Synthesis, solvatochromism, and biological activity of novel azo dyes bearing 2-pyridone and benzimidazole moieties

Article abstract of DOI:10.3906/kim-1711-97

New azo dyes bearing 2-pyridone and benzimidazole moieties were prepared using diazotization of 4-(1H-benzo[d]imidazol-2-yl)aniline and coupling of the obtained diazonium salt with substituted 3-cyano-2-pyridones. Obtained compounds were characterized via UV-Vis, FT-IR, and ¹ H and ¹³ C NMR spectroscopy as well as by elemental analysis data. The UV-Vis spectra of the synthesized dyes were measured in thirteen solvents of different properties at room temperature. Solvatochromism and tautomerism of novel azo dyes were discussed. An MTT (3,4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test was performed to prove the biocompatibility of the investigated dyes. The investigated dyes exhibited satisfying antiproliferative activities against both tumor cell lines, MDA-MB-231 and HCT-116, demonstrating the potent capacity for treatment of tumors.

Full text of DOI:10.3906/kim-1711-97

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 896 – 907
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1711-97
Research Article
Synthesis, solvatochromism, and biological activity of novel azo dyes bearing
2-pyridone and benzimidazole moieties
2
1
Duˇsan MIJIN1,∗, Biljana BOZIC NEDELJKOVIC , Bojan BOZIC , Ilijana KOVRLIJA ,
ˇ ´
´
ˇ ´2
1
Jelena LADAREVIC , Gordana USCUMLIC
1
´
ˇ ´
´
1Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia
2Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia
Received: 30.11.2017
•
Accepted/Published Online: 02.04.2018
•
Final Version: 01.06.2018
Abstract: New azo dyes bearing 2-pyridone and benzimidazole moieties were prepared using diazotization of 4-(1H-
benzo[d]imidazol-2-yl)aniline and coupling of the obtained diazonium salt with substituted 3-cyano-2-pyridones. Ob-
tained compounds were characterized via UV-Vis, FT-IR, and 1 H and 13 C NMR spectroscopy as well as by elemental
analysis data. The UV-Vis spectra of the synthesized dyes were measured in thirteen solvents of different properties
at room temperature. Solvatochromism and tautomerism of novel azo dyes were discussed. An MTT (3,4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test was performed to prove the biocompatibility of the investigated dyes.
The investigated dyes exhibited satisfying antiproliferative activities against both tumor cell lines, MDA-MB-231 and
HCT-116, demonstrating the potent capacity for treatment of tumors.
Key words: UV-Vis spectroscopy, azo dyes, biocompatibility, antiproliferative activity, tautomerism
1. Introduction
Heterocyclic compounds can be found in many natural and synthetic compounds. They have a wide range of
applications, mostly in pharmaceuticals, as well as in color chemistry. Among these compounds, benzimidazole
and 2-pyridone have gained wide interest due to their biological, clinical, and industrial applications.
Benzimidazole in natural organic compounds is present as N -ribosyl-dimethylbenzimidazole and it can
be found in vitamin B12 as an axial ligand for cobalt1 as well as in DNA and RNA molecules.2 Various
benzimidazole derivatives exhibit different pharmacological activities such as antihypertensive,3 antiviral,4
antiulcer,5 antifungal,6 antibacterial,7 anticancer,8 and antihistaminic.9 The pharmaceutical activities of this
class of compounds have been the subject of numerous review papers10−16 in which the structures of compounds
were discussed along with their activity.
