New 1,2,4-triazole-Chalcone hybrids induce Caspase-3 dependent apoptosis in A549 human lung adenocarcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2018.03.073

A series of novel 1, 2, 4-triazole/chalcone hybrids was prepared and identified with different spectroscopic techniques. The prepared compounds showed remarkable cytotoxic activity against different cancer cell lines. Compounds 24, 25, 27, 41 and 47 had shown the highest cytotoxicity among the tested compounds against human lung adenocarcinoma A549 cells with IC₅₀ ranging from 4.4 to 16.04 μM compared to cisplatin with IC₅₀ of 15.3 μM. Flow cytometric analysis of the tested compounds showed an increase in the number of apoptotic cells in a dose-dependent manner. The further mechanistic study demonstrated that 1, 2, 4-triazole-chalcone hybrids induced apoptosis via increased level of proapoptotic protein Bax, release of cytochrome c from mitochondria and activation of caspase-3/8/9 proteins. However, general caspase inhibition by the pan-caspase inhibitor, z-VAD-fmk, significantly decreased the apoptosis induced by the tested hybrids, suggesting dependency of apoptosis on activation of the caspase-3 pathway.

Full text of DOI:10.1016/j.ejmech.2018.03.073

Accepted Manuscript
New 1,2,4-triazole-Chalcone hybrids induce Caspase-3 dependent apoptosis in A549
human lung adenocarcinoma cells
Fatma F. Ahmed, Amer Ali Abd El-Hafeez, Samar H. Abbas, Dalia Abdelhamid,
Mohamed Abdel-Aziz
PII:
S0223-5234(18)30316-7
10.1016/j.ejmech.2018.03.073
EJMECH 10338
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 September 2017
Revised Date: 23 March 2018
Accepted Date: 24 March 2018
Please cite this article as: F.F. Ahmed, A.A. Abd El-Hafeez, S.H. Abbas, D. Abdelhamid, M.
Abdel-Aziz, New 1,2,4-triazole-Chalcone hybrids induce Caspase-3 dependent apoptosis in A549
human lung adenocarcinoma cells, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.073.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
New 1,2,4-Triazole-Chalcone Hybrids Induce Caspase-3 Dependent
Apoptosis in A549 Human Lung Adenocarcinoma Cells
a
b,c,**,
a*
a
Fatma F. Ahmed , Amer Ali Abd El-Hafeez
, Samar H. Abbas , Dalia Abdelhamid ,
a
Mohamed Abdel-Aziz
a
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.
b
Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National Cancer
Institute, Cairo University, Cairo, 11796, Egypt.
c
Pharmacotherapy Department, Graduate School of Biomedical and Health Sciences, Hiroshima
University, Hiroshima, Japan.
*
Corresponding Author
Samar H. Abbas; Phone: (+20) 100-542-4005. Fax: (+20) 086-236-9075. E-mail:
drsamar77@hotmail.com, samar_hafez@mu.edu.eg.
*
* Corresponding Author
Amer Ali Abd El-Hafeez; Phone: (+81)-80-4556-7496, Fax: (+81)-82-424-7000,
E-mail: amer.ali@nci.cu.edu.eg
Abstract
A series of novel 1, 2, 4-triazole/chalcone hybrids was prepared and identified with
different spectroscopic techniques. The prepared compounds showed remarkable
cytotoxic activity against different cancer cell lines. Compounds 24, 25, 27, 41 and 47
had shown the highest cytotoxicity among the tested compounds against human lung
adenocarcinoma A549 cells with IC ranging from 4.4 to 16.04 µM compared to
5
0
cisplatin with IC of 15.3 µM. Flow cytometric analysis of the tested compounds
5
0
showed an increase in the number of apoptotic cells in a dose-dependent manner. The
further mechanistic study demonstrated that 1, 2, 4-triazole-chalcone hybrids induced
apoptosis via increased level of proapoptotic protein Bax, release of cytochrome c
from mitochondria and activation of caspase-3/8/9 proteins. However, general caspase
inhibition by the pan-caspase inhibitor, z-VAD-fmk, significantly decreased the
apoptosis induced by the tested hybrids, suggesting dependency of apoptosis on
activation of the caspase-3 pathway.
1

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Key words
1
, 2, 4-triazole; chalcone; caspase-3; apoptosis; A549 human lung; NCI.
1
. Introduction
Nowadays, cancer is the third leading cause of the death globally [1]. Development of
drugs for cancer treatment based on the ability to block cell proliferation and/or to
induce apoptosis has received great efforts in the last years [2]. Apoptosis is a form of
physiological cell death that is essential for normal tissue development and
homeostasis [3,4]. This process can clearly be identified by characteristic changes in
the cells (i.e., caspase activation, DNA fragmentation and cell fragmentation through
the formation of apoptotic bodies) [5]. Chalcones are important class of flavonoids
due to their broad spectrum of biological activities including anticancer [6-9],
antioxidant [10,11], antimicrobial [12,13], anticonvulsant [14,15], anti-inflammatory
[
16,17], antiviral [18,19], and antimalarial [20,21] activities. Chalcones exhibited
remarkable anticancer activity against cancer cells and this activity may be attributed
to induction of apoptosis [22], blocking cell cycle progression in the G2/M phase
[
23], anti-estrogenic activity [24], inhibition of tubulin polymerization [25] and
inhibition of angiogenesis [22]. Chalcone derivative I (Fig. 1) exhibited promising
anticancer activity with IC50 ranging from 5.25 to14.49 µg/mL against different
cancer cell lines and it induced apoptosis through the intrinsic and extrinsic pathways
in MCF-7 cells [8]. Chalcones IIa-b (Fig. 1) showed cytotoxic activity against
leukemia cell lines through induction of apoptosis by activating the intrinsic pathway
through reduction of the mitochondrial membrane potential, reduction in Bcl-2
expression, increase in Bax expression and increase in the active caspase-3 [26].
Moreover, amino chalcone derivative III (Fig. 1) exhibited remarkable anticancer
activity through induction of apoptosis via increasing the death receptors expression
TNF-related apoptosis-inducing ligand (TRAIL-R1 and TRAIL-R2) and also
activation of p21, Bad, Bim, Bid, Bax, Smac, caspase-3, and caspase-8 and reducing
the antiapoptotic markers livin, XIAP, and HSP27 [27]. Additionally, naphthyl
chalcones derivatives IV (Fig. 1) showed anticancer activity through the apoptotic
pathway by activation of caspase-8, caspase-9, and caspase-12 and increase CHOP
expression [28].
2

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Fig. 1. Structures of compounds I-V.
On the other hand, triazoles are important class of heterocyclic compounds which
have been studied extensively due to their numerous biological activities such as
antibacterial [29], antifungal [30], antimicrobial [31], antioxidant [32], analgesic [33],
.
anticancer [34-36], .anticonvulsant [37],.antiviral [38] and.anti-inflammatory [39,40]
activities. Triazoles are readily able to bind to variable receptors and enzymes in
biological systems by diverse non-covalent interaction [41,42]. The anti-proliferative
activity of 1,2,4-triazole derivative V (Fig. 1) was attributed to cell cycle arrest in the
G2/M phase, tubulin polymerization inhibition, mitochondrial depolarization, and
induction of apoptosis through activation of caspase-3 [43]. Moreover, efforts have
been made in the last decades to improve the anticancer activity of the drugs, to solve
the drug resistance, lack of selectivity and side effects of other drugs. One of these
efforts was the hybridization between different chalcone derivatives and heterocyclic
derivatives such as piperazine [44], quinolone [45], quinolone [46], coumarin [47,48],
quinazolinone [49] and 1,2,3-triazoles [50-52] derivatives aiming to obtain synergistic
anticancer activity. For example, chalcone/1,2,3-triazole hybrid VI (Fig. 2) showed
cytotoxic activity with an IC50 value of 13.03 µM against HeLa cells relative to
cisplatin as a reference drug [51]. Also, chalcone-1,2,3-triazole hybrid VII (Fig. 2)
showed remarkable anticancer activity with IC of 1.52 µM against SK-N-SH cell
5
0
line through induction of apoptosis [52]. Additionally, epipodophyllotoxin/chalcone
hybrid VIII (Fig. 2) exhibited remarkable anticancer activity with IC ranging from
5
0
0
.35 to 12.45µM, with high selectivity against SW-620 and SKN-SH cell lines [53].
3

