Novel pyridinium based ionic liquids with amide tethers: Microwave assisted synthesis, molecular docking and anticancer studies

Article abstract of DOI:10.1016/j.molliq.2019.04.143

The ionic liquids are remarkable molecules with exceptional properties and applications. The microwave assisted green method was used to synthesize (yield, up to 95%)a new series of pyridinium based ionic liquids (ILs); utilizing molecular hybridization of 2- and/or 3-methyl pyridine and several fluorinated phenyls. The characterization of the synthesized ILs was carried out using different spectroscopic techniques (¹H, ¹³C, ¹⁹F, ³¹P, ¹¹B NMR and mass). The DNA binding constants (Kb)of the ionic liquids were in the range of 1.3 × 10³ to 1.52 × 10⁸ M^?1; indicating good binding tendencies of the ionic liquids with DNA. All the ionic liquids intercalated with DNA through minor grooves via covalent, intercalative and electrostatic bondings. All the reported ionic liquids showed >50% anticancer activities with two lung cancer cell lines i.e. A549 and H-1229. Moreover, the maximum proliferation inhibitions were 99.16 to 91.24% for 4a series and 99.23 to 99.69% for 5a series of the ionic liquids. The docking studies indicated quite good binding affinities (?4.6 to ?4.9 kcal/mol.)of the reported ionic liquids with DNA. The experimental results of DNA bindings were in good agreement with those of docking ones. The percentage inhibitions were excellent for 4a, 4e and 5n and, hence, these ionic liquids may be the future anti-cancer agents.

Full text of DOI:10.1016/j.molliq.2019.04.143

Accepted Manuscript
Novel pyridinium based ionic liquids with amide tethers:
Microwave assisted synthesis, molecular docking and anticancer
studies
Ateyatallah Aljuhani, Mohamed Reda Aouad, Nadjet Rezki,
Omar Abdulaziz Aljaldy, Salsabeel A. Al-Sodies, Mouslim
Messali, Imran Ali
PII:
S0167-7322(19)32058-6
DOI:
Reference:
https://doi.org/10.1016/j.molliq.2019.04.143
MOLLIQ 10866
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
10 April 2019
23 April 2019
26 April 2019
Please cite this article as: A. Aljuhani, M.R. Aouad, N. Rezki, et al., Novel pyridinium
based ionic liquids with amide tethers: Microwave assisted synthesis, molecular docking
and anticancer studies, Journal of Molecular Liquids, https://doi.org/10.1016/
j.molliq.2019.04.143
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ACCEPTED MANUSCRIPT
Novel pyridinium based ionic liquids with amide tethers: Microwave assisted
synthesis, molecular docking and anticancer studies
Ateyatallah Aljuhani,a Mohamed Reda Aouad,a, Nadjet Rezki,a,b Omar Abdulaziz Aljaldy,a
Salsabeel A. Al-Sodies,a Mouslim Messali,a and *Imran Alia,c
aDepartment of Chemistry, Faculty of Science, Taibah University, Al-Madinah Al-
Munawarah 30002, Saudi Arabia
bDepartment of Chemistry, Faculty of Science, University of Science and Technology
Mohamed Boudiaf, Laboratoire de Chimie and Electrochimie des Complexes Metalliques
(LCECM) USTO-MB, P.O. Box 1505, El M‘nouar, Oran 31000, Algeria
CDepartment of Chemistry, Jamia Millia Islamia (A Central University), New Delhi-
110025, India
Abstract
The ionic liquids are remarkable molecules with exceptional properties and applications.
The microwave assisted green method was used to synthesize (yield, up to 95%) a new series of
pyridinium based ionic liquids (ILs); utilizing molecular hybridization of 2- and/or 3-methyl
pyridine and several fluorinated phenyls. The characterization of the synthesized ILs was carried
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out using different spectroscopic techniques (1H, C, F, P, B NMR and mass). The DNA
binding constants (Kb) of the ionic liquids were in the range of 1.3 × 103 to 1.52 × 108 M−1;
indicating good binding tendencies of the ionic liquids with DNA. All the ionic liquids
intercalated with DNA through minor grooves via covalent, intercalative and electrostatic
bondings. All the reported ionic liquids showed more than 50% anticancer activities with two
lung cancer cell lines i.e. A549 and H-1229. Moreover, the maximum proliferation inhibitions
were 99.16 to 91.24% for 4a series and 99.23 to 99.69% for 5a series of the ionic liquids. The
docking studies indicated quite good binding affinities (-4.6 to -4.9 kcal/mol.) of the reported
ionic liquids with DNA. The experimental results of DNA bindings were in good agreement with
those of docking ones. The percentage inhibitions were excellent for 4a, 4e and 5n and, hence,
these ionic liquids may be the future anti-cancer agents.
Keywords: Pyridinium ionic liquids, Green syntheses, DNA binding, Docking, Anticancer
study.
*Correspondence: Department of Chemistry, Faculty of Science, Taibah University, Al-
Madinah
Al-Munawarah
30002,
Saudi
Arabia;
drimran_ali@yahoo.com;
drimran.chiral@gmail.com

