1,2,3-Triazole-Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic investigation of apoptosis induction in multiple myeloma RPMI-8226

Article abstract of DOI:10.1016/j.ejmech.2020.112062

A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and SR leukemia cell lines by 99.73% and 94.95% at 10 μM, respectively. Also, it inhibited the growth of M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test concentration. 4d showed IC₅₀ values less than 1 μM on six types of tumor cells and high selectivity index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound 4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner. Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.

Full text of DOI:10.1016/j.ejmech.2020.112062

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1,2,3-Triazole-Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic
investigation of apoptosis induction in multiple myeloma RPMI-8226
Heba F. Ashour, Laila A. Abou-zeid, Magda A. -A. El-Sayed, Khalid B. Selim
PII:
S0223-5234(20)30029-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112062
EJMECH 112062
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 November 2019
Revised Date: 8 January 2020
Accepted Date: 9 January 2020
Please cite this article as: H.F. Ashour, L.A. Abou-zeid, M.A..-A. El-Sayed, K.B. Selim, 1,2,3-Triazole-
Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic investigation of apoptosis
induction in multiple myeloma RPMI-8226, European Journal of Medicinal Chemistry (2020), doi: https://
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1,2,3-Triazole-Chalcone hybrids: synthesis, in vitro cytotoxic activity and
mechanistic investigation of apoptosis induction in multiple myeloma RPMI-8226
Heba F. Ashour1,2, Laila A. Abou-zeid1,3, Magda A. -A. El-Sayed*,1,2, Khalid B. Selim*,1
1Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt.
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University, New Dammeitta,
Egypt.
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Delta University, Gamsaa, Egypt.
*Corresponding author: M. A. -A. El-Sayed (e-mail: magdaaziz1@yahoo.com) and K. B. Selim (e-mail:
khbselim@mans.edu.eg)
tel: +2050-2247496 fax: +2050-2247496
Abstract
A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a
panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-
dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and
SR leukemia cell lines by 99.73% and 94.95% at 10 µM, respectively. Also, it inhibited the growth of
M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test
concentration. 4d showed IC50 values less than 1 µM on six types of tumor cells and high selectivity
index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and
gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity
against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound
4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner.
Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway
through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.
Keywords Antiproliferative activity, 1,2,3-triazole, chalcone, cell cycle analysis, apoptosis, ROS, NCI-
60
1. Introduction
Cancer is a life threatening disease and is the major cause of death throughout the world [1].
Development of effective cancer therapies, based on preventing cell proliferation and/or inducing
apoptosis, is a great challenge to the researchers. Despite major advances, multiple myeloma (MM)
remains an incurable malignancy. MM is a hematologic malignancy characterized by a clonal expansion
of plasma cells in bone marrow, accounting for 1% of all malignancies and 10% of hematologic
malignancies [2]. Combination therapy has been used as an initial treatment for MM patients and
involves normally two or three anti-myeloma drugs which have different mechanisms of action and
work synergistically including alkylating agents such as melphalan or cyclophosphamide,
corticosteroids such as prednisolone or dexamethasone, proteosome inhibitors such as bortezomib,
carfilzomib or ixazomib and recent approved drugs (Figure 1) such as venetoclax as Bcl-2 inihibitor,
panobinostat as histone deacetylase (HDAC) inhibitor or selinexor as nuclear export inhibitor [3]. Even
though those treatments improved the outcome of MM patients, MM often remains incurable due to the
1

development of drug resistance governed by the bone marrow microenvironment [4]. Therefore,
targeting new pathways, via synthesis of chemotherapeutics as small molecules, is needed to overcome
this resistance and to increase their potency.
Figure 1. Drugs approved in the treatment of multiple myeloma
Several studies have introduced many mechanisms, in treatment of leukemia, triggered to induce anti-
proliferative effect against MM RPMI-8226 cells, including: i) down regulation of the expression of
HDACs such as HDAC1, HDAC2, HDAC3 and HDAC6 [5, 6, 7]; ii) inhibition of nuclear factor-kappa
B (NF-κB) [8, 9]; iii) inhibition of epidermal growth factor receptors (EGFRs) family [10]; iv)
regulation of Bcl-2 and Bax as well as suppression of vascular endothelial growth factors (VEGFs) [11];
v) direct binding to microtubules in endothelial cells [12]; vi) disturbing the mitochondrial pathway [13]
and via accumulation of reactive oxygen species (ROS) [14].
Chalcone is an α,β-unsaturated ketone that represented a central core for a variety of important bioactive
molecules which possess versatile biological activities including anticancer [15], antimicrobial [16],
antituberculosis [17], antiinflammatory [18], antioxidant [19], antimalarial [20], antidiabetic [21] and
antihypertensive [22]. Structural modification of chalcones was proved to be successful strategy in
developing new anticancer agents differ in their potency and mechanism of action as a result of various
interactions with various target proteins related to cytotoxic activity such as HDACs, EGFRs, VEGFRs,
microtubules, topoisomerases, NF-κB. [23]. Furthermore, it was well-explored that compounds
containing chalcone moiety induce apoptosis and cell cycle arrest at G2/M phase by triggering
mitochondrial apoptotic pathway through activation of caspases and elevation of Bax/Bcl-2 ratio [24].
For example, as shown in Figure 2, Flavokawain B [25] was reported to induce G2/M cell-cycle arrest
and apoptosis in human oral and gastric carcinoma through generation of intracellular ROS.
Isobavachalcone [26] and JAI-51 [27] were reported to induce apoptotic cell death through increasing
Bax/Bcl-2 ratio and activation of caspases 3 and 9 in the mitochondrial pathway. MBL-II-58 [27] was
2

reported to induce ROS production and autophagic cell death. Moreover, triazolyl [28] and quinolyl-
thienyl chalcones [29] were reported as antitubulin and antiangiogenic agents, respectively.
OMe O
OH
O
OMe O
N
HO
OMe
HO
OH
MeO
OMe
JAI-51
Me
Flavokawain B
Isobavachalcone
O
MeO
MeO
N
N
N
O
OMe O
OMe
R
Me
S
OMe
N
N
Cl
MeO
OMe
R
Quinolyl-thienyl chalcones
Triazolyl chalcones
MBL-II-58
Figure 2. Chalcone derivatives with anticancer activity.
Furthermore, 1,2,3-triazoles have attained considerable attention owing to their diverse biological
activities, attractive physicochemical properties and ease of synthesis [30]. 1,2,3-Triazole is one of the
key structural units found in a large variety of bioactive molecules including anticancer, antimicrobial,
antifungal, antiviral, antiparasitic, antitubercular, antidiabetic, antiinflammatory, analgesic,
anticonvulsant, antidepressant [31]. These azoles have the ability to bind to different biological targets
(e.g. receptors and enzymes) by diverse non-covalent interactions [31]. In addition, 1,2,3-triazole ring is
highly stable to metabolic degradation and can be used as a bioisostere for amide bond, disulphide bond,
ester bond, carboxylic acid, aromatic rings and olefins rigid analogs [32].
Based on the earlier findings, molecular hybridization between chalcones and different bioactive
heterocyclic moieties was carried out in an attempt to get new effective anticancer molecules with
increased selectivity, lower drug resistance and/or less side effects. Thus, in the current work,
hybridization between the chalcone moiety of Flavokawain B and the bioactive 1,2,3-triazole core was
performed to give novel bioactive hybrids (Figure 3). All the synthesized compounds were evaluated for
their in vitro antiproliferative activities against a panel of NCI-60 cancer cell lines. The most potent
compound was further investigated to measure its apoptotic effect and to discover its mechanistic
pathway through measuring its effect on HDACs, NF-κB, EGFR, VEGFR, topoisomerase 2, tubulin,
Bax, Bcl-2, PARP-1, caspases 3, 7, 9 and ROS in leukemia RPMI-8226 cells.
3

