Synthesis and antiproliferative activity of ammonium and imidazolium ionic liquids against T98G brain cancer cells

Article abstract of DOI:10.3390/molecules171213727

Four ammonium and imidazolium ionic liquids (ILs) have been synthesized and screened against the T98G cell line (brain cancer) and HEK normal cells. Treatment induced metabolic cell death (MTT), growth inhibition, clonogenic inhibition were studied as cellular response parameters. Treatment with ILs enhanced growth inhibition and cell death in a concentration dependent manner in both the T98G and HEK cell lines. At higher concentrations (>0.09 mg/mL) the cytotoxic effects of the ILs were highly significant. An inhibitory effect on clonogenic capacity was also observed after cell treatment. Amongst all ILs IL 4 (BMIMCl) exhibited potent activity against T98G brain cancer cells. Despite potent in-vitro activity, all ILs exhibited less cytotoxicity against the normal human HEK cells at all effective concentrations.

Full text of DOI:10.3390/molecules171213727

Molecules 2012, 17, 13727-13739; doi:10.3390/molecules171213727
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antiproliferative Activity of Ammonium and
Imidazolium Ionic Liquids against T98G Brain Cancer Cells
Nagendra Kumar Kaushik, Pankaj Attri, Neha Kaushik and Eun Ha Choi *
Plasma Bioscience Research Center, Kwangwoon University, Seoul 139701, Korea;
E-Mails: kaushik.nagendra@kw.ac.kr (N.K.K.); chem.pankaj@gmail.com (P.A.);
neha.bioplasma@gmail.com (N.K.)
* Author to whom correspondence should be addressed; E-Mails: ehchoi@kw.ac.kr or
Kaushik.nagendra@gmail.com; Tel.: +82-10-2240-5236; Fax: +82-2-940-5664.
Received: 22 October 2012; in revised form: 13 November 2012 / Accepted: 14 November 2012 /
Published: 22 November 2012
Abstract: Four ammonium and imidazolium ionic liquids (ILs) have been synthesized and
screened against the T98G cell line (brain cancer) and HEK normal cells. Treatment
induced metabolic cell death (MTT), growth inhibition, clonogenic inhibition were studied
as cellular response parameters. Treatment with ILs enhanced growth inhibition and cell
death in a concentration dependent manner in both the T98G and HEK cell lines. At higher
concentrations (>0.09 mg/mL) the cytotoxic effects of the ILs were highly significant. An
inhibitory effect on clonogenic capacity was also observed after cell treatment. Amongst
all ILs; IL 4 (BMIMCl) exhibited potent activity against T98G brain cancer cells. Despite
potent in-vitro activity, all ILs exhibited less cytotoxicity against the normal human HEK
cells at all effective concentrations.
Keywords: ionic liquid; T98G brain cancer cells; HEK non-malignant cells
1. Introduction
In the race to synthesize new pharmceutical drugs, ionic liquids (ILs) have attracted a great deal of
attention amongst in the scientific community due to their variety of potential pharmaceutical
applications. ILs represents a big success for industrial and engineering chemistry at the beginning of
the 21st century. ILs are defined as thermally stable salts synthesized by combining organic cations
with a wide variety of anions. The huge interest in ILs has led to a broad and diverse range of ions

