Functionalized imidazolium-based ionic liquids: biological activity evaluation, toxicity screening, spectroscopic, and molecular docking studies

Article abstract of DOI:10.1007/s00044-020-02631-3

A series of long-chain imidazolium-based ionic liquids (ILs) 1-dodecyl-3-methylimidazolium chloride (1), 1,3-bis(octyloxycarbonylmethyl)imidazolium chloride (2) and 1-dodecyloxycarbonylmethyl-3-methyloxycarbonylmethylimidazolium chloride (3), were synthesized and evaluated as antimicrobials against a wide range of bacteria and fungi. Toxicological risks of selected compounds were assessed using the biomodels of various organizational and functional levels. All compounds demonstrated significant antibacterial and antifungal activity. The toxicity results indicate that ILs containing an ester functional group in the alkyl side chain exhibited much lower toxicity to D. magna and acetylcholinesterase inhibition than ILs with long alkyl chain without polar substituents, while toxicity toward Danio rerio was on a par. The HSA-binding properties of ILs have been investigated by FT-IR spectroscopy technique and the evidences have suggested that the test compounds could induce the protein unfolding and changes in the secondary structure of HSA. The docking studies were carried out to provide structural insights of the ILs–HSA-binding interactions. The docked compounds exhibit a high binding affinity to HSA and the hydrogen bonding, hydrophobic and electrostatic interactions played a major role in the process. ILs 2 and 3 may be perspective for further investigation as potential low-toxic biocides with high antimicrobial activity against reference and clinical multidrug-resistant strains.

Full text of DOI:10.1007/s00044-020-02631-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02631-3
ORIGINAL RESEARCH
Functionalized imidazolium-based ionic liquids: biological activity
evaluation, toxicity screening, spectroscopic, and molecular docking
studies
1
Ivan V. Semenyuta1 Diana Hodyna1 Alla D. Ocheretniuk2 Sergey I. Vdovenko3
● ● ●
●
●
●
●
●
Maria M. Trush
Sergiy P. Rogalsky4 Larisa E. Kalashnikova5 Volodymyr Blagodatnyi6 Oleksandr L. Kobzar2 Larisa O. Metelytsia1
●
Received: 16 June 2020 / Accepted: 3 September 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of long-chain imidazolium-based ionic liquids (ILs) 1-dodecyl-3-methylimidazolium chloride (1), 1,3-bis
(octyloxycarbonylmethyl)imidazolium chloride (2) and 1-dodecyloxycarbonylmethyl-3-methyloxycarbonylmethylimidazolium
chloride (3), were synthesized and evaluated as antimicrobials against a wide range of bacteria and fungi. Toxicological risks of
selected compounds were assessed using the biomodels of various organizational and functional levels. All compounds
demonstrated significant antibacterial and antifungal activity. The toxicity results indicate that ILs containing an ester functional
group in the alkyl side chain exhibited much lower toxicity to D. magna and acetylcholinesterase inhibition than ILs with long
alkyl chain without polar substituents, while toxicity toward Danio rerio was on a par. The HSA-binding properties of ILs have
been investigated by FT-IR spectroscopy technique and the evidences have suggested that the test compounds could induce the
protein unfolding and changes in the secondary structure of HSA. The docking studies were carried out to provide structural
insights of the ILs–HSA-binding interactions. The docked compounds exhibit a high binding affinity to HSA and the hydrogen
bonding, hydrophobic and electrostatic interactions played a major role in the process. ILs 2 and 3 may be perspective for further
investigation as potential low-toxic biocides with high antimicrobial activity against reference and clinical multidrug-resistant
strains.
●
●
●
●
●
Keywords Ionic liquids Antimicrobial activity Toxicity Human serum albumin Spectroscopy Molecular docking
* Maria M. Trush
Introduction
maria.m.trush@gmail.com
In recent years, there has been great interest in studying the
1
Department of Medical and Biological Researches, V.P. Kukhar
unique physical, chemical, and biological properties of ionic
liquids (ILs), which allow to use them as active pharma-
ceutical agents, for designing multifunctional materials, in
different processes and production technologies [1]. In
particular, ILs find their applications in chemical separa-
tions [2], electrochemistry [3, 4], organic chemistry [5],
polymer chemistry [6], biocatalysis [7], and for biomass
conversion [8], solar and thermal energy conversion [9],
biological and medical procedures [10, 11], and others.
Due to the wide use of ILs, increasing attention has been
paid to their potential toxicological effects on humans and
the environment. The risk of air pollution by ILs is minimal
because of their nonvolatile characteristics, but in large-
scale industrial applications they may be released into the
aquatic environment through accidental spills or via efflu-
ents [11].
