An efficient method for the synthesis of new derivatives of 2,4,6-triarylpyridines as cytotoxic agents

Article abstract of DOI:10.1007/s11164-019-04025-6

New series of 2,4,6-triarylpyridines derivatives 5a–f and 6a–d were designed, synthesized and evaluated for their cytotoxic activity against breast cancer cell lines. Three-component reaction between acetophenones, ammonium acetate and 4-methyl or 2-methy

Full text of DOI:10.1007/s11164-019-04025-6

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-019-04025-6
An efcient method for the synthesis of new derivatives
of 2,4,6‑triarylpyridines as cytotoxic agents
Bahram Ahmadi Baloutaki1 · Mohammad Hosein Sayahi2 ·
Mohammad Nikpassand1 · Hassan Kefayati1
Received: 29 April 2019 / Accepted: 12 October 2019
© Springer Nature B.V. 2019
Abstract
New series of 2,4,6-triarylpyridines derivatives 5a–f and 6a–d were designed, syn-
thesized and evaluated for their cytotoxic activity against breast cancer cell lines.
Three-component reaction between acetophenones, ammonium acetate and 4-methyl
or 2-methylpyridine in the presence of iodine in DMSO lead to title 2,4,6-triar-
ylpyridines. This method allows the simple preparation of the desired products using
readily available reagents under mild reaction conditions in good yields. Synthesized
compounds were investigated for their cytotoxic activities against MDA-MB-231,
MCF-7 and T-47D cell lines. Most of the synthesized compounds showed remark-
able cytotoxicity, comparable to standard drug etoposide.
Keywords Acetophenones · 2-Methylpyridine · 4-Methylpyridine ·
2,4,6-Triarylpyridines
Introduction
The pyridine substructure is one of the most prevalent heterocycles found in natu-
ral products, pharmaceuticals, and functional materials [1]. The 2,4,6-triarylpyri-
dines are signifcant building blocks in supramolecular chemistry [2] and are also
important because of their biological activities [3, 4]. Also, the quaternary 2,4,6-tri-
arylpyridinium derivatives have signifcant synthetic applications such as in nucleo-
philic reaction [5], C-alkylation of β-diketones [6], electrophilic amination [7], and
azepine synthesis [8]. Therefore, the development of novel and efcient methods
* Mohammad Hosein Sayahi
sayahymh@pnu.ac.ir
* Mohammad Nikpassand
Nikpassand@iaurasht.ac.ir
1
Department of Chemistry, Rasht Branch, Islamic Azad University, Rasht, Iran
2
Department of Chemistry, Payame Noor University (PNU), 19395-3697, Tehran, Iran
Vol.:(0123456789)
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B. A. Baloutaki et al.
for the synthesis of pyridine scafolds with diverse functionalities and substituents
remains an area of current interest, which has been studied in recent year [9–13].
Several series of 2,4,6-triaryl pyridines (Kröhnke pyridines) [14] have been
reported that exhibited excellent cytotoxic activities [14–16]. These derivatives
were synthesized using various methods and procedures. Traditionally, these com-
pounds have been synthesized through the reaction of N-phenacylpyridinium salts
with α,β-unsaturated ketones in the presence of NH4OAc [17, 18]. However, the
pyridinium salts and the unsaturated ketones have to be synthesized frst, and this
method is relatively expensive. So, several improved methods have been developed
for the synthesis of these pyridines such as the reaction of a ketoketene dithioacetals
with methyl ketones in the presence of NH4OAc [19], the reaction of N-phosphiny-
lethanimines with aldehydes [20], the addition of lithiated β-enaminophosphonates
to chalcones [21], the solvent-free reaction between acetophenones, benzaldehydes,
and NH4OAc in the presence of sodium hydroxide [22], the one-pot reaction of ace-
tophenones, benzaldehydes, and NH4OAc without catalyst under microwave irradia-
tion [23], and the iodine-mediated reaction between alkyl-branched amino acids and
acetophenones [24].
Considering the importance of Kröhnke pyridines as anticancer agents, cytotoxic
activates of all synthesized compounds were evaluated against three breast cancer
cell lines MDA-MB-231, MCF-7, and T-47D [14].