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1. Introduction
The ionic liquids are remarkable molecules having exceptional properties and
applications [1]. It is well known that about 50% pharmaceuticals in market are ionic liquids due
to the combination of a pharmaceutically active cations and anions making them good and easily
soluble drugs in human body. Besides, the negligible vapor pressure, good hydrophobicity,
solvent miscibility, polarity, high chemical, thermal and electrochemical stabilities made the
ionic liquids as the best pharmaceutical molecules [2]. Therefore, the chemistry of ionic liquids
has become a subject of an increasing interest in the organic synthesis [3]. The considerable
efforts have been made towards the design and synthesis of these important classes of organic
molecules [4-9]. These have been explored extensively for their antibacterial [10], antifungal
[11], anti-inflammatory [12] and anticancer [13] properties. The quaternization of sp2 nitrogen of
heterocyclic components is one of the most efficient approaches in the ionic liquids synthesis [3].
Notably, pyridine derivatives are very useful scaffolds gaining wide popularity in the ionic
liquids synthesis [14,15].
Cancer is a composite, aggressive, heterogeneous and horrible disease killing millions of
people globally [16-22]. As per the GLOBOCAN assessment, 18.1 million new cases and 9.6
million deaths were recorded worldwide in 2018 due to cancer alone [23]. Therefore, there is a
great necessity to fight with this lethal disease. Among various modes, chemotherapy is
considered as the best approach due to its fast and targeted action [24]. But unfortunately, the
chemical compounds show severe toxicities to the normal cells. Besides, the solubility is another
problem restricting the use of chemotherapy. Due to these problems, the search of new molecules
for the chemotherapy is an area of growing and demanding research. The stratagem of the
coherent design of new bioactive molecules has become the goal of many researchers in the
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medicinal chemistry. The molecular clubbing of two or more pharmacophore units is the
promising approach to develop promising target scaffolds in the drugs design [25,26]. These
goals have been molecular hybridization of small and simple cores in one frame work.
The microwave assisted organic synthesis; with green advantages like eco-friendly, good
yields and reducing reaction times; is the need of today for organic synthetic chemists [26,27].
The modification of the counter anions to evaluate their synergetic effects in the synthesis of
ionic liquids is a subject of great interest [25-36]. The prevailing goals of this study are the
development of novel pyridinium ionic liquids as anticancer agents via green synthesis, DNA
binding, molecular docking and anticancer studies.
2. Experimental section
2.1. Chemicals and reagents
All the chemicals, solvents and reagents used were of analytical grade and used without
further purification. These include the following chemicals.
Fine Chemicals: 3-Fluoro-4-methylaniline, 2-fluoro-4-iodoaniline and 2,4,5-
trifluoaniline (BDH Chemicals Ltd), 3-methylpyridine, 4-methylpyridine and triethylamine
(Sigma Aldrich), chloroacetyl chloride, potassium hexafluorophosphate, sodium
tetrafluoroborate and sodium trifluoroacetate (Across).
Solvents: Dichloromethane, ethylacetate, hexane, acetonitrile, chloroform and
ethylacetate (Sigma-Aldrich).
2.2 Instruments used
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The newly synthesized ILs were fully characterized by H, C, F, P, B-NMR and
MS spectroscopic methods. The melting points of the reported compounds were measured on a
melt-temp apparatus (SMP10). TLC was performed on UV fluorescent Silica gel Merck 60 F254
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plates and viewed in a UV chamber (254 nm). The eluent used was ethyl acetate-hexane (1:2
v/v). SHIMADZU FT-IR-8400S spectrometer was used for recording IR spectra. The NMR
spectra were run with Bruker spectrotometer (400 MHz) with TMS as internal reference. ESI
mass spectral data were obtained by a Finnigan LCQ spectrometer. A controllable single-mode
microwave reactor (CEM Discovery) was used to carry out the microwave-assisted reactions.
The microwave reactor was equipped with a magnetic stirrer as well as pressure, temperature,
and power controls and a nitrogen cooling system. The maximum operating pressure of the
reactor was 2 x 106 Pa with power set as 300 W.
2.3 Synthesis of ionic liquids
The various ionic liquids were synthesized as per the procedure given.
2.3.1 Synthesis of 2-chloro-N-(substituted phenyl)acetamides 2a-c
The appropriate fluorinated aniline (3-fluoro-4-methylaniline, 2-fluoro-4-iodoaniline
and/or 2,4,5-trifluoaniline) 1a-c (10 mmol), triethylamine (12 mmol), chloroacetyl chloride (12
mmol) in dichloromethane (40 mL) were allowed to stir at 0 oC for 2 hr. The reaction was stirred
at room temperature for 6 hrs, until the consumption of the starting materials. The resulting solid
was filtered off, washed with water and re-crystallized with ethanol to afford the corresponding
2-chloro-N-(substituted phenyl)acetamides 2a-c.
2.3.2 Synthesis of pyridinium ionic liquid halides 4a-f:
To a mixture of 3-methyl and/or 4-methyl pyridine 3a,b (10 mmol), sodium iodide (10
mmol) and acetonitrile (30 mL) were added with constant stirring and adding with 2-chloro-N-
(3-fluoro-4-methylphenyl)acetamide, 2-chloro-N-(2-fluoro-4-iodophenyl)acetamide and/or 2-
chloro-N-(2,4,5-trifluorophenyl)acetamide 3a-c (10 mmol). The mixture was stirred at reflux for
18-20 hrs, until the consumption of the starting material was complete. The solvent was removed
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under reduced pressure. The product formed was collected by filtration and/or extracted by
chloroform to afford the desired ionic liquids 4a-f.
2.3.2.1 Microwave procedure
3-Methyl and/or 4-methyl pyridine 3a,b (1 mmol), sodium iodide (1 mmol), the
appropriate amounts of 2-chloro-N-(substituted phenyl)acetamides 2a-c (1 mmol) and
acetonitrile (5 mL) were placed in closed borosilicate glass vessel fitted with a silicone cap and
exposed to irradiation for 15-20 min using a microwave reactor. The reaction was processed as
described in the conventional procedure outlined earlier to give the same pyridinium ionic liquid
halides 4a-f.
2.3.3 Metathesis the synthesis of imidazolium ILs tethering fluorinated counter anion 5a-r:
2.3.3.1 Conventional procedure
A mixture of a suspension of ionic liquids 4a-f (1 mmol) and the appropriate metal salts
potassium hexafluorophosphate, sodium tetrafluoroborate and/or sodium trifluoroacetate (1.1
mmol) in acetonitrile (20 mL) were stirred at reflux for 24 h. After cooling, the reaction mixture
was filtered to remove solid metal halide. Acetonitrile was removed under reduced pressure. The
product formed was collected by filtration and/or extracted by chloroform to afford the desired
ionic liquids 5a-r.
2.3.3.2 Microwave procedure
Pyridinium halides 4a-f (1 mmol), the appropriate metal salts (1.1 mmol) and acetonitrile
(5 mL) were placed in closed borosilicate glass vessel fitted with a silicone cap and exposed to
irradiation for 20 min using a microwave reactor. The reaction was processed as described in the
conventional procedure outlined earlier to give the same pyridinium ionic liquids 5a-r.
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2.4 Characterization
The synthesized ionic liquids are characterized by various spectroscopic methods as
described below.
2.4.1 Characterization of 2-chloro-N-(substituted phenyl)acetamides 2a-c:
2-Chloro-N-(3-fluoro-4-methylphenyl)acetamide (2a)
Brown solid, mp: 155-156 oC. 1H NMR (400 MHz, DMSO-d6): δH = 2.17 (s, 2H, NCH2),
4.27 (s, 2H, NCH2), 7.22 (d, 2H, J = 4.0 Hz, Ph-H), 7.51-7.54 (m, 1H, Ph-H), 10.49 (s, 1H,
CONH). 13C NMR (100 MHz, DMSO-d6): δC = 14.1 (NCH2), 43.9 (NCH2), 106.6, 115.3, 119.7,
132.0, 138.3, 159.4, 161.8 (Ph-C), 165.2 (C=O). 19F NMR (377 MHz, DMSO-d6): δF = (-115.95
to -115.91) (m, 1F, Ph-F). LCMS (M+) 201.0 found for C9H9ClFNO.
2-Chloro-N-(2-fluoro-4-iodophenyl)acetamide (2b)
o
1
Colorless crystals, mp: 161-162 C. H NMR (400 MHz, DMSO-d6): δH = 4.45 (s, 2H,
13
NCH2), 7.55 (d, 1H, J = 8.0 Hz, Ph-H), 7.68-7.75 (m, 2H, Ph-H), 10.19 (s, 1H, CONH). C
NMR (100 MHz, DMSO-d6): δC = 43.5 (NCH2), 124.8, 125.8, 126.1, 133.9, 152.4, 154.9 (Ph-C),
19
165.7 (C=O). F NMR (377 MHz, DMSO-d6): δF = -122.17 to -122.12 (m, 1F, Ph-F). LCMS
(M+) 312.9 found for C8H6ClFINO.
2-Chloro-N-(2,4,5-trifluorophenyl)acetamide (2c)
o
1
Colorless crystals, mp: 118-119 C. H NMR (400 MHz, DMSO-d6): δH = 4.36 (s, 2H,
NCH2), 7.69 (td, 1H, J = 8.0, 12.0 Hz, Ph-H), 7.97-8.04 (m, 1H, Ph-H), 10.32 (s, 1H, CONH).
13C NMR (100 MHz, DMSO-d6): δC = 43.4 (NCH2), 106.6, 112.4, 122.9, 144.7, 146.9, 147.2,
19
150.6 (Ph-C), 165.9 (C=O). F NMR (377 MHz, DMSO-d6): δF = -141.78 to -141.63 (m, 1F,
Ph-F), -139.14 to -139.03 (m, 1F, Ph-F), -125.62 to -125.54 (m, 1F, Ph-F). LCMS (M+) 223.0
found for C8H5ClF3NO.
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2.4.2 Characterization of pyridinium IL halides 4a-f
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium iodide (4a)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.60 (s, 2H,
NCH2), 7.20-7.29 (m, 2H, Ph-H), 7.51 (d, 1H, J = 12.0 Hz, Ph-H), 8.15 (dd, 1H, J = 8.0 Hz, 16.0
Hz, Ph-H), 8.56 (d, 1H, J = 8.0 Hz, Ph-H), 8.89 (d, 1H, J = 8.0 Hz, Ph-H), 8.97 (s, 1H, Ph-H),
10.88 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 14.1 (CH3), 18.3 (CH3), 62.3
(NCH2), 106.5, 115.2, 119.9, 127.3, 132.3, 137.9, 138.5, 144.2, 146.2, 147.1, 159.5, 161.9 (Ph-
19
C, C=N), 163.7 (C=O). F NMR (377 MHz, DMSO-d6): δF = (-115.65 to -115.59) (m, 1F, Ph-
F). LCMS (M+)-I− 259.1 found for C15H16FN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium iodide (4b)
1H NMR (400 MHz, DMSO-d6): δH = 2.52 (s, 3H, CH3), 5.70 (s, 2H, NCH2), 7.56 (d, 1H,
J = 8.0 Hz, Ph-H), 7.71-7.76 (m, 2H, Ph-H), 8.15 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.56 (d, 1H, J
= 8.0 Hz, Ph-H), 8.93 (d, 1H, J = 8.0 Hz, Ph-H), 9.02 (s, 1H, Ph-H), 10.76 (s, 1H, CONH). 13C
NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 64.4 (NCH2), 89.0, 124.7, 124.9, 125.5, 125.8,
127.3, 133.9, 138.5, 144.2, 144.2, 147.1, 152.2, 154.7 (Ph-C, C=N), 164.4 (C=O). 19F NMR
(377 MHz, DMSO-d6): δF = -122.25 to -122.20 (m, 1F, Ph-F). LCMS (M+)-I− 371.0 found for
C14H13FIN2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium iodide (4c)
1H NMR (400 MHz, DMSO-d6): δH = 2.53 (s, 3H, CH3), 5.66 (s, 2H, NCH2), 7.78 (td,
1H, J = 8.0, 12.0 Hz, Ph-H), 7.98-8.05 (m, 1H, Ph-H), 8.15 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.56
(d, 1H, J = 8.0 Hz, Ph-H), 8.89 (d, 1H, J = 8.0 Hz, Ph-H), 8.97 (s, 1H, Ph-H), 10.81 (s, 1H,
13
CONH). C NMR (100 MHz, DMSO-d6): δC= 18.3 (CH3), 62.3 (NCH2), 106.7, 111.7, 112.0,
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127.3, 138.6, 144.2, 146.2, 147.2 (Ph-C, C=N), 164.7 (C=O). F NMR (377 MHz, DMSO-d6):
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δF = -141.46 to -141.31 (m, 1F, Ph-F), -138.92 to -138.81 (m, 1F, Ph-F), -126.01 to -125.93 (m,
1F, Ph-F). LCMS (M+)-I− 281.2 found for C14H12F3N2O+.
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-4-methylpyridin-1-ium iodide (4d)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.66 (s, 3H, CH3), 5.57 (s, 2H,
NCH2), 7.20-7.29 (m, 2H, Ph-H), 7.51 (dd, 1H, J = 4.0, 12.0 Hz, Ph-H), 8.06 (d, 2H, J = 8.0 Hz,
Ph-H), 8.88 (d, 2H, J = 8.0 Hz, Ph-H), 10.79 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
δC = 14.1 (CH3), 22.0 (CH3), 61.7 (NCH2), 106.2, 106.5, 115.2, 119.8, 128.2, 132.2, 137.9,
19
145.8, 159.5, 160.2, 161.9 (Ph-C, C=N), 164.0 (C=O). F NMR (377 MHz, DMSO-d6): δF = -
115.63 to -115.58 (m, 1F, Ph-F). LCMS (M+)-I− 259.2 found for C15H16FN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-4-methylpyridin-1-ium iodide (4e)
1H NMR (400 MHz, DMSO-d6): δH = 2.65 (s, 3H, CH3), 5.63 (s, 2H, NCH2), 7.57 (d, 1H,
J = 8.0 Hz, Ph-H), 7.71-7.77 (m, 2H, Ph-H), 8.06 (d, 2H, J = 8.0 Hz, Ph-H), 8.88 (d, 2H, J = 4.0
Hz, Ph-H), 10.76 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC= 22.0 (CH3), 61.8
(NCH2), 88.6, 124.7, 124.9, 125.5, 125.8, 128.3, 145.8, 152.1, 154.6, 160.3 (Ph-C, C=N), 164.6
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(C=O). F NMR (377 MHz, DMSO-d6): δF = -122.45 to -122.40 (m, 1F, Ph-F). LCMS (M+)-I−
371.0 found for C14H13FIN2O+.
4-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium (4f)
1H NMR (400 MHz, DMSO-d6): δH = 2.66 (s, 3H, CH3), 5.64 (s, 2H, NCH2), 7.78 (td,
1H, J = 8.0, 12.0 Hz, Ph-H), 7.97-8.06 (m, 3H, Ph-H), 8.89 (d, 2H, J = 8.0 Hz, Ph-H), 10.79 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.7 (NCH2), 106.4, 106.7,
106.9, 111.7, 111.9, 122.7, 128.3, 144.6, 160.3 (Ph-C, C=N), 165.0 (C=O). 19F NMR (377 MHz,
DMSO-d6): δF = -141.46 to 141.31 (m, 1F, Ph-F), -138.99 to -138.88 (m, 1F, Ph-F), -125.99 to -
125.91 (m, 1F, Ph-F). LCMS (M+)-I− 281.2 found for C14H12F3N2O+.
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2.4.3 Characterization of pyridinium ILs 5a-r
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
tetrafluoroborate (5a)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.59 (s, 2H,
NCH2), 7.20-7.29 (m, 2H, Ph-H), 7.51 (dd, 1H, J = 4.0, 12.0 Hz, Ph-H), 8.15 (dd, 1H, J = 8.0,
12.0 Hz, Ph-H), 8.56 (d, 1H, J = 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 4.0 Hz, Ph-H), 8.96 (s, 1H, Ph-
13
H), 10.83 (s, 1H, CONH). C NMR (100 MHz, DMSO-d6): δC = 14.1 (CH3), 18.3 (CH3), 62.3
(NCH2), 106.2, 106.5, 115.2, 119.9, 127.3, 132.2, 137.8, 138.6, 144.2, 146.2, 147.1, 159.5, 161.9
11
(Ph-C, C=N), 163.7 (C=O). B NMR (128 MHz, DMSO-d6): δB = -1.32 to -1.31 (m, 1B, BF4).
19F NMR (377 MHz, DMSO-d6): δF = -115.64 to -115.57 (m, 1F, Ph-F); -148.23 (d, 4F, BF4).
−
LCMS (M+)-BF4 259.1 found for C15H16FN2O+.
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
hexafluoro-
phosphate (5b)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.59 (s, 2H,
NCH2), 7.20-7.29 (m, 2H, Ph-H), 7.51 (dd, 1H, J = 4.0, 12.0 Hz, Ph-H), 8.15 (dd, 1H, J = 8.0,
12.0 Hz, Ph-H), 8.56 (d, 1H, J = 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 8.0 Hz, Ph-H), 8.96 (s, 1H, Ph-
13
H), 10.82 (s, 1H, CONH). C NMR (100 MHz, DMSO-d6): δC= 14.1 (CH3), 18.3 (CH3), 62.3
(NCH2), 106.2, 106.5, 115.2, 119.9, 127.3, 132.2, 137.8, 138.6, 144.2, 146.2, 147.1, 159.5, 161.9
31
(Ph-C, C=N), 163.7 (C=O). P NMR (162 MHz, DMSO-d6) δP = -157.45 to -131.04 (sept, 1P,
PF6). 19F NMR (377 MHz, DMSO-d6): δF = -69.18 (d, 6F, PF6), -115.62 to -115.57 (m, 1F, Ph-
−
F). LCMS (M+)-PF6 259.1 found for C15H16FN2O+.
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1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium trifluoroacetate
(5c)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.60 (s, 2H,
NCH2), 7.20-7.29 (m, 2H, Ph-H), 7.51 (d, 1H, J = 12.0 Hz, Ph-H), 8.15 (dd, 1H, J = 4.0, 12.0
Hz, Ph-H), 8.56 (d, 1H, J = 8.0 Hz, Ph-H), 8.89 (d, 1H, J = 8.0 Hz, Ph-H), 8.97 (s, 1H, Ph-H),
10.87 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 15.3 (CH3), 19.5 (CH3), 63.5
(NCH2), 107.4, 107.7, 116.4, 121.1, 128.5, 133.4, 139.7, 145.4, 147.4, 148.3, 160.7 (Ph-C,
C=N), 164.9 (C=O). 19F NMR (377 MHz, DMSO-d6): δF = -72.20 (s, 3F, CF3), -114.44 to -
114.39 (m, 1F, Ph-F). LCMS (M+)-CH3COO− 259.2 found for C15H16FN2O+.
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-4-methylpyridin-1-ium
tetrafluoro-
borate (5d)
1H NMR (400 MHz, DMSO-d6): δH = 2.18 (s, 3H, CH3), 2.66 (s, 3H, CH3), 5.57 (s, 2H,
NCH2), 7.19-7.29 (m, 2H, Ph-H), 7.50 (d, 1H, J = 12.0 Hz, Ph-H), 8.06 (d, 2H, J = 8.0 Hz, Ph-
H), 8.88 (d, 2H, J = 4.0 Hz, Ph-H), 10.79 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC =
14.1 (CH3), 22.0 (CH3), 61.7 (NCH2), 106.2, 106.5, 115.2, 119.8, 128.2, 132.2, 137.9, 145.8,
159.5, 160.2, 161.9 (Ph-C, C=N), 164.0 (C=O). 11B NMR (128 MHz, DMSO-d6): δB = -1.32 to -
19
1.31 (m, 1B, BF4). F NMR (377 MHz, DMSO-d6): δF = -115.64 to -115.57 (m, 1F, Ph-F); -
−
148.21 (d, 4F, BF4). LCMS (M+)-BF4 259.2 found for C15H16FN2O+.
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-4-methylpyridin-1-ium
hexafluoro-
phosphate (5e)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.66 (s, 3H, CH3), 5.55 (s, 2H,
NCH2), 7.19-7.29 (m, 2H, Ph-H), 7.50 (dd, 1H, J = 4.0, 12.0, Hz, Ph-H), 8.06 (d, 2H, J = 8.0 Hz,
Ph-H), 8.87 (d, 2H, J = 4.0 Hz, Ph-H), 10.78 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
9