Institute of Bioorganic Chemistry and Petrochemistry, National
Academy of Science of Ukraine, 1 Murmanska Str., Kyiv 02660,
Ukraine
2
3
4
5
6
Department of Bioorganic Mechanisms, V.P. Kukhar Institute of
Bioorganic Chemistry and Petrochemistry, National Academy of
Science of Ukraine, 1 Murmanska Str., Kyiv 02660, Ukraine
Department of Fine Organic Synthesis, V.P. Kukhar Institute of
Bioorganic Chemistry and Petrochemistry, National Academy of
Science of Ukraine, 1 Murmanska Str., Kyiv 02660, Ukraine
Laboratory of Modification of Polymers, V.P. Kukhar Institute of
Bioorganic Chemistry and Petrochemistry, National Academy of
Science of Ukraine, 1 Murmanska Str., Kyiv 02660, Ukraine
Department of Biomedical Engineers, National Technical
University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”,
37 Victory Ave., Kyiv 03056, Ukraine
Shupyk National Medical Academy of Postgraduate Education, 9
Dorohozhytska Str., Kyiv 04112, Ukraine

Medicinal Chemistry Research
C12H25
ILs are well-known for their strong cytotoxic action
against different cellular test systems such as the IPC-81
[12–14], PC12 [15], MCF-7 [16], HeLa [17], HT-29 and
CaCo-2 [18, 19], AGS and A549 [20], and other cell lines.
A number of phytotoxicity studies have been conducted
on the microalga Selenastrum capricornutum [21, 22],
marine macroalga Ulva lactuca [23], duckweed Lemna
minor [15, 24], wheat Triticum aestivum, garden cress
Lepidium sativum [25], and others.
N
N
+
N
120 oC
C12H25Cl
+
(C12C1IM-Cl)
Cl
N
CH3
CH3
Scheme 1 Synthesis of ionic liquid C12C1IM–Cl
Materials and methods
The toxicity researches of ILs to invertebrates are mostly
based on the use of Daphnia magna [22, 26, 27], however,
a number of studies have also used the snail Physa acuta
[28], the roundworm Caenorhabditis elegans [29] and the
zebra mussel Dreissena polymorpha [30] as biomodels for
toxicity testing.
The zebrafish (Danio rerio) as a vertebrate model plays a
key role in ecotoxicology and is currently widely used for
in vivo toxicity studies [22, 31]. A number of other verte-
brate species were examined for ILs toxicity such as the
common carp Cyprinus carpio [32], the goldfish Carassius
auratus [33], the frog Rana nigromaculata [34] and, of
course, rats and mice [35, 36].
The toxicological behavior of ILs can be evaluated by
investigating their effects on enzymes and proteins, as
shown by a number of studies using acetylcholinesterase
(AChE) from the electric eel Electrophorus electricus
[37, 38], HIV-1 integrase [39], bovine α-chymotrypsin [40],
adenosine monophosphate deaminase [41], luciferase from
Photinus pyralis (PpyLuc) [42], human serum albumin
(HSA) [43, 44], and bovine serum albumin [45, 46].
It should be noted that ILs exhibit different toxic effects,
depending on the nature of the biological system under
investigation [47]. The aqueous solubility, lipophilicity, and
susceptibility to hydrolysis of the structural elements of
salts are essential for their toxicity [10]. Thus, ecotox-
icological tests using environmental (biological) objects and
organisms of different trophic levels allow to evaluate the
consistency of the structure–activity relationships across
different biological targets.
In this article, ester-functionalized long-chain
imidazolium-based ILs have been synthesized and eval-
uated for their antimicrobial activity against eight different
strains of bacteria and fungi. In addition, their potential
ecotoxicological risks have been studied using D. rerio and
D. magna as test vertebrate and invertebrate organisms, and
AChE inhibitory activity of tested ILs was also evaluated.
Besides, the influence of ILs on the conformational char-
acteristics of the multifunctional transport protein HSA was
assessed by FT-IR spectroscopy and molecular docking
simulation. The findings of the study can be useful in the
development of new low-toxic biocidal agents with a wide
range of antimicrobial activity.
Materials
HSA was purchased from Sigma-Aldrich Chemicals Com-
pany (USA). The HSA and compound solutions were pre-
pared in 10 mM phosphate buffer (pH 7.4). The working
concentration of protein was 50 µM.
Following chemicals were used for the synthesis of ILs:
chloroacetyl chloride (98%), methyl chloroacetate (99%), 1-
octanol (98%), 1-dodecanol (99%), 1-chlorododecane
(97%), 1-methylimidazole (99%), imidazole (99%), acet-
onitrile (99.8%), ethyl acetate (99.8%), hexane (95%),
benzene (99.8%). All chemicals were purchased from
Sigma-Aldrich.
Methods
Synthesis of ionic liquids
1-dodecyl-3-methylimidazolium chloride (C12C1IM–Cl) (1)
The IL C12C1IM-Cl was synthesized according to Scheme 1.
The stirred mixture of 1-chlorododecane (10 g, 0.05 mol)
and 1-methylimidazole (3.1 g, 0.037 mol) was heated at
120 °C for 24 h. The reactionary mixture solidified after
cooling to room temperature. The product was purified by
recrystallization from hexane-ethyl acetate mixture (3:1 (v/v))
(Yield 6.8 g, 64%).