Experimental
All chemicals were purchased from Merck and Fluka companies. All yields refer
1
to isolated products. H and 13C NMR spectra were recorded on a Bruker, Rhein-
stetten, Germany (at 500 and 400 MHz) NMR spectrometer using tetramethylsilane
(TMS) as internal standard. Elemental analyses for C, H, and N were performed
using a Heraeus CHN-O-Rapid analyzer. Melting points were determined in a capil-
lary tube and are not corrected. The progress of reaction was followed with TLC
using silica gel SILG/UV 254 and 365 plates. All products are known compounds,
and their structures were deduced by 1H and 13C NMR spectroscopy.
General procedure for the preparation of compounds 5a–f and 6a–d
A solution of 4-methylpyridine 1 or 2-methylpyridine 2 (1 mmol) and iodine
(1 mmol) in 3 mL DMSO was heated at 100 °C for 2 h. Then, acetophenones 3
(2 mmol) and ammonium acetate 4 (1 mmol) were added to the reaction mixture and
stirring was continued for 3 h. Upon completion of the reaction, monitored by TLC,
the reaction mixture was diluted with 10 mL aqueous saturated solution of sodium
thiosulphate and extracted with dichloromethane (3×4 mL). Combined organic
layers were washed with brine solution, dried over anhydrous Na2SO4, solvent was
removed under reduced pressure and the resultant crude product was purifed by
silica gel column chromatography using hexane and ethyl acetate (7:1) as eluents to
obtain the desired products 5a–f and 6a–d.
1 3

An efcient method for the synthesis of new derivatives of…
All of synthesized compounds were characterized by their physical constant,
1
comparison with authentic samples, IR, H NMR, 13C NMR spectroscopy and ele-
mental analysis.
1
2,6‑diphenyl‑4,4′‑bipyridine (5a) H NMR (500.0 MHz, DMSO-d6): δ 7.39–7.61 (m,
6H, 6CH), 8.07 (d, J=8.0 Hz, 4H, 4CH), 8.26 (s, 2H, 2CH), 8.55 (d, J=6.0 Hz,
2H, 2CH), 9.08 (d, J=6.5 Hz, 2H, 2CH) ppm. 13C NMR (125.7 MHz, DMSO-d6):
δ 125.3 (4CH), 127.2 (2C), 128.1 (2CH), 130.0 (2CH), 131.5 (2CH), 131.8 (4CH),
135.2 (C), 144.9 (2C), 145.1 (2C), 159.3 (2C) ppm. Anal Calc. for C22H16N2: C,
85.69; H, 5.23; N, 9.08. Found: C, 85.73; H, 5.21; N, 9.11.
1
2,6‑di‑o‑tolyl‑4,4′‑bipyridine (5b) H NMR (500.0 MHz, DMSO-d6): δ 2.26 (s, 6H,
2CH3), 7.16–7.33 (m, 4H, 4CH), 7.46 (d, J=7.5 Hz, 2H, 2CH), 8.01 (d, J=8.0 Hz,
2H, 2CH), 8.17 (s, 2H, 2CH), 8.23 (d, J=7.5 Hz, 2H, 2CH), 8.94 (d, J=6.0 Hz,
2H, 2CH) ppm. 13C NMR (125.7 MHz, DMSO-d6): δ 18.7 (2CH3), 109.3 (2CH),
119.7 (2CH), 120.6 (C), 122.5 (2C), 125.6 (2CH), 126.1 (2CH), 126.6 (2CH),
129.4 (2CH), 130.9 (2CH), 137.0 (C), 144.9 (2C), 158.2 (2C) ppm. Anal Calc. for
C24H20N2: C, 85.68; H, 5.99; N, 8.33. Found: C, 85.72; H, 6.02; N, 8.37.
1
2,6‑di‑p‑tolyl‑4,4′‑bipyridine (5c) H NMR (500.0 MHz, DMSO-d6): δ 2.33 (s, 6H,
2CH3), 7.21 (dd, J=5.5, 3.0 Hz, 4H, 4CH), 8.06 (d, J=8.0 Hz, 4H, 4CH), 8.26
(d, J=5.0 Hz, 2H, 2CH), 8.46 (s, 2H, 2CH), 9.10 (d, J=6.5 Hz, 2H, 2CH) ppm.
