Synthesis and anticancer activity of new azo compounds containing extended π-conjugated systems

Article abstract of DOI:10.1007/s11696-017-0140-9

A series of novel azo compounds with extended π-conjugated systems were prepared by azo coupling reaction compounds trans-2-(4′-aminostyryl)-thiophene, 1-(4-aminophenyl)-4-phenyl-1,3-butadiene and 4-amino-4′-methoxystilbene with some phenols. The compounds were evaluated for their cytotoxicity against breast cancer adenocarcinoma (MCF-7), cervix adenocarcinoma (HeLa) and human embryonic kidney (HEK 293) cell lines using the MTT assay. The results showed all derivatives had more toxic effects than tamoxifen. Of all the compounds tested, the azo product obtained from coupling trans-2-(4′-Aminostyryl)-thiophene with 2-naphthol (compound 5b) exhibited the potent in vitro antiproliferative activity with IC₅₀ 27 ± 1 and 18 ± 0 μg/mL against MCF-7 and HeLa cell lines, respectively, while it was devoid of any cytotoxicity against normal HEK 293 cells even at 200 μg/mL.

Full text of DOI:10.1007/s11696-017-0140-9

Chem. Pap.
DOI 10.1007/s11696-017-0140-9
ORIGINAL PAPER
Synthesis and anticancer activity of new azo compounds
containing extended p-conjugated systems
Esmail Rezaei-Seresht1 Erfan Mireskandari1 Mitra Kheirabadi2
•
•
•
Hamid Cheshomi3 Hasan Rezaei-Seresht4 Leila Sadat Aldaghi3
•
•
Received: 25 September 2016 / Accepted: 30 January 2017
Ó Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract A series of novel azo compounds with extended
p-conjugated systems were prepared by azo coupling
reaction compounds trans-2-(40-aminostyryl)-thiophene,
1-(4-aminophenyl)-4-phenyl-1,3-butadiene and 4-amino-
40-methoxystilbene with some phenols. The compounds
were evaluated for their cytotoxicity against breast cancer
adenocarcinoma (MCF-7), cervix adenocarcinoma (HeLa)
and human embryonic kidney (HEK 293) cell lines using
the MTT assay. The results showed all derivatives had
more toxic effects than tamoxifen. Of all the compounds
tested, the azo product obtained from coupling trans-2-(40-
Aminostyryl)-thiophene with 2-naphthol (compound 5b)
exhibited the potent in vitro antiproliferative activity with
IC50 27 1 and 18 0 lg/mL against MCF-7 and HeLa
cell lines, respectively, while it was devoid of any cyto-
toxicity against normal HEK 293 cells even at 200 lg/mL.
Introduction
Breast cancer is the leading cause of malignancy among
women. Main cause of breast cancer is overexpression of
estrogen receptor alpha (ERa) (Hayashi et al. 2003).
Therefore, ERa is used as a target for prevention of
breast cancer. Tamoxifen (Fig. 1) is a commercially
available drug which is currently used for the treatment
of several types of breast cancer in women, and as a
hormone treatment for male breast cancer (Pettit and
Lippert 2000). Tamoxifen is a stilbene-based compound
that binds with the estrogen receptor and blocks its
function and prevents the proliferation of breast cells or
in other words, prevents from breast cancer (Fisher et al.
1998). Although anti-estrogenic therapies are highly
valuable to improve the prognosis of breast cancer
patients, a large portion of breast tumors develop resis-
tance towards anti-hormonal drugs (Josefsson and Lein-
ster 2010; Musgrove and Sutherland 2009). Therefore,
new compounds are required to replace the anti-hormone
drugs such as tamoxifen which is subjected to resistance
by tumors.
Keywords Breast cancer MCF-7 Á Anticancer activity Á
Synthesis Á Azo compounds
Azo compounds constitute one of the main chemical
classes of organic compounds owing to their diverse
applications in various fields, such as dying textile fibers,
biomedical studies, liquid crystalline displays, lasers,
electro-optical devices and ink jet printer (Peters and
Freeman, 1991; Gregory 2012; Catino and Farris 1985).
They also show a variety of interesting biological
activities, including antibacterial (Awad et al. 1998;
Phillips et al. 2009; Ibrahim et al. 1991), pesticidal
(Jarrahpour et al. 2004), and antiseptic (Samadhiya and
Halve 2001) activities. Recently, some of azo com-
pounds have been introduced as potent vesicular gluta-
mate transporters inhibitors (Favre-Besse et al. 2014).
