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

Article abstract of DOI:10.1039/c6ra16182k

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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Toxicity reduction of imidazolium-based ionic liquids by the
oxygenation of the alkyl substituent
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
a
b
c
M. Vraneš, A. Tot, S. Jovanović-Šanta, M. Karaman, S. Dožić, K. Tešanović, V. Kojić and S.
a
Gadžurić
DOI: 10.1039/x0xx00000x
In this work five different salicylate based ionic liquids are prepared in order to study their toxicity, namely: 1-butyl-3-
www.rsc.org/
methylimidazolium salicylate, [bmim][Sal], 1-(4-hydroxy-2-oxybutyl)-3-methylimidazolium salicylate, [OHC
-(3-hydroxypropyl)-3-methylimidazolium salicylate, [OHC mim][Sal], 1-etoxyethyl-3-methylimidazolium salicylate,
OC mim][Sal] and imidazolium salicylate [Im][Sal]. For that purpose aquatic organisms (Artemia salina) and human non-
2 2
OC mim][Sal],
1
3
[C
2
2
tumor cell line (normal fetal lung fibroblasts, MRC-5) are used. Introduction of the polar groups (in the form of hydroxyde
and/or ether group) in the alkyl side chain of the imidazolium cation and their influence on the reduction of the ionic
liquid's toxicity is also demonstrated. The results indicate that both, toxicity against A. salina and cytotoxicity against
healthy cell line of lipophobic ionic liquids are significantly lower comparing with non-functionalized analogues and the
same order of magnitude as the reference standard sodium salicylate. These facts open the possibility of designing new
non-toxic ionic liquids that can be used as active pharmaceutical ingredients in the liquid form, adjusting only lipophilicity
of the cations introducing polar oxygen groups in the side alkyl chain of the cation.
The ionic liquids are known as the salts with the tunable
properties and the high thermal stability, which gives the new
1
. Introduction
Pharmaceutical companies currently rely on crystalline solid
forms for the delivery of active pharmaceutical ingredients
perspective in the pharmaceutical industry. There are several
8, 13-18
strategies how the ionic liquids can be used as the APIs:
(
APIs) for the reasons of their high purity, thermal stability,
1
.
API-ILs as pharmaceuticals prepared as single or dual-
active liquid salts of APIs by liquefaction using the
oligomeric ions or by formation of liquid co-crystals;
Synthesis of new API-ILs starting from existing API-IL
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 an addition of external
surfactant or iii) ILs designed as hydrophilic-lipophilic
balanced (HLB) hydrotropes that keep the APIs dissolved
once added to water.
1
-3
synthetic methods and easy handling. Nowadays, many
therapeutics are used as the adequate salts, as well as
pharmaceutical formulations in the preclinical phase of
development, due to their better physical properties, better
solubility, bioavailability, permeability and drug delivery
2.
3.
2
-6
potential. 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 the polimorphism or pseudopolimorphism, since the
1
,2,5,6
each form shows different solubility and bioavailability.
To
exceed this problems, the liquid forms of the drugs are
proposed to be used. Thus, ionic liquids (ILs) as the salts with
o
the melting temperatures below 100 C with the biologicaly
Application of the ionic liquids as potential drugs is still
restricted due to the lack of the data concerning their toxicity
8,19,20
and biodegradability.
It was shown that many commercial
active components (cation or/and anion) were recently
6
studied as the potential APIs.
ILs are toxic. Thus, synthesis of the new ILs with
environmentally friendly cations and biologically active anions
such as aminoacids is proposed. For that purpose, many
-12,13
choline-based ILs were synthesized, but most of them show
21-24
a.
University of Novi Sad, Faculty of Sciences, Department of Chemistry,
Biochemistry and Environmental Protection, Trg D. Obradovića 3, 21000 Novi Sad
thermal, hydrolytical and electrochemical unstability.
At
(
Serbia)
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 toxicity of the imidazolium ion applying fine tuning of
the essential properties by the variation of the alkyl
b.
c.