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O
O
OCH
3
S
S
N
O
N
N
O
OCH3
OH
N
OC H
2
5
N
N
N
N
OCH
3
VI
HOH
2
C
O
VII
O
N
N
O
O
F
N
3
H CO
O
O
HO
VIII
OCH
3
Fig, 2. Structures of compounds VI-VIII.
However, 1,2,3- triazole-chalcone hybrids showed synergistic anticancer activities
with apoptotic capacity, these hybrids lack its ability to activate the caspase-3
apoptotic pathway in low micromolar concentrations. A series of 1,2,3- triazole-
chalcone was synthesized that can activate caspase-3 at 50 µM [54]. Recently, the
research has been partially shifted toward 1,2,4-triazole nucleus that has been found to
exhibit anticancer activity due to their interaction at the active site of a receptor as
hydrogen bond acceptor and as a donor [55,56]. However, the apoptotic capacity of
the 1,2,4-triazole nucleus through caspase-mediated pathway as well as its
hybridization with chalcones to augment this apoptotic potency are completely
unknown. Herein, we aimed to clarify and augment the apoptotic induction capacity
of the 1,2,4-triazole nucleus through the caspase-3-mediated pathway via synthesis a
novel series of 1,2,4-triazole-chalcone hybrids through S-alkylation of 1,2,4-triazoles
with different acetylated chalcones as shown in Fig. 3. The chemical structures were
designed so that these analogues will possess different electron donating or electron
withdrawing substituents. The prepared compounds were evaluated for their cytotoxic
activity against different cell lines. Moreover, the mechanistic studies of the
anticancer activity of the tested compounds against A549 human lung
adenocarcinoma cells were also evaluated. To the best of our knowledge, this is the
first report that explains the apoptotic potency of 1,2,4-triazole-chalcone hybrids, at
low micromolar concentrations, via a caspase-3-mediated pathway.
4

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Fig. 3. Pharmacophore of the target compounds.
2
. Results and discussion
.1.Chemistry
2
The target compounds and intermediates were prepared as outlined in Schemes 1.
The benzoic acid derivatives 1a-e were converted to ethyl esters 2a-e via Fisher
esterification [57] using absolute ethanol and conc. H SO . The obtained esters were
2
4
then converted to the corresponding hydrazides 3a-e using hydrazine hydrate [58, 59],
Scheme 1. Heating at reflux of equimolar amounts of the hydrazides 3a-e and
allyl/phenyl isothiocyanate in ethanol affords the corresponding 1,4-disubstituted
thiosemicarbazides [58,60] which were used as crude products for the next step. The
1
,2,4-triazole-3-thiol derivatives 4a-j were prepared by intramolecular cyclization of
the 1,4-disubstituted thiosemicarbazides in presence of aqueous 2N KOH followed by
the acidification with concentrated hydrochloric acid [59].
Synthesis of the acetylated chalcones starts with a reaction of various aromatic
aldehydes 5a-d with p-aminoacetophenone through usual Clasien Schmidt
condensation to form the corresponding chalcones 6a-d. The acetylated chalcone
intermediates 7a-d were prepared as reported [7, 46] by reaction of the appropriate
chalcone with bromoacetyl bromide using potassium carbonate as a base in
dichloromethane. Alkylation of triazole derivatives 4a-j with acetylated chalcone
derivatives 7a–d was achieved in acetonitrile in the presence of TEA to afford the
1
13
target compounds 8-47 in a good yield [46]. H NMR and C NMR confirmed the
1
formation of target compounds. H NMR spectra of compounds 8-47 showed a singlet
signal at δ 3.96-4.32 ppm related to (S-CH -CO) of the linker. Furthermore, The two
2
5

ACCEPTED MANUSCRIPT
chalcone protons appear at the aromatic region as doublet signals at δ 7.35-7.64 ppm
and 7.60-7.82 ppm with coupling constant J = 15.00-15.84 Hz, The amide proton NH
appears as singlet signals at δ 10.83-11.22 ppm. Moreover, compounds 8-27 showed
characteristic allyl signals as a doublet at δ 4.53-4.62 ppm related to (N-CH2-
CH=CH2) with coupling constant J = 4.00-4.86 Hz, doublet of doublet at δ 5.03-5.13
ppm and 5.32-5.45 ppm for (N-CH -CH=CH ), with coupling constant J = 17.10-
2
2
1
7.22 Hz and 9.72-11.04 Hz, respectively. Due to restricted rotation around the
double bond; Multiplet signal appears at δ 5.87-6.02 ppm related to (N-CH2-
1
CH=CH ). On the other hand; allyl signals are disappeared in H NMR spectra for the
2
compounds 28-47 but the aromatic protons are increased by five protons of the phenyl
1
3
ring. The C NMR spectra of compounds 8-47 revealed the presence of two carbonyl
groups appeared δ 188.43-189.25 ppm and δ 166.15-168.15 ppm related to C=O of
chalcone and N-C=O, a characteristic signal of SCH appears at 35.81-37.54 ppm.
2
Moreover, NCH signal appears at δ 45.90-47.60 ppm in compounds 8-27.
2
6

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Compd.
R1
H
R2
H
Compd.
18, 38
19, 39
20, 40
21, 41
22, 42
23, 43
24, 44
25, 45
26, 46
27, 47
R1
H
R2
8
, 28
, 29
4-OCH
4-OCH
4-OCH
4-OCH
4-OCH
3
3
3
3
3
9
4-Cl
4-OCH
H
4-Cl
4-OCH
3
10, 30
11, 31
12, 32
13, 33
14, 34
15, 35
16, 36
17, 37
3
H
3,4-di-OCH
H
3,4-di-OCH
3
3
3,4,5-tri-OCH
H
3,4,5-tri-OCH
3
3
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
H
3,4,5-tri-OCH
3,4,5-tri-OCH
3,4,5-tri-OCH
3,4,5-tri-OCH
3,4,5-tri-OCH
3
3
3
3
3
4-Cl
4-Cl
4-OCH
3,4-di-OCH
3,4,5-tri-OCH
4-OCH
3,4-di-OCH
3,4,5-tri-OCH
3
3
3
3
3
3
Scheme1:-Synthesis of the target compounds 8-47.
2
2
2
.2. Biological evaluation
.2.1. Cytotoxic assays
.2.1.1. In vitro one dose assay
Compounds 9-11, 13-19, 21-27, 29-35, 37-41, 43-45 and 47 were selected by the
National Cancer Institute (NCI), USA for in vitro anticancer screening. Compounds
were screened at 10 µM dose using Sulforhodamine B colorimetric assay, against full
NCI 60 cell lines derived from nine tumor subpanels, including leukemia, lung, colon,
melanoma, renal, prostate, CNS, ovarian, and breast cancer cell lines.
Results showed that the tested compounds had a promising anticancer activity (Table
1, 2, supporting information) against human cancer cells especially compounds 24,
25, 27, 41 and 47 (mean growth inhibition percentages were 82.79, 108.20, 65.81,
61.60 and 92.19%, respectively (Fig. 80, supporting information)).
Furthermore, eighteen compounds; 9, 11, 13-15, 17, 18, 22-25, 27, 31, 41, 43-45 and
4
7 exhibited remarkable anticancer activity against RPMI-8226 cell line with cell
growth inhibition% ranging from 68.90 to 133.10%. Additionally, compounds; 9, 11,
1
3, 14, 17, 18, 22-25, 27, 31, 32, 34, 40, 41, 43, 44 and 47 showed remarkable
anticancer activity against HCT-116 cell line with cell growth inhibition% ranging
from71.98–191.71%.
On The other hand; only the compounds; 11,13, 18, 22-25, 27, 41, 44 and 47
7