Figure 3. Molecular hybridization of chalcone scaffold with 1,2,3-triazole.
2. Results and discussion
2.1. Chemistry
°
Scheme 1. Synthesis of hybrids 4a–k. Reagents and conditions: (a) HCl/NaNO2, 0 C; NaN3; (b)
acetylacetone, Na, MeOH, rt, 24 h; (c) Ar-CHO, NaOH, EtOH, rt, 24-72 h.
As depicted in Scheme 1, the starting aryl azides 2a, b were prepared according to the reported methods
[33] via the reaction of the corresponding aryl amine 1a, b with sodium nitrite under acidic condition at
0 °C followed by treating with sodium azide. The chloroazide 2a was extracted by ethyl acetate after 24
h of stirring, while the nitroazide 2b precipitated from the reaction mixture. The key intermediates,
acetyl 1,2,3-triazoles 3a, b were prepared by treating the azides 2a, b with acetylacetone in presence of
sodium methoxide [34]. The target triazole-chalcone hybrids 4a–k were synthesized by conventional
aldol condensation between aromatic aldehydes (e.g. 4-dimethylaminobenzaldehyde, 4-
4

flourobenzaldehyde, furfural, 3,4-dimethoxybenzaldehyde, isovanillin or vanillin) and acetyl triazole
derivatives 3a, b in presence of ethanolic solution of sodium hydroxide. New hybrids were produced in
46–92% yield as pure compounds and their structures were characterized by spectroscopic analysis (1H
and 13C-NMR) as described in the experimental section.
2.2. Biological evaluation
2.2.1. In vitro one-dose antiproliferative screening
The newly synthesized hybrids 4a–k were screened against a panel of 60 cell lines of different cancer
types (leukemia, non small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal
cancer, prostate cancer and breast cancer) at the National Cancer Institute (NCI), USA [35] in order to
detect their growth inhibition percentages (GI%) at a single dose concentration (10 µM) on cancer cell
lines. Additionally, the selectivity ratio on a particular cell line with regard to all NCI-60 cell line (Table
1) was recorded.
Data analysis of growth inhibition assay showed that the majority of the compounds exhibited potent
anticancer activity against leukemia cell lines rather than other cell lines. Compounds 4d and 4f have
broad spectrum cytotoxic activity against the tested cell lines, particularly against leukemia cell lines.
Compounds 4b and 4h showed potent activity against leukemia cell line RPMI-8226 with GI% of 88
and 86, respectively. Compound 4i showed high activity against leukemia CCRF-CEM with GI% value
of 81. The rest of the tested compounds 4c, 4e, 4g, 4j and 4k showed low to moderate growth inhibitory
activities. Compound 4d has excellent activity against leukemia cell lines (K-562, RPMI-8226, SR),
melanoma M14 and breast MCF7 cell lines with GI% range of 82–100; colon cancer HCT-116 and
prostate cancer PC-3 with GI% values of 68 and 60, respectively. Moreover, compound 4f showed
moderate to high activity against leukemia cell lines with GI% range of 47–92; however, inhibitory
activity for colon cancer HCT-116 and breast cancer MCF7 are 74 and 73, respectively.
Many tested hybrids showed high potency with great selectivity on specific cell lines. For example,
compounds 4b and 4h having para flouro phenyl-chalcone moiety showed strong GI% against leukemia
RPMI-8226 cell line of 88% and 86%, respectively, with high selectivity ratio equal to 11.95 fold and
7.51 fold, respectively. While, compound 4i with furfuryl-chalcone moiety showed strong GI% against
leukemia CCRF-CEM cell line equal to 81.25% with remarkable selectivity ratio of 12.48 fold.
Compound 4f having para hydroxy and meta methoxy groups on the phenyl-chalcone moiety showed
strong GI% over 4 cell lines (leukemia HL-60 (TB), leukemia CCRF-CEM, colon HCT-116 and breast
cancer MCF7) equal to 92.41%, 76.46%, 73.77% and 73.16%, respectively, with selectivity ratio around
3 fold with regard to all NCI-60 cell lines. It also exerted good activity over K-562, RPMI-8226 and SR
leukemia cell lines with GI% values of 65.06%, 57.21% and 56.22%, respectively. Compound 4d
possessing 3, 4-dimethoxyphenyl chalcone moiety was the most active in our series. It showed strong
GI% over 5 cell lines, RPMI-8226 leukemia, SR leukemia, K-562 leukemia, M14 melanoma and MCF7
breast cancer cell lines reached to 99.73%, 94.95% , 83.17%, 87.26% and 81.97%, respectively, with
selectivity ratio of around 3 fold with regard to all NCI-60 cell lines. It also exerted good activity against
HCT-116 colon and PC-3 prostate cancer cell lines with GI% values of 68.01% and 59.88%,
respectively.
5

Figure 4. SAR study of triazole-chalcone hybrids.
As illustrated in Figure 4, the results showed that the derivatives substituted with the weak electron
withdrawing chloro atom were more potent cytotoxic agents than the derivatives substituted with the
strong electron withdrawing nitro group, particularly the para chloro compound 4d exhibited the highest
% mean of inhibition value over the 60 cell line panel equal to 27.19% in comparison to the para nitro
compound 4j which exhibited 3.15%. Moreover, switching the positions of the high polar OH and the
less polar OCH3 groups (from para to meta position and vice versa) on the phenyl ring of 4f and 4e led
to diminish the % mean of inhibition from 25% (for 4f) to 4.9% (for 4e), indicating the important
cytotoxic effect of the meta position of OCH3 group in 4f. Confirming the importance of the para chloro
substituent, replacing Cl atom in 4e with NO2 group in the corresponding 4k quenched the activity to
give the weakest active compound among the series with 0.23% mean of inhibition. Such results suggest
that the electronic and steric properties of the substituents play an important role in the binding affinity
of chalcones to their cellular target(s). As a general statement, the presence of meta OCH3 group on the
right side and para Cl atom on the left side of the hybrid was proved to be essential for general
anticancer activity of the tested series.
Table 1. Percentages of growth inhibition (GI%) induced by the synthesized compounds at 10 µM
concentration on NCI-60 cell line panel and the selectivity ratio on the most sensitive cell lines.
Subpanel
cancer
Lines
%Growth Inhibition (GI%)
cell
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
Leukemia
9.43
11.46
13.62
9.29
CCRF-CEM
HL-60(TB)
K-562
24.22
24.76
21.83
17.03
8
14.54
17.1
27.56
13.33
6.93
9.18
14.44
14.3
2.79
2.33
2.1
33.21 36.91
28.32 16.87
26.26 15.71
14.29 20.52
49.25
54.02
83.17
38.68
76.46
92.41
65.06
47.54
81.25
4.69
NI
11.73
14.49
18.76
MOLT-4
1.37
2.76
6

