Design, synthesis, in-silico and in-vitro evaluation of di-cationic pyridinium ionic liquids as potential anticancer scaffolds

Article abstract of DOI:10.1016/j.molliq.2018.06.045

An array of dicationic pyridinium ionic liquids (DILs) based hydrazone linkage were designed and synthesized via the quaternization of the appropriate bispyridine hydrazone with different phenacyl halides and led to the formation of halogenated DILs, which undergo metathesis reaction to give the specific task dicationic pyridinium liquids carrying fluorinated counter anions (PF₆ ^?, BF₄ ^?, CF₃COO^?). The newly synthesized DILs were well characterized using whole spectroscopic data. The Anticancer evaluation of DILs against breast and colon cancer cell lines revealed that compound 22 appears to be the most active compound in the series with IC₅₀ in the two-digit micromolar range. The in-vitro anticancer results were further supported by in-silico molecular docking studies revealing the highest potency of compound 22. The docking analysis demonstrated good docking score and binding affinities of the synthesized compounds on the target protein PI3Kinase.

Full text of DOI:10.1016/j.molliq.2018.06.045

Accepted Manuscript
Design, synthesis, in-silico and in-vitro evaluation of di-cationic
pyridinium ionic liquids as potential anticancer scaffolds
Nadjet Rezki, Mouslim Messali, Salsabeel A. Al-Sodies, Arshi
Naqvi, Sanaa K. Bardaweel, Fawzia F. Al-blewi, Mohamed R.
Aouad, El Sayed H. El Ashry
PII:
DOI:
Reference:
S0167-7322(18)31719-7
doi:10.1016/j.molliq.2018.06.045
MOLLIQ 9243
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
1 April 2018
4 June 2018
11 June 2018
Please cite this article as: Nadjet Rezki, Mouslim Messali, Salsabeel A. Al-Sodies, Arshi
Naqvi, Sanaa K. Bardaweel, Fawzia F. Al-blewi, Mohamed R. Aouad, El Sayed H. El
Ashry , Design, synthesis, in-silico and in-vitro evaluation of di-cationic pyridinium ionic
liquids as potential anticancer scaffolds. Molliq (2017), doi:10.1016/j.molliq.2018.06.045
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ACCEPTED MANUSCRIPT
Design, Synthesis, In-silico and In-vitro Evaluation of Di-Cationic Pyridinium
Ionic Liquids as Potential Anticancer Scaffolds
Nadjet Rezkia,b, Mouslim Messalia,*, Salsabeel A. Al-Sodiesa, Arshi Naqvia, Sanaa K.
Bardaweelc,*, Fawzia F. Al-blewia, Mohamed R. Aouada, El Sayed H. El Ashryd
aDepartment of Chemistry, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah, 30002, Saudi Arabia.
bDepartment of Chemistry, Faculty of Sciences, University of Sciences and Technology Mohamed Boudiaf,
Laboratoire de Chimie et Electrochimie des Complexes Metalliques (LCECM) USTO-MB, P.O. Box 1505, El
M‘nouar, Oran 31000, Algeria.
cDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Amman 11942, Jordan.
dDepartment of Chemistry, Faculty of Science, Alexandria University, Alexandria, Egypt.
ABSTRACT
An array of dicationic pyridinium ionic liquids (DILs) based hydrazone linkage were designed
and synthesized via the quaternization of the appropriate bispyridine hydrazone with different
phenacyl halides and led to the formation of halogenated DILs, which undergo metathesis
reaction to give the specific task dicationic pyridinium liquids carrying fluorinated counter anions
-
-
(PF6 , BF4 , CF3COO-). The newly synthesized DILs were well characterized using whole
spectroscopic data. The Anticancer evaluation of DILs against breast and colon cancer cell lines
revealed that compound 22 appears to be the most active compound in the series with IC50 in the
two-digit micromolar range. The in-vitro anticancer results were further supported by in-silico
molecular docking studies revealing the highest potency of compound 22. The docking analysis
demonstrated good docking score and binding affinities of the synthesized compounds on the
target protein PI3Kinase.
Keywords: Ionic liquids synthesis, quaternization, metathesis, dicationic ionic liquids,
hydrazones, anticancer activity, molecular docking
1.
Introduction
Cancer is one of the most compelling public health concerns worldwide. Based on the most
recent statistics released by the World Health Organization (WHO) in 2015, it was estimated that
about 8.8 million people passed away from cancer in that year [1]. Chemotherapy remains to be
considered as the standard regime for cancer treatment [2, 3]. Despite of the improvements that
have been consistently achieved in the field of chemotherapy over the last two decades, the
search for new anticancer agents with significantly superior efficacy/safety ratio continues.

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Indeed, the major focus of drug discovery has been directed nowadays to develop novel
anticancer agents that minimize the toxicity associated with the commonly used chemotherapy.
Yet, one major concern for drug discovery is the ability to turn new chemical moieties into
drugs. Many small molecules with very promising features are currently deemed as technically
undruggable or at the very least as tremendously difficult to optimize by medicinal chemists [4].
Another infuriating problem is that for medicines which have been successful and have shown
significant therapeutic effectiveness, resistance often emerges, as demonstrated lately by
crizotinib and vemurafenib [5, 6].
During the last two decades, Ionic liquids (ILs) have emerged as new class of compounds
with characteristic properties and the capability to modify their physiochemical properties [7-9].
In fact, ILs become omnipresent in the recent medicinal chemistry [10]. Several studies have
reported the exploration of IL as anticancer agents [11-16]. Interestingly, ILs with their adjustable
physicochemical properties brought a new prospect of creating new therapeutic agents with
tailored anticancer activity and diminished toxicity toward the human normal cells.
An extensive effort has been engaged to produce novel ionic liquids with tuning properties.
Recently, a type of ILs namely dicationic ionic liquids (DILs), in which the cation candidates
joining together via either a rigid or a flexible spacer has been reported [17, 18]. They were often
found to have higher melting points than monocationic ILs with the same anion and sometimes
are solids at room temperature [17]. The physicochemical properties such as thermal stability,
density, viscosity, and solubility behaviours can be tuned to a greater extent in DILs than the
monocationic analogues by changing the cation, anion and linker [19]. Therefore, they have high
potential to be used in numerous applications
[20-27].
Additionally, hydrazones have attracted increasing interest in organic synthesis [28-30].
Considerable attention have been devoted on the designing and the synthesis of such compounds
as inexhaustible resource of new molecules with diverse chemical and biological activities [31-
33].
On-to-date demand of pyridine ILs is obvious judging from their endless synthetic value for
construction of pharmacological scaffolds. 34-38 Based on these observation and in our endeavour
to increase the synergetic effect of some hydrazones based pyridine rings as continuation of our
interest in design and synthesis of novel functionalized ionic liquids tethered hydrazone linkage

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and their biological potency [39, 40], herein we report an efficient synthetic transformation for
designing a new class of dicationic liquids derived from bispyridine hydrazone, which often
generated new anticancer scaffold for further optimization.
2. Results and discussions
2.1.Chemistry
The synthetic methods carried out in this work were outlined in scheme 1. The two
precursors bispyridine hydrazone 2 and 3 were easily synthesized via the thermal condensation of
isonicotinohydrazide 1 with nicotinaldehyde and/or isonicotinaldehyde in boiling ethanol in the
presence of few drops of hydrochloric acid (Scheme 1) [40].
The strategy adopted for the construction of diionic liquid tethering two pyridinium cations
linked via a hydrazone spacer involved quaternization and metathesis reaction. Thus, the
nucleophilic alkylation of hydrazones 2 and/or 3 with substituted phenacyl bromides in refluxing
acetonitrile gave successfully the DILs 4-9 in good yields (88-94 %).
PLEASE INSERT SCHEME 1
The NMR data of the synthesized derivative 7 was taken as model to discuss and confirm the
quaternization reaction. Its 1H NMR spectrum showed the two singlets at δH 3.91 ppm integrated
for six protons of the two methoxy groups (-OCH3) (Figure 1). The spectrum also showed the
presence of the methylene protons (2xNCH2) as doublet at δH 6.63 ppm. The imine proton
(HC=N) was observed as two singlets at δH 8.81 and 8.86 ppm and integrated as one proton, with
ration of 1:3. Same ratio was observed for the NH proton splited into two singlets at δH 13.11 and
13.28 ppm. Moreover, the pyridinium protons were recorded with the same isomeric
ratio to that displayed by NH and HC=N groups. Such pairing of signals was previously
confirmed in our reported work to be due to the E/cis and E/trans geometrical isomerism
about
the
carbonylamide
group
(CONH)
and
the
imine
bond
(HC=N) [42, 43]. The extra eight aromatic protons recorded at their usual chemical shifts
belonging the two aromatic rings of the phenacyl moiety (See experimental section).
PLEASE INSERT FIGURE 1