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exhibited remarkable anticancer activity against MCF7 cell line with cell growth
inhibition% ranging from 68.59 – 108.34%.
2
.2.1.2 In vitro five-dose assay
Compounds 26, 27, 29, 43 and 49 were selected for advanced five-dose testing
against the full panel of 60 human tumor cell lines. All the 60 cell lines representing
nine tumor subpanels were incubated at five different concentrations (0.01, 0.1, 1, 10
and 100 µM). This revealed their great potency against almost cell lines.
Data of five dose assay results showed that the five selected compounds exhibited a
remarkable and broad-spectrum antitumor activity against all cell lines used with GI50
ranging from 0.15 to 23.30 µM (Table 1) with no selectivity (Tables 3-7, supporting
information).
8

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Table (1): GI of compounds 24, 25, 27, 41 and 47 against 60 cell lines of 9 different cancer
5
0
panels tested using NCI's in vitro five dose anticancer assay.
Panel/Cell
Line
GI (µM)
Compound
27
Panel/Cell
Line
GI (µM)
Compound
5
0
50
2
4
25
41
47
24
25
27
41
47
Leukemia
Melanoma
CCRF-CEM 2.38
0.34 3.33 1.85 nd
0.32 2.88 2.57 nd
0.40 3.44 3.02 nd
0.33 4.22 2.26 nd
0.15 1.51 1.72 0.27
0.23 2.34 0.72 0.32
LOX IMVI 1.66 0.21 2.29
MALME-3M 6.69 1.28 11.10 1.75 nd
1.46 nd
HL-60(TB)
K-562
2.65
1.70
2.66
M14
MDA-MB-435
2.43 0.32 2.96
1.58 0.27 2.72
1.80 nd
1.91 nd
MOLT-4
RPMI-8226 0.25
SR
1.52
Non-Small Cell Lung Cancer
A549/ATCC 3.61
0.46 6.46 14.90 nd
SK-MEL-2 4.52 1.78 14.80 5.03 nd
SK-MEL-28 4.23 0.71 3.29
SK-MEL-5 2.53 0.42 4.27
5.78 nd
1.81 nd
12.0 nd
2.26 nd
UACC-257
UACC-62
6.38 nd
7.45
EKVX
2.92
3.76
2.47
1.94
2.70
1.33 4.03
0.28 5.16
3.05
4.36
nd
2.94 0.85 3.31
HOP-62
HOP-92
NCI-H226
NCI-H23
1.84
Ovarian Cancer
6.58 12.50 1.56
2.06 IGROV1
2.76 0.33 3.42
2.41 0.25 2.98
3.10 0.56 5.67
4.75 1.37 3.97
2.79 0.46 5.94
1.85
1.72
1.96
14.00
3.09
nd
0.22 1.98
0.43 3.41
3.92
1.80
nd
nd
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
nd
1.80
nd
NCI-H322M 4.89
0.60 6.85 13.60 nd
NCI-H460
NCI-H522
3.41
0.34 3.76
5.05
nd
nd
NCI/ADR-
RES
nd
nd
3
.25
0.39 6.90
1.48
2 .05
4.74 1.01 15.30 2.66
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
SK-OV-3
Renal Cancer
786-0
6.01 0.64 5.08
13.10
4.92
2.36
0.47
2.68
2.33
1.97
3.00
0.40 4.94
0.18 2.05
0.15 2.09
0.22 1.86
0.24 2.66
0.30 2.82
0.36 3.37
2.10
2.97
1.17
1.80
1.86
2.24
1.96
nd
nd
nd
nd
nd
nd
nd
2.16 0.31 1.85
2.47 4.11 7.40
3.67 3.43 4.16
1.47 0.20 2.16
3.43 0.46 4.74
1.78
14.70
2.38
1.66
2.25
1.05
1.35
1.61
1.33
nd
A498
ACHN
RXF 393
SN12C
KM12
SW-620
TK-10
7.87 1.28 23.30 14.60
9.30
nd
CNS cancer
SF-268
UO-31
1.98 0.21 4.18
1.48
4.32
4.49
0.32 3.99
1.89
nd
Breast Cancer
SF-295
0.40 5.25 12.20 1.90 MCF7
0.38 0.20 1.83
0.38 0.42 3.81
1.41 nd
MDA-MB231/
ATCC
SF-539
nd
2
3
.96
.64
0.25 2.52
1.80
1.73 4.09
SNB-19
2.18 HS 578T
0.35 4.30
0.22 2.71
0.23 2.15
3.50
2.60
1.75
5.81 0.74 4.66
2.40 0.21 2.14
2.24 0.39 5.44
7.68 20.40
1.60 nd
2.24 nd
SNB-75
U251
1.79
nd
0.43 BT-549
0.53 T-47D
MDA-MB-
Prostate Cancer
4
68
3.47 0.32 2.45
1.96 nd
PC-3
3.20
3.73
0.56 4.29
0.44 6.09
3.09
3.21
nd
nd
DU-145
nd= not detected
9

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2
.2.1.3 In vitro Anti-proliferative assay on A549 cells
Independently, all the synthesized compounds 8-47 were tested for their inhibitory
effect on human non-small cell lung cancer A549 cell growth using BrdU
incorporation assay. A549 cells were exposed to different concentrations (0.5, 1, 5,
1
0, 25, 50 or 100 µM) from chalcone/ 1,2,4-triazole hybrids 8-47 or the reference
cisplatin for 24 h using DMSO as a negative control. Data were summarized in
Table2. Twenty-five compounds out of forty showed significant cell growth
inhibition on A549 cells (IC = 4.4-79.6 µM) in a dose-dependent manner, while
5
0
fifteen compounds had very weak or no anticancer activity (IC50 > 100 µM) in
comparison with cisplatin as a positive control (IC = 15.3 µM). In particular, in
5
0
agreement with the NCI results, compounds 24, 25, 27, 41 and 47 exhibited the
highest activities against A549 cells (IC = 6.06, 4.4, 7.55, 16.04 and 8.04 µM,
5
0
respectively).
In other words, through a simple structure-activity relationship (SAR) analysis, we
found that hybrids containing N4-allyl triazole were more active than that containing
N4-phenyl triazole. In addition, substitution in both phenyl rings was essential for
anticancer activity. Also, for optimum activity in the allyl triazole hybrids; R must be
2
3
,4,5-trimethoxy groups and the phenyl ring must be substituted with mono
substitution either electron donating or electron withdrawing (OCH or Cl) good in p-
3
position but not exclusive as in case of unsubstituted ring, the activity decreased
slightly, while in case of 3,4-dimethoxy substitution, the activity decreased to great
extent but the activity retained in case of trimethoxy substitution. On the other hand;
for optimum activity in the phenyl triazole hybrids; R and R must be 3,4,5-
1
2
trimethoxy groups. The activity decreased slightly if the phenyl ring substituted with
mono substitution either electron donating or electron withdrawing (OCH or Cl) in p-
3
position. From these results, it is obvious that 1,2,4-triazole/chalcone hybrids
especially compounds 24, 25, 27, 41 and 47 had the ability to suppress the cell
proliferation in human cancer cells especially A549cells.
1
0

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Table (2): Cytotoxic activity of triazole-chalcone hybrids, 8-47, against human lung
adenocarcinoma A549 cell line for 24 h.
Compound
IC50
µM)± SD
>100
>100
>100
23.15±1.3
28.2±0.81
22.7±0.3
>100
44.15±2.5
>100
23.6±1.5
24.6±0.42
>100
79.6±3.08
>100
16.6±1.6
21.8±0.9
6.06±0.5
4.4±0.3
59.7±2.4
7.55±0.8
15.3±1.04
Compound
IC50
(µM)± SD
>100
19.43±1.7
24.02±0.9
30.7±1.05
>100
49.82±2.2
>100
21.9±1.8
>100
19.58±0.58
>100
49.25±3.45
46.72±1.85
16.04±1.32
>100
(
8
9
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
>100
20.82±0.78
31.5±1.27
>100
8.04±0.59
Cisplatin
2
.2.2. Triazole-chalcone hybrids induced apoptosis in A549 human lung
adenocarcinoma cells
Apoptosis plays an important role in hemostasis process. Balance between survival
and apoptosis is critical for the maintenance of physiologic functions. Imbalance
toward the survival in cell results in cancer development and resistance to anticancer
therapies such as radiotherapy and chemotherapy. Apoptosis is typically accompanied
by the activation of a class of death proteases (caspases) [61]. The activation of the
caspase cascade is involved in chemical-and agent-induced apoptosis [62]. Caspase-3
has been shown to be a key component involved in the underlying mechanisms of
apoptosis and lies on the action of the initiator caspases including caspase-8 and
caspase-9 for its action [63]. Caspase-8 is usually activated through the extrinsic
pathway triggered by tumor necrosis factor receptor and Fas/CD95 receptor [64]. In
contrast, caspase-9 is activated through the intrinsic pathway, which can be initiated
by translocation of Bax into mitochondria [65]. This is followed by an increase in
mitochondrial membrane permeability and a release of cytochrome c from
mitochondria into the cytosol. In the cytosol, cytochrome c activates caspase-9, which
1
1