13C NMR (125.7 MHz, DMSO-d6): δ 20.8 (2CH3), 121.7 (2C), 124.4 (2C), 124.5
(C), 127.5 (4CH), 127.6 (4CH), 128.4 (2CH), 128.6 (2CH), 132.1 (2CH), 139.2 (C),
159.3 (2C) ppm. Anal Calc. for C24H20N2: C, 85.68; H, 5.99; N, 8.33. Found: C,
85.71; H, 5.96; N, 8.29.
1
2,6‑bis(4‑fuorophenyl)‑4,4′‑bipyridine(5d) H NMR (500.0 MHz, DMSO-d6):
3
4
δ 7.52–7.60 (m, 4H, 4CH), 8.13 (dd, JHH =4.5, JFH =4.5 Hz, 4H, 4CH), 8.25
(d, J=4.5 Hz, 2H, 2CH), 8.51 (s, 2H, 2CH), 9.17 (s, 2H, 2CH) ppm. 13C NMR
(125.7 MHz, DMSO-d6): δ 122.7 (2CH), 123.5 (4CH), 129.7 (4CH), 132.1
(C), 132.3 (4CH), 133.5 (C), 134.1 (4CH), 143.4 (2C), 151.1 (2C), 163.9 (d,
1JC-F =253.9 Hz, 2C) ppm. Anal Calc. for C22H14F2N2: C, 76.73; H, 4.10; N, 8.13.
Found: C, 76.76; H, 4.07; N, 8.15.
1
2,6‑bis(2‑chlorophenyl)‑4,4′‑bipyridine (5e) H NMR (500.0 MHz, DMSO-d6): δ
7.32–7.58 (m, 6H, 6CH), 8.12 (dd, J=8.2, 6.0 Hz, 2H, 2CH), 8.25 (d, J=8.0 Hz,
2H, 2CH), 8.55 (d, J=6.0 Hz, 2H, 2CH), 9.52 (d, J=6.5 Hz, 2H, 2CH) ppm. 13C
NMR (125.7 MHz, DMSO-d6): δ 116.0 (2CH+CH), 116.3 (CH), 120.4 (CH), 120.6
(CH), 124.6 (2CH), 126.1 (CH), 126.9 (CH), 127.4 (2CH), 129.9 (C), 131.8 (CH),
131.9 (CH), 137.0 (2C), 146.5 (C), 146.6 (2C), 149.9 (2C) ppm. Anal Calc. for
C22H14Cl2N2: C, 70.04; H, 3.74; N, 7.43. Found: C, 70.07; H, 3.72; N, 7.46.
1
2,6‑bis(4‑bromophenyl)‑4,4′‑bipyridine (5f) H NMR (500.0 MHz, DMSO-d6): δ
7.57 (s, 4H, 4CH), 8.14 (s, 2H, 2CH), 8.27 (s, 4H, 4CH), 8.65 (s, 2H, 2CH), 9.15 (s,
1 3

B. A. Baloutaki et al.
2H, 2CH) ppm. 13C NMR (125.7 MHz, DMSO-d6): δ 126.0 (4CH), 127.0 (4CH),
128.7 (C), 129.8 (2C), 130.3 (2CH), 130.9 (2CH), 131.5 (2CH), 133.9 (2C), 136.7
(C), 159.5 (2C) ppm. Anal Calc. for C22H14Br2N2: C, 56.68; H, 3.03; N, 6.01.
Found: C, 56.72; H, 3.06; N, 5.97.
1
2′,6′‑diphenyl‑2,4′‑bipyridine (6a) H NMR (500.0 MHz, DMSO-d6): δ 7.06 (t,
J=7.0 Hz, 1H, CH), 7.16 (t, J=8.0 Hz, 2H, 2CH), 7.20 (d, J=8.0 Hz, 4H, 4CH),
7.44 (d, J=9.0 Hz, 1H, CH), 7.91 (t, J=6.0 Hz, 1H, CH), 8.05 (d, J=8.0 Hz, 4H,
4CH), 8.46 (s, 2H, 2CH), 9.20 (d, J=7.5 Hz, 1H, CH) ppm. 13C NMR (125.7 MHz,
DMSO-d6): δ 123.9 (C), 124.0 (2C), 127.5 (4CH), 127.6 (4CH), 128.3 (2CH), 128.4
(2CH), 132.5, 136.0, 137.9 and 140.9 (4CH), 143.6 (C), 143.8 (2C) ppm. Anal Calc.
for C22H16N2: C, 85.69; H, 5.23; N, 9.08. Found: C, 85.72; H, 5.27; N, 9.11.