White solid, m. p. 46–47 °C. 1H NMR (400 MHz,
CDCl3): δ = 0.88 (t, 3H, CH3), 1.25 (m, 18H, СH3(CH2)9),
1.9 (m, 2H, NCH2CH2), 4.13 (s, 3H, NCH3), 4.31 (t, 2H,
NCH2), 7.4 (d, 1H, C4–H), 7.58 (d, 1H, С5–H), 10.56 (br s,
1H, C2–H).
1,3-bis(octyloxycarbonylmethyl)imidazolium chloride (IM
(CH2COOC8)2–Cl) (2)
The IL was synthesized according to Scheme 2. 20 g
(0.15 mol) of 1-octanol was dissolved in 100 ml of dry
benzene and 25 g (0.18 mol) of potassium carbonate was
added to the solution. To the stirred mixture was added
dropwise 20.5 g (0.18 mol) of chloroacetyl chloride. The

Medicinal Chemistry Research
C6H6
Scheme 2 Synthesis of ionic
liquid IM(CH2COOC8)2–Cl
+
ClCH2COCl
C8H17OH
ClCH2COOC8H17
CH2COOC8H17
K2CO3
N
N
85 oC
_
+
N
+
2 ClCH2COOC8H17
(IM(CH2COOC8)2-Cl)
Cl
N
H
CH3CN, K2CO3
CH2COOC8H17
C6H6
K2CO3
C12H25OH
ClCH2COCl +
ClCH2COOC12H25
CH2COOCH3
N
N
N
85 oC
CH3CN, K2CO3
85 oC
CH3CN
_
+
N
ClCH2COOC12H25
ClCH2COOCH3
+
+
Cl
N
H
N
CH2COOC12H25
CH2COOC12H25
(IMCH2COOC12CH2COOC1-Cl)
Scheme 3 Synthesis of ionic liquid IMCH2COOC12CH2COOC1–Cl
reaction was carried out for 6 h at room temperature. The
reactionary mixture was washed successively with water
(200 ml) and saturated water solution of sodium hydro-
carbonate (2 × 100 ml). The solution was dried over calcium
chloride overnight. The solvent was distilled off at normal
conditions. The product was dried in vacuum 5 mbar at
60 °C for 12 h. Octyl chloroacetate was obtained as trans-
parent liquid.
To the solution of imidazole (5 g, 0.07 mol) and octyl
chloroacetate (32 g, 0.15 mol) in 50 ml of acetonitrile was
added potassium carbonate (11 g, 0.08 mol). The mixture
was stirred and heated to a slight boil for 24 h. After cooling
to room temperature the residue of inorganic salts was fil-
tered off. The solvent was removed by distillation. The
product was purified by recrystallization from ethyl acetate
(Yield 16.2 g, 52%).
To the solution of imidazole (5 g, 0.07 mol) and dodecyl
chloroacetate (20 g, 0.08 mol) in 40 ml of acetonitrile was
added potassium carbonate (11 g, 0.08 mol). The mixture was
stirred and heated to a slight boil for 24 h. After cooling to
room temperature the residue of inorganic salts was filtered
off. The solvent was removed by distillation. The inter-
mediate product (1-dodecyloxycarbonylmethylimidazole)
was purified by recrystallization from hexane-ethyl acetate
mixture (4:1, v/v) (Yield 14.8 g, 72%).
White solid, m. p. 94–95 °C. 1H NMR (400 MHz,
CDCl3): δ = 0.87 (t, 3H, CH3), 1.25 (m, 18H, СH3(CH2)9),
1.62 (m, 2H, NCH2CH2), 4.16 (t, 2H, –COOCH2), 4.69 (s,
2H, NCH2), 6.95 (d, 1H, C4–H), 7.09 (d, 1H, С5–H), 7.51
(br s, 1H, C2–H).
The solution of 1-dodecyloxycarbonylmethylimidazole (5 g,
0.017 mol) and methyl chloroacetate (2 g, 0.018 mol) in 20 ml
of acetonitrile was heated to gentle boil for 24 h. The solvent
was removed by distillation. The final product was purified by
recrystallization from ethyl acetate (Yield 4 g, 58%).
White solid, m. p. 129–130 °C. 1H NMR (400 MHz,
CDCl3): δ = 0.86 (t, 6H, CH3), 1.26 (m, 20H, СH3(CH2)5),
1.65 (m, 4H, –COOCH2CH2), 4.17 (t, 4H, –COOCH2), 5.4
(s, 4H, NCH2), 7.54 (d, 2H, C4–H, С5–H), 10.46 (br s, 1H,
C2–H).
1
White solid, m. p. >230 °C (dec.). H NMR (400 MHz,
CDCl3): δ = 0.85 (t, 3H, CH3), 1.23 (m, 18H, СH3(CH2)9),
1.65 (m, 2H, –COOCH2CH2), 3.78 (s, 3H, COOCH3), 4.15
(t, 2H, –COOCH2), 5.37 (s, 2H, NCH2), 5.48 (s, 2H, NCH2),
7.6 (d, 1H, C4–H), 7.71 (d, 1H, С5–H), 10.2 (br s, 1H, C2–H).