University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology,
Trg Dositeja Obradovića 3, 21000 Novi Sad (Novi Sad)
Institute of Oncology Sremska Kamenica, Institutski Put 4, 21204 Sremska
Kamenica (Serbia).
tel:(+381) 21 4852744
fax:(+381) 21 454 065
2
5
email: milan.vranes@dh.uns.ac.rs
Electronic Supplementary Information (ESI) available: NMR and IR spectra of
substituents in the position N1 or N3 of the imidazolium ring.
†
newly synthesized ionic liquids. See DOI: 10.1039/x0xx00000x
Introducing the oxygen atom in the side alkyl chain decreases
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lipophilicity due to a presence of the polar groups, decreasing number: 616-47-7,
Journal Name
ω
≥ 0.99), hydrochloric acid (Sigma Aldrich,
2
5-27
in the same time toxicity of the ILs.
the ether and/or hydroxide functions increases solubility of the
ILs in water and the solubility of the salts in ILs, promoting Synthesis of 1-butyl-3-methylimidazolium salicyalte ionic
Also, the presence of CAS number:7647-01-0), acetone (Lachner, 67-64-1)
Five different salicylate based ionic liquids are synthesized.
2
6,28
33
them to the excellent candidates for API-ILs synthesis.
Second strategy is to obtain biologically active ILs by ILs,
selection of the adequate anion. One of the best studied [C OC
anions is the salicylate which in the form of acetyl-salicylic acid corresponding
liquid, [bmim][Sal], is described in our previous paper. Other
namely: [OHC OC mim][Sal], [OHC mim][Sal],
mim][Sal] and [Im][Sal] were prepared starting from the
chloride salts: [OHC OC mim][Cl],
OC mim][Cl] and [Im][Cl] according to the
It is well known that the aspirin is only synthetic path presented in Figure 1.
3-chloro-1-propanol (or 2-(2-chloroetoxy)ethanol or 2-
2
2
3
2
2
2
2
(
Aspirin) is one of the most commonly used medicaments in [C
3
OHmim][Cl], [C
2
2
5
,7,29,30
the world.
partialy soluble in water (0.33 g in 100 ml of water) and in the
acidic stomach enviroment, which results in undissolved chloroethyl ether) and 1-methylimidazole were added to a
particles adhering to the gastrointestinal mucosea causing round-bottom flask. Ethyl acetate was used as a solvent, and
2
9-31
irritation and gastric distress.
It is obvious that better drug 3-chloro-1-propanol (or 2-(2-chloroetoxy)ethanol or 2-
delivery can be achieved applying the salicylate in the liquid chloroethyl ether) was added in 10% excess. The mixture was
o
form. Salicylic acid is also known as a cytotoxin with a kept under the reflux for 48 h at 70 C with stirring, until two
prominent anticancer effect. Also, some salicylate based ionic phases were formed. The top phase, containing unreacted
3
2
liquids show a luminescence properties, which facilitates its starting material was removed. The bottom phase was washed
tracking and marking in the body. four times with new portion of ethyl acetate. The products
Taking into account all above mentioned facts, in this were obtained in the liquid state, and additionally stored
paper we synthesized a series of ionic liquids with imidazolium under vacuum with P for the next 72 h.
cations functionalized with ether and/or hydroxide groups and Non-substitued imidazolium chloride, [Im][Cl], was
2
O
5
salicylate anion, in order to study their toxicity and compare synthesized by the potentiometric acid-base titration. The
obtained results with the unsubstituted analogues of 1-butyl- reaction was conducted by slow addition of HCl (0.1053
–
3
3
-methylimidazolium salicylate. In such way, the influence of mol·dm ) to a water solution of imidazole with constant
the oxygen functions in the substituent on the imidazole ring stirring, until achieving adequate pH. Additionaly, IL was dried
on the ILs toxicity will be discussed. under vacuum in order to remove water for the next 24 h and
obtained solid [Im][Cl] was stored under P
2 5
O .