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The structure of DILs 7 was also supported by its 13C NMR spectrum (Figure 2), which
revealed the presence of the methoxy carbons at δC 56.34 ppm. The spectrum also showed the
appearance of two sets of signals for each peak, supporting the geometrical isomerism.
In addition, the methylene carbons appeared as two signals at δC 66.58 and 66.70 ppm,
respectively. The presence of E/cis and E/trans diastereomers of the C=N and C=O groups of the
hydrazone linkage were also recorded as two signals at δC 160.04 and 164.78 ppm, respectively.
The presence of molecular ion peak at 684.4041 [M+] in the electron impact mass spectrum
of compound 7, confirmed the quaternization reaction of hydrazone 3 with methoxy phenacyl
bromide.
PLEASE INSERT FIGURE 2
The specific task DiILs 10-18 were obtained in satisfactory yields (85-92 %), according to
the metathesis protocol. In this step, the bromide anion of the synthesized DILs 4-9 were
-
-
subjected to anion exchange with an appropriate anions (PF6 , BF4 , CF3COO-), through their
thermal treatment with metal salts in acetonitrile resulted in the formation of specific task
dicationic pyridinium ionic liquids 10-29. The introduction of such anion was confirmed by the
1
whole NMR analysis, which revealed that no change in their H and 13C NMR data compared to
their corresponding precursors 4-9. Accordingly, their structures were established based on their
31P, 11B, 19F NMR and mass spectra analyses.
31
31
The structure of compound 19 was supported by the P and 19F NMR spectra. Its P NMR
spectrum showed a characteristic multiplet between δP -157.39 to -131.04 ppm relative to the
-
phosphorus atom in the PF6 anion (Figure 3).
PLEASE INSERT FIGURE 3
19
On the other hand, the doublet recorded in the F NMR spectrum at -69.13 ppm confirmed
-
the bromide (Br-) anion exchange with hexafluorophosphate anion (PF6 ) yielding the compound
19 (Figure 4).
PLEASE INSERT FIGURE 4

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-
Moreover, the presence of tetrafluoroborate anion (BF4 ) as counter anion in the compound 20
was confirmed by the resonance of a multiplet between δB (-1.31) to (-1.29) ppm in the 11B
19
spectrum (Figure 5) and two doublets at δF -148.18 and -148.12 ppm in its F NMR spectrum
(Figure 6), respectively.
PLEASE INSERT FIGURE 5
PLEASE INSERT FIGURE 6
PLEASE INSERT FIGURE 7
19
The singlet observed at δF -73.49 ppm in the F NMR spectrum being related to the fluorine
atom in trifluoroacetate anion form (CF3COO-) confirming the formation of DiIL 21 (Figure 7).
2.1. Biological Screening
2.2.1. Anticancer Activity
The in vitro anticancer activities of the synthesized compounds were examined against four
human cancer cell lines, characterizing tumors of different origins. The human breast
adenocarcinoma cell line MCF-7, the human ductal breast epithelial tumor cell line T47D, the
human mammary gland/breast tumor cell line MDA231, and the human colorectal
adenocarcinoma cell line Caco-2 were incubated in culture media with varying concentrations of
the examined compound and the impact of treatment was measured using the MTT assay. The
IC50 values obtained for the tumor cells under investigation are summarized in Table 1. The
results indicate that out of the 26 synthesized derivatives, compounds 8, 22, 23, 24, 2 and 2
exhibited considerable cancer cell growth inhibition against all examined cancer types.
Interestingly, compound 22 demonstrates the most potent activity amongst the examined series
with IC50 value ranging between 59-64 µM on the different cell lines.
PLEASE INSERT TABLE 1

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Phosphoinositide 3-kinases (PI3Ks) belongs to the family of lipid kinases and plays a major
role in the promotion of cellular growth and survival of various types of cancer [44]. The PI3Ks
are the vital regulators of apoptosis in cancer cells. The phosphorylation of phosphatidylinositol
is catalyzed by PI3Ks at the 3′-OH position of the inositol ring, producing secondary messenger
lipid i.e phosphatidylinositol-3,4,5-trisphosphate. The serine/threonine kinase AKT is activated
by this trisphosphate, which regulates several signaling pathways in turn, thus, controlling
survival of cell, cell proliferation, apoptosis, and motility [45]. PI3Ks are divided into three
classes based on their structural features and substrate specificity [46]. The most studied class I
PI3Ks are further grouped into 4 isoforms: p110, p110, p110, and p110 [48]. PI3KCA gene
encoding p110 is the most frequently mutated gene in human tumors and has emerged as an
attractive target for tumor therapy [48]. Also, some hydrazone moieties have been reported to
exhibit marked PI3kinase p110α inhibition [49].
Thus, in order to explain the anticancer properties of the synthesized ionic liquids on breast
cancer (MCF-7, MDA 231 and T47D) and colon cancer (Caco-2) cell lines, in-silico molecular
docking approach was undertaken to get more insights into the binding mode of the synthesized
compounds with PI3K p110α domain (PDB ID: 2RD0). Remarkably, all investigated cell lines
have been reported to express PI3Ks [50-53].
2.2.2. In-silico molecular docking studies
Considering the promising in-vitro anticancer activities, it was thought worthwhile to predict if
the targets have analogous binding mode to the PI3K protein receptor, hence inculcating both in-
silico and in-vitro screening results. In order to determine the best in-silico conformation,
comparative and automated docking studies of newly synthesized dicationic pyridinium ionic
liquids on PI3K as the target protein was performed. To satisfy this purpose, AutoDock 4.0, the
Lamarckian genetic algorithm docking program was employed. There are two main programs in
AutoDock, one is auto grid which is used to precalculate grid maps of interaction energies for
various atom types of ligand with a macromolecule and second one is autodock that performs the
docking of the ligand to specified grids. We performed molecular docking on the identified active
site residues i.e ILE800, LEU807, LEU814, TYR836, GLY837, CYS838, ILE848 in the p110
PI3K [54]. The above residues are almost 100% identical among the p110 isoforms and are
highly conserved in the PI3K family [55]. All the synthesized compounds 2-27 were docked.

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Figure. 8 shows the docked images of some selected ligands 13 and 22 including the hydrazones
2 and 3. Table 2 shows the minimum binding energy and Inhibition constant of all the docked
compounds. The docking study revealed that the ligands exhibited bonds with one or more amino
acids in the receptor active pocket with good binding energy toward the target protein ranging
from -11.88 to -6.37 kJ/mol and calculated inhibition constant ranges from 16.40 nM
(nanomolar) to 205.52 uM (micromolar).
PLEASE INSERT FIGURE 8
PLEASE INSERT TABLE 2
3. Experimental
3.1.Chemical characterization and synthesis
Melting points were recorded on a Stuart Scientific SMP1 apparatus (Stuart, Red Hill, UK)
and are uncorrected. The NMR spectra were measured with a Bruker spectrometer (600 and 400
Brucker, Fällanden, Switzerland) using Tetramethylsilane (TMS) (0.00 ppm) as an internal
standard. High resolution mass spectroscopy (HRMS) was carried out using a LCMS/MS
impact II.
Synthesis of N'-(pyridinylmethylene)isonicotino hydrazide derivatives 2 and /or 3. To a
mixture of isonicotinic acid hydrazide (1) (1 mmol) in ethanol (25 mL) was added 4-
pyridinecarboxaldehyde and/or 3-pyridinecarboxaldehyde (1.2 mmol) and few drops of
hydrochloric acid, then the mixture was refluxed for 1 h. After cooling, the excess of solvent was
removed under reduced pressure. The product formed was recrystallized from ethanol to furnish
the desired compounds.
(E)-N'-(Pyridin-4-ylmethylene)isonicotinohydrazide (2). It was obtained as yellow crystals
1
in 92 % yield; mp: 223–224 C (Lit. mp: 224–225 ºC) [41]. H NMR (400 MHz, DMSO-d6): δH
= 7.57 (d, 0.25H, J = 4 Hz, Ar-H), 7.71 (d, 0.25H, J = 4 Hz, Ar-H), 7.82 (d, 1.75H, J = 8 Hz, Ar-
H), 7.90 (dd, 1.75H, J = 4 Hz, 8 Hz,, Ar-H), 8.14 (s, 0.2H, H-C=N), 8.55 (s, 0.8H, H-C=N), 8.66
(d, 0.25H, J = 4 Hz, Ar-H), 8.74 (d, 1.75H, J = 4 Hz, Ar-H), 8.79 (d, 0.25H, J = 4 Hz, Ar-H),
13
8.84 (dd, 1.75H, J = 4 Hz, 8 Hz,, Ar-H), 12.47 (s, 0.2H, CONH), 12.58 (s, 0.8H, CONH). C
NMR (100 MHz, DMSO-d6): δC = 121.32, 121.62, 121.76, 123.28, 140.14, 142.58, 146.21,