RPMI-8226
SR
28.66
47.33
5.17
6.88
4.01
20.08
1.48
6.38
16.28
26.25
2.91
NI
37.68 87.98
99.73
94.95
57.21
56.22
86.33
45.42
20.18
32.7
Non-Small Cell Lung Cancer
NCI-H226
0.33
10
4.87
0.65
8.63
11.14
8.05
7.01
5.6
1.01
30.04
Colon Cancer
HCT-116
HCT-15
HT29
KM12
SW-620
1.84
3.93
11.26 10.15
6.53
NI
7.49
10.91
10.33
18.86
11.67
8.74
NI
13.03
NI
NI
4.95
1.27
11.4
6.51
5.08
5.65
NI
17.07
22.04
16.28
2.25
0.38
4.22
0.02
11.1
0.25
1.94
4.58
NI
NI
2.31
68.01
41.06
34.27
45.77
33.06
73.77
46.43
41.02
41.55
30.27
46.87
4.18
NI
3.34
NI
15.9
NI
3.65
Melanoma
LOX IMVI
M14
NI
NI
NI
13.88
29.66
87.26
7.01
9.47
3.18
9.34
9.16
26.01
NI
35.83
NI
14.86
2.26
0.76
44.44
27.64
18.22 17.59
MDA-MB-
435
SK-MEL-5
UACC-62
NI
NI
NI
NI
NI
NI
25.37
7.48
2.34
12.88
NI
NI
12.06
1.35
1.21
40.12
NI
NI
15.31
NI
NI
16.48
3.68
1
8.53
32.02
31.88
41.17
50.78
4.89
30.69
11.05 26.54
Ovarian Cancer
IGROV1
OVCAR-3
NI
NI
NI
NI
2.31
NI
5.46
NI
20.34
37.43
NI
NI
2.58
NI
NI
NI
NI
NI
0.39
NI
30.18
15.51
Renal Cancer
786-0
NI
6.13
7.26
27.77 14.85
24.15 23.53
27.28 33.47
7.6
3.19
5.68
14.4
NI
10.37
19.79
12.84
12.45
23.47
34.16
6.45
15.48
9.17
24
9.61
9.25
3.23
9.61
11.89
8.5
6.13
40.87
NI
3.95
10.47
9.41
34.18
4.27
10.97
22.04
CAKI-1
SN12C
UO-31
8.22
4.45
18.72
42.24
31.46
46.69
25.34
20.15
Prostate Cancer
10.42
Breast Cancer
PC-3
9.34
13.59
15.68
4.68
11.94
6.01
59.88
36.7
MCF7
28.59
9.01
15.52
7.2
6.1
15.43
3.84
18.02
10.32
13.08
35.18
12.33
13.32
10.32
9.88
5.23
NI
2.17
81.97
43.99
32.11
73.16
29.45
27.51
54.72
15.07
17.19
BT-549
T-47D
MDA-MB-
468
7.35
16.45 17.78
5.6
NI
NI
8.02
1.68
27.03
NI
5.39
1.12
6.51
NI
0.41
0.23
33.89
Mean Inhibition
4.9 25
4.78
7.36
7.9
27.19
6.28
11.5
3.15
Selectivity Ratio on the Most sensitive Cell Line
(GI% on the sensitive cell line/Mean Inhibition)
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
HCT-116
M14
MCF7
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11.95
-----
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3.06
-----
3.67
3.49
-----
3.21
3.01
-----
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-----
3.06
3.70
-----
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-----
2.95
-----
2.93
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7.51
-----
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-----
12.48
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*The bold red and blue figures indicate strong (GI% > 70%) and moderate (GI% 30-70%) anticancer activity,
respectively; NI, no inhibition.
7

*Non sensitive cell lines which show GI% < 30% for all synthesized compounds were not mentioned.
*Mean inhibition and selectivity ratio were calculated with regard to GI% values over all NCI-60 cell lines
.
Figure 5 shows the cytotoxic effect of para chloro substituent versus para nitro substituent for
compounds 4d and 4j towards the most sensitive cell lines in comparison to methotrexate and gefitinib
as reference drugs. The cytotoxic activity of the reference drugs was obtained from NCI data ware house
index [36]. Compound 4d showed higher activity than methotrexate and gefitinib against the leukemia
cell lines RPMI-8226, SR and K-562. Moreover, it showed higher or comparable activity against breast
MCF7, colon HCT-116, prostate PC-3 and melanoma M14 cancer cell lines. These results indicate that
compound 4d can be considered as a potential lead compound for future development of broad spectrum
anticancer agents.
Figure 5. A comparison between 4d (with chloro atom) and 4j (with nitro group). GI % of in vitro
subpanel tumor cell lines, Leukemia: K-562, RPMI-8226, SR, Colon Cancer: HCT-116, Prostate
Cancer: PC-3, Breast Cancer: MCF7 and Melanoma: M14 in comparison to methotrexate and
gefitinib.
2.2.2. In vitro cytotoxicity screening (IC50) against cancer and human normal cell lines and selectivity
index (SI)
As shown in Table 2, the IC50 of compounds 4b, 4d, 4f, 4h and 4i were determined to assess their
cytotoxic activity against several cancer cells versus corresponding human normal cells in order to
evaluate their selectivity index (SI). It is accepted that the tested compound which has SI value more
than three exhibits selective cytotoxicity towards the cancer cells rather than the normal cells, while the
compound which has SI value less than three exhibits general toxicity for cancer and normal cells [37,
38]. The results showed that: i) compounds 4b and 4h exhibited IC50 values in nanomolar range against
leukemia RPMI-8226 cell line as well as high SI values on cancer cells rather than normal cells equal to
114.43 folds and 45.19 folds, respectively; ii) compound 4i also showed IC50 values in nanomolar range
against leukemia CCRF-CEM cell line as well as high SI value of 103.6 folds; iii) compound 4f showed
IC50 values in nanomolar range over two cancer cell lines, HL(60)-TB leukemia and CCRF-CEM
leukemia cell lines with high SI values of 27.1 folds and 14.26 folds, respectively. It also showed IC50
values in submicromolar range over two cancer cell lines, MCF7 breast and HCT-116 colon cancer cell
lines with high SI values of 22.08 folds and 13.38 folds, respectively; iv) compound 4d, the most potent
8