1-dodecyloxycarbonylmethyl-3-
methyloxycarbonylmethylimidazolium chloride
(IMCH2COOC12CH2COOC1–Cl) (3)
Antimicrobial assay
The IL was synthesized according to Scheme 3.
Dodecyl chloroacetate was synthesized using the similar
method as octyl chloroacetate except 1-dodecanol was used.
Antibacterial and antifungal properties of the ILs were
tested using standard disk diffusion method in Mueller-

Medicinal Chemistry Research
Hinton agar and Sabouraud agar, respectively [48]. The
tested bacterial strains were Staphylococcus aureus ATCC
25923, S. aureus ampicillin-resistant strain isolated from
biomaterial, Escherichia coli АТСС 25922 and its
ampicillin-resistant isolate, Pseudomonas aeruqinosa
АТСС 27853, and fungal strains were Candida albicans
ATCC 10231 M885, clinically isolated fluconazole-
resistant test strains of C. albicans and Candida krusei. A
0.5 McFarland standard was used as a reference to adjust
the turbidity of microbial suspensions. The microbial load
was 1 × 105 colony-forming units in 1 ml. All ILs were
investigated in concentrations of 1.0 and 0.1%. The samples
were dissolved in water. The tested compounds (0.02 ml)
were applied to paper disks (6 mm). Several paper disks
were placed on each agar plate and incubated at 37 °C for
24 h. The experiments were repeated three times. The
antimicrobial activity of ILs was assessed by measuring the
diameter of the growth inhibition zone on agar plates
(in mm).
(7 test fish for each concentration) were exposed to ILs
solutions at concentrations of 0.01, 0.1, 1.0, 10.0, 15.0,
and 20.0 mg/L. The test concentrations were selected
based on data from the preceding acute toxicity tests and
preliminary test results in the range of 0.1–100 mg/L. The
mortality was checked every 24, 48, 72, and 96 h. Each
experiment was replicated three times. The obtained data
were statistically processed using the software package
STATISTICA 6 for Windows.
Acetylcholinesterase inhibition assay
AChE from electric organ of E. electricus, acetylthiocholine
iodide as substrate and Ellman’s reagent (5,5′-dithiobis-(2-
nitrobenzoic acid) were purchased from Sigma-Aldrich.
Anticholinesterase activity of the tested compounds was
investigated in a model system which consisted of 25 mM
phosphate buffer (pH 7.48), 0.1 mM acetylthiocholine
iodide, 1 mM DTNB, 1% DMSO, and water. The total
volume of the mixture was 0.5 ml. The inhibitory action of
the compounds was determined by modified Ellman’s
method [49]. The inhibitory activity was expressed as the
concentration that inhibited 50% of the AChE activity
(IC50). The reaction mixture was preincubated for 5 min at
25 °C and then reaction was started by adding the enzyme.
Spectrophotometric study was performed at a wavelength of
412 nm.
Acute toxicity test on Daphnia magna
The acute toxicity test of the investigated compounds to D.
magna was estimated by calculating the median lethal dose
(LD50) which is the concentration of a chemical that causes
death to 50% of the tested organisms. The tests were per-
formed according to the OECD 202 guideline (2004) in a
light incubator with the 22 1 °C temperature and 16:8 h
light:dark photoperiod. D. magna neonates (6–24-h old)
were used for the controls and for the geometric series of
concentrations of each test compound according to the 48-h
acute toxicity test procedure without food or organic
extract. Five neonates were randomly taken and placed in a
50 mL glass beaker containing 30 mL of test solution. The
mortality of the neonates in each beaker was observed after
48-h that being the endpoint for effect calculation. Animals
not able to swim within 15 s after gentle agitation of the test
vessel were considered to be immobilized. The sensitivity
of the D. magna to the reference toxicant potassium
dichromate (K2Cr2O7) was determined as well. Experiments
were run in triplicate. The averaged LD50 values in mg/L
were determined using STATISTICA 6 program.
Spectral study
1H NMR technique was used to characterize the structure of
synthesized ILs. The spectra were recorded with a Varian
Gemini-2000 (400 MHz) NMR spectrometer at 23 °C.
The FT-IR spectra were registered on Bruker VERTEX
70 FT-IR spectrometer equipped with KBr beam splitter,
RT-DLaTGS detector and diamond attenuated total reflec-
tion accessory at ambient temperature. The spectrometer was
continuously purged with dry air during the measurements.
The absorption spectra were obtained in the wavenumber
rage of 4000–400 cm−1 and spectral resolution 2 cm−1. A
spectrum was taken as an average of 100 scans to increase
the signal to noise ratio. Base-line correction, normalization
and band deconvolution were performed for all the spectra
by OPUS software. The FT-IR spectrum of free HSA and
HSA–IL complexes were obtained in the region of
1400–1800 cm−1. The FT-IR spectrum of free HSA was
obtained by subtracting the absorption spectrum of the
phosphate buffer from the spectrum of the protein solution.