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149.21, 149.48, 150.38 (Ar-C), 162.08, 162.10 (C=N, C=O). HRMS (ESI) m/z = 226.1414
[M+].
(E)-N'-(Pyridin-3-ylmethylene)isonicotinohydrazide (3). It was obtained as pale yellow
crystals in 95 % yield; mp: 230–231 ºC (Lit. mp: 232–233 C).41 1H NMR (600 MHz, DMSO-
d6): δH = 7.43 (dd, 0.2 H, J = 6 Hz, Ar-H), 7.52 (dd, 0.8H, J = 6 Hz, 12 Hz, Ar-H), 7.68 (d, 0.2 H,
J = 6 Hz, Ar-H), 7.84 (dd, 1.8 H, J = 6 Hz, 12 Hz, Ar-H), 7.89 (d, 0.2H, J = 6 Hz, Ar-H), 8.15 (s,
0.2 H, H-C=N), 8.18 (dd, 0.8 H, J = 6 Hz, 12 Hz, Ar-H), 8.53 (s, 0.8H, H-C=N), 8.57 (d, 0.2H, J
= 6 Hz, Ar-H), 8.65 (dd, 0.8H, J = 6 Hz, 12 Hz, Ar-H), 8.72 (s, 0.2H, Ar-H), 8.75 (d, 0.2H, J = 6
Hz, Ar-H), 8.81 (dd, 1.8H, J = 6 Hz, 12 Hz,Ar-H), 8.89 (s, 0.8H, Ar-H), 12.20 (s, 0.2H, CONH),
13
12.25 (s, 0.8H, CONH). C NMR (150 MHz, DMSO-d6): δC = 122.01; 123.58; 124.43; 124.53;
130.29; 130.42; 133.86; 134.10; 140.74; 141.96; 142.43; 146.79; 149.38; 149.40; 150.83; 151.74
(Ar-C), 162.23; 168.46 (C=N, C=O). HRMS (ESI) m/z = 226.1418 [M+].
General Procedures for the Synthesis of dipyridinium ionic liquids tethered hydrazones 4-9.
A mixture of compound 2 and/or 3 (1 mmol) in acetonitrile (30 mL) and the appropriate phenacyl
bromide (2.1 mmol) was refluxed for 6-12 h. The solvent was reduced by evaporation under
reduce pressure, the precipitate formed was collected by filtration and/or extraction by
chloroform to afford the desired dicationic liquids 4-9.
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-4-((2-(1-(2-(4-methoxyphenyl-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium bromide (4). It was obtained as syrup in 88
1
% yield. H NMR (400 MHz, DMSO-d6): δH = 3.92 (s, 6H 2×OCH3), 6.58 (d, 4H, J = 8 Hz,
2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.09 (d, 4H, J = 8 Hz, Ar-H), 8.29 (d, 0.5H, J = 4 Hz,
Ar-H), 8.40 (d, 0.5H, J = 4 Hz, Ar-H), 8.52 (s, 0.25H, H-C=N), 8.57 (d, 1.5H, J = 8 Hz, Ar-H),
8.71 (d, 1.5H, J = 4 Hz, Ar-H), 8.78 (s, 0.75H, H-C=N), 8.97 (d, 0.5H, J = 4 Hz, Ar-H), 9.07 (d,
1.5H, J = 8 Hz, Ar-H), 9.19 (d, 0.5H, J = 4 Hz, Ar-H), 9.28 (d, 1.5H, J = 4 Hz, Ar-H), 13.37 (s,
0.25H, CONH), 13.42 (s, 0.75H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 56.32
(2×OCH3), 66.12; 66.76 (2×NCH2), 141.92; 125.13; 126.75; 127.56; 131.25; 131.30; 145.04;
147.23; 147.76; 147.89; 149.83 (Ar-C), 160.54; 164.79 (C=N, C=O), 189.08; 189.46
(2×CH2C=O). HRMS (ESI) m/z = 684.3530 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazone )methyl)pyridin-1-ium bromide (5). It was obtained as syrup in 90 %

ACCEPTED MANUSCRIPT
yield. 1H NMR (400 MHz, DMSO-d6): δH = 6.67 (d, 4H, J = 8 Hz, 2×NCH2), 7.78 (d, 4H, J = 4
Hz, Ar-H), 8.13 (d, 4H, J = 8 Hz, Ar-H), 8.33 (d, 0.5H, J = 4 Hz, Ar-H), 8.43 (d, 0.5H, J = 4 Hz,
Ar-H), 8.54 (s, 0.25H, H-C=N), 8.59 (d, 1.5H, J = 4 Hz, Ar-H), 8.75 (d, 1.5H, J = 4 Hz, Ar-H),
8.83 (s, 0.75H, H-C=N), 9.01 (d, 0.5H, J = 4 Hz, Ar-H), 9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.23 (d,
0.5H, J = 4 Hz, Ar-H), 9.31 (d, 1.5H, J = 4 Hz, Ar-H), 13.39 (s, 0.25H, CONH), 13.48 (s, 0.75H,
CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.41; 67.02 (2×NCH2), 124.89; 125.20; 126.85;
127.60; 129.80; 129.84; 130.68; 130.72; 132.72; 132.81; 140.03; 140.11; 145.03; 146.92; 147.22;
147.36; 147.88; 149.91; 149.97 (Ar-C), 160.51; 164.79 (C=N, C=O), 189.08; 189.44
(2×CH2C=O). HRMS (ESI) m/z = 693.2168 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium bromide (6). It was obtained as syrup in 90 % yield.
1H NMR (400 MHz, DMSO-d6): δH = 6.67 (d, 4H, J = 8 Hz, 2×NCH2), 7.79 (d, 4H, J = 4 Hz, Ar-
H), 8.33 (d, 4H, J = 8 Hz, Ar-H), 8.49 (d, 0.5H, J = 4 Hz, Ar-H), 8.55 (d, 0.5H, J = 4 Hz, Ar-H),
8.58 (s, 0.25H, H-C=N), 8.63 (d, 1.5H, J = 4 Hz, Ar-H), 8.70 (d, 1.5H, J = 4 Hz, Ar-H), 8.73 (s,
0.75H, H-C=N), 9.03 (d, 0.5H, J = 4 Hz, Ar-H), 9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.18 (d, 0.5H, J
= 4 Hz, Ar-H), 9.25 (d, 1.5H, J = 4 Hz, Ar-H), 12.90 (s, 0.25H, CONH), 12.97 (s, 0.75H,
CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.60; 67.24 (2×NCH2), 122.23; 124.68; 124.98;
126.73; 130.23; 131.20; 138.64; 138.74; 140.02; 143.57; 147.08; 147.82; 150.23; 150.96; 151.10
(Ar-C), 159.98; 162.99 (C=N, C=O), 190.30; 190.67 (2×CH2C=O). HRMS (ESI) m/z =
714.3592 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-3-((2-(1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium bromide (7). It was obtained as yellow
1
crystals (EtOH) in 89 % yield; mp: 86-88 C. H NMR (400 MHz, DMSO-d6): δH = 3.91 (s, 6H
2×OCH3), 6.63 (d, 4H, J = 8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.09 (d, 4H, J = 8 Hz,
Ar-H), 8.32 (dd, 0.25H, J = 8 Hz, Ar-H), 8.41 (dd, 0.75H, J = 8 Hz, Ar-H), 8.57 (d, 0.25H, J = 4
Hz, Ar-H), 8.74 (d, 1.75H, J = 8 Hz, Ar-H), 8.81 (s, 0.2H, H-C=N), 8.86 (s, 0.8H, H-C=N), 9.07
(d, 1H, J = 4 Hz, Ar-H), 9.12 (d, 1H, J = 8 Hz, Ar-H), 9.20 (d, 0.25H, J = 4 Hz, Ar-H), 9.31 (d,
1.75H, J = 8 Hz, Ar-H), 9.32 (s, 0.25H, Ar-H), 9.56 (s, 0.75H, Ar-H), 13.11 (s, 0.25H, CONH),
13
13.28 (s, 0.75H, CONH). C NMR (100 MHz, DMSO-d6): δC = 56.34 (2×OCH3), 66.58; 66.70
(2×NCH2), 114.94; 126.68; 126.71; 126.78; 127.56; 128.46; 131.30; 134.18; 144.02; 144.54;