ACCEPTED MANUSCRIPT
leads to apoptotic cell death. Both pathways activate caspase-3, eventually causing
apoptosis [66]. To study in depth the bioactivities of triazole-chalcone hybrids,
compounds 24, 25, 27, 41 and 47, against A549 cells, the cancer cells were treated
with vehicle alone as a control or with one of the five testing compounds at different
concentrations (1, 5 and 10 µM). After 24 h, the samples were double-stained with
annexin V and PI. The percentages of cell populations at various stages of apoptosis
were exhibited in Fig. 4. (A and B). The five compounds increased both early and late
apoptotic cells percentage in a dose-dependent manner. At 10 µM, the total apoptotic
cells percentages (early + late, annexin V positive cells) were higher in compounds
2
4, 25 and 27 (78.6, 54.4 and 66.1%, respectively) than in compounds 41 and 47 (24.2
and 42.3 %, respectively) in comparison with control (9.5%). It is obvious from these
results that hybrids containing allyl triazole (24, 25 and 27) were more apoptotic
inducers than hybrids containing phenyl triazole (41 and 47) which confirm our
previous cytotoxicity assay results. Evidently, these data pointed out triazole-chalcone
hybrids having the ability to induce apoptosis in A549 cells at low micromolar.
1
2

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Fig.4. (A) Flow cytometric analysis of A549 cells treated with different concentrations (0, 1, 5,
1
0 µM) of triazole-chalcone hybrids, (24, 25, 27, 41 and 47) for 24 h. (B) Apoptotic cell % of
early, late and total (early + late) apoptotic cells after incubation of A549 cells with different
concentrations (0, 1, 5, 10 µM) of triazole-chalcone hybrids, (24, 25, 27, 41 and 47) for 24 h.
The values are expressed as the mean ± SD of three different experiments. *, P < 0.05 and **, P
<
0.005 indicate a significant difference compared with vehicle treatment (control).
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3

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2
3
.2.3. Triazole-chalcone hybrids induced apoptosis through activation of caspase-
, -8 and -9
The effect of triazole-chalcone hybrids 24, 25, 27, 41 and 47 on the expression of pro-
apoptotic markers (Bax, cytochrome c and caspases 3, 8 and 9) was tested to elucidate
signal transduction pathway through which these compounds fulfill their apoptotic
effect as shown in Fig.5. (A-C). All tested compounds upregulated Bax protein in a
dose-dependent manner. As a consequence of Bax activation, cytochrome c is also
released from the mitochondria and increased in the cytosol, in a dose-dependent
manner, after treatment with the mentioned compounds. Furthermore, cleaved
caspase-3, -8, and -9 were increased by triazole-chalcone hybrids treatment for 24 h
in a dose-dependent manner. From these results, it is observed that the ability of the
allyl triazole compounds (24, 25 and 27) to upregulate the tested apoptotic markers
was higher than the phenyl triazole compounds (41 and 47). Furthermore, it was
observed that although compound 47 always had higher activities than 43 but its
ability to upregulate cleaved caspase 8 was very weak.
Furthermore, triazole-chalcone hybrids activated both extrinsic and intrinsic apoptotic
pathways. The studied compounds activated the extrinsic pathway via activation of
caspase-8 while activated the intrinsic pathway through Bax activation, the release of
cytochrome c from mitochondria and caspase-9 activation. The two apoptotic
pathways activated the most effector caspase-3 leading to apoptosis in A549 cells.
Triazole alone can induce apoptosis; these results in agreement with the previous
reports that showed triazole-chalcone hybrids induce apoptosis through the
mitochondria and/or death receptor pathways in several cancer cell lines [67, 54].
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4

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Fig.5. (A) Western blotting analysis of the pro-apoptotic marker proteins, Bax,
cytochrome c and cleaved caspase-3/8 and 9 in A549 cells treated with 0, 1, 5, or 10
µM triazole-chalcone hybrids, (24, 25, 27, 41 and 47). β-actin served as a loading
control. (B-C) The relative expression (fold/ β-actin) of Bax, cytochrome c and
cleaved caspase-3/8 and 9 compared to β-actin and quantified by Image J. The values
are expressed as the mean ± SD of three different experiments.
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5

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2
.2.4. Pan-caspase inhibitor, z-VAD-fmk, decreased triazole-chalcon hybrids-
induced caspase-3 activation in A549 cells
To investigate the relationship between apoptosis induced by triazole-chalcone
hybrids in A549 cells and caspase-3, pan-caspase inhibitor z-VAD-fmk was tested.
Representative results are shown in Fig.6A, and quantitative data from 3 different
experiments are summarized in Fig.6B. The cleaved Caspase-3 level after triazole-
chalcone hybrids treatment in the presence of z-VAD-fmk was significantly decreased
at 24 h after treatment, as compared with triazole chalcone hybrids treatment alone.
Fig.6. (A) Western blotting analysis of cleaved caspase-3 in A549 cells treated with 0,
or 10 µM triazole-chalcone hybrids, (24, 25, 27, 41 and 47) in the absence or presence
of Pan-caspase inhibitor (z-VAD-fmk) for 24 h. β-actin served as a loading control.
(B) The relative protein expression levels of cleaved caspase-3 compared to β-actin
and quantified by Image J. The values are expressed as the mean ± SD of three
different experiments. **, P < 0.005 indicates a significant difference vs. triazole-
chalcone hybrids-treated cells.
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6

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2
.2.5. Triazole-chalcone hybrids induce Caspase-3 dependent apoptosis
The effect of the general caspase inhibitor, z-VAD-fmk, on the triazole chalcone
hybrids- induced apoptosis was determined by Annexin V assay (Fig. 7A-B).
Triazole-chalcone hybrids treatment for 24 h in the presence of z-VAD-fmk
significantly reduced the total apoptotic cells (early+late) in all tested compounds.
The great effect was found in the presence of compound 25, 27, 41 and 47 where the
total apoptotic cells percentage reduced from 53.8 to 20.09%, 64.4 to 26.46%, 24.04
to 15.1%, and 44.4 to 13.33%, respectively. However, z-VAD-fmk significantly
reduced the apoptotic cells percentage, induced by compound 24, from 78.23 to
5
0.67 % which still higher than control (11.85 %). These results indicated that
triazole-chalcone hybrids induced apoptosis via caspase-3 dependent pathway.
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7

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Fig.7. (A) Flow cytometric analysis of A549 cells treated with 0 or 10 µM of triazole-
chalcone hybrids, (24, 25, 27, 41 and 47) with or without z-VAD-fmk for 24 h.
(B) Apoptotic cells % of early, late and total (early + late) apoptotic cells after
incubation of A549 cells with 0 or 10 µM of triazole-chalcone hybrids, (24, 25, 27, 41
and 47) with or without z-VAD-fmk for 24 h. The values are expressed as the mean ±
SD of three different experiments. **, P < 0.005 indicates a significant difference vs.
triazole-chalcone hybrids treated cells.
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8