derivative, showed IC50 values in nanomolar range over six cancer cell lines, RPMI-8226 leukemia, SR
leukemia, K-562 leukemia, M14 melanoma, MCF7 breast and HCT-116 colon cancer cell lines, and
showed IC50 value in submicromolar range over PC-3 prostate cancer cell line, however, it showed
much higher IC50 values (12.88 µM to 30.09 µM) on the corresponding normal cell lines indicating the
high selectivity of compound 4d towards cancer cells rather than normal cells reached to 45.53 folds in
RPMI-8226 leukemia, 30.35 folds in SR leukemia, 34.69 folds in K-562 leukemia, 43.61 folds in M14
melanoma, 104.17 folds in MCF7 breast cancer, 65.96 folds in HCT-116 colon cancer and 4.37 folds in
PC-3 prostate cancer. In summary, the results of cytotoxicity point to the potent broad spectrum
anticancer activity with the high safety of compound 4d.
Table 2. Cytotoxic activity (IC50, µM) and Selectivity Index (SI) of compounds 4b, 4d, 4f, 4h and 4i.
Cytotoxicity of compound 4b
Cancer cells (IC50, Normal cells (IC50,
M)a
Leukemia RPMI-
SIb
114.43
SIb
µ
µ
M)a
56.07 ± 1.07
Blood
0.49 ± 0.02
8226
PCS-800-011
Cytotoxicity of compound 4d
M)a M)a
Cancer cells (IC50, Normal cells (IC50, µ
µ
Leukemia RPMI-
8226
0.64 ± 0.02
45.53
Blood
PCS-800-011
29.14 ± 1.4
Leukemia SR
Leukemia K-562
Melanoma M14
Breast MCF7
Colon HCT116
Prostate PC3
0.96 ± 0.04
0.84 ± 0.07
0.69 ± 0.01
0.24 ± 0.01
0.26 ± 0.01
2.95 ± 0.14
30.35
34.69
43.61
104.17
65.96
4.37
Skin WS1
30.09 ± 1.7
25.00 ± 1.1
17.15 ± 1.5
12.88 ± 0.9
Breast MCF 10A
Colon CCD-18
Prostate HPrEC
Cytotoxicity of compound 4f
M)a M)a
Cancer cells (IC50, Normal cells (IC50, µ
SIb
µ
Leukemia CCRF-
CEM
0.76 ± 0.01
14.26
Blood
PCS-800-011
10.84 ± 0.66
Leukemia Hl-60 (TB)
Leukemia K-562
Breast MCF7
0.40 ± 0.005
10.84 ± 0.4
1.76 ± 0.04
2.87 ± 0.06
27.1
1
Breast MCF 10A 38.86 ± 1.14
Colon CCD-18 38.39 ± 0.95
M)a
Normal cells (IC50,
22.08
13.38
Colon HCT116
Cytotoxicity of compound 4h (IC50,
Cancer cells (IC50,
M)a
Leukemia RPMI-
µ
SIb
µ
µ
M)a
35.25 ± 0.81
Blood PCS-800-
011
0.78 ± 0.04
8226
45.19
Cytotoxicity of compound 4i
Cancer cells (IC50, Normal cells (IC50,
M)a
Leukemia CCRF- 0.45 ± 0.008
SIb
µ
µ
M)a
46.62 ± 1.23
Blood PCS-800-
103.6
9

CEM
011
aCytotoxicity (IC50,
µ
M): The concentration of compound that inhibit 50% of the cell growth after
48 h of drug exposure measured by MTT assay. Each value was shown as mean ± SD of three
experiments; bSelectivity Index (SI) = IC50 for normal cells/IC50 for cancer cells.
2.2.3. Physicochemical properties and Lipinski’s rule of five
The bioavailability of the compound determines its ability to cross the biological barriers in order to
reach its target sites. It is influenced by many parameters such as solubility, membrane permeability, as
well as active uptake and transport within the organism [39]. The “rule-of-five” introduced by Lipinski
[40] summarized the molecular features and properties that are associated with orally active drugs in
humans, and additional related criteria were added later on by Veber [41]. These parameters have been
widely used as a filter for new potential drugs and the violation of more than one of these rules may
indicate problems in the drug bioavailability. Therefore, the physicochemical parameters of compound
4d, the most compound as anticancer, were predicted (Table 3) using Molin spiration software [42]. The
calculation showed that compound 4d complies with these rules which indicated the drug-likeness
properties of this compound.
Table 3. Reference values of Lipinski's and Veber's rules as well as predicted physicochemical
properties of compound 4d, 4f and Gefitinib.
cLogP
MW
PSA
-
HBD
HBA
nRotB
-
≤ 10
Lipinskia
≤
5
≤
500
-
≤
5
≤
10
-
Veberb
-
≤
140
66.26
77.25
68.75
-
Compound 4dc
Compound 4fc
Gefitinib
3.79
3.48
4.19
383.83
369.81
446.91
0
1
1
6
6
7
6
5
8
b
aLipinski's rule of five for pharmaceuticals; Veber added more criteria to Lipinski's rule of five;
cPredicted physicochemical properties; cLogP: calculated lipophilicity; MW: molecular weight; PSA:
polar surface area; HBD: number of hydrogen bond donor; HBA: number of hydrogen bond acceptor;
nRotB: number of rotatable bounds.
2.2.4. Mechanistic investigation of inducing cytotoxicity in multiple myeloma RPMI-8226
Since compound 4d showed high potency against leukemia cells, good safety profile and drug-likeness
properties along with easy synthetic pathway (in only 3 steps), it is indicated that compound 4d could be
a promising lead for future development of active antileukemic agents. So, in this study, we focused our
efforts to find out the biological target for compound 4d inside RPMI-8226 cells and its mechanism of
inducing cytotoxicity in order to offer new insights in treating MM. Therefore, we investigated the
activity of compound 4d on a variety of proteins and enzymes targets which previously reported to
induce cytotoxicity in RPMI-8226 cell line as well as its effect on some proteins which regulate the cell
cycle progression.
10

2.2.4.1. In vitro enzyme inhibition assays
As illustrated in Figure 6, we measured the activity of compound 4d on class I HDACs (1, 2, 3 and 8)
and class IIb (HDAC6) using velcade as a reference drug. The results showed that the effect of 4d on
several types of HDAC is weak except on HDAC8 as it shows high activity (IC50 = 0.047 µM) in
comparison to velcade (IC50 = 0.072 µM). Moreover, the effect of compound 4d against several
molecular targets including NF-κB, EGFR, VEGFR2, topoisomerase 2 and tubulin were screened where
compound 4d showed lower inhibitory activities in comparison to the reference drugs as shown in
Figure 6. These results indicate that the previous enzymes and proteins might be not the molecular
targets for 1,2,3-triazole-chalcone hybrid 4d to induce cytotoxicity inside MM RPMI-8226 cells.
Figure 6. Assessment of activity of compound 4d against HDACs, NF-κB, EGFR, VEGFR2, topoisomerase 2
and tubulin in RPMI-8226 cells. HDAC, histone deacetylase; NF-κB, nuclear factor kappa B; EGFR, epidermal
growth factor receptor; VEGFR, vascular endothelial growth factor receptor.
2.2.4.2. Measuring the expression of apoptotic and anti-apoptotic markers
Permeabilization of mitochondrial outer membrane is controlled by a balance between the proteins of
the Bcl-2 family [43], which composed of pro-apoptotic proteins such as Bax, Bim, Bad, Bid and Puma
and anti-apoptotic proteins such as Bcl-2, Mcl-1 and Bcl-XL [44, 45]. In human MM, the chemo-
resistance correlates with elevated levels of Bcl-2 which is commonly observed in this disease [46, 47].
Also, Apoptosis is known to be inhibited when Bcl-2 increases and forms a Bcl-2–Bax heterodimer, and
induced when Bax increases and forms a homodimer [48]. Bax homodimers can increase mitochondrial
membrane permeability and leakage of cytochrome C into the cytoplasm, resulting in a caspase
dependent apoptosis [49] in which caspase-9 is activated and leads naturally to caspase-3 activation in
the latter [50] which in turn induces apoptosis that can be identified by the proteolytical cleaved caspase-
3 and caspase-7 [51]. Based on that, as shown in, Figure 7, the study was further extended to investigate
the effect of compound 4d to provoke apoptosis by affecting the expression of apoptotic and anti-
apoptotic markers in RPMI-8226 cells. Compound 4d significantly increased the expression levels of
11

the active apoptotic markers including caspase-3, caspase-7, caspase-9 and Bax by about 100, 4.5, 8.5,
5.3 folds, respectively, in comparison to the control. Moreover, 4d strongly decreased the level of the
anti-apoptotic marker Bcl-2. These results showed the activation of caspase-9 which is the upstream of
caspases-3 and 7, besides the down regulation of Bcl-2 and upregulation of Bax leading to elevated
Bax/Bcl-2 ratio suggests that compound 4d induces apoptosis in human MM RPMI-8226 cells through
mitochondrial-mediated pathway (Figure 8).
Figure 7. Assessment of activity of compound 4d against Bax, Bcl-2, PARP-1 and Casp. 3, 7, 9 in RPMI-8226
cells. Casp., Caspase; PARP-1, poly (ADP-ribose) polymerase.
Figure 8. Effect of compound 4d on apoptosis via mitochondrial-mediated pathway in RPMI-8226
cells.
12

