Synthesis, cytotoxicity, docking study, and tubulin polymerization inhibitory activity of novel 1-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)- 1h-1,2,4-triazole-3-carboxanilides

Article abstract of DOI:10.1002/ardp.201400096

A series of novel 1-(3,4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2, 4-triazole-3-carboxylic acid derivatives (4a-n) were synthesized and evaluated for their in vitro cytotoxic activity against the growth of four different human cell lines (hepatoca

Full text of DOI:10.1002/ardp.201400096

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1
Full Paper
Synthesis, Cytotoxicity, Docking Study, and
Tubulin Polymerization Inhibitory Activity of Novel
1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
1H-1,2,4-triazole-3-carboxanilides
Omar M. Aly1, Eman A. Beshr1, Raed M. Maklad1, Muhamad Mustafa1, and Amira M. Gamal-Eldeen2
1
Faculty of Pharmacy, Medicinal Chemistry Department, Minia University, Minia, Egypt
Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo,
2
Egypt
A series of novel 1-(3,4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxylic acid
derivatives (4a–n) were synthesized and evaluated for their in vitro cytotoxic activity against the growth
of four different human cell lines (hepatocarcinoma HepG2, breast adenocarcinoma MCF-7, colon
carcinoma DLD-1, and leukemia HL-60). The anilides of m-anisidine 4e, o-anisidine 4f, and 3,5-
difluoroaniline 4l demonstrated best results on MCF-7 cells and mean IC50 values of 7.79, 10.79, and
13.20 mM, respectively. The compounds produced a significant reduction in cellular microtubules at a
concentration of 25 mg/mL, for microtubule loss. Molecular modeling studies involving compounds 4d,
4e, 4f, and 4l with the colchicine binding site of a,b-tubulin revealed hydrogen bonding and
hydrophobic interactions with several amino acids in the colchicine binding site of b-tubulin.
Keywords: Cytotoxicity / Docking study / 1,2,4-Triazole / Tubulin
Received: March 10, 2014; Revised: March 29, 2014; Accepted: May 5, 2014
DOI 10.1002/ardp.201400096
Introduction
tubulin binding agents. They include natural products as well
as synthetic small molecules (Fig. 1) [1, 2]. Among all the
colchicine site agents, combretastatin A-4 (CA-4) has received
special attention in the last few years [3, 4]. In addition to its
potent cytotoxicity and inhibitory activity on tubulin
polymerization, CA-4 is one of the few anti-microtubule
agents reported to have selective vascular disrupting activi-
ty [5, 6]. CA-4 and its water-soluble prodrug, combretastatin
A-4 phosphate (CA-4P), are selectively cytotoxic to rapidly
proliferating tumor vasculature than normal blood vessels,
resulting in reduced blood flow to tumor and eventual
hemorrhagic necrosis [7, 8]. The anti-tumor and anti-vascular
activities of CA-4 analogs have been demonstrated in both
animal and human studies [9–12]. Many analogs of CA-4 have
been designed to study the structure–activity relationship of
the molecule in order to enhance both the cytotoxic and
selective vascular disrupting activities [13–24].
Tubulin polymerization inhibition is the subject of both
research and clinical trials, specially those tubulin polymeri-
zation inhibitors that have the ability of vascular disrupting
activity. Tubulin is the building block of microtubules, which
are important in cellular functions such as cell transport,
movement, separation of chromosomes during mitosis in
addition to the cytoskeleton structure. Anti-microtubule
agents are anti-tumor agents that can be categorized into
three classes: (i) microtubule-stabilizing agents, (ii) vinca site
binding agents, and (iii) colchicine site binding agents. Of the
three classes of anti-microtubule agents, colchicine site agents
are the only class of agents that do not have a representative
drug in clinical use for cancer. However, these agents are
structurally the most diverse group among the three classes of
Promoted with the above-mentioned studies and as a
continuation of our research interest in the synthesis
and biological activities of novel tubulin polymerization
inhibitors [25, 26], the present study is concerned with the
Correspondence: Dr. Omar M. Aly, Faculty of Pharmacy, Medicinal
Chemistry Department, Minia University, Minia 61519, Egypt.
E-mail: omarsokkar@mu.edu.eg
Fax: þ20 86 2369075
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O. M. Aly et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
relationship study of 1,5-diaryl-1H-1,2,4-triazole-3-carboxylic
acid derivatives. The ability of the prepared compounds was
evaluated for both in vitro cytotoxic properties on four
different cancer cell lines and tubulin polymerization
inhibitory activity. Furthermore, molecular modeling studies
of the most active tubulin inhibitor with the colchicine
binding site of a,b-tubulin were also performed.
Results and discussion
Chemistry
The synthetic route used to synthesize 1-(3,4-dimethoxy-
phenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carbox-
anilides 4a–n is outlined in Scheme 1. Key starting compound,
2-(3,4,5-trimethoxybenzamido)acetic acid 1, was prepared in
good yield (85%) by the reaction of glycine with 3,4,5-
trimethoxybenzoyl chloride in 10% NaOH. Heating of
compound 1 with acetic anhydride afforded the correspond-
ing compound 2. The synthesis of the key intermediate 3 was
carried out using Kuskov like reaction through coupling of
Figure 1. Examples of colchicine and combretastatin analogs.