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3
. Conclusion
A series of novel 1,2,4-triazole/chalcone hybrids was prepared and identified with
different spectroscopic techniques. The prepared compounds showed remarkable
cytotoxic activity against different cancer cell lines. Compounds 24, 25, 27, 41 and 47
had shown the highest cytotoxicity among the tested compounds against human lung
adenocarcinoma A549 cells with an IC range from 4.40 to 16.04 µM compared to
5
0
cisplatin with IC50 of 15.3 µM. Primary in vitro one dose anticancer and BrdU
incorporation assays confirmed that compounds 24, 25, 27, 41 and 47 exhibited the
highest activities against human cancer cells. The results indicated that the hybrids
containing allyl triazole were more active than that containing phenyl triazole. Also, it
was observed that the apoptosis was induced in a dose-dependent manner.
Furthermore, triazole-chalcone hybrids activated both extrinsic and intrinsic apoptotic
pathways. The studied compounds activated the extrinsic pathway via activation of
caspase-8 while activated the intrinsic pathway through Bax activation, the release of
cytochrome c from mitochondria and caspase-9 activation. Additionally, the apoptotic
cells percentages in all tested compounds were significantly reduced after using z-
VAD-fmk which indicated the dependency of triazole-chalcone hybrids-induced
apoptosis on the caspase-3 pathway. In summary, 1,2,4-triazole/chalcone hybrids
induced caspase-3 dependent apoptosis through both extrinsic and intrinsic pathways
in A549 cells. The finding that 1,2,4-triazole/chalcone hybrids had an anticancer
effect is meaningful for the treatment of lung cancer as an alternative to conventional
chemotherapy, which has many side effects. However, further study with in vivo
models is needed to clarify the efficacy of 1,2,4-triazole/chalcone hybrids as an
anticancer agent.
4
4
. EXPERIMENTAL SECTION
.1.Chemistry section:
•
Chemicals and solvents that were used for the analytical grade. The progress
of the reactions was monitored by thin layer chromatography pre-coated
Merck silica gel 60 F254 aluminum sheets.
•
Melting points were determined on Stuart electro-thermal melting point
apparatus and were uncorrected.
1
9

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1
•
H spectra were recorded on JEOL JNM-GX-600 spectrometer (600 MHz);
Japan and Burker AG, Switzerland, 500 MHz, Faculty of Pharmaceutical
Sciences, Umm Al-Qura University, Mecca, Saudi Arabia; chemical shift (δ)
in ppm relative to TMS (δ=0 PPM) as internal standard and CDCl as a
3
solvent. Chemical shifts (δ) are expressed in parts per million (ppm) and
coupling constants (J) are expressed in Hertz. The signals are designated as
follows: s, singlet; d, doublet; t, triplet; m, multiplet.
1
3
•
C spectra were recorded on JEOL JNM-GX-150 MHz; Japan and Burker
AG, Switzerland and 125 MHz, faculty of Pharmaceutical Sciences, Umm Al-
Qura University, Mecca, Saudi Arabia using TMS as the reference standard
and CDCl as a solvent. Chemical shifts (δ) are expressed in parts per million
3
(ppm).
•
•
The purity of the compounds was checked by HPLC.
Elemental analyses were recorded on Shimadzu GC/ MS-QP5050A, The
Regional center for Mycology and Biotechnology, Al-Azhar University,
Egypt.
4
.1.1. General procedure for the synthesis of substituted ethylbenzoate 2a-e [57].
A mixture of the appropriate substituted benzoic acid 1a-e (10 mmol), absolute
ethanol (20 mL) and concentrated sulfuric acid (2 mL) was heated under reflux for 12-
1
8 h. Excess solvent was removed under reduced pressure; The residue extracted with
ether (2 X 50 mL) and washed with saturated NaHCO (2 X 20 mL). The ether layer
3
was dried over anhydrous magnesium sulphate, and the ether was evaporated under
vacuum to give the ethyl ester derivatives 2a-e.
4
.1.2. General procedure for the synthesis of substituted benzohydrazides 3a-e
[
58, 59].
A solution of the isolated esters 2a–e (10 mmol) in ethanol (20 mL), hydrazine
hydrate (97 %, 3 mL) was added and heated under reflux for 5-8 h. After cooling, the
formed precipitate was filtered off, washed with water, dried, and crystallized from
ethanol.
Benzohydrazide (3a) [59].
o
o
White solid (77.2 % yield); mp 110-112 C (Reported mp = 112-114 C).
2
0

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4
-Chloro-benzohydrazide (3b) [68].
White solid (79.3% yield); mp 195-197 C (Reported mp = 189-191 C).
-Methoxybenohydrazide (3c) [59].
White solid (80.2 % yield); mp 134-137 ºC (Reported mp =136-140ºC)
,4-Dimethoxybenzohydrazide (3d) [59].
White solid (72.7 % yield); mp 145-146 ºC as reported.
o
o
4
3
3
,4,5-Trimethoxybenzohydrazide (3e) [59].
o
White solid (72.9% yield); mp 158-160 C as reported.
4
.1.3. General procedure for the synthesis of 4-allyl/phenyl-5-aryl-4H-1,2,4-
triazole-3-thiol derivatives (4a-j) [59, 60].
Equimolar quantities of the benzohydrazides 3a-e (0.1 mol) and allyl/ phenyl
isothiocyanate (0.1 mol) in 125 mL of absolute ethanol were heated under reflux for 4
h. The solvent was evaporated under vacuum. The resulting solid was filtered off,
dried and used for the following step as a crude product. A mixture of the crude
products (10mmol) and 100 mL of 2 N NaOH was heated under reflux for 3 h. The
reaction mixture was cooled and acidified to pH 2 with concentrated HCl. The solid
that precipitated was filtered off, washed with water, and recrystallized from 95%
ethanol [59].
4
-Allyl-3-phenyl-4H-1,2,4-triazole-3-thiol (4a) [36].
o
o
White solid (78.6% yield); mp 120-121 C (Reported mp = 118-119 C).
4
-Allyl-3-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (4b) [ 69].
o
o
White solid (77.9% yield); mp 176-178 C (Reported mp = 180-181 C).
4
-Allyl-3-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (4c) [36].
o
o
White solid (69.5% yield); mp 119-120 C (Reported mp = 120-121 C).
4
-Allyl-3-(3,4-dimethoxyphenyl)-4H-1,2,4-triazole-3-thiol (4d) [36].
o
o
White solid (70.2% yield); mp 123-124 C (Reported mp = 121-123 C).
-Allyl-3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-3-thiol (4e) [ 70].
4
o
o
White solid (70.3% yield); mp 202-203 C (Reported mp = 200-201 C).
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1

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3
,4-Diphenyl-4H-1,2,4-triazole-3-thiol (4f) [ 59].
o
o
White solid (67.9% yield); mp 281-283 C (Reported mp = 280-283 C).
3
-(4-Chlorophenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (4g) [71].
o
o
White solid (77.0% yield); mp 239-241 C (Reported mp = 243-245 C).
3
-(4-Methoxyphenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (4h) [59].
o
o
White solid (72.8% yield); mp 281-283 C (Reported mp = 284-285 C).
3
-(3,4-Dimethoxyphenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (4i) [59].
o
o
White solid (69.7% yield); mp 237-238 C (Reported mp = 234-235 C).
3
-(3,4,5-Trimethoxyphenyl)-4-phenyl-4H-1,2,4-triazole-3-thiol (4j) [59].
o
o
White solid (78.5% yield); mp 209-211 C (Reported mp = 210-211 C).
4
1
.1.4. General procedure for the synthesis of 1-(4-aminophenyl)-3-arylprop-2-en-
-one (6a-d) [7].
An equimolar amount of p-aminoacetophenone (13.51 gm, 0.1 mol) and the
appropriate aldehyde 5a-d (0.1mol), were dissolved in a minimum amount of ethanol,
aqueous NaOH (0.25 mol, 60%) was added dropwise. The reaction mixture was
stirred in ice bath for 30 min then at rt until the precipitate was formed within 3 h. The
precipitate was filtered off and washed thoroughly with cold distilled water and cold
methanol (2x20 mL). The product was recrystallized from absolute ethanol. The
structure of the product was confirmed by mp.
1
-(4-Aminophenyl)-3-phenyl prop-2-en-1-one (6a) [46].
o
o
Buff solid (77.8% yield); mp 156-157 C (Reported mp = 157-158 C).
1
-(4-Aminophenyl)-3-(4-chlorophenyl)prop-2-en-1-one (6b) [7].
o
o
Yellow solid (80.3% yield); mp 159-160 C (Reported mp = 158-159 C).
1
-(4-Aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (6c) [7].
o
o
Yellow solid (67.3% yield); mp 115-116 C (Reported mp = 114-115 C).
1
-(4-Aminophenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6d) [7].
o
Yellow solid (70.4% yield); mp 166-168 C as reported.
2
2