1
2′,6′‑di‑p‑tolyl‑2,4′‑bipyridine (6b) H NMR (500.0 MHz, DMSO-d6): δ 2.33 (s,
6H, 2CH3), 7.11 (d, J=8.5 Hz, 4H, 4CH), 7.28 (t, J=7.5 Hz, 1H, CH), 7.45 (d,
J=9.5 Hz, 1H, CH), 7.92 (d, J=4.5 Hz, 1H, CH), 8.06 (d, J=8.0 Hz, 4H, 4CH),
8.31 (s, 2H, 2CH), 9.12 (d, J=4.5 Hz, 1H, CH) ppm. 13C NMR (125.7 MHz,
DMSO-d6): δ 123.9 (C), 124.2 (2C), 126.4 (2C), 127.5 (4CH), 127.6 (4CH), 128.4
(2CH), 128.6, 132.1, 136.3 and 138.9 (4CH), 144.1 (C), 159.3 (2C) ppm. Anal Calc.
for C24H20N2: C, 85.68; H, 5.99; N, 8.33. Found: C, 85.65; H, 6.02; N, 8.30.
1
2′,6′‑bis(4‑3fuorophenyl)‑2,4′‑bipyridine (6c) H NMR (500.0 MHz, DMSO-d6): δ
3
7.26 (dd, JHH =7.0, JFH =7.0 Hz, 4H, 4CH),7.58 (d, J=9.5 Hz, 1H, CH), 7.74 (t,
J=8.0 Hz, 1H, CH), 8.14 (dd, 3JHH =7.0, 4JFH =6.5 Hz, 4H, 4CH), 8.23 (d, J=7.5 Hz,
1H, CH), 8.47 (s, 2H, 2CH), 9.10 (d, J=5.0 Hz, 1H, CH) ppm. 13C NMR (125.7 MHz,
DMSO-d6): δ 122.5 (2C), 124.2 (C), 127.9 (4CH), 128.2 (2CH), 129.2 (4CH), 131.0,
132.9, 137.1 and 137.4 (4CH), 138.6 (C), 143.0 (2C), 151.5 (2C) ppm. Anal Calc. for
C22H14F2N2: C, 76.73; H, 4.10; N, 8.13. Found: C, 76.72; H, 4.07; N, 8.16.
1
2′,6′‑bis(4‑bromophenyl)‑2,4′‑bipyridine (6d) H NMR (500.0 MHz, DMSO-d6):
δ 7.11 (d, J=8.0 Hz, 1H, CH), 7.24 (t, J=7.0 Hz, 1H, CH), 7.52 (d, J=8.9 Hz,
4H, 4CH), 7.93 (t, J=8.0 Hz, 1H, CH), 8.12 (d, J=8.0 Hz, 4H, 4CH), 8.42 (s, 2H,
2CH), 9.12 (d, J=6.5 Hz, 1H, CH) ppm. 13C NMR (125.7 MHz, DMSO-d6): δ
121.1 (2C), 121.2 (2CH), 123.5 (2CH), 123.6 (2CH), 124.5 (2CH), 124.6 (2CH),
132.5 (CH), 136.0 (2CH), 137.9 (2C) and 140.9 (CH), 143.6 (2C), 143.8 (C), 157.3
(C) ppm. Anal Calc. for C22H14Br2N2: C, 56.68; H, 3.03; N, 6.01. Found: C, 56.68;
H, 2.99; Br, 34.31; N, 6.04.