synthesis of novel 1-(3,4-dimethoxyphenyl)-5-(3,4,5-trimethox-
yphenyl)-1H-1,2,4-triazole-3-carboxylic acid derivatives with
the objective of discovering novel and potent anti-tubulin
agents and as a starting point of a full structure–activity
H3CO
H3CO
H3CO
H3CO
NaNO2, HCl
NH2
N2 Cl
°
–5 to 0 C
O
NHCH2COOH
O
N
O
C
NaOAc, 0−5°C
Ac2
O
H3CO
OCH3
heat
OCH3
H3CO
H3CO
N2 Cl
OCH3
OCH3
1
2
H3CO
H3CO
H3CO
H3CO
H3CO
O
O
N
N
R
H
N
N
C
R
N
O
N
H2N
N
H
H3CO
H3CO
4a: R = H, 4b: R = 4-CH3
AcOH, NaOAc, reflux
4a−n
H3CO
OCH3
OCH3
OCH3
, 4c: R = 2-CH , 4d: R = 4-OCH , 4e: R = 3-OCH
3
3
3
4f: R = 2-OCH3, 4g: R = 3,4-di-OCH3, 4h: R = 4-Br, 4i: R = 4-F, 4j: R = 2,3-di-F
3
4k:R = 3,4-di-F, 4l: R = 3,5-di-F, 4m: R = 2,3,4-tri-fluoro, 4n: R = 2,3-benzo
Scheme 1. Synthesis of the target compounds 4a–n.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxanilides
3
the diazonium salt of 3,4-dimethoxyaniline with the active
methylene of 2 in presence of sodium acetate. The IR spectrum
of 3 revealed the presence of strong stretching band at
p-anisidine, p-bromoaniline, and 4-fluoroaniline, respectively,
showed decreased activity in comparison with the unsub-
stituted anilide of aniline 4a or that of 1-naphthyl amine 4n. In
conclusion, from all anti-tumor experiments, it is clear that
anilides with m-anisidine 4e and o-anisidine 4f are multi-
potential antitumor compounds, since both of them possessed
high cytotoxic effect against liver cancer cells HepG2, breast
cancer MCF-7 cells, and leukemia HL-60 cells. The highest
promising cytotoxic compound is 4e, which had IC50 values of
15.43, 7.97, and 12.97 mM against HepG2, MCF-7 and HL-60
cells, respectively, compared with values of 0.014–0.403 mM for
3-amino-deoxyCA-4 [29].
1780 cmꢀ1 of the C O group of the lactone ring.
–
–
According to Sawdey rearrangement [27, 28], 4-(2-(3,4-
dimethoxyphenyl)hydrazono)-2-(3,4,5-trimethoxyphenyl)oxa-
zol-5(4H)-one 3 by a variety of nucleophiles opened the
oxazoline ring to form acyclic intermediates, which then
recyclized via loss of water affording triazole derivatives.
Reaction of 3 with primary aromatic amines in mild acidic
medium formed the target anilides 4a–n in 53.01–70.20%
yield. The physical and spectral data are listed in the
Experimental section.
Effect of compounds 4a–n on tubulin polymerization
The effect of the synthesized compounds on tubulin
polymerization was done to investigate whether the cytotox-
icity is related to the interaction with tubulin or not. Tubulin
was evaluated in MCF-7 cell line after treating with 25 mg/mL
of each compound for 48 h using indirect ELISA. The
treatment of breast carcinoma MCF-7 cells revealed that
compounds 4a, 4e, 4f, 4g, 4i, 4k, 4l, and 4m showed a
promising inhibitory effect of cellular tubulin, as concluded
from their percentage of inhibition (around 50%), as shown in
Fig. 2, while the other tested compounds showed a variable
but lower inhibitory effect. Compound 4e was the most
effective inhibitory compound in MCF-7 cells.
Biological investigations
Anti-tumor activity against four different human cancer cell
lines
Using MTT assay, the effect of the tested compounds on the
viability of different human cancer cell lines were studied after
48 h of incubation. The treatment of hepatocellular carcinoma
HepG2 cells, leukemia HL-60 cells, colon cancer DLD-1 cells,
and breast cancer MCF-7 cells with gradual concentrations of
different compounds revealed that compounds 4e and 4f
possessed the highest promising cytotoxic effect against
HepG2 cells, compounds 4e, 4f, and 4l demonstrated cytotoxic
effect against HL-60 cells, and the tested compounds showed
weak cytotoxic effect on DLD-1 cells. Test compounds 4e, 4f, 4g,
4k, 4l, and 4m possessed the highest promising cytotoxic
effect on MCF-7 cells as concluded from their IC50 values,
as shown in Table 1. Anilides of the para-substituted anilines
like 4b, 4d, 4h, and 4i, which are anilides of p-toluidine,
Immunofluorescence localization of tubulin in liver cancer
cells
The localization of tubulin was explored in HepG2 cell
line after treatment with selected promising compounds
Table 1. Collective calculated IC50 (mM) from linear equation of dose–response curve for compounds 4a–n against hepatocarcinoma
HepG2, breast adenocarcinoma MCF-7, colon carcinoma DLD-1, and leukemia HL-60 cells.
a)
IC50 (mM)
Compound
no.