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known to support IL formation. One of the most appealing features of ILs for pharmaceutical
applications is that they are highly customisable materials that can be specially made with pre-selected
characteristics by varying the cations and anions of which they are comprised. Combinations of different
cations and anions result in various ILs which provide a wide range of hydrophobicity/hydrophilicity,
acidity/basicity, viscosities, etc. [1,2]. IL strategies can take advantage of the dual nature (discrete ions)
to realize enhancements which may include controlled solubility (e.g., both hydrophilic and
hydrophobic ILs are possible), bioavailability or bioactivity, stability, elimination of polymorphism,
new delivery options (e.g., slow release or the IL-API as ‘solvent’), or even customized pharmaceutical
cocktails [3]. ILs having an unique protective metabolic roles can act as an antioxidants [4], protect
macromolecules, enhance protein folding and regulate cell volume [5,6]. ILs have also attracted the
scientific community’s due to their pharmaceutical properties such as antimicrobial [7–12], antiseptic
or antifouling actions [13], acetylcholinesterase (AChE) inhibition [14], AMP deaminase inhibition [15],
delivery of anti-inflammatory drugs [16], local anesthetic [3], anti-nociceptive [3], anticholinergic [3]
and anticancer activities [13,17], and in protein formulations [18–21]. Enzymes suspended in the ILs
could be reused three times, with less than 10% loss of activity per cycle without influencing
enantioselectivity [22]. Based on these interesting properties, ILs appear ready to provide a new
research outlook in the field of medicinal chemistry. However the toxicity of some ILs has not been
explored much in applications where this property can be desirable and lead to a variety of
pharmaceutical applications [23]. Carson et al. have reported the broad spectrum antibioflim activity of
1-alkyl-3-methylimidazolium chloride ILs against a variety of clinically important microbes [24].
Therefore, ILs with their tunable properties and toxicities could potentially be designed as anti-cancer,
anti-viral and other therapeutic agents [13]. If a therapeutic response were seen, then the major
advantage of ionic liquids would be in tuning their toxicity while tailoring the physio-chemical and
pharmacological properties necessary for the desired therapeutic application [13].
The present work is in continuation of our search for biological significant properties of ILs based
on extensive studies reported earlier by Malhotra and Kumar [13,17]. In the present study, the
anticancer activity of four ILs against T98G brain cancer cells was described. Cytotoxicity of the
synthesized ILs on normal cells were assessed using the HEK cell line, which resembles developing
neuron and neuronal stem cells and is also mentioned as a good model for neuroscience studies [25].
Based on this background, we have used HEK normal cells as a model for brain normal cells in this study.
2. Results and Discussion
2.1. Synthesis
We have synthesized and studied the anticancer effects of ammonium and imidazolium ILs. The
1
structure of ILs 1, 2 and 3 are clearly supported by their H-NMR and microanalysis data. Figure 1
shows the prepared ILs, triethylammonium sulfate (TEAS, 1), triethylammonium phosphate
(TEAP, 2), 1-methylimidazolium chloride ([MIM]Cl, 3) and 1-butyl-3-methylimidazolium chloride
([BMIM]Cl, 4).

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Figure 1. Structure of ammonium and imidazolium ILs. Brown, white, red, blue, pink,
golden and green colored balls represent carbon, hydrogen, oxygen, nitrogen, phosphorus,
sulphur and chlorine atoms, respectively. Small pink balls represent the lone pairs on
oxygen atoms.
2.2. MTT Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay is
widely accepted as a reliable way to measure the cell proliferation rates and cell death. The data
obtained by the MTT assay show that ILs 1, 2, 3 and 4 had inhibitory effects on the growth of T98G
and HEK cells in dosage-dependent manners (Figures 2 and 3). ILs 1, 2, 3, and 4 can inhibit 50%
T98G cell growth (IC50) with a range of 0.78–0.09 mg/mL after 24, 48 and 72 h treatment (Figure 2).
The maximum inhibitory effect was shown by IL 4 on T98G cells after 72 h of treatment, however IL
3 also effectively inhibits the growth of T98G cells at all the concentrations tested (0.78–0.09 mg/mL)
and in time dependent manner (Figure 2). Overall IL 4 was the most potent one, having a significant
inhibitory effect on T98G cells growth. All ILs are less toxic on HEK cells at all effective
concentrations as compared with T98G cells (Figure 3).
Figure 2. % Viability evaluated from MTT assay on T98G brain cancer cells treated with
ILs for 24, 48, and 72 h. All values are considered significant, when p < 0.05.

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Figure 2. Cont.
Figure 3. % Viability evaluated from MTT assay on normal HEK cells treated with ILs for
24, 48, and 72 h. All values are considered significant, when p < 0.05.