For the net interaction effect, the difference spectra of
(HSA–IL complex) were generated using the featureless
region of the HSA solution 1800–2100 cm−1 as an internal
standard [50]. The spectral differences were used to inves-
tigate the nature of HSA–IL interaction.
Acute toxicity test on Danio rerio
The acute toxicity of ILs to the D. rerio (zebrafish) was
assessed measuring their lethal effect after 96-h exposure
in a static test, according to the procedure 203 of the
OECD (1992). The experimental zebrafish had an average
body length of 11.8 0.1 mm and a weight 2.6 0.2 g.
During the experiment, fish were kept in glass aquaria
with well-aerated water (pH 7.3 0.3) at 26.5 °C. Fish
were not fed during the acute toxicity tests. Test organisms

Medicinal Chemistry Research
Molecular docking
Results and discussion
We used AutoDock Tools (ADT) (ver. 1.5.6) [51] to pre-
pare the docking compatible structure formats of the pro-
tein, ligands and grid box creation. The crystal structure of
the HSA (PDB code 1AO6) [52] was used in our study. The
structure of A-subunit of HSA was selected and stored as a
pdb file by Accelrys DS (ver. 2.5.5) [53]. To add all polar
hydrogens in ADT, we used the noBondOrder method and
renumbered atoms to include the new hydrogens. The par-
tial charges were calculated and added using Gasteiger
method. The prepared file was saved in PDBQT format. We
used the ChemAxon Marvin Sketch 5.3.735 program [54]
for the creation and optimization of ligand structures. These
structures were saved in Mol2 format. The optimization of
ligand structures and energy minimization were performed
by AM1 semi-empirical quantum-mechanical method
(MOPAC2016) [55]. Partial charges and torsions angles of
ligands were changed by ADT, and the resulting files were
saved in PDBQT format. Thus, the prepared protein and
ligands were used as inputs for molecular docking using the
program AutoDock Vina 1.1.2 [56]. AutoGrid was used for
the preparation of the grid map using a grid box. The box
center (x = 25.915, y = 35.711, z = 34.227) was set to the
atom CD2 of the tryptophan amino acid residue (Trp214) in
the original pdb file. A grid of 30 × 30 × 30 points was used
with grid spacing of 1 Å. The software package Accelrys
DS was used for illustration and to study protein–ligand
interactions.
Antimicrobial studies
All compounds tested exhibited broad spectrum anti-
microbial activity. The results of the mean and standard
deviation of the diameter of inhibition zones are summar-
ized in Table 1.
It can be summarized that the long-chain imidazolium-
based IL C12C1IM–Cl (1) was more efficient in inhibiting
the growth of both gram-positive and gram-negative bac-
teria. The diameters of the inhibition zones of bacterial
growth varied within 29–33 mm for the ATCC cultures S.
aureus, E. coli, and P. aeruginosa in the concentration 1.0%
(0.7 µМ on a disk). Ampicillin-resistant isolate S. aureus
was more sensitive to compound 1 than E. coli (AMPR)
with the diameters of corresponding inhibition zones of
about 21 and 15 mm, respectively. When the amount of
tested compound was reduced to 0.1%, zones of growth
inhibition were in the range from 15 to 24 mm for standard
ATCC strains, and 9–13 mm for clinical isolates.
Compounds 2 and 3 containing polar ether functional
groups in the alkyl chains are characterized by lower anti-
bacterial activity. The diameters of the zones of growth
inhibition for the aforecited active compounds in the con-
centration 1.0% (0.5 µМ on a disk) against S. aureus ATCC
25923 were 21–22 mm, against E. coli АТСС 25922 were
16–25 mm, for P. aeruginosa АТСС 27853 constituted
only 14–16 mm. The previously mentioned substances were
Table 1 Antimicrobial activity of long-chain imidazolium ionic liquids (average of three measurements)
Sample
C (%)
Diameter of the inhibitory zone (mm)
Bacteria
Fungi
S.aa
S.ab
E.cc
E.cd
P.ae
С.af
С.ag
С.kh
1
2
3
1.0
0.1
1.0
0.1
1.0
0.1
33.7 0.9
21.7 0.9
22.0 0.9
na
21.3 0.6
13.3 0.3
15.3 0.3
na
29.3 0.3
15.0 0.6
16.7 0.3
na
15.3 0.6
9.3 0.6
10.3 0.6
na
30.7 0.6
24.0 0.6
14.0 0.6
na
53.3 0.9
37.7 0.3
17.0 0.3
9.0 0.3
28.0 0.3
17.7 0.6
13.3 0.3
9.3 0.6
63.7 0.3
38.3 0.3
21.7 0.3
15.3 0.3
23.3 0.3
14.0 0.6
21.3 0.3
na
15.3 0.6
na
25.0 0.9
na
na
16.7 0.3
na
16.3 0.6
12.7 0.6
15.0 0.6
10.0 0.3
na
na no activity
aStaphylococcus aureus ATCC 25923
bStaphylococcus aureus (ampicillin-resistant (AMPR) isolate)
cEscherichia coli АТСС 25922
dEscherichia coli (ampicillin-resistant (AMPR) isolate)
ePseudomonas aeruginosa АТСС 27853
fCandida albicans ATCC 10231
gCandida albicans (fluconazole-resistant isolate)
hCandida krusei (fluconazole-resistant isolate)

Medicinal Chemistry Research
Table 2 Acute toxicity of long-
chain imidazolium ionic liquids
to D. magna and D. rerio
Sample
D. magna
D. rerio
LD50, mg/L*
Aquatic toxicity**
LD50, mg/L*
Aquatic toxicity**
1
<0.01
(І)
4.72 0.1
2.15 0.2
7.5 0.51
0.018 0.01
(ІV)
(ІV)
(ІV)
(ІІ)
2
0.81 0.29
2.08 0.51
<0.01
(ІII)
(ІV)
(І)
3
CPC
*All values are presented as the mean standard deviation (р ≤ 0.05)
**Aquatic toxicity rating scale (by Passino and Smith): <0.01, supertoxic (І); 0.01–0.1, extremely toxic (ІІ);
0.1–1, highly toxic (ІІІ); 1 to 10, moderately toxic (ІV); 10–100, slightly toxic (V); 100–1000, practically
harmless (VІ)
Table 3 AChE inhibitory potential of long-chain imidazolium ionic
liquids
considerably less active against ampicillin-resistant S. aur-
eus (diameter of the zone ≈ 15.5 mm) and almost inactive
against E. coli isolate. With a decrease in concentration to
0.1%, there was a complete loss of antimicrobial properties.