In vitro cytotoxicity assay
Three breast cancer cell lines (MDA-MB-23, MCF-7, and T-47D, 5×104 cells/mL)
in 96-well culture plates were incubated for 48 h with diferent concentrations of
2,4,6-triaryl pyridines 5a–f and 6a–d dissolved in DMSO/ethanol as solvent (the
fnal volume of solvent/medium was less than 1% in all experiments). Etoposide and
solvent were used as positive and negative controls, respectively. After incubation,
1 3

An efcient method for the synthesis of new derivatives of…
the solvent/medium was removed and 200 μL of MTT solution (1 mg/mL, fnal
concentration) was added to all wells. After 4 h of treatment, the culture medium
was replaced with DMSO (100 μL) to each well. The absorbance was determined at
492 nm with a multi-well plate reader (Gen5, Power wave xs2, BioTek, America).
The IC50 values (all experiments were performed at least three times and) for all
compounds were calculated by nonlinear regression analysis [25, 26].
Docking study
The docking study was carried out using AutoDock Tools (version 1.5.6), and the
pdb structure of 4FM9 was taken from the protein data bank. Subsequently, water
and additional molecules were removed from the enzyme structure. The 3D struc-
ture of compound 5e was depicted using MarvineSketch 5.8.3, 2012, and Che-
mAxon (http://www.chemaxon.com). After AutoDock format of topoisomerase
II and compound 5e were prepared. AutoDock format of enzyme was used as an
input for the AUTOGRID program. The center of the grid box was placed in coor-
dinates x=27.42, y=25.50, and z=30.68 and the dimensions of the grid box were
set at 60×60×The fexible ligand docking system was carried out by 25 runs of
the AUTODOCK search by the Lamarckian genetic algorithm. The highest binding
energy conformation of the compound 5e was selected by analyzing the interactions
between topoisomerase II and this compound, and the results were visualized using
Discovery Studio (4.0 Client, Figs. 1 and 2).
Results and discussion
Chemistry
In order to achieve a more efcient synthetic process, minimize by-products,
decrease the number of separate reaction steps, improving the yields and reaction
times and also in extending our research on the synthesis and study of heterocy-
clic and pharmaceutical compounds, [27–33] in this work, at frst, the reaction of
4-methylpyridine 1 (1 mmol), acetophenone 3a (1 mmol), ammonium acetate 4 in
Fig. 1 Compound 5e in the
active site of topoisomerase II
1 3

B. A. Baloutaki et al.
Fig. 2 Binding mode of the compound 5e in the active site of topoisomerase II
the presence of iodine was investigated in various reaction temperature and solvents
(Table 1). Although the 2,4,6-triarylpyridine 5a was not generated in the absence of
DMSO (entry 1), the reaction lead to desired product 5 in 75% yield in presence of
DMSO (entry 2).
We examined the reaction in various solvents such as DMF, THF, toluene, dichlo-
roethane (DCE), and CH3CN (entries 3–7), and it was found that the best yield was
achieved in DMSO. Also, we tested various amount of catalyst iodine for the reac-
tion as 0.5 and 0.2 equiv. per 1 mmol substrate and the 2,4,6-triarylpyridine 5a was
produced in 60% and 51% yield, respectively (entries 8, 9). The reaction did not
preceded in better yield at 120 °C and 80 °C (entries 10–11).
Encouraged by these results, the reactions for produce desired 2,4,6-triaryl pyri-
dines 5a–f and 6a–d were performed using 4-methylpyridine 1 or 2-methylpyridine
2, ammonium acetate 4 and various acetophenones 3 in the presence of 1 equiv. of
iodine at 100 °C in 3 mL DMSO (Scheme 1). The structures of the isolated products
5a–f and 6a–d were assigned on the basis of 1H and 13C NMR spectroscopy.
A proposed mechanism for the formation of the target 2,4,6-triarylpyridine
derivatives is depicted in Scheme 2. Initially, the methylpyridine (1 or 2) iodinates
by iodide to produce iodinated intermediate 7. This intermediate undergoes the
Kornblum oxidation [34, 35] at 100 °C in DMSO to give aldehyde 8. Next, ace-
tophenones 3 may condense with the aldehyde 8 to generate α,β-unsaturated com-
pound 9. Alternatively, the condensation of NH4OAc with acetophenones 3 leads
to the formation of enamine intermediate 10. The reaction of enaminone 10 and
1 3

An efcient method for the synthesis of new derivatives of…
Table 1 Optimization of the synthesis of 2,4,6-triarylpyridine 5a
Entry
I2 (equiv.)
