Novel phenethylimidazolium based ionic liquids: Design, microwave synthesis, in-silico, modeling and biological evaluation studies

Article abstract of DOI:10.1016/j.molliq.2020.113778

An eco-friendly preparation method for the novel bioactive imidazolium ionic liquids halides (ILs) was developed under microwave-assisted conditions. Synthesized ILs were characterized by spectroscopic techniques. Selected ILs were investigated for their antimicrobial activity against highly resistant Gram-positive and Gram-negative bacterial strains. Overall, 3-(2-chlorobenzyl)-1-phenethyl-1H-imidazol-3-iumchloride (4) showed high antimicrobial activity against the Staphylococcus aureus strain in the inhibition zone tests and displayed low MIC and MBC levels against almost all tested bacteria. Furthermore, the ILs were screened in vitro against human hepatocellular carcinoma (HepG-2), human breast adenocarcinoma (MCF-7), and human colon carcinoma (Caco-2) cell lines. The screening results showed excellent to moderate anticancer activity across the ILs. Among the synthesized ILs, Overall, 3-(2-chlorobenzyl)-1-phenethyl-1H-imidazol-3-ium chloride (4) and 1-phenethyl-3-(3-phenoxypropyl)-1H-imidazol-3-ium bromide (7) were found to exhibit the most promising ant proliferative effects and had the lowest IC₅₀ values. The docking study suggested strong interaction of ILs with DNA with binding energy ranging from ?4.9 to ?4.1 kcal/mol. ILs 4 and 7 were most strongly bonded with ?4.9 and ?4.8 kcal/mol binding energy; confirming in vitro anticancer results.

Full text of DOI:10.1016/j.molliq.2020.113778

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Novel phenethylimidazolium based ionic liquids: Design,
microwave synthesis, in-silico, modeling and biological
evaluation studies
Abderrahim Titi, Saud M. Almutairi, Abdulwahed F. Alrefaei,
Salim Manoharadas, Bakheet A. Alqurashy, Pramod K. Sahu,
Belkheir Hammouti, Rachid Touzani, Mouslim Messali, Imran
Ali
PII:
S0167-7322(20)33679-5
DOI:
https://doi.org/10.1016/j.molliq.2020.113778
MOLLIQ 113778
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
6 June 2020
2 July 2020
7 July 2020
Please cite this article as: A. Titi, S.M. Almutairi, A.F. Alrefaei, et al., Novel
phenethylimidazolium based ionic liquids: Design, microwave synthesis, in-silico,
modeling and biological evaluation studies, Journal of Molecular Liquids (2020),
https://doi.org/10.1016/j.molliq.2020.113778
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Date: 07/022020
Prof. W. Schröer
Editor-in-Chief
Journal of Molecular Liquids
Dear Prof. Schröer,
Good day and Greetings…
Thank you very much for giving us a chance to revise our manuscript. The manuscript is
revised as per the suggestions made by the learned reviewers. The changes made are highlighted
in red color. The revised manuscript is uploaded herewith for publication in Journal Molecular
Liquids.
Ref: MOLLIQ_2020_3529
Title: Novel phenethylimidazolium based ionic liquids: Design, microwave synthesis, in-
silico, modeling and biological evaluation studies
Novelty:
 Economic microwave synthesis of novel phenethylimidazolium based ionic liquids.
 Antimicrobial activity of the synthesized ILs against resistant Gram-positive and Gram-
negative bacteria.
 Anticancer screening of the synthesized ILs against MCF-7, HepG-2 and CACO2 cell
lines.
 In-silico physico-chemical analysis to predict molecular properties.
 Docking with DNA to determine the working mechanism.
Looking forward to hearing from you a positive response.

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Thanking you with regards,
Yours sincerely,
Prof. & Dr. Imran Ali
PhD, C Chem, FRSC, London (UK), Ambassador of Bentham.Science Publisher.
Editor-in-Chief:
- Current Chromatography, Bentham Science Publisher.
- Nanomedicine and Drug Delivery, Glausius Scientific Press.
Editor:
- Journal of Taibah University for Science, Taylor & Francis.
- Advanced Materials & Technologies, Tambov State Technical University, Tambov, Russia.
- Int. J. Environ. Issues, Hanrell International, Zimbawe, South Africa.
Associate and Section Editor: 05 Journals
On the Editorial Board: 40 Journals.
Address:
Department of Chemistry
Jamia Millia Islamia (Central University), Jamia Nagar, New Delhi - 110025, India.
Phone and Whats App: 0091-9211458226
Web: http://jmi.ac.in/iali2
Public URL: https://scholar.google.co.in/citations?user=ukpmKoQAAAAJ&hl=en