The results of the antifungal assay indicate that the
compound 1 exhibited strong activity against C. albicans
ATCC 10231 with the diameters of inhibition zones up to
54 mm at concentration 1.0% (0.7 µМ on a disk) and up to
38 mm at concentration 0.1%. Similar results were noted for
fluconazole-resistant C. krusei. The about two-times lower
activity of compound 1 was observed against fluconazole-
resistant isolate of C. albicans.
According to the obtained results, C. albicans ATCC
10231 and its isolate were more tolerant toward the com-
pounds 2 and 3 with ether functional groups in the alkyl
chains then C. krusei isolate. The diameters of the zones of
inhibited growth for these compounds in the concentration
1.0% (0.5 µМ on a disk) on the strains of C. albicans varied
within 13.3–15.0 mm as compared to 21.7–23.3 mm against
C. krusei.
Sample
IC50, μM
1
32.4 3.26
>100
2
3
>100
CPC
3.99 0.14
0.81 0.29 mg/L. It should be noted that cationic disin-
fectant CPC was categorized as supertoxic according to
classification Passino and Smith [57].
The results demonstrated that all test samples can be
classified as moderately toxic (ІV) substances toward D.
rerio according to the acute toxicity rating scale. The
highest toxic effect was observed for compound 2 with a
LD50 2.15 0.2 mg/L. Whereas the less toxic were com-
pounds 1 and 3 with the LD50 values being 4.72 and
7.50 mg/L, respectively. For comparison, the toxic activity
of the cationic surfactant CPC was also investigated in our
A reduction in the concentration to 0.1% of all tested
compounds resulted in a 30–40% loss of the antifungal
activity, but even in the case of functionalized imidazolium
ILs they were still effective.
study. According to the obtained LD50 values, CPC (LD50
=
0.018 0.01 mg/L) could be classified as extremely toxic
(ІІ) to fish, which agrees with known literature data [58, 59].
This indicated that CPC could cause deleterious effect to the
population of D. rerio and indirectly disturb freshwater
ecosystem functions, which once again confirms the rele-
vance of our study.
The ILs did not significantly affect AChE activity (see
Table 3). Compounds 1 showed the highest AChE inhibi-
tory activity with IC50 values of 32.4 3.26 μM. Com-
pounds 2 and 3 containing ether groups exhibited weak
inhibitory effects on activity of AChE with IC50 values
above 100 μM. CPC demonstrated the most potent AChE
inhibition with IC50 of 3.99 0.14 μM.
Acute toxicity assessment
The results of acute toxicity testing of the ILs on D. magna
and D. rerio are presented in Table 2.
The acute toxicity results of studied ILs toward D.
magna indicated the dependence of toxicity on ILs structure
(Table 2). The most toxic with LD50 value < 0.01 mg/L was
imidazolium-based IL 1 containing alkyl chain length of 12
carbon atoms which could possibly explain the high anti-
microbial activity of this compound. The introduction of
polar ether functional groups into alkyl chains reduced the
toxicity of imidazolium ILs. The LD50 value was 2.08
0.51 mg/L for compound 3 which allows it to be classified
as moderately toxic. IL 2 with two identical ester-
functionalized alkyl chains C8 was highly toxic with LD50
FTIR spectra of ILs–HSA complexes
In this work, we have studied the interactions of some
functionalized imidazolium salts with HSA by means of FT-

Medicinal Chemistry Research
(a)
IR spectroscopy. FT-IR spectra were recorded in the mid-
range, which covers the frequency range from 4000 to
400 cm−1. The wavelength region includes bands of three
conformational sensitive vibrations within the protein
backbone, namely, Amide I, Amide II, and Amide III.