2.2.4.3. Intracellular ROS accumulation assay
Reactive oxygen species (ROS) such as H2O2 and superoxide can cause cytochrome c (Cyt c) release
from mitochondria and induce apoptosis through the mitochondrial pathway [52]. As illustrated in
Figure 9, compound 4d stimulates ROS accumulation (86.22 Pg/ml) in RPMI-8226 cells slightly higher
than gefitinib (80.29 Pg/ml) and 3 folds higher than the control cells. The results indicate that ROS
accumulation can be considered as one of the major cause of apoptosis caused by compound 4d.
Figure 9. Effect of 4d and gefitinib on intracellular ROS accumulation in RPMI-8226 cells.
In order to confirm the correlation between 4d-induced apoptosis and intracellular ROS accumulation in
RPMI-8226 cells, we performed cell cycle analysis of RPMI-8226 cells treated with different
concentration of compound 4d (0.6, 3 and 6 µM) for 24 h, then ROS scavenger N-acetylcysteine (NAC)
was added to the 4d set (6 µM) and its effect on the apoptosis rate was examined. The cell cycle analysis
(Figure 10) showed that 4d induced apoptosis via aggregation of MM RPMI-8226 cells in G2/M phase
in a dose-dependent manner, where the aggregation percentage increased from 10.32% in the control
cells to 19.47%, 44.18% and 49.01% in 0.6, 3 and 6 µM 4d-treated cells, respectively, leading to
apoptosis by 15.32%, 27.56% and 34.69%, respectively. However, the aggregation of RPMI-8226 cells
in G2/M phase reduced significantly from 49.01% in 6 µM 4d-treated cells to 33.52% after addition of
NAC, leading to significant reduction in apoptosis from 34.69% to 14.89% (Figures 10 and 11). The
Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) double staining (Figures
12 and 13) showed that the apoptotic rate in the late apoptotic quadrant Q2 decreased from 21.74% in 6
µM 4d-treated cells to 2.5% after addition of NAC. From the above investigations, it is obvious that the
principal mechanism in the 4d-induced cell death is the apoptosis induction which is initiated mainly by
ROS accumulation in RPMI-8226 cells treated with 4d.
13

Figure 10. Flow cytometry analysis of cell cycle phase distribution in RPMI-8226 cells after treatment
with different concentration of 4d for 24 h as well as after addition of NAC. Histogram (A-E) represents
propidium iodide staining of RPMI-8226 cells treated with vehicle control (DMSO) (A), compound 4d
(0.6, 3 and 6 µM) (B-D) or compound 4d (6 µM) in presence of NAC (E).
Figure 11. Bar diagram shows the cell distribution in the subG1, G0/G1, S and G2/M phases for RPMI-
8226 cells treated with vehicle control and 4d (0.6, 3 and 6 µM with or without NAC). NAC: N-
acetylcysteine.
14

Figure 12. Annexin V-FITC/PI double staining for detection of apoptosis in RPMI-8226 cell after
treatment with different concentration of 4d for 24 h as well as after addition of NAC. Q4 quadrant
represent early apoptosis; Q2 quadrant represent late apoptosis; Q1 quadrant represent dead (necrotic)
cells; Q3 quadrant represent live cells. Summation of early and late apoptosis represents total apoptosis.
15

Figure 13. Bar diagram shows % apoptosis in RPMI-8226 cells treated with vehicle control and 4d (0.6,
3 and 6 µM with or without NAC). NAC: N-acetylcysteine. Summation of early and late apoptosis
represents total apoptosis.
From the previous mechanistic evaluation, we can conclude that triazole-chalcone 4d exerts its
anticancer effect through induction of apoptosis in MM RPMI-8226 cells, which caused by triggering
mitochondrial apoptotic pathway. This apoptotic pathway is initiated by ROS accumulation, increasing
Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.
3. Conclusion
New hybrids of 1,2,3-triazole-chalcone were synthesized and evaluated for their antiproliferative
activities against a panel of NCI-60 cancer cell lines. Compound 4d having 3,4-dimethoxyphenyl moiety
was the most potent anticancer agent with broad spectrum activity against RPMI-8226 leukemia, SR
leukemia, K-562 leukemia, M14 melanoma, MCF7 breast, HCT-116 colon and PC-3 prostate cancer cell
lines. Mechanistic evaluation of hybride 4d in RPMI-8226 cells was studied through measuring its effect
on HDACs, NF-κB, EGFR, VEGFR, topoisomerase 2, tubulin, Bax, Bcl-2, caspases 3, 7, 9 and PARP-
1. Compound 4d causes cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant
manner via triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing
Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9. The current study evidently identified the potential
of compound 4d to be a promising lead for future development of active anticancer agents, and may
offer new insights in treating multiple myeloma RPMI-8226.
4. Experimental
4.1. General
Melting points were determined on Stuart melting point apparatus and were uncorrected. IR spectra
were recorded on Thermo Fisher SCIENTIFIC Nicolet IS10 Spectrometer (
ν
in cm–1). Mass
1
spectrometry (EI) m/z analyses were performed using Hewlett Packard 5988 spectrometer. The H and
13C NMR spectra were recorded on a Joel 500 MHz spectrometer, 1H spectra were run at 500 MHz and
13C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d6), chemical shifts are
quoted in δ as parts per million (ppm) using solvent peak as standard. TLC was carried out using
Kieselgel 60 F254 sheets (Merck, Darmstadt, Germany), the developing solvents were ethyl
acetate/petroleum ether (3:1) or dichloromethane/methanol (20:1) and the spots were visualized using
UV lamp at 366or 254 nm. Compounds 1a and 1b were purchased, while compounds 2a, 2b [33], 3a
and 3b [34] were prepared according to the reported procedures.
4.2. General procedure for synthesis of compounds 4a-k
To a suspension of compound 3 (0.01 mol) in EtOH (95%, 20 ml), substituted aldehyde (0.01 mol) was
added and stirred in an ice bath. A solution of NaOH (6 gm) in EtOH (95%, 30 ml) was added to the
mixture dropwise with continuous stirring for 30 min. The reaction mixture was stirred at room
temperature for 24–72 hours, the completion of the reaction was judged by TLC. After that it was
diluted with ice-cold water (40 ml) and neutralized with HCl solution. The separated solids were
filtered, washed with cold water and dried to give the titled compound 4a-k without further purification.
16