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145.33; 147.24; 147.83; 147.85 (Ar-C), 160.04, 164.78 (C=N, C=O), 189.10, 189.33
(2×CH2C=O). HRMS (ESI) m/z = 684.4014 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium bromide (8). It was obtained as colorless crystals
(EtOH) in 92 % yield; mp: 94-95 ºC. 1H NMR (400 MHz, DMSO-d6): δH = 6.66 (d, 4H, J = 8 Hz,
2×NCH2), 7.79 (d, 4H, J = 8 Hz, Ar-H), 8.13 (d, 4H, J = 8 Hz, Ar-H), 8.33 (dd, 0.25H, J = 4 Hz,
8 Hz, Ar-H), 8.41 (dd, 0.75H, J = 4 Hz, 8 Hz, Ar-H), 8.58 (d, 0.25H, J = 4 Hz, Ar-H), 8.75 (d,
1.75H, J = 8 Hz, Ar-H), 8.81 (s, 0.2H, H-C=N), 8.85 (s, 0.8H, H-C=N), 9.06 (d, 1H, J = 8 Hz,
Ar-H), 9.11 (d, 1H, J = 8 Hz, Ar-H), 9.20 (d, 0.25H, J = 8 Hz, Ar-H), 9.30 (d, 1.75H, J = 8 Hz,
Ar-H), 9.32 (s, 0.2H, Ar-H), 9.56 (s, 0.8H, Ar-H), 13.11 (s, 0.25H, CONH), 13.27 (s, 0.75H,
CONH). 13C NMR (100 MHz, DMSO-d6): δC = 66.89; 66.98 (2×NCH2), 126.76; 127.65; 128.32;
128.54; 129.81; 129.83; 130.71; 132.73; 132.80; 134.23; 140.03; 140.11; 144.35; 144.58; 145.22;
147.19; 147.31; 147.83; 148.01 (Ar-C), 160.04; 164.78 (C=N, C=O), 190.08; 190.29
(2×CH2C=O). HRMS (ESI) m/z = 693.2262 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium bromide (9). It was obtained as colorless crystals in
1
94 % yield; mp: 98-99 C. H NMR (400 MHz, DMSO-d6): δH = 6.67 (d, 4H, J = 4 Hz,
2×NCH2), 8.34 (d, 4H, J = 8 Hz, Ar-H), 8.42 (dd, 0.75H, J = 4 Hz, 8 Hz, Ar-H), 8.51 (d, 4H, J =
8 Hz, Ar-H), 8.59 (d, 0.25H, J = 4 Hz, Ar-H), 8.67 (s, 0.2H, H-C=N), 8.76 (d, 1.75H, J = 8 Hz,
Ar-H), 8.85 (s, 0.8H, H-C=N), 9.03 (d, 1H, J = 4 Hz, Ar-H), 9.08 (d, 1H, J = 8 Hz, Ar-H), 9.18
(d, 0.25H, J = 4 Hz, Ar-H), 9.27 (d, 1.75H, J = 8 Hz, Ar-H), 9.29 (s, 0.2H, Ar-H), 9.54 (s, 0.8H,
Ar-H), 13.14 (s, 0.25H, CONH), 13.38 (s, 0.75H, CONH). 13C NMR (100 MHz, DMSO-d6): δC
= 67.12; 67.25 (2×NCH2), 124.70; 126.67; 126.82; 128.50; 128.58; 13.24; 130.27; 134.29;
138.56; 138.74; 144.69; 145.11; 147.15; 147.85; 148.16; 151.15 (Ar-C), 160.05; 160.09 (C=N,
C=O), 190.28; 190.49 (2×CH2C=O). HRMS (ESI) m/z = 714.4627 [M+].
General procedure for the synthesis of task specific dipyridinium ILs 10-27. A mixture of
ionic liquid 4-9 (1 mmol) and metal salts: potassium hexafluorophosphate, sodium
tetrafluoroborate, and/or sodium trifluoroacetate (2.5 mmol) in acetonitrile (20 ml) was refluxed
for 16 h. After cooling, the product was isolated by extraction or filtration to give the dicationic
liquids 10-27.

ACCEPTED MANUSCRIPT
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-4-((2-(1-(2-(4-methoxyphenyl-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium hexafluorophosphate (10). It was obtained
as pale yellow crystals (EtOH) in 87 % yield; mp: 100-102 C. 1H NMR (400 MHz, DMSO-d6):
δH = 3.91 (s, 6H 2×OCH3), 6.57 (d, 4H, J = 8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.07
(d, 4H, J = 8 Hz, Ar-H), 8.29 (d, 0.5H, J = 4 Hz, Ar-H), 8.40 (d, 0.5H, J = 4 Hz, Ar-H), 8.52 (s,
0.25H, H-C=N), 8.57 (d, 1.5H, J = 8 Hz, Ar-H), 8.71 (d, 1.5H, J = 4 Hz, Ar-H), 8.78 (s, 0.75H,
H-C=N), 8.97 (d, 0.5H, J = 4 Hz, Ar-H), 9.07 (d, 1.5H, J = 8 Hz, Ar-H), 9.19 (d, 0.5H, J = 4 Hz,
13
Ar-H), 9.26 (d, 1.5H, J = 4 Hz, Ar-H), 12.95 (s, 0.25H, CONH), 12.98 (s, 0.75H, CONH). C
NMR (100 MHz, DMSO-d6): δC = 56.31 (2×OCH3), 66.11; 66.56 (2×NCH2), 141.91; 125.11;
126.74; 127.56; 131.24; 131.30; 145.08; 147.23; 147.76; 147.90; 149.83 (Ar-C), 160.54; 164.84
(C=N, C=O), 189.08; 189.45 (2×CH2C=O). 31P NMR (162 MHz, DMSO-d6): δP = (-157.39) to (-
19
131.04) (m, 2P, 2×PF6). F NMR (377 MHz, DMSO-d6): δF = -69.13 (d, 12F, 2×PF6). HRMS
(ESI) m/z = 814.5801 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-4-((2-(1-(2-(4-methoxyphenyl-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium tetrafluoroborate (11). It was obtained as
1
white needless (EtOH) in 86 % yield; mp: 92-93 C. H NMR (400 MHz, DMSO-d6): δH = 3.92
(s, 6H 2×OCH3), 6.54 (d, 4H, J = 8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.09 (d, 4H, J =
8 Hz, Ar-H), 8.28 (d, 0.5H, J = 4 Hz, Ar-H), 8.38 (d, 0.5H, J = 4 Hz, Ar-H), 8.52 (s, 0.25H, H-
C=N), 8.58 (d, 1.5H, J = 4 Hz, Ar-H), 8.69 (d, 1.5H, J = 4 Hz, Ar-H), 8.73 (s, 0.75H, H-C=N),
8.94 (d, 0.5H, J = 4 Hz, Ar-H), 9.05 (d, 1.5H, J = 8 Hz, Ar-H), 9.17 (d, 0.5H, J = 4 Hz, Ar-H),
13
9.25 (d, 1.5H, J = 4 Hz, Ar-H), 13.37 (s, 0.25H, CONH), 13.42 (s, 0.75H, CONH). C NMR
(150 MHz, DMSO-d6): δC = 56.31 (2×OCH3), 66.12; 66.76 (2×NCH2), 141.92; 125.13; 126.75;
127.56; 131.25; 131.30; 145.04; 147.23; 147.76; 147.89; 149.83 (Ar-C), 160.54; 164.79 (C=N,
C=O), 189.08; 189.46 (2×CH2C=O). 11B NMR (128 MHz, DMSO-d6): δB = (-1.31) to (-1.29) (m,
2B, 2×BF4). 19F NMR (377 MHz, DMSO-d6): δF = -148.20, -148.15 (2d, 8F, 2×BF4). HRMS
(ESI) m/z = 698.1808 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-4-((2-(1-(2-(4-methoxyphenyl-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium trifluoroacetate (12). It was obtained as
syrup in 90 % yield. 1H NMR (400 MHz, DMSO-d6): δH = 3.92 (s, 6H 2×OCH3), 6.58 (d, 4H, J =
8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.09 (d, 4H, J = 8 Hz, Ar-H), 8.30 (d, 0.5H, J = 8
Hz, Ar-H), 8.40 (d, 0.5H, J = 4 Hz, Ar-H), 8.53 (s, 0.25H, H-C=N), 8.58 (d, 1.5H, J = 4 Hz, Ar-