HepG2
MCF-7
DLD-1
HL-60
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
30.97 ꢁ 4.5
151.43 ꢁ 4.62
31.07 ꢁ 2.27
58.39 ꢁ 2.65
15.43 ꢁ 0.91
15.94 ꢁ 1.12
33.50 ꢁ 2.5
25.39 ꢁ 2.31
164.83 ꢁ 4.1
71.18 ꢁ 3.5
50.28 ꢁ 1.1
7.79 ꢁ 0.82
10.79 ꢁ 0.93
8.40 ꢁ 1.11
26.66 ꢁ 1.03
30.52 ꢁ 2.32
45.32 ꢁ 2.25
14.34 ꢁ 0.45
13.20 ꢁ 1.51
14.08 ꢁ 0.09
28.62 ꢁ 1.24
26.22 ꢁ 1.75
173.88 ꢁ 3.32
38.32 ꢁ 2.9
28.01 ꢁ 3.5
132.52 ꢁ 2.22
27.27 ꢁ 1.9
68.29 ꢁ 2.11
27.59 ꢁ 1.81
21.64 ꢁ 1.12
39.67 ꢁ 1.99
167.86 ꢁ 2.91
176.72 ꢁ 2.95
40.37 ꢁ 3.01
54.29 ꢁ 1.0
53.22 ꢁ 1.42
12.97 ꢁ 0.88
11.49 ꢁ 1.01
30.28 ꢁ 1.65
102.97 ꢁ 2.01
151.02 ꢁ 4.01
34.02 ꢁ 1.23
42.85 ꢁ 1.25
13.13 ꢁ 0.61
66.24 ꢁ 1.78
27.80 ꢁ 1.12
127.63 ꢁ 1.87
163.12 ꢁ 5.61
36.20 ꢁ 1.8
46.77 ꢁ .099
19.55 ꢁ 1.02
70.89 ꢁ 1.56
30.39 ꢁ 0.98
38.01 ꢁ 1.19
79.80 ꢁ 1.11
54.06 ꢁ 1.56
a)
IC50 ¼ compound concentration required to inhibit tumor cell viability by 50%. Data are expressed as the mean ꢁ SE from the dose–
response curves of at least three experiments.
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O. M. Aly et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Figure 2. Level of tubulin in MCF-7 cells after treatment for 24 h by
tested compounds. Black bars represent inactive compounds, and
gray bars represent promising tubulin inhibiting compounds (around
50% inhibition).
Figure 4. Colchicine binding site between a and b subunits of
tubulin.
that revealed an inhibitory effect on tubulin as
indicated by ELISA. Cells were treated with 25 mg/mL
of each compound for 48 h, and then submitted to
immunofluorescence labeling and analysis under fluores-
cence microscope. The treatment of hepatocellular
carcinoma HepG2 cells revealed that compound 4f
showed a moderate inhibitory effect on cellular tubulin
formation (Fig. 3), as concluded from the inhibition of the
fluorescence intensity (about 65% of that of the original
control tubulin).
Molecular docking study results with tubulin protein
Inspection of the colchicines binding site revealed that
colchicine site is mostly buried in the intermediate domain of
the b subunit. Colchicine also interacts with loop T5 of the
neighboring a subunit (Fig. 4) [30].
All the tested compounds (4e, 4f, and 4l) showed similar
binding to colchicines and inhibition of tubulin protein. The
results of interaction energies with tubulin protein are shown
in Table 2. Molecular docking simulation of compounds 4e
and 4f into tubulin protein active site was done. They got
Figure 3. Fluorescence intensity (IFU) of tubulin localization in HepG2 cells after treatment with 4f for 24 h compared to control cells.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxanilides
5
Table 2. Interaction energies of compounds (4d, 4e, 4f, and 4l)
In addition, several molecular interactions were considered
with tubulin protein.
to be responsible for the observed affinity. Two hydrogen
bonds between carbonyl group of the ligand and Ala250b
were found and the other between O in dimethoxyphenyl of
the ligand and Ser178a. Also, there were van der Waals
interactions between the benzene ring of the ligand and
Lys254b, Asn249b, and Gln11a.
Compound 4l showed different flipped binding mode than
compounds 4e and 4f as the more lipophilic difluorophenyl
ring being oriented in the hydrophobic pocket, as shown in
Fig. 5C.
Compound no.
DG (kcal/mol)
4d
4e
4f
4l
ꢀ10.4857
ꢀ15.3932
ꢀ13.6294
ꢀ10.4586
stabilized at the colchicine-binding site of tubulin by
hydrophobic and hydrogen bond interactions (Fig. 5A and B).
The trimethoxyphenyl is oriented at hydrophobic pocket
composed of Leu248b, Ala316b, Val318b, Met259b, Ile378b,
and Ala354b. The 3 and 4 OCH3-substitutions can form
hydrogen bonds with Cys241 in b-subunit (3.82 or 4.98 Å).
The para substituent is out the pocket of the DAMA–
colchicine–tubulin complex as shown in Fig. 5D, which may
explain the decreased cytotoxic and tubulin polymerization
inhibitory activity in the para substituted anilides.
Figure 5. 2D representation of docking of compounds 4e (A), 4f (B), 4l (C), and 4d (D) into the colchicine binding site in the tubulin protein.
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O. M. Aly et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Conclusions
0.018 mol) for 2 h. The reaction mixture was cooled and poured
into ice water (50 mL). The formed precipitate was filtered off,
dried, and recrystallized from aqueous methanol.
A group of novel 1-(4-methoxyphenyl)-5-(3,4,5-trimethoxy-
phenyl)-1H-1,2,4-triazole-3-carboxanilides was prepared and
characterized by different spectroscopic and elemental
analysis techniques. Only ortho and meta substituted anilides
showed the highest activity while the para substitution
showed a decrease in the activity. The compounds showed the
highest degree of tubulin polymerization inhibition, only
those compounds of highest cytotoxic activity. In conclusion,
the prepared 1-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
1H-1,2,4-triazole-3-carboxylic acid derivatives are revealed as
promising anticancer agents, stimulating the study of their
full structure–activity relationship.
1-(3,4-Dimethoxyphenyl)-N-phenyl-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4a
Brown crystals (3.28 g, 67% yield); m.p. 90–92°C; IR (KBr) (cmꢀ1):
3315 (NH), 1691 (CO), 1595 (C N); 1H NMR (300 MHz, CDCl3) d
–
–
(ppm): 3.69 (s, 6H, 2OCH3), 3.84 (s, 9H, 3OCH3), 6.84 (s, 2H, Ar–H),
7.12–7.17 (m, 3H, Ar–H), 7.37–7.56 (m, 4H, Ar–H), 7.86 (d, 2H,
J ¼ 8.10 Hz, Ar–H), 10.50 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) d
(ppm): 57.73 and 62.07 (OCH3), 107.76, 115.91, 121.83, 123.32,
125.29, 129.01, 129.73, 131.47, 139.16, 140.01,153.42, 155.33,
–
156.65, and 158.08 (aromatic carbons), 160.75 (C O). Anal. calcd.
