipophobic ionic liquids are significantly lower than non-functionalized analogues, and the same order of magnitude as the reference standard, sodium salicylate. The most significant impact on the reduction of the toxicity shows the introduction of a hydroxide function at the terminal position of the alkyl substituent of the imidazolium cation, while the introduction of a larger amount of oxygen does not contribute to reduced toxicity. Bearing in mind the obtained results, this opens the possibility of designing new, non-toxic ILs, adjusting only the lipophilicity of the cations by introducing polar oxygen groups into the side alkyl chain.

Acknowledgements

This work was financially supported by the Ministry of Education, Science and Technological Development of Republic of Serbia under project contract ON172012.

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Footnote

† Electronic supplementary information (ESI) available: NMR and IR spectra of newly synthesized ionic liquids. See DOI: 10.1039/c6ra16182k

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