Obtained chloride ionic liquids were transfered into
salicylates by the addition of the equimolar amount of sodium
salicylate using acetone as a solvent. Resulting solution was
stirred and refluxed for 12 h. After that, the white precipitate
2
. Experimental section
All chemicals for ILs synthesis were used without purification
as purchased from the manufacturer: 1-methylimidazole
(
NaCl) was removed and the acetone solution of the ionic
(
Sigma Aldrich, CAS number: 616-47-7,
ether (Sigma Aldrich, CAS number: 628-34-2,
chloro-1-propanol (Sigma Aldrich, CAS number: 627-30-5,
ω
≥ 0.99), 2-chloroethyl
liquid was obtained. Acetone is removed by the evaporation
o
under the vacuum at 70 C for 1 h achieving the constant mass.
ω
≥ 0.99), 3-
ω
≥
Water content was determined by the Karl-Fischer titration
and chloride content by the ion chromatography. It was found
that water content was less than 200 ppm and chloride
content less than 8.3 ppm in all synthesized ionic liquids.
0
6
1
5
2
.98), 2-(2-chloroethoxy)ethanol (Sigma Aldrich, CAS number:
28-89-7,
41-78-6,
ω
ω
≥ 0.99), ethyl acetate (Sigma Aldrich, CAS number:
≥ 0.998), sodium salicylate (Reanal, CAS number:
≥ 0.995), imidazole (Sigma Aldrich, CAS number:
4-21-7;
ω
88-32-4,
ω
≥ 0.99), N-methylimidazole (Sigma Aldrich, CAS
-
O
O
Na-Sal
acetone
12h
+
+
ethyl acetate
o
+
+
-
R
R1
R2
1
R1
R2
N
N
N
N
-
OH
+
R
2
Cl
N
N
Cl
4
8h, 70 C
NaCl
H_
_
CH3
R =
1
;
H_
HO
HO
R =
2
_
H C
O
_
_
;
CH
2
3
CH
2
O
CH
2
;
;
Figure 1. Synthetic paths for: [OHC
2
OC
2
mim][Sal], [OHC
3
mim][Sal]; [C
2 2
OC mim][Sal] and [Im][Sal]
1 13
For additional characterization the IR and NMR spectra internal standard for calibrating chemical shift for H and C.
1
(
Figures S1 and S2 in Supporting information) of the newly
H homodecoupling and 2D COSY method were used routinely
1
3
synthesized ionic liquids were recorded. NMR spectra were for the assignation of the obtained NMR spectra. C spectra
o
recorded in D
2
O at 25 C on a Bruker Advance III 400 MHz were assigned by selective decoupling technique.
spectrometer. Tetramethylsilane was used as accepted
2
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6RA16182K
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[
OHC
2
OC
2
mim][Sal]
CH
OH); 3.58 (m, 2H, 1284 (stretching ν CC); 1376 (stretching ν C-O); 1166
); 3.67 (t, 2H, (stretching ν C-O); 1144 and 1026 (stretching ν C-O from ether
4.7, group); 806 (in-plane bending mode δ CC);
OH,); 6.77 (d, 1H, J3′,4′ = 8.2 Hz, H-3′); 6.10 (t, [OHC mim][Sal]
3
) 1585 (in-plane vibrations of imidazolium ring); 1483 and
1
H NMR (D
2
O): 3.45 (m, 2H, NCH
2
CH
OH); 3.67 (s, 3H,
OH); 4.13 (t,
2 2 2
OCH CH
NCH
NCH
2
2
2
CH
2
2
2
OCH
OCH
OCH
2
2
2
C
H
2
2
2
CH
3
C
H
CH
CH
2H,
J
=
NC
H
CH
3
1
7
6
H, J3′,4′ = 7.5 Hz, H-5′); 7.17 and 7.25 (2xs, 2H, H-4 and H-5); 3260 (stretching OH); 2954 (asym. stretching ν CH
3
); 2880
.29 (m, 1H, H-4′); 7.67 (dd, 1H, J4′,6′ = 1.3 Hz, J5′,6′ = 7.8 Hz, H- (sym. stretching ν CH ); 3081 (sym. stretching ν HC(2); 3144
3
′); 8.44 (s, 1H, H-2)
(sym. stretching ν HC((4)C(5)H); 1585 (in-plane vibrations of
OH); imidazolium ring); 1483 and 1284(stretching ν CC); 1376
OH); (stretching ν C-O); 1166 (stretching ν C-O); 806 (in-plane
1
3
C NMR (D
3.08 (NCH
4.51 (NCH
2
O): 38,63 (NCH
CH OCH OH); 70.99 (NCH
CH
3
); 51.77 (N
CH
2
CH
2
OCH
OCH
2
CH
CH
2
6
7
1
1
2
2
2
C
H
2
2
CH
2
2
2
2
2
O
C
H
2
CH
2
OH); 118.97 (C-3′); 120.75 (C-1′); bending mode δ CC); 702 (out-of-plane deformation γ CC)
22.03 (C-5′); 125.17 and 126.07 (C-5 and C-4); 133.18 (C-6′);
36.66 (C-4′); 138.83 (C-2); 162.47 (C-2′); 177.80 (C=O).