–
for C26H26N4O6 (490.19): C, 63.66; H, 5.34; N, 11.42. Found: C,
63.38; H, 5.0; N, 11.56.
Experimental
1-(3,4-Dimethoxyphenyl)-N-(p-tolyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4b
Pale brown crystals (2.77 g, 55% yield); m.p. 75–77°C; IR (KBr)
Chemistry
(cmꢀ1): 3331 (NH), 1691 (CO), 1589 (C N); 1H NMR (300 MHz,
Melting points were determined on Stuart electrothermal
melting point apparatus and were uncorrected. IR spectra were
recorded on Nicolet iS5 FT-IR spectrometer at Minia University.
NMR spectra were carried out using a Bruker Avance 300 MHz
NMR spectrometer, using TMS as internal reference. Chemical
shifts (d values are given in parts per million (ppm) relative to
CDCl3 (7.29 for proton and 76.9 for carbon) or DMSO-d6 (2.50 for
proton and 39.50 for carbon) and coupling constants (J) in Hertz.
Splitting patterns are designated as follows: s, singlet; d, doublet;
t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. The
progress of reactions and the purity of the prepared compounds
were monitored by thin-layer chromatography (TLC) using Merck
9385 pre-coated aluminum plate silica gel (Kieselgel 60) 5 cm
ꢂ 20 cm plates with a layer thickness of 0.2 mm. The spots were
detected by exposure to UV-lamp at l ¼ 254 nm. Elemental
analysis was performed on Vario El Elementar CHN Elemental
analyzer; organic microanalysis section, Cairo University, Giza,
Egypt and the results were within ꢁ0.4% of the theoretical values.
–
–
CDCl3) d (ppm): 2.32 (s, 3H, CH3), 3.69 (s, 6H, 2OCH3), 3.84 (s, 9H,
3OCH3), 6.78 (s, 2H, Ar–H), 6.89 (q, 2H, Ar–H), 7.01 (s, 1H, Ar–H),
7.16 (d, 2H, J ¼ 8.1 Hz, Ar–H), 7.62 (d, 2H, J ¼ 8.4 Hz, Ar–H), 8.96 (s,
1H, CONH). 13C NMR (75 MHz, CDCl3) d (ppm): 20.84 (ArCH3),
56.01, 56.14, 56.21, 60.86 (OCH3), 106.25, 109.17, 110.75, 118.24,
121.75, 129.51, 130.65, 134.23, 134.79, 149.47, 149.85, 153.08,
–
154.71, and 156.08 (aromatic carbons), 156.59 (C O). Anal. calcd.
–
for C27H28N4O6 (504.53): C, 64.27; H, 5.59; N, 11.10. Found: C,
64.43; H, 5.57; N, 11.33.
1-(3,4-Dimethoxyphenyl)-N-(o-tolyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4c
Pale brown powder (2.87 g, 57% yield); m.p. 80–83°C; IR (KBr)
(cmꢀ1): 3341 (NH), 1690 (CO), 1585 (C N); 1H NMR (300 MHz,
–
–
CDCl3) d (ppm): 2.38 (s, 3H, CH3), 3.69 (s, 6H, 2OCH3), 3.90 (s, 9H,
3OCH3), 6.80 (s, 2H, Ar–H), 6.87–6.95 (p, 2H, Ar–H), 7.10 (t, 2H,
Ar–H), 7.24 (q, 2H, Ar–H), 8.10 (d, 1H, J ¼ 8.1 Hz, Ar–H), 8.96 (s, 1H,
CONH). 13C NMR (75 MHz, CDCl3) d (ppm): 17.69 (ArCH3), 56.03,
56.16, 56.23, and 60.89 (OCH3), 106.32, 109.25, 110.79, 118.32,
121.83, 122.46, 125.23, 126.86, 130.43, 135.20, 149.47, 149.88,
–
Synthesis of 4-(2-(3,4-dimethoxyphenyl)hydrazono)-2-
(3,4,5-trimethoxyphenyl)oxazol-5(4H)-one 3
Trimethoxyhippuric acid 1 (0.26 mol, 70.00 g) in acetic anhydride
(100 mL) was heated for 30 min or until a clear solution of 2 was
obtained; this solution was cooled to room temperature (solution
A). To a cold solution of 3,4-dimethoxyaniline (0.2 mol, 30.64 g) in
5 N HCl (60 mL) in an ice-salt bath ꢀ5 to 0°C, a solution of sodium
nitrite (0.26 mol, 17.94 g) in water (30 mL) was added in a
dropwise manner. The reaction mixture was left for 10 min
(solution B). Solution A was added to solution B in presence of
anhydrous sodium acetate (0.36 mol, 30 g). The reaction mixture
was stirred at 0–10°C for 2 h; the formed precipitate was filtered
off and dried (dark red solid, yield 70%).
153.08, 154.78, and 156.08 (aromatic carbons), 156.79 (C O). Anal.
–
calcd. for C27H28N4O6 (504.53): C, 64.27; H, 5.59; N, 11.10. Found:
C, 64.5; H, 5.27; N, 11.41.