The 2-pyridone structure (dominant tautomer in pyridinol-pyridone tautomerism of 2-hydroxypyridine)
is also present in DNA and RNA molecules.2 2-Pyridone derivatives possess pharmacological and a wide range
of therapeutic activities.17 They exhibit antimicrobial,18 antiviral,19 anti-HIV,20 anticancer,21 cardiotonic,22
and vasorelaxant activities.23 Pharmaceutically active derivatives obtained by a combination of benzimidazole
and antimicrobial active 2-pyridone rings have also been synthesized and their potential biological applications
have been observed.24
∗Correspondence: kavur@tmf.bg.ac.rs
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MIJIN et al./Turk J Chem
Azo dyes represent the largest group of colored organic compounds due to the number of variations
in the chemical structure and their wide commercial applications.25,26 They can be used for the dyeing of
natural and synthetic fibers and for coloring plastics, rubber, foods, paints, varnishes, printing inks, drugs, and
cosmetics. A number of dyes contain different heterocyclic moieties including benzimidazole and 2-pyridone
cores. Benzimidazole-based dyes are used for dyeing of polyester27 and polyamide fibers.28 On the other hand,
pyridone azo dyes are more abundant. These dyes are used as disperse dyes that are characterized by low
aqueous solubility. Disperse dyes are usually applied to hydrophobic fibers like polyester and nylon. Some
of the pyridone azo dyes are commercial products.29 A synthesis of azo dyes containing benzimidazole and a
2-pyridone ring from 4-(4-methoxyphenyl)glutaconic anhydride was also reported.30
In this work, we have prepared two dyes from 4-(1H -benzo[d]imidazol-2-yl)aniline and two 3-cyano-
6-hydroxy-2-pyridones having different substituents in position 4 (methyl and phenyl) using a diazo-coupling
reaction in order to examine the solvatochromic behavior in various solvents. The tautomerism of the synthesized
dyes was also investigated. In addition, a study of the biological activities of the synthesized compounds was
performed.
2. Results and discussion
The aryl azo pyridone dyes studied in this work were prepared according to the synthetic route given in Figure 1.
The synthesis started with the preparation of 4-(1H-benzo[d]imidazol-2-yl)aniline (1) from o-phenylenediamine
and p-aminobenzoic acid31,32 and continued with the diazotization of 1. The prepared diazonium salt was then
coupled with prepared pyridones,33,34 using a well-known procedure.35 The structures of new compounds were
characterized via UV-Vis, FT-IR, and 1 H and 13 C NMR spectroscopy as well as elemental analysis data.
The analysis of FT-IR spectra of compounds 3a and 3b suggests the existence of a hydrazone form in
the solid state. The conclusion was made due to the detection of two intense carbonyl bands (1619–1655 cm−1
and a broad band ascribed to the N–H proton (3424–3426 cm−1).
)
The NMR data obtained for compounds 3a and 3b at ambient temperature in DMSO-d6 are given in
Section 3. According to the analysis of NMR spectra the obtained dyes exist as hydrazone tautomers in DMSO-
d6 . This was concluded by 1 H NMR shifts assigned to the imino proton of the hydrazone group, which are in
the range of 14.55–14.71 ppm. This is in accordance with the literature data of the aryl azo pyridone dyes.33,34
The existence of the hydrazone form was further confirmed by the signals of two carbonyl groups in 13 C NMR
spectra observed in the range of 160.7–161.9 ppm.
2.1. Solvatochromism
The UV-Vis spectra of the synthesized dyes were measured in the range of 300–600 nm in thirteen solvents
of different properties at room temperature (Table 1). Aryl azo pyridone dyes were characterized by azo-
hydrazone tautomerism due to the proton exchange between a hydroxy group in the pyridone moiety and an
azo group.29 Possible tautomeric forms of the investigated dyes are given in Figure 2. Structure I is the azo
form while Structure II represents the hydrazone form of the investigated dyes. The addition of a base leads to
deprotonation and an anion is formed (Structure III). The formed anion is a resonance hybrid of two canonical
structures: hydrazone (IIIa) and azo (IIIb).36−38 According to the data given in Table 1 and the UV-Vis spectra
in Figures 3 and 4, the studied pyridone azo dyes exist generally in the hydrazone form, in the range of 438–457
nm for 3a and 434–468 nm for 3b, except in dipolar proton-accepting solvents (DMF, DMSO; Figure 4) where
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MIJIN et al./Turk J Chem
NH2
NH2
a
O
N
+
NH2
NH2
N
H
HO
1
b
N
+
N2
N
H
R
O
R
CN
O
O
CN
O
c
d
+
O
HO
N
H
H2N
2a R=Me
2b R=Ph
CN
R
N
N
N
H
N
O
N
3a R=Me
HO
H
3b R=Ph
Figure 1. Reagents and conditions: a) H3 PO3 / 180–200 ◦ C; b) NaNO2 , HCl / 0–5 ◦ C; c) KOH, alcohol / reflux,
d) KOH / 0–5 ◦ C.