4.2.1. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-[4-(dimethylamino)phenyl]prop-2-en-1-one
4a
(
)
1
Orange solid; Yield = 72%; m.p. = 192-195 ºC; H NMR (500 MHz, DMSO-d6): δ = 7.78 (d, J = 15.9
Hz, 1H), 7.75 (d, J = 15.9 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 9.0 Hz, 2H), 7.66 (d, J = 8.9
Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 3.02 (s, 6H), 2.59 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ = 183.3,
152.5, 144.6, 143.7, 138.8, 135.1, 134.3, 131 (2C), 130.1 (2C), 127.6 (2C), 121.9, 117.1, 112.2 (2C),
39.5 (2C), 10.2; MS (EI) m/z (Chemical Formula: C20H19ClN4O): 366.9 (56.26%) [M+]; IR (KBr, cm–1):
3447, 3101, 2909, 2813, 1653 (C=O
825.
α,β-unsaturated), 1568, 1528, 1498, 1431, 1366, 1181, 1033, 997,
4.2.2. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-(4-fluorophenyl)prop-2-en-1-one (4b
)
1
Pale yellow solid; Yield = 65%; m.p. = 195-197 ºC; H NMR (500 MHz, DMSO-d6): δ = 7.99 (d, J =
16.0 Hz, 1H), 7.94 (dd, J = 8.6, 5.7 Hz, 2H), 7.87 (d, J = 16.0 Hz, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.73 (d,
J = 9.0 Hz, 2H), 7.32 (t, J = 8.8 Hz, 2H), 2.61 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ = 183.8, 164
(d, 1JCF = 249.4 Hz), 143.6, 142.6, 139.8, 135.4, 134.4, 131.7 (d, 3JCF = 8.8 Hz, 2C), 131.5 (d, 4JCF =
2
2.4 Hz), 130.3 (2C), 127.8 (2C), 123, 116.7 (d, JCF = 21.8 Hz, 2C), 10.4; MS (EI) m/z (Chemical
Formula: C18H13ClFN3O): 342.01 (26.58%) [M+]; IR (KBr, cm–1): 3449, 3073, 1667 (C=O
α,β-
unsaturated), 1590, 1498, 1422, 1363, 1227, 1187, 1159, 1028, 999, 977, 833.
4.2.3. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-(2-furyl)prop-2-en-1-one (4c
)
1
Pale yellow solid; Yield = 60%; m.p. = 146-148 ºC; H NMR (500 MHz, DMSO-d6): δ = 7.95 (d, J =
1.0 Hz, 1H), 7.76 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.73 (d, J = 9.0 Hz, 2H), 7.67 (d, J =
15.8 Hz, 2H), 7.13 (d, J = 3.4 Hz, 1H), 6.71 (dd, J = 3.4, 1.8 Hz, 1H), 2.60 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ = 183.2, 151.3, 146.9, 143.4, 139.5, 135.2, 134.2, 130.2 (2C), 129.8, 127.7 (2C), 119.8,
118.1, 113.6, 10.2; MS (EI) m/z (Chemical Formula: C16H12ClN3O2): 313.98 (14.39%) [M+]; IR (KBr,
cm–1): 3424, 3098, 3059, 1660 (C=O
1032, 996, 836, 753.
α,β-unsaturated), 1600, 1557, 1501, 1424, 1363, 1269, 1211,
4.2.4. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4d
)
1
Yellow solid; Yield = 61%; m.p. = 166-168 ºC; H NMR (500 MHz, DMSO-d6): δ = 7.91 (d, J = 15.9
Hz, 1H), 7.82 (d, J = 15.9 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.73 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 1.7
Hz, 1H), 7.40 (dd, J = 8.4, 1.7 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 2.61 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ = 183.7, 151.8, 149.4, 144.1, 143.6, 139.4, 135.2, 134.3, 130.2 (2C),
127.7 (2C), 127.6, 124, 120.8, 112.1, 111, 56 (2C), 10.3; MS (EI) m/z (Chemical Formula:
C20H18ClN3O3): 384.04 (21.87%) [M+]; IR (KBr, cm–1): 3447, 3070, 2997, 2968, 2936, 2836, 1658
(C=O α,β-unsaturated), 1581, 1509, 1424, 1339, 1257, 1134, 1028, 996, 980, 840.
4.2.5. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-(3-hydroxy-4-methoxy-phenyl)prop-2-en-1-
one (4e
)
1
Brown solid; Yield = 70%; m.p. = 236-239 ºC; H NMR (500 MHz, DMSO-d6): δ = 9.36 (s, 1H), 7.79
(d, J = 15.8 Hz, 1H), 7.76 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 8.9 Hz, 2H), 7.73 (d, J = 8.9 Hz, 2H), 7.26
(d, J = 1.7 Hz, 1H), 7.24 (dd, J = 8.2, 1.7 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 3.83 (s, 3H), 2.59 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ = 183.1, 150.6, 146.9, 143.6, 143.2, 138.9, 134.8, 133.9, 129.8 (2C),
127.3 (2C), 127.2, 122.3, 119.9, 114, 112.1, 55.6, 9.7;MS (EI) m/z (Chemical Formula: C19H16ClN3O3):
369.31 (100%) [M+]; IR (KBr, cm–1): 3351, 3011, 2957, 2930, 2839, 1657 (C=O
1588, 1550, 1505, 1439, 1271, 1034, 984, 830.
α,β-unsaturated),
17