1-(3,4-Dimethoxyphenyl)-N-(4-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4d
Yellowish brown powder (3.08 g, 59% yield); m.p. 184–185°C; IR
1
(KBr) (cmꢀ1): 3340 (NH), 1691 (CO), 1592 (C N); H NMR (300 MHz,
–
–
CDCl3) d (ppm): 3.69 (s, 6H, 2OCH3), 3.84 (s, 12H, 4OCH3), 6.78 (s,
2H, Ar–H), 6.86 (d, 2H, J ¼ 9 Hz, Ar–H), 6.89 (d, 1H, J ¼ 2.1 Hz,
Ar–H), 6.92 (d, 1H, J ¼ 3.9 Hz, Ar–H), 7.02 (d, 1H, J ¼ 1.8 Hz, Ar–H),
7.65 (d, 2H, J ¼ 9 Hz, Ar–H), 8.91 (s, 1H, CONH). 13C NMR (75 MHz,
CDCl3) d (ppm): 55.42, 56.03, 56.16, 56.23, and 60.87 (OCH3),
106.25, 109.19, 110.75, 114.18, 118.25, 121.47, 121.81, 130.53,
130.70, 149.47, 149.85, 153.10, 154.70, 156.15, and 156.51
–
General procedure for the synthesis of 1-(3,4-di-
methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-
triazole-3-carboxanilides (4a–n)
A mixture of compound 3 (4.15 g, 0.01 mol) and appropriate
primary aromatic amine (0.01 mol) was refluxed in acetic acid
(50 mL) in the presence of anhydrous sodium acetate (1.5 g,
(aromatic carbons), 156.54 (C O). Anal. calcd. for C27H28N4O7
–
(520.53): C, 62.30; H, 5.42; N, 10.76. Found: C, 62.79; H, 4.93; N,
10.90.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxanilides
7
1-(3,4-Dimethoxyphenyl)-N-(3-methoxyphenyl)-5-(3,4,5-
Ar–H), 7.69 (s, 2H, Ar–H), 9.01 (s, 1H, CONH). 13C NMR (75 MHz,
CDCl3) d (ppm): 55.98, 56.11, 56.18, 60.83, 106.22, 109.12, 110.75,
115.50, 115.80, 118.20, 121.48, 133.35, 139.88, 149.46, 149.88,
153.07, 154.78, 155.80, and 161.03 (aromatic carbons), 156.68
–
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4e
Yellowish powder (3.07 g, 59% yield); m.p. 78–81°C; IR (KBr)
(cmꢀ1): 3351 (NH), 1691 (CO), 1589 (C N); 1H NMR (300 MHz,
–
–
(C O). Anal. calcd. for C26H25FN4O6 (508.18): C, 61.90; H, 4.80; N,
–
CDCl3) d (ppm): 3.72 (s, 6H, 2OCH3), 3.84 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.72 (d, 1H, J ¼ 7.2 Hz,
Ar–H), 6.81 (s, 2H, Ar–H), 6.90 (d, 1H, J ¼ 8.4 Hz, Ar–H), 6.93 (d, 1H,
J ¼ 2.1 Hz, Ar–H), 7.05 (s, 1H, Ar–H), 7.27 (d, 2H, J ¼ 9 Hz, Ar–H),
7.53 (s, 1H, Ar–H), 9.02 (s, 1H, CONH). 13C NMR (75 MHz, CDCl3) d
(ppm): 55.25, 56.05, 56.16, 56.23, and 60.87 (OCH3), 105.56,
106.29, 109.17, 110.79, 118.24, 121.75, 130.65, 138.53, 149.49,
149.88, and 153.10, 155.98, and 156.71 (aromatic carbons), 160.14
–
11.11. Found: C, 61.51; H, 4.84; N, 11.27.
N-(2,3-Difluorophenyl)-1-(3,4-dimethoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4j
Dark brown powder (2.98 g, 57% yield); m.p. 74–76°C; IR (KBr)
(cmꢀ1): 3351 (NH), 1691 (CO), 1589 (C N); 1H NMR (300 MHz,
–
–
CDCl3) d (ppm): 3.70 (s, 6H, 2OCH3), 3.86 (s, 9H, 3OCH3), 6.79 (s, 2H,
Ar–H), 6.86–6.98 (m, 4H, Ar–H), 7.01–7.12 (m, 1H, Ar–H), 8.30 (t,
1H, Ar–H), 9.26 (s, 1H, CONH). 13C NMR (75 MHz, CDCl3) d (ppm):
56.05, 56.18, 56.24, and 60.89 (OCH3), 106.32, 109.22, 110.84,
112.17, 116.74, 118.34, 121.58, 130.63, 149.54, 150.00, 153.12,
(C O). Anal. calcd. for C27H28N4O7 (520.53): C, 62.30; H, 5.42; N,
–
10.76. Found: C, 62.55; H, 5.19; N, 11.11.
1-(3,4-Dimethoxyphenyl)-N-(2-methoxyphenyl)-5-(3,4,5-
–
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4f
155.01, and 155.54 (aromatic carbons), 156.81 (C O). Anal. calcd.
–
Reddish brown powder (3.22g, 62% yield); m.p. 170–172°C; 1H NMR
(300MHz, CDCl3) d (ppm): 3.68 (s, 6H, 2OCH3), 3.84 (s, 6H, 2OCH3),
3.90 (s, 6H, 2OCH3), 6.80 (s, 2H, Ar–H), 6.85–7.10 (m, 6H, Ar–H), 8.57
(dd, 1H, J ¼ 1.5 and 1.5 Hz, Ar–H), 9.57 (s, 1H, CONH). 13C NMR
(75 MHz, CDCl3) d (ppm): 55.70, 55.98, 56.11, 56.18, and 60.83
(OCH3), 106.34, 109.27, 109.91, 110.74, 118.32, 120.10, 121.02,
121.91, 148.22, 149.41, 149.79, 153.00, 154.71, and 156.35 (aromatic
–
for C26H24F2N4O6 (526.49): C, 59.31; H, 4.59; N, 10.64. Found: C,
59.36; H, 4.29; N, 10.80.