[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
2 2 2 3
CH OCH CH ); 3.43 (q, vibrations of imidazolium ring); 1488 (stretching ν CC) 1383
[C
2
OC
2
mim][Sal]
O): 1.05 (t, 3H, J = 7.0 NCH
H, J = 7.1, NCH CH OC CH ); 3,66 (t, 2H, NCH OCH
3
.73 (s, 3H, NC ); 4.14 (t, 2H, NC CH OCH CH ); 6.80 (d, 1H, plane bending mode δ CC); 667 (out-of-plane deformation γ
1
H NMR (D
2
2
3
2
2
H
2
3
2
CH
2
2 3
CH ); (stretching ν C-O); 1463 and 1346 (stretching ν C-N); 809 (in-
H
3
H
2
2
2
J
3′,4′ = 8.3 Hz, H-3′); 6.84 (t, 1H, J3′,4′ = 7.6 Hz, H-5′); 7.23 and CC)
.27 (2xs, 2H, H-4 and H-5); 7.31 (m, 1H, H-4′); 7.72 (bd, 1H,
5′,6′ = 7.8 Hz, H-6′); 8.47 (s, 1H, H-2)
7
J
1
2.1 Toxicity assays
3
C NMR (D
2
O): 16.83 (NCH
2
CH
69.39
); 74.51 (NCH
2
OCH
(NCH
CH
2
CH
3
); 38,39 (NCH
CH );
3
); 51.76
70.38
2
.1.1 Toxicity on aquatic microcrustaocean A. salina
(N
CH
2
CH
2
OCH
OCH
2
CH
CH
3
);
2
CH
2
O
CH
2
3
(NCH
2
CH
2
2
3
2
2
O
C
H
2
CH
3
); 118.94 (C-3′);
Mean lethal concentration (LC50) was determined by a
3
modification of the in vitro test described by Parra et al.
4
1
20.88 (C-1′); 121.99 (C-5′); 125.12 and 126.19 (C-5 and C-4);
33.19 (C-6′); 136.59 (C-4′); 138.70 (C-2); 162.54 (C-2′); 177.71
1
Culture of Artemia salina (Great Salt Lake Artemia Cysts,
(
C=O).
OHC
H NMR (D
.1, NCH CH
NC CH CH OH); 6.84-6.94 (m, 2H, H-3′ and H-5′); 7.29 and
Ogden UT, USA) was incubated in the artificial sea water (ASW)
o
at temperature of 30 C under the constant illumination and
[
3
mim][Sal]
O): 2.00 (m, 2H, NCH CH
3
OH); 3.78 (s, 3H, NCH ); 4.15 (t, 2H, J = 7.2,
1
2
2
C
H
2
2
OH,); 3.55 (t, 2H, J =
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. Test was 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). Then,
microplates were incubated at 30 °C under the constant
illumination and the mortality of the larvae was monitored
after 24 and 48 h.
6
2
2
CH
2
H
2
2
2
7
.34 (2xs, 2H, H-4 and H-5); 7.40 (m, 1H, H-4′); 7.75 (d, 1H, J5′,6′
8.1 Hz, H-6′); 8.54 (s, 1H, H-2)
=
1
3
C NMR (D
CH CH
2
O): 34.35 (NCH
2
C
H
2
CH
2
OH); 38,39 (NCH
3
); 49.19
(N
C
H
2
2
2
OH); 60.64 (NCH
2
CH
2 2
CH OH); 119.04 (C-3′);
1
20.74 (C-1′); 122.14 (C-5′); 124.99 and 126.33 (C-5 and C-4);
33.24 (C-6′); 136.77 (C-4′); 138.63 (C-2); 162.44 (C-2′); 178.08
1
(C=O).