4.2.6. (E)-1-[1-(4-Chlorophenyl)-5-methyl-triazol-4-yl]-3-(4-hydroxy-3-methoxy-phenyl)prop-2-en-1-
one (4f
)
Yellow solid; Yield = 59%; m.p. = 164 -167 ºC; 1H NMR (500 MHz, DMSO-d6): δ = 9.82 (s, 1H), 7.85
(d, J = 15.8 Hz, 1H), 7.79 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.73 (d, J = 9.0 Hz, 2H), 7.41
(d, J = 1.7 Hz, 1H), 7.27 (dd, J = 8.1, 1.7 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H), 3.87 (s, 3H), 2.60 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ = 183.3, 150.1, 148.1, 144.2, 143.2, 138.9, 134.8, 133.9, 129.8 (2C),
127.3 (2C), 125.8, 123.9, 119.4, 115.8, 111.6, 55.7, 9.9;MS (EI) m/z (Chemical Formula:
C19H16ClN3O3): 369.21 (100%) [M+]; IR (KBr, cm–1): 3434, 3063, 2977, 2942, 1662 (C=O
α,β-
unsaturated), 1581, 1507,1426, 1263,1209, 1029, 991, 830.
4.2.7. (E)-3-[4-(Dimethylamino)phenyl]-1-[5-methyl-1-(4-nitrophenyl)triazol-4-yl]prop-2-en-1-one (4g
)
Orange solid; Yield = 92%; m.p. = 247-249 ºC; 1H NMR (500 MHz, DMSO-d6): δ = 8.50 (d, J = 9.0 Hz,
2H), 8.02 (d, J = 9.0 Hz, 2H), 7.80 (d, J = 15.8 Hz, 1H), 7.76 (d, J = 15.8 Hz, 1H), 7.67 (d, J = 8.9 Hz,
13
2H), 6.78 (d, J = 8.9 Hz, 2H), 3.03 (s, 6H), 2.68 (s, 3H); C NMR (125 MHz, DMSO-d6): δ = 183.6,
153.4, 147.7, 144.8, 144, 140.3, 139.1, 130.9 (2C), 126.9 (2C), 125.7 (2C), 121.6, 117.1, 112.3 (2C),
39.5 (2C), 10.4; MS (EI) m/z (Chemical Formula: C20H19N5O3): 377.12 (100%) [M+]; IR (KBr, cm–1):
3424, 3093, 2910, 1651 (C=O
1177, 1033, 996, 856.
α,β-unsaturated), 1597, 1567, 1525 (NO2), 1433, 1366, 1344 (NO2),
4.2.8. (E)-3-(4-Fluorophenyl)-1-[5-methyl-1-(4-nitrophenyl)triazol-4-yl]prop-2-en-1-one (4h
)
1
Pale yellow solid; Yield = 62%; m.p. = 196-199 ºC; H NMR (500 MHz, DMSO-d6): δ = 8.51 (d, J =
9.0 Hz, 2H), 8.03 (d, J = 9.0 Hz, 2H), 8.00 (d, J = 16.0 Hz, 1H), 7.96 (dd, J = 8.7, 5.6 Hz, 2H), 7.89 (d, J
= 16.0 Hz, 1H), 7.33 (t, J = 8.8 Hz, 2H), 2.69 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ = 183.7, 164
1
3
4
(d, JCF = 249.4 Hz), 148.3, 143.7, 142.7, 140.2, 140, 131.6 (d, JCF = 8.6 Hz, 2C), 131.4 (d, JCF =
2
2.8 Hz), 127 (2C), 125.5 (2C), 122.9, 116.6 (d, JCF = 21.7 Hz, 2C), 10.4; MS (EI) m/z (Chemical
Formula: C18H13FN4O3): 352 (100%) [M+]; IR (KBr, cm–1): 3447, 3093, 1666 (C=O α,β-unsaturated),
1595, 1522 (NO2), 1426, 1343 (NO2), 1231, 1031, 997, 977, 858, 832.
4.2.9. (E)-3-(2-Furyl)-1-[5-methyl-1-(4-nitrophenyl)triazol-4-yl]prop-2-en-1-one (4i
)
1
Pale yellow solid; Yield = 92%; m.p. = 232-234 ºC; H NMR (500 MHz, DMSO-d6): δ = 8.51 (d, J =
9.0 Hz, 2H), 8.02 (d, J = 9.0 Hz, 2H), 7.97 (d, J = 1.4 Hz, 1H), 7.78 (d, J = 15.7 Hz, 1H), 7.69 (d, J =
15.7 Hz, 1H), 7.15 (d, J = 3.4 Hz, 1H), 6.73 (dd, J = 3.4, 1.7 Hz, 1H), 2.68 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ = 183.3, 151.4, 147.1, 143.8, 140.3, 139.9, 130.2, 127.1 (2C), 126.8, 125.6 (2C), 119.9,
118.4, 113.8, 10.5; MS (EI) m/z (Chemical Formula: C16H12N4O4): 324.05 (100%) [M+]; IR (KBr, cm–
1): 3448, 3084, 1659 (C=O α,β-unsaturated), 1596, 1556, 1530 (NO2), 1502, 1425, 1347 (NO2), 1285,
981, 860, 756.
4.2.10. (E)-3-(3,4-Dimethoxyphenyl)-1-[5-methyl-1-(4-nitrophenyl)triazol-4-yl]prop-2-en-1-one (4j
)
Yellow solid; Yield = 91%; m.p. = 223-225 ºC; 1H NMR (500 MHz, DMSO-d6): δ = 8.51 (d, J = 9.0 Hz,
2H), 8.02 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 15.9 Hz, 1H), 7.84 (d, J = 15.9 Hz, 1H), 7.45 (d, J = 1.8 Hz,
1H), 7.41 (dd, J = 8.4, 1.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H), 3.83 (s, 3H), 2.68 (s, 3H); 13C
NMR (125 MHz, DMSO-d6): δ = 183.1, 151.4, 149.1, 147.8, 143.6, 143.3, 139.7, 138.9, 127.1, 126.3
(2C), 124.8 (2C), 123.2, 120.5, 111.9, 111.1, 55.5 (2C), 9.7; MS (EI) m/z (Chemical Formula:
18

C20H18N4O5): 394.10 (52.36%) [M+]; IR (KBr, cm–1): 3448, 3088, 2994, 2838, 1658 (C=O
α,β-
unsaturated), 1587, 1521 (NO2), 1424, 1344 (NO2), 1263, 1239, 1139, 1033, 981, 856.
4.2.11. (E)-3-(3-Hydroxy-4-methoxy-phenyl)-1-[5-methyl-1-(4-nitrophenyl)triazol-4-yl]prop-2-en-1-one
(4k)
Yellow solid; Yield = 46%; m.p. = 268-272 ºC; 1H NMR (500 MHz, DMSO-d6): δ = 9.38 (s, 1H), 8.49
(d, J = 9.0 Hz, 2H), 8.01 (d, J = 9.0 Hz, 2H), 7.80 (d, J = 15.8 Hz, 1H), 7.74 (d, J = 15.8 Hz, 1H), 7.27
(d, J = 1.8 Hz, 1H), 7.24 (dd, J = 8.2, 1.8 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 3.83 (s, 3H), 2.67 (s, 3H);
13C NMR (125 MHz, DMSO-d6): δ = 182.8, 150.3, 147.6, 146.6, 143.5, 143.1, 139.6, 138.9, 126.8,
126.3 (2C), 124.8 (2C), 122.1, 119.5, 113.7, 111.8, 55.3, 9.7; MS (EI) m/z (Chemical Formula:
C19H16N4O5): 380.34 (100%) [M+]; IR (KBr, cm–1): 3380, 3089, 2939, 2839, 1659 (C=O
α,β-
unsaturated), 1586, 1524 (NO2), 1504, 1432,1343 (NO2), 1268, 1031, 980, 855.
4.3. Biological evaluation
4.3.1. Cancer cell line screening at NCI
The synthesized compounds were screened against a panel of 60 cancer cell lines derived from nine
tumor types at the National Cancer Institute (NCI), Bethesda, Maryland, USA, applying the standard
NCI protocol reported elsewhere [53]. The compounds were tested at a single dose concentration of 10
µM, and the one-dose data were reported as a mean graph of the percent growth of the treated cells. The
number reported for the one-dose assay is growth relative to the no-drug control, and relative to the time
zero number of cells. Using the seven absorbance measurements [time zero, (Tz), control growth, (C),
and test growth in the presence of drug at the five concentration levels (Ti)], the percent growth is
calculated at each of the drug concentrations levels. Percent growth inhibition is calculated as:
•
•
[(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz
[(Ti − Tz)/Tz] × 100 for concentrations for which Ti<Tz.
4.3.2. Cytotoxicity MTT assay for compounds 4b, 4d, 4f, 4h and 4i
Cancer cell lines (RPMI-8226, SR, K-562, M14, MCF7, HCT-116 and PC-3) and normal cell lines
(PCS-800-011, WS1, MCF 10A, CCD-18Co and HPrEC) were used to verify the cytotoxicity of
compounds 4b, 4d, 4f, 4h and 4i using the MTT assay. The principle of this assay is the transformation
of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate
dehydrogenase in viable cells [54]. Cell lines were cultured in RPMI-1640 medium with 10% fetal
bovine serum. The antibiotics added were 100 µg/ml streptomycin and 100 units/ml penicillin at 37º C
in a 5% CO2 incubator. The cell lines were planted in a 96-well plate at a density of 1.0x104 cells/ well
at 37º C for 48 h under 5% CO2 [55]. After incubation, the cells were handled with several
concentrations of the synthesized compounds and incubated for 24 h. After that, 20 µl of MTT solution
at 5 mg/ml was included and incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 µl is
supplemented into each well to dissolve the purple formazan made. The absorbance was measured at a
wavelength of 570 nm using a plate reader (EXL 800 USA), and IC50 values were determined.
19