ACCEPTED MANUSCRIPT
H), 8.72 (d, 1.5H, J = 8 Hz, Ar-H), 8.79 (s, 0.75H, H-C=N), 8.98 (d, 0.5H, J = 4 Hz, Ar-H), 9.07
(d, 1.5H, J = 4 Hz, Ar-H), 9.20 (d, 0.5H, J = 4 Hz, Ar-H), 9.28 (d, 1.5H, J = 4 Hz, Ar-H), 13.37
(s, 0.25H, CONH), 13.43 (s, 0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 56.32
(2×OCH3), 66.11; 66.75 (2×NCH2), 141.91; 125.13; 126.75; 127.56; 131.25; 131.30; 145.03;
147.23; 147.75; 147.89; 149.83 (Ar-C), 160.54; 164.85 (C=N, C=O), 189.09; 189.45
19
(2×CH2C=O). F NMR (377 MHz, DMSO-d6): δF = -73.78 (s, 6F, 2×CF3). HRMS (ESI) m/z =
750.5615 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-chloro phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)
hydrazono) methyl)pyridin-1-ium hexafluorophosphate) (13). It was obtained as pale yellow
crystals (EtOH) in 91 % yield; mp: 78-80 C. 1H NMR (400 MHz, DMSO-d6): δH = 6.62 (d, 4H,
J = 8 Hz, 2×NCH2), 7.78 (d, 4H, J = 4 Hz, Ar-H), 8.11 (d, 4H, J = 8 Hz, Ar-H), 8.31 (d, 0.5H, J
= 4 Hz, Ar-H), 8.41 (d, 0.5H, J = 4 Hz, Ar-H), 8.53 (s, 0.25H, H-C=N), 8.59 (d, 1.5H, J = 4 Hz,
Ar-H), 8.73 (d, 1.5H, J = 4 Hz, Ar-H), 8.85 (s, 0.75H, H-C=N), 9.01 (d, 0.5H, J = 4 Hz, Ar-H),
9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.23 (d, 0.5H, J = 4 Hz, Ar-H), 9.27 (d, 1.5H, J = 4 Hz, Ar-H),
13.39 (s, 0.25H, CONH), 13.48 (s, 0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.41;
67.02 (2×NCH2), 124.89; 125.20; 126.85; 127.60; 129.80; 129.84; 130.68; 130.72; 132.72;
132.81; 140.03; 140.11; 145.03; 146.92; 147.22; 147.36; 147.88; 149.91; 149.97 (Ar-C), 160.51;
164.79 (C=N, C=O), 189.08; 189.44 (2×CH2C=O). 31P NMR (162 MHz, DMSO-d6): δP = (-
157.39) to (-131.04) (m, 2P, 2×PF6). 19F NMR (377 MHz, DMSO-d6): δF = -69.15 (d, 12F,
2×PF6). HRMS (ESI) m/z = 823.3839 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium tetrafluoroborate (14). It was obtained as syrup in
90 % yield. 1H NMR (400 MHz, DMSO-d6): δH = 6.63 (d, 4H, J = 8 Hz, 2×NCH2), 7.78 (d, 4H, J
= 4 Hz, Ar-H), 8.12 (d, 4H, J = 8 Hz, Ar-H), 8.31 (d, 0.5H, J = 4 Hz, Ar-H), 8.41 (d, 0.5H, J = 4
Hz, Ar-H), 8.54 (s, 0.25H, H-C=N), 8.59 (d, 1.5H, J = 4 Hz, Ar-H), 8.73 (d, 1.5H, J = 4 Hz, Ar-
H), 8.79 (s, 0.75H, H-C=N), 8.98 (d, 0.5H, J = 8 Hz, Ar-H), 9.07 (d, 1.5H, J = 4 Hz, Ar-H), 9.20
(d, 0.5H, J = 4 Hz, Ar-H), 9.29 (d, 1.5H, J = 8 Hz, Ar-H), 13.38 (s, 0.25H, CONH), 13.44 (s,
0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.39, 67.01 (2×NCH2), 124.87; 125.20;
126.82; 127.60; 129.80; 130.67; 130.71; 132.71; 132.80; 140.03; 140.12; 145.04; 147.21; 147.34;
11
147.89; 149.93; 149.98 (Ar-C), 160.53; 164.79 (C=N, C=O), 190.06; 190.42 (2×CH2C=O). B

ACCEPTED MANUSCRIPT
NMR (128 MHz, DMSO-d6): δB = (-1.30) to (-1.28) (m, 2B, 2×BF4). 19F NMR (377 MHz,
DMSO-d6): δF = -148.21 and -148.16 (2d, 8F, 2×BF4). HRMS (ESI) m/z = 707.0516 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium trifluoroacetate (15). It was obtained as yellow
crystals (EtOH) in 89 % yield; mp: 78-79 C. 1H NMR (400 MHz, DMSO-d6): δH = 6.67 (d, 4H,
J = 8 Hz, 2×NCH2), 7.78 (d, 4H, J = 4 Hz, Ar-H), 8.13 (d, 4H, J = 8 Hz, Ar-H), 8.33 (d, 0.5H, J
= 4 Hz, Ar-H), 8.43 (d, 0.5H, J = 4 Hz, Ar-H), 8.54 (s, 0.25H, H-C=N), 8.59 (d, 1.5H, J = 4 Hz,
Ar-H), 8.75 (d, 1.5H, J = 4 Hz, Ar-H), 8.84 (s, 0.75H, H-C=N), 9.01 (d, 0.5H, J = 4 Hz, Ar-H),
9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.23 (d, 0.5H, J = 4 Hz, Ar-H), 9.31 (d, 1.5H, J = 4 Hz, Ar-H),
13.39 (s, 0.25H, CONH), 13.49 (s, 0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.41,
67.02 (2×NCH2), 124.89; 125.19; 126.84; 127.60; 129.80; 129.84; 130.69; 130.72; 132.72;
132.81; 140.03; 140.11; 145.03; 146.91; 147.22; 147.35; 147.88; 149.91; 149.97 (Ar-C), 160.51;
164.79 (C=N, C=O), 190.08; 190.44 (2×CH2C=O). 19F NMR (377 MHz, DMSO-d6): δF = -74.05
(s, 6F, 2×CF3). HRMS (ESI) m/z = 759.4248 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium hexafluorophosphate (16). It was obtained as syrup
1
in 92 % yield. H NMR (400 MHz, DMSO-d6): δH = 6.67 (d, 4H, J = 8 Hz, 2×NCH2), 7.79 (d,
4H, J = 4 Hz, Ar-H), 8.33 (d, 4H, J = 8 Hz, Ar-H), 8.49 (d, 0.5H, J = 4 Hz, Ar-H), 8.55 (d, 0.5H,
J = 4 Hz, Ar-H), 8.58 (s, 0.25H, H-C=N), 8.63 (d, 1.5H, J = 4 Hz, Ar-H), 8.70 (d, 1.5H, J = 4
Hz, Ar-H), 8.73 (s, 0.75H, H-C=N), 9.03 (d, 0.5H, J = 4 Hz, Ar-H), 9.09 (d, 1.5H, J = 4 Hz, Ar-
H), 9.18 (d, 0.5H, J = 4 Hz, Ar-H), 9.25 (d, 1.5H, J = 4 Hz, Ar-H), 12.90 (s, 0.25H, CONH),
12.97 (s, 0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.59; 67.23 (2×NCH2), 122.21;
124.69; 124.73; 124.98; 126.72; 130.22; 130.27; 138.64; 138.74; 147.08; 147.82; 150.66; 151.00;
151.11 (Ar-C), 159.96; 162.99 (C=N, C=O), 190.30; 190.67 (2×CH2C=O). 31P NMR (162 MHz,
19
DMSO-d6): δP = (-148.61) to (-139.82) (m, 2P, 2×PF6). F NMR (377 MHz, DMSO-d6): δF = -
69.15 (d, 12F, 2×PF6). HRMS (ESI) m/z = 844.4078 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium tetrafluoroborate (17). It was obtained as syrup in
86 % yield. 1H NMR (400 MHz, DMSO-d6): δH = 6.68 (d, 4H, J = 8 Hz, 2×NCH2), 7.80 (d, 4H, J
= 4 Hz, Ar-H), 8.33 (d, 4H, J = 8 Hz, Ar-H), 8.49 (d, 0.5H, J = 4 Hz, Ar-H), 8.55 (d, 0.5H, J = 4
Hz, Ar-H), 8.60 (s, 0.25H, H-C=N), 8.63 (d, 1.5H, J = 4 Hz, Ar-H), 8.70 (d, 1.5H, J = 4 Hz, Ar-