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ILs 1 and 2 are less toxic to the HEK cells with 75–99% viability at range of 0.39–0.09 mg/mL
concentration at all incubation time intervals, which further increased by decreasing concentration of
ILs (Figure 3). For further growth kinetic studies, we have selected ILs 1 and 4 on the basis of its
significant toxicity, against T98G brain cancer cells.
2.3. Growth Kinetics Assay
Figure 4 shows the growth kinetics of T98G cells treated with ILs 1 and 4. Cell proliferation
kinetics have been studied at 24, 48, 72 h after IL treatment, following trypsinization and counting
total cells per plate by using a trypan blue dye and a hemocytometer. Data obtained from the growth
kinetics assay shows that the ILs 1 and 4 have an inhibitory effect on the growth of T98G cells. IL 1
shows this inhibitory effect only in a concentration dependent manner, however IL 4 shows an
inhibitory effect in a concentration as well as in an incubation time dependent manner (Figure 4). It was
also noted that the cells exposed to 0.097 mg/mL concentration of IL 4 shows less significant (p > 0.04)
growth inhibitory effects than those treated with 0.19, 0.39 and 0.78 mg/mL concentration (p < 0.04).
Maximum effect was shown by 0.78 mg/mL concentration of IL 4; where it inhibits the growth of cells
up to 61% at 72 h after treatment and its viability range was 39.9% (p = 0.03). In the case of 0.19 and
0.39 mg/mL exposures of IL 4, we found 56–53% cells death (p < 0.04) at 72 h after treatment.
Whereas in case of IL 1, we found only 16–37% cell death (p < 0.05) at 0.39 and 0.78 mg/mL
concentrations, at all incubation time after treatment. Overall, growth kinetics assay results also shows
that IL 4 is having maximum inhibitory effect on T98G cells. Cell morphology analysis have revealed
that size and density of viable cells were also affected by treatment of IL 4 (Figure 5).
Figure 4. Growth kinetics of T98G cells at 24, 48 and 72 h after treatment by ILs at
0.097–0.78 mg/mL concentrations. Untreated cells are taken as control and all values given
as mean (±SE) of three independent experiments, n = 3. All values are considered significant,
when p < 0.05.

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Figure 5. Morphology of T98G cells. treated with IL 4 at 0.19 mg/mL concentration after
incubation for 24 h. Control is untreated cells.
2.4. Clonogenic Assay
The clonogenic assay shows the effect of ILs 1, 2, 3 and 4 on the colony-forming capacity and
survival of exponentially growing T98G cells. We have used the clonogenic assay to confirm our
growth inhibition results with the ILs. The clonogenic assay or colony formation assay is an in vitro
assay based on the ability of a single cell to grow into a colony. Only a fraction of seeded cells retain
the capacity to produce colonies after cytotoxic drug treatment. It observed in Figure 6, the surviving
fraction of T98G cells has been drastically decreased after treatment with ILs.
Figure 6. Effect of ILs on the colony-forming capacity or clonogenic survival of
exponentially growing T98G cell lines studied by a clonogenic assay. Data presented are
mean values from three independent observations, n = 3. All values are considered
significant, when p < 0.05.
All IL treatments enhanced cell death and also inhibited the colony-formation capability in the
T98G cell population in a concentration dependent manner. After treatments with different
concentrations (0.097–0.78 mg/mL), the surviving fractions of T98g cells declined, as evidenced by
the reduction in the number of colonies formed. Even at low doses (0.097, 0.19 and 0.39 mg/mL)

Molecules 2012, 17
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exposure to IL 4 results in a significant decline (p < 0.04) in colony survival and their corresponding %
survival were found to be 56, 19.2 and 4.8, respectively. However, a more significant drastic decline
(p = 0.04) in their survival fraction could be observed after exposure to 0.78 mg/mL treatment of IL 4
and their survival fraction were found to be 0.8. IL 1 also shows higher inhibitory effect on T98G cells,
and at doses of 0.097, 0.19, 0.39 and 0.78 mg/mL exposure showed % survival rates of 81, 60.8, 32
(p = 0.046) and 9.6 (p = 0.007), respectively. This shows that these treatments are significantly
inhibiting the colony formation capabilities of brain cancer cells at all the dosages. The most drastic
clonogenic inhibitory effect was found after treatment with 0.39 and 0.78 (p < 0.05) mg/mL
concentrations of ILs.
2.5. In Silico Pharmacokinetics
For a molecule to be a potential drug, besides having a good biological activity, it must have good
pharmacokinetic properties in biological systems. To access the pharmacokinetic profile of the
synthesized molecules, we used well validated in silico tools: Osiris, Chemaxon and Catalyst. These
tools have been validated with almost 7,000 drug molecules available on the market.
The analysis of theoretical toxicity risks for the ILs using the OSIRIS program shows that all ILs
were less toxic and can be used as therapeutic molecules (Figure 7).
Figure 7. In silico drug safety analysis for ILs by the Osiris software. High score is an
indicator of safe drugs (Score 1 is a highly safe drug indicator).
As these ILs are considered for oral delivery, they were submitted to the analysis of Lipinski ‘rule
of five’, druglikness and drug score by using the Catalyst software (Table 1). Our results pointed that
all effective ILs fulfill this rule and their druglikness property such as molecular weight (118–199),
LogP (0.12–1.5), nHBA (1–2), nHBD (0) and number of rotatable bonds (rotb) (0–3) were better than
those of commercial drugs (Table 1). Finally, we evaluated all ILs as potential drugs by calculating
druglikeness and drug-score. Druglikeness, which is related to the similarity with trade drugs
(−0.35–−1.29). Drug score of all ILs were in the range of 0.128–0.560. Among the all ILs, 4 showed
the best values of drug-score (0.56) with a lower toxicity risk, which points it out for further exploration.