& Esmail Rezaei-Seresht
e.rezaei@hsu.ac.ir
1
Department of Chemistry, School of Sciences, Hakim
Sabzevari University, Sabzevar, Iran
2
Department of Biology, School of Sciences, Hakim Sabzevari
University, Sabzevar, Iran
3
Cellular and Molecular Research Center, Sabzevar University
of Medical Sciences, Sabzevar, Iran
4
Traditional and Complementary Medicine Research Center,
Sabzevar University of Medical Sciences, Sabzevar, Iran
123

Chem. Pap.
Synthesis
4-Nitrobenzyl triphenylphosphonium bromide (2)
This compound was prepared as described previously
(Moran et al. 2009). Mp = 275–277 °C [lit. (Wiederrecht
et al. 1995) = 275 °C].
O
N
General procedure for synthesis of compounds
1a–c (Moran et al. 2009)
Fig. 1 Chemical structure of tamoxifen
Salt 2 (3.90 g, 8.15 mmol) and aldehyde (10.35 mmol)
were added to dichloromethane (150 mL) and stirred at
room temperature. A sodium hydroxide solution (50% w/v)
was added dropwise to the stirred solution (0.4 mL NaOH
solution per 1 mmol of aldehyde). The solution was stirred
at room temperature for 1.5 h and then washed with brine
and water. The solvent was removed in vacuo to yield the
crude product. Finally, recrystallization from the appro-
priate solvent gave the pure compound 1.
Singh et al. (2014) have reported two novel series of
1,2,3-triazole linked azo dyes with good activity against
six microbial strains. Moreover, the anticancer and
antitumor activities of azo compounds have been the
subject of several studies: for example some azo-based
prodrugs have indicated activity against human col-
orectal cancer cell lines (Sharma et al. 2013), azo dyes
derived from Sudan I and Sudan III have shown
chemoprotective activity in vivo (De Long et al. 1986),
and thiophene-based azo dyes have exhibited good
antitumor activity (Gouda et al. 2016).
(E)-2-(4-nitrostyryl)thiophene (1a)
This compound was prepared as described in the general
procedure. Yield 39%, yellow solid. Mp 172–174 °C
(EtOH) [lit. (Freund 1953) = 168–169 °C]. IR (KBr,
cm-1) m = 1332 and 1502 (NO2 stretching bands).
Therefore, the easy and successful synthesis of azo
compounds by the azo coupling reaction, and recent con-
siderations on the anticancer activity of azo compounds
(Sharma et al. 2013; Song et al. 2012; Kantar et al. 2015)
prompted us to synthesize and evaluate anticancer activity
of some new azo compounds. Here, we report the synthesis
and bioactivities of a series of new azo compounds pos-
sessing an extended p-conjugated moiety as anticancer
agents.
1-Nitro-4-((1E,3E)-4-phenylbuta-1,3-dien-1-
yl)benzene (1b)
This compound was prepared as described in the general
procedure. Yield 33%, yellow solid. Mp = 171–172 °C
(EtOH) [lit. (Bergmann and Weinberg 1941) = 172 °C].
IR (KBr, cm-1) m = 1334 and 1512 (NO2 stretching
bands).
Experimental
Chemicals
(E)-1-methoxy-4-(4-nitrostyryl)benzene (1c)
This compound was prepared as described in the general
All solvents were redistilled prior to use. Reagents and
starting materials were purchased from Merck and used as
received. All reactions were monitored by TLC on com-
mercially available precoated glass plates (silica gel
60F254, Merck) and the plates were visualized in UV light
(254 nm). Melting points were recorded on an Elec-
trothermal-9200 melting point apparatus and are uncor-
rected. The IR spectra were obtained on a 8400 Shimadzu
spectrometer and only noteworthy absorptions are listed.
1H NMR and 13C NMR spectra were recorded on a Bruker
Avance 300 MHz instrument using TMS as internal ref-
erence. Elemental analysis data were obtained using a
LECO TruSpec analyzer.
procedure.
Yield
25%,
bright
yellow
solid.
Mp = 148–150 °C (EtOH–H2O, 1:1, v/v) [lit. (Manecke
and Lu¨uttke 1970) = 150–151 °C]. IR (KBr, cm-1
m = 1328 and 1504 (NO2 stretching bands).
)
General procedure for synthesis of compounds 4a–c
Reduction of compound 1 was performed according to the
literature procedure (Gao et al. 2012). A solution of
ammonium chloride (0.58 g, 10.80 mmol) in water (3 mL)
was added to a solution of compound 1 (5.20 mmol) in
acetone (15 mL). After heating the mixture to boiling
123

Chem. Pap.