ACCEPTED MANUSCRIPT
δC = 14.1 (CH3), 22.0 (CH3), 61.7 (NCH2), 106.2, 106.5, 115.2, 119.9, 128.2, 132.3, 137.7,
145.8, 160.2, 161.6 (Ph-C, C=N), 164.0 (C=O). 31P NMR (162 MHz, DMSO-d6) δP = -157.38 to
19
-130.48 (sept, 1P, PF6). F NMR (377 MHz, DMSO-d6): δF = -69.18 (d, 6F, PF6), -115.62 to -
−
115.57 (m, 1F, Ph-F). LCMS (M+)-PF6 259.1 found for C15H16FN2O+.
1-(2-((3-Fluoro-4-methylphenyl)amino)-2-oxoethyl)-4-methylpyridin-1-ium trifluoroacetate
(5f)
1H NMR (400 MHz, DMSO-d6): δH = 2.19 (s, 3H, CH3), 2.66 (s, 3H, CH3), 5.56 (s, 2H,
NCH2), 7.19- 7.29 (m, 2H, Ph-H), 7.50 (dd, 1H, J = 4.0, 12.0 Hz, Ph-H), 8.06 (d, 2H, J = 8.0 Hz,
Ph-H), 8.87 (d, 2H, J = 4.0 Hz, Ph-H), 10.79 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
δC= 14.1 (CH3), 22.0 (CH3), 61.7 (NCH2), 106.2, 106.5, 115.2, 119.8, 128.2, 132.2, 137.9,
145.8, 159.5, 160.2, 161.9 (Ph-C, C=N), 164.0 (C=O). 19F NMR (377 MHz, DMSO-d6): δF = -
73.45 (s, 3F, CF3), -115.63 to -115.58 (m, 1F, Ph-F). LCMS (M+)-CH3COO− 259.1 found for
C15H16FN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium tetrafluoroborate
(5g)
1H NMR (400 MHz, DMSO-d6): δH = 2.52 (s, 3H, CH3), 5.64 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.73-7.78 (m, 2H, Ph-H), 8.14 (dd, 1H, J = 8.0, 12.0 Hz, Ph-H), 8.55 (d, 1H, J
= 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 8.0 Hz, Ph-H), 8.96 (s, 1H, Ph-H), 10.67 (s, 1H, CONH). 13C
NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.4 (NCH2), 88.7, 124.7, 124.9, 125.3, 125.8,
127.3, 134.0, 138.6, 144.2, 146.2, 147.1, 152.1, 154.6 (Ph-C, C=N), 164.4 (C=O). 11B NMR
(128 MHz, DMSO-d6): δB = -1.32 to -1.30 (m, 1B, BF4). 19F NMR (377 MHz, DMSO-d6): δF = -
−
122.62 to -122.57 (m, 1F, Ph-F); -148.22 (d, 4F, BF4). LCMS (M+)-BF4 371.0 found for
C14H13FIN2O+.
10