Molecules 2012, 17
Table 1. Pharmacokinetic parameters (Catalyst, Chemaxon and Osiris softwares).
13734
ILs
1
2
3
4
nHba nHbd nrotb
MW
199
199
118
174
cLog P Druglikeness Drug Score
1
1
2
2
0
0
0
0
3
3
0
3
1.5
1.5
−1.29
−1.28
−0.35
−0.79
0.130
0.128
0.560
0.641
0.12
1.14
3. Experimental
3.1. General
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), bisbenzimidazole
derivative Hoechst-33342 (bisbenzimide(2-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinylpiperazinyl]-
2,5-bi-1H-benzimidazole)trihydrochloride), N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]
(HEPES) buffer, propidium iodide (PI), ribonuclease-A (RNase-A), Tris-hydrochloride, propidium
iodide (PI) were obtained from the Sigma Chemical Co. (Yongin, Korea). Trypsin-EDTA were
obtained from the Gibco (Grand Island, NY, United States). Antibiotic-antimycotic solution and
phosphate buffer saline (PBS) were obtained from Welgene (Daegu, Korea). Dulbecco’s modified
phosphate buffered saline (PBS), Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum
(FBS) were obtained from Hyclone (Logan, UT, USA). Triethylamine, sulfuric acid, phosphoric acid,
1-methylimidazole and 1-chlorobutane was obtained from Sigma–Aldrich (Yongin, Korea). 1-methyl
imidazolium chloride ([MIM]Cl, 3) were also obtained from Sigma–Aldrich (Yongin, Korea), are of
high purity, were used without further purification. All solvents and acids used in the present study
were of analytical grade and obtained from Sigma-Aldrich (Yongin, Korea). Instruments used in the
present study are Synergy HT plate reader (Biotek, USA), Nikon Eclipse Ti (Nikon, Japan), Jeol-500
NMR (Japan).
3.2. Preparation of ILs 1–4
All the ammonium and imidazolium families of ILs were synthesized in our laboratory. The
synthesis of ILs was described in our previous papers [26–28].
Synthesis of triethylammonium sulfate (TEAS, 1). The synthesis of TEAS was carried out in a 250 mL
round-bottomed flask, which was immersed in a water-bath and fitted with a reflux condenser.
Sulphuric acid (1 mol) was added dropwise to triethylamine (1 mol) at 60 °C for 1 h. The reaction
mixture was heated at 80 °C under vigorous stirring for 2 h to ensure that the reaction had proceeded to
completion. The reaction mixture was then dried at 80 °C until the weight of the residue remained
constant. The sample was analysed by Karl Fisher titration which revealed very low levels of water
(below 70 ppm). The purity of the IL was estimated by 1H-NMR spectroscopy. The yield of TEAS was
198 g. 1H-NMR (CDCl3): 1.3 (t, 9H), 3.16 (m, 6H), 5.04 (s, 1H). Melting point: 90 °C.
Synthesis of triethylammonium phosphate (TEAP, 2). The synthesis of TEAP was carried out in a
250 mL round-bottomed flask, which was immersed in a water-bath and fitted with a reflux condenser.
Phosphoric acid (1 mol) was added dropwise to triethyl amine (1 mol) at 70 °C for 1 h. The reaction
mixture was heated at 80 °C under vigorous stirring for 2 h to ensure that the reaction proceeded to