N-(3,4-Difluorophenyl)-1-(3,4-dimethoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4k
Brown powder (2.79 g, 53% yield); m.p. 73–75°C; IR (KBr) (cmꢀ1):
3339 (NH), 1694 (CO), 1589 (C N); 1H NMR (300 MHz, CDCl3) d
–
–
carbons), 156.63 (C O). Anal. calcd. for C27H28N4O7 (520.53): C,
–
(ppm): 3.69 (s, 6H, 2OCH3), 3.85 (s, 6H, 2OCH3), 3.91 (s, 3H, OCH3),
6.77 (s, 2H, Ar–H), 6.89 (s, 2H, Ar–H), 7.00 (s, 1H, Ar–H), 7.13 (d, 1H,
J ¼ 9.3 Hz, Ar–H), 7.31 (s, 1H, Ar–H), 7.84 (s, 1H, Ar–H) 9.01 (s, 1H,
CONH). 13C NMR (75 MHz, CDCl3) d (ppm): 56.05, 56.18, 56.24, and
60.91 (OCH3), 106.27, 109.14, 110.80, 115.50, 117.17, 118.25,
121.57, 149.56, 150.00, 153.15, 154.93, and 155.59 (aromatic
62.30; H, 5.42; N, 10.76. Found: C, 62.64; H, 5.16; N, 11.0.
N,1-bis(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-
1H-1,2,4-triazole-3-carboxamide 4g
Dark red powder (3.40 g, 62% yield); m.p. 83–85°C; IR (KBr) (cmꢀ1):
1
–
–
carbons), 156.76 (C O). Anal. calcd. for C26H24F2N4O6 (526.49): C,
3339 (NH), 1690 (CO), 1589 (C N); H NMR (300 MHz, CDCl ) d (ppm):
–
–
3
59.31; H, 4.59; N, 10.64. Found: C, 59.09; H, 4.39; N, 10.43.
3.71 (s, 6H, 2OCH3), 3.87 (s, 9H, 3OCH3), 3.93 (s, 6H, 2OCH3), 6.80 (s,
2H, Ar–H), 6.857–6.921 (m, 3H, Ar–H), 7.03 (s, 1H, Ar–H), 7.16 (d, 1H,
J ¼ 8.4Hz, Ar–H), 7.58 (s, 1H, Ar–H), 8.91 (s, 1H, CONH). 13C NMR
(75 MHz, CDCl3) d (ppm): 55.98, 56.13, 56.24, 56.29, 60.78, and 60.97
(OCH3), 104.79, 106.35, 106.86, 109.20, 110.85, 111.40, 118.27,
121.88, 130.76, 131.06, 146.13, 153.10, and 153.21 (aromatic
–
N-(3,5-Difluorophenyl)-1-(3,4-dimethoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4l
Yellowish brown powder (3.09 g, 59% yield); m.p. 70–72°C; IR
1
(KBr) (cmꢀ1): 3332 (NH), 1690 (CO), 1589 (C N); H NMR (300 MHz,
–
–
carbons), 156.64 (C O). Anal. calcd. for C28H30N4O8 (550.56):
–
CDCl3) d (ppm): 3.67 (s, 6H, 2OCH3), 3.84 (s, 6H, 2OCH3), 3.90 (s, 3H,
OCH3), 6.58 (s, 1H, Ar–H), 6.75 (s, 2H, Ar–H), 6.88 (s, 2H, Ar–H), 6.99
C, 61.08; H, 5.49; N, 10.18. Found: C, 61.08; H, 5.27; N, 10.32.
(s, 1H,Ar–H), 7.36 (d, 2H, J ¼ 6.9 Hz, Ar–H), 9.13 (s, 1H, CONH). 13
C
N-(4-Bromophenyl)-1-(3,4-dimethoxyphenyl)-5-(3,4,5-
NMR (75 MHz, CDCl3) d (ppm): 56.01, 56.14, 56.21, 60.87, and
(OCH3), 102.74, 103.12, 106.27, 109.11, 110.79, 118.24, 121.43,
139.40, 149.52, 150.00, 153.12, 154.99, and 155.37 (aromatic
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4h
Light blue powder (3.29 g, 58% yield); m.p. 170–172°C; IR (KBr)
(cmꢀ1): 3345 (NH), 1695 (C O), 1589 (C N); 1H NMR (300MHz,
–
–
–
–
–
–
carbons), 156.84 (C O). Anal. calcd. for C26H24F2N4O6 (526.49): C,
59.31; H, 4.59; N, 10.64. Found: C, 59.44; H, 4.54; N, 10.60.
CDCl3) d (ppm): 3.67 (s, 6H, 2OCH3), 3.83 (s, 6H, 2OCH3), 3.89 (s, 3H,
OCH3), 6.76 (s, 2H, Ar–H), 6.85 (d, 2H, J ¼ 8.7Hz, Ar–H), 6.99 (s, 1H,
Ar–H), 7.44 (d, 2H, J ¼ 8.7Hz, Ar–H), 7.64 (d, 2H, J ¼ 8.7 Hz, Ar–H),
9.01 (s, 1H, CONH). 13C NMR (75 MHz, CDCl3) d (ppm): 56.00, 56.13,
56.19, and 60.84 (OCH3), 106.25, 109.12, 110.74, 117.10, 118.22,
121.30, 136.42, 139.90, 149.46, 149.88, 153.07, 154.81, and 155.72
–
1-(3,4-Dimethoxyphenyl)-N-(2,3,4-trifluorophenyl)-5-
(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide
4m
1
Yellowish brown powder (3.12 g, 57% yield); m.p. 70°C; H NMR
(aromatic carbons), 156.68 (C O). Anal. calcd. for C26H25BrN4O6
–
(300 MHz, CDCl3) d (ppm): 3.67 (s, 6H, 2OCH3), 3.84 (s, 6H, 2OCH3),
3.88 (s, 9H, 3OCH3), 6.77 (s, 2H, Ar–H), 6.81–7.04 (m, 4H, Ar–H),
8.17–8.25 (m, 1H, Ar–H), 9.12 (s, 1H, CONH). 13C NMR (75 MHz,
CDCl3) d (ppm): 55.98, 56.14, 56.19, and 60.84 (OCH3), 106.25 (CH),
109.15 (CH), 110.79 (CH), 111.66 (CH), 115.75 (CH), 118.29 (CH),
121.47 (CH), 139.98, 149.51, 149.98, 153.07, 154.99, and 155.31
(aromatic carbons), 156.87 (CO). Anal. calcd. for C26H23F3N4O6
(544.48): C, 57.35; H, 4.26; N, 10.29. Found: C, 57.19; H, 3.98; N,
10.51.