Solvent
Temperature (°C) Time (h)
Yield of 4a (%)a, b
1
2
3
4
5
6
7
8
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.5
0.2
0.5
0.5
–
100
100
100
66
100
80
5
5
5
5
5
5
5
5
5
5
5
No reaction
DMSO
DMF
THF
Toluene
DCE
CH3CN
DMSO
DMSO
DMSO
DMSO
75
15
10
25
28
35
60
51
70
62
80
100
100
120
80
9
10
11
aReaction conditions: use of 4-methylpyridines (1 mmol), acetophenone (2 mmol), ammonium acetate
(1 mmol) and appropriate amount of iodine; solvent (3 mL); related temperature; 5 h
bIsolated yield
α,β-unsaturated compound 9 afords intermediate 11 which undergoes ring closing
condensation to give dihydopyridine 12. Finally, the dihydopyridine 9 oxidizes to
corresponding 2,4,6-triarylpyridine 5a–f and 6a–d.
Scheme 1 New method for the synthesis of 2,4,6-triarylpyridines 5a–f and 6a–d
1 3

B. A. Baloutaki et al.
Scheme 2 Proposed mechanism for formation 2,4,6-triarylpyridine 5a–f and 6a–d
Cytotoxicity
The synthesized 2,4,6-triaryl pyridines 5a–f and 6a–d were evaluated for in vitro
cytotoxicity against breast cancer cell lines MDA-MB-231, MCF-7 and T-47D using
the MTT assay [36]. The IC50 values of these compounds in comparison with stand-
ard drug etoposide are listed in Table 1. Results of the MTT assay demonstrated that
compounds 5e, 5b, 5d and 5c with IC50 values 8.74 0.7, 9.45 0.4, 11.59 0.7 and
17.18 0.4 respectively were more potent than etoposide (IC50 =22.56 0.53 μM)
against MDA-MB-231 cells. Moreover, compound 6d was as potent as etopo-
side against the latter cell line. Compounds 5d, 5e, and 6b (IC50s=8.12 0.54,
13.1 0.54, and 16.81 0.65 μM, respectively) showed high cytotoxic efects
against MCF-7 cell line in comparison etoposide (IC50 =17.76 0.68 μM). Among
the synthesized compounds, cytotoxic activities of the compounds 5b, 5d, 5e, 6a,
6b, and 6d (IC50 values in the range of 8.61 0.32–13.97 0.4 μM) were superior to
that of standard drug etoposide (IC50 =21.18 0.63 μM) against T-47D cells. Com-
pound 5f was also as potent as etoposide against T-47D cells (Table 2).
To obtain an optimized cytotoxic agent, 4-methylpyridine 1 (series 5a–f), 2-meth-
ylpyridine 2 (series 6a–d) and substituted acetophenones 3 were used for the synthe-
sis of the target 2,4,6-triaryl pyridines.
In the 4-methylpyridine series 5a–f, 2,6-diphenyl-4-(pyridin-4-yl)pyridine 5a
showed moderate cytotoxic efect against all three cell lines studied. Introduc-
tion of methyl substituents on 2-positions of 2 and 6-phenyl rings, as in com-
pound 5b, led to a signifcant increase in cytotoxicity against MDA-MB231 and
T-47D. Changing the position of the methyl groups in the mentioned phenyl rings
from C-2 to C-4, producing 5c diminished the activity against MDA-MB231 and
loss of cytotoxic activity against MCF-7 and T-47D cells. Among the synthesized
compounds, 2,6-bis(4-fuorophenyl)-4-(pyridin-4-yl)pyridine 5d and 2,6-bis(2-
chlorophenyl)-4-(pyridin-4-yl)pyridine 5e of 4-methylpyridine series showed
excellent cytotoxic efects against all the studied cell lines. It is worthy to note
that compounds 5d and 5e were the most potent compounds against MCF-7 and
MDA-MB231 cells, respectively. The comparison of IC50 value of 4-fuoro com-
pound 5d with their 4-bromo analog 5f revealed that the fuorine atom is more
efective than bromine atom in this series.