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completion. The reaction mixture was then dried at 80 °C until the weight of the residue remained
constant. The sample was analysed by Karl Fisher titration which revealed very low levels of water
1
(below 70 ppm). The purity of IL was estimated by H-NMR spectroscopy. The yield of TEAP was
198 g. 1H-NMR (DMSO-d6): δ (ppm) 1.18 (t, 9H), 3.06 (m, 6H), 6.37 (s, 1H). Melting point: 92 °C.
1-Methyl imidazolium chloride ([MIM]Cl, 3). Please see 3.1 (General Section).
Synthesis of 1-Butyl-3-methylimidazolium Chloride ([BMIM]Cl, 4). To a vigorously stirred solution of
1-methylimidazole (1.25 mol) in toluene (125 mL) at 0 °C was added 1-chlorobutane (1.38 mol). The
solution was heated to reflux at 110 °C for 24 h, after which it was placed in a freezer at −20 °C for 12 h.
The toluene was decanted and the remaining viscous oil/semi-solid was recrystallized from acetonitrile
and then repeatedly recrystallized from ethyl acetate to yield a white crystalline solid, which was dried
1
in vacuo to give [BMIM]Cl in approximately 86% yield. H-NMR (400 MHz, DMSO-d6) : 10.54
(1H, s), 7.55 (1H, m), 7.40 (1H, m), 4.26 (2H, t, J = 7.3 Hz, 4.11 (3H, s), 1.82 (2H, m), 1.30 (2H, m),
0.89 (3H, t, J = 7.3 Hz). Melting point: 67 °C.
3.3. Human Cell Culture
T98G malignant (brain cancer) and Human Embryonic Kidney (HEK) non-malignant cells used in
the present studies were purchased from the Korean Cell Line Bank (KCLB), Seoul, Korea. We
cultured these cell lines in 75 cm2 culture flasks (SPL, Pocheon, Korea) using Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids,
1% glutamine, penicillin (100 IU/mL) and streptomycin (100 µg/mL) according to the manufacturer’s
instructions. All cultures were maintained at 37 °C, 95% relative humidity and 5% CO2. Prior to each
cytotoxicity test, the cells were harvested using trypsin–ethylenediamine tetraacetic acid (EDTA)–PBS
solution (with 0.25% trypsin–0.05 mM according to the distributor’s instructions) and diluted at a
density of 105 cells/mL for assays. Stock cultures were passaged every third day after harvesting the
cells with 0.05% trypsin and seeding 8 × 103 cells/cm2 in tissue culture flasks to maintain the cells in
the exponential phase. All experiments were carried out in exponentially growing cells. The cell
suspension was seeded into 24-well plates (SPL) at 100 µL/well, and incubated for approximately
20–24 h before tests in order to reach confluency.
3.4. In Vitro Metabolic Viability Assay
Cells were seeded in 24-well plates at a concentration of 104 cells/well in 200 μL of complete media
and incubated for 24 h at 37 °C in 5% CO2 atmosphere to allow for cell adhesion. All assays were
performed in two independent sets of quadruplicate tests. Control group containing without treatment
was run in each assay. Following after 24, 48 and 72 h of exposure of cells to IL, each well will be
carefully rinsed with 200 μL PBS buffer. Cytotoxicity were assessed using the MTT (3-[4,5-
dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. MTT solutions 20 μL (5 mg mL−1 dd
H2O) along with 200 μL of fresh, complete media were added to each well and plates were incubated
for 3 h [29–32]. Following incubation, the medium were removed and the purple formazan precipitate
in each well were sterilized in 200 μL DMSO. Absorbances were measured using microplate reader at