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4
.1.5.
General
procedure
for
synthesis
of
2-bromo-N-(4-(3-
arylacryloyl)phenyl)acetamides (7a-d) [46].
To a stirred mixture of the appropriate chalcone 6a-d (6.30mmol) in dichloromethane
20 mL) and potassium carbonate (0.18gm, 1.302 mmol) in 100 mL water in an ice
(
bath. bromoacetyl bromide (1.856g, 9.20 mmol) in 30 mL dichloromethane was
added in a dropwise manner with stirring over 30 min. Stirring was continued for 2h
at 0ᴼC and at RT overnight. The reaction mixture was extracted with dichloromethane
(2 x 60 mL) and the organic layer was washed with distilled water (2x40 mL), dried
over anhydrous sodium sulphate, filtered, evaporated under vacuum and the residue
was recrystallized from ethanol.
2
-Bromo-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide (7a) [46].
o
o
Pale yellow powder (70.0% yield); mp 157-159 C (Reported mp = 157-158 C).
2
-Bromo-N-(4-(E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (7b) [7].
o
o
Pale yellow powder (71.5% yield); mp 191-192 C (Reported mp = 190-192 C).
-Bromo-N-(4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)acetamide (7c) [46].
2
o
o
Pale orange crystal (69.70% yield); mp 155-156 C (Reported mp = 160-162 C).
2
-Bromo-N-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide (7d)
[
46].
o
o
Yellow powder (78.0% yield); mp 166-167 C (Reported mp= 160-162 C).
.1.6. General Procedure for synthesis of 2-(4-allyl/phenyl-5-phenyl-4H-1,2,4-
4
triazol-3-ylthio)-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide derivatives (8-47).
An equimolar mixture of 4a-j (0.09 mmol) and compound 7a-d (0.09 mmol) in
acetonitrile. TEA (0.18 mmol) was added as a base. The reaction mixture was stirred
at room temperature until the precipitate formed. The formed precipitate was filtrated
off then the precipitate was crystallized with acetonitrile to afford the target
compounds [46].
2
3

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4
.1.6.1. 2-(4-Allyl-5-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-
phenylacryloyl)-phenyl)acetamide (8).
1
Orange crystal (0.29g, 80% yield); mp 140-143ᴼC; H NMR (600 MHz, CDCl ) δ
3
(
ppm): 4.08 (2H, s, SCH ), 4.54 (2H, d, J = 4.00 Hz, NCH ), 5.03 (1H, d, Jtrans=17.22
2
2
Hz, N-CH CH=CH ), 5.32 (1H, d, J =11.04 Hz, N-CH CH=CH ), 5.88-5.89 (1H, m,
2
2
cis
2
2
N-CH CH=CH ), 7.35-7.36 (3H, m, Ar-H), 7.45-7.50 (4H, m, Ar-H), 7.57-7.58 (3H,
2
2
m, Ar-H + C-H=CH), 7.71-7.76 (4H, m, Ar-H + CH=CH), 7.93 (2H, d, J = 8.22 Hz,
13
Ar-H), 11.00 (1H, s, NH); C NMR (150 MHz, CDCl ) δ (ppm): 37.18, 47.04, 97.32,
3
1
1
18.93, 119.13, 121.74, 126.00, 128.32, 128.43, 128.84, 129.04, 129.69, 130.01,
30.65, 133.60, 134.84, 142.64, 144.25, 152.60, 156.41, 166.94, 188.97; Anal. Calcd.
For C H N O S (480.58): C, 69.98; H, 5.03; N, 11.66, Found: C, 70.25; H, 5.11; N,
2
8
24
4
2
1
1.95.
4
.1.6.2. 2-(4-Allyl-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-
phenyl- acryloyl)phenyl)acetamide (9).
1
Off white powder (0.32g, 84% yield); mp:219-221ᴼC; H NMR (600 MHz, CDCl ) δ
3
(
ppm): 4.03 (2H, s, SCH ), 4.55 (2H, d, J=4.86 Hz, NCH ), 5.07 (1H, d, Jtrans=17.16
2
2
Hz, N-CH CH=CH ), 5.37 (1H, d, J =10.32 Hz, N-CH CH=CH ), 5.89-5.93 (1H,
2
2
Cis
2
2
m, N-CH CH=CH ), 7.39-7.40 (3H, m, Ar-H + C-H=CH), 7.41-7.51 (3H, m, Ar-H),
2
2
7
.56 (2H, d, J=8.94 Hz, Ar-H),7.60 (1H, d, J = 15.00 Hz, C-H=CH), 7.61-7.62 (1H,
m, Ar-H), 7.73-7.78 (3H, m, Ar-H), 7.97 (2H, d, J = 8.94 Hz, Ar-H), 10.96 (1H, s,
1
3
NH); C NMR (150 MHz, CDCl ) δ (ppm): 36.90, 47.15, 119.18, 121.81, 124.55,
3
1
1
28.43, 128.93, 129.52, 129.78, 129.83, 130.00, 130.43, 130.48, 133.69, 134.98,
37.14, 142.58, 144.39, 153.27, 155.62, 166.94, 188.99; Anal. Calcd. For
C H ClN O S (515.03): C, 65.30; H, 4.50; N, 10.88. Found: C, 65.57; H, 4.33; N,
28
23
4
2
1
1.23.
4
.1.6.3. 2-(4-Allyl-5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-
phenyl- acryloyl)phenyl)acetamide (10)
1
Pale brown powder (0.34g, 84% yield); mp: 214-216ᴼC; H NMR (600MHz, CDCl )
3
δ (ppm): 3.84 (3H, s, OCH ), 4.05 (2H, s, SCH ), 4.54 (2H, d, J = 4.08 Hz, NCH ),
3
2
2
5
.06 (1H, d, Jtrans = 17.16 Hz, NCH -CH=CH ), 5.35 (1H, d, J =10.26 Hz, N-
2 2 Cis
CH CH=CH ), 5.89-5.92 (1H, m, N-CH CH=CH ), 7.00 (2H, d, J=8.94 Hz, Ar-H),
2
2
2
2
2
4