Among them Amide I is most widely used and provides
information on protein secondary structure [60, 61] and its
changes due to protein interactions with ILs [62].
Results of quantitative analysis of albumin secondary
structure and adducts with imidazolium salts are shown in
Fig. 1 and Table 4.
1720
1700
1680
1660
Wavenumber cm-1
1640
1620
1600
(b)
This study demonstrated that native HSA consisted of 46.9%
α-helix (1655 cm−1), 25.2% β-sheet parallel (1621 cm−1),
24.3% random coil (1641 cm−1), and small percentage (0.6%)
of β-antiparallel structure (see Fig. 1 and Table 4).
In case of HSA–C12C1IM–Cl system, the influence of long
alkyl substituent on the imidazolium salt interaction with
HSA is seen from a comparison of the changes in secondary
structure of the protein. This interaction invokes generation of
the β-antiparallel form (appears at 1700 cm−1). Percentage of
both turn and α-helix forms significantly increases whereas
content of random coil and β-sheet parallel decreases.
At the same time the interaction of compound IM
(CH2COOC8)2-Cl (2), containing two symmetric ester-
functionalized C8 alkyl chains, with HSA led to a slight
decrease in both wavenumber and quantitatively of β-sheet
parallel forms in protein secondary structure (from 25.2 to
18.9%). While the percentage of random coil and turn
structure increases at the same time with the main bands
shift to lower wavenumbers. The total amount of α-helix
forms in the protein structure was not changed significantly.
1720
1700
1680
1660
Wavenumber cm-1
1640
1620
1600
1580
(c)
1720
1700
1680
1660
1640
1620
1600
Wavenumber cm-1
(d)
Interaction
of
HSA
with
compound
IMCH2COOC12CH2COOC1–Cl (3) results in increase of turn
structure percentage (from 0.6 to 2.1%) and α-helix form
(from 46.9 to 70.3%). The percentages of random coil and
β-sheet parallel form decreased with a concurrent shift to lower
wavenumbers due to intermolecular hydrogen bond formation.
From the experimental results, it can be concluded that in
all complexes of HSA with studied ionic salts percentage of
β-sheet parallel structure is significantly lower than in free
HSA. However, there is the appearance of the turn form and,
in some cases, the increase in α-helix content which is often an
important determinant of protein-binding interactions. Thus,
the analysis of the obtained IR spectra showed that the shift in
peak positions of amide bands of HSA is the result of con-
formational changes in the secondary structure of the protein
due to formation of intermolecular hydrogen bonds. This can
be interpreted as HSA-binding activity of imidazolium salts.
1720
1700
1680
1660
Wavenumber cm-1
1640
1620
1600
Fig. 1 Curve-fitting of amide I region (1720–1600 cm−1) of FTIR
spectrum: a free HSA; b HSA–IL 1 complex; c HSA–IL 2 complex; d
HSA–IL 3 complex
previous research [43, 44, 63], in the current study ILs were
docked to the hydrophobic pocket located in subdomain IIA
of HSA (Sudlow’s Site I).
Molecular docking of the HSA–ILs complexes
The molecular docking of compound 1 (Fig. 2) indicates
the formation of different stabilizing interactions of the
imidazole ring such as the electrostatic with Lys195
(3.79 Å), the hydrophobic π-alkyl with Ala291 (4.88 Å) and
The molecular docking studies provide more detailed
information about ligand–protein interactions. Based on

Medicinal Chemistry Research
Table 4 Peak frequencies (cm−1
and percentage (%) of the main
absorption bands of the FT-IR
spectra of HSA before and after
interaction with the
)
System
HSA
Assignment
β-Antiparallel
(1687–1700)
Turn
(1672–1687)
α-Helix
Random coil
(1627–1643)
β-Sheet parallel
(1610–1627)
(1643–1672)
investigated ILs
–
1686 (0.6)
1655 (46.9)
1641 (24.3)
1633 (17.7)
1621 (25.2)
1619 (7.6)
HSA–IL 1 1700 (5.3)
1681 (12.7)
1668 (3.4)
1652 (53.3)
HSA–IL 2
HSA–IL 3
–
–
1674 (4.1)
1679 (2.1)
1659 (22.8)
1645 (26.4)
1632 (27.8)
1634 (20.6)
1603 (18.9)
1617 (7.0)
1666 (12.8)
1653 (57.5)
Fig. 2 Molecular docking of
imidazolium ionic liquid 1 into
the active site of HSA
the hydrogen bonds with Glu292 (3.41 Å), Ser192 (3.67 Å),
Glu153 (3.76 Å). The long alkyl chain C12 of the ligand
connects with the amino acids Leu234 (3.75 Å), Leu219
(3.95 Å), Ile264 (4.83 Å), and Phe223 (5.02 Å) through four
hydrophobic alkyl and π-alkyl interactions.