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570 nm and results were expressed as % viability which is directly proportional to metabolic active
cell number. Percentage (%) viability were calculated as:
% Viability = OD in sample well/OD in control well × 100
3.5. Growth Kinetics Assay
Cells were seeded at 8,000 cells/cm2 in 100 mm Petri disc or flask, and their proliferation kinetics
studied at 24, 48, 72 h after treatment with the ILs, following trypsinization and counting total cells per
flask/disc using a hemocytometer [29].
3.6. Clonogenic Assay
Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of
a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay
essentially tests every cell in the population for its ability to undergo “unlimited” division [33]. The
clonogenic assay is the method of choice to determine cell reproductive death after treatment with
ionizing radiation, but we can also be used to determine the effectiveness of drug molecules. Only a
fraction of seeded cells retains the capacity to produce colonies before or after treatment, cells will be
seeded out in appropriate dilutions to form colonies in 1–3 weeks. After harvesting with 0.05% trypsin,
150–400 (depending on the treatment) cells will be plated 10–14 h before treatment in DMEM at 37 °C.
Cultured cells will be treated with doses 20 to 100 ug/mL of ILs. After the treatment, cells will be
incubated in dark under humidified, 5% CO2 atmosphere at 37 °C for 8–10 days to allow colony
formation. Colonies will be fixed with methanol and will be stained with 1% crystal violet. Colonies of
more than 50 cells will be counted and the surviving fraction (SF) will calculated. Clonogenic survival
curves will be constructed from three independent experiments by least-squares regression fitting
averaged survival levels.
3.7. In Silico Pharmacokinetic Screening
To evaluate pharmacokinetic profile descriptors such as cLog P (octanol/water partition coefficient)
and Log S (water solubility) were calculated using the Osiris Property Explorer on-line system
available at [34]. The ILs were submitted to in silico ADMET (absorption, distribution, metabolism,
excretion, and toxicity) screening, using the Osiris program. Values of druglikeness are based on the
occurrence frequency of each fragment of the molecule in commercial drugs while the drug-score
evaluates the IL’s potential to qualify for a drug and is related to topological descriptors, fingerprints of
molecular druglikeness, structural keys and other properties such as cLog P and molecular mass [35,36].
In silico theoretical safety analysis is also evaluated using the Osiris software, where a score of 1 means a
drug is safe and a score <1 means a drug molecule is theoretically toxic for use. The pharmacokinetic
profile, important for a good oral bioavailability of a IL, was also evaluated according to Lipinski’s
‘rule-of-five’ using the Catalyst and Chemaxon softwares, which analyse features that a drug should
present to allow the absorption and permeation across the membranes and states molecular
weight <500 Daltons (Da), calculated octanol/water partition coefficient (cLog P) < 5, number of
hydrogen-bond acceptors (nHba) < 10, and number of hydrogen-bond donors(nHbd) < 5 as well as a
fifth rule added later, which infers the number of rotatable bonds < 10 [37].

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3.8. Statistical Analysis
Data have been expressed as means ± SD. Statistical analysis was done by student T-tests. Results
were considered significant when their p-value < 0.05.
4. Conclusions
The obtained results with ILs against human T98G brain cancer cells were compared with HEK
non-malignant cells. These ILs were more efficacious on T98G cancer cells and less toxic to HEK
non-malignant cells at effective concentrations. These ILs may be regarded as lead molecules for a
new class of cytotoxic agents effective against T98G brain cancer cells with good drug delivery
capability because of their small size and capable of chemosensitization. Finally we can conclude that
ILs could have the broad dosage ranges of activity against the human T98G brain cancer cells. IL 4
shows significant toxicity on T98G cells and the least toxicity on normal Human Embryonic Kidney
(HEK) cells at effective concentrations. IL 4 also inhibits the clonogenic capacity of T98G brain
cancer cells in a concentration and time dependent manner. IL 4 presents the overall best parameters
including: (a) high activity against T98G brain cancer cell line; (b) low cytotoxicity on HEK cells at
effective concentrations; (c) low toxicity risks in in silico analysis; (d) good oral bioavailability
according to the Lipinski ‘rule of five’; and (e) better druglikeness and drug-score values, nearly
similar to commercial drugs. Studies on the mechanism by which the IL 4 induces antiproliferative
effects on T98G brain cancer cell lines are ongoing.
Acknowledgments
The present research has been conducted by the Research Grant of SRC program of National
Research Foundation of Korea (20100029418) and Kwangwoon University in 2012.
References
1. Stoimenovski, J.; MacFarlane, D.R.; Bica, K.; Rogers, R.D. Crystalline vs. ionic liquid salt forms
of active pharmaceutical ingredients: A position paper. Pharm. Res. 2010, 27, 521–526.
2. Rogers, R.D.; Seddon, K.R. Ionic Liquids: Industrial Applications to Green Chemistry;
ACS Symp. Ser.: Washington, DC, USA, 2002.
3. Hough, W.L.; Smiglak, M.; Rodrıguez, H.; Swatloski, R.P.; Spear, S.K.; Daly, D.T.; Pernak, J.;
Grisel, J.E.; Carliss, R.D.; Soutullo, M.D.; et al. The third evolution of ionic liquids: Active
pharmaceutical ingredients. New J. Chem. 2007, 31, 1429–1436.
4. Yu, M.; Wang, S.-H.; Luo, Y.-R.; Han, Y.-W.; Li, X.-Y.; Zhang, B.-J.; Wang, J.-J. Effects
ofthe1-alkyl-3-methylimidazolium bromide ionic liquids on the antioxidant defense system of
Daphnia magna. Ecotox. Environ. Saf. 2009, 72, 1798–1804.
5. Attri, P.; Venkatesu, P. Influence of protic ionic liquids on the structure and stability of
succinylated Con A. Int. J. Biol. Macromol. 2012, 51, 119–128.
6. Attri, P.; Venkatesu, P.; Kumar, A. Water and a protic ionic liquid acted as refolding additives for
chemically denatured enzymes. Org. Biomol. Chem. 2012, 10, 7441–7636.