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Novel phenethylimidazolium based ionic liquids: Design, microwave
synthesis, in-silico, modeling and biological evaluation studies
Abderrahim Titi a, Saud M. Almutairi b, Abdulwahed F Alrefaei c, Salim Manoharadas d,
Bakheet. A. Alqurashy e, Pramod K. Sahu f, Belkheir Hammouti a, Rachid Touzani a,
Mouslim Messali g, Imran Ali g, h*
a Laboratory of Applied and Environmental Chemistry(LCAE), Mohammed First University, Oujda, Morocco
b King Abdulaziz City for Science and Technology, Riyadh 11442, P.O. Box 6086, Saudi Arabia.
c
Department of Zoology, King Saud University, College of Science, P. O. Box 2455, Riyadh 11451, Saudi Arabia
d Central Laboratory, College of Science, King Saud University, Riyadh, Saudi Arabia
e
Department of Basic Science and Technologies Community Faculty, Taibah University 30002, Al-Madina Al
Mounawara, Saudi Arabia
f School of Study in Chemistry, Jiwaji University, Gwalior 474011, Madhya Pradesh, India
g Department of Chemistry, Taibah University, 30002, Al-Madina Al-Mounawara, Saudi Arabia.
h Department of Chemistry, Jamia Millia Islamia (A Central University), New Delhi, 110025, India
Abstract:
An eco-friendly preparation method for the novel bioactive imidazolium ionic liquids
halides (ILs) was developed under microwave-assisted conditions. Synthesized ILs were
characterized by spectroscopic techniques. Selected ILs were investigated for their antimicrobial
activity against highly resistant Gram-positive and Gram-negative bacterial strains. Overall, 3-(2-
chlorobenzyl)-1-phenethyl-1H-imidazol-3-iumchloride (4) showed high antimicrobial activity
against the Staphylococcus aureus strain in the inhibition zone tests and displayed low MIC and
MBC levels against almost all tested bacteria. Furthermore, the ILs were screened in vitro
against human hepatocellular carcinoma (HepG-2), human breast adenocarcinoma (MCF-7), and
human colon carcinoma (Caco-2) cell lines. The screening results showed excellent to moderate
anticancer activity across the ILs. Among the synthesized ILs, Overall, 3-(2-chlorobenzyl)-1-
phenethyl-1H-imidazol-3-ium chloride (4) and 1-phenethyl-3-(3-phenoxypropyl)-1H-imidazol-3-
ium bromide (7) were found to exhibit the most promising ant proliferative effects and had the
lowest IC50 values. The docking study suggested strong interaction of ILs with DNA with
binding energy ranging from -4.9 to -4.1 kcal/mol. ILs 4 and 7 were most strongly bonded with -
4.9 and -4.8 kcal/mol binding energy; confirming in vitro anticancer results.
Keywords: Novel ionic liquids; Microwave-assisted; In silico prediction; In vitro anticancer
activity; DNA modeling.
*Corresponding author: E-mail address: drimran_ali@yahoo.com; drimran.chiral@gmail.com

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1. Introduction
Nowadays, scientists have focused on developing alternatives to environmentally harmful
compounds due to greater environmental consciousness. In this regard, imidazolium-based halide
ionic liquids (ILs) are alternatives that have great properties including zero-vapor pressure,
excellent conductivity, remarkable chemical and electrochemical stabilities and no flammability
[1-3]. The composition of ILs contain anions such as halides or fluorinated anions [4–5].
Scientists have synthesized a variety of ILs using different combinations of cations and anions.
Recently, ILs have been widely used in many applications, such as media for electrodeposition
of metals [6], a catalyst and biocatalyst [7–8], and potential corrosion inhibitors [9–11]. ILs have
also been used in the polymer [12] and food chemical science [13], as well as electrolytes for
batteries [14,15] and in many other areas of research. Moreover, ILs have also been investigated
for their biological activity and toxicity [16] and have been found to display broad activity
against both environmental and clinically important microorganisms [17,18]. Environmentally
benign approaches, such as microwave irradiation, have gained attention in recent years for the
clean synthesis of ILs. With such an approach, the reaction time could be reduced compared with
that required in the conventional preparation of ILs [19,20].
On the other hand, there is a great demand for the search of new antibacterial agents as
bacteria are becoming antibiotics resistant continuously. Besides, scientists are exploring new
anticancer drugs due to the lack of proper and exact medicine for treating cancer; especially at
the late stage of cancer [21-26]. ILs have been found as bioactive molecules [27,28] and
expected to be good candidates for many biological actions (medicines). In view of these facts,
the efforts are made to synthesize a variety of new ILs via most eco-friendly microwave
synthesis. These ILs were characterized by various spectroscopic techniques and tested as
antibacterial and anticancer agents. Finally, DNA docking was carried out with the most active
ILs to understand the mechanism of their biological activities. The results of these findings are
given in this article.
2. Results and discussion
Novel room temperature imidazolium-based ionic liquids were synthesized using
microwave irradiation to reduce or eliminate the related costs, disposal requirements, and

