Design and synthesis of novel quinoline/chalcone/1,2,4-triazole hybrids as potent antiproliferative agent targeting EGFR and BRAFV600E kinases

Article abstract of DOI:10.1016/j.bioorg.2020.104510

New quinoline / chalcone hybrids containing 1,2,4-triazole moiety have been designed, synthesized and their structures elucidated and confirmed by various spectroscopic techniques. The designed compounds showed moderate to good activity on different NCI 60 cell lines in a single-dose assay with a growth inhibition rate ranging from 50% to 94%. Compounds 7b, 7d, 9b, and 9d were the most active compounds in most cancer cell lines with a growth inhibition percent between 77% and 94%. Newly synthesized hybrids were evaluated for their anti-proliferative activity against a panel of four human cancer cell lines. Compounds 7a, 7b, 9a, 9b, and 9d showed promising antiproliferative activities. These compounds were further tested for their inhibitory potency against EGFR and BRAF^V600E kinases with erlotinib as a reference drug. The molecular docking study of compounds 7a, 7b, 9a, 9b, and 9d revealed nice fitting into the active site of EGFR and BRAF^V600E kinases. Compounds 7b, 9b, and 9d displayed the highest binding affinities and similar binding pattern to those of erlotinib.

Full text of DOI:10.1016/j.bioorg.2020.104510

Journal Pre-proofs
Design and Synthesis of Novel quinoline/chalcone/1,2,4-triazole hybrids as
potent antiproliferative agent targeting EGFR and BRAFV600E kinases
Aliaa M. Mohassab, Heba A. Hassan, Dalia Abdelhamid, Ahmed M. Gouda,
Bahaa G. M. Youssif, Hiroshi Tateishi, Mikako Fujita, Masami Otsuka,
Mohamed Abdel-Aziz
PII:
S0045-2068(20)31808-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104510
YBIOO 104510
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
15 June 2020
10 October 2020
19 November 2020
Please cite this article as: A.M. Mohassab, H.A. Hassan, D. Abdelhamid, A.M. Gouda, B. G. M. Youssif, H.
Tateishi, M. Fujita, M. Otsuka, M. Abdel-Aziz, Design and Synthesis of Novel quinoline/chalcone/1,2,4-triazole
hybrids as potent antiproliferative agent targeting EGFR and BRAFV600E kinases, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.104510
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Design and Synthesis of Novel quinoline/chalcone/1,2,4-triazole hybrids as
potent antiproliferative agent targeting EGFR and BRAFV600E kinases
Aliaa M. Mohassab a, Heba A. Hassan a*, Dalia Abdelhamida, Ahmed M. Gouda b, Bahaa G. M.
Youssif c*, Hiroshi Tateishi d, Mikako Fujita d, Masami Otsuka d,e, Mohamed Abdel-Aziza
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia 61519,
Egypt, bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-
C
Suef 62514, Egypt, Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
d
Assiut University, Assiut 71526, Egypt., Medicinal and Biological Chemistry Science Farm
Joint Research Laboratory, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi,
Chuo-ku, Kumamoto, Kumamoto 862-0973, Japan., e Department of Drug Discovery, Science
Farm Ltd., 1-7-30 Kuhonji, Chuo-ku, Kumamoto, Kumamoto 862-0976, Japan.
Short running title: Synthesis and antiproliferative activity of new quinoline/chalcone/1,2,4-
triazole hybrid.
*To whom correspondence should be addressed:
Mohamed Abdel-Aziz, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Minia
University, 61519-Minia, Egypt.
Tel.:(002)-01003311327
E-mail address: Abulnil@hotmail.com
Heba A. Hassan, Ph.D. Department of Medicinal Chemistry, Faculty of Pharmacy, Minia
University, 61519-Minia, Egypt.
Tel.:(002)-01068390918
E-mail address: heba.hasan@mu.edu.eg
Bahaa G. M. Youssif, Ph.D. Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy, Assiut University, Assiut 71526, Egypt.
Tel.: (002)-01098294419
E-mail address: bgyoussif@ju.edu.sa, bahaa.youssif@pharm.aun.edu.eg
1

Abstract
New quinoline / chalcone hybrids containing 1,2,4-triazole moiety have been designed,
synthesized and their structures elucidated and confirmed by various spectroscopic techniques.
The designed compounds showed moderate to good activity on different NCI 60 cell lines in a
single-dose assay with a growth inhibition rate ranging from 50% to 94%. Compounds 7b, 7d, 9b,
and 9d were the most active compounds in most cancer cell lines with a growth inhibition percent
between 77% and 94%. Newly synthesized hybrids were evaluated for their anti-proliferative
activity against a panel of four human cancer cell lines. Compounds 7a, 7b, 9a, 9b, and 9d showed
promising antiproliferative activities. These compounds were further tested for their inhibitory
potency against EGFR and BRAFV600E kinases with erlotinib as a reference drug. The molecular
docking study of compounds 7a, 7b, 9a, 9b, and 9d revealed nice fitting into the active site of
EGFR and BRAFV600E kinases. Compounds 7b, 9b, and 9d displayed the highest binding affinities
and similar binding pattern to those of erlotinib.
Key words: Quinoline, triazole, chalcone, EGFR, BRAFV600E, Apoptosis, Docking.
2

Graphical Abstract
3

Highlights

A series of quinoline/chalcone hybrids containing 1,2,4-triazole moiety was synthesized
and tested by NCI for their anticancer activity.
 Compounds 7b, 7d, 9b, and 9d were the most active ones with a growth inhibition percent
between 77 and 94%.
 In vitro antiproliferative activity was evaluated using MTT assay.
 7a, 7b, 9a, 9b, and 9d showed promising antiproliferative activities and were evaluated
against EGFR and BRAFV600E
.
 A docking study of compounds 7a, 7b, 9a, 9b, and 9d showed good binding affinities
towards EGFR and BRAFV600E
.
4