the hydrazone-anion equilibrium has been observed. The introduction of a phenyl group in position 4 of the
pyridone moiety causes a bathochromic shift in comparison to a dye with a methyl group in same position. The
bathochromic shift is small due to the spatial orientation of the phenyl group to the pyridone moiety and the
absence of planarity.39 Solvent polarity, on the other hand, has an almost negligible impact on the absorption
maxima of the investigated dyes, but a slightly hypsochromic effect, with increase of polarity, can be observed.
Moreover, a moderate bathochromic effect from ampholytic to aprotic solvents is also observed. Also, in solvents
acting as a proton donors, such as acetic acid, a hypsochromic shift is observed with respect to proton-acceptor
solvents (DMF, DMSO).
It is well known that the structure of the dye influences the acid-base equilibrium36−38 as presented
in Figure 2 as well as the relative positions of UV-Vis maxima of hydrazone and anionic forms. In dipolar
proton-accepting solvents aryl azo pyridone dyes can easily dissociate and an anion form can appear, as shown
in Figures 3 and 4. The same can be achieved by the addition of a base. On the other hand, in acetic acid
(proton-donating solvents) the hydrazone form is favorable (Figure 3). This can be achieved also by the addition
of an acid. Thus, the concentrated hydrochloric acid and potassium hydroxide solution (1%) were added in
drops to an ethanol solution of dyes in order to observe the position of hydrazone and anion forms. The UV-Vis
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MIJIN et al./Turk J Chem
N
N
N
N
R
R
H
H
N
CN
O
N
O
CN
O
N
N
H
HO
N
H
N
H
I
II
_
+H+
H+
_
+H+
H+
N
N
N
N
R
R
H
H
N
CN
O
N
CN
O
N
N
_
_
O
N
H
O
N
H
3a R=Me
3b R=Ph
IIIa
IIIb
Figure 2. Possible tautomeric forms of the investigated aryl azo pyridone dyes.
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Methanol
THF
Acetic acid
Acetonitrile
DMF
DMSO
300
400
500
600
λ, nm
Figure 3. UV-Vis spectra of 3b in selected solvents.
Figure 4. UV-Vis spectra of 3a and 3b in DMF and
DMSO.
spectra of 3a in ethanol and acid/basic solution are presented in Figure 5. It can be observed that the addition
of concentrated hydrochloric acid has a small hypsochromic shift and a change in shape, indicating that the dye
is not completely in the hydrazone form. On the other hand, the addition of a potassium hydroxide solution
leads to deprotonation and the formation of anion/hydrazone equilibrium. Higher base quantities do not shift
the equilibrium completely to the anion form, which indicates the high stability of the hydrazone form. UV-Vis
maxima of anionic forms are at higher wavelengths in comparison to UV-Vis maxima of the hydrazone form, as
published before.36−38
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Table 1. Summary of the UV–Vis absorption bands for the synthesized dyes.
λmax, nm (log εa)
3b
Hydrazone:anion ratio
Solvent\compound 3a
3a
1:0
1:0
3b
1:0
1:0
Methanol
Ethanol
449 (4.39)
452 (4.68)
438 (4.30),
446 (4.60)
461 (4.64)
434 (4.28),
i-Propanol
0.56:0.44 0.33:0.67
457 (sh) (4.28) 460 (sh) (4.23)
i-Butanol
Cyclohexanol
Dioxane
455 (4.27)
455 (3.75)
457 (4.51)
449 (4.88)
468 (4.54)
463 (4.60)
468 (4.49)
462 (4.47)
460 (4.62)
460 (4.47)
451 (4.76)
439 (4.52),
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
1:0
Methylene chloride 442 (4.19)
THF
456 (4.72)
454 (4.65)
452 (4.69)
445 (4.76)
438 (4.43),
Acetone
Acetonitrile
Acetic acid
DMF
0.61:0.39 0.64:0.36
1:0 0.96:0.04
473 (sh) (4.29) 477 (sh) (4.33)
447 (4.42),
455 (4.82)
DMSO
474 (sh) (4.35)
aε, dm3 mol−1 cm−1
.