[
Im][Sal]
H NMR (D O): 6.35-6.45 (m, 2H, H-3′ and H-5′); 6.83-6.93 (m,
H, H-4, H-5 and H-4′); 7.34 (dd, 1H, J5′,6′ = 7.8 Hz, J4′,6′ = 0.9 Hz,
H-6′); 8.10 (s, 1H, H-2)
2.1.1 In vitro antiproliferative assay
1
2
Cell lines and cell culture. Human non-tumor cell line
3
(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 was
supplemented with 10% of fetal calf serum (FCS, Sigma) and
1
3
2
C NMR (D O): 118.62 (C-3′); 120.34 (C-1′); 121.21 (C-5′);
1
21.62 (C-5 and C-4); 132.84 (C-6′); 135.52 (C-2); 136.26 (C-4′);
62.14 (C-2′); 177.81 (C=O).
1
−1
−1
antibiotics: 105 IU mL of penicillin and 100 μg·mL of
Infrared spectra were recorded as neat samples from
streptomycin (ICN Galenika). Cell line was cultured in flask
2
Costar, 25 cm ) at 37 °C in a 100% humidity atmosphere
–1
(
4000-650) cm on a Thermo-Nicolet Nexus 670 spectrometer
fitted with a Universal ATR Sampling Accessory.
OHC OC mim][Sal]
500-3300 (H-bond OH); 3145 (sym. stretching ν HC((4)C(5)H));
094 (sym. stretching ν HC(2)); 2867 (sym. stretching νCH );
584 (in-plane vibrations of imidazolium ring); 1484 (stretching
(
containing 5% of CO . Only viable cells were used in the assays.
2
[
2
2
Cell viability was determined by the trypan blue dye exclusion
assay.
3
3
1
3
Antiproliferative activity. Antiproliferative activity of the
tested salicylate derivatives was evaluated by tetrazolium
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
colorimetric
MTT
([3-(4,5-dimethylthiazol-2-yl)-2,5-
35
diphenyltetrazolium bromide]) assay. Cells were exposed to
the test compounds during 72 h in concentrations ranged from
(
in-plane bending mode δ CC)
OC mim][Sal]
142 (sym. stretching ν HC((4)C(5)H)); 3077 (sym. stretching ν
); 2871 (sym. stretching ν
[C
2
2
–8
0
–4 –1
to 10 mol·L . Reference compound used in the MTT
1
3
assay was sodium salicylate. Exponentially growing cells were
HC(2)); 2974 (asym. stretching ν CH
3
This journal is © The Royal Society of Chemistry 20xx
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harvested, counted by trypan blue exclusion test, seeded onto metals and pesticides toxicity.
Journal Name
3
4,37,38
Also, this test is often
9
6-well plates at a density of 5000 cells/well and allowed to used for testing of general toxicity of compounds before their
3
7,39
stand overnight. Solutions of the tested compounds in medium commercial use .
In this work, it was used for the lethal
–
1
10 μmol·L /well) were added so final concentrations ranging concentration (LC50) determination.
(
–
from 10 to 10 mol·L . After 72 h tretament period, cells
8
–4
–1
In order to investigate toxicity against human healthy cells,
viability was determined by the addition of 10 μL of sterile MRC-5 cell line was used, which is commonly applied in
–1
40
MTT solution (5 mg·mL ). The precipitated formazan crystals vaccine development, as a transfection host in virology
4
1
42
were solubilized with acidifed 2-propanol (100 μL of 0.04 research, and for in vitro cytotoxicity testing. In order to
–1
mol·L HCl in 2-propanol) and the absorbance was recorded evaluate the cytotoxicity of the synthesized salicylate ionic
Multiscan MCC340, Labsystems) at 540 and 690 nm after a liquids, obtained IC50 values were compared with the results
(
few minutes incubation at room temperature. Wells obtained for non-funcionalized ionic liquid [bmim][Sal] and for
containing cells without tested compounds were used as the sodium salicylate. First reference compound is highly toxic,
control. Wells without cells, containing only complete medium while second one is commonly used in medicine as an
and MTT were used as blank. Cytotoxicity (CI) was calculated analgetic, antipyretic, and non-steroidal anti-inflammatory
according to the formula:
drug which antitumor potential due to inducing necrosis
4
and/or apoptosis in cancer cell.