ACCEPTED MANUSCRIPT
H), 8.74 (s, 0.75H, H-C=N), 9.03 (d, 0.5H, J = 4 Hz, Ar-H), 9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.20
(d, 0.5H, J = 4 Hz, Ar-H), 9.27 (d, 1.5H, J = 4 Hz, Ar-H), 12.91 (s, 0.25H, CONH), 12.98 (s,
0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.60, 67.23 (2×NCH2), 122.23; 124.67;
124.98; 126.72; 127.47; 130.23; 130.28; 138.64; 138.74; 140.00; 143.58; 147.07; 147.81; 150.39;
150.96; 151.09 (Ar-C), 159.97; 163.00 (C=N, C=O), 190.30; 190.66 (2×CH2C=O). 11B NMR
(128 MHz, DMSO-d6): δB = (-1.31) to (-1.29) (m, 2B, 2×BF4). 19F NMR (377 MHz, DMSO-d6):
δF = -148.21 and -148.16 (2d, 8F, 2×BF4). HRMS (ESI) m/z = 728.1631 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-4-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium trifluoroacetate (18). It was obtained as yellow
crystals (ETOH) in 85 % yield; mp: 91-93 C. 1H NMR (400 MHz, DMSO-d6): δH = 6.64 (d, 4H,
J = 8 Hz, 2×NCH2), 7.79 (d, 4H, J = 4 Hz, Ar-H), 8.32 (d, 4H, J = 8 Hz, Ar-H), 8.49 (d, 0.5H, J
= 4 Hz, Ar-H), 8.56 (d, 0.5H, J = 4 Hz, Ar-H), 8.58 (s, 0.25H, H-C=N), 8.63 (d, 1.5H, J = 4 Hz,
Ar-H), 8.69 (d, 1.5H, J = 4 Hz, Ar-H), 8.86 (s, 0.75H, H-C=N), 9.01 (d, 0.5H, J = 4 Hz, Ar-H),
9.09 (d, 1.5H, J = 4 Hz, Ar-H), 9.17 (d, 0.5H, J = 4 Hz, Ar-H), 9.24 (d, 1.5H, J = 4 Hz, Ar-H),
12.92 (s, 0.25H, CONH), 12.95 (s, 0.75H, CONH). 13C NMR (150 MHz, DMSO-d6): δC = 66.60,
67.24 (2×NCH2), 122.23; 124.68; 124.98; 126.73; 130.23; 131.20; 138.64; 138.74; 140.02;
143.57; 147.08; 147.82; 150.23; 150.96; 151.10 (Ar-C), 159.98; 162.99 (C=N, C=O), 190.30;
190.67 (2×CH2C=O). 19F NMR (377 MHz, DMSO-d6): δF = -73.61 (s, 6F, 2×CF3). HRMS (ESI)
m/z = 780.5815 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-3-((2-(1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono)methyl)pyridin-1-ium hexafluorophosphate (19). It was obtained
as yellow crystals (EtOH) in 90 % yield; mp: 102-104 C. 1H NMR (400 MHz, DMSO-d6): δH =
3.92 (s, 6H 2×OCH3), 6.67 (d, 4H, J = 8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.09 (d,
4H, J = 8 Hz, Ar-H), 8.30 (dd, 0.25H, J = 8 Hz, Ar-H), 8.39 (dd, 0.75H, J = 4 Hz, Ar-H), 8.52 (s,
0.2H, H-C=N), 8.57 (d, 0.25H, J = 4 Hz, Ar-H), 8.70 (d, 1.75H, J = 4 Hz, Ar-H), 8.79 (s, 0.8H,
H-C=N), 9.05 (d, 1H, J = 4 Hz, Ar-H), 9.06 (d, 1H, J = 4 Hz, Ar-H), 9.16 (d, 0.25H, J = 4 Hz,
Ar-H), 9.27 (d, 1.75H, J = 8 Hz, Ar-H), 9.32 (s, 0.2H, Ar-H), 9.53 (s, 0.8H, Ar-H), 13.11 (s,
0.25H, CONH), 13.23 (s, 0.75H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 56.33
(2×OCH3) 66.68, 66.71 (2×NCH2), 114.92; 126.66; 126.68; 126.76; 128.46; 131.25; 131.28;
134.20; 144.16; 144.59; 145.24; 147.23; 147.27; 147.85; 147.93 (Ar-C), 160.08; 164.79 (C=N,
C=O), 189.10; 189.32 (2×CH2C=O). 31P NMR (162 MHz, DMSO-d6): δP = (-157.39) to (-

ACCEPTED MANUSCRIPT
19
131.04) (m, 2P, 2×PF6). F NMR (377 MHz, DMSO-d6): δF = -69.13 (d, 12F, 2×PF6). HRMS
(ESI) m/z = 814.5142 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-3-((2-(1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono)methyl)pyridin-1-ium tetrafluoroborate (20). It was obtained as
syrup in 90 % yield. 1H NMR (400 MHz, DMSO-d6): δH = 3.91 (s, 6H 2×OCH3), 6.61 (d, 4H, J =
8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.08 (d, 4H, J = 8 Hz, Ar-H), 8.31 (dd, 0.25H, J =
8 Hz, Ar-H), 8.40 (dd, 0.75H, J = 8 Hz, Ar-H), 8.57 (d, 0.25H, J = 4 Hz, Ar-H), 8.72 (d, 1.75H, J
= 4 Hz, Ar-H), 8.79 (s, 0.2H, H-C=N), 8.83 (s, 0.8H, H-C=N), 9.07 (d, 1H, J = 8 Hz, Ar-H), 9.10
(d, 1H, J = 8 Hz, Ar-H), 9.18 (d, 0.25H, J = 4 Hz, Ar-H), 9.29 (d, 1.75H, J = 8 Hz, Ar-H), 9.30
13
(s, 0.2H, Ar-H), 9.55 (s, 0.8H, Ar-H), 13.11 (s, 0.25H, CONH), 13.26 (s, 0.75H, CONH). C
NMR (100 MHz, DMSO-d6): δC = 56.33 (2×OCH3), 66.58; 66.70 (2×NCH2), 114.92; 126.67;
126.77; 127.57; 128.46; 131.26; 134.17; 144.05; 144.55; 145.31; 147.23; 147.28; 147.83 (Ar-C),
160.04; 164.78 (C=N, C=O), 189.10; 189.33 (2×CH2C=O). 11B NMR (128 MHz, DMSO-d6): δB
19
= (-1.31) to (-1.29) (m,2B, 2×BF4). F NMR (377 MHz, DMSO-d6): δF = -148.17, -148.12 (2d,
8F, 2×BF4). HRMS (ESI) m/z = 898.1998 [M+].
1-(2-(4-Methoxyphenyl)-2-oxoethyl)-3-((2-(1-(2-(4-methoxyphenyl)-2-oxoethyl)pyridin-1-
ium-4-carbonyl) hydrazono) methyl)pyridin-1-ium trifluoroacetate (21). It was obtained as
1
colorless needless (EtOH) in 88 % yield; mp: 82-84 C. H NMR (400 MHz, DMSO-d6): δH =
3.92 (s, 6H 2×OCH3), 6.55 (d, 4H, J = 8 Hz, 2×NCH2), 7.22 (d, 4H, J = 8 Hz, Ar-H), 8.08 (d,
4H, J = 8 Hz, Ar-H), 8.30 (dd, 0.25H, J = 8 Hz, Ar-H), 8.39 (dd, 0.75H, J = 8 Hz, Ar-H), 8.51 (s,
0.2H, H-C=N), 8.56 (d, 0.25H, J = 4 Hz, Ar-H), 8.69 (d, 1.75H, J = 4 Hz, Ar-H), 8.77 (s, 0.8H,
H-C=N), 9.07 (d, 1H, J = 4 Hz, Ar-H), 9.12 (d, 1H, J = 8 Hz, Ar-H), 9.15 (d, 0.25H, J = 4 Hz,
Ar-H), 9.25 (d, 1.75H, J = 4 Hz, Ar-H), 9.32 (s, 0.2H, Ar-H), 9.52 (s, 0.8H, Ar-H), 13.11 (s,
0.25H, CONH), 13.28 (s, 0.75H, CONH). 13C NMR (100 MHz, DMSO-d6): δC = 56.34
(2×OCH3), 66.58; 66.70 (2×NCH2), 114.94; 126.68; 126.71; 126.78; 127.56; 128.46; 131.30;
134.18; 144.02; 144.54; 145.33; 147.24; 147.83; 147.85 (Ar-C), 160.04; 164.78 (C=N, C=O),
189.10; 189.33 (2×CH2C=O). 19F NMR (377 MHz, DMSO-d6): δF = -73.49 (s, 6F, 2×CF3).
HRMS (ESI) m/z = 750.5984 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium hexafluorophosphate (22). It was obtained as syrup
1
in 89 % yield. H NMR (400 MHz, DMSO-d6): δH = 6.57 (d, 4H, J = 8 Hz, 2×NCH2), 7.79 (d,