ACCEPTED MANUSCRIPT
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
hexafluoro-
phosphate (5h)
1H NMR (400 MHz, DMSO-d6): δH = 2.52 (s, 3H, CH3), 5.64 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.73-7.78 (m, 2H, Ph-H), 8.14 (dd, 1H, J = 8.0, 12.0 Hz, Ph-H), 8.55 (d, 1H, J
= 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 4.0 Hz, Ph-H), 8.96 (s, 1H, Ph-H), 10.67 (s, 1H, CONH). 13C
NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.4 (NCH2), 88.7, 124.7, 124.9, 125.4, 125.8,
127.3, 134.0, 138.6, 144.2, 146.2, 147.1, 154.6 (Ph-C, C=N), 164.4 (C=O). 31P NMR (162 MHz,
DMSO-d6) δP = -157.40 to -131.16 (sept, 1P, PF6). 19F NMR (377 MHz, DMSO-d6): δF = -69.17
−
(d, 6F, PF6), -122.60 to -122.55 (m, 1F, Ph-F). LCMS (M+)-PF6 371.0 found for C14H13FIN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
(5i)
trifluoroacetate
1H NMR (400 MHz, DMSO-d6): δH = 2.52 (s, 3H, CH3), 5.65 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.73-7.77 (m, 2H, Ph-H), 8.14 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.55 (d, 1H, J =
13
8.0 Hz, Ph-H), 8.89 (d, 1H, J = 8.0 Hz, Ph-H), 8.97 (s, 1H, Ph-H), 10.67 (s, 1H, CONH). C
NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.4 (NCH2), 88.8, 124.7, 124.9, 125.4, 125.8,
127.3, 134.0, 138.6, 144.2, 146.2, 147.1, 154.6 (Ph-C, C=N), 164.4 (C=O). 19F NMR (377 MHz,
DMSO-d6): δF = -73.46 (s, 3F, CF3), -122.59 to -122.54 (m, 1F, Ph-F). LCMS (M+)-CF3COO−
371.0 found for C14H13FIN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium tetrafluoroborate
(5j)
1H NMR (400 MHz, DMSO-d6): δH = 2.65 (s, 3H, CH3), 5.61 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.72-7.77 (m, 2H, Ph-H), 8.04 (d, 2H, J = 4.0 Hz, Ph-H), 8.87 (d, 2H, J = 4.0
Hz, Ph-H), 10.64 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.8
11