3-46
Asample
CI(%) =1− Acontrol ⋅100
Obtained toxicity results are presented in Table 1 and in
the Figures 2 and 3.
(
1)
Data analysis. Two independent experiments were Table 1. Toxicity of the studied salicylate salts
conducted in quadruplicate for each concentration of tested
compound. Mean values and standard deviations ( ) were
Salt
LC50 (A. salina)/μM IC50 (MRC-5)/μM
σ
1
2
3
4
5
6
Sodium salicylate
[bmim][Sal]
8.87
*
**
27.54
**
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
[OHC
[C OC
[OHC
[Im][Sal]
2
OC
mim][Sal]
mim][Sal]
2
mim][Sal]
8.41
8.18
10.18
*
2
2
**
3
6
compound was determined by median effect analysis.
3
**
**
*
Very high toxicity at low concentrations, LC50 could not be determined.
3
. Results and discussion
**IC50 is not detected in the investigated concentration range meaning that these
compounds can be consider as non-toxic.
Described tests on aquatic microorganisms (Artemia salina)
and on human non-tumor cell line (normal fetal lung
fibroblasts, MRC-5) were performed in order to investigate
influence of the alkyl chain substituent on the ILs toxicity.
Assay based on toxicity against Artemia salina is considered as
rapid, convenient, low cost and one of the most reliable
methods for the preliminary detection of mycotoxines, heavy
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.
1
00
100
95
8
0
60
90
40
85
20
80
0
75
0
5
10
15
20
25
30
35
0
5
10 15 20 25 30 35 40 45 50 55
c (Im-Sal) /
c (OHC OC mim-Sal) /
ꢀ
M
ꢀM
2
2
a)
b)
4
| J. Name., 2012, 00, 1-3
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100
70
60
50
40
30
20
10
0
8
0
60
40
20
0
0
5
10
15
20
25
30
M
35
40
45
0
5
10
15
20
25
30
c (OHC mim-Sal) /
ꢀ
c (C Omim-Sal) /
ꢀ
M
3
4
c)
d)
1
00
1
00
80
60
8
6
4
2
0
0
0
0
0
4
0
2
0
0
0
5
10
15
20
25
30
35
0
10
20
30
40
50
60
c (Bmim-Sal) /
ꢀM
c (Na-Sal) /
ꢀ
M
e)
Figure 2. A. salina mortality in function of concentration of: a) [OHC
4 hours; ○, aꢀer 48 hours)
f)
3 4
mim][Sal], b) [Im][Sal], c) [OHC mim][Sal], d) [C Omim][Sal],e) [bmim][Sal], f) Na-Sal (■, after
2
OC
2
2
The introduction of the oxygen in the form of a hydroxy and/or observation is in the agreement with those reported by Stolte
2
7,47
ether group in the side alkyl chain of the imidazolium ion leads et al.
2 2
In the case of [OHC OC mim][Sal] the introduction of
to a significant reduction of ILs toxicity on A. salina, wherein OH group in the alkyl chain with the existing ether oxygen had
the LC50 values of these newly synthesized ILs after 48 h were a lower impact on reducing toxicity, comparing introduction of
found to be similar as the LC50 obtained for the reference OH group in the non-functionalized alkyl chain.
sodium salicylate. A significant reduction of toxicity can be
In the case of healthy lung cells, MRC-5, it was observed
observed comparing the results obtained for [bmim][Sal] and that only [bmim][Sal] expressed significant toxicity, its IC50 was
ILs with the oxygen in the alkyl substituent (Table 1, Figure 2). 27.54 µM (Table 1). Otherwise, oxygen-modified ILs were not
It can be seen that introduction of the oxygen in the form of toxic against healthy cell line MRC-5. The citotoxicity tests
the hydroxide group in [OHC
on reducing toxicity then the presence of oxygen in the form of (0.01 to 100) µmol·mL and the results are presented
ether group in the side alkyl chain of [C OC mim][Sal]. This graphically in Figure 3.