2
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
3
1
300
400
500
600
λ, nm
Figure 5. UV-Vis spectra of 3a in ethanol at different pH values (1- pure ethanol; 2- with the addition of 1 drop of
HCl; 3- with the addition of 1 drop of KOH).
2.2. Biological activity of the investigated aryl azo pyridone dyes
2.2.1. Biocompatibility
As a preliminary biocompatibility test, the MTT40 test was used to reveal the new synthesized dyes’ biocom-
patibility. The effect of the synthesized dyes on the viability of MRC-5 cells during 24 h was measured. The
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MIJIN et al./Turk J Chem
results from biocompatibility analysis show a very good biocompatibility of the tested dyes in concentrations
of 0.01, 0.1, 1, and 10 µM while the viability of MRC-5 cells in the presence of exanimated dyes was around
100%. The color of the dye solution at concentrations higher than 10 µM was not adequate for performing the
color MTT assay that was used in this investigation. This is because there are no results about biocompatibility
at higher concentrations. The present biocompatibility analysis clearly demonstrated the nontoxic effect of the
investigated dyes on the normal human fibroblast cell line in the concentration range from 0.01 to 10 µM, qual-
ifying them as suitable compounds for potential application in the treatment of some pathological conditions
in humans.
2.3. Antiproliferative activity
According to the good biocompatibility of the investigated dyes and the potential for anticancer activity of
compounds with similar structure shown in literature,41 the antiproliferative activity of the investigated dyes
against cancer cell lines HCT-116 and MDA-MB-231 has been tested.40
The assessment of the in vitro antiproliferative activity of the dyes against the human breast cancer
MD-MB-231 cell line was performed, and the results are presented in Figure 6. It can be noticed that both dyes
expressed antiproliferative activities against MD-MB-231 cells. Dye 3b exhibited greater potential for inhibition
of MD-MB-231 cell proliferation compared to dye 3a, which expressed antiproliferative activity only at higher
concentrations (10 and 1 µM). The influence of dye structure on proliferative activity was observed.
Figure 6. The effect of the investigated dyes at concentrations of 0.01, 0.1, 1, and 10 µM on the inhibition of proliferation
of MDA-MB-231 cells (*P < 0.05 vs. control nontreated cells).
Furthermore, the noted antiproliferative activity of the dyes against the human breast cancer MD-MB-231
cell line was checked against another cell line, colon cancer HCT-116, and these results are presented in Figure
7. The antiproliferative activities of dyes against those cells were not as satisfactory as the detected activities
against the MD-MB-231 cells. Both dyes exhibited slight potential for inhibition of HCT-116 cell proliferation
at lower concentrations (0.1 and 0.01 µM). These results indicate that the antiproliferative activity of the
investigated dyes was determined by the type of tumor cells.
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Figure 7. The effect of the investigated dyes at concentrations of 0.01, 0.1, 1, and 10 µM on the inhibition of proliferation
of HCT-116 cells (*P < 0.05 vs. control nontreated cells).
2.4. ADMET factor profiling
One of the most important factors in the design of biologically therapeutic species is their oral bioavailability.
In order to determine which of the investigated compounds possess pharmacokinetic activity, their structural
properties have been tested by the empirical ‘rule of five’ established by Lipinski.42 The rule states that most
‘drug-like’ molecules have to satisfy the following four structural features: molecular weight lower than 500; a
maximum of 5 atoms that are proton-donors of the hydrogen bonds (–OH and –NH); a maximum of 10 atoms
(N and O) that are hydrogen bond acceptors; and a calculated value of the logarithm of the octanol–water
partition coefficient (log P) lower than 5. Molecules satisfying these rules may potentially exhibit good in vivo
permeability.43 Additional structural features that have to be considered in attempts to propose a corresponding
SAR model of investigated molecules are number of rotatable bonds lower than 8 and low polar surface area
2
˚
(<140 A ).