4-((E)-(4-((E)-2-(thiophen-2-
yl)vinyl)phenyl)diazenyl)benzene-1,3-diol (5a)
point, the oil bath was removed and zinc (1.17 g,
18.00 mmol) was added in small portions. Once the reac-
tion subsided, an additional amount of zinc (0.58 g,
9.00 mmol) was added and the reaction mixture refluxed
for 2 h. The precipitate was filtered off while hot and the
filtrate was evaporated until an aqueous solution remained.
This was basified with 2 M NaOH aqueous solution until
pH 10, followed by extraction of the suspension with
dichloromethane. The organic phase was separated, dried
(Na2SO4) and the solvent was evaporated to give an orange
solid of compound 4.
This compound was prepared according to the general
procedure using 4a and resorcinol. Yield 40%.
Mp = 182–185 °C (EtOH–H2O, 2:1, v/v). IR (KBr, cm-1
)
m = 1097, 1224, 1492, 1595, 3301, 3529. 1H NMR
(300 MHz, acetone-d6, ppm): d 6.45 (d, J = 2.5 Hz, 1H),
6.65 (dd, J = 2.5, 8.8 Hz, 1H), 7.03–7.08 (m, 4H), 7.25 (d,
J = 3.3, 1H), 7.42 (d, J = 5.1 Hz, 1H), 7.57 (d,
J = 16.1 Hz, 1H), 7.72–7.77 (m, 3H), 7.85 (d, J = 8.6 Hz,
2H). 13C NMR (75 MHz, acetone-d6, ppm): d 103.2, 114.9,
122.8, 126.1, 127.0, 127.9, 128.8, 129.6, 130.6, 131.9,
133.6, 140.6, 142.7, 150.2, 156.6, 163.5. CHN analysis
(%): calculated: C, 67.06; H, 4.38; N, 8.69; found: C,
66.86; H, 4.19; N, 8.31.
(E)-4-(2-(thiophen-2-yl)vinyl)aniline (4a)
This compound was prepared as described in the general
procedure.
Mp = 122–125 °C
[lit.
(Freund
1953) = 128–129 °C]. IR (KBr, cm-1) m = 3442 and 3353
(NH2 stretching bands).
1-((E)-(4-((E)-2-(thiophen-2-
yl)vinyl)phenyl)diazenyl)naphthalen-2-ol (5b)
4-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)aniline (4b)
This compound was prepared according to the general
procedure using 4a and 2-naphthol. Yield: 79%.
Mp = 138–140 °C (MeOH–EtOH, 1:2, v/v). IR (KBr,
cm-1) m = 925, 1184, 1247, 1488, 1558, 1588, 3008, 3342,
This compound was prepared as described in the general
procedure. Mp = 125–127 °C [lit. (Lee et al.
1999) = 123–125 °C]. IR (KBr, cm-1) m = 3375 and 3330
(NH2 stretching bands).
1
3501. H NMR (300 MHz, acetone-d6, ppm): d 6.97 (d,
J = 9.3 Hz, 1H), 7.02–7.09 (m, 2H), 7.20–7.24 (m, 2H),
7.38–7.62 (m, 4H), 7.73–7.78 (m, 3H), 7.88–7.92 (m, 2H),
8.67 (d, J = 8.0 Hz, 1H), 15.78 (s, 1H). 13C NMR
(75 MHz, acetone-d6, ppm): d 115.6, 122.0, 122.4, 125.6,
126.2, 128.1, 128.6, 128.8, 129.1, 129.6, 130.6, 131.1,
131.9, 133.2, 133.6, 140.6, 141.4, 142.7, 143.2, 175.8.
CHN analysis (%): calculated: C, 74.13; H, 4.52; N, 7.86;
found: C, 74.34; H, 4.35; N, 7.69.
(E)-4-(4-methoxystyryl)aniline (4c)
This compound was prepared as described in the general
procedure. Mp = 160–163 °C [lit. Papper et al.
2014) = 162–164 °C]. IR (KBr, cm-1) m = 3380 and 3292
(NH2 stretching bands).