(569.40): C, 54.84; H, 4.43; N, 9.84. Found: C, 54.54; H, 4.07; N, 10.02.
1-(3,4-Dimethoxyphenyl)-N-(4-fluorophenyl)-5-(3,4,5-
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4i
Yellowish brown powder (3.01 g, 59% yield); m.p. 78–80°C; IR
1
(KBr) (cmꢀ1): 3350 (NH), 1691 (CO), 1589 (C N); H NMR (300 MHz,
–
–
CDCl3) d (ppm): 3.67 (s, 6H, 2OCH3), 3.83 (s, 6H, 2OCH3), 3.89 (s, 3H,
OCH3), 6.76 (s, 2H, Ar–H), 6.87 (s, 2H, Ar–H), 7.03 (t, 3H, J ¼ 8.7 Hz
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8
O. M. Aly et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1-(3,4-Dimethoxyphenyl)-N-(naphthalen-1-yl)-5-(3,4,5-
each concentration and the average was calculated. Data were
expressed as the percentage of relative viability compared with
the untreated cells compared with the vehicle control, with
cytotoxicity indicated by <100% relative viability. Percentage of
relative viability was calculated using the following equation:
[absorbance of treated cells/absorbance of control cells)] ꢂ 100.
Then the half maximal inhibitory concentration (IC50) was
calculated from the equation of the dose–response curve (linear
regression).
trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide 4n
Reddish brown powder (3.79g, 70% yield); m.p. 88–90°C; IR (KBr)
1
(cmꢀ1): 3336 (NH), 1696 (CO), 1589 (C N); H NMR (300 MHz, CDCl )
–
–
3
d (ppm): 3.71 (s, 6H, 2OCH3), 3.88 (s, 6H, 2OCH3), 3.92 (s, 3H, OCH3),
6.84 (s, 2H, Ar–H), 6.92 (m, 2H, Ar–H), 7.06 (s, 1H, Ar–H), 7.53 (m, 3H,
Ar–H), 7.73 (d, 1H, J¼ 8.4 Hz, Ar–H), 7.89 (d, 1H, J ¼ 9Hz, Ar–H),
8.04 (d, 1H, J ¼ 7.5Hz, Ar–H), 8.28 (d, 1H, J ¼ 7.2Hz, Ar–H), 9.56
(s, 1H, CONH). 13C NMR (75MHz, CDCl3) d (ppm): 56.09, 56.19,
56.28, and 60.92 (OCH3), 106.39, 109.30, 110.84, 118.37, 120.03,
120.49, 121.85, 125.72, 125.92, 126.00, 126.28, 128.79, 130.76,
131.72, 134.06, 149.54, 149.95, 153.15, 153.93, and 156.26
–
Y ¼ aꢃX þ b
ð0:5 ꢀ bÞ
(aromatic carbons), 157.29 (C O). Anal. calcd. for C30H28N4O6
–
IC50
¼
(540.56): C, 66.66; H, 5.22; N, 10.36. Found: C, 66.51; H, 5.0; N,
10.34.
a
where drug concentrations x1, x2, …, xn and growth inhibition
y1, y2, …, yn.
Biology
Cell culture
Cell lysate preparation
Human hepatocarcinoma cell line (HepG2), breast adenocarci-
noma cell line (MCF-7), colon carcinoma (DLD-1), and leukemia
(HL-60), which were purchased from ATCC, USA, were used
to evaluate the cytotoxic effect of the tested samples. Cells
were routinely cultured in Dulbecco’s modified Eagle’s
medium (DMEM). Media was supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, containing 100 units/mL
of penicillin G sodium, 100 units/mL of streptomycin sulfate,
and 250 ng/mL amphotericin B. Cells were maintained at
sub-confluence at 37°C in humidified air containing 5% CO2.
For sub-culturing, monolayer cells were harvested after trypsin/
EDTA treatment at 37°C. Cells were used when confluence
had reached 75%. Tested samples were dissolved in dimethyl
sulfoxide (DMSO), and then diluted thousand times in the
assay to begin with the indicated concentration. All cell culture
material was obtained from Cambrex BioScience (Copenhagen,
Denmark). All chemicals were from Sigma/Aldrich (USA),
except mentioned. All experiments were repeated three
times, unless mentioned.
Cell lysate was prepared to be used in ELISA. The treated and
untreated cells were trypsinized, washed, and centrifuged for
10 min at 1000g. Cell pellet was lysed in 0.5 mL of ice-cold lysis
buffer [50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA,
20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, and 10 mg/
mL leupeptin (pH 7.4)]. Cell lysates were passed through a 21-
gauge needle to break up cell aggregates, and then centrifuged at
14,000g for 15 min at 4°C. The supernatants (total cell lysate) were
submitted to ELISA and lipid peroxides estimation. The total
protein content of the lysates was measured according to Smith
et al. [32], by bicinchoninic acid (BCA) and using bovine serum
albumin (BSA) as a standard (data not mentioned).