In accordance with the biological data and ADMET profiling, 3b has been highlighted as a promising
antiproliferative agent against MD-MB-231 cells (Table 2). The main difference between 3a and 3b is in the log
P-value, which seems to be crucial for the determination of the biological activity. Therefore, further studies
have to be directed towards the synthesis of novel 3b derivatives, which may potentially exhibit excellent
biological activities against different cancer cells.
3. Experimental
3.1. Materials and methods
All chemical reagents were obtained commercially from Aldrich (USA), Fluka (Germany), Fischer (USA), and
Merck (Germany) and were used without any further purification. The melting points were determined in
capillary tubes using a Stuart SMP30 melting point system (UK). The IR spectra were obtained using a Bomem
MB-Series 100 Fourier transform-infrared (FT-IR) spectrophotometer (USA) in the form of KBr pellets. The
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Table 2. Evaluation of drug candidates. All parameters are obtained from the program Molinspiration Cheminformatics
2017.
Molecular
weigh
Hydrogen bonds
Donorsa Acceptorsb bonds
Rotatable Topological polar
No.
miLog P
2
˚
surface area/A
126.80
3a
3b
370.37
3.11
4.46
< 5
3
8
3
432.44
3
8
4
126.80
Ideal compound < 500
< 5
< 10
< 8
< 140
a
A donor indicates any O–H or N–H group.
bAn acceptor indicates any O or N, including those in donor groups.
1 H and 13 C spectra were taken on a Varian Gemini 2000 (USA; 200 Hz and 50 Hz, respectively) in deuterated
dimethyl sulfoxide (DMSO-d6) with tetramethylsilane (TMS) as an internal standard at room temperature
(25 ◦ C). Elemental analysis was performed using a Vario EL III elemental analyzer (Germany). The ultraviolet-
visible (UV-Vis) absorption spectra were recorded on a Shimadzu 1700 spectrophotometer (Japan) in the region
of 300–600 nm at a concentration 1 × 10−5 mol dm−3 . The obtained dyes were analyzed by use of a high-
performance liquid chromatograph (Surveyor, Thermo Fisher Scientific, USA) coupled to a linear ion trap mass
spectrometer (LTQ XL, Thermo Fisher Scientific, USA). The flow rate of the mobile phase was 0.3 mL min−1
,
and the mobile phase contained 40% water, 50% methanol, and 10% formic acid (0.1%). Mass spectra and MS2
spectra were obtained by the use of electrospray ionization in the positive ionization mode.
3.2. Synthesis
3.2.1. Synthesis of 4-(1H -benzo[d]imidazol-2-yl)aniline (1)
4-(1H -Benzo[d]imidazol-2-yl)aniline was synthesized from o-phenylenediamine and p-aminobenzoic acid in the
presence of an acid as a dehydrating agent.31,32
A mixture of o-phenylenediamine (30 mmol) and p-aminobenzoic acid (45 mmol) was refluxed in
o-phosphoric acid (30 mL) at 180–200 ◦ C for 4 h on an oil bath. After that, the reaction mixture was
cooled to 50–60 ◦ C and poured onto crushed ice. The reaction mixture was then neutralized with a sodium
hydroxide solution (10%, 70 mL) and the precipitate was collected by filtration. The raw product was washed
with an excess 10% sodium hydroxide solution and water and then dried. The product was further purified by
crystallization from ethanol. Mp: 248.1–249.8 ◦ C (lit. mp: 248 ◦ C44 ; yield = 55%; IR (KBr) νmax /cm−1
:
3438 (NH benzimidazole), 3360, 3216 (NH2 aminophenyl), 3050 (CH arom), 1621 (C=N), 1605 (C=C arom).
1 H NMR (DMSO-d6) δ/ppm: 5.66 (2H, s, NH2 aminophenyl), 6.73 (2H, d, J = 8 Hz, Ar), 7.14–7.20 (2H, m,
Ar), 7.51–7.58 (2H, m, Ar), 7.91 (2H, d, J = 8 Hz, Ar), 12.42 (1H, brs, NH benzimidazole).
3.2.2. Synthesis of 2-pyridones
2-Pyridones were prepared according to literature procedures33,34 from an ester and cyanoacetamides.