ACCEPTED MANUSCRIPT
4H, J = 8 Hz, Ar-H), 8.12 (d, 4H, J = 8 Hz, Ar-H), 8.30 (dd, 0.25H, J = 4 Hz, 8 Hz, Ar-H), 8.40
(dd, 0.75H, J = 8 Hz, Ar-H), 8.57 (d, 0.25H, J = 4 Hz, Ar-H), 8.69 (d, 1.75H, J = 8 Hz, Ar-H),
8.72 (s, 0.8H, H-C=N), 8.79 (s, 0.2H, H-C=N), 9.03 (d, 1H, J = 8 Hz, Ar-H), 9.07 (d, 1H, J = 8
Hz, Ar-H), 9.13 (d, 0.25H, J = 4 Hz, Ar-H), 9.23 (d, 1.75H, J = 8 Hz, Ar-H), 9.32 (s, 0.2H, Ar-
H), 9.51 (s, 0.8H, Ar-H), 13.10 (s, 0.25H, CONH), 13.15 (s, 0.75H, CONH). 13C NMR (100
MHz, DMSO-d6): δC = 66.96; 66.99 (2×NCH2), 126.73; 127.73; 128.32; 128.51; 129.82; 129.85;
130.71; 132.70; 132.78; 134.26; 140.08; 140.15; 144.35; 144.65; 145.08; 147.15; 147.31; 147.87;
148.15 (Ar-C), 160.11; 164.78 (C=N, C=O), 190.05; 190.27 (2×CH2C=O). 31P NMR (162 MHz,
19
DMSO-d6): δP = (-157.39) to (-131.04) (m, 2P, 2×PF6). F NMR (377 MHz, DMSO-d6): δF = -
69.13 (d, 12F, 2×PF6). HRMS (ESI) m/z = 823.5163 [M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-chloro phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)
hydrazono) methyl)pyridin-1-ium tetrafluoroborate (23). It was obtained as white crystals
1
(EtOH) in 87 % yield; mp: 78-80 °C. H NMR (400 MHz, DMSO-d6): δH = 6.64 (d, 4H, J = 8
Hz, 2×NCH2), 7.79 (d, 4H, J = 8 Hz, Ar-H), 8.12 (d, 4H, J = 8 Hz, Ar-H), 8.33 (dd, 0.25H, J = 8
Hz, Ar-H), 8.41 (dd, 0.75H, J = 4 Hz, 8 Hz, Ar-H), 8.58 (d, 0.25H, J = 4 Hz, Ar-H), 8.74 (d,
1.75H, J = 8 Hz, Ar-H), 8.81 (s, 0.2H, H-C=N), 8.83 (s, 0.8H, H-C=N), 9.06 (d, 1H, J = 4 Hz,
Ar-H), 9.08 (d, 1H, J = 4 Hz, Ar-H), 9.18 (d, 0.25H, J = 4 Hz, Ar-H), 9.28 (d, 1.75H, J = 8 Hz,
Ar-H), 9.31 (s, 0.2H, Ar-H), 9.55 (s, 0.8H, Ar-H), 13.11 (s, 0.25H, CONH), 13.25 (s, 0.75H,
CONH). 13C NMR (100 MHz, DMSO-d6): δC = 66.87; 66.98 (2×NCH2), 126.76; 127.66; 128.54;
129.82; 129.84; 130.68; 130.70; 132.72; 132.79; 134.22; 140.04; 140.11; 144.39; 144.59; 145.20;
147.19; 147.29; 147.84; 148.02 (Ar-C), 160.06; 164.09 (C=N, C=O), 190.08; 190.30
(2×CH2C=O). 11B NMR (128 MHz, DMSO-d6): δB = (-1.30) - (-1.28) (m, 2B, 2×BF4). 19F NMR
(377 MHz, DMSO-d6): δF = -148.17 and -148.12 (2d, 8F, 2×BF4). HRMS (ESI) m/z = 707.1026
[M+].
1-(2-(4-Chlorophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-chlorophenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl) hydrazono) methyl)pyridin-1-ium trifluoroacetate (24). It was obtained as white
crystals (EtOH) in 90 % yield; mp: 93-94 C. 1H NMR (400 MHz, DMSO-d6): δH = 6.64 (d, 4H,
J = 8 Hz, 2×NCH2), 7.79 (d, 4H, J = 8 Hz, Ar-H), 8.12 (d, 4H, J = 8 Hz, Ar-H), 8.33 (dd, 0.25H,
J = 8 Hz, Ar-H), 8.41 (dd, 0.75H, J = 4 Hz, 8 Hz, Ar-H), 8.58 (d, 0.25H, J = 4 Hz, Ar-H), 8.74
(d, 1.75H, J = 8 Hz, Ar-H), 8.81 (s, 0.2H, H-C=N), 8.83 (s, 0.8H, H-C=N), 9.06 (d, 1H, J = 4 Hz,

ACCEPTED MANUSCRIPT
Ar-H), 9.08 (d, 1H, J = 4 Hz, Ar-H), 9.18 (d, 0.25H, J = 4 Hz, Ar-H), 9.28 (d, 1.75H, J = 8 Hz,
Ar-H), 9.31 (s, 0.2H, Ar-H), 9.55 (s, 0.8H, Ar-H), 13.11 (s, 0.25H, CONH), 13.25 (s, 0.75H,
CONH). 13C NMR (100 MHz, DMSO-d6): δC = 66.87; 66.98 (2×NCH2), 126.76; 127.66; 128.54;
129.82; 129.84; 130.68; 130.70; 132.72; 132.79; 134.22; 140.04; 140.11; 144.39; 144.59; 145.20;
147.19; 147.29; 147.84; 148.02 (Ar-C), 160.06; 164.09 (C=N, C=O), 190.08; 190.30
19
(2×CH2C=O). F NMR (377 MHz, DMSO-d6): δF = -73.86 (s, 6F, 2×CF3). HRMS (ESI) m/z =
759.4167 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium hexafluorophosphate (25). It was obtained as syrup
1
in 90 % yield. H NMR (400 MHz, DMSO-d6): δH = 6.65 (d, 4H, J = 4 Hz, 2×NCH2), 8.34 (d,
4H, J = 8 Hz, Ar-H), 8.42 (dd, 0.75H, J = 8 Hz, Ar-H), 8.51 (d, 4H, J = 8 Hz, Ar-H), 8.66 (s,
0.2H, H-C=N), 8.71 (d, 0.25H, J = 8 Hz, Ar-H), 8.76 (d, 1.75H, J = 4 Hz, Ar-H), 8.84 (s, 0.8H,
H-C=N), 9.06 (d, 1H, J = 4 Hz, Ar-H), 9.07 (d, 1H, J = 8 Hz, Ar-H), 9.16 (d, 0.25H, J = 4 Hz,
Ar-H), 9.26 (d, 1.75H, J = 8 Hz, Ar-H), 9.29 (s, 0.2H, Ar-H), 9.54 (s, 0.8H, Ar-H), 13.14 (s,
13
0.25H, CONH), 13.38 (s, 0.75H, CONH). C NMR (100 MHz, DMSO-d6): δC = 67.24; 67.28
(2×NCH2), 124.71; 126.67; 126.83; 128.57; 128.58, 13.26; 130.27; 134.29; 138.63; 138.73;
144.74; 145.08; 147.85; 148.15; 148.16, 151.15 (Ar-C), 160.04; 160.09 (C=N, C=O), 190.28;
190.50 (2×CH2C=O). 31P NMR (162 MHz, DMSO-d6): δP = (-157.39) to (-131.04) (m, 2P,
2×PF6). 19F NMR (377 MHz, DMSO-d6): δF = -69.14 (d, 12F, 2×PF6). HRMS (ESI) m/z =
844.4387 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium tetrafluoroborate (26). It was obtained as pale
yellow crystals (EtOH) in 88 % yield; mp: 99-100 C. 1H NMR (400 MHz, DMSO-d6): δH = 6.58
(d, 4H, J = 4 Hz, 2×NCH2), 7.79 (d, 4H, J = 8 Hz, Ar-H), 8.12 (d, 4H, J = 8 Hz, Ar-H), 8.30 (dd,
0.25H, J = 4 Hz, 8 Hz, Ar-H), 8.40 (dd, 0.75H, J = 8 Hz, Ar-H), 8.57 (d, 0.25H, J = 4 Hz, Ar-H),
8.69 (d, 1.75H, J = 4 Hz, Ar-H), 8.73 (s, 0.8H, H-C=N), 8.77 (s, 0.2H, H-C=N), 9.05 (d, 1H, J =
4 Hz, Ar-H), 9.07 (d, 1H, J = 8 Hz, Ar-H), 9.14 (d, 0.25H, J = 4 Hz, Ar-H), 9.24 (d, 1.75H, J = 8
Hz, Ar-H), 9.26 (s, 0.2H, Ar-H), 9.52 (s, 0.8H, Ar-H), 13.09 (s, 0.25H, CONH), 13.15 (s, 0.75H,
CONH). 13C NMR (100 MHz, DMSO-d6): δC = 67.24; 67.28 (2×NCH2), 124.71; 126.67; 126.83;
128.57; 128.58; 13.26; 130.27; 134.29; 138.63; 138.73; 144.74; 145.08; 147.85; 148.15; 148.16;
151.15 (Ar-C), 160.04; 160.09 (C=N, C=O), 190.28; 190.50 (2×CH2C=O). 11B NMR (128 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): δB = (-1.31) to (-1.29) (m,2B, 2×BF4). 19F NMR (377 MHz, DMSO-d6): δF = -148.17,
-148.12 (2d, 8F, 2×BF4). HRMS (ESI) m/z = 728.1147 [M+].
1-(2-(4-Nitrophenyl)-2-oxoethyl)-3-((2-(1-(2-(4-nitro
phenyl)-2-oxoethyl)pyridin-1-ium-4-
carbonyl)hydrazono) methyl)pyridin-1-ium trifluoroacetate (27). It was obtained as syrup in 88
% yield. 1H NMR (400 MHz, DMSO-d6): δH = 6.65 (d, 4H, J = 4 Hz, 2×NCH2), 8.34 (d, 4H, J =
4 Hz, J = 8 Hz, Ar-H), 8.43 (dd, 0.75H, J = 4 Hz, J = 8 Hz, Ar-H), 8.51 (d, 4H, J = 8 Hz, Ar-H),
8.61 (s, 0.2H, H-C=N), 8.69 (d, 0.25H, J = 4 Hz, Ar-H), 8.73 (d, 1.75H, J = 4 Hz, Ar-H), 8.79 (s,
0.8H, H-C=N), 9.07 (d, 1H, J = 4 Hz, Ar-H), 9.09 (d, 1H, J = 8 Hz, Ar-H), 9.16 (d, 0.25H, J = 4
Hz, Ar-H), 9.26 (d, 1.75H, J = 8 Hz, Ar-H), 9.27 (s, 0.2H, Ar-H), 9.54 (s, 0.8H, Ar-H), 13.12 (s,
13
0.25H, CONH), 13.24 (s, 0.75H, CONH). C NMR (100 MHz, DMSO-d6): δC = 67.24; 67.28
(2×NCH2), 124.71; 126.67; 126.83; 128.57; 128.58; 130.26; 130.27; 134.29; 138.63; 138.73;
144.74; 145.08; 147.85; 148.15; 148.16; 151.15 (Ar-C), 160.04; 160.09 (C=N, C=O), 190.28;
190.50 (2×CH2C=O). 19F NMR (377 MHz, DMSO-d6): δF = -73.87 (s, 6F, 2×CF3). HRMS (ESI)
m/z 780.5824 [M+].
3.2. Biological screening
Cell Lines and Culture Conditions. The human breast adenocarcinoma cell line MCF-7, the
human ductal breast epithelial tumor cell line T47D, the human mammary gland/breast tumor cell
line MDA231, the human Colorectal Adenocarcinoma cell line Caco-2, were purchased from
ATCC. Cell lines were cultured in high glucose Dulbecco’s modified eagle medium (DMEM)
(Invitrogen, USA) containing 10 % heat inactivated fetal bovine serum (HI-FBS) (Invitrogen), 2
mmol L–1 of L-glutamine, 50 U mL–1 of penicillin and 50 μg mL–1 of streptomycin. Cell lines
were maintained at 37 °C in a 5 % CO2 atmosphere of 95% humidity.
Cell proliferation by MTT assay. Viable cell number was verified using the 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Purple
formazan product is yielded from the metabolically active cells via reduction of the yellow
tetrazolium
dye
[MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt] and released into the intracellular compartment.
Afterward, the quantity of the formed formazan salt was determined by measuring it absorbance
at wavelength 490. Cells were seeded at density of 8x103 to ensure exponential growth
throughout the experimental period and a linear relationship between absorbance and cell
number.