ACCEPTED MANUSCRIPT
7
.38-7.39 (3H, m, Ar-H), 7.49 (1H, d, J = 15.78 Hz, C-H=CH), 7.54 (2H, d, J =8.94
Hz, Ar-H), 7.60-7.61 (2H, m, Ar-H), 7.74 (2H, d, J = 8.94 Hz, Ar-H),7.76 (1H, d,
1
3
J=15.78 Hz, C-H=CH), 7.95 (2H, d, J =8.94 Hz, Ar-H),11.14 (1H, s, NH), C NMR
150 MHz, CDCl ) δ (ppm): 37.07, 47.06, 55.35, 97.39, 114.55, 118.23, 118.97,
(
3
1
1
19.19, 121.85, 128.40, 128.90, 129.80, 130.01, 130.78, 133.56, 134.98, 142.72,
44.29, 152.51, 156.48, 161.47, 167.12, 189.00; Anal. Calcd. For C H N O S
2
9
26
4
3
(510.61): C, 68.21; H, 5.13; N, 10.97. Found: C, 68.49; H, 5.06; N, 11.21.
4
.1.6.4. 2-(4-Allyl-5-(3,4-dimethoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-
3
-phenyl acryloyl)phenyl)acetamide (11)
1
Brown Crystal ( 0.34g, 78.90% yield); mp: 117-120ᴼC; H NMR (500 MHz, CDCl ),
3
δ (ppm): 3.91 (3H, s, OCH ), 3.95 (3H, s, OCH ), 4.11 (2H, s, SCH ), 4.61 (2H, d,
3
3
2
J=4.07 Hz, NCH ), 5.12 (1H, d, Jtrans = 17.10 Hz, N-CH CH=CH ), 5.41 (1H, d, J =
Cis
2
2
2
1
0.40 Hz, N-CH CH=CH ), 5.92-6.02 (1H, m, N-CH CH=CH ), 6.98 (1H, d, J=8.20
2 2 2 2
Hz, Ar-H), 7.17 (2H, d, J = 8.20 Hz, Ar-H), 7.21 (1H, s, Ar-H), 7.42-7.43 (2H, m, Ar-
H), 7.53 (1H, d, J = 15.70 Hz, C-H=CH), 7.64-7.65 (2H, m, Ar-H), 7.78-7.84 (3H, m,
1
3
Ar-H + C-H=CH), 8.00 (2H, d, J = 8.20 Hz, Ar-H), 11.12 (1H, s, NH); C NMR (125
MHz, CDCl ) δ (ppm): 37.25, 47.41, 56.05, 56.13, 111.25, 111.65, 112.71, 119.03,
3
1
1
19.26, 121.20, 121.85, 128.44, 128.96, 129.82, 130.46, 130.78, 134.97, 142.72,
44.41, 149.45, 151.22, 152.70, 155.96, 156.31, 166.93, 189.00; Anal. Calcd. For
C H N O S (540.36): C, 66.65; H, 5.22; N, 10.36. Found: C, 66.94; H, 5.36; N,
30
28
4
4
1
0.62.
4
.1.6.5.
2-(4-Allyl-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-
((E)-3-phenylacryloyl)phenyl)acetamide (12)
1
Pale yellow Crystal (0.35g, 90.20% yield); mp =189-191ᴼC; H NMR (600 MHz,
CDCl ) δ (ppm): 3.82 (6H, s, 2OCH ), 3.88 (3H, s, OCH ), 4.05 (2H, s, SCH ), 4.58
3
3
3
2
(
(
(
2H, d, J = 4.04 Hz, NCH ), 5.11 (1H, d, J
= 17.16 Hz,N-CH CH=CH ), 5.39
2
trans 2 2
1H, d, JCis = 9.72 Hz, N-CH CH=CH ), 5.94-5.99 (1H, m, N-CH CH=CH ), 6.82
2
2
2
2
2H, s, Ar-H), 7.36-7.37 (3H, m, Ar-H), 7.49 (1H, d, J = 15.84 Hz, C-H=CH), 7.59-
.60 (2H, m, Ar-H), 7.74-7.76 (3H, m, Ar-H + C-H=CH), 7.95 (2H, d, J = 7.56 Hz,
7
1
3
Ar-H), 11.05 (1H, s, NH ); C NMR (150 MHz, CDCl ) δ (ppm): 37.24, 47.46,
3
5
1
6.46, 61.15, 106.01, 119.02, 119.43, 121.10, 122.00, 128.64, 129.14, 130.01, 130.70,
31.26, 133.87, 135.19, 140.32, 142.86, 144.60, 153.12, 153.95, 156.84, 167.22,
2
5

ACCEPTED MANUSCRIPT
1
89.18; Anal. Calcd. For C H N O S (570.66): C, 65.25; H, 5.30; N, 9.82. Found:
31
30
4
5
C, 65.57; H, 5.18; N, 10.06.
4
.1.6.6.
2-(4-Allyl-5-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
Chlorophenyl)- acryloyl)phenyl)acetamide (13)
1
Off white powder (0.30 g, 82% yield); mp 219-221ᴼC; H NMR (600 MHz, CDCl ) δ
3
(
ppm): 4.05 (2H, s, SCH ), 4.56 (2H, d, J = 4.08 Hz, NCH ), 5.08 (1H, d, J
=
2
2
trans
1
7.16 Hz, N-CH CH=CH ), 5.36 (1H, d, J = 10.32 Hz, N-CH CH=CH ), 5.89-5.92
2 2 Cis 2 2
(
1H, m, N-CH CH=CH ), 7.35 (2H, d, J = 8.94 Hz, Ar-H), 7.46 (1H, d , J = 15.78 Hz,
2 2
C-H=CH), 7.50-7.52 (3H, m, Ar-H), 7.53 (2H, d, J = 8.22 Hz, Ar-H), 7.61 (2H, d, J =
.22 Hz, Ar-H), 7.69 (1H, d, J = 15.78 Hz, C-H=CH), 7.74 (2H, d, J = 8.94 Hz, Ar-
8
1
3
H), 7.94 (2H, d, J = 8.94 Hz, Ar-H), 11.11 (1H , s, NH); C NMR (150 MHz, CDCl )
3
δ (ppm): 36.74, 46.89, 118.97, 122.00, 125.84, 128.19, 128.29, 128.92, 129.31,
1
1
29.58, 129.65, 130.38, 131.01, 133.18, 133.38, 136.00, 142.44, 142.54, 152.71,
56.35, 166.84, 188.43; Anal. Calcd. For C H ClN O S (515.03): C, 65.30; H, 4.50;
2
8
23
4
2
N, 10.88. Found: C, 65.19; H, 4.57; N, 11.21.
4
.1.6.7.
2-(4-Allyl-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
chloro- phenyl)acryloyl)phenyl)acetamide (14)
1
Off white powder (0.42 g, 85.01% yield); mp: 178-180ᴼC; H NMR (500 MHZ,
CDCl ) δ (ppm): 4.17 (2H, s, SCH ), 4.60-4.62 (2H, m, NCH ), 5.12 (1H, d, J
=
trans
3
2
2
1
7.16 Hz, N-CH CH=CH ), 5.43 (1H, d, Jcis = 10.32 Hz, N-CH CH=CH ), 5.91-6.00
2 2 2 2
(
1H, m, N-CH CH=CH ), 7.41 (2H, d, J = 7.10 Hz, Ar-H), 7.49-7.53 (3H, m, Ar-H +
2 2
CH=CH), 7.58-7.62 (4H, m, Ar-H), 7.75 (1H, d, J = 15.60 Hz, CH=CH), 7.82 (2H, d,
13
J = 7.80 Hz, Ar-H), 8.00 (2H, d, J =7.80 Hz, Ar-H), 11.06 (1H, s, NH); C NMR
125 MHz, CDCl ) δ (ppm): 37.16, 47.51, 118.46, 119.50, 122.32, 123.38, 124.05,
(
3
1
1
26.57, 128.30, 129.05, 129.33, 130.77, 133.30, 136.44, 137.47, 142.47, 143.51,
45.31, 152.69, 155.51, 166.78, 188.67; Anal. Calcd. For C H Cl N O S (549.47):
2
8
22
2
4
2
C, 61.20; H, 4.04; N, 10.20. Found: C, 61.48; H, 3.98; N, 10.47.
4
.1.6.8. 2-(4-Allyl-5-(4-methoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
chloro- phenyl)acryloyl)phenyl)acetamide (15)
1
Off white powder (0.29 g, 79.5% yield); mp: 195-197ᴼC; H NMR (600 MHz,
CDCl ) δ (ppm): 3.84 (3H, s, OCH ), 4.05 (2H, s, SCH ), 4.54 (2H, d, J = 4.04 Hz ,
3
3
2
2
6