General procedure for synthesis of compounds 5a–e
4-((E)-(4-((1E,3E)-4-phenylbuta-1,3-dien-1-
yl)phenyl)diazenyl)benzene-1,3-diol (5c)
A freshly prepared cold (ca. 5 °C) solution of sodium nitrite
(0.37 g, 5.50 mmol) in water (3 mL) was added portionwise
to a cold (ca. 5 °C) stirred solution of compound 4
(5.00 mmol) in aqueous HCl (50% v/v, 4 mL) at such a rate
so as to maintain the temperature below ca. 10 °C. Upon
completion of the addition, the cold solution was stirred for
20–25 min. The cold solution of the diazonium salt was
slowly added to a cold stirred solution/suspensionofaphenol
(resorcinol or 2-naphthol) (5.00 mmol) in aqueous NaOH
(10% w/v, 5 mL). The resulting cold intense red-orange
suspension was stirred for 1.5 h. The precipitated crude
product was collectedby vacuum filtration, washed well with
water and a minimum of diethyl ether, and then dried at
40 °C. Finally, recrystallization from the appropriate solvent
gave the pure compound 5.
This compound was prepared according to the general
procedure using 4b and resorcinol. Yield: 24%.
)
Mp = 109–112 °C (EtOH–H2O, 1:1, v/v). IR (KBr, cm-1
m = 981, 1103, 1155, 1473, 1596, 1664, 3016, 3257, 3442.
1H NMR (300 MHz, acetone-d6, ppm): d 6.45 (d,
J = 2.5 Hz, 1H), 6.55 (dd, J = 2.5, 8.8 Hz, 1H), 6.71 (d,
J = 15.2 Hz, 1H), 6.81–7.04 (m, 3H), 7.12–7.27 (m, 4H),
7.37–7.50 (m, 4H), 7.61 (d, J = 8.4 Hz, 2H), 7.85 (d,
J = 8.4 Hz, 2H). 13C NMR (75 MHz, acetone-d6, ppm): d
103.2, 114.9, 122.7, 126.1, 126.4, 127.0, 127.5, 127.9,
128.5, 129.2, 132.6, 137.3, 138.4, 150.0, 156.6, 163.5.
CHN analysis (%): calculated: C, 77.17; H, 5.30; N, 8.18;
found: C, 77.11; H, 5.23; N, 7.92.
123

Chem. Pap.
1-((E)-(4-((1E,3E)-4-phenylbuta-1,3-dien-1-
yl)phenyl)diazenyl)naphthalen-2-ol (5d)
dissolve the formazan crystals. The absorbance at
A540 nm was determined on a ELISA microplate reader
(Awareness, USA). IC50 values were determined from log
plots of percent of control vs. concentration. Each com-
pound was assayed twice in quadruplicate.
This compound was prepared according to the general pro-
cedure using 4b and 2-naphthol. Yield: 36%.
)
Mp = 79–81 °C (MeOH–EtOH, 1:2, v/v). IR (KBr, cm-1
1
m = 983, 1134, 1234, 1492, 1569, 3008, 3188, 3415. H
NMR (300 MHz, acetone-d6, ppm): d 6.74 (d, J = 15.2 Hz,
1H), 6.97–7.11 (m, 3H), 7.25–7.36 (m, 3H), 7.49–7.62 (m,
4H), 7.68 (d, J = 7.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H),
7.88–7.94 (m, 3H), 8.60 (d, J = 8.0 Hz, 1H), 15.82 (s, 1H).
13C NMR (75 MHz, acetone-d6, ppm): d 115.6, 122.0, 122.4,
125.6, 126.2, 126.4, 127.5, 128.1, 128.5, 128.6, 129.1, 129.2,
131.1, 132.6, 133.2, 137.3, 138.4, 141.4, 143.2, 175.8. CHN
analysis(%): calculated: C, 82.95; H, 5.36; N, 7.44; found: C,
82.74; H, 5.28; N, 7.26.
Statistical analysis
Experimental values are presented as the mean standard
deviation (SD) for four independent experiments. Analysis
of variance (ANOVA) was performed using GraphPad
Prism 6 software. Statistical significance was defined when
P \ 0.05 (Scheme 1).
Results and discussion
Chemistry
4-((E)-(4-((E)-4-methoxystyryl)phenyl)diazenyl)
benzene-1,3-diol (5e)
At first, we prepared 4-nitro compounds 1 via the Wittig
reaction of the phosphonium salt 2 with 4-methoxyben-
zaldehyde (3a), cinnamaldehyde (3b) and thiophene-2-car-
baldehyde (3c). The trans isomer was easily separated from
the cis and other impurities by recrystallization in ethanol or
ethanol–water. In the case of cinnamaldehyde, the Wittig
product was a 1,4-diarylbutadiene system. An anticoccidial
activity for some 1,4-diarylbutadienes has also been reported
by Gage et al. (2003). Compounds 1a–c were reduced to the
corresponding 4-amino compounds 4a–c in high yields
using Zn/NH4Cl. These compounds were then used as
suitable substrates for diazo coupling reaction. Compounds
4a–c were readily coupled with two phenols, including
resorcinol and 2-naphthol, to yield the azo compounds 5a–
e. The structure of azo products 5 was confirmed by spectral
This compound was prepared according to the general
procedure using 4c and resorcinol. Yield: 38%.