6-Hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (2a)
A mixture of ethyl acetoacetate (0.012 mol), cyanoacetamide (0.019 mol), and potassium hydroxide (0.014 mol)
in methanol (20 mL) was mixed and heated under reflux for 1 h. The reaction mixture was cooled and filtrated.
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The obtained crystals were dissolved in hot water and filtrated, and after cooling to room temperature, the
solution was acidified with hydrochloric acid. The formed crystals were isolated by filtration, then washed with
water and methanol. The product was further purified by crystallization from acetone. Mp: 315.1–316.2 ◦ C
(lit. mp: 315–316 ◦ C;35 yield = 50%; IR (KBr) νmax /cm−1 : 3420 (OH), 3367 (NH), 2224 (CN), 1603 (C =
O). 1 H NMR (DMSO-d6) δ/ppm: 2.23 (3H, s, CH3), 5.59 (1H, s, Ar).
3.2.2.2. 6-Hydroxy-2-oxo-4-phenyl-1,2-dihydropyridine-3-carbonitrile (2b)
A mixture of ethyl benzoylacetate (10 mmol), cyanoacetamide (10 mmol), and potassium hydroxide (10 mmol)
was heated under reflux in ethanol (10 mL) for 20 h. After cooling to room temperature, the product was isolated
by filtration and then purified by crystallization from ethanol. Mp: 279.1–280.0 ◦ C (lit. mp: 280 ◦ C);45 yield
= 55%; IR (KBr) νmax /cm−1 : 3418 (OH), 3320 (NH), 2227 (CN), 1653 (C=O); 1 H NMR (DMSO-d6) δ/ppm:
6.27 (1H, s, Pyr); 7.40–7.61 (5H, m, Ar); 11.73 (1H, s, OH).
3.2.3. Synthesis of dyes
Both azo compounds were synthesized according to the procedure described as published before35 starting
from 4-(1H-benzo[d]imidazol-2-yl)aniline. The products were purified by crystallization from N ,N -dimethyl
formamide. Both dyes were obtained as dark red solids.
General procedure: The corresponding 2-pyridone (3 mmol) was first dissolved in 4 mL of sodium
hydroxide solution (3 mmol) and then cooled to 0–5 ◦ C. A diazonium solution was prepared by the addition of
a sodium nitrate solution (3.3 mmol) in water (1.5 mL) to a solution of 4-(1H-benzo[d]imidazol-2-yl)aniline (3
mmol) in conc. hydrochloric acid (1.1 mL) cooled to 0–5 ◦ C. The 2-pyridone solution was then slowly added
to the cold diazonium solution for 10 min and stirred at 0–5 ◦ C for 4 h. The formed solid was isolated by
filtration, washed with ethanol, and then dried (Figure 1).
3.2.3.1. 5-((4-(1H -Benzo[d]imidazol-2-yl)phenyl)diazenyl)-6-hydroxy-4-methyl-2-oxo-1,2-dihyd-
ropyridine-3-carbonitrile (3a)
Yield: 41%; mp >330 ◦ C; FT-IR (KBr, νmax /cm−1): 3426 (NH on hydrazone form), 2222 (CN), 1655, 1640
(C=O on heterocyclic); 1 H NMR (DMSO-d6) δ/ppm: 2.61 (3H, s, CH3), 7.25–7.32 (2H, m, Ar-H), 7.63–7.72
(2H, m, Ar-H), 7.89 (2H, d, J = 8.8 Hz, Ar-H), 8.32 (2H, d, J = 8.8 Hz, Ar-H), 12.15 (1H, s, NH pyridone),
13.05 (1H, s, NH imidazole), 14.71 (1H, s, NH hydrazone); 13 C NMR (50 MHz, DMSO-d6) δ/ppm: 17.0 (Me),
101.6 (Py), 115.4 (CN), 118.1 (Ar), 122.8 (Ar), 124.7 (Py), 126.8 (Ar), 128.3 (Ar), 130.5 (Ar), 143.0 (Ar), 151.9
(Ar), 160.7 (Py), 161.2 (Py), 161.9 (Py); Anal. Calcd for C20 H14 N6 O2 (370.12): C, 64.86; H, 3.81; N, 22.69.