ACCEPTED MANUSCRIPT
(NCH2), 88.7, 124.9, 125.4, 125.8, 128.3, 134.0, 145.8, 152.1, 154.6, 160.3 (Ph-C, C=N), 164.6
(C=O). 11B NMR (128 MHz, DMSO-d6): δB = -1.32 to -1.30 (m, 1B, BF4). 19F NMR (377 MHz,
−
DMSO-d6): δF = -122.54 to -122.50 (m, 1F, Ph-F); -148.22 (d, 4F, BF4). LCMS (M+)-BF4 371.0
found for C14H13FIN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
hexafluoro-
phosphate (5k)
1H NMR (400 MHz, DMSO-d6): δH = 2.65 (s, 3H, CH3), 5.61 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.72-7.77 (m, 2H, Ph-H), 8.04 (d, 2H, J = 4.0 Hz, Ph-H), 8.87 (d, 2H, J = 8.0
Hz, Ph-H), 10.64 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.8
(NCH2), 88.7, 124.7, 124.9, 125.4, 125.8, 128.3, 134.0, 145.8, 152.1, 154.6, 160.3 (Ph-C, C=N),
164.6 (C=O). 31P NMR (162 MHz, DMSO-d6) δP = -157.38 to -131.10 (sept, 1P, PF6). 19F NMR
(377 MHz, DMSO-d6): δF = -69.17 (d, 6F, PF6), -122.55 to -122.51 (m, 1F, Ph-F). LCMS (M+)-
−
PF6 371.2 found for C14H13FIN2O+.
1-(2-((2-Fluoro-4-iodophenyl)amino)-2-oxoethyl)-3-methylpyridin-1-ium
(5l)
trifluoroacetate
1H NMR (400 MHz, DMSO-d6): δH = 2.65 (s, 3H, CH3), 5.62 (s, 2H, CH2), 7.57 (d, 1H, J
= 8.0 Hz, Ph-H), 7.72-7.77 (m, 2H, Ph-H), 8.06 (d, 2H, J = 12.0 Hz, Ph-H), 8.87 (d, 2H, J = 4.0
Hz, Ph-H), 10.64 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.8
(NCH2), 88.8, 124.7, 124.9, 125.4, 125.8, 128.3, 134.0, 145.8, 152.1, 154.6, 160.3 (Ph-C, C=N),
164.6 (C=O). 19F NMR (377 MHz, DMSO-d6): δF = -73.46 (s, 3F, CF3), -122.55 to -122.50 (m,
1F, Ph-F). LCMS (M+)-CF3COO− 370.9 found for C14H13FIN2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium
(5m)
tetrafluoroborate
12

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, DMSO-d6): δH = 2.53 (s, 3H, CH3), 5.66 (s, 2H, CH2), 7.77 (td, 1H,
J = 8.0, 12.0 Hz, Ph-H), 7.98-8.06 (m, 1H, Ph-H), 8.15 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.56 (d,
2H, J = 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 4.0 Hz, Ph-H), 8.96 (d, 1H, J = 4.0 Hz, Ph-H), 10.81 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.3 (NCH2), 106.5, 106.9,
111.8, 112.3, 122.5, 127.3, 138.6, 144.2, 144.6, 146.2, 147.2 (Ph-C, C=N), 164.7 (C=O). 11B
NMR (128 MHz, DMSO-d6): δB = -1.31 to -1.29 (m, 1B, BF4). 19F NMR (377 MHz, DMSO-d6):
δF = -126.05 to -125.96 (m, 1F, Ph-F), -138.94 to -138.82 (m, 1F, Ph-F), -141.48 to -141.32 (m,
−
1F, Ph-F), -148.25 (d, 4F, BF4). LCMS (M+)-BF4 281.1 found for C14H12F3N2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium
hexafluoro-
phosphate (5n)
1H NMR (400 MHz, DMSO-d6): δH = 2.52 (s, 3H, CH3), 5.65 (s, 2H, CH2), 7.77 (td, 1H,
J = 8.0, 12.0 Hz, Ph-H), 7.97-8.04 (m, 1H, Ph-H), 8.14 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.55 (d,
2H, J = 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 4.0 Hz, Ph-H), 8.95 (d, 1H, J = 4.0 Hz, Ph-H), 10.81 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.3 (NCH2), 106.5, 106.9,
111.8, 112.3, 122.5, 127.3, 138.6, 144.2, 144.6, 146.2, 147.2 (Ph-C, C=N), 164.7 (C=O). 31P
NMR (162 MHz, DMSO-d6) δP = -157.39 to -131.06 (sept, 1P, PF6). 19F NMR (377 MHz,
DMSO-d6): δF = -69.18 (d, 6F, PF6), -126.04 to -125.96 (m, 1F, Ph-F), -138.92 to -138.81 (m,
−
1F, Ph-F), -141.44 to -141.36 (m, 1F, Ph-F). LCMS (M+)-PF6 281.2 found for C14H12F3N2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium trifluoroacetate (5o)
1H NMR (400 MHz, DMSO-d6): δH = 2.53 (s, 3H, CH3), 5.66 (s, 2H, CH2), 7.77 (td, 1H,
J = 8.0, 12.0 Hz, Ph-H), 7.97-8.04 (m, 1H, Ph-H), 8.15 (dd, 1H, J = 4.0, 8.0 Hz, Ph-H), 8.56 (d,
2H, J = 8.0 Hz, Ph-H), 8.88 (d, 1H, J = 4.0 Hz, Ph-H), 8.96 (s, 1H, Ph-H), 10.81 (s, 1H, CONH).
13C NMR (100 MHz, DMSO-d6): δC = 18.3 (CH3), 62.3 (NCH2), 106.5, 106.9, 111.8, 112.3,
13