)
Mp = 115–118 °C (MeOH–H2O, 1:1, v/v). IR (KBr, cm-1
1
m = 945, 1159, 1240, 1492, 1550, 3296, 3492. H NMR
(300 MHz, acetone-d6, ppm): d 3.82 (s, 3H), 6.41 (d,
J = 2.5 Hz, 1H), 6.54 (dd, J = 2.5, 8.7 Hz, 1H), 6.94 (d,
J = 8.7 Hz, 2H), 7.03 (d, J = 16.5 Hz, 1H), 7.16 (d,
J = 16.5 Hz, 1H), 7.36 (s, 2H), 7.43–7.48 (m, 5H), 7.57 (d,
J = 8.7 Hz, 2H). 13C NMR (75 MHz, acetone-d6, ppm): d
55.5, 103.2, 114.9, 122.8, 126.1, 127.0, 127.9, 128.5,
128.9, 129.4, 130.7, 135.2, 140.8, 150.2, 156.6, 160.7,
163.5. CHN analysis (%): calculated: C, 72.82; H, 5.24; N,
8.09; found: C, 72.79; H, 5.13; N, 8.01.
1
Biological methods
data, particularly by NMR spectra. For example, the H
NMR spectrum of compound 5a shows an upfield aromatic
doublet at d 6.45 ppm with a meta coupling constant
(J = 2.5 Hz) belonging to the proton located between the
two hydroxyl groups and a doublet of doublets signal at d
6.65 ppm with both meta and ortho coupling constants
(J = 2.5, 8.8 Hz, respectively) belonging to the proton
located meta to a hydroxyl group and para to other hydroxyl.
These two signals clearly indicate the presence of the
resorcinol moiety in the structure of 5a. On the other hand,
the two doublet signals at 7.25 and 7.42 ppm with unusual
3J values of 3.3 and 5.1 Hz correspond to H-3 and H-5 of a
2-substituted thiophene, respectively. The configuration of
the alkene C=C bond of compound 5a can be easily deduced
by the observed coupling constant (J = 16.1 Hz) for the
doublet signal at d 7.57 ppm, confirming the E configuration
The human breast cancer adenocarcinoma (MCF-7), cer-
vical cancer (HeLa) and human embryonic kidney HEK
293 cells were maintained in DMEM (Dulbecco’s Modified
Eagle Medium) supplemented with 5% fetal bovine serum,
2 mM glutamine, and 100 units/mL penicillin/strepto-
mycin. Cells were incubated at 37 °C in a 5% CO2 atmo-
sphere. The MTT (methyl thiazolyl tetrazolium)
colorimetric assay was used to determine growth inhibi-
tion. Cells were plated in 96-well plates and allowed to
attach for 24 h. All derivatives were dissolved in DMSO at
10 mM. Cells in quadruplicate wells were exposed to the
individual all derivatives at 5, 10, 20, 50, 75, 100, 130, 175
and 200 lg/mL for 48 h. After 48 h, the medium was
aspirated and 100 lL of 1 mg/mL MTT solution (Sigma
Chemical Co.) diluted in serum-free medium was added to
each well. After 4 h of incubation, the MTT solution was
removed, and 100 lL of DMSO was added to each well to
1
of the alkene bond. Moreover, inspection of the H NMR
spectra of these compounds revealed that in compounds 5b
and 5d containing.
123

Chem. Pap.
Y
Br
PPh3
Br
YCHO
PPh3
:
=
=
1a Y
Benzene
Wittig reaction
N
O2
N
1
O2
N
O2
S
2
Zn Reduction
:
1b Y
Y
Y
ArOH
N
:
=
1c Y
Diazo coupling
Ar
N
H2N
OMe
5
4
OH
OH
:
=
5c
=
=
=
=
=
Ar
Ar
Ar
Y
;
:
=
=
Y
Y
;
5a
Ar
S
S
OH
OH
OH
;
OH
:
5d
=
=
:
Y
5b
Ar
;
;
OH
:
5e
Y
OMe
OH
Scheme 1 Synthesis of azo compounds 5a-e
The 2-naphthol moiety, there is a highly downfield
resonance that it appears at 15.78 and 15.82 ppm, respec-
tively. We performed a literature survey to find cause of
this uncommon chemical shift for the phenolic protons in
5b and 5d. Surprisingly, we found several reports indi-
cating the presence of a highly downfield proton signal in
...