4.3.3. In vitro enzyme inhibitory assays (against HDAC, NF-κB, EGFR, VEGFR-2, topoisomerase-2 and
tubulin) as well as intracellular ROS accumulation assay of compound 4d in RPMI-8226 cell line
In vitro enzyme inhibitory assay of compound 4d was performed against five HDAC isoforms (HDAC1,
2, 3, 8, 6) using ELISA assay kits (Mybiosource, Inc.), NF-κB using ELISA assay kits(ab176648–NFĸB
p65 Total Simple Step ELISA™ Kit), EGFR using ELISA kit for EGFR (cloud clone SEA757Hu 96
Tests), VEGFR-2 using ELISA kit for VEGFR-2 (RayBio_Human VEGFR2), topoisomerase-2 using
ELISA kit for TOP2B (human DNA topoisomerase 2-beta, MBS942146) and tubulin using ELISA kit
for TUBb (Cloud-Clone Corp., SEB870Hu 96 Tests Enzyme-linked Immunosorbent Assay Kit) as well
as intracellular ROS accumulation assay using ELISA kit for ROS (EIAab ROS eia kit). The procedure
of the used kit was done according to the manufacturer's instructions. Briefly, 96-well microtiter plates
were used for RPMI-8226 growing cells in which trypsinization, counting and seeding of cells at the
proper densities were carried out followed by incubation at 37 ºC in a humidified atmosphere for 24 h.
The standards, 4d compound, and the positive reference (Velcade as HDAC inhibitor; Gefitinib as NF-
κB and EGFR inhibitor; Sorafenib as VEGFR-2 inhibitor; Doxorubicin as topoisomerase-2 inhibitor;
Combretastatin as tubulin inhibitor; Gefitinib as ROS generator) were adjusted to nominated
concentrations. Then 100 µL of sample or standard was added to each well of the 96-well microtiter
plates, and incubated for 2 h at 37 ºC. After aspiration of the solution, prepared Detection Reagent A
was added in 100 µL to each well and incubated for 1 h at 37 ºC. Washing was performed, then prepared
Detection Reagent B was added in 100 µL, and incubation was continued for another 1 h at 37 ºC.
Afterthat, Five washings were performed followed by addition of 3,3',5,5'-tetramethylbenzidine (TMB)
substrate solution in 90 µL and incubation for 15–25 min at 37 ºC. Next, Stop solution was added in 50
µL. Cells were exposed to different concentrations of compound 4d and refrence for 72 h. Optical
density (O.D.) was measured at 450 nm using microplate reader. The values of % activity versus a series
of compound concentrations (with semi-log decrease in concentration) were then plotted using non-
linear regression analysis of sigmoidal dose-response curve, and the concentration that induces 50% of
maximum inhibition (IC50) of HDAC isoforms, NF-κB, EGFR, VEGFR-2, topoisomerase-2 and tubulin
were determined.
4.3.4. Measurement of the effect of compound 4d on the level of BAX, Bcl-2, caspases 3, 7, 9 and PARP-
1 in RPMI-8226 cell line
The level of BAX (apoptotic marker), Bcl-2 (anti-apoptotic marker), caspases 3, 7, 9 and PARP-1
(apoptotic markers) were assessed using ELISA colorimetric kits (ELISA kit for BAX (DRG® Human,
EIA-4487), ELISA kit for Bcl-2 (Invitrogen Zymed®), ELISA kit for caspases (Invetrogen Casp3,
Casp7, Casp9 (Active) eia kit) and ELISA kit for PARP-1(Abcam PARP-1 Human eia kit ab110215)).
The procedure of the used kit was done according to the manufacturer's instructions. Briefly, RPMI-
8226 Cells (obtained from American Type Culture Collection) were grown in RPMI 1640 containing
10% fetal bovine serum at 37°C. Cells were Plated in a density of 1.2–1.8 × 10,000 cells/well in a
volume of 100 µl complete growth medium + 100 ul of the compound 4d per well in a 96-well plate for
24 hours before the assay and lysed with Cell Extraction Buffer. This lysate was diluted in Standard
Diluent Buffer over the range of the assay and measured for human active BAX, Bcl-2, caspase-3,
caspase-7, caspase-9 or PARP-1 content.
4.3.5. Cell cycle analysis of compound 4d in RPMI-8226 cells
20

The RPMI-8226 cells were treated with compound 4d at three concentrations (IC50 (x), 5x and 10x) in
absence of NAC, and at concentration 10x of compound 4d in presence of NAC for 24 h. After
treatment, the cells were washed twice with ice-cold phosphate buffer saline (PBS), collected by
centrifugation, and fixed in ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with 0.1 mg/mL
RNase, stained with 40 mg/mL propidium iodide (PI), and analyzed by flow cytometry using
FACSCalibur (Becton Dickinson) [56]. The cell cycle distributions were calculated using Cell-Quest
software (Becton Dickinson).
4.3.6. Annexin-V-FITC apoptosis assay of compound 4d in RPMI-8226 cells
Apoptosis was determined by staining the cells with Annexin V fluorescein isothiocyanate (FITC) and
counterstaining with PI using the Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San
Diego, CA) according to the manufacturer's instructions. Briefly, 4 × 106 cell/T 75 flask were exposed to
compounds 4d at three concentrations (IC50 (x), 5x and 10x) in absence of NAC, and at concentration
10x of compound 4d in presence of NAC for 24 h. The cells then were collected by trypsinization and
0.5 × 106 cells were washed twice with PBS and stained with 5 µL Annexin V-FITC and 5 µL PI in 1 ×
binding buffer for 15 min at room temperature in the dark. Analyses were performed using FACS
Calibur flow cytometer (BD Biosciences, San Jose, CA).
4.4. Statistical analysis
In this study part of the data is presented as mean ± SD of three individual experiments performed in
triplicate. Non-parametric one-way analysis of variance (ANOVA) followed by a Dunnett post-hoc test
was applied for Statistical analysis using the SPSS software (version 18.0). P < 0.05 was regarded
statistically significant.
Acknowledgments
Our sincere thanks to the National Cancer Institute (NCI), USA, for carrying out the in vitro
antiproliferative analysis.
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Highlights:
• A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in
vitro against a panel of NCI-60 human cancer cell lines.
• Compound 4d with 3, 4-dimethoxyphenyl chalcone moiety was the most potent
anticancer agent.
• Compound 4d showed IC50 values less than 1 µM on 6 cancer cell lines with high
Selectivity Index (SI).
• Compound 4d induce cell cycle arrest in RPMI-8226 cell line at G2/M phase and
trigger mitochondrial apoptotic pathway through accumulation of ROS, up regulation
of BAX, down regulation of Bcl-2 and activation of caspases 3, 7, 9.

Declaration of interests
x The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: