Synthesis, Antiproliferative and Cytotoxic Activities, DNA Binding Features and Molecular Docking Study of Novel Enamine Derivatives

Article abstract of DOI:10.1002/cbdv.202000139

Novel enamine derivatives were synthesized from the reaction of lactone and chalcones and their antiproliferative and cytotoxic activities against six cancer cell lines (e. g., HeLa, HT29, A549, MCF7, PC3 and Hep3B) and one normal cell lines (e. g., FL) were investigated along with their mode of interactions with CT-DNA. Most of the enamine derivatives with IC₅₀ values of 86–168 μM demonstrated much stronger antiproliferative activity than the starting molecules against the cancer cells. While, among the enamine derivatives, four compounds displayed higher cytotoxic potency than the control drugs (5-fluorouracil and cisplatin) against the Hep3B cell lines, these compounds did not exhibit any significant toxicity against normal cells, FL. The UV/VIS spectral data suggest that eight compounds cause hypochromism with a slight bathochromic shift (～6 nm), indicating that they bind to the DNA by way of an intercalative or minor groove binding mode. The binding constants of the compounds are in the range of 0.1×103 M^?1–2.3×104 M^?1. The antiproliferative activity of studied enamine derivatives could possibly be due to their DNA binding as well as their cytotoxic properties. In addition to these assays, the chalcones and enamine derivatives were investigated by molecular docking to calculate the synergistic effect of antiproliferative activities against six human cancer cell lines.

Full text of DOI:10.1002/cbdv.202000139

Title: Synthesis, antiproliferative, cell cytotoxicity activity, DNA
binding features and molecular d ocking study of novel enamine
derivatives
Authors: meliha Burcu gürdere, Ali Aydın, belkız yencilek, Fatih Ertürk,
oğuz özbek, Sultan Erkan, Yakup BUDAK, and Mustafa
Ceylan
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000139
Link to VoR: https://doi.org/10.1002/cbdv.202000139

10.1002/cbdv.202000139
Chemistry & Biodiversity
Synthesis, antiproliferative, cell cytotoxicity activity, DNA binding
features and molecular docking study of novel enamine derivatives
Meliha Burcu GÜRDEREa*, Ali AYDINb, Belkız YENCİLEKa, Fatih ERTÜRKc, Oğuz ÖZBEKa, Sultan ERKANd, Yakup BUDAKa,
Mustafa CEYLANa
aDepartment of Chemistry, Faculty of Arts and Sciences, Tokat Gaziosmanpaşa University, 60250 Tokat, Turkey
b Department of Basic Medical Science, Faculty of Medicine, Yozgat Bozok University, 66100, Yozgat, Turkey
cVocational School, Occupational Health and Safety Programme, Istanbul Arel University,34100, Istanbul,Turkey
dChemistry and Chemical Processing Technologies, Yıldızeli Vocational School, Sivas Cumhuriyet University 58140 Sivas/Turkey
Corresponding author: e-mail: burcugurdere@gmail.com
Novel enamine derivatives (7a-o) were synthesized from the reaction of lactone (6) and chalcones (3a-
o) and their antiproliferative and cell cytotoxicity activities against six cancer cell lines (e.g., HeLa,
HT29, A549, MCF7, PC3 and Hep3B) and one normal cell line (e.g., FL) were investigated along with
their mode of interactions with CT-DNA. Most of the enamine derivatives (7a-o) with 86-168 µM IC50
values demonstrated much stronger antiproliferative activity than the starting molecules (3a-o) against
the cancer cells. While, among the enamine derivatives, the compounds 7e, 7f, 7k and 7l displayed
higher cytotoxic potency than the control drugs (5-fluorouracil and cisplatin) against the Hep3B cell
line, these compounds did not exhibit any significant toxicity against normal cells, FL. The UV-Vis
spectral data suggest that the compounds 7c, 7e, 7i, 7l, 7m, 3d, 3i and 3o cause hypochromism with a
slight bathochromic shift (~6 nm), indicating that they bind to the DNA by way of an intercalative or
minor groove binding mode. The binding constants of the compounds are in the range of 0.1 × 103 M-
1-2.3 × 104 M-1. The antiproliferative activity of studied enamine derivatives could possibly be due to
their DNA binding as well as their cytotoxic properties. In addition to these assays, compounds 3a-3o
and 7a-7o were investigated by molecular docking to calculate the synergistic effect of antiproliferative
activities against six human cancer cell lines.
Keywords: Lactone, chalcone, enamine, antiproliferative activity, cytotoxicity activity, molecular
docking
Introduction
Cancer prevalence is an increasing problem for human health and life.[1] Cancer is a complex
disease that varies from one patient to another in terms of manifestation, progression and
outcomes. Cancer was the 7th-8th in the order of diseases leading to death at the beginning of
the century; but, today it is second cause of death following cardiovascular diseases in many
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10.1002/cbdv.202000139
Chemistry & Biodiversity
countries.[2] Treatment methods commonly used in cancer are surgery, radiotherapy and
chemotherapy.[3] In addition to these standard treatments, hormone treatments, biological
treatment methods and targeted therapies are used less frequently. It is not realistic to talk about
the existence of a single definitive treatment method; because, each method has its own
advantages and disadvantages; with cancer being a highly heterogeneous disease, treatment
options can vary from patient to patient.[4] The development of anticancer agents with better
treatment efficiency and fewer clinical side effects have attracted increased attention of the
medicinal chemists as the number of cancer patients is growing rapidly.[5]
Lactones, widely found in nature, have received considerable attention because of their
interesting pharmacological activities.[6] Especially, -methylene--butyrolactones have
displayed anticancer properties.[7-10] In addition, enamines obtained from the reaction of
aldehydes and ketones with secondary amines are important compounds with strong biological
properties such as antibacterial,[11] anticolvunsant, anti-inflammatory,[12] and anticancer
effects.[13] Moreover, natural and synthetically obtainable chalcones are among the most
frequently studied compounds during the last 50 years due to their wide range of biological
activities. The chalcone derivatives have been reported to have potent anticancer activity with
low side effects and better solubility for therapeutic applications.[14-18] The studies mentioned
above have shown us that derivatives of chalcones and lactones show excellent therapeutic
effects in the treatment of many diseases, especially cancer. In addition, these derivatives have
attracted the attention of researchers due to their broad antitumor spectrum, low toxicity values
against non-cancer cells and immune-boosting effects. In addition, these publications have
demonstrated the molecular mechanisms of anti-tumor potential of chalcones and lactones
derivatives and have shown that these derivatives can inhibit angiogenesis, induce caspase-
dependent apoptotic cell death, and regulate the expression of pro-apoptotic proteins. Therefore,
it is of high importance to design and synthesize a hybrid molecule (enamine) consisting of a
series of chalcones and lactones and to examine the mechanisms of action of these novel
molecules on the tumor cells.
Considering the biological importance of lactones and chalcones, we have designed and
synthesized a series of new hybrid molecules (enamine derivatives) by bringing the lactone and
the chalcone units together with an enamine bridge. Moreover, all the synthesized compounds
were evaluated for their antiproliferative activities against six human cancer cell lines (HeLa
(cervical cancer), HT29 (colorectal adenocarcinoma), A549 (lung carcinoma), MCF7 (breast
adenocarcinoma), PC3 (prostate adenocarcinoma), Hep3B (hepatocellular carcinoma) and
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10.1002/cbdv.202000139
Chemistry & Biodiversity
against a non-cancer cell line, FL (normal amnion epithelial cell). Among them, five
compounds, 7c, 7d, 7e, 7f and 7h displayed higher antiproliferative activity compared to the
control drugs, 5-Fluorouracil and Cisplatin. In addition, molecular docking study was carried
out to understand the binding interactions of the compounds. The binding energies between the
target proteins and the studied compounds were shown to be parallel with the experimental
results. We observed that the new enamine derivatives synthesized in the current study initiated
apoptosis in cancer cells. Enamine derivatives may possibly lead to cancer cell death in two
ways. First, they may activate the apoptotic genes by directly binding to DNA. Our DNA
binding experiment showed that these molecules can bind to DNA as much as control agents.
Secondly, they may activate apoptotic proteins present in the cytoplasm. Molecular docking
studies have shown that enamine derivatives can interact with several proteins (sirtuin 1,
psathyrella asperospora, epidermal growth factor receptor, and fibroblast growth factor 19)
related to apoptosis and cell cycle regulation.
Results and Discussion
Chemistry
4-Amino-chalcone derivatives (3a-o) were synthesized from the Claisen-Schmidt condensation
of the benzaldehyde derivatives (1a-o) with 4-aminoacetophenone (2) according to the
[19-22]
published procedure
(Scheme 1). In the case of 4-substituted benzaldehyde (1k-n) (-
methyl, -methoxy, -bromo and -chloro), the related Schiff-base derivatives were the main
products in these reactions. Due to this situation, the 4-substituted chalcone derivatives (3k-n)
were obtained by a different protocol. Firstly, the nitro-chalcone derivatives (5k-n) were
synthesized by the addition of the benzaldehyde derivatives (1k-n) to 4-nitroacetophenone (4).
Then, the reduction of the nitro group to amino group with SnCl2 gave the 4-substituted
chalcone derivatives (3k-n) (Scheme 1).
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4-Aminochalcones (3a-o) were reacted with lactone (6) in toluene in the presence of
AcOH to obtain lactone-enamine derivatives (7a-o) in the reflux conditions for 24 hours.
However, the yield of p-substituted lactone enamines (7k-n) was very low. For the synthesis of
the enamines (7k-n) in higher yields, the lactone enamine 8 was obtained from the reaction of
4-aminoacetophenone (2) with lactone (6), followed by the addition of p-substituted
benzaldehydes (1k-n) in the basic medium leading to p-substituted lactone enamines (7k-n) in
good yields (Scheme 2, Table 1).
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10.1002/cbdv.202000139
Chemistry & Biodiversity
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10.1002/cbdv.202000139
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Antiproliferative studies
Inhibition of cellular proliferation
The antiproliferative effects of the chalcones (3a-o) and enamine derivatives (7a-o) were
assayed against human cancer cell lines; HeLa (cervical cancer), HT29 (colorectal
adenocarcinoma), A549 (lung carcinoma), MCF7 (breast adenocarcinoma), PC3 (prostate
adenocarcinoma), Hep3B (hepatocellular carcinoma) and a non-cancer cell line, FL (normal
amnion epithelial cell). In these tests, cisplatin and 5-fluorouracil (5-FU) were used as reference
anticancer drugs. IC50 values were calculated via Excel software with the use of absorbance
data in treated and control cells and the results were given in Table 2.
According to IC50 values, all chalcone derivatives (3a-o) showed higher activity than 5-
FU against all cell lines. Compounds 3e, 3i and 3o exhibited lower activity compared to
Cisplatin, the others displayed higher activity than Cisplatin against HeLa cell line. Compounds
3d, 3g, 3h, 3j, 3m and 3n demonstrated higher activity, the others showed lower activity than
Cisplatin against HT29 cell line. While compounds 3a and 3i showed almost the same activity
with Cisplatin, the other compounds exhibited higher activity relative to Cisplatin against A549
cell line. All compounds (3a-o) showed higher activity compared to Cisplatin against MCF7
cell line. Compounds 3e-g, 3l and 3m displayed higher activity, the others demonstrated lower
activity than Cisplatin against PC3 cell line. Except compounds 3i, 3j and 3o, all the other
compounds exhibited higher activity than Cisplatin against Hep3B cell lines The addition of
compound 3d resulted in higher activity compared to reference chemotherapeutic agents
(Cisplatin and 5-FU) against all the cell lines. Among the most active chalcone derivatives (3a-
o) towards HeLa, HT29, A549, MCF7, PC3 and Hep3B cells were compounds 3b (IC50
=
110.36 µM), 3g (IC50 = 94.91 µM), 3m (IC50 = 49.24 µM), 3d (IC50 = 67.15 µM), 3m (IC50 =
72.79 µM) and 3m (IC50 = 59.21 µM), respectively.
Among the enamine derivatives (7a-o), while compounds 7k and 7l showed lower
activity than 5-FU against HeLa cell line, all the other compounds exhibited higher activity than
5-FU against all cell lines. Except compounds 7b and 7k-n, the other compounds displayed
higher activity compared to Cisplatin against HeLa cell line. Except compounds 7a, b and 7k-
n, all the other compounds displayed higher activity than Cisplatin against HT29 cell line.
Except compounds 7b and 7j-n, all the other compounds displayed higher activity compared to
Cisplatin against A549 cell line. Except compounds 7b, 7h and 7k-m, all the other compounds
demonstrated higher activity relative to Cisplatin against MCF7 cell line. While the compounds
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10.1002/cbdv.202000139
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7c-i and 7o resulted in higher activity than Cisplatin against PC3 cell line, the activity of other
compounds was lower relative to Cisplatin. All compounds exhibited higher activity than
Cisplatin against Hep3B cell line. Addition of the compounds 7c-g led to higher activity
compared to standards (Cisplatin and 5-FU) against all cell lines. Among the enamine
derivatives (7a-o), the most active compounds towards HeLa, HT29, A549, MCF7, PC3 and
Hep3B cells were determined as 7d (IC50 = 80.54 µM), 7d (IC50 = 102.03 µM), 7h (IC50 = 99.47
µM), 7o (IC50 = 80.81 µM), 7d (IC50 = 69.92 µM) and 7g (IC50 = 44.31 µM), respectively.
When the chalcones (3a-o) and the enamine derivatives (7a-o) were compared, the
chalcone derivatives 3b and 3k-n showed higher activity than the enamine derivatives (7b and
7k-n) against all cancer cell lines except Hep3B. All enamine derivatives (7a-o) except 7m
exhibited higher activity than their corresponding chalcones (3a-o) against Hep3B cell line.
Moreover, compounds 7a, 7c-j and 7o displayed higher activity than the corresponding
chalcone derivatives against the Hela cancer cell lines. While the chalones 3a and 3g,h showed
higher activity than enamines 7a and 7g,h against HT29 cell line, the enamines 7c-e, 7i,j and
7o showed higher activity than chalcones 3c-e, 3i,j and 3o. In addition, the enamines 7a, 7c,
7e-h and 7o were more active than the chalcones 3a, 3c, 3e-h and 3o against A549 cancer cell
lines. However, the enamines 7a, 7c, 7f-g, 7i,j and 7o had more activity than the chalcones 3a,
3c, 3f-g, 3i,j and 3o against MCF7 cancer cell lines. The enamines except 7b and 7k-n
exhibited considerably higher activity compared to chalcones against PC3 cancer cell lines.
Generally, most of the enamines (except 7a, 7k-n) displayed higher activity than the chalcones
against especially Hela, PC3 and Hep3B cancer cell lines. The results significantly highlight
that the enamine derivatives (7a-o) may possibly act as selective antiproliferative agents for
cancer treatment, especially against HeLa, PC3 and Hep3B cancer cell lines.
Furthermore, normal cells (FL cell line) were more sensitive to chalcone derivatives
(3a-o) (IC50 = ~ 20-100 µM) with exceptions of the 7a, 7i, 7j and 7o, compared to the cancer
cells. These results point to the fact that enamine derivatives (7a-o) may possibly act as
selective antiproliferative agents which can be evaluated for their use in the treatment of cancer.
Antiproliferative activity of enamine derivatives (7a-o) was similar between normal FL
cells and cancer cells (Table 2). However, FL cells may have possibly lower mitochondrial
activities than cancer cells and the MTT assay measures only mitochondrial activity of live
cells. It is based on the principle that FL cells have decreased mitochondrial activity and are
possibly exposed to increased antiproliferative effect. LDH (lactate dehydrogenase) based
cytotoxicity assays were also used in addition to MTT Assay to overcome these limitations.
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Table 2. IC50 (μM) values of the novel compounds containing lactone, chalcone and enamine in a single
structure in human cell lines
HeLa
HT29
183.27
147.07
168.21
120.76
152.72
173.33
94.91
A549
217.57
155.54
180.73
100.15
189.39
143.57
133.33
126.71
208.46
156.67
150.44
129.84
49.24
MCF7
165.23
91.31
PC3
146.81
121.15
136.83
100.31
106.28
109.61
126.68
151.17
226.56
150.84
116.81
101.65
72.79
Hep3B
146.31
114.03
141.41
63.52
FL
130.21
74.50
3a
3b
3c
3d
3e
3f
135.65
110.36
138.41
114.48
192.54
149.58
157.79
138.43
204.23
154.38
120.73
117.88
132.00
155.07
194.02
118.21
558.56
107.78
80.54
114.72
67.15
107.85
64.35
98.32
97.20
98.98
121.26
152.13
173.51
177.17
156.76
91.52
101.43
147.40
143.62
200.86
177.76
151.49
119.06
59.21
101.89
66.78
3g
3h
3i
101.53
201.89
133.60
168.94
142.00
126.57
124.89
160.00
326.55
383.32
135.02
102.03
114.56
110.37
116.78
112.37
124.26
107.07
446.43
408.49
404.45
382.87
112.81
77.60
144.91
111.03
61.65
3j
3k
3l
71.69
60.95
3m
3n
3o
7a
7b
7c
7d
7e
7f
78.77
44.58
109.57
143.78
142.94
332.14
113.04
113.23
129.97
124.63
101.96
99.47
167.88
186.30
91.33
134.26
178.41
100.28
412.48
95.37
134.12
171.69
56.61
27.83
110.33
95.47
432.91
94.46
101.98
60.95
259.63
107.37
86.45
93.55
69.92
60.23
109.15
119.13
104.20
120.79
127.29
114.86
570.93
516.16
542.88
462.79
126.60
100.50
102.67
87.10
74.46
45.29
112.79
103.40
82.78
76.59
45.11
7g
7h
7i
92.84
44.31
401.81
100.08
106.07
108.51
473.59
417.06
408.87
80.81
109.63
136.01
121.79
318.93
436.99
327.05
317.35
93.13
56.07
111.91
102.30
93.41
376.62
455.90
484.24
481.10
382.07
311.31
100.64
147.52
131.82
123.05
93.69
7j
7k
7l
421.63
407.36
307.75
102.11
74.74
7m
7n
7o
76.25
50.37
98.14
Cisplatin
5FU
169.99
476.62
136.66
507.38
203.32
538.13
213.32
576.56
109.99
530.44
163.32
484.31
176.66
461.25
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Cytotoxic activity of the compounds
The cytoplasmic lactate dehydrogenase (LDH) released to the medium from damaged plasma
membranes is a marker of an increased cell cytotoxicity. Thus, cell cytotoxicity can be
indirectly estimated by measuring the amount of lactate dehydrogenase in the medium with
Roche’s cytotoxicity detection kit. The cytotoxic activities of the enamine derivatives (7a-o)
and the chalcones (3a-o) were evaluated after the overnight treatment of cells with the IC50
concentrations of the compounds. Compounds 7b and 7k-m exhibited over 50% cytotoxicity
against normal FL cells, while all other compounds 3a-o and 7a-o showed cytotoxicity in the
range of 4% to 38% (Table 3). Some of the enamine derivatives (7a, 7c-j and 7o) in the LDH
assay were shown to have strong antiproliferative effects on cancer cells (IC50 ~ 44.31-147.52
µM), especially in PC3 and Hep3B cells, and were non-toxic to normal FL cells.
Comparison of MTT and LDH assays demonstrated that 7c exhibited higher optimal
antiproliferative and cytotoxic effects in comparison with both its structural isomer 3c and the
others against cancer and normal cells (Table 2 and 3). The fact that 7c is a significantly potent
antiproliferative compound with IC50 values of 107.78, 135.02, 113.04, 94.46, 95.37, and 60.95
µM towards the growth of HeLa, HT29, A549, MCF7, PC3, and Hep3B cells, respectively,
implies that 7c may be selected as a candidate for further preclinical trials. Tables 2 and 3 also
indicate that the compounds 7d and 7e (IC50 = 80.54, 109.15 µM, respectively), the compounds
7a and 7i (IC50 = 91.33, 100.08 µM, respectively), and the compounds 7d and 7h (IC50 = 69.92,
109.63 µM, respectively) probably displayed strong antiproliferative effects against HeLa,
MCF7 and PC3 cells, respectively, but were not cytotoxic to FL normal cells. This is an
indication that 7d and 7e, the 7a and 7i, and the 7d and 7h were quite toxic for HeLa, MCF7
and PC3 cell lines, respectively, at IC50 concentration, but were safe for FL normal cells,
therefore these compounds may show promise in further preclinical studies.
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Table 3. % Cytotoxicity of the novel compounds at IC50 concentrations
HeLa
28
HT29
29
A549
32
MCF7
23
PC3
42
Hep3B
13
12
10
8
FL
15
15
18
29
7
3a
3b
3c
3d
3e
3f
25
20
31
10
28
32
34
25
22
38
37
33
28
12
26
43
25
32
19
22
11
9
34
35
25
32
24
4
3g
3h
3i
37
28
22
62
14
38
34
29
36
17
7
15
5
34
25
33
>50
20
38
39
39
35
40
25
24
28
34
25
10
11
25
>50
31
32
36
34
25
38
25
28
>50
>50
>50
30
18
3j
30
41
27
17
29
3k
3l
27
24
32
9
29
41
32
34
11
25
3m
3n
3o
7a
7b
7c
7d
7e
7f
31
30
18
12
10
9
45
36
34
27
32
29
33
20
9
43
26
29
25
35
20
>50
>50
50
27
28
33
>50
36
>50
40
>50
38
>50
35
8
33
27
22
32
33
7
45
26
25
25
26
6
42
20
20
40
32
4
7g
7h
7i
39
47
23
23
25
6
50
34
11
>50
32
39
4
42
34
>50
>50
>50
>50
>50
>50
34
39
20
18
8
7j
41
43
40
33
7k
7l
>50
>50
>50
>50
39
>50
>50
>50
>50
29
34
>50
>50
>50
>50
21
>50
>50
>50
20
10
4
7m
7n
7o
7
15
Cell morphology
The effects of enamine derivatives (7a-o) on the HeLa, HT29 and MCF7 cells were investigated
morphologically and the images of the treated cells compared to untreated control cells were
analyzed. The images in Figures 1-3 indicate that untreated control cells displayed a normal
cell morphology resembling those of fibroblasts or epithelial cells. On the other hand, the
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images obtained via phase contrast microscopy demonstrate that these compounds caused cell
detachment from the surface of the flask in a dose dependent manner (Figure 1-3). Moreover,
HeLa, HT29 and MCF7 cells exposed to enamine derivatives (7a-o) displayed some certain
morphological changes at middle and high concentrations (25-50 µg/mL) including cell
shrinkage, cytoplasmic blebs and spikes, formation of abnormal globular structures and
apoptotic bodies, as well as cell rounding and floating (this indicates that the cells are dead)
(Figure 1-3). In general, concentrations of 37.5 µg/mL and above resulted in cell separation
together with insufficient adhesion leading to smaller cells and decreased cell numbers.
Figure 1. The analysis of cellular morphology of HeLa cells following treatments with compounds 7a,7b-7k and
3a,3b-3k. Morphological changes were observed under the phase-contrast microscopy following treatments at
concentrations 15, 20, 25, 37.5 and 50 µg/mL of 7a,7b-7k and 3a,3b-3k for 24 h.
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In addition, the weak confluency, lower cell size and abnormal cellular morphology
implied that reduced cell viability resulted in apoptosis. In low concentrations (< 20 µg/mL),
majority of the treated cells maintained their astrocyte-like or normal fibroblast-like appearance
similar to the control cells. Treatment of cells with the compounds at higher concentrations (>
37.5 µg/mL) led to cell morphologies similar to those of apoptotic cells as well as cells with
cellular damage observed in affected growth (Figure 1-3). We also observed that the images at
the bottom display some crystals grown from test compounds after which they were added to
the cell medium. However, the interaction between compounds and fetal bovine serum or other
components in the cell medium may cause the formation of these crystals.
Figure 2. Analysis of cellular morphology of HT29cells after 7a,7b-7k and 3a,3b-3k treatments. Morphological
changes were observed under the phase-contrast microscopy following treatments at concentrations 15, 20, 25,
37.5 and 50 µg/mL of 7a,7b-7k and 3a,3b-3k for 24 h.
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Figure 3. Analysis of cellular morphology of MCF7 cells after treatments with compounds 7a,7b-7k and 3a,3b-
3k Morphological changes were observed under the phase-contrast microscopy after treating cells with
concentrations 15, 20, 25, 37.5 and 50 µg/mL of 7a,7b-7k and 3a,3b-3k for 24 h.
Spectral analysis of the compounds-DNA interactions
The mode of interactions of compounds with DNA has been evaluated by using a
spectrophotometric method which compares UV-Vis absorption spectra of the free compound
and compound-DNA adducts. Figure 4 and 5 depict the interaction of these compounds with
CT-DNA. UV-spectra of the compounds in Tris–HCl buffer at pH 7.4 were measured
separately. One intensive peak was observed in the UV-spectra of 3a, 3b, 3d, 3i, 3k, 3l, and 3o
at ca 365 nm and the less intensive peak observed for 3e, 3f, 3g, 3j, 3m, and 3n at ca 365 nm.
3c exhibited two peaks at ca 365 nm and 320 nm. However, 7b and 7l displayed fewer intensive
peaks at ca 365, 370 nm, respectively. 7k, 7c, 7d, 7e, 7f, 7i, 7j, 7m, and 7o displayed one
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10.1002/cbdv.202000139
Chemistry & Biodiversity
intensive peak at ca 355, 370, 360, 385, 370, 385, 380, 375, and 355 nm, respectively.
Interestingly, we observed two peaks for 7a, 7g, 7h, and 7n at ca 390 and 315 nm. The spectra
of the compounds were determined in the range of 50-250 µM of DNA at 50 µM concentration
of the compound at physiological pH. The spectral alterations marked the formation of the
compound-DNA adduct.
Table 4. UV–Vis absorption data of 7a-o and 3a-o in the absence and presence of ct DNA.
Kb (M−1)
4.1 x 103
0.7 x 103
2.3x 103
1.8 x 103
1.1 x 104
1.7 x 104
0.8 x 103
2.1 x 104
4.0 x 103
1.7 x 104
8.3x 103
0.1 x 103
1.2 x 104
1.4 x 104
3.6 x 103
6.7 x 103
1.8 x 104
2.0 x 103
1.2 x 104
2.3 x 104
1.7 x 104
1.6 x 103
6.7 x 103
3.9 x 103
3.3 x 103
1.1 x 104
1.0 x 104
3.4 x 103
4.3 x 103
5.8 x 103
Hypochromicity (%)
Hyperchromicity (%)
3a
3b
3c
3d
3e
3f
-
29.16
21.91
17.92
-
-
-
13.85
-
24.46
22.13
35.67
13.87
-
-
3g
3h
3i
-
-
18.86
3j
-
26.09
22.88
33.33
17.31
41.80
-
3k
3l
-
-
3m
3n
3o
7a
7b
7c
7d
7e
7f
-
-
29.00
-
10.51
20.12
-
-
23.72
-
28.29
-
19.00
-
8.63
1.44
16.18
-
7g
7h
7i
-
-
41.01
7j
-
2.61
35.64
-
7k
7l
-
30.00
7m
7n
7o
23.51
-
-
-
41.11
12.61
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10.1002/cbdv.202000139
Chemistry & Biodiversity
Adding increasing concentrations of DNA on fixed compound concentration led to a
gradual increase or decrease in the absorption intensity, resulting in a typical hyperchromicor
hypochromic effect, respectively (Table 4) (Figure 4 and 5). The hyperchromic effect due to
the unstacking of DNA may show the formation of stable compound-DNA complex via
electrostatic interaction or groove binding mode. However, hypochromic effect due to stacking
interactions in the DNA helix may display the formation of compound-DNA complex via the
intercalative binding mode. Thus, the absorption bands of 7c, 7e, 7i, 7l, and 7m exhibited
23.72%, 19.0%, 41.01%, 30.0%, 23.51%, and hypochromism, respectively, with a slight
bathochromic shift (~6 nm) (Table 4) (Figure 4 and 5). The absorption bands of 3d, 3i, and 3o
also measured 13.85%, 18.86%, and 29.0% hypochromism, respectively (Table 4). The rest of
7a-o and 3a-o caused the hyperchromic effect in their absorption bands (Table 4).
Figure 4. UV-Visible absorption spectra of 50 µM of each compound in the absence and presence of 50 μM (a),
100 μM (b), 150 μM (c), 200 μM (d) and 250 μM DNA (d). Note: The direction of arrow demonstrates the
increasing concentrations of DNA. Inner graph is the plot of A0/(A - A0) vs. 1/[DNA]to find the binding constant
of the complex-DNA adduct.
The binding constant (Kb) of the compounds–DNA adduct can be determined from the
alteration in absorption bands together with increasing concentrations of DNA. Benesi-
Hildebrand equation, A0/A-A0=G/H–G-G+G/H–G-Gx1/K[DNA] was used to calculate the
binding constants, where K is the binding constant, A0 and A represent the absorbance of each
compound and its adduct with DNA and G and H–G denote the absorption coefficients of the
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10.1002/cbdv.202000139
Chemistry & Biodiversity
compounds and the compound-DNA adducts. The binding constant can be obtained from the
intercept-to-slope ratio of A0/(A - A0) vs. 1/[DNA] plots (Table 4).
The Kb values of the compounds-DNA adduct are mostly consistent with the data
obtained using the MTT assay. The 7e depicted the strongest interactions with CT-DNA at
physiological pH. Overall, the DNA binding affinity (Kb) has this order: 7e > 3h > 7b > 7f = 3f
= 3j > 3n > 7d = 3m > 7k = 3e > 7l > 3k > 7h = 7a > 7o > 7n > 3a > 3i > 7i > 3o > 7m > 7j >
3c > 7c > 3d > 7g > 3g > 3b > 3l. Generally, the 7a-o compounds exhibited equal DNA binding
activity comparable to that of 3a-o. This is an indication that antiproliferative effects of 7a-o
could be estimated based on their different action mechanisms as well as their DNA binding
features. Kb value 5.73 × 104 M−1 and 9.7 × 104 M−1 of cis-platin and 5-FU, respectively, were
observed to be slightly greater than those of these compounds at the same pH.[23, 24]
Figure 5. UV-Visible absorption spectra of 50 µM of each compound in the absence and presence of
50 μM (a), 100 μM (b), 150 μM (c), 200 μM (d) and 250μM DNA (d). Note: The direction of arrow
demonstrates increasing concentrations of DNA. Inside graph is the plot of A0/(A - A0) vs. 1/[DNA]to
find the binding constant of the complex-DNA adduct.
Molecular Docking Analyses
The relationship between drugs and target proteins can be determined by molecular docking
analyses. The target protein for HeLa (cervical cancer), HT29 (colorectal adenocarcinoma),
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10.1002/cbdv.202000139
Chemistry & Biodiversity
A549 (lung carcinoma), MCF7 (breast adenocarcinoma), PC3 (prostate adenocarcinoma) and
Hep3B (hepatocellular carcinoma) cell lines were chosen as the following with their PDB ID:
4I5I,[25] 5MB4,[26] 1M17,[27] 3HY3,[28] 3V49[29] and 5VND,[30] respectively. Sirtuin-1 is one of
the histone deacetylase enzymes involved in the inactivation of p53 gene. However, the p53
gene produces a protein to kill the tumor cell. As cancer occurs, mutations in p53 gene occurs
and this produces high sirtuin-1 content.[31] Human colon cancer contains Psathyrella
asperospora with strong antiproliferative effects against HT29 cell. PAL is expressed on the
cell surface of terminal N-acetyl-D-glucosamine (GlcNAc) found on glycoconjugates. Since
terminal GlcNAc is a characteristic abnormal epitope of cancer cells, it is a very interesting
antigen to be targeted for diagnosis and treatment.[32-35] A combination of PAL sequence and
surface plasmon resonance (SPR) analysis is used for the amino acid sequence and three-
dimensional structure of PAL. An in-depth characterization of PAL is considered using a
structural basis for the specificity determined from the three-dimensional structure of the PAL
/ GlcNAc complex at amino acid sequence and solubility. Controlling cell life in the treatment
of lung cancer has identified genetic and regulatory deviations that suppress cell death, promote
cell division and induce tumorigenesis. One of these discoveries is the epidermal growth factor
receptor (EGFR). EGFR is a transmembrane receptor tyrosine kinase protein expressed in some
normal epithelial, mesenchymal and neurogenic tissues. Overexpression of EGFR has been
reported in cancer and has been involved in the pathogenesis of many human malignancies,
including NSCLC.[36,37] 1M17 is the crystal structure of the epidermal growth factor receptor
(EGFRK) containing forty amino acids from the carboxyl terminal tail of the kinase domain.[38]
Prostate Cancer (PCa) is the most commonly diagnosed cancer in men. PCa growth is
mainly due to activation of the androgen receptor by androgens. PCa treatment may include
surgery, hormonal therapy and the use of oral chemotherapeutic drugs. The structure based
molecular insertion approach revealed the findings of (E)-N'-((1-chloro-3,4-
dihydronaphthalen-2-yl)methylene) benzohydrazide derivatives; 3V49) .[29] Hepatocellular
carcinoma (HCC), which accounts for 90% of all primary liver cancers, is an important health
problem.[39] Recent genomic studies of HCC have identified several oncogenic pathways,
including mutations in the TERT promoter of CTNNB1, TP53 and ARID1A.[40] These studies
also identify other genes, including fibroblast growth factor 19 (11q13). Three chromosomal
loci containing genes containing fibroblast growth factor 19 (FGF19) and cyclin D1 (CCND1)
suggest that FGF19 acts as a potential driver oncogen in HCC.[41] H3B-6527 is a highly
selective and covalent small molecule inhibitor of FGFR4; and H3B-6527 monotherapy of HCC
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10.1002/cbdv.202000139
Chemistry & Biodiversity
with increased FGF19 expression and H3B-6527 combination therapy with CDK4/6 inhibitor
were evaluated in clinical trials for the treatment of HCC. The crystal structure of H3B-6527
attached to the ATP pocket can be found with PDB ID of 5VND.[30] The synthesis compounds
and target proteins were docked with each other. The interaction energies were given in Table
5.
Table 5. The interaction energies (kcal/mol) between mentioned molecules and six human
cancer cell lines
4I5I
-246.51
-255.55
-261.63
-271.95
-242.29
-265.10
-261.55
-281.84
-244.40
-255.46
-252.98
-261.29
-265.83
-257.05
-242.49
-269.48
-264.93
-325.37
-283.45
-271.27
-311.26
-293.98
-299.05
-266.74
-270.77
-269.83
-269.69
-271.25
-292.28
-328.64
5MB4
-243.94
-238.25
-268.78
-250.66
239.56
1M17
-17.92
-19.75
-16.08
-14.41
-32.21
-38.23
-26.57
-31.26
-11.81
-13.71
-19.68
-21.90
-29.32
-21.90
-13.58
-20.68
-24.52
-29.16
-29.09
-24.76
-25.01
-16.88
-28.34
-21.03
-29.30
-26.96
-29.34
-30.86
-25.74
-29.71
3HY3
-235.65
-276.30
-261.22
-343.97
-262.26
-244.95
-267.92
-259.14
-259.36
-256.39
-263.93
-292.99
-294.12
-267.47
-239.10
-290.19
-293.12
-294.27
249.94
3V49
-250.72
-229.04
-235.08
-244.42
-229.57
-244.95
-229.57
-238.75
-209.68
-227.16
-243.54
-233.45
-235.65
-227.08
-213.01
-297.25
-291.34
-295.64
-284.76
-285.89
-314.73
-289.88
-289.04
-271.86
-299.93
-290.23
-313.32
-292.32
-281.95
-290.09
5VND
-73.43
3a
3b
3c
3d
3e
3f
-76.61
-75.67
-78.07
-71.43
-244.64
-239.02
-236.78
-333.72
-228.17
-267.11
-248.61
-245.93
-238.55
-229.05
-288.82
-312.54
-327.76
-303.18
-291.83
-306.17
-298.49
-313.93
-294.43
-336.94
-295.78
-301.15
-319.68
-305.64
-320.37
-79.69
3g
3h
3i
-74.79
-76.63
-56.52
3j
-66.17
3k
3l
-76.81
-83.39
3m
3n
3o
7a
7b
7c
7d
7e
7f
-88.62
-119.74
-63.31
-133.46
-122.02
-154.23
-121.43
-132.28
120.24
-123.25
-134.76
-101.97
-120.44
-135.59
-125.88
-132.72
-131.53
-107.35
-279.94
-290.32
-283.03
-319.85
-286.59
-289.39
-289.52
-289.12
-291.33
-294.60
-281.68
7g
7h
7i
7j
7k
7l
7m
7n
7o
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10.1002/cbdv.202000139
Chemistry & Biodiversity
The docking results show that the binding affinities of the enamine derivatives (7a-o)
with the target proteins are higher than the binding affinities of the chalcone derivatives (3a-o).
7c, 7j and 7o exhibit interaction energy in the top row. The binding energy between 5MB4
target protein and 7j is -336.94 kcal/mol. In addition, the other highest binding energy is
between 3HY3 target protein and 3d. The docking binding mode of these target proteins and
related compounds were given in Figure 6. When the experimental and docking results are
evaluated it can be concluded that the studied compounds show a similar tendency towards
cancer cell lines.
Figure 6. The docking binding mode between the target protein and 7j and 3d.
There are hydrogen bonds, polar and hydrophobic interactions between the 3d drug candidate
molecule and the 3HY3 target protein. H-bond forms between nitrogen atom with ILE111 and
PRO112 amino acid residues. There is polar interaction between the oxygen atom and TRP109,
GLN113, TYR152, THR107 and PHE85. They performed hydrophobic interaction between
PRO81, TYR83, PHE85 and ILE111 with the carbon atoms of the 3d compound. There are
hydrogen bonds, pi- pi, polar and hydrophobic interactions between the 7j drug candidate
molecule and the 5MB4 target protein.
In the 7j compound, H-bonding occurred between nitrogen atom ILE224 and ASP169 amino
acids, in addition, between oxygen and THR223 amino acid. The number of hydrogen bonds is
higher than the 3d compound. In addition, pi-pi interaction between carbon and GLN7, polar
interaction between oxygen and LEU168, ASP169 and ALA225, moreover hydrophobic
interactions with amino acid residues ALA225 and ILE224 were observed.
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10.1002/cbdv.202000139
Chemistry & Biodiversity
Conclusions
In this study, a new series of enamine derivatives (7a-o) derived from lactone (6) and chalcones
(3a-o) have been successfully synthesized. The antiproliferative, cytotoxic and DNA binding
properties of the compounds (7a-o) and (3a-o) were also investigated. Here, we focused on the
certain molecules with stronger antiproliferative effects with less toxicity as well as good DNA
binding properties. The studies revealed that 7c displayed relatively optimal antiproliferative
(IC50 = ~ 101.10 µM) and low cytotoxic activity (31%) similar to cisplatin and 5-FU indicating
that the compound may be a promising candidate as an antiproliferative agent. The findings
point out that modification of the 3c by the substitute of lactone and chalcone results in desired
molecular activation and strongly enhanced pharmacological potency. Furthermore, the
interaction of the 7c with CTDNA implied that 7c has the optimal binding parameters with 2.0
× 103 Kb value and the hypochromism value of 23.72% of the absorption band at ca. 285 nm.
Overall, this study introduced new enamine derivatives (7a-o) as candidate lead molecules for
further preclinical tests as antiproliferative agents. The binding energies between the target
proteins and the compounds studied were almost in agreement with the experimental results.
Considering structure-activity relationship (SAR), the compound 3b (m-CH3 which do not
contain electron pair) and p-substituted compounds 7k-n exhibited the lowest activity against
all cancer cell lines except Hep3B. The o- and m-substituted compounds 7c-g and 7o contain
thiophene ring displayed very good activity against all cancer cell lines. Also, the compound
7a contain o-CH3 substituent, furan ring contains compound 7i and ji demonstrated generally
good activity. Moreover, the electron donor methoxy (-OCH3) group at the o- and m-position
increased activity.
Experimental Section
Chemistry
All chemicals and solvents were obtained from Merck (Germany) and Fluka (Germany).
1
Melting points were measured using an Electrothermal 9100 apparatus. H and 13C NMR
spectra were recorded with a Bruker Avance DPX-400 instrument. Elemental analyses were
obtained from a LECO CHNS 932 Elemental Analyzer.
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10.1002/cbdv.202000139
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Synthesis of chalcone derivatives 3a-j and 5k-n
Amino-chalcones 3a-j and nitro-chalcone derivatives 5k-n were synthesized in accordance
with published procedures [19-22]
.
Reduction of 5k-n to 3k-n
SnCl2.2H2O (5 mmol) was added to a solution of 5k-n (1 mmol) in EtOH (15 mL) which was
then refluxed for 2 h, after which a water solution of NaHCO3 (5%, 25 mL) was added drop
wise to the reaction mixture during a period of 45 min. The reaction mixture was than extracted
with chloroform, the organic layer was dried over Na2SO4 and the solvent was removed via
rotavapor. The crude product was crystallized in a CH2Cl2/hexane (3/7) mixture.
General procedure for the synthesis of 7a-o
To solution of chalcones 3a-o (1 mmol) in 10 mL of toluene, (6) 3-acetyldihydrofuran-2(3H)-
one (1 mmol) was added and refluxed for 24 h. Then, the mixture was diluted with CH2Cl2 (50
mL) and dried over Na2SO4. The solvent was evaporated, and the crude product was crystallized
in CHCl3/hexane (3/7) mixture. The purity of these compounds was determined via TLC and
their structures were confirmed using IR, 1H NMR, and 13C NMR.
Synthesis of (E)-3-(1-((4-acetylphenyl)amino)ethylidene)dihydrofuran-2(3H)-one (8)
The p-aminoacetophenone 2 (1 mmol) was dissolved in 10 mL of toluene followed by the
addition of 3-acetyldihydrofuran-2(3H)-one (1 mmol) (6). After the mixture was stirred for 15
min at RT, 2 mL of acetic acid was added and refluxed for 24 h. Then, the reaction mixture was
diluted with CH2Cl2 (50 mL) and dried with Na2SO4. The solvent was removed, and the crude
product was crystallized in CH2Cl2/hexane (3/7) mixture. The compound 8 was obtained in
good yield (70%). Structure of the compound was confirmed by IR, 1H NMR, and 13C NMR.
(E)-3-(1-((4-acetylphenyl)amino)ethylidene)dihydrofuran-2(3H)-one (8). Yield:
70%. M.p. 140-143 °C. IR (KBr, cm-1): 3202, 2992, 2976, 2927, 2865, 1673, 1629, 1595, 1567,
1518, 1479, 1451, 1438, 1389, 1360, 1319, 1271, 1241, 1222, 1180, 1118, 1020. 1H NMR (400
MHz, CDCl3):  10.27 (bs, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 4.39 (t, J =
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10.1002/cbdv.202000139
Chemistry & Biodiversity
13
8.0 Hz, 2H), 2.95 (t, J = 8.0 Hz, 2H), 2.58 (s, 3H), 2.18 (s, 3H). C-NMR (100 MHz, CDCl3,
ppm):  196.7, 173.6, 151.3, 143.8, 132.2, 129.8 (2C), 121.2 (2C), 93.0, 65.4, 26.4, 26.3, 18.2.
Anal. calc. for C14H15NO3: C, 68.56; H, 6.16; N, 5.71. Found: C, 68.48; H, 6.07; N, 5.69.
Synthesis of 7k-n
The chalcones 7k-n were synthesized by published procedures.[19-22]
(E)-3-(1-((4-((E)-3-(o-tolyl)acryloyl)phenyl)amino)ethylidene)dihydrofuran-
2(3H)-one (7a). Yield: 90%. M.p. 116-119 °C. IR (KBr, cm-1): 2975, 2911, 2866, 1687, 1653,
1644, 1608, 1584, 1519, 1482, 1391, 1361, 1336, 1319, 1281, 1253, 1219, 1186, 1119, 1043,
1024. 1H NMR (400 MHz, CDCl3, ppm):  10.32 (bs, 1H, -NH), 8.14 (d, J = 15.2 Hz, 1H), 8.05
(d, J = 8.4 Hz, 2H), 7.72 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 15.2 Hz, 1H), 7.35-7.33 (m, 1H),
7.29-7.25 (m, 2H), 7.15 (d, J = 8.4 Hz, 2H), 4.41 (t, J = 8.4 Hz, 2H), 2.97 (t, J = 8.4 Hz, 2H),
2.51 (bs, 3H, -CH3), 2.21 (bs, 3H, -CH3). 13C-NMR (100 MHz, CDCl3, ppm):  188.6, 173.6,
151.3, 143.7, 142.1, 138.3, 134.0, 133.3, 130.9, 130.2 (2C), 130.0, 126.4, 126.3, 122.7, 121.5
(2C), 92.9, 65.4, 26.4, 19.9, 18.2. Anal. calc. for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found:
C, 75.96; H, 6.01; N, 3.98.
(E)-3-(1-((4-((E)-3-(m-tolyl)acryloyl)phenyl)amino)ethylidene)dihydrofuran-
2(3H)-one (7b). Yield: 80%. M.p. 126-130 °C. IR (KBr, cm-1): 2861, 2360, 1692, 1643, 1603,
1
1589, 1520, 1491, 1357, 1315, 1278, 1242, 1217, 1185, 1120, 1023. H NMR (400 MHz,
CDCl3, ppm):  10.32 (bs, 1H, -NH), 8.05 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 15.6 Hz, 1H), 7.55
(d, J = 15.6 Hz, 1H), 7.48 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 7.2 Hz,
1H), 7.15 (d, J = 8.4 Hz, 2H), 4.41 (t, J = 8.0 Hz, 2H), 2.97 (t, J = 8.0 Hz, 2H), 2.43 (bs, 3H, -
13
CH3), 2.21 (bs, 3H, -CH3). C-NMR (100 MHz, CDCl3, ppm):  188.7, 173.6, 151.3, 144.7,
143.6, 138.6, 134.9, 133.3, 131.3, 130.0, 129.0 (2C), 128.8, 125.7, 121.5, 121.4 (2C), 92.9,
65.4, 26.4, 21.3, 18.2. Anal. calc. for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found: C, 75.89;
H, 5.93; N, 3.88.
(E)-3-(1-((4-((E)-3-(2-methoxyphenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7c). Yield: 85%. M.p. 140-145 °C. IR (KBr, cm-1): 3186, 3050, 2989, 2900,
2831, 1689, 1629, 1600, 1591, 1483, 1456, 1413, 1359, 1330, 1256, 1210, 1183, 1169, 1120,
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10.1002/cbdv.202000139
Chemistry & Biodiversity
1
1091, 1018. H NMR (400 MHz, CDCl3, ppm,):  10.23 (bs, 1H, -NH), 7.95 (d, J = 8.4 Hz,
2H), 7.72 (d, J = 15.2 Hz, 1H), 7.45 (d, J = 15.2 Hz, 1H), 7.25 (d, J = 7.6 Hz, 2H), 7.19-7.16
(m, 1H), 7.09-7.08 (m, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.92-6.87 (m, 1H), 4.31 (t, J = 8.4 Hz,
2H), 3.79 (s, 3H, -OCH3), 2.88 (t, J = 8.4 Hz, 2H), 2.12 (bs, 3H, -CH3). 13C-NMR (100 MHz,
CDCl3):  188.7, 173.7, 159.9, 151.4, 144.4, 143.7, 136.3, 133.2, 130.1 (2C), 130.0, 121.9,
121.5 (2C), 121.1, 116.2, 113.5, 93.0, 65.5, 55.4, 26.4, 18.3. Anal. calc. for C22H21NO4: C,
72.71; H, 5.82; N, 3.85. Found: C, 72.69; H, 5.78; N, 3.70.
(E)-3-(1-((4-((E)-3-(3-methoxyphenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7d). Yield: 69%. M.p. 135-138 °C. IR (KBr, cm-1): 3199, 3118, 3073,
2997, 2907, 2857, 2836, 1686, 1651, 1606, 1591, 1524, 1493, 1450, 1429, 1413, 1361, 1339,
1314, 1257, 1214, 1193, 1175, 1122, 1081, 1026. 1H NMR (400 MHz, CDCl3, ppm):  10.22
(bs, 1H, -NH), 7.94 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 15.6 Hz, 1H), 7.43 (d, J = 15.6 Hz, 1H),
7.27 (t, J = 7.8 Hz, 2H), 7.19-7.16 (m, 1H), 7.09-7.04 (m, 2H), 6.91-6.88 (m, 1H), 4.31 (t, J =
7.8 Hz, 2H), 3.79 (s, 3H, -OCH3), 2.87 (t, J = 7.8 Hz, 2H), 2.11 (s, 3H, -CH3). 13C-NMR (100
MHz, CDCl3):  188.6, 173.6, 159.9, 151.3, 144.3, 143.7, 136.3, 133.2, 130.2 (2C), 129.9,
121.9, 121.4 (2C), 121.0, 116.2, 113.4, 93.0, 65.4, 55.3, 26.4, 18.2. Anal. calc. for
C22H21NO4: C, 72.71; H, 5.82; N, 3.85. Found: C, 72.66; H, 5.77; N, 3.79.
(E)-3-(1-((4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7e). Yield: 75%. M.p. 145-148 °C. IR (KBr, cm-1): 3194, 2974, 2909, 2868,
1687, 1643, 1603, 1585, 1518, 1491, 1470, 1439, 1412, 1388, 1356, 1338, 1315, 1272, 1254,
1219, 1183, 1119, 1022. 1H NMR (400 MHz, CDCl3, ppm):  10.32 (bs, 1H, -NH), 8.20 (d, J =
15.6 Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.78-7.65 (m, 1H), 7.51 (d, J = 15.6 Hz, 1H), 7.47-7.45
(m, 1H), 7.37-7.33 (m, 2H), 7.14 (d, J = 8.4 Hz, 2H), 4.40 (t, J = 8.0 Hz, 2H), 2.96 (t, J = 8.0
Hz, 2H), 2.21 (bs, 3H, -CH3). 13C-NMR (100 MHz, CDCl3):  188.5, 173.6, 151.2, 143.8, 140.2,
135.4, 133.3, 132.9, 131.3, 130.3 (2C), 130.2, 127.8, 127.0, 124.4, 121.3 (2C), 93.1, 65.4, 26.3,
18.2. Anal. calc. for C21H18ClNO3: C, 68.57; H, 4.93; N, 3.81. Found: C, 68.44; H, 4.87; N,
3.75.
(E)-3-(1-((4-((E)-3-(3-chlorophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7f). Yield: 70%. M.p. 135-138 °C. IR (KBr, cm-1): 3054, 2913, 1685, 1644,
1603, 1583, 1452, 1420, 1377, 1353, 1334, 1316, 1275, 1252, 1212, 1182, 1119, 1049, 1026.
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1H NMR (400 MHz, CDCl3, ppm):  10.33 (bs, 1H, -NH), 8.04 (d, J = 8.8 Hz, 2H), 7.76 (d, J
= 15.6 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 15.6 Hz, 1H), 7.40-7.35 (m, 2H), 7.15 (d,
13
J = 8.8 Hz, 2H), 4.41 (t, J = 7.8 Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 2.21 (bs, 3H, -CH3). C-
NMR (100 MHz, CDCl3):  188.1, 173.6, 151.2, 143.9, 142.6, 136.8, 134.9, 132.8, 131.1, 130.2
(2C), 130.2, 127.8, 126.8, 122.8 (2C), 121.4, 93.2, 65.4, 26.3, 18.2. Anal. calc. for
C21H18ClNO3: C, 68.57; H, 4.93; N, 3.81. Found: C, 68.51; H, 4.83; N, 3.77.
(E)-3-(1-((4-((E)-3-(2-bromophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7g). Yield: 75%. M.p. 143-145 °C. IR (KBr, cm-1): 3332, 1759, 1654, 1603,
1
1463, 1337, 1312, 1270, 1220, 1180, 1049, 1024. H NMR (400 MHz, CDCl3, ppm):  10.33
(bs, 1H, -NH), 8.15 (d, J = 15.6 Hz, 1H), 8.03 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 7.8 Hz, 1H),
7.66 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 15.6 Hz, 1H), 7.39 (t, J = 7.2 Hz, 1H), 7.29-7.25 (m, 1H),
7.14 (dd, J = 8.8 Hz, 2H), 4.41 (t, J = 7.6 Hz, 2H), 2.96 (t, J = 7.6 Hz, 2H), 2.21 (bs, 3H, -CH3).
13C-NMR (100 MHz, CDCl3):  188.5, 173.6, 151.2, 137.1, 143.8, 142.8, 135.1, 133.5, 130.5,
130.2 (2C), 127.8, 127.7, 125.8, 124.7, 121.3 (2C), 93.1, 65.4, 26.4, 18.3. Anal. calc. for
C21H18BrNO3: C, 61.18; H, 4.40; N, 3.40. Found: C, 61.03; H, 4.36; N, 3.37.
(E)-3-(1-((4-((E)-3-(3-bromophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7h). Yield: 76%. M.p. 162-165 °C. IR (KBr, cm-1): 3050, 1752, 1686, 1644,
1602, 1583, 1520, 1478, 1452, 1417, 1377, 1353, 1334, 1315, 1275, 1252, 1213, 1182, 1119,
1049, 1026. 1H NMR (400 MHz, CDCl3, ppm):  10.33 (bs, 1H, -NH), 8.04 (d, J = 8.8 Hz, 2H),
7.82 (bs, 1H), 7.75 (d, J = 15.6 Hz, 1H), 7.58-7.52 (m, 3H), 7.34-7.30 (m, 1H), 7.15 (d, J = 8.4
Hz, 2H), 4.41 (t, J = 7.8 Hz, 2H), 2.97 (t, J = 7.8 Hz, 2H), 2.21 (bs, 3H, -CH3). 13C-NMR (100
MHz, CDCl3, ppm):  188.2, 173.8, 142.5, 137.1, 133.1, 130.7, 130.4, 130.1 (2C), 127.2, 123.0,
122.8, 121.4 (2C), 93.2, 65.4, 26.4, 18.2. Anal. calc. for C21H18BrNO3: C, 61.18; H, 4.40; N,
3.40. Found: C, 61.14; H, 4.37; N, 3.32.
(E)-3-(1-((4-((E)-3-(furan-2-yl)acryloyl)phenyl)amino)ethylidene)dihydrofuran-
2(3H)-one (7i). Yield: 86%. M.p. 130-135 °C. IR (KBr, cm-1): 3441, 3418, 1638, 1603, 1587,
1546, 1517, 1476, 1438, 1387, 1336, 1297, 1281, 1257, 1234, 1178, 1148, 1015. 1H NMR (400
MHz, CDCl3, ppm):  10.31 (bs, 1H, -NH), 8.04 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 15.2 Hz, 1H),
7.55-7.54 (m, 1H), 7.48 (d, J= 15.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 3.2 Hz, 1H),
6.55-6.53 (m, 1H), 4.40 (t, J = 8.0 Hz, 2H), 2.96 (t, J = 8.0 Hz, 2H), 2.20 (bs, 3H, -CH3). 13C-
NMR (100 MHz, CDCl3, ppm):  188.0, 173.6, 151.7, 151.3, 144.8, 143.6, 133.2, 130.3, 129.9
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10.1002/cbdv.202000139
Chemistry & Biodiversity
(2C), 121.4, 118.9, 116.1, 112.6 (2C), 92.9, 65.4, 26.4, 18.2. Anal. calc. for C19H17NO4: C,
70.58; H, 5.30; N, 4.33. Found: C, 70.45; H, 5.23; N, 4.21.
(E)-3-(1-((4-((E)-3-(p-tolyl)acryloyl)phenyl)amino)ethylidene)dihydrofuran-
2(3H)-one (7k). Yield: 76%. M.p. 160-163 °C. IR (KBr, cm-1): 2972, 2920, 2864, 1684, 1639,
1602, 1586, 1509, 1489, 1449, 1414, 1353, 1336, 1308, 1274, 1220, 1185, 1176, 1119, 1022.