Evaluation of tubulin
Tubulin level was estimated in cell lyastes by indirect ELISA. All
steps of indirect ELISA were followed according to the method
modified by Gamal-Eldeen et al. [33], with some modifications, in
which 96-well flat bottom polystyrene plates were coated with cell
lysates samples (100 mL/well). After overnight incubation at 4°C,
the plates were washed and then 200 mL of blocking buffer was
delivered into each well. The plates were covered and incubated
for 1.5 h at 37°C and after a washing step, rabbit polyclonal
antibody to tubulin (Abcam, Inc., Cambridge, MA, USA) was
adjusted to 0.2 mg/mL and dispensed as (100 mL/well); after 1 h
incubation at 37°C the plates were washed and the 100 mL/well of
polyclonal goat anti-rabbit peroxidase-conjugate diluted 1:1000
was added and the plates were incubated for another 1 h at 37°C.
At the end of the incubation period, the plates were washed, and
the substrate solution was prepared (equal volumes of 30,5,50-
tetramethyl benzidine (TMB) and H2O2; Kirkegaard and Perry
Labs, Gaithersburg, MD). Color development was stopped by
the addition of 100 mL/well of stopping buffer (1 M HCl). The color
intensity was measured at 450 nm using the microplate reader
(FLUOstar OPTIMA; BMG Labtech GmbH, Offenburg, Germany).
The levels of tubulin in the cell lysate were expressed as
milliabsorbance.
Anti-tumor activity
Cytotoxicity of tested samples was measured against each cell line
using the MTT cell viability assay. MTT (3-[4,5–dimethylthiazole-2-
yl]-2,5-diphenyltetrazolium bromide) assay is based on the ability
of active mitochondrial dehydrogenase enzyme of living cells to
cleave the tetrazolium rings of the yellow MTT and form a dark
blue, insoluble formazan crystal, which is largely impermeable to
cell membranes, resulting in its accumulation within healthy
cells. Solubilization of the cells results in the liberation of crystals,
which are then solubilized. The number of viable cells is directly
proportional to the level of soluble formazan dark blue color.
The extent of the reduction of MTT was quantified by measuring
the absorbance at 570 nm [31]. Briefly, cells (0.5 ꢂ 105 cells/well),
in serum-free media, were plated in a flat bottom 96-well
microplate, and treated with 20 mL of serial concentrations of the
tested samples for 48 h at 37°C, in a humidified 5% CO2
atmosphere. After incubation, media were removed and 40 mL of
MTT solution (5 mg/mL of MTT in 0.9% NaCl) in each well was
added and incubated for an additional 4 h. MTT crystals were
solubilized by adding 180 mL of acidified isopropanol/well and
the plate was shaken at room temperature, followed by
photometric determination of the absorbance at 570 nm using
microplate ELISA reader. Triplicate repeats were performed for
Immunofluorohistochemical evaluation of tubulin
Immunohistochemical detection of tubulin in fixed cells was
followed according to the method originally developed by
Kawahira [34], with some modifications.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxanilides
9
Reagents preparation: 0.1 M Citrate buffer pH to 6.0: 9 mL of
0.1 M citric acid solution was added to 41 mL of 1 M sodium
citrate solution and the volume was adjusted to 500 mL by
deionized water.
Antigen retrieval solution: 50 mL of 0.1 M citrate buffer, 500 mL
of Triton-100, and 250 mL of Tween-20 were mixed together and
the final volume was adjusted to 500 mL by deionized water.
Blocking solution: 50 mL of 0.1 M citrate buffer, 500 mL of
Triton 100, 250 mL of Tween-20, and 25 mL of FBS were mixed
together and the final volume was adjusted to 500 mL by
deionized water.
structure using all default items. Dummy atoms were created
from the obtained alpha spheres.
Docking of the target molecules to tubulin protein in
colchicine binding site
Docking of the conformation database of the target compounds
was done using MOE-Dock software. The following methodology
was generally applied: the enzyme active site file was loaded and
the Dock tool was initiated. The program specifications were
adjusted to: Dummy atoms as the docking site, triangle matcher
as the placement methodology to be used. London dG as scoring
methodology to be used was adjusted to its default values. The
MDB file of the ligand to be docked was loaded and Dock
calculations were run automatically. The obtained poses were
studied and the poses showing best ligand–enzyme interactions
were selected and stored for energy calculations.
Procedure: Slides of fixed cells were rinsed in three changes of
PBS. Antigen retrieval step, by which the availability of the
antigen for interaction with a specific antibody is maximized,
was performed by immersing slides in antigen retrieval solution
and incubated in a water bath at 95–99°C for 20 min. Afterwards,
slides were directly transferred to pre-cooled antigen retrieval
solution placed at ꢀ4°C for 5 min. Non-specific binding of the
antibody is prevented by incubating the slides in blocking
solution at 37°C for 30 min. Slides were then incubated for
30 min at 37°C with rabbit anti-human tubulin antibody (1:500)
diluted with blocking solution. Excess antiserum was rinsed from
the slide by immersing in cold buffer for two changes of 5–10 min
each. Slides were then incubated at 37°C with goat FITC-anti
rabbit IgG (1:2500) diluted with blocking solution. The slides were
rinsed in the enzyme substrate till the color developed. Images
were visualized using a fluorescence microscope (Axiostar Plus,
Zeiss, Goettingen, Germany) equipped with image analyzer and
digital camera (PowerShot A20, Canon, USA).
The authors acknowledge the Science and Technology Development
Fund (STDF) Egypt (Project No. 2943 Basic and Applied Research) for
funding this work.
The authors have declared no conflict of interest.
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