3
mim][Sal], had a greater impact were performed in the ionic liquid concentration range from
–1
2
2
60
45
30
15
0
6
0
45
30
15
0
0
.01
0.1
1
10
100
1000
0.01
0.1
1
10
100
1000
c (OHC OC mim-Sal) /
ꢀM
c (Im-Sal) / ꢀM
2
2
a)
b)
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0
60
45
30
15
0
45
30
15
0
0.01
0.1
1
10
100
1000
0.01
0.1
1
10
100
1000
c (OHC mim-Sal) /
ꢀM
c (C Omim-Sal) /
ꢀ
M
3
4
c)
d)
6
0
5
0
5
0
60
45
30
15
0
4
3
1
0.01
0.1
1
10
M
100
1000
0.01
0.1
1
10
100
1000
c (Bmim-Sal) /
ꢀ
c (Na-Sal) / ꢀM
e)
f)
3 4
mim][Sal], b) [Im][Sal], c) [OHC mim][Sal], d) [C Omim][Sal],e) [bmim][Sal], f) Na-Sal
Figure 3. MRC-5 citotoxicity in function of concentration of: a) [OHC
2
OC
2
(
symbol+lines - experimental values; dashed red lines - calculated values)
It is known that structure of the cation has the greater oxygenation of the alkyl chain does not decrease toxicity of the
2
5,27,47-49
impact on the ILs toxicity.
From the results presented ionic liquids, which is in agreement with the results reported
5
3
in this manuscript it is obvious that alkyl side chain is the by Samori et al.
primary factor that affects the toxicity of imidazolium based Ionic liquids examined in this work, contains biological active
ionic liquids, namely change of its polarity. This phenomenon anion – salicylate, and cation as a drug carrier. Due to
can be explained by the assumption that the lipophilic cations dissociation of the ionic compounds, cation and anion can be
are adsorbed or intercalated in the cell membrane, causing delivered separately to the cells. Thus, decreasing the
"perturbations" in the membrane (expansion or swelling, lipophilicity of the cation, its toxicity and bioavailability can be
incerase in fluidity, lowering of the phase transition reduced without affecting bioavailability of salycilate anion.
temperature and alteration of the ion permeability of the
2
5,49-52
membrane).
The presented results indicate that the
Conclusions
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 (Figure 3e). Introduction of the oxygen in the
alkyl substituent increases polarity of the ion, while
lipophilicity decreases. This weakens the interactions between
lipid cell membrane and ionic liquid which significantly reduces
the toxicity (Figures 3a, 3c and 3d). Lower toxicity of the ionic
liquid which in the side chain contains hydroxide group 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 cellular
membrane and its reactivity, namely hydrophilicity/
hydrophobicity, is mainly responsible for the toxicity of the
In this paper the toxicity of newly synthesized salicylate
based ionic liquids has been investigated using aquatic
organisms (Artemia 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 then 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 a terminal position of the alkyl substituent of the
imidazolium cation, while the introduction of a larger number
of oxygen does not contribute to reduced toxicity. Bearing in
mind the obtained results, it opens the possibility of designing
new non-toxic ILs adjusting only lipophilicity of the cations
introducing polar oxygen groups in the side alkyl chain.
whole ionic liquid. Ionic liquid [C
the alkyl chain has a more hydrophobic methyl group
compared to [OHC mim][Sal] where side chain terminates with
distinctly polar OH group, explaining thus the higher toxicity of
OC mim][Sal]. Also, it can be observed that additional
2 2
OC mim][Sal] at the end of
3
[C
2
2
6
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2
9. K. Bica, C. Rijksen, M. Nieuwenhuyzena and R.D. Rogers, Phys.
Chem. Chem. Phys., 2010, 12, 2011.
This work was financially supported by the Ministry of 30. L. Alfonso, G. Ai, R. Spitale and G.J. Bhat, Br. J. Cancer, 2014,
Acknowledgements
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