ACCEPTED MANUSCRIPT
122.5, 127.3, 138.6, 144.2, 144.6, 146.2, 147.2 (Ph-C, C=N), 164.7 (C=O). 19F NMR (377 MHz,
DMSO-d6): δF = -75.31 (d, 3F, CF3), -126.01 to -125.92 (m, 1F, Ph-F), -138.91 to -138.78 (m,
1F, Ph-F), -141.46 to -141.31 (m, 1F, Ph-F). LCMS (M+)-CF3COO− 281.1 found for
C14H12F3N2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium
(5p)
tetrafluoroborate
1H NMR (400 MHz, DMSO-d6): δH = 2.66 (s, 3H, CH3), 5.63 (s, 2H, NCH2), 7.77 (td,
1H, J = 8.0, 12.0 Hz, Ph-H), 7.96-8.06 (m, 3H, Ph-H), 8.88 (d, 2H, J = 8.0 Hz, Ph-H), 10.79 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.7 (NCH2), 106.4, 106.7,
106.9, 111.7, 111.9, 122.7, 128.3, 144.6, 160.3 (Ph-C, C=N), 165.0 (C=O). 11B NMR (128 MHz,
DMSO-d6): δB = -1.31 to -1.29 (m, 1B, BF4). 19F NMR (377 MHz, DMSO-d6): δF = -125.96 to -
125.88 (m, 1F, Ph-F), -138.98 to -138.86 (m, 1F, Ph-F), -141.46 to -141.33 (m, 1F, Ph-F), -
−
148.24 (d, 4F, BF4). LCMS (M+)-BF4 281.0 found for C14H12F3N2O+.
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium
hexafluoro-
phosphate (5q)
1H NMR (400 MHz, DMSO-d6): δH = 2.66 (s, 3H, CH3), 5.63 (s, 2H, NCH2), 7.77 (td,
1H, J = 8.0, 12.0 Hz, Ph-H), 7.97-8.06 (m, 3H, Ph-H), 8.88 (d, 2H, J = 8.0 Hz, Ph-H), 10.79 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.7 (NCH2), 106.4, 106.7,
106.9, 111.7, 111.9, 122.7, 128.3, 144.6, 160.3 (Ph-C, C=N), 165.0 (C=O). 31P NMR (162 MHz,
DMSO-d6) δP = -157.38 to -131.03 (sept, 1P, PF6). 19F NMR (377 MHz, DMSO-d6): δF = -69.17
(d, 6F, PF6), -126.03 to -125.94 (m, 1F, Ph-F), -139.00 to -138.89 (m, 1F, Ph-F), -141.46 to -
−
141.31 (m, 1F, Ph-F). LCMS (M+)-PF6 281.2 found for C14H12F3N2O+.
14

ACCEPTED MANUSCRIPT
3-Methyl-1-(2-oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)pyridin-1-ium trifluoroacetate (5r)
1H NMR (400 MHz, DMSO-d6): δH = 2.66 (s, 3H, CH3), 5.63 (s, 2H, NCH2), 7.77 (td, 1H, J
= 8.0, 12.0 Hz, Ph-H), 7.97-8.06 (m, 3H, Ph-H), 8.88 (d, 2H, J = 6.6 Hz, Ph-H), 10.79 (s, 1H,
13
CONH). C NMR (100 MHz, DMSO-d6): δC = 22.0 (CH3), 61.7 (NCH2), 106.4, 106.7, 106.9,
111.7, 111.9, 122.7, 128.3, 144.6, 160.3 (Ph-C, C=N), 165.0 (C=O). 19F NMR (377 MHz,
DMSO-d6): δF = -71.50 (s, 3F, CF3), -125.97 to -125.89 (m, 1F, Ph-F), -138.96 to -138.85 (m,
1F, Ph-F), -141.46 to -141.31 (m, 1F, Ph-F). LCMS (M+)-CF3COO− 280.9 found for
C14H12F3N2O+.
2.5 DNA binding studies
The DNA binding experiments were done in Tris-buffer (10-2 M, pH 7.2). Initially, the
concentration of DNA was ascertained by recording the absorption spectrum of CT-DNA
solution. This solution gave UV absorbance at 230/260 nm (=1.8), indicated free nature of DNA,
with ε value equal to 6600 M−1 cm−1 [37]. The different compounds and DNA solutions were
kept at 4 ˚C. The compounds were firstly dissolved in 0.1% DMSO and then diluted with Tris-
buffer so that the concentrations become 2.4 × 10-4 M. After individual addition of various
concentrations (1.2 – 1.5 × 10-5 M) of DNA, the absorption spectra were recorded. The binding
constants (Kb) were determined with help of Benessi-Hilderbrand equation (1) [38].
[DNA]
[DNA]
1








…………………….. (1)
a  f
a  f
K b  f
a ,
b
where, absorption coefficients;
f , and
are related to Aobs/[compound], extinction
coefficient for compound in free and extinction coefficient for compound in fully bound form.
15

ACCEPTED MANUSCRIPT
The binding constants (Kb) for different compounds were ascertained by slopes and the
[DNA]
vs[DNA]


a  f
intercepts of the plots of
.
2.6 Anticancer studies
The anti-proliferative testing of the synthesized compounds was done with two lung
cancer cell lines i.e. A549 and H-1229 by MTT assay. Briefly, the cells were seeded (density of
8000 cells/well) in a 96 well plate and set to incubate. At about 60-70% confluency, the cells
were treated with concentrations (5.0, 10.0, 15 and 20.0 mM) of the reported compounds and
allowed to incubate for next 24 hrs. The cells were assayed by adding 15.0 µL (5.0 mg/mL
MTT). At 37 ˚C of incubation for 4 hrs, the respective media from each well were aspirated. The
cells were re-suspended in 100 µL of DMSO and the plate immediately covered with aluminum
foil, followed by gentle shaking on a shaker for about 15 min. Absorbance was recorded at 540
nm [39] and the percent inhibition in proliferation was calculated by the formula in Eq.2
% Inhibitaion = [AControl - ASample)/AControl] x 100
2.7 Docking procedure
- 2
The docking studies were performed by Intel dual CPU (1.86 GHz) with Windows XP
operating system. The 3D structure of ligand was drawn by using Marwin sketch. The so
obtained 3D structure was converted to the pdb file format. Ligand preparation was done by
assigning Gastegier charges, merging non-polar hydrogens, and saving it in PDBQT file format
using AutoDock Tools (ADT) 4.0 [40]. X-ray crystal structure of DNA (PDB ID: 1bna), was
obtained from the Protein Data Bank (http://www.rcsb.org/pdb). Using AutoDock Tools (ADT)
4.0, receptors were saved in PDB file format leaving heteroatoms (water). Gastegier charges
were assigned to receptor and saved in PDBQT file format using ADT. Preparation of parameter
files for grid and docking was done using ADT. Docking was performed with AutoDock 4.0
16