1 3

An efcient method for the synthesis of new derivatives of…
2-Methylpyridine derivative 6a without substituents on the 2 and 6-phenyl
rings showed no activity against MDA-MB231 and MCF-7 cells; however, it
showed good activity against T-47D. Compound 6b having 4-methyl group on
2 and 6-phenyl rings did not show activity against MDA-MB231. On the other
hand, this compound showed high cytotoxic activity against MCF-7 and T-47D.
Among the synthesized compounds, compound 6b was the most potent com-
pound against T-47D. Replacement of methyl groups of compound 6b with
fuorine atom, as in compound 6c, led to loss of cytotoxic activity. In contrast,
4-bromo derivative 6d was a good cytotoxic agent against all the three cell lines
MDA-MB-231, MCF-7 and T-47D.
The cytotoxicity of the most potent compound 5e, as the most potent com-
pound against MDA-MB231, was also evaluated against normal cell line HDF
[34, 35]. The result revealed that compound 5e did not show cytotoxic activity
against HDF (IC50 > 100 µM).
Table 2 Structures and in vitro cytotoxic activities of 2,4,6-triaryl pyridines 5a–f and 6a–d against breast
cancer cell lines
Product
R
Cytotoxicity (IC50 µM)a
MDA-MB231
MCF-7
T-47D
5a
H
30.9 0.7
9.45 0.4
17.18 0.4
11.59 0.7
8.74 0.7
>100
>100
>100
>100
22.9 0.8
28.6 0.87
30.71 0.9
>100
8.12 0.54
13.1 0.54
>100
>100
9.13 0.43
>100
43.40 1.2
13.01 0.1
>100
9.10 0.4
9.61 0.63
5b
2-CH3
4-CH3
4-F
2-Cl
4-Br
H
4-CH3
4-F
4-Br
5c
5d
5e
5f
6a
17.73 0.67
8.61 0.32
>100
13.97 0.4
21.18 0.63
6b
6c
6d
16.81 0.65
17.76 0.68
Etoposide
–
22.56 0.53
aData are expressed as Mean SE (three independent experiments)
1 3

B. A. Baloutaki et al.
Docking studies
Several cytotoxic agents whit 2,4,6-triarylpyridine scafold were reported that
displayed high inhibitory activity against topoisomerase II [16]. Therefore, to fnd
a possible interaction mode of the compound 5e as one of the most potent com-
pound against studied cancer cell lines, molecular modeling was performed in the
active site of topoisomerase II (PDB code: 4FM9). As can be seen in Fig. 1, the
active site binding pocket of topoisomerase II includes protein residues and DNA
bases [37].
Detailed binding mode of compound 5e in the active site of this enzyme revealed
that this compound as well ftted in the active site of enzyme and interacted with
both protein and DNA parts (Fig. 2). 2-Chlorophenyl moieties of the compound 5e
interacted with DNA bases cytosine (DC5) and adenine (DA6) via a hydrophobic
interaction and a π-anion interaction. These moieties also formed two hydrophobic
interactions with Glu503 and Ala505, and one of them created a π-anion interac-
tion with Arg487. The latter amino acid established a hydrophobic interaction with
4-pyridine group. 4-Pyridine group also interacted with Glu506 through a π-anion
interaction. Furthermore, the central pyridine moiety formed a hydrophobic interac-
tion with Ala505.
Conclusions
In conclusion, new derivatives of 2,4,6-triaryl pyridine 5a–f and 6a–d were synthe-
sized and evaluated as cytotoxic agents. For the synthesis of target compounds, an
efcient procedure and simple starting materials were used and these compounds.
The cytotoxic assay was done against MDA-MB-231, MCF-7, and T-47D using
MTT assay. Among the synthesized compounds 5e, 5b, 5d and 5c against MDA-
MB-231, compounds 5d, 5e, and 6b against MCF-7, and compounds 5b, 5d, 5e, 6a,
6b, and 6d against T-47D were more potent than etoposide. Compound 5e as one of
the most potent compounds against all three studied cell lines was docked into active
site of topoisomerase II. Obtained results revealed that this compound interacted as
well with both amino acids and DNA bases in the active site of topoisomerase II.
Acknowledgements Financial support from the Research Council of Islamic Azad University of Rasht
branch is sincerely acknowledged.
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