O
H
N
N
S
1
the H NMR spectrum of the 1-arylazo-2-naphthols due to
existence of an azo-hydrazo tautomerism where in the
hydrazo form is the preferable tautomer (Ball and Nicholls
1982; Ferreira et al. 2013; Rauf et al. 2015). For example,
Sudan I dye which is a familiar 1-arylazo-2-naphthol
compound and structurally similar to compounds 5b and
5d, affords the hydrazo form as the predominant one
(Ferreira et al. 2013). Therefore, because of the
intramolecular N–HÁÁÁO hydrogen bonding forming a six-
membered ring in the hydrazo form (Fig. 2), the NH proton
signal appears as a highly deshielded resonance at 15.78
(for 5b) or 15.82 ppm (5d).
Fig. 2 Representation of the hydrazo form of compound 5b (dashed
lines indicate the hydrogen bonding)
chemotherapy; therefore, all the compounds were evalu-
ated for possible cytotoxicity in normal cells, HEK 293 that
shown in (Fig. 3). Of the active molecules, three com-
pounds 5a, 5c and 5d induced significant cell death in HEK
293 cells. Compounds 5b and 5e showed no cytotoxic
effect on HEK 293 cells, suggesting that the molecules are
particularly active against breast cancer and cervical cancer
cell lines (Table 1).
Biological activity
Antiproliferative activity against breast cancer (MCF-7)
Cytotoxicity towards normal cells
All the targeted compounds were screened for their
antiproliferative activity on breast cancer cell lines (MCF-7
cells) using MTT assay. The results are shown in Fig. 4.
Specific killing of the cancer cells without affecting the
normal cell growth is a key safety feature of cancer
123

Chem. Pap.
100
90
80
70
60
50
40
30
20
10
0
100
80
60
40
20
0
0
50
5a
100
150
200
250
0
50
100
150
200
250
-20
Concentraꢀon (μg/mL)
Concentraꢀon (μg/mL)
5a
5b
5c
5d
5e
TMX
5b
5c
5d
5e
TMX
Fig. 3 Cytotoxic effect of compounds 5a–e and tamoxifen (TMX) on
HEK 293 cells were determined by MTT assay. HEK 293 cells were
treated with 5a–e at the concentration of 5–200 lg mL-1 for 48 h.
The viability percentage of cells was indicated
Fig. 5 Cytotoxic effect of compounds 5a–e and tamoxifen (TMX) on
HeLa cells were determined by MTT assay. HeLa cells were treated
with 5a–e at the concentration of 5–200 lg/mL for 48 h. The viability
percentage of cells was indicated
azo compounds containing the thiophene moiety (5a and
5b) were the most active, while the derivatives having the
1,3-butadiene and 4-methoxyphenyl moieties (5c–e) were
the least active ones.
Table 1 Comparison of antiproliferative activity (IC50) compound
5a-e with tamoxifen against MCF-7, HeLa and HEK 293
Compound
IC50 (lg/mL)a
MCF-7
HeLa
HEK 293
5a
24
27
1
1
2
0
7
2
42
18
11
79
46
54
5
0
1
4
2
3
31
2
Antiproliferative activity against cervical cancer (HeLa)
5b
[200
54
Cytotoxicity of compounds 5a–e was evaluated using MTT
assay (Fig. 5). HeLa cells were incubated with different
concentrations of compound 5 for 48 h. The results showed
that compounds 5b and 5c have the best inhibitory effects
on Hela cell line (Table 1).
5c
44
9
2
5d
51
51
5e
113
91
169 12
142
Tamoxifen
9
a
Cytotoxicity (IC50) for 5a–e and tamoxifen in a panel of various
cancer cell lines, including breast cancer adenocarcinoma cell line
(MCF-7), cervical cancer cell line (HeLa) and human embryonic
kidney (HEK 293) cells. The MTT colorimetric assay was used to
determine growth inhibition. Cells were plated in 96-well plates and
after 48 h of treatment; the values are averaged from at least four
independent experiments
Conclusions
In conclusion, we synthesized five new azo molecules
containing an extended p-conjugated moiety, and their
anticancer activity was investigated using MTT method.
Compound 5b with inhibitory effects better than tamoxifen
as commercial drug on estrogen receptor alpha (ERa) in
the variety of cancers was introduced as a good potent drug
candidate. In addition, compound 5b which has the thio-
phene and 2-naphthol rings, showed the best cytotoxicity
(IC50) activity against HeLa cell line; while it had the least
cytotoxicity against HEK 293 cells.