Found: C, 64.05; H, 3.79; N, 22.21. ESI-MS (m/z, positive mode) = 371.10 (molecular ion peak), 209.10 (base
ion peak).
3.2.3.2. 5-((4-(1H -Benzo[d]imidazol-2-yl)phenyl)diazenyl)-6-hydroxy-2-oxo-4-phenyl-1,2-dihyd-
ropyridine-3-carbonitrile (3b)
Yield: 45%; mp 300.7–301.5 ◦ C; FT-IR (KBr, νmax /cm−1): 3424 (NH on hydrazone form), 2225 (CN), 1639,
1619 (C=O on heterocyclic); 1 H NMR (DMSO-d6) δ/ppm: 7.18–7.26 (2H, m, Ar-H), 7.48–7.70 (7H, m, Ar-H),
7.38 (2H, d, J = 8 Hz, Ar-H), 8.12 (2H, d, J = 10 Hz, Ar-H), 12.03 (1H, s, NH pyridone), 12.99 (1H, s, NH
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MIJIN et al./Turk J Chem
imidazole), 14.55 (1H, s, NH hydrazone); 13 C NMR (DMSO-d6) δ/ppm: 101.1 (Py), 115.2 (Ar), 115.4 (CN),
117.6 (Ar), 123.0 (Ar), 124.5 (Py), 127.2 (Ar), 128.1 (Ar), 129.8 (Ar), 130.3 (Ar), 133.0 (Ar), 138.7 (Ar), 142.7
(Ar), 150.3 (Ar), 160.9 (Py), 161.2 (Py), 161.8 (Py); Anal. Calcd for C25 H16 N6 O2 (432.13): C, 69.44; H, 3.73;
N, 19.43. Found: C, 68.25; H, 3.65; N, 18.95. ESI-MS (m/z, positive mode) = 433.20 (molecular ion peak),
209.10 (base ion peak).
3.3. Biological evaluation
3.3.1. Compounds and solutions
MTT was dissolved (5 mg cm−3) in phosphate-buffered saline (pH 7.2) and filtered (0.22 µm) before use. RPMI
1640 cell culture medium, fetal bovine serum (FBS), and MTT were purchased from Sigma Chemical Company
(USA).
3.3.2. Cell lines
Normal human fibroblast (MRC-5), human colon cancer (HCT-116), and human breast cancer (MDA-MB-
231) cell lines were maintained in monolayer cultures using nutrient medium RPMI 1640 with 10% FBS and
antibiotics.
3.3.3. Biocompatibility assay
The biocompatibility testing of newly synthesized compounds is the first step in their biological characterization.
The normal human fibroblast cell line (MRC-5) was used for the evaluation of biocompatibility. MRC-5 cells
were seeded in 96-well microtiter plate and cultivated in full culture media for 24 h. After that, the investigated
compound was added to cells in final concentrations of 0.01, 0.1, 1, 10, and 100 µM, except in the control
wells, where only the medium was added. Thus, prepared cell cultures were incubated for an additional 24 h.
At the end of this period, the MTT test,46 as modified by Ohno and Abe,40 was used for measuring the cell
viability/proliferation.
3.4. Treatment of cell lines for antiproliferative in vitro screening
The target cell lines HCT-116 and MDA-MB-231 were seeded in quadruplicates into 96-well, flat-bottomed
microtiter plates in 0.1 cm3 of culture medium. After cell adaptation and the adherence of both cell lines, 0.1
cm3 of the investigated compound was added to cells in final concentrations of 0.01, 0.1, 1, 10, and 100 µM,
except in the control wells, where only the medium was added to the cells and was incubated for an additional
24 h. The effect of the investigated compounds on cell survival was determined at the end of the incubation
period by the MTT test40 as described in the previous paragraph. The antiproliferative effect of the compounds
was expressed as a percentage of viable cells.
Acknowledgment
This work was financially supported by Ministry of Education, Science, and Technological Development, Re-
public of Serbia, under Grants No. 172013 and 173052.
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