The results of molecular docking of IL 2 (Fig. 3) show
that the imidazole ring of the ligand generates an electro-
static (4.01 Å) with Lys199 and hydrophobic π-alkyl
(4.04 Å) with Ala291 interactions. The carboxyl groups of
the ligand participate in the interaction with amino acids
Lys199 (3.19 Å), Arg222 (3.07 Å), Glu292 (3.39 Å),
Ala191 (3.22 Å, 2.82 Å) by formation of hydrogen bonds.
Stabilization of the ligand alkyl chains occurs due to
hydrophobic interactions with amino acids Ile290 (4.46 Å),
Ala261 (3.68 Å), Arg257 (4.53 Å), Ala191 (4.46 Å), and
Lys195 (4.94 Å).
(4.50 Å), Leu219 (4.52 Å), and Ile264 (5.46 Å) though
the five hydrophobic alkyl interactions. Furthermore, the
carboxyl groups of the ligand are linked by hydrogen
bonds with Lys195 (2.80 Å), Lys199 (3.06 Å), His242
(3.46 Å), and the hydrophobic alkyl interaction with
Arg218 (5.11 Å).
Table 5 shows that all ligand–protein complexes are
quite stable due to the high predicted binding energy ΔG. In
particular, the highest predicted binding affinity is observed
for HSA–IL 2 complex with the ΔG value of −7.2 kcal/
mol. The predicted binding energy of other protein–ligand
complexes is in the range of −6.8 to −7.1 kcal/mol. The
results indicate that non‐specific interactions such as elec-
trostatic and hydrophobic attractions as well as hydrogen
bonding are involved in the binding of the structural ele-
ments of ligands and protein.
The results of molecular docking of compound 3
(Fig. 4) indicate the formation of the electrostatic inter-
actions between the imidazole ring and residues Lys199
(3.95 Å), Arg222 (3.95 Å), and the hydrophobic π–alkyl
interactions with Ala291(5.35 Å) and Arg218 (5.07 Å).
The long alkyl chain C12 of the ligand is associated with
amino acids Leu234 (3.92 Å), Leu238 (4.29 Å), Leu260
Conclusions
In this paper, we report the synthesis and antimicrobial
evaluation of some long-chain imidazolium-based ILs.
Among them, compound 1 containing one long alkyl chain

Medicinal Chemistry Research
Fig. 3 Molecular docking of
imidazolium ionic liquid 2 into
the active site of HSA
Fig. 4 Molecular docking of
imidazolium ionic liquid 3 into
the active site of HSA
Table 5 Interaction types and free binding energy of studied protein–ligand complexes
Sample ΔG (kcal/mol) Hydrogen bond
Hydrophobic interaction
Electrostatic
interaction
1
2
–6,8
–7,2
Glu292 (3.41 Å) Ser192 (3.67 Å)
Glu153 (3.76 Å)
Ala291 (4.88 Å) Leu234 (3.75 Å) Leu219 (3.95 Å) Ile264
(4.83 Å) Phe223 (5.02 Å)
Lys195
(3.79 Å)
Ala191 (2.82 Å) Ala191 (3.22 Å)
Arg222 (3.07 Å) Glu292 (3.38 Å)
Lys199 (3.19 Å)
Ala291 (4.04 Å) Ile290 (4.46 Å) Arg257 (4.53 Å) Ala191
(4.46 Å) Lys195 (4.94 Å) Ala261 (3.68 Å)
Lys199
(4.01 Å)
3
–7,1
Lys195 (2.80 Å) Lys199 (3.06 Å)
His242 (3.46 Å)
Ala291 (5.35 Å) Arg218 (5.07 Å) Leu234 (3.92 Å), Leu238 Lys199
(4.29 Å), Leu260 (4.50 Å), Leu219 (4.52 Å) Ile264 (5.46 Å) (3.95 Å)
Arg218 (5.11 Å)
Arg222
(3.45 Å)

Medicinal Chemistry Research
displayed significant growth inhibitory action against all
microbial species and at the same time the highest toxicity
value for D. magna (toxicity class I) and AChE inhibition
(IC50 value of 32.4 3.26 μM). The introduction of two ester‐
functionalized alkyl chains in the structures of ILs 2 and 3
resulted in a significant reduction of their toxic potential
toward D. magna (toxicity classes III and IV) and the AChE
inhibitory activity with IC50 values above 100 μM, while
antimicrobial activity decreased only by 35%. It should be
noted that Candida species were more sensitive than bacteria
to the action of all studied compounds. All tested ILs
demonstrated similar moderate toxicity to zebrafish (toxicity
class IV). The results obtained from FT-IR spectroscopy
analysis showed that the test compounds could induce the
protein unfolding and changes in the secondary structure of
HSA. Molecular docking results indicated that all tested
compounds showed a high binding affinity to HSA with
predicted binding energies in the range from −6.8 to
−7.2 kcal/mol. Hydrogen bonds, electrostatic and hydro-
phobic interactions play a significant role in the formation
and the stabilization of these ligand–protein complexes. Thus,
ILs 2 and 3 are original objects for further studies as potential
biocides with moderate toxicity and high antimicrobial
potential, including against resistant strains.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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