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hazards associated with volatile organic synthesis methods. Additionally, select synthesized
ILs were evaluated for their antibacterial and antitumor activity.
2.1. Chemistry
The ILs were synthesized via the conventional procedure. First, 1-phenethyl-1H-
imidazole was synthesized by treating imidazole with butyl bromide in acetonitrile in the
presence of t-BuOK. Then, the initial phenethylimidazolium based derivatives (1–5)
containing aliphatic chains were prepared by treating 1-phenethyl-1H-imidazole with the
appropriate alkyl halides in toluene at 80 °C for 18 h (Scheme 1).
Scheme 1. (i) N-alkylation of 1-phenethyl-1H-imidazole leading to ILs 1-17: conventional preparation and
microwave irradiation conditions. CP: RBr, toluene, 80 °C, 18 h; MW: RBr, toluene, 80 °C, 20 min.

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The quaternization reaction led to the production of the imidazolium bromides 1–5 when
the nitrogen lone pair of 1-phenethyl-1H-imidazole reacted with the alkyl halides. The resulting
yields of the oils were 69%–79% (Table 1). Microwave irradiation reactions were used for the
synthesis of 1-phenethyl-1H-imidazolium-based ILs 1–5. 1-Phenethyl-1H-imidazole and the
appropriate alkyl halide were treated with microwave irradiation in a closed vessel. IL formation
was confirmed when two phases were formed, as the ILs are insoluble in toluene. A short
irradiation period was required for completion, and good yields were achieved.
Table 1. IL yields from the conventional and microwave-assisted preparation methods.
Yield (%) of the Quaternization Step
Ionic
liquid
1
Alkyl halide
RX
Ph(CH2)3Br
Ph(CH2)2Br
PhCH2Br
CP
71
72
73
69
69
72
72
73
78
76
79
78
74
70
73
72
70
MW
87
88
89
86
87
85
83
87
90
87
89
88
89
86
89
88
84
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
(2-Cl)-PhCH2Cl
(4-F)-PhCH2Cl
PhO(CH2)4Br
PhO(CH2)3Br
PhO(CH2)2Br
(2-Cl)-PhCOCl
(4-Cl)-PhCOCl
EtO2CCH2Cl
EtO2C(CH2)2Br
EtO2C(CH2)3Cl
EtO2C(CH2)4Br
EtO2C(CH2)4Br
MeCO2(CH2)4Br
HO(CH2)4Cl
Conventional procedure (CP) and microwave (MW) irradiation conditions.
To establish the structures of ILs 1–5, 1-phenethyl-3-(3-phenylpropyl)-1H-imidazol-3-ium
bromide (1) was chosen for the discussion of spectral data. The structure of 1-phenethyl-3-(3-
phenylpropyl)-1H-imidazol-3-ium bromide (1) was confirmed using 1H NMR. A quintet at 2.11
ppm and one multiplet around 1.27 ppm were due to the methylene protons of CH3CH2CH2, and
a quintet around 1.86 ppm was due to the methylene protons of CH2CH2CH2. Since IL 1
contained 4 other methylene groups, the protons from these groups corresponded to triplets at
2.53 and 3.17 ppm for the CH2CH2Ph protons and 4.19 and 4.55 ppm for the NCH2CH2 protons.
The singlet at 9.90 was due to the aromatic region around the NCHN imidazolium proton. The