ACCEPTED MANUSCRIPT
(Scripps Research Institute, USA) considering all the rotatable bonds of ligand as rotatable and
receptor as rigid [41]. Grid box size of 60 × 80 × 114 Å with 0.375 Å spacing was used that
included the whole DNA. Docking to macromolecule was performed using an empirical-free
energy function and Lamarckian Genetic Algorithm, with an initial population of 150 randomly
placed individuals, a maximum number of 2500000 energy evaluations, a mutation rate of 0.02,
and crossover rate of 0.80. Fifty independent docking runs were performed for each ligand and
serum-ligand adduct for lowest free energy of binding conformation from the largest cluster and
saved in PDBQT format.
3. Results and discussion
3.1 Synthetic of ionic liquids
The selected 2-chloro-N-(3-fluoro-4-methylphenyl)acetamide, 2-chloro-N-(2-fluoro-4-
iodophenyl)acetamide and 2-chloro-N-(2,4,5-trifluorophenyl)acetamide 3a-c used in this study
were synthesized via the condensation of the appropriate fluorinated aniline 1a-c with
chloroacetyl chloride in the presence of triethylamine as catalyst. The synthetic protocol for all
the molecules is shown in scheme 1.
In our investigations, the designing of halogenated pyridinium ionic liquids was studied
and found to be easily obtained via the alkylation of 3-methyl and/or 4-methylpyridine with the
appropriate fluorinated phenylacetamide chlorides 2a-c, in refluxing acetonitrile in the presence
of sodium iodide, under both conventional and microwave conditions.
The structural elucidation of the new pyridinium iodides 4a-f was carried out on the basis
1
1
of their H, 13C, 19F NMR and mass spectrometric data. The H, 13C NMR spectra of all the
synthesized ILs 4a-f agreed with their assigned structures. Thus, their 1H NMR spectra revealed
the presence of two diagnostic singlets between 5.57-5.70 ppm and 10.76-10.88 ppm attributed
17

ACCEPTED MANUSCRIPT
to the NCH2 and CONH protons of the acetamide linkage, respectively. The spectra also
exhibited extra aromatic protons in their appropriate chemical shifts (see experimental section).
In their 13C-NMR spectra, the appearance of new signal in the aliphatic region at 61.7-64.4 ppm
assigned to the -NCH2 carbon and a diagnostic amide carbonyl carbon at 163.7-165.0 ppm
provided additional evidences for the success of the quaternization reaction. The 19F NMR
19
results were also in the agreement with the proposed structures. Thus, the F NMR spectra of
ILs 4a, 4b, 4d and 4e showed a multiplet between -122.45 to -115.58 ppm belonging to one
aromatic fluorine atom. In contrast, three multiplets were recorded in the 19F NMR spectra of ILs
4c and 4f from -141.46 to -125.91 ppm attributed to three aromatic fluorine atoms; thus,
confirming the presence of trifluorophenyl group. The structures, yields and reaction times in
conventional and microwave assisted syntheses are given in Table 1.
Under the optimized conventional versus metathesis conditions, the reaction of the
pyridinium IL halides 4a-f with three fluorinated metal salts (including NaBF4, KPF6 and
CF3COONa) led to the formation of task specific ionic liquids tethering fluorinated counter
anions 5a-r (Table 2). The composition and structure of the designed ILs 5a-r were confirmed
1
13
19
31
11
by usual spectroscopic experiments such as H, C, F, P, B NMR and mass analysis. No
1
changes were recorded on the H and 13C NMR spectra of 5a-r as compared to their
corresponding halogenated ionic liquids 4a-f. However, their structures were efficiently
19
31
11
established from their F, P, B NMR and Mass analysis. Thus, the significant doublets (at -
19
11
148.25 to -148.21 ppm) and multiplet (at -1.32 to -1.29 ppm) were recorded on the F and B
-
NMR spectra, respectively; assigning to the tetrahydroborate anion (BF4 ). These results
31
confirmed the success of the metathethical reaction. On the other hand, the P and 19F NMR
spectra exhibited characteristic septets and doublets near (-157.45 to -131.03 ppm) and (-69.18 to
18