160
140
120
100
80
60
40
20
0
0
50
5a
100
150
200
250
-20
-40
-60
Concentraꢀon (μg/mL)
5b 5c 5d
5e
TMX
References
Fig. 4 Cytotoxic effect of compounds 5a–e and tamoxifen (TMX) on
MCF-7 cells were determined by MTT assay. MCF-7 cells were
treated with 5a–e at the concentration of 5–200 lg/mL for 48 h. The
viability percentage of cells was indicated
Awad IM, Aly AA, Abdel RA, Ahmed SH (1998) Synthesis of some
5-azo(40-substituted benzene-sulphamoyl)-8-hydroxyquinolines
with antidotal and antibacterial activities. J Inorg Biochem
33:77–89. doi:10.1016/0162-0134(88)80036-9
Compounds 5a, 5b, 5d and 5e were found to inhibit the
growth of MCF-7 cells with IC50 less than 50 lM
(Table 1). A close observation of the data showed that the
Ball P, Nicholls CH (1982) Azo-hydrazone tautomerism of hydrox-
yazo compounds—a review. Dyes Pig 3:5–26. doi:10.1016/
0143-7208(82)80010-7
123

Chem. Pap.
Bergmann F, Weinberg MZ (1941) Remarks on the synthesis of
substituted stilbenes and diphenylbutadienes. J Org Chem
6:134–139. doi:10.1021/jo01201a013
monotherapy, sequenced therapy and extended therapy. Breast J
19:76–83. doi:10.1016/j.breast.2009.12.010
Kantar C, Akal H, Kaya B, Islamoglu F, Tu¨rk M, Sasmaz S (2015)
Novel phthalocyanines containing resorcinol azo dyes; synthesis,
determination of pKa values, antioxidant, antibacterial and
anticancer activity. J Organomet Chem 783:28–39. doi:10.
1016/j.jorganchem.2014.12.042
Lee IS, Seo H, Chung YK (1999) Preparation of (thiophene)
manganese tricarbonyl cations for nonlinear optics. Organome-
tallics 18:1091–1096. doi:10.1021/om980922z
Catino S, Farris
E (1985) Concise encyclopedia of chemical
technology. Wiley, New York
De Long MJ, Prochaska HJ, Talalay
P (1986) Induction of
NAD(P)H:quinone reductase in murine hepatoma cells by
phenolic antioxidants, azo dyes, and other chemoprotectors: a
model system for the study of anticarcinogens. Proc Natl Acad
Sci USA 83:787–791
¨
Manecke G, Lu¨uttke S (1970) Uber Synthesen einiger oligomerer
Favre-Besse F-C, Poirel O, Bersot T, Kim-Grellier E, Daumas S, El
Mestikawy S, Acher FC, Pietrancosta N (2014) Design, synthesis
and biological evaluation of small-azo-dyes as potent vesicular
glutamate transporters inhibitors. Eur J Med Chem 78:236–247.
doi:10.1016/j.ejmech.2014.03.056
substituierter Arylenvinylene. Chem Ber 103:700–707. doi:10.
1002/cber.19701030308
Moran BW, Anderson FP, Devery A, Cloonan S, Butler WE,
Varughese S, Draper SM, Kenny PT (2009) Synthesis, structural
characterisation and biological evaluation of fluorinated ana-
logues of resveratrol. Bioorg Med Chem 17:4510–4522. doi:10.
1016/j.bmc.2009.05.007
Musgrove EA, Sutherland RL (2009) Biological determinants of
endocrine resistance in breast cancer. Nature Rev Cancer
9:631–643. doi:10.1038/nrc2713
Ferreira GR, Costa Garcia H, Couri MRC, Dos Santos HF, de Oliveira
LFC (2013) On the azo/hydrazo equilibrium in sudan I azo dye
derivatives.
jp310229h
J Phys Chem A 117:642–649. doi:10.1021/
Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M,
Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly
M, Wieand S, Tan-Chiu E, Ford L, Wolmark N (1998)
Tamoxifen for prevention of breast cancer: report of the
National Surgical Adjuvant Breast and Bowel Project P-1 Study.
J Natl Cancer Inst 90:1371–1388. doi:10.1093/jnci/90.18.1371
Freund W (1953) The reaction of diazonium salts with b-2-
thienylacrylic acid. J Chem Soc 42:2889–2891. doi:10.1021/
j100021a069
Papper V, Pokholenko O, Wu Y, Zhou Y, Jianfeng P, Steele TW,
Marks RS (2014) Novel photochrome aptamer switch assay
(PHASA) for adaptive binding to aptamers.