Molecules 2012, 17
13738
7. Hough-Troutman, W.L.; Smiglak, M.; Griffin, S.; Reichert, W.M.; Mirska, I.; Jodynis-Liebert, J.;
Adamska, T.; Nawrot, J.; Stasiewicz, M.; Rogers, R.D.; et al. Ionic liquids with dual biological
function: Sweet and anti-microbial, hydrophobic quaternary ammonium-based salts. New J.
Chem. 2009, 33, 26–33.
8. Pernak, J.; Goc, I.; Mirska, I. Antimicrobial activities of protic ionic liquids with lactate anion.
Green Chem. 2004, 6, 323–329.
9. Demberelnyamba, D.; Kim, K.-S.; Choi, S.; Park, S.Y.; Lee, H.; Kim, C.J.; Yoo, I.D. Synthesis
and antimicrobial properties of imidazolium and pyrrolidinonium salts. Bioorg. Med. Chem. 2004,
12, 853–857.
10. Pernak, J.; Sobaszkiewicz, K.; Mirska, I. Anti-microbial activities of ionic liquids. Green Chem.
2003, 5, 52–56.
11. Pernak, J.; Feder-Kubis, J. Synthesis and properties of chiral ammonium-based ionic liquids.
Chem. Eur. J. 2005, 11, 4441–4449.
12. Pernak, J.; Sobaszkiewicz, K.; Foksowicz-Flaczyk, J. Ionic liquids with symmetrical dialkoxy
methyl-substituted imidazolium cations. Chem. Eur. J. 2004, 10, 3479–3485.
13. Kumar, V.; Malhotra, S.V. Study on the potential anti-cancer activity of phosphonium and
ammonium-based ionic liquids. Bioorg. Med. Chem. Lett. 2009, 19, 4643–4646.
14. Stock, F.; Hoffman, J.; Ranke, J.; Ondruschka, B.; Jastorff, B. Effects of ionic liquids on the
acetylcholinesterase—A structure–activity relationship consideration. Green Chem. 2004, 6, 286–290.
15. Skladanowski, A.C.; Stepnowski, P.; Kleszczynski, K.; Dmochowska, B. AMP deaminase in vitro
inhibition by xenobiotics A potential molecular method for risk assessment of synthetic nitro- and
polycyclic musks, imidazolium ionic liquids and N-glucopyranosyl ammonium salts. Environ.
Toxicol. Pharmacol. 2005, 19, 291–296.
16. Hosseinzadeh, F.; Mahkam, M.; Galehassadi, M. Synthesis and characterization of ionic liquid
functionalized polymers for drug delivery of an anti-inflammatory drug. Des. Monomers Polym.
2012, 15, 379–388.
17. Malhotra, S.V.; Kumar, V. A profile of the in-vitro antitumor activity of imidazolium-based ionic
liquids. Bioorg. Med. Chem. Lett. 2010, 20, 581–585.
18. Fujita, K.; MacFarlane, D.R.; Forsyth, M. Protein solubilising and stabilising ionic liquids.
Chem. Commun. 2005, 4804–4806.
19. Von Hagen, J.; Michelsen, U. Use of ionic liquids for protein extraction. U.S. Patent
US7470778B2, 2008.
20. Fujita, K.; Forsyth, M.; MacFarlane, D.R.; Reid, R.W.; Elliott, G.D. Unexpected improvement in
stability and utility of cytochrome c by solution in biocompatible ionic liquids. Biotechnol.
Bioeng. 2006, 94, 1209–1213.
21. Fujita, K.; MacFarlane, D.R.; Forsyth, M.; Yoshizawa-Fujita, M.; Murata, K.; Nakamura, N.;
Ohno, H. Solubility and stability of cytochrome c in hydrated ionic liquids: Effect of Oxo acid
residues and kosmotropicity. Biomacromolecules 2007, 8, 2080–2086.
22. Schöfer, S.H.; Kaftzik, N.; Wassrescheid, P.; Kragl, U. Enzyme catalysis in ionic liquids: Lipase
catalysed kinetic resolution of 1-phenylethanol with improved enantioselectivity. Chem. Commun.
2001, 425–426.