ACCEPTED MANUSCRIPT
-
-69.17 ppm), respectively, belonging to the PF6 anion as counter anion. In addition, the fluorine
atoms of CF3COO- anion were observed as a distinct singlet ranging between -75.31 to -71.50
19
ppm in their F NMR analysis. These observations supported the halide anion exchange with
trifluoroacetate anion (CF3COO-). The remaining multiplets signals recorded in all 19F NMR
spectra were between -141.48 and -114.39 ppm; attributing to the aromatic fluorine atoms.
Further structural elucidation was confirmed by their mass spectral analyses, which was in
accordance with the proposed structures.
3.2 DNA binding studies of ionic liquids
All the absorption experiments involving interactions of the reported compounds with
CT-DNA were executed in tris-buffer having physiological pH. The UV spectra of CT-DNA in
buffer showed two absorbance peaks at 260 and 280 nm in the ratio of 1.9:1 that indicated
protein free nature of DNA [42]. Using molar absorption coefficient ε260 = 6600 L mol-1 cm-1, the
concentration of DNA was determined by UV absorption [43]. For elimination of DNA
absorbance an equal amount of DNA was added to both the reference and the other solutions.
The absorption titration experiments were conducted by varying the concentration of DNA with
the fixed concentrations of the compounds.
DNA is one of the important pharmacological targets of drugs in various diseases
particularly cancer. Although, there are several techniques used to study the drugs and DNA
interactions but, absorption spectroscopy is simple and powerful tool. The absorption spectra of
the most active ionic liquids 4a, 4e and 5n are given in Figure 1 while the others are reported in
the supplementary information. In the absence of CT-DNA, the compounds exhibited absorbance
in the range of 260-300 nm in tris-buffer at physiological pH. With the addition of the different
concentrations (1.2 – 1.5 × 10-5 M) of CT-DNA to the fixed concentration of compounds (2.4 ×
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10-5 M), the absorption intensity of the compounds showed hypochromism with slight and
moderate changes in the shape and position of absorption bands. Generally, hyperchromism and
hypochromism are the spectral features observed during the spectrophotometric titration of small
molecules with CT-DNA [44-47]. Hypochromism is usually characterized by non-covalently
intercalative bindings of the compounds with DNA [48], whereas hypochromism associated with
non-intercalative binding modes; most probably electrostatic or hydrogen bondings [49].
Furthermore, the hyperchromism of the compounds indicated bindings to DNA it its grooves.
The binding constants of the reported compounds were calculated by using modified Benesi–
Hildebrand equation [50].
In the present study, almost all the compounds showed small red shifts. The absorption
spectra of compound 4a in both the absence [2.4 × 10-4 M] and the presence [1.8-3.8 × 10-4 M]
of CT-DNA are shown in Figure 1. It is clear from the absorption spectra that moderate
bathochromism and hypochromism at 270 nm were observed when 4a was titrated with various
concentrations of CT-DNA. These spectral changes indicated that 4a had been interacted with
CT-DNA via non-covalently intercalations, which might be due to the stacking interactions
between the two chromophores. Similarly, for compound 4d, the absorption peak at 269 nm was
observed, but with addition of various DNA concentrations, the absorption peak underwent
strong hypochromism and slight bathochromism. In case of other compounds (4b, 4c, 4e, 4f and
5a-r) the similar changes (hypochromic and bathochromic shifts) were observed that showed the
strong interactions of the compounds with CT-DNA. The intrinsic DNA binding constants were
calculated for all the compounds 4a-f and 5a-r and ranged from 1.3 × 103 to 1.52 × 108 M−1
(Table 3). In addition to the hypochromism and bathochromism changes, the isosbestic points
were also appeared in DNA binding spectra for some compounds (4e, 4f, 5b and 5h). These were
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also the indications of the moderate bindings of the compounds with DNA. Finally, from the
above observed spectral changes in all the compounds and high values of Kb suggested that the
compounds have strong affinity with CT-DNA. These can be considered as non-covalent binders
[51-53].
3.3 Anticancer activities of ionic liquids
In vitro percentage inhibitions of two lung cancerous cells i.e. A549 and H-1229 were
studied at 5.0, 10.0, 15.0 and 20.0 mM concentrations of the reported compounds. The results are
given in Figures 2 and 3, respectively. Originally, the molecules were dissolved in 0.1 % DMSO
and natural cells with DMSO were utilized as vehicle control. Then accumulation of the
molecules was waited till the control cells reached at inactive phase. The cells were counted after
24 hrs. For all the compounds, the proliferation inhibitions for both the cells were augmented
with increasing amount of the compounds. The results indicated the significant inhibition in
cancer cell proliferations. The two sets of the compounds i.e. 4a-f and 5a-r were screened. The
maximum percentage inhibitions of 4a-f series with A549 and H-1229 cell lines were 99.16%
(4a) and 91.24%, (4e), respectively. The order of the anticancer activities with these cell lines
was 4a > 4e > 4b > 4d > 4c and 4e > 4c > 4a > 4f > 4b > 4d. The maximum percentage
inhibitions of 5a-r series with A549 and H-1229 cell lines were 99.23% (5n) and 99.69% (5n),
respectively. The order of the anticancer activities with these cell lines was 5n > 5f > 5h > 5i >
5g > 5a > 5k > 5m > 5e > 5d > 5c > 5p > 5b > 5i > 5g > 5j > 5o > 5r and 5n > 5p > 5q > 5h > 5c
> 5g > 5f > 5r > 5m > 5k > 5a > 5o > 5i > 5j > 5i > 5d > 5b > 5e. All the compounds showed
more than 50% inhibition of the lung cancer cell lines. Moreover, the maximum proliferation
inhibitions were 99.16 to 91.24% for 4a series and 99.23 to 99.69% for 5a series of the ionic
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liquids. The percentage inhibition was found to be excellent (4a, 4e and 5n) and, hence, these
ionic liquids may be the future molecules for treating cancer.
3.4 Docking studies of ionic liquids
The attempts were made to determine the interactions and mechanism of action of the
reported molecules with DNA. The different binding affinities obtained are given in Table 4.
Basically, the binding study of only organic molecules with DNA is carried out. The present
article describes twenty-four compounds but the basic organic structures are of six different
types 4a-f. Therefore, the binding study was carried out with six structures only. The binding
affinities of the ionic liquids with DNA ranged from -4.6 to -4.9 kcal/mol because of the
different structures of the organic moieties. Besides, the hydrogen bond formation with DNA
was also based on the different structures of the molecules. The hydrogen bonds formed with
DNA were one (4a), two (4b,c) and three (4d-f). After hydrogen bond formation study, the
hydrophobic interactions were seen using ligplot software. The common residues of DNA
involved with ionic liquids were dc9, dc13, dc14, dc15, dg10, dg14, dg16, c1, c2, c3, c4, c5, c6,
c7, c8, c7, c9, c10, c11, c13, c14 and c15. The interactions models of the most active compound
(4a) is given in Figure 4, while the rest in supplementary information. It is clear from the docked
models that all the compounds preferred DNA minor grooves. During the process of DNA
interaction, it was also observed that all the compounds oriented themselves in such a fashion
that their active sites were inside the minor grooves. Overall, the experimental results of DNA
binding were in good agreement with those of docking studies. It is very important to mention
here that all twenty-four ionic liquids have only six different organic moieties while these differ
with respect to the anionic parts. In spite of only six organic moieties, all the twenty-four
compounds showed the different anticancer activities. It is due to the fact that the different
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ionization of ionic liquids provided different concentrations of six organic moieties. It is similar
to the different interactions of cis- and trans-platin isomers [54].
4. Significance of microwave assisted synthesis of ionic liquids
It was noticeable that the excellent yields obtained were 92-95 % within short reaction
times (15-20 min), when the quaternization of the same picoline 3a and/or 3b was carried out
under MWI (Table 1). Microwave irradiated (MWI) versus thermal metathetical anion exchange
was also occurred smoothly; when the designed pyridinium IL halides 4a-f were treated with the
appropriate fluorinated metal salts (NaBF4, KPF6 and CF3COONa). This led to the formation of
task specific ionic liquids tethering fluorinated counter anions 5a-r. Under the thermal
conditions, 24 hrs were required to complete the anion exchange furnishing the formation of 5a-r
in 80-86 % yields. The general metathesis reaction worked well in very short time (20 min) for
the anion exchange using MWI and yielded the target task ionic liquids 5a-r in excellent yields
(up to 97 %; Table 2).
5. Conclusion
Really, ionic liquids are remarkable molecules with excellent properties and applications.
The reported ionic liquids were obtained in good yields (up to 95%) with microwave assisted
green approach syntheses. The DNA binding constants (Kb) were in the range of 1.3 × 103 to
1.52 × 108 M−1. All the ionic liquids intercalated with DNA through the minor grooves via
covalent, intercalative and electrostatic bindings. All the reported ionic liquids showed more than
50% anticancer activities with two lung cancer cell lines i.e. A549 and H-1229. Moreover, the
maximum proliferation inhibitions were 99.16 to 91.24% for 4a series and 99.23 to 99.69% for
5a series of the ionic liquids. The docking studies indicated quite good binding affinities (-4.6 to
-4.9 kcal/mol.) of the reported ionic liquids with DNA. The experimental results of DNA
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bindings were in good agreement with those of docking studies. The percentage inhibition was
found to be excellent (4a, 4e and 5n) and, hence, these ionic liquids may be the future molecules
for treating cancer.
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