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1H NMR spectrum for 1 also contained two additional signals at 7.05 and 7.20 ppm
corresponding to the imidazolium ring protons (NCHCHN).
In the 13C NMR spectrum of IL 1, the five signals at δC 49.3 ppm were due to the
imidazolium ring, and the peak at 50.9 ppm was due to NCH2. The peaks at 31.4, 32.1, 29.8, and
36.3 ppm corresponded to the three other methylene groups. Furthermore, the signals
corresponding to the aromatic carbons were in the range of 121.9–139.6 ppm. The findings from
the 13C NMR spectrum were confirmed with APT-13C-NMR. Under similar reaction conditions,
ILs 6–17 were synthesized. These ILs were modified with different substituents, such as ethers,
ketones, esters, and alcohols. The last IL synthesized in this work was 3-(4-hydroxybutyl)-1-
phenethyl-1H-imidazol-3-ium chloride (17). The structures of ILs 6–17 were also confirmed by
spectroscopic analysis. Two ILs were selected from 6–17, namely, 3-(4-chlorobenzoyl)-1-
phenethyl-1H-imidazol-3-ium chloride (10) and 3-(4-ethoxy-4-oxobutyl)-1-phenethyl-1H-
imidazol-3-ium chloride (13), for the discussion of the spectral data to verify the quaternization
reaction.
13C-NMR analysis was used to establish the structure of IL 10. A signal at 160.6 ppm
corresponded to the carbonyl group (CO). In the H-NMR spectrum, IL 13 showed a triplet and a
quartet around 1.19 and 4.06 ppm, respectively. Furthermore, the C-NMR spectrum of IL 13 had
a peak at 172.1 ppm corresponding to the carbonyl group (CO) and peaks at 60.8 ppm and 14.1
ppm corresponding to the CH2 and CH3 of the ester group (OCH2CH3), respectively. The 13C
NMR spectrum of IL 13 showed peaks at 48.8 and 51.0 ppm due to the 2 NCH2CH2 carbons.
The functional groups of ILs 10 and 13 were also confirmed by FT-IR spectroscopy. The
1
absorption bands at 1685 and 1742 cm⁻ were designated to the ketone and ester carbonyl
groups (C=O), respectively.
2.2. Antimicrobial activity
The inhibition zone (IZ) assay of the compounds was performed against bacterial strains such
as Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, Escherichia coli MC4100,
and Escherichia coli XL-1 Blue. As shown in Table 2, the most potent activity was exhibited by
4against S. aureus, with an IZ diameter of 22 mm. An IZ diameter of 19 mm was observed
against S. aureus with 1. Interestingly, 1, 2, 4, and 5 exhibited antibacterial activity against all
the tested bacterial strains, particularly with strong activity against Gram-positive bacterial

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strains. However, no activity was observed from 16 and 17, apart from the moderate activity
against E. coli MC4100. In comparison with 16 and 17, 15 had moderate antimicrobial activity
against all the bacterial strains.
Table 2. IZ diameter measurements (mm) of select ILs against various bacterial strains.
K.
P.
E. coli
MC4100
E. coli
XL1-Blue
Sample
S. aureus
B. subtilis
pneumoniae
aeruginosa
19
14
22
16
9
9
12
0
12
9
12
13
0
0
9
0
0
11
10
12
13
0
9
12
0
8
6
9
10
0
0
8
0
0
10
12
14
14
8
11
13
9
10
10
14
13
0
6
9
0
0
1
2
4
5
11
14
15
16
17
0
0
7
The IZ assay results prompted us to compare the minimum inhibitory concentration
(MIC) of the ILs against the bacterial strains with that of the antibiotic control. IL 4, which had
high antimicrobial activity against the S. aureus strain in the IZ tests, had MIC and minimum
bactericidal concentration (MBC) levels of 12 and 24 µg/ml, respectively (Table 3).
Interestingly, the activity of 4 was higher than that of the antibiotic control (Ampicillin: AMP),
as the MIC and MBC of AMP against S. aureus were both 24 µg/ml. Moreover, ILs 16 and 17
did not show any MIC or MBC against the tested strains besides E. coli MC4100. The maximum
MIC or MBC tested among the ILs was 384 µg/ml, which was exhibited by ILs 2, 11, and 17
against P. aeruginosa, E. coli, and E.coli, respectively.
Table 3. MIC and MBC levels of select ILs against various bacterial strains.
K.
E. coli
MC4100
E. coli
XL1-Blue
Sample
S. aureus
B. subtilis
P. aeruginosa
pneumoniae
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
12
24
12
48
96
96
48
-
12
24
24
48
192
96
96
-
96
192
96
96
-
-
48
-
96
192
96
96
-
-
48
-
96
192
96
96
-
192
96
-
96
384
96
192
-
192
96
-
192
384
192
96
-
192
384
192
96
-
96
12
24
48
384
24
192
24
24
96
384
24
48
48
24
48
-
384
96
-
48
48
24
48
-
384
96
-
1
2
4
5
11
14
15
16
17
AMP
-
-
192
-
-
192
-
-
24
24
192
384
96
192
384
96
-
24
-
24
-
24
-
24
-
-
-
-
-
96
-
96
-
-