24:1581–1591. doi:10.1007/s10895-014-1441-9
J Fluoresc
Peters AT, Freeman HS (1991) Colour chemistry: the design and
synthesis of organic dyes and pigments. Elsevier Applied
Science, New York
Gage JL, Kirst HA, O’Neil D, David BA, Smith CK II, Naylor SA
(2003) Synthesis and evaluation of a series of 1,4-diarylbutadi-
enes for anticoccidial activity. Bioorg Med Chem 11:4083–4091.
doi:10.1016/S0968-0896(03)00330-4
Gao F, Ye X, Li H, Zhong X, Wang Q (2012) Evidence for two-
photon absorption-induced ESIPT of chromophores containing
hydroxyl and imino groups. ChemPhysChem 13:1313–1324.
doi:10.1002/cphc.201100885
Gouda MA, Eldien HF, Girges MM, Berghot MA (2016) Synthesis
and antitumor evaluation of thiophene based azo dyes incorpo-
rating pyrazolone moiety. J Saudi Chem Soc 20:151–157.
doi:10.1016/j.jscs.2012.06.004
Gregory P (2012) High-technology applications of organic colorants.
Springer Science & Business Media, New York
Hayashi S, Eguchi H, Tanimoto K, Yoshida T, Omoto Y, Inoue A,
Yoshida N, Yamaguchi Y (2003) The expression and function of
estrogen receptor alpha and beta in human breast cancer and its
clinical application. Endocr Relat Cancer 10:193–202. doi:10.
1677/erc.0.0100193
Ibrahim S, El-Gahami M, Khafagi Z, El-Gyar S (1991) Structure and
antimicrobial activity of some new azopyrazolone chelates of
Ni(II) and Cu(II) acetates, sulfates, and nitrates. J Inorg Biochem
43:1–7. doi:10.1016/0162-0134(91)84063-F
Jarrahpour A, Motamedifar M, Pakshir K, Hadi N, Zarei M (2004)
Synthesis of novel azo Schiff bases and their antibacterial and
antifungal activities. Molecules 9:815–824. doi:10.3390/
91000815
Josefsson ML, Leinster SJ (2010) Aromatase inhibitors versus
tamoxifen as adjuvant hormonal therapy for oestrogen sensitive
early breast cancer in post-menopausal women: meta-analyses of
Pettit GR, Lippert JW 3rd (2000) Antineoplastic agents 429.
Syntheses of the combretastatin A-1 and combretastatin B-1
prodrugs. Anticancer Drug Des 15:203–216
Phillips OA, Udo EE, Abdel-Hamid ME, Varghese R (2009)
Synthesis and antibacterial activity of novel 5-(4-methyl-1H-1,
2, 3-triazole) methyl oxazolidinones. Eur J Med Chem
44:3217–3227. doi:10.1016/j.ejmech.2009.03.024
Rauf MA, Hisaindee S, Saleh N (2015) Spectroscopic studies of keto-
enol tautomeric equilibrium of azo dyes. RSC Adv
5:18097–18110. doi:10.1039/C4RA16184J
Samadhiya S, Halve A (2001) Synthetic utility of Schiff bases as
potential herbicidal agents. Orient J Chem 17:119–122
Sharma R, Rawal RK, Gaba T, Singla N, Malhotra M, Matharoo S,
Bhardwaj TR (2013) Design, synthesis and ex vivo evaluation of
colon-specific azo based prodrugs of anticancer agents. Bioorg
Med Chem Lett 23:5332–5338. doi:10.1016/j.bmcl.2013.07.059
Singh H, Sindhu J, Khurana JM, Sharma C, Aneja K (2014)
Syntheses, biological evaluation and photophysical studies of
novel 1, 2, 3-triazole linked azo dyes. RSC Adv 4:5915–5926.
doi:10.1039/c3ra44314k
Song YM, Ha YM, Kim J-A, Chung KW, Uehara Y, Lee KJ, Chun P,
Byun Y, Chung HY, Moona HR (2012) Synthesis of novel azo-
resveratrol, azo-oxyresveratrol and their derivatives as potent
tyrosinase inhibitors. Bioorg Med Chem Lett 22:7451–7455.
doi:10.1016/j.bmcl.2012.10.050
Wiederrecht GP, Svec WA, Niemczyk MP, Wasielewski MR (1995)
Femtosecond transient grating studies of chlorophylls and a
chlorophyll-based electron donor-acceptor molecule. J Phys
Chem 99:8918–8926. doi:10.1021/j100021a069
123