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Anti-proliferative assay. The examined compounds were first dissolved in a volume of
DMSO to provide a final concentration of 1 M stock solution. This stock solution was further
diluted in culture media to yield the various concentrations used in this study. Final concentration
of DMSO was maintained constant in wells and never exceeded 0.1%. Cells were treated with the
various concentrations, in three triplicates for each concentration, and incubated at 37 °C in a 5 %
CO2 incubator, for 48 h. At the end of the treatment time, MTT assay was carried out as
previously described [39].
In-silico molecular docking studies. In order to find out the more effective inhibitors out of
the synthesized compounds by the structure based drug designing for PI3K, in-silico molecular
docking approach was employed. The 3D atomic coordinates of the ligands were created using
ACD/Labs-Chemsketch 12.0 software. Their geometries were cleaned and generated as the
corresponding pdb ligand files. 2RD0 (PDB ID) was analyzed as a most suitable pure three-
dimensional crystal structure of p110α subunit of PI3K retrieved from the protein data bank
(PDB) (Source: www.rcsb.org/pdb/). The Graphical User Interface program namely ‘‘Auto-Dock
Tools (ADT, 1.5.6)’’ was used in preparing, running and analyzing the docking simulations.
Energy of the molecules was minimized using Dundee PRODRG2 server [56]. The docking
analyses of the energy minimized compounds were carried out by means of the Autodock v4. 0
program [57]. The ligand and the receptor protein were treated using the united-atom
approximation, Kollman united-atom charges were assigned and only polar hydrogens were
added to the receptor protein. Gasteiger charge was assigned to the ligands, non-polar hydrogens
were merged and the internal degrees of freedom and torsions were set. We have made the grids
and adjusted the number of points in X, Y, Z-axis in order to cover the entire active site of
protein. Electrostatic and affinity maps for all the atom types present were computed with a grid
spacing of 0.375 Å. The Lamarckian Genetic Algorithm (LGA) was chosen to search for the best
conformers in populations of 150 individuals with 0.02 mutation rate in 10 docking trials with
cross-over rate of 0.8. Other docking parameters were set to the software’s default values. The
results were evaluated by sorting the different compounds with respect to the binding energy
predicted. A cluster analysis which is based on root mean square deviation values, that takes the
starting geometry as the reference, was performed and the conformation of the more populated
cluster possessing the lowest energy was considered to be the most trustable solution. The lowest
binding energy and the inhibitory constant (ki) values of the analyzed ligands were extracted

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from this statistical mechanical analysis. The ligand protein interaction was visualized using
UCSF Chimera 1.11.2.
4. Conclusion
This study explores the designing and the synthesis of dicationic pyridinium (DILs)
incorporating the hydrazone linkage starting from bis-pyridine hydrazones through their
quaternization reaction with several phenacyl bromide. The resulted ionic liquids undergo
metathesis process via the introduction of some fluorinated anions yielding the task-specific
dicationic pyridinium ionic liquids. The structures of newly synthesized DILs were evidenced by
conventional spectroscopic experiments. The obtained compounds were screened for their in-
vitro anticancer properties. Compound 22 is the most active against all tested cell lines. These
results were further supported by in-silico studies revealing good docking scores and lesser
binding energies towards PI3Kinase as target protein. The targeted ligands exhibited bonds with
one or more amino acids in the active pocket site of the receptor with good binding energies
toward the target protein. Compound 22 manifested highest Minimum binding energy score i.e -
11.88 kJ/mol and lowest concentration of 16.40 nM as calculated inhibition constant. Therefore,
the in-silico analysis propounds the importance of evaluating the prediction accuracy of scoring
functions employed in AutoDock and unveiled excellent correlation between binding free energy,
calculated inhibition constant (Ki) and IC50 values. Hence, this investigation validated the
importance of drug design studies and viability of computational tools in correlating experimental
values with the computational ones.
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Scheme 1. Synthetic strategies for the synthesis of di-cationic pyridinium hydrazone ionic liquids
4-27.
Figure 1. 1H NMR spectrum of compound 7.
Figure 2. 13C NMR spectrum of compound 7.
Figure 3. 31P NMR spectrum of compound 19.
Figure 4. 19F NMR spectrum of compound 19.
Figure 5. 11B NMR spectrum of compound 20.
Figure 6. 19F NMR spectrum of compound 20.
Figure 7. 19F NMR spectrum of compound 21.
Figure 8. Docking results of some selected compounds 2, 3, 13 and 22 with hydrogen bonding
with one or another amino acid present in the active site of PI3K (PBD:2RD0).

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Table 1. Anticancer activities against four examined cell lines. Data are expressed as IC50 (µM)
±SD.*
Compound
IC50
IC50
IC50
IC50
No
2
3
T47D MCF7 MDA231 Caco-2
192±17 211±19 188±13 196±19
165±13 189±16 175±15 171±11
>300
>300
>300
>300
>300
>300
>300
>300
>300
149±9
82±5
97±6
>300
>300
>300
>300
>300
>300
>300
>300
>300
158±8
>300
>300
>300
161±14
89±8
103±14
>300
>300
>300
>300
>300
>300
>300
>300
>300
156±11
110±9
4
5

7
8
9
156±16 154±15
83±9
105±8
>300
>300
>300
>300
>300
>300
>300
>300
>300
88±7
99±7
>300
>300
>300
>300
>300
>300
>300
>300
>300
10
11
12
13
14
15
1
17
18
19
20
21
22
23
24
25
2
27
163±9 169±17
112±13 109±11 113±12
161±16 173±12 169±10 165±17
62±6
71±4
86±9
96±4
94±6
59±2
77±5
89±8
95±6
93±4
59±4
68±5
91±5
96±5
99±5
108±9
64±3
72±5
84±3
98±7
101±8
102±11 110±12
105±10
* Each experiment was repeated 3 times. IC50 concentrations were obtained from the dose–
response curves using Graph Pad Prism Software 5 (San Diego California USA,
www.graphpad.com)

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Table 2. Binding Energy and Inhibition Constant of the docked compounds 2-27.
Compound
Minimum binding
energy (kJ mol-1)
-6.37
Inhibition Constant
(ki)
No
2
3
4
5

7
8
9
10
11
12
13
14
15
1
17
18
19
20
21
22
23
24
25
2
27
19.55 uM
10.35 uM
32.45 uM
94.32 uM
48.10 uM
15.07 uM
48.10 nM
175.23 nM
165.51 uM
111.62 uM
16.40 uM
87.68 uM
105.40 uM
67.83 uM
103.51 uM
83.38 uM
205.52 uM
3.47 uM
-6.80
-10.68
-10.56
-9.78
-10.00
-11.25
-9.53
-9.55
-10.20
-10.62
-9.36
-10.00
-10.88
-10.05
-9.78
-10.06
-9.54
-10.53
-10.25
-11.88
-11.20
-10.37
-10.17
-10.00
-9.89
5.76 uM
453.72 nM
16.40 nM
25.62 nM
67.83 nM
87.68 nM
84.38 nM
1.50 uM