ACCEPTED MANUSCRIPT
NCH ), 5.07 (1H, d, J
= 17.16 Hz, N-CH CH=CH ), 5.35 (1H, d, J = 10.26 Hz,
2
trans
2
2
Cis
N-CH CH=CH ), 5.89-5.92 (1H, m, N-CH CH=CH ), 6.99 (2H, d, J = 8.94 Hz, Ar-
2
2
2
2
H), 7.35 (2H, d, J = 8.28 Hz, Ar-H), 7.46 (1H, d, J = 15.78 Hz, C-H=CH), 7.52-7.54
(4H, m, Ar-H), 7.68 (1H, d, J = 15.78 Hz, C-H-CH), 7.73 (2H, d, J = 8.22 Hz, Ar-H),
1
3
7
.93 (2H, d, J = 8.22 Hz, Ar-H), 11.10 (1H, s, NH); C NMR (150 MHz, CDCl ) δ
3
(ppm): 37.08, 47.06, 55.35, 114.55, 118.22, 118.97, 119.20, 122.22, 129.16, 129.55,
1
1
29.80, 130.01, 130.78, 133.18, 133.48, 136.21, 142.72, 142.84, 152.49, 156.48,
61.47, 167.11, 188.64; Anal. Calcd. For C H ClN O S (545.05): C, 63.90; H, 4.62;
2
9
25
4
3
N, 10.28. Found: C, 64.23; H, 4.70; N, 10.45.
4
.1.6.9. 2-(4-Allyl-5-(3,4-dimethoxyphenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-
(4-chlorophenyl)acryloyl)phenyl)acetamide (16)
1
Off white Crystal (0.31 g, 79.04% yield); mp: 138-139ᴼC; H NMR (500 MHz,
CDCl ), δ (ppm): 3.92 (3H, s, OCH ), 3.96 (3H, s, OCH ), 4.19 (2H, s, SCH ), 4.64
3
3
3
2
(
2H, d, J=4.07 Hz, NCH ), 5.13 (1H, d, J
= 17.20 Hz, N-CH CH=CH ), 5.41 (1H,
trans 2 2
2
d, JCis = 10.40 Hz, N-CH CH=CH ), 5.92-6.01 (1H, m, N-CH CH=CH ), 6.99 (1H, d,
2
2
2
2
J = 8.20 Hz, Ar-H), 7.19 (1H, d, J = 8.20 Hz, Ar-H), 7.23 (1H, s, Ar-H), 7.40 (2H, d,
J = 8.20 Hz, Ar-H), 7.50 (1H, d, J = 15.60 Hz, C-H=CH), 7.58 (2H, d, J = 8.20 Hz,
Ar-H), 7.75 (1H, d, J = 15.60 Hz, CH=C-H), 8.85 (2H, d, J = 8.30 Hz, Ar-H), 7.99
1
3
(
2H, d, J = 8.30 Hz, Ar-H), 11.22 (1H, s, NH); C NMR (125 MHz, CDCl ) δ (ppm):
3
3
1
1
6
7.31, 47.60, 56.08, 56.16, 111.28, 111.43, 111.69, 117.33, 119.28, 121.27, 122.24,
29.23, 129.60, 129.80, 133.46, 133.48, 136.30, 142.84, 142.87, 149.54, 151.17,
51.46, 152.95, 156.11, 166.75, 188.69; Anal. Calcd. For C H N O S (575.08) C,
3
0
27
4
4
2.66; H, 4.73; N, 9.74. Found: C, 62.49; H, 4.81; N, 9.98.
4
.1.6.10. 2-(4-Allyl-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4triazol-3-ylthio)-N-(4-
((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (17)
1
White powder (0.32 g, 85% yield); mp: 207-209ᴼC; H NMR (600 MHz, CDCl ) δ
3
(
ppm): 3.83 (6H, s, 2OCH ), 3.86 (3H, s, OCH ), 4.06 (2H, s, SCH ), 4.58 (2H, d, J =
3 3 2
4
1
.10 Hz, NCH ), 5.11 (1H, d, Jtrans = 17.16 Hz, N-CH CH=CH ), 5.38 (1H, d, J
=
2
2
2
Cis
0.26 Hz, CHCH ), 5.95-5.96 (1H, m, N-CH CH=CH ), 6.81 (2H, s, Ar-H), 7.32 (2H,
2
2
2
d, J = 8.22 Hz, Ar-H), 7.43 (1H, d, J = 15.84 Hz, C-H=CH), 7.51 (2H, d, J = 8.22 Hz,
Ar-H), 7.66 (1H, d, J = 15.84 Hz,CH =C-H), 7.71 (2H, d, J = 8.28 Hz, Ar-H), 7.91
1
3
(
2H, d, J = 8.28 Hz, Ar-H), 11.06 (1H, s, NH); C NMR (150 MHz, CDCl ) δ (ppm):
3
2
7

ACCEPTED MANUSCRIPT
3
7.06, 47.17, 56.17, 60.85, 105.75, 118.73, 119.15, 121.10, 122.09, 129.10, 129.51,
29.70, 130.10, 131.01, 133.39, 136.17, 140.03, 142.72, 142.90, 152.76, 153.66,
56.53, 166.90, 188.50; Anal. Calcd. For C H ClN O S (605.10): C, 61.53; H, 4.83;
1
1
3
1
29
4
5
N, 9.26. Found: C, 61.76; H, 4.79; N, 9.44.
4
.1.6.11.
2-(4-Allyl-5-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
methoxyphenyl)- acryloyl)phenyl)acetamide (18)
1
Pale yellow powder (0.35 g, 84% yield); mp: 199-200ᴼC; H NMR (600MHz, CDCl )
3
δ (ppm): 3.80 (3H, s, OCH ), 4.07 (2H, s, SCH ), 4.54 (2H, d, J = 4.14 Hz, NCH ),
3
2
2
5
.03 (1H, d , Jtrans = 17.16 Hz, N-CH CH=CH ), 5.31 (1H, d, J = 10.32 Hz, N-
2 2 Cis
CH CH=CH ), 5.87-5.89 (1H, m, N-CH CH=CH ), 6.87 (2H, d, J = 8.94 Hz, Ar-H),
2
2
2
2
7
.35 (1H, d, J = 15.78 Hz, C-H=CH), 7.43-7.47 (3H, m, Ar-H), 7.54 (2H, d, J = 8.94
Hz, Ar-H), 7.57 (2H, d, J = 7.56 Hz, Ar-H), 7.71 (1H, d, J = 15.78 Hz, CH=C-H),
7
.75 (2H, d, J = 8.94 Hz, Ar-H), 7.92 (2H, d, J = 8.94 Hz, Ar-H), 10.98 (1H, s, NH);
1
3
C NMR (150 MHz, CDCl ) δ (ppm): 36.93, 45.71, 55.01, 114.03, 118.68, 118.83,
3
1
1
19.16, 125.74, 127.31, 128.19, 128.39, 128.80, 129.01, 129.85, 130.39, 133.51,
42.19, 143.87, 152.37, 156.15, 161.27, 166.66, 188.76; Anal. Calcd. For
C H N O S (510.61), C, 68.21; H, 5.13; N, 10.97. Found: C, 68.57; H, 5.03; N,
29
26
4
3
1
1.26.
4
.1.6.12. 2-(4-Allyl-5-(4-chlorophenyl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
methoxy- phenyl)acryloyl)phenyl)acetamide (19)
1
Pale yellow powder (0.35 g, 81.10% yield); mp: 231-232ᴼC; H NMR (600 MHZ,
CDCl ) δ (ppm): 3.84 (3H, s, OCH ), 4.00 (2H, s, SCH ), 4.54 (2H, d, J = 4.14 Hz,
3
3
2
NCH ), 5.08 (1H, d, J = 17.16 Hz, N-CH CH=CH ), 5.38 (1H, d, J = 10.32 Hz, N-
2
trans
2
2
CH CH=CH ), 5.89-5.92 (1H, m, N-CH CH=CH ), 6.92 (2H, d, J = 8.84 Hz, Ar-H),
2
2
2
2
7
.38 (1H, d, J = 15.84 Hz, C-H=CH), 7.49 (2H, d, J = 8.84 Hz, Ar-H ), 7.56-7.58 (4H,
m, Ar-H), 7.74-7.77 (3H, m, Ar-H + CH=C-H ), 7.98 (2H, d, J = 8.22 Hz, Ar-H),
13
1
1
1
0.92 (1H, s, NH); C NMR (150 MHz, CDCl3) δ (ppm): 36.80, 47.17, 55.36,
14.40, 119.17, 119.28, 119.55, 125.55, 127.75, 129.55, 129.80, 130.21, 130.46,
33.90, 134.05, 137.18, 142.37, 144.28, 153.33, 155.65, 161.80, 166.96, 189.08;
Anal. Calcd For C H ClN O S (545.05) : C, 63.90; H, 4.62; N, 10.28 .Found: C,
2
9
25
4
3
6
4.16;H, 4.51; N,10.59.
2
8