1. INTRODUCTION
Cancer is a major global public health concern [1]. In developed countries, it presents a huge
burden on society and economy. The incidence of cancer increases as population growth and aging
increase, as well as the prevalence of other established risk factors [2]. It is estimated that there
will be a 3-fold increase in the incidence of cancer in Egypt by 2050 compared to 2013 statistics
[3]. Medicinal chemists are therefore faced with a challenging task of developing new anti-cancer
agents to overcome the spread of cancer, either in Egypt or around the world [4].
Quinoline derivatives are common, broad-spectrum drugs [5]. Quinoline moiety can be recognized
in many bioactive compounds including anti-cancer [6], anti-inflammatory [7, 8], antiviral [9],
antimicrobial [10], and anti-parasitic agents [11]. Recently, several research papers have described
quinoline derivatives with anti-cancer activity, which can be observed by an increasing number of
published reviews of the chemistry and biological activity of anti-cancer quinolines [6, 12, 13].
Quinoline derivatives act as anti-cancer agents through a variety of mechanisms, including
inhibition of cell growth, promoting factors such as tubulin polymerization, topoisomerase,
tyrosine kinase, proteasome, tyrosine kinase, and DNA repair [4,14].
Recently, due to their effective therapeutic importance, attention has been paid to the chemistry of
triazoles and their derivatives [15]. The 1,2,4-triazole ring could be considered as an ester, amide,
carboxylic acid, and other heterocycle isostere [16]. The 1,2,4-triazole-containing ring system is a
common pharmacophore that has been incorporated into a wide range of therapeutically interesting
active compounds. For example, anti-cancer [17], anti-inflammatory [18], anti-microbial [19],
analgesic [20], anti-viral, [21] and anti-convulsant [22] active compounds.
5

Chalcones are an important category of flavonoid family derived from synthetic or natural
compounds [23]. Due to their versatile therapeutic activities, such as anti-cancer [24, 25], the
production of chalcones as privileged scaffolds in medicinal chemistry has attracted great interest.
Chalcones are proposed to cause cell-cycle disruption and apoptosis, inhibit tubulin
polymerization, inhibit unique kinases that are essential for cancer cell proliferation and survival
[26].
Over the last three decades, many hybrid molecules have been of great importance in the
development of new drugs and have undergone clinical trials for the treatment of various diseases
[27, 28]. Hybridization strategy has been used to develop new anti-cancer drugs by fusing more
than two or more active pharmacophores in a single hybrid molecule with synergistic anti-cancer
activity [29]. Hybrid molecules are designed to enhance the biological spectrum and efficacy,
overcome drug cross resistance, and reduce potential toxicity compared to the parent drugs. For
example, Hybridization of quinoline ring with 1,2,4-triazole ring in compounds I and II (Fig. 1)
were found to induce apoptosis and inhibit cellular growth and proliferation of SB-590885-
sensitive and SB-590885-resistant 451Lu melanoma cell lines, harboring BRAFV600E mutation, via
inhibition of STAT3 phosphorylation with IC50 values ranging from 4.7 µM to 17 µM [30].
Hybridization of 1,2,4-triazole ring with chalcone moiety in compound III exhibited significant
growth inhibition and induced caspase-3 dependent apoptosis in A549 human lung
adenocarcinoma cells with IC50 value of 4.4 µM relative to cisplatin with IC50 value of 15.3 µM
[25] (Fig. 1).
6

On the other hand, a growing number of research studies have been focused on the optimization
and incorporation of quinoline and chalcone scaffolds into chemotherapeutic agents as it has been
reported that combining quinoline and chalcone moieties in one compact structure leads to more
potent cytotoxic activity [31, 32]. For instance, compound IV, a quinoline-chalcone hybrid, was
reported to have a remarkable cytotoxic activity against different cancer cell lines. Compound IV
could induce G2/M cell cycle arrest and apoptosis in A549 cancer cells (Fig. 1) [4].
Following the previous study on the importance of hybrid molecules in the treatment of different
types of cancer and pathways, Herein, we report the design and synthesis of certain novel quinoline
/ chalcone derivatives (7a-f, 8a-f, and 9a-f, Fig. 1) that incorporate quinoline, chalcone and triazole
rings into a single compact structure for synergistic anti-cancer activity, manage drug resistance
development, and reduce possible side effects. Prepared compounds have different substitutions
for the electron donating and the electron withdrawing groups for the SAR study of these
compounds. The synthesized derivatives were evaluated for NCI cytotoxic assay and in-vitro
antiproliferative activity against four different cancer cell lines: pancreas cancer cell line (Panc-1),
breast cancer cell line (MCF-7), colon cancer cell line (HT-29), and epithelial cancer cell line (A-
549). Furthermore, the most potent compounds were evaluated for their ability to inhibit EGFR
and BRAFV600E as a potential mechanism for these compounds. The most potent derivatives were
docked into the ATP binding sites of EGFR and BRAFV600E
.
7

Fig. 1. Structures of representative hybrids I-IV and newly hybrids 7a-f, 8a-f, and 9a-f
2. RESULTS AND DISCUSSION
2.1. Chemistry
The sequence of reactions used to synthesize quinoline-chalcone hybrids 7a-f, 8a-f and 9a-f is
obtained in Scheme 1. Synthesis of 2-(substituted phenyl) quinoline-4-carboxylic acids 1a–c was
performed by refluxing isatin with an appropriate acetophenone in aqueous ethanol [33]. Separate
acids were refluxed with absolute ethanol in the presence of conc. H2SO4 as a dehydrating agent
providing the corresponding esters 2a-e [34]. Heating at reflux temperature of ethyl esters 2a-e
with hydrazine monohydrate afforded carbohydrazide derivatives 3a-c [35]. The appropriate
carbohydrazide 3a-e was heated at reflux temperature with allyl or phenyl isothiocyanate in
8

ethanol, then aqueous 2N NaOH was added followed by acidification with conc. HCl affording
1,2,4-triazole-3-thiol derivatives 4a-f [36]. Structures of the formed compounds have been
1
confirmed by their reported melting points, H NMR and 13C NMR spectra. The acetylated
chalcone intermediates 6a-c were prepared according to the reported procedure [37] by reaction
of the appropriate chalcone 5a-c with bromoacetyl bromide using potassium carbonate as a base
in dichloromethane. The separated compounds were coupled with 1,2,4-triazole-3-thiol derivatives
4a-f in presence of acetonitrile and triethylamine (TEA) as base affording target compounds 7a-f,
8a-f, and 9a-f. The structure of these novel compounds has been validated by NMR spectroscopy
and mass spectral analysis.
9

SH
COOH
N
O
COY
N
N
N
a
b
N
d
O
R'
N
H
R
R
N
1a-c
2a-c, Y = OCH2CH3
3a-c, Y = NHNH2
R
c
4a-f
R= H, Cl, OCH3
R
R'
4a
4b
4c
4d
4e
4f
H
H
Ph
Allyl
Ph
Allyl
Ph
Allyl
4-Cl
4-Cl
4-OCH3
4-OCH3
O
H2N
O
R"
R"
O
e
Br
f
N
O
H
6a-c
5a-c
R"= a) Cl, b) OCH3, c) 3,4,5-Tri-OCH3
R''
O
NH
S
N
N
N
R'
For R, R', and R'' see Table 1
N
R
7a-f (R"= Cl)
8a-f (R"= OCH3)
9a-f (R"= 3,4,5-TriOCH3)
Scheme1: Synthesis of quinoline-chalcone hybrids 7a-f, 8a-f, and 9a-f
Reagent and reaction conditions: (a) Appropriate acetophenone, EtOH, Reflux, 9–18 h; (b)
EtOH, Conc. H2SO4, reflux 10 h; (c) NH2NH2, EtOH, reflux 3-7 h; (d) Appropriate isocyanate,
EtOH, reflux, 5 h; (e) Bromoacetyl bromide, K2CO3, rt, 18 h; (f) TEA, Acetonitrile, rt, 4-8 h.
10

The 1H NMR spectrum of 8f as a representative example showed the appearance of the amidic NH
signal and four sets of doublets at 8.04 and 7.82, and 7.08 and 6.96 ppm, which is indicative of
aromatic p-disubstitution, 3 signals attributed to the allyl protons in the form of multiplet (1H) at
5.83-5.77 ppm, two sets of doublets at 5.32 and 5.05 (2H), and doublet signal (2H) at 4.49 ppm.
In addition to singlet signal of two protons corresponding to SCH2 and two signals with three
13
proton integrations each at 3.92 (s) and 3.88 (s) ppm attributed to two methoxy. The C NMR
spectrum of 8f showed the appearance of two carbonyl carbon signals at δ 188.99 (CH=CHCO),
166.77 (NHCO), a resonance at 36.99 ppm which was assigned to methylene carbon of SCH2, and
two resonances at 142.30 and 124.60 ppm which were assigned to CH=CH as well as the allyl
carbons. The structure was also confirmed by HRESI-MS with [M+H]+ ion at m/z 668.2379 which
correlates with the molecular weight and molecular formula C39H34N5O4S of 8f.
11

2.2. Evaluation of biological Activities
2.2.1. Screening of cytotoxic activity by NCI
A total of eighteen compounds were selected by the National Cancer Institute (NCI) for anticancer
screening according to the protocol of the Drug Evaluation Branch of the National Cancer Institute,
Bethesda, USA [38]. In-vitro anticancer screening was carried out in 60 cell lines of nine different
cancer cell types: leukemia, ovarian, renal, prostate, melanoma, lung, CNS, colon, and breast
cancers. Compound 7b exhibited the most promising anti-cancer activity among all the tested
compounds. It showed remarkable inhibitory activity against leukemia (K-562, MOLT-4, and SR),
colon (HCT-15 and SW-620), ovarian (OVCAR-8), and breast (MCF7) cancer cell lines with
percent growth inhibition in the range of 72.37-94.60% (supplementary data). In addition,
compound 7b lead to complete cell death against colon (HCT-116), melanoma (LOX IMVI), and
ovarian (IGROV1) cancer cell line. Compound 9b had percent growth inhibition of 78.72% and
79.57% against breast (MCF7) and colon (HCT-116), respectively (supplementary data).
Compound 7d exhibited moderate growth inhibition (66%) specifically against colon (HCT-116)
cell line although it had a weak overall mean growth. Compound 9d had an overall weak mean
growth percent inhibition of 82% against all cancer cell lines, but he specifically caused complete
cell death against leukemia (RPMI-8226) cell line and exhibited moderate growth inhibition ~ 56%
against leukemia (MOLT-4), breast (MCF7), renal (UO-31 and RXF 393), ovarian (OVCAR-8),
and colon (HCT-116 and HCT-15) cancer cell lines. Also, compounds 8d, 9c, and 9f with weak
overall mean growth percent inhibition of 90%, 94.30%, and 90.05%, respectively
(supplementary data). Nevertheless, compounds 8d and 9c had moderate growth inhibition of
55% and 41%, respectively against renal (UO-31) cell line. While compound 9f experienced
moderate growth inhibition of 45% and 50% against leukemia (RPMI-8226 and SR) cell lines,
12

respectively (supplementary data). From the abovementioned results it could be generally
detected that quinoline-chalcone derivatives with R” = 4-Cl or 3,4,5-TriOCH3 substituents
introduced more potent growth inhibitory activity against most of cancer cell lines on the contrary
with other derivatives with R”= 4-OCH3 that exhibited weak activity.
2.2.2 Cell viability assay
Cell viability testing using human mammary epithelial cell line (MCF-10A) was performed. with
MCF-10A cells were treated with compounds 7a-f, 8a-f, and 9a-f for 4 days and cell viability was
assessed using a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazol (MTT) test. All compounds
were found to be non-toxic, most of which had a cell viability of over 82% at a concentration of
50 μM.
2.2.3. Antiproliferative effects
Propidium iodide (PI) tests were used to evaluate the antiproliferative activities of all selected
compounds in four cancer cell lines, pancreatic cancer cell line (Panc-1), breast cancer cell line
(MCF-7), cell line colon cancer (HT-29) and epithelial line cancer cell (A-549) and doxorubicin
was used as the reference compound. GraphPad Prism software (GraphPad Software, San Diego,
CA, USA) was used to calculate the median inhibition concentration (IC50) for all compounds.
Based on IC50, an interesting relationship between analogs and their structural characteristics was
observed.
As illustrated in Table 1, five most active compounds 7a, 7b, 9a, 9b, and 9d among both (4-
phenyl-5-(2-phenyl-quinolin-4-yl)-1,2,4-triazol-3-ylsulfanyl-acetamide (R' = Ph and R'' = Cl) and
(4-allyl-5-(2-phenyl-quinolin-4-yl)-1,2,4-triazol-3-ylsulfanyl-acetamide (R' = Allyl and R'' =3,4,5-
TriOCH3 ) derivatives exhibited potent inhibition of cancer cells growth. After compound 7b,
13

almost comparable and extreme anti-cancer activity was observed between 9b and 9d compounds
with both GI50 cell lines of 3.625 μM and 4.750 μM, respectively. While compound 7b showed
the highest activity among all new compounds against cancer cell growth with GI50 3.325 µM.
When comparing the effects on different cell lines of the individual compound, the findings of
these most active compounds were almost similar with a slight difference in GI50 values from all
four-cancer cell lines. All other compounds had weak to moderate effects on cancer cell growth.
Compound 7b ( R = H, R' = Allyl and R'' = Cl) displayed the highest anticancer potential and it
was bearing allyl-triazole backbone while on the other side compound 7a ( R = H, R' = Ph and R''
= Cl) was bearing same substitution pattern as compared to 7b and difference was phenyl-triazole
moiety but it showed almost 2.5 folds less activity. Obviously, it can be concluded that all seven
compounds belonging to allyl-triazole backbone (7b, 7d, 8b, 8d, 8f, 9b, and 9f) were better
inhibitors of cancer cell growth as compared to other derivatives (7a, 7c, 8c, 8e, 9a, 9c, and 9e)
bearing same substitutions but different backbone of phenyl-triazole.
Presence of different functional groups on terminal 3-phenyl acryloyl moiety (R'') exhibited a
remarkable change in activity as compared to various groups on phenyl moiety in the quinoline
ring (R). The presence of the 3,4,5-trimethoxy group on the 3-phenyl acryloyl ring showed a
significant difference from the other groups and was found to be responsible for maximum activity
after chloro group at same position. It was analyzed that compound 7b bearing chloro substitution
at 3-phenyl acryloyl ring and unsubstituted (R = H) phenyl ring of quinoline moiety was
discovered best antiproliferative agent and second most significant substitution was the presence
of 3,4,5-trimethoxy group at phenyl ring of 3-phenyl acryloyl (Compound 9b) with best
inhibitory property. Moreover, the mean GI50 values of derivatives 7e, 7f, 8e, 8f, 9e, and 9f were
the lowest among the tested compounds, suggesting the importance of the para substitution in the
14

phenyl tail of quinoline moiety for the antiproliferative activity and correlating with higher
antiproliferative effects and the activity increased with (R) in the order of H > Cl > OCH3. Five
most potent compounds (7a, 7b, 9a, 9b, and 9d) among the above-mentioned series were selected
for anticancer mechanistic experiments including their effect on BRAFV600E and EGFR-TK.
Table 1. Antiproliferative activities of compounds 7a-f, 8a-f, and 9a-f
O
R''
O
NH
S
N
N
N
R'
N
R
7a-f, 8a-f, and 9a-f
R''
Compd.
Cell
viability
Anti-proliferative activity IC50± SEM (µM)
R
R’
A-549
MCF-7
Panc-1
HT-29
Average
%
7a
7b
7c
7d
7e
7f
H
H
Ph
Allyl
Ph
Cl
Cl
Cl
Cl
Cl
Cl
82
6.1±0.8
7.5±1.2
8.2±1.2
7.8±1.4
7.400
91
79
89
85
82
87
84
86
96
89
91
3.6±0.05
31.6±3.8
15.2±0.6
26.8±3.2
29.4±3.6
20.4±3.2
23.5±0.2
30.1±3.6
11.3±2.5
25.5±2.6
24.5±2.6
3.3±0.08
35.4±2.7
14.9±0.3
26.5±2.7
28.5±2.8
19.5±2.7
23.1±0.1
29.5±2.8
12.9±1.8
25.6±2.2
23.6±2.2
2.9±0.02 3.5±0.02
36.0±1.9 34.7±3.2
15.8±1.5 14.9±0.8
31.7±3.1 28.2±3.4
32.6±3.5 29.2±2.2
21.7±3.1 21.2±3.4
23.3±0.2 23.9±0.6
32.6±3.5 29.8±2.2
12.5±2.3 12.2±1.4
25.6±2.9 25.8±1.4
24.6±2.9 24.8±1.4
3.325
34.425
15.200
28.300
29.925
20.700
23.450
30.500
12.225
25.375
24.375
4-Cl
4-Cl
Allyl
Ph
4-OCH3
4-OCH3
H
Allyl
Ph
8a
8b
8c
8d
8e
8f
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
Allyl
Ph
4-Cl
4-Cl
Allyl
Ph
4-OCH3
4-OCH3
Allyl
15

9a
H
H
Ph
Allyl
Ph
Tri-MeO
Tri-MeO
Tri-MeO
Tri-MeO
Tri-MeO
Tri-MeO
--
90
89
84
92
90
89
--
6.9±0.3
3.9±0.6
16.4±1.3
4.7±0.4
17.7±2.5
9.9±0.7
6.8±1.6
3.2±0.08
15.4±5.2
4.9±0.6
15.6±2.9
9.4±1.0
6.6±1.5
3.6±1.2
6.9±1.1
3.8±0.2
6.800
3.625
9b
9c
4-Cl
18.6±2.5 16.9±3.7
4.5±0.2 4.9±0.2
16.825
4.750
9d
4-Cl
Allyl
Ph
9e
4-OCH3
4-OCH3
17.8±1.9 17.8±1.6
10.6±0.5 10.4±1.6
17.225
10.075
1.136
9f
Allyl
--
Doxorubicin
1.21 ± 0.80 0.90± 0.62 1.41 ± 0.58 1.01 ± 0.82
2.2.4. EGFR inhibitory activity assay
An assessment of the EGFR inhibitory ability of the most potent five derivatives was performed,
and findings are included in Table 2. The results from this study support the findings of both
cytotoxic activity by NCI and cancer cell-based assays. All investigated compounds 7a, 7b, 9a,
9b, and 9d exhibited inhibition of EGFR with IC50 ranging from 1.3 to 4.8 µM. According to data
presented, three derivatives (9d, 7b, and 9b) selected from allyl-triazole backbone were found to
be most potent in comparison to the positive control erlotinib (IC50 = 0.08±0.04 µM). This study
demonstrates that these compounds are effective EGFR inhibitors and may be used for future
development as lead compounds.
2.2.5. BRAFV600E inhibitory activity
The BRAFV600E inhibitory ability of five most active synthesized compounds 7a, 7b, 9a, 9b, and
9d were evaluated in vitro. All compounds analyzed showed IC50 within the range of 1.1 to 6.9
μM according to data in Table 2. It is noteworthy that two of allyl-triazole backbone (9d and 9b)
with the presence of R'' = 3,4,5-tri-methoxy have the highest inhibitory potential for BRAFV600E
and were also effective against the growth of cancer cells. The results of this assay show that the
compounds examined are possible anticancer agents and effectively inhibit the BRAF enzyme.
16

Table 2. Effects of compounds 7a, 7b, 9a, 9b and 9d on EGFR and BRAFV600E
Compd
R
R'
R''
EGFR
inhibition
BRAFV600E
inhibition
IC50 ± SEM
(µM)
IC50 ± SEM
(µM)
7a
7b
H
H
Ph
Allyl
Ph
Cl
Cl
4.8±1.7
1.3±1.2
3.7±1.5
2.1±0.4
2.8±0.8
0.08±0.04
6.9±1.7
3.8±1.5
5.7±1.6
1.6±1.4
1.1±0.6
0.06±0.04
9a
H
Tri-MeO
Tri-MeO
Tri-MeO
--
9b
H
Allyl
Allyl
--
9d
4-Cl
--
Erlotinib
2.2.6. Cell cycle analysis and apoptosis detection
2.2.6.1. Cell cycle analysis
Studies have been conducted on the effect of compound 7b on growth and apoptosis in the Panc-
1 cell cycle. Panc-1 cells were incubated with an IC50 concentration (2.9 μM) of 7b for 24 h. The
results of the study (Fig. 2) reveal that the pre-G1 apoptosis rate of compound 7b on Panc-1 was
27.15%. In G2/M phase, a high percent of cell accumulation was observed in Panc-1 treated with
7b (34.36%) indicating cell cycle arrest at G2/M transition.
17

Fig. 2. Cell cycle analysis in Panc-1 cell line treated with compound 7b
2.2.6.2. Apoptosis assay
Analysis of the Panc-1 cell cycle showed that pre-G1 apoptosis signalling occurred after 7b
treatment. Annex V/ PI was labelled in cells and incubated 24 hours to check 7b for apoptosis
cells. Early and late apoptosis studies have shown that 7b with 2.99% necrosis is likely to cause
significant apoptosis, (Table. 3).
Table 3. Apoptosis detection of 7b in Panc-1 cancer cell line.
Compound
Apoptosis
Early
8.34
Necrosis
Total
27.15
1.79
Late
15.82
0.47
7b/panc-1
2.99
0.3
Control/panc-1
1.02
18

2.3. Docking study into EGFR and BRAFV600E
In the current study, the biological evaluation of the new compounds (7a, 7b, 9a, 9b, and 9d)
revealed inhibitory activity against EGFR and BRAFV600E kinases, Table 2. Accordingly, we have
performed a molecular docking study to evaluate the binding modes, orientations and interactions
of these compounds into the ATP binding sites of EGFR (pdb code: 1M17) (38) and BRAFV600E
(pdb code: 3OG7) (39). The crystal structures of the two kinases were obtained from the Protein
Data Bank (https://www.rcsb.org/structure).
AutoDock 4.2 (40) was used in the docking study. Ligand and protein files were prepared
according to the previous reports (41, 42). Grid and docking parameter files were prepared by
AutoDock tools (ADT) following the previous reports (43, 44). Visualization of the 2/3D binding
modes of the new compounds was performed by Discovery Studio Visualizer (45). To validate the
docking procedures, the co-crystallized ligands, erlotinib and vemurafenib were initially re-docked
into the active site of EGFR and BRAFV600E, respectively. The results revealed superposition of
erlotinib and vemurafenib over the native ligands, Fig. 3. Moreover, the re-docked ligands
exhibited similar binding interactions to those of the corresponding co-crystallized ligands
Fig. 3. Binding mode/interactions of redocked/co-crystallized erlotinib and vemurafenib into their
corresponding kinases: A) binding mode of the redocked erlotinib (shown as sticks, colored by
element) into the ATP binding site of EGFR (PDB code: 1M17) overlaid with the co-crystallized
19

ligand (show as line, colored in blue) with RMSD of 1.44 Å; B) binding mode of redocked
vemurafenib (shown as sticks, colored by element) into the ATP binding site of BRAFV600E (pdb
code: 3OG7) overlaid with the co-crystallized ligand (show as line, colored in blue) with RMSD
of 1.44 Å. Receptor shown as hydrogen bond surface.
The molecular docking analyses revealed that the new compounds adopted binding conformations
into the active site of EGFR in which the chalcone-bearing side chain are folded toward the triazole
ring inside the front hydrophobic pocket of the two kinases. this conformation allows the new
compounds to occupy small volume and fit into the active site of EGFR without any steric clashes
or unfavorable interactions. Moreover, the new compounds exhibited higher binding affinities
(Gb = -9.94 to -11.43 kcal/mol) for the five compounds (7a, 7b, 9a, 9b, and 9d) compared to
erlotinib (Gb = -7.39 kcal/mol), Table 4. However, the tested compounds exhibited weaker
inhibitory activity against EGFR compared to erlotinib. Accordingly, other parameters such as
binding interactions and conformations of the new compounds were investigated to understand
their inhibitory activities. Among the docked derivatives, compound 7b exhibited the highest
binding free energy (Gb = -11.43 kcal/mol) toward EGFR compared to -7.39 kcal/mol for
erlotinib, Table 4. These results were matched with the in vitro inhibitory activity of the new
compounds against EGFR, Table 2.
Investigation of the binding orientation of compound 7b revealed that it adopted an orientation in
which the 2-phenyl group occupy the hydrophobic pocket like the cyanophenyl group of erlotinib.
The quinoline ring of compound 7b overlaid partially over the quinazoline ring in erlotinib. The
4-(3-(4-chlorophenyl)acryloyl)phenyl moiety in compound 7b is folded toward the triazole ring
which decreases the molecular volume of the compound and allowed it to occupy the front
hydrophobic pocket in EGFR, Fig. 4.
20

Table 4. Results of the docking of compounds 7b and 9d into EGFR (pdb: 1M17) [54] and
BRAFV600E (pdb code: 3OG7) [55] in comparison to its co-crystallized ligand.
b
HBsc Atoms in H-bonding
Length d
(Å)
a
Kinase
(pdb)
Ligand
Ki
Gb
EGFR
(1M17)
Pyridine N OH of Thr766
3.01
2.98
2.12
2.61
2.45
2.89
7b
-11.43 4.19 nM
3
7
Ph-NH f
C=O
NH of Cys773
NH of Met769
SH of Cys751
C=O of Thr766
C=O of Gln767
Gly695, Asp776, Thr830,
Asp831
CONH
CONH
CONH
9d
-9.94
51.45 nM
e
3/5-OCH3
1.72-2.86
Met769
CH-2 e
C=O
NH
C=O
4-OCH3
C=O
S
3-OCH3
3-OCH3
Pyridine N NH of Cys532
SO2
SO2
Met769
1.64
2.06
2.45
2.89
2.49
2.20
1.83
2..87
2.73
2.29
2.00
2.55
1.65
AQ4
-7.39
-9.84
3.84 µM
2
3
Gln767 e
BRAFV600E 7b
(3OG7)
61.08 nM
NH2 of Lys483
C=O of Gln530
NH of Asp594
NH2 of Lys483
NH of Cys532
NH of Ser536
Asn581
9d
-9.94
-9.89
51.6 nM
5
3
e
e
Asp594
032
55.89 nM
NH of Phe595
NH of Gly596
a Binding free energy (kcal/mol); b Inhibition constant; c number of hydrogen bond; d bond distance
e
(angstrom); carbon hydrogen bond; AQ4, erlotinib ([6,7-bis(2-methoxy-ethoxy)quinazoline-4-
yl]-(3-ethynylphenyl)amine); 032, vemurafenib (N-(3-{[5-(4-chlorophenyl)-1H-pyrrolo[2,3-
b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane-1-sulfonamide)
Investigation of the binding interactions of compound 7b revealed two conventional hydrogen
bonds with Thr766 and Cys773 compared to only one hydrogen bonds for erlotinib. Compound
7b also exhibited one carbon hydrogen bond with CYS773 and one pi-sulfur interaction with
Lys721. In addition, compound 7b showed several types of hydrophobic interactions with
hydrophobic residues such as Ala719, Met742, Met769, and Phe771 in EGFR, Fig. 4.
21

Fig. 4. Binding modes/interactions of compound 7b into EGFR (PDB code: 1M17): A) 3D binding
mode of compound 7b into the active site of EGFR, the co-crystallized erlotinib shown as orang
line, receptor shown as hydrogen bond surface, hydrogen atoms were omitted for clarity; B) 2D
binding mode of compound 7b into EGFR showing different types of interactions with amino acids
in the active site, hydrogen atoms were omitted for clarity.
In addition, the docking results revealed that compounds 7a and 9a have lower binding affinities than
compounds 7b and 9b. Moreover, compound 9a showed slightly higher affinity toward EGFR than
compound 7a, Table 4. These results were matched with the in vitro inhibitory activity of the four
compounds, Table 2. On the other hand, compounds 7a and 9a exhibited slightly binding affinities
compared to that of compound 9d although they were less active in vitro. These results could be
attributed to the higher number of hydrogen bonds observed with compound 9d. In addition.
The new compounds (7a, 7b, 9a, 9b, and 9d) were docked into BRAFV600E (pdb code: 3OG7)[55].
The results of the docking study revealed that the five high binding affinities (Gb = -9.11 to -
9.94 kcal/mol) which were comparable to that of vemurafenib (Gb = -9.89 kcal/mol), Table 4.
Compound 9d displayed the highest binding free energy (Gb = of -9.94 kcal/mol) toward
BRAFV600E compared to -9.89 kcal/mol for vemurafenib, Table 4. Compound 9d displayed three
conventional hydrogen bonds with Lys483, Cys532 and Ser536 with bond length in the range of
22

1.83-2.87 Å. In addition, compound 9d showed two carbon hydrogen bonds between with Asn581
Asp594 amino acids. One pi-sulfur interaction with Phe583 and several hydrophobic interactions
with Ile463, Leu514, and Tyr538 were also observed for compound 9d, Fig. 5. The high binding
affinity and the interaction pattern of compound 9d could account for its high activity against
BRAFV600E, Table 2.
Figure 5: Binding modes/interactions of compound 9d into BRAFV600E (pdb code: 3OG7): A) 3D
binding mode of compound 9d into the active site of BRAFV600E, vemurafenib shown as brown
line, receptor shown as hydrogen bond surface, hydrogen atoms were omitted for clarity; B) 2D
binding mode of compound 9d into BRAFV600E showing different types of interactions with amino
acids in the active site, hydrogen atoms were omitted for clarity.
The binding modes, orientations, and interactions of compound 7a, 9a, and 9b into the active site
of EGFR and BRAFV600E are provided in supplementary data (Figs. S1-8).
23

3. CONCLUSION
Eighteen novel quinoline / chalcone hybrids containing 1,2,4-triazole moiety have been designed,
synthesised, and submitted to NCI for an anticancer activity assessment. The newly synthesized
hybrids were tested in vitro against a panel of cancer cell lines and EGFR and BRAFV600E
anticancer targets. Some of the compounds analysed had a significant inhibitory anti-proliferative
effect. The most potent compounds were 7a, 7b, 9a, 9b and 9d. Compounds 9d, 7b, and 9b
selected from allyl-triazole backbone were found to be most potent in comparison to the positive
control erlotinib. The results of BRAFV600E inhibitory assay show that the compounds examined
are possible anticancer agents and effectively inhibit the BRAFV600E enzyme. Compound 7b
induced apoptosis and demonstrated cell cycle arrest in G2/M phase. To understand the results of
the kinases inhibitory activities of compounds 7a, 7b, 9a, 9b, and 9d, a molecular docking study
was performed into the active sites of EGFR and BRAFV600E. The results revealed nice fitting into
the active sites of the two kinases. The new compounds also exhibited higher binding free energies
toward EGFR than the native ligand, erlotinib. Moreover, these compounds displayed binding
affinities toward BRAFV600E comparable to that of vemurafenib.
Acknowledgment
Not applicable
Conflicts of interest
The authors declare no conflict of interest
24

4. EXPERIMENTAL
4.1. Chemistry
General details: See Appendix A
4.1.1. General procedure for the synthesis of compounds (7a-f, 8a-f, and 9a-f).
An equimolar mixture of compounds 4a-f, compounds 6a-c and TEA (1.2 mmol) in acetonitrile
(50 mL) was stirred at RT for 4-8 h. The resulting precipitate is filtered off, dried and then applied
to Silica gel 60 N to obtain pure product using the DCM/EA (9:1) as eluent system to afford the
pure novel products 7a-f, 8a-f, and 9a-f.
4.1.1.1.
2-(4-Phenyl-5-(2-phenylquinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
chlorophenyl)acryloyl)phenyl)acetamide (7a).
Pale yellow solid (61%); m.p. 261-262 oC; IR (FT-IR, cm-1): 3328 (NH), 1698 (C=O), 1659 (CO-
NH); 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.85 (s, 1H, NH), 8.22 (d, J = 9.0 Hz, 2H, Ar-H),
8.14 (d, J= 8.4 Hz, 1H, Ar-H), 8.11 (d, J = 7.8 Hz, 2H, Ar-H), 8.07 (s, 1H, Ar-H), 8.03 (d, J = 9.0
Hz, 1H, Ar-H), 8.00 (d, J = 15.6 Hz, 1H, CH=CH), 7.95 (d, J = 8.4 Hz, 2H, Ar-H), 7.84–7.81 (m,
3H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H, CH=CH), 7.64–7.62 (m, 1H, Ar-H), 7.55–7.53 (m, 3H, Ar-
H), 7.53–7.50 (m, 4H, Ar-H), 7.47–7.43 (m, 3H, Ar-H), 4.40 (s, 2H, S-CH2); 13C NMR (151 MHz,
DMSO-d6) δ (ppm): 187.46, 166.18, 155.19, 152.06, 151.85, 147.82, 147.81, 143.26, 141.95,
137.77, 134.97, 133.76, 133.17, 133.12, 132.50, 130.50, 130.05, 129.98, 129.78, 129.53, 128.93,
128.91, 127.48, 127.32, 126.98, 125.53, 124.52, 122.76, 120.39, 118.56, 37.00; MS
(FAB) m/z 678.3 (M+H)+; HRMS (FAB). Calcd for C40H29O2N5ClS: 678.1730. Found: 678.1729.
25

4.1.1.2.
2-(4-Allyl-5-(2-phenylquinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
chlorophenyl)acryloyl)phenyl)acetamide (7b).
Yellow solid (79%); m.p. 237-238 oC; IR (FT-IR, cm-1): 2969 (NH), 1690 (C=O), 1660 (CO-NH);
1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.82 (s, 1H, NH), 8.31 (d, J = 7.2 Hz, 1H, Ar-H), 8.30
(s, 1H, Ar-H), 8.21–8.19 (m, 3H, Ar-H), 7.99 (d, J = 15.6 Hz, 1H, CH=CH), 7.94 (d, J = 8.4 Hz,
2H, Ar-H), 7.89–7.85 (m, 2H, Ar-H), 7.82 (d, J = 9.0 Hz, 2H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H,
CH=CH), 7.64–7.62 (m, 1H, Ar-H), 7.59 (d, J = 7.8 Hz, 2H, Ar-H), 7.55–7.53 (m, 3H, Ar-H),
5.90–5.82 (m, 1H, CH2-CH=CH2), 5.13 (d, J = 10.8 Hz, 1H, CH=CH2 ), 4.84 (d, J = 16.8 Hz, 1H,
CH=CH2), 4.64 (d, J = 4.8 Hz, 2H, CH2-CH), 4.35 (s, 2H, S-CH2); 13C NMR (151 MHz, DMSO-
d6) δ (ppm): 187.95, 166.84, 156.10, 152.51, 151.59, 148.49, 143.71, 142.45, 138.39, 135.45,
134.24, 134.13, 133.02, 132.98, 132.63, 131.09, 130.98, 130.53, 130.19, 129.43, 129.42, 128.08,
127.77, 125.65, 125.32, 123.25, 120.14, 119.07, 118.01, 47.21, 38.30; MS (FAB) m/z 642.1
(M+H)+; HRMS (FAB). Calcd for C37H29O2N5ClS: 642.1730. Found: 642.1730.
4.1.1.3.
2-(5-(2-(4-Chlorophenyl)quinolin-4-yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-
((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (7c).
Pale yellow solid (72%); m.p. 281-282 oC; IR (FT-IR, cm-1): 3330 (NH), 1697 (C=O), 1660 (CO-
NH); 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.84 (s, 1H, NH), 8.21 (d, J = 8.4 Hz, 2H, Ar-H),
8.12–8.08 (m, 5H, Ar-H), 7.99 (d, J = 15.6 Hz, 1H, CH=CH), 7.94 (d, J = 8.4 Hz, 2H, Ar-H), 7.84–
7.81 (m, 3H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H, CH=CH), 7.63 (t, J = 8.4 Hz, 1H, Ar-H), 7.60 (d, J
= 8.4 Hz, 2H, Ar-H), 7.54 (d, J = 8.4 Hz, 2H, Ar-H), 7.52–7.50 (m, 2H, Ar-H), 7.46 – 7.42 (m,
3H, Ar-H), 4.40 (s, 2H, S-CH2); 13C NMR (151 MHz, DMSO-d6) δ (ppm): 187.94, 166.65, 154.46,
152.57, 152.28, 148.21, 143.74, 142.44, 137.13, 137.03, 135.45, 134.25, 133.92, 133.54, 132.99,
131.13, 130.98, 130.53, 130.25, 130.01, 129.47, 129.42, 129.20, 128.19, 127.77, 126.00, 125.08,
26

123.25, 120.78, 119.05, 37.50; MS (FAB) m/z 712.0 (M+H)+; HRMS (FAB). Calcd for
C40H28O2N5Cl2S: 712.1341. Found: 712.1335.
4.1.1.4. 2-(4-Allyl-5-(2-(4-chlorophenyl)quinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-
(4-chlorophenyl)acryloyl)phenyl)acetamide (7d).
Off-white solid (78%); m.p. 255-256 oC; IR (FT-IR, cm-1): 3244 (NH), 1698 (C=O), 1658 (CO-
NH); 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.82 (s, 1H, NH), 8.34 (d, J = 9.0 Hz, 2H, Ar-H),
8.32 (s, 1H, Ar-H), 8.22–8.19 (m, 3H, Ar-H), 7.99 (d, J = 15.6 Hz, 1H, CH=CH), 7.94 (d, J = 8.4
Hz, 2H, Ar-H), 7.90–7.84 (m, 2H, Ar-H), 7.82 (d, J = 9.0 Hz, 2H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H,
CH=CH), 7.66–7.63 (m, 3H, Ar-H), 7.54 (d, J = 8.4 Hz, 2H, Ar-H), 5.87–5.80 (m, 1H, CH2-
CH=CH2), 5.11 (d, J = 10.5 Hz, 1H, CH=CH2 ), 4.83 (d, J = 17.2 Hz, 1H, CH=CH2), 4.64 (d, J =
13
4.2 Hz, 2H, CH2-CH), 4.35 (s, 2H, S-CH2); C NMR (151 MHz, DMSO-d6) δ (ppm): 187.46,
166.34, 154.42, 151.93, 151.13, 147.92, 143.22, 141.97, 136.71, 134.99, 134.13, 133.84, 133.76,
132.53, 132.10, 130.88, 130.73, 130.49, 130.04, 129.69, 128.98, 128.93, 128.27, 125.18, 124.92,
122.75, 119.59, 118.58, 117.57, 46.70, 37.79; MS (FAB) m/z 676.4 (M+H)+; HRMS (FAB). Calcd
for C37H28O2N5Cl2S: 676.1341. Found: 676.1316.
4.1.1.5. 2-(5-(2-(4-Methoxyphenyl)quinolin-4-yl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-
((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (7e).
Pale yellow solid (68%); m.p. 281-282 oC; IR (FT-IR, cm-1): 3310 (NH), 1697 (C=O), 1657 (CO-
NH); 1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.84 (s, 1H, NH), 8.21 (d, J = 8.4 Hz, 2H, Ar-H),
8.09 (d, J = 7.8 Hz, 1H, Ar-H), 8.05 (d, J = 8.4 Hz, 1H, Ar-H), 8.02 (d, J = 9.6 Hz, 3H, Ar-H),
7.99 (d, J = 15.6 Hz, 1H, CH=CH), 7.95 (d, J = 8.4 Hz, 2H, Ar-H), 7.83 (d, J = 9.0 Hz, 2H, Ar-
H), 7.78 (t, J = 7.8 Hz, 1H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H, CH=CH), 7.58 (t, J = 7.8 Hz, 1H, Ar-
H), 7.55 (d, J = 8.4 Hz, 2H, Ar-H), 7.52 (d, J = 8.4 Hz, 2H, Ar-H), 7.47–7.42 (m, 3H, Ar-H), 7.07
27

(d, J = 9.0 Hz, 2H, Ar-H), 4.39 (s, 2H, S-CH2), 3.84 (s, 3H, OCH3); 13C NMR (151 MHz, DMSO-
d6) δ (ppm): 187.95, 166.67, 161.41, 155.36, 155.34, 152.46, 152.42, 148.30, 143.74, 142.43,
135.45, 134.25, 133.61, 133.47, 132.99, 130.97, 130.85, 130.69, 130.53, 130.24, 129.78, 129.41,
128.95, 127.79, 127.47, 125.94, 124.68, 123.26, 120.49, 119.05, 114.81, 55.83, 37.51; MS
(FAB) m/z 707.9 (M+H)+; HRMS (FAB). Calcd for C41H31O3N5ClS: 708.1836. Found: 708.1846.
4.1.1.6.
2-(4-Allyl-5-(2-(4-methoxyphenyl)quinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-
((E)-3-(4-chlorophenyl)acryloyl)phenyl)acetamide (7f).
White solid (95%); m.p. 235-236 oC; IR (FT-IR, cm-1): 3163 (NH), 1689 (COCH3), 1660 (CONH);
1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.82 (s, 1H, NH), 8.28 (d, J = 8.4 Hz, 2H, Ar-H)), 8.23
(s, 1H, Ar-H), 8.21 (d, J = 8.4 Hz, 2H, Ar-H), 8.15 (d, J = 8.4 Hz, 1H, Ar-H), 7.98 (d, J = 15.6 Hz,
1H, CH=CH), 7.94 (d, J = 8.4 Hz, 2H, Ar-H), 7.84–7.80 (m, 4H, Ar-H), 7.73 (d, J = 15.6 Hz, 1H,
CH=CH), 7.58 (t, J = 8.4 Hz, 1H, Ar-H), 7.54 (d, J = 8.4 Hz, 2H, Ar-H), 7.13 (d, J = 8.4 Hz, 2H,
Ar-H), 5.89–5.83 (m, 1H, CH2-CH=CH2), 5.13 (d, J = 10.2 Hz, 1H, CH=CH2), 4.84 (d, J = 17.4
Hz, 1H, CH=CH2), 4.63 (d, J = 4.2 Hz, 2H, CH2-CH), 4.35 (s, 2H, S-CH2), 3.87 (s, 3H, O-CH3);
13C NMR (151 MHz, DMSO-d6) δ (ppm): 187.52, 166.35, 161.01, 157.94, 155.31, 152.13, 150.98,
148.03, 141.93, 134.96, 133.78, 133.50, 133.48, 132.12, 130.87, 130.46, 130.00, 129.47, 128.92,
128.77, 128.25, 127.08, 127.02, 125.08, 124.50, 119.26, 118.63, 117.57, 114.37, 55.36, 46.71,
37.88; MS (FAB) m/z 672.2 (M+H)+; HRMS (FAB). Calcd for C38H31O3N5ClS: 672.1836. Found:
672.1854.
4.1.1.7.
2-(4-Phenyl-5-(2-phenylquinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
methoxyphenyl)acryloyl)phenyl)acetamide (8a).
White solid (89%); m.p. 275-276 oC; IR (FT-IR, cm-1): 3176 (NH), 1693 (C=O), 1655 (CO-NH);
1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.85 (s, 1H, NH), 8.19 (d, J = 8.4 Hz, 2H, Ar-H), 8.14
28

(d, J = 8.4 Hz, 1H, Ar-H), 8.11 (d, J = 8.4 Hz, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 8.04 (d, J = 8.4 Hz,
2H, Ar-H), 7.86 (d, J = 8.4 Hz, 2H, Ar-H), 7.84–7.81 (m, 4H, Ar-H), 7.72 (d, J = 15.6 Hz, 1H,
CH=CH), 7.62 (t, J = 7.8 Hz, 1H, Ar-H), 7.54–7.47 (m, 5H, Ar-H+CH=CH), 7.45–7.40 (m, 3H,
Ar-H), 7.04 (d, J = 8.4 Hz, 2H, Ar-H), 4.39 (s, 2H, S-CH2), 3.84 (s, 3H, OCH3); 13C NMR (151
MHz, DMSO-d6) δ (ppm): 187.96, 166.62, 161.78, 155.69, 152.54, 152.33, 148.30, 143.92,
143.47, 138.26, 133.68, 133.61, 133.38, 131.86, 131.16, 130.99, 130.47, 130.30, 130.01, 129.40,
127.98, 127.90, 127.81, 127.47, 126.01, 125.01, 120.88, 119.98, 119.04, 114.90, 114.14, 55.87,
37.51; MS (FAB) m/z 673.9 (M+H)+; HRMS (FAB). Calcd for C41H32O3N5S: 674.2226. Found:
674.2241.
4.1.1.8.
2-(4-Allyl-5-(2-phenylquinolin-4-yl)-4H-1,2,4-triazol-3-ylthio)-N-(4-((E)-3-(4-
methoxyphenyl)acryloyl)phenyl)acetamide (8b).
White solid (90%); m.p. 205-207 oC; IR (FT-IR, cm-1): 3161 (NH), 1689 (C=O), 1654 (CO-NH);
1H NMR (600 MHz, DMSO-d6) δ (ppm): 10.79 (s, 1H, NH), 8.31 (d, J = 8.4 Hz, 2H, Ar-H), 8.29
(s, 1H, Ar-H), 8.20 (d, J = 8.4 Hz, 1H, Ar-H), 8.18 (d, J = 9.0 Hz, 2H, Ar-H), 7.88 (d, J = 8.4 Hz,
2H, Ar-H), 7.85 (d, J = 9.0 Hz, 2H, Ar-H), 7.83–7.80 (m, 3H, Ar-H+CH=CH), 7.72 (d, J = 15.6
Hz, 1H, CH=CH), 7.63 (t, J = 7.8 Hz, 1H, Ar-H), 7.60–7.56 (m, 2H, Ar-H), 7.54 – 7.53 (m, 1H,
Ar-H), 7.03 (d, J = 8.4 Hz, 2H, Ar-H), 5.89–5.83 (m, 1H, CH2-CH=CH2), 5.13 (d, J = 10.2 Hz,
1H, CH=CH2), 4.84 (d, J = 17.4 Hz Hz, 1H, CH=CH2), 4.65 (d, J = 4.8 Hz, 2H, CH2-CH), 4.35 (s,
2H, S-CH2), 3.84 (s, 3H, OCH3); 13C NMR (151 MHz, DMSO-d6) δ (ppm): 187.96, 166.79,
161.78, 156.11, 152.51, 151.59, 148.49, 143.93, 143.42, 138.39, 134.14, 133.42, 132.62, 131.16,
131.10, 130.52, 130.31, 130.19, 129.44, 128.09, 127.89, 127.77, 125.65, 125.32, 120.14, 119.96,
119.06, 118.00, 114.90, 55.87, 47.21, 38.32; MS (FAB) m/z 638.2 (M+H)+; HRMS (FAB). Calcd
for C38H32O3N5S: 638.2226. Found: 638.2246.
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