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2.3.Cytotoxic activity of ILs 1–10 against tumor cell lines
A previously reported method by the Regional Center for Mycology & Biotechnology at
Al-Azhar University, Egypt [29] was used to evaluate the cytotoxicity in terms of cell growth
and cell viability. The results are illustrated in Figures 1–3. The screening was performed with
various IL dosages. The screening results showed reductions in bacterial survival when the IL
dosage was increased. However, there was minimal improvement with increased dosage within
the range of 50–125 µM. Moreover, ILs 4 and 7 were observed to exhibit higher antiproliferative
activity within the range of 50–125 µM.
(a)
(b)
Figure 1 (a) and (b) Cytotoxic activities of ILs 1–5 and 6–10, respectively, against HepG-2 cells.

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(a)
(b)
Figure 2 (a) and (b). Cytotoxic activities of ILs 1–5 and 6−10, respectively, against MCF-7 cells.
(a)
(b)
Figure 3 (a) and (b) Cytotoxic activities of ILs 1–5 and 6–10, respectively, against Caco-2 cells.
Screening results showed that the IC50 values ranged from 3.87 to 224 µM. The results
indicated that IL 10 exhibited poor performance overall (Table 4). Based on the IC50 values, it
was observed that ILs 4 and 7 demonstrated good activity, with IC50 values ranging from 3.87 to
7.41 µM. Although, the activities of these ILs were lower than that of 5-FU, which was used as a

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positive control. ILs 1 and 5, with IC50 values ranging from 4.94 to 16 µM, also showed good
activity against two of the three cell lines compared to 5-FU.
Table 4. IC50 (μg/mL)values of ILs 1–10 against HepG-2, MCF-7, and Caco-2.
Cell line
HepG-2
MCF-7
Caco-2
5-FU
1
2
3
4
5
6
7
8
9
10
15.1 16.1
25.2 25.5
9.93
26
22.5 40 206
30.9 30.7 224
17.4 42.3 153
9.11 8.59
3.87 4.94
6.53 13.3
4.45
7.41
6.46
17
16
13.8 21 19.4
7.21 14.5
5.72
6.9
2.4.In silico analysis of ILs 1–17
In the drug designing and development process, Lipinski’s Rule of Five is generally used
to predict the bioavailability of the target drug. It is assumed that compounds that follow
Lipinski’s Rule of Five will likely be orally active. Drug property descriptors such as molecular
weight (MW) and topological surface area (TPSA) [30,31]were calculated; the results are
summarized in Table 5. Molecules that have drug applications usually have molecular weights of
less than 500 Da [32, 33]. All ILs did not violate Lipinski’s Rule of Five and were expected to be
orally active. It was assumed that all ILs were easily diffused, absorbed, and transported due to
having molecular weights less than 500 Da [34, 35]. The TPSA (Total Polar Surface Area)
values of the ILs were within the range 8.82–35.12 A° and were well below the limit of 160 A°.
Permeability property of ILs 1–17 considered as liphophilicity (Log P value) were ranging from -
2.46 to 0.09 (<5) [36], table 6 and TPSA were calculated as described [37], table 5. Calculated
Log P and TPSA were in the acceptable limit, and all the compounds qualified as drug
candidates. The calculated values of the molecular properties and bioactivity scores (TPSA,
GPCR (G-Protein coupled receptor) ligand, ICM (Ion Channel modulator), KI (Kinase inhibitor),
NRL (Nuclear receptor ligand), PI (Protease inhibitor), EI (Enzyme inhibitor) are summarized in
Table 5. The molecule has considered biological active possess bioactivity score more than 0.00,
values -0.50 to 0.00 are expected to be moderately active and considered as inactive if the score
is less than -0.50 [38]. From table 5, it is clear that the investigated compounds were biologically
active and produced the physiological actions by interacting with GPCR ligands, nuclear
receptor ligands, inhibit protease and other enzymes.

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Table 5. In silico physicochemical property predictions of ILs 1–17
Calculation of Molecular Properties
Calculation of Bioactivity Scores
Compd.
TPSA
NONH
NV
VOL
GPCRL
ICM
KI
NRL
PI
EI
8.82
0
0
295
0.24
0.43
−0.43
−1.11
−0.25
0.34
1
2
8.82
8.82
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7
4
4
21
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
3
3
278
261
275
266
321
304
287
277
277
251
268
287
302
318
285
248
459
96
0.04
0.22
0.16
0.25
0.21
0.12
0.06
0.14
0.15
−0.22
−0.01
0.10
0.14
0.15
0.17
0.13
0.20
−1.95
0.04
−3.94
−2.10
0.49
0.59
0.42
0.54
0.41
0.28
0.19
0.33
0.34
0.19
0.38
0.45
0.44
0.42
0.51
0.64
−0.20
−1.24
−0.47
−3.99
−3.27
−0.61
−0.73
−0.65
−0.63
−0.38
−0.49
−0.55
−0.38
−0.38
−0.95
−0.74
−0.66
−0.60
−0.56
−0.59
−0.62
−0.07
−1.90
−0.71
−4.00
−3.04
−1.27
−1.32
−1.21
−1.20
−1.00
−1.06
−1.09
−0.89
−0.90
−1.47
−1.33
−1.29
−1.20
−1.12
−1.25
−1.56
0.32
−0.45
−0.35
−0.41
−0.32
−0.23
−0.32
−0.38
−0.17
−0.11
−0.60
−0.40
−0.34
−0.27
−0.23
−0.27
−0.47
0.67
0.37
0.44
0.26
0.40
0.27
0.22
0.25
0.14
0.16
0.26
0.22
0.28
0.28
0.27
0.36
0.42
0.66
−1.19
0.25
−3.95
−2.42
3
8.82
4
8.82
5
18.05
18.05
18.05
25.89
25.89
35.12
35.12
35.12
35.12
35.12
35.12
29.05
206
6
7
8
9
10
11
12
13
14
15
16
17
SD1
SD2
Amp
Van
RD
66
−2.50
0.61
−2.66
0.87
112
299
1207
756
530
−4.01
−2.89
−3.90
−1.61
220
SD1: Doxorubicin; SD2: Fluorouracil; Amp: Ampicillin; Van: Vancomycin, and RD: Rifampicin
The in silico toxicity study is necessary to assess whether a drug is safe for
administration. To confirm the toxicity of the synthesized ionic liquids, mutagenicity and
carcinogenicity were calculated in silico, and the results were tabulated in Table 6. As shown in
Table 6, all compounds were safe for carcinogenicity and mutagenicity, except for 16 which was
safe for mutagenicity only.

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Table 6. In silico calculated toxicity risks and logP values of ILs (1–17).
Toxicity Risks[a]
cLogP
Compd.
Mw(g/mol)
Mut
Tum
Irri
Rep
1
2
291
277
263
297
271
321
307
293
311
311
259
273
287
301
315
287
245
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
−0.12
−0.57
−0.91
−0.31
−0.81
−0.09
−0.54
−0.99
0.09
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
0.08
−2.46
−2.00
−1.55
−1.10
−0.64
−1.55
−2.03
+++
Highly toxic: (---), slightly toxic: (++), not toxic (+++).[a] Mut: Mutagenic,
Tum: Tumorigenic, Irrit: Irritant, and RE: Reproductive effective.
2.4 Modeling
Generally, it is known that most of the anticancer agents work by interacting with DNA
[39-41] because mostly caner is occurred by DNA mutation. The modeling study was carried out
only for 1-10 ILCs as they were found to be the most active. The results are given in Tables 7
and it is clear from this table that the ILCs interacted with DNA differently. The binding energies
of the ILCs with DNA ranged from -4.9 to -4.1 kcal/mol. The common residues involved were
dc13, dg14, dc15, dg16, N7, dt8 and dg10. The diverse binding energies of the ILCs with DNA
were observed due to their dissimilar configuration. The docking studies showed that the
connections among the ILCs and DNA were due to hydrophobic interactions. Besides, hydrogen
bonding may be responsible for these interactions. All ILs are π electron-rich molecules due to
the presence of benzene rings. Therefore, π-π interactions may be a major contribution. It is
important to mention here that only the cation (organic moiety) of ILs interacted with DNA. All

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the molecules have benzene rings with nitrogen, chlorine, fluorine and oxygen atoms. The
structures of these ILs are almost similar with very little difference. Therefore, the different
binding of ILs may be because of the different steric fitting into DNA major groves.
Interestingly, the cationic moiety of ILs is attached to DNA molecules by the phosphate negative
site of DNA. The strength of the interaction was based on binding energy. ILCs 4 and 7
interacted with DNA stronger than other ones with -4.9 and -4.8 kcal/mol binding energy. Based
on these results, ILs 4, and 7 should be more active and effective in cancer treatment because
they bind more tightly with DNA as compared to other ILs. And of course, these results are in
good agreement with those obtained by anticancer activities. The representative interactions of
the most active 4 and 7 ILCs with DNA are shown in Figure 4 while the rest figures are given in
supplementary information. These figures also reflected that the ILCs interacted with DNA
through major grooves.
Table 7. The DNA modeling results of ILs 1-10.
ILs
1.
Binding energies
(kcal/mol)
Hydrophobic interactions
Helix A
Helix B
Dg10::C17,C20,C16,C18
dc9::C17,C7,C19
dt8::C7,C8,C11
Dc9::C4,C16,C1
dg10::C12,C14
dt8::C4,C1,C2
Dc15::C6,C10,C20,C1,C7
dg14::C10,C13 dc16::C1
-4.2
-4.4
-4.3
-4.9
-4.6
2.
3.
4.
5.
Dg16::C1,C2
dc15::C1,C7,C19,C3,C8,C11
Dt8::C5,C4
Dc15::C18,C3,C2,C1
dg14::C10,C13
dc9::C4,C8,C7
dg10::C18,C17
Dt8::C4,C1,C2,C3
dc9::C2,C7,C1,C4
dg10::C12
Dg14::C15,C14
dc15::C13,C10,C5,C4,C15,C14,C6
dg16::C4,C5
Dt8::C12,C8
Dc15::C14,C13
dc9::C7,C9,C12,C3,C4
dg14::C16,C1,C6,C10
dg10::C10,C3,C7
dc13::C11,C2
6.
7.
8.
9.
Dc9::C17,N1,C19,C8
dt8::C19,C7,C9,C8
dg10::C18,C12,C21
Dg10::C20,C15,C16
dc9::C16,C18,C7
dt8::C7,C9,C8,C3
Dc9::C7,C16,C15
dg10::C14,C18,C15
dt8::C8,C9,C7
Dg14::O,C20,C13,C12
dc15::C11,C15,C21,C8
dg16::C3
Dc15::C11,C6,C20,C7
dg14::C14,C11,OC17
dc16::C1
-4.1
-4.8
-4.1
-4.5
Dc15::C10,C18,C7,C5
dg14::C10,O
Dc9::C8
dt8::C4
Dg14::C14,C15,C7,C3
dc15::C15,C8,C18,C13
dc13::C11,C7,C3

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dc16::C4
10.
Dc9::C14,C16,C5
dt8::C3,C5
dg10::C17,C14,C15
Dc13::C9,C13,C1
dc15::C17,C10,C1,C2
dg16::C3,C5
-4.4
(a)
(b)
Figure 4. DNA docking models of the most active ILCs (a): 4 and (b): 7.
3. Materials and methods
3.1. Experimental
Minimal amounts of reagents and solvents were used. The synthesized ILs were
13
characterized by spectroscopic analysis. 1H NMR (400 MHz) and C NMR (100 MHz) spectra
were measured at room temperature with CDCl3 as the solvent. Chemical shifts (δ) were reported
in ppm to a scale calibrated to the internal standard, tetramethylsilane (TMS). IR spectra were
recorded in NaCl disc on a Shimadzu 8201 PC, FT-IR spectrophotometer (υmax in cm−1). The
elemental analyses were given by using the 2400 Series II CHNS/O Elemental Analyzer. The
microwave-assisted reactions were performed using a microwave oven with a temperature
controller (750 W) and an upper-temperature limit of 100 °C, with nitrogen gas being used as the
cooling agent when the temperature inside the reaction chamber increased.

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3.2. Synthesis
3.2.1. General procedures for the synthesis of imidazolium halides (1–17) using the
microwave method
The alkyl halides (1.1 eq) were added to a solution of 1-pentyl-1H-imidazole (1 eq) in
toluene. The solution was then treated with irradiation for 20 min in a closed vessel at 80 °C
using a CEM Microwave. The completion of the reaction was indicated by the formation of an
oil or solid phase from the initially clear homogenous mixture composed of N,N-pentylimidazole
and the alkyl halide in toluene. The product was either filtered or extracted, and then washed
with ethyl acetate. The IL/salt was then dried at reduced pressure.
3.2.2. General procedure for the synthesis of imidazolium halides (1–17) using conventional
irradiation
The alkyl bromides (1.1 eq) were added to a solution of 1-pentylimidazole (1 eq) in
toluene. The corresponding solutions were placed in a closed vessel at room temperature and
stirred at 80 °C for 18 h. After which, the IL was filtered and washed with ethyl acetate. The IL
was then filtered again, and ethyl acetate was evaporated to obtain the pure ionic liquid.
3.3 Modeling
DNA is the most likely target of anticancer drugs [42,43] and, hence, a modeling study of
ILs with DNA was carried out to understand the mechanism of action. The docking studies of the
selected 1-10 compounds were done by Intel® dual CPU (1.86 GHz) with Windows XP
operating system. Marwin Sketch software was utilized to draw the structures of 1-10
compounds. The structures were cleaned to 3D and saved in PDB file format [44]. After that, the
structure of DNA (pdb ID: 1bna) was downloaded from the protein data bank. Using AutoDock
Tools (ADT) 4.2 the structure of DNA to be docked was prepared by assigning Gastegier
charges, merging non-polar hydrogen atoms and saving it in PDBQT file format. Docking was
performed with AutoDock 4.2 (Scripps Research Institute, USA) considering all the rotatable
bonds of the ligand as rotatable and the receptor as rigid [45]. Using the same tool, 1-10 ILs (as
ligands) were edited to be saved in PDBQT formate. The grid box size of 60 × 80 × 110 Aᵒ with
0.375 Aᵒ spacing was used. After saving both files in PDBQT formate, Vina software was used
to get binding energy/affinity between receptor (DNA) and ligand (1-10 compounds). After
using Vina software, the output file was opened in PyMOL to carry out the molecular docking,

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virtual screening and binding site analysis and to get an image of interaction and the bond length
of the hydrogen bond between DNA and 1-10 compounds.
4. Conclusion
Seventeen novel RTILs were prepared through quaternization reactions by using microwave
irradiation instead of the conventional preparation method. The structures of the ILs were
1
13
characterized by FT-IR, H NMR, C NMR, and mass spectroscopies. We determined that the
use of microwave irradiation was simple and could both shorten the preparation time and
improve the yield of target molecules, thereby fulfilling some principles of green chemistry such
as the atomic economy. Based on IC50 values, we determined that ILs 4 and 7 demonstrated good
activity with IC50 values of 3.87–7.41 µM. In silico toxicity prediction results showed that all
compounds were safe for carcinogenicity and mutagenicity, except for 16which was safe for
mutagenicity only. In silico prediction results also proved that all the ILs satisfied Lipinski’s
Rule of Five to qualify as drug candidates. Molecular property calculations showed that all
compounds had LogP values within the acceptable range to be eligible for lipophilicity. TPSA
values of ILs were within the range of 8.82–35.12 A° and were well below the limit of 160 A°.
The rest of the calculated properties were in good agreement, and all the compounds qualified as
drug candidates. DNA modeling results indicated a strong binding of ILs with DNA with binding
energy ranging from -4.9 to -4.1 kcal/mol. The results confirmed that the biological activity of
ILS is through DNA binding.
Supplementary Materials: Characterization data of all newly synthesized ILS 1-17 included all
1H-NMR, 13C-NMR, DEPT-13C-NMR spectra are associated with this article. Supporting
information contains also the methods used for the determination of IZ, MIC and MBC, in-silico
analysis and modeling figures.
Acknowledgment: The authors are thankful for the University Mohamed Premier for their
financial support. We extend our appreciation to the Deanship of Scientific Research at King
Saud University for funding a part of this work through research group no. RG-1441-307, and
we thank the Researchers Support Services Unit at King Saud University for their technical
support.

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Credit author statement
Abderrahim Titi: performed the synthesis of compounds and analyzed the data; Saud M.
Almutairi: performed the synthesis of compounds and analyzed the data; Abdulwahed F
Alrefaei: carried out the biological tests; Salim Manoharadas: carried out the biological tests;
Bakheet. A. Alqurashy: analyzed the data; Pramod K. Sahu: performed in-silico analysis,
Software; Belkheir Hammouti: Reviewing; Rachid Touzani: Reviewing; Mouslim Messali:
Conceptualization, Methodology, Visualization, Validation, Writing- Original draft preparation,
Methodology, Software, Investigation, Supervision Imran Ali: Conceptualization, Methodology,
Software, Visualization, Writing- Reviewing and Editing, Validation, Investigation, Supervision.

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☒The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
Novel phenethylimidazolium based ionic liquids: Design, microwave synthesis,
in-silico, modeling and biological evaluation studies

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Graphical Abstract
In-silico

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Highlights
 Economic microwave synthesis of novel phenethylimidazolium based ionic liquids.
 Antimicrobial activity of the synthesized ILs against resistant Gram-positive and Gram-negative
bacteria.
 Anticancer screening of the synthesized ILs against MCF-7, HepG-2 and CACO2 cell lines.
 In-silico physico-chemical analysis to predict molecular properties.
 Docking with DNA to determine working mechanism.