Molecules 2012, 17
13739
23. Ranke, J.; Stolte, S.; Stormann, R.; Arning, J.; Jastorff, B. Design of sustainable chemical
products-the example of ionic liquids. Chem. Rev. 2007, 107, 2183–2206.
24. Carson, L.; Chau, P.K.W.; Earle, M.J.; Gilea, M.A.; Gilmore, B.F.; Gorman, S.P.; McCann, M.T.;
Seddon, K.R. Antibiofilm activities of 1-alkyl-3-methylimidazolium chloride ionic liquids.
Green Chem. 2009, 11, 492–497.
25. Shaw, G.; Morse, S.; Ararat, M.; Graham, F.L. Preferential transformation of human neuronal
cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 2002, 16, 869–871.
26. Attri, P.; Reddy, P.M.; Venkatesu, P.; Kumar, A.; Hofman, T. Measurements and Molecular
Interactions for N,N-dimethylformamide with Ionic Liquid Mixed Solvents. J. Phys. Chem. B
2010, 114, 6126–6133.
27. Attri, P.; Venkatesu, P.; Kumar, A. Temperature effect on the molecular interactions between
ammonium ionic liquids and N,N-dimethylformamide. J. Phys. Chem. B 2010, 114, 13415–13425.
28. Attri, P.; Venkatesu, P.; Hofman, T. Temperature dependence measurements and structural
characterization of trimethyl ammonium ionic liquids with a highly polar solvent. J. Phys. Chem. B
2011, 115, 10086–10097.
29. Kaushik, N.K.; Kim, Y.H.; Han, Y.G.; Choi, E.H. Effect of jet plasma on T98G human brain
cancer cells. Curr. Appl. Phys. 2013, 13, 176–180.
30. Wahab, R.; Kaushik, N.K.; Verma, A.K.; Mishra, A.; Hwang, I.H.; Yang, Y.B.; Shin, H.S.; Kim, Y.S.
Fabrication and growth mechanism of ZnO nanostructures and their cytotoxic effect on human brain
tumor U87, cervical cancer HeLa, and normal HEK cells. J. Biol. Inorg. Chem. 2011, 16, 431–442.
31. Mishra, A.; Jung, H.; Park, J.W.; Kim, H.K.; Kim, H.; Stang, P.J.; Chi, K.W. Anticancer activity
of self-assembled molecular rectangles via arene-ruthenium acceptors and a new unsymmetrical
amide ligand. Organometallics 2012, 31, 3519−3526.
32. Kaushik, N.K.; Kim, H.S.; Chae, Y.J.; Lee, Y.N.; Kwon, G.C.; Choi, E.H.; Kim, I.T. Synthesis
and anticancer activity of di(3-thienyl)methanol and di(3-thienyl)methane. Molecules 2012, 17,
11456–11468.
33. Kaushik, N.K.; Uhm, H.S.; Choi, E.H. Micronucleus formation induced by dielectric barrier
discharge plasma exposure in brain cancer cells. Appl. Phys. Lett. 2012, 100, 0841021–0841024.
34. Organic Chemistry Portal. Available online: http://www.organic-chemistry.org/prog/peo/
(accessed on 6–13 October 2012).
35. Tekto, I.V. Computing chemistry on the web. Drug Discov. Today 2005, 10, 1497–1500.
36. Wenlock, M.C.; Austin, R.P.; Barton, P.; Davis, A.M.; Leeson, P.D. A comparison of
physicochemical property profiles of development and marketed oral drugs. J. Med. Chem. 2003,
46, 1250–1256.
37. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development settings.
Adv. Drug. Del. Rev. 2001, 4, 3–26.
Sample Availability: Samples of all the ILs are available from the authors.
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