1H NMR (400 MHz, CDCl3):  10.21 (bs, 1H, -NH), 7.93 (d, J = 8.8 Hz, 2H), 7.73-7.71 (m,
1H), 7.48-7.39 (m, 3H), 7.15 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 4.30 (t, J = 7.6 Hz,
13
2H), 2.86 (t, J = 7.6 Hz, 2H), 2.32 (s, 3H, -CH3), 2.10 (s, 3H, -CH3). C-NMR (100 MHz,
CDCl3, ppm):  188.7, 173.6, 151.4, 144.5, 141.0, 132.2, 130.0 (2C), 129.7(2C), 129.6, 128.4
(2C), 128.2, 121.5 (2C), 120.6, 92.8, 65.4, 26.4, 21.6, 18.2. Anal. calc. for C22H21NO3: C, 76.06;
H, 6.09; N, 4.03. Found: C, 75.97; H, 5.94; N, 3.94.
(E)-3-(1-((4-((E)-3-(4-methoxyphenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7l). Yield: 74%. M.p. 175-179 °C. IR (KBr, cm-1): 2965, 2903, 2863, 1686,
1646, 1598, 1571, 1509, 1422, 1390, 1359, 1318, 1293, 1277, 1250, 1221, 1172, 1121, 1026.
1H NMR (400 MHz, CDCl3):  10.30 (bs, 1H, -NH), 8.03 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 15.6
Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 15.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 2H), 6.96 (d,
J = 8.8 Hz, 2H), 4.40 (t, J = 7.8 Hz, 2H), 3.88 (s, 3H, -OCH3), 2.96 (t, J = 7.8 Hz, 2H), 2.20 (s,
13
3H, -CH3). C-NMR (100 MHz, CDCl3, ppm):  188.7, 173.6, 161.6, 151.4, 144.3, 143.4,
133.6, 130.2 (2C), 129.9 (2C), 127.6, 121.5 (2C), 119.3, 114.4 (2C), 92.7, 65.4, 55.4, 26.4, 18.2.
Anal. calc. for C22H21NO4: C, 72.71; H, 5.82; N, 3.85. Found: C, 72.66; H, 5.77; N, 3.79.
(E)-3-(1-((4-((E)-3-(4-chlorophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7m). Yield: 81%. M.p. 184-188 °C. IR (KBr, cm-1): 2909, 1685, 1644, 1607,
1567, 1518, 1489, 1450, 1407, 1359, 1337, 1317, 1252, 1222, 1184, 1120, 1088, 1024. 1H NMR
(400 MHz, CDCl3, ppm):  10.33 (bs,1H, -NH), 8.03 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 15.6 Hz,
1H), 7.60 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 15.6 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.14 (d, J =
13
8.8 Hz, 2H), 4.40 (t, J = 7.6 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H), 2.21 (s, 3H, -CH3). C-NMR
(100 MHz, CDCl3, ppm):  188.2, 173.6, 151.2, 143.8, 142.9, 136.3, 133.4, 132.9, 130.0 (2C),
129.5 (2C), 129.2 (2C), 122.0, 121.3 (2C), 93.1, 65.4, 26.3, 18.3. Anal. calc. for C21H18ClNO3:
C, 68.57; H, 4.93; N, 3.81. Found: C, 68.52; H, 4.86; N, 3.70.
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10.1002/cbdv.202000139
Chemistry & Biodiversity
(E)-3-(1-((4-((E)-3-(4-bromophenyl)acryloyl)phenyl)amino)ethylidene)dihydro
furan-2(3H)-one (7n). Yield: 73%. M.p. 191-195 °C. IR (KBr, cm-1): 2974, 2908, 2861, 1682,
1645, 1609, 1586, 1487, 1392, 1360, 1316, 1277, 1252, 1222, 1180, 1122, 1071, 1049, 1027.
1H NMR (400 MHz, CDCl3, ppm):  10.33 (bs, 1H, -NH), 8.03 (d, J = 8.8 Hz, 2H), 7.77 (d, J =
15.6 Hz, 1H), 7.59-7.52 (m, 5H), 7.14 (d, J = 8.4 Hz, 2H), 4.41 (t, J = 7.8 Hz, 2H), 2.97 (t, J =
13
7.8 Hz, 2H), 2.21 (s, 3H, -CH3). C-NMR (100 MHz, CDCl3, ppm):  188.3, 173.6, 151.2,
143.8, 142.9, 133.8, 132.9, 132.2 (2C), 130.1 (2C), 129.7 (2C), 124.7, 122.1, 121.4 (2C), 93.1,
65.4, 26.4, 18.3. Anal. calc. for C21H18BrNO3: C, 61.18; H, 4.40; N, 3.40. Found: C, 61.09; H,
4.32; N, 3.34.
(E)-3-(1-((4-((E)-3-(thiophen-2-
yl)acryloyl)phenyl)amino)ethylidene)dihydrofuran-2(3H)-one (7o). Yield: 78%. M.p. 139-
142 °C. IR (KBr, cm-1): 2917, 2862, 1685, 1637, 1597, 1451, 1414, 1374, 1359, 1277, 1244,
1219, 1180, 1121, 1050, 1026. 1H NMR (400 MHz, CDCl3, ppm):  10.31 (bs, 1H, -NH), 8.01
(d, J = 8.4 Hz, 2H), 7.96 (d, J = 15.6 Hz, 1H), 7.44 (d, J = 5.2 Hz, 1H), 7.38-7.37 (m, 1H), 7.30
(d, J = 15.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 7.11-7.10 (m, 1H), 4.40 (t, J = 7.8 Hz, 2H), 2.96
13
(t, J =7.8 Hz, 2H), 2.20 (s, 3H, -CH3). C-NMR (100 MHz, CDCl3):  188.0, 173.6, 151.3,
143.7, 140.4, 136.8, 133.2, 131.9, 129.9 (2C), 128.6, 128.3, 121.5 (2C), 120.4, 92.9, 65.4, 26.4,
18.2. Anal. calc. for C19H17NO3S: C, 67.24; H, 5.05; N, 4.13. Found: C, 67.12; H, 4.91; N, 4.08.
Materials and Methods
Pharmacology
Preparation of cell culture
The antiproliferative potential of the compounds was investigated on cancerous HeLa (ATCC®
CCL2™), HT29 (ATCC® HTB38™), MCF7 (ATCC® HTB22™), PC3 (ATCC® CRL1435™),
A549 (ATCC® CCL185™) and Hep3B (ATCC® HB8064™) and normal FL cells (ATCC®
CCL62™). The cell lines were cultured in a cell medium (Dulbecco’s modified eagle’s)
enriched with 10% (v/v) fetal bovine serum (FBS) and 2% (v/v) Penicillin-Streptomycin
(10,000 U/mL). First, the old medium was removed out of the flask while the cells had reached
approximately 80% confluency. Next, the cells were detached from the flask surface using 4-5
mL of trypsin-EDTA solution, and then centrifugation was performed to remove trypsin.
Afterwards, the cell pellet was suspended with 4 mL of DMEM working solution and was
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10.1002/cbdv.202000139
Chemistry & Biodiversity
counted to obtain a final concentration of 5.0 × 104 cells/mL which was then inoculated into
wells (100 μL cells/well).
Cell proliferation assay (MTT assay)
A cell suspension containing approximately 1.0 × 104 cells in 100 µL was seeded into the wells
of 96-well culture plates. The cells were treated overnight with the compounds and control drug,
cisplatin and 5-fluorouracil (5-FU), dissolved in sterile DMSO (max 0.5% of DMSO) at final
concentrations of 2.5, 5, 10, 15, 20, 25, 37,5, and 50 µg/mL at 37 °C with 5% CO2 . The final
volume of the wells was completed to 200 µL with the addition of medium.
Cell proliferation assay was evaluated using MTT (yellow tetrazolium MTT (3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay.[42] An MTT stock solution (5 mg
of MTT/mL of distilled water) was filter sterilized and kept at -20 °C until further use. The cells
were exposed to a MTT reagent for 4 h to form MTT formazan dye. This step was followed by
the addition of dye dissolved in DMSO with Sorenson’s buffer for 30 min at room temperature
after which the plate was measured at 560 nm, with 690 nm as a reference wavelength interval,
using a microplate reader. Each experiment was repeated at least three times for each cell line.
Cytotoxic activity assay
The cytotoxicity of the compounds, cisplatin and 5-fluorouracil on cells was determined
through the use of a Lactate Dehydrogenase Assay Kit in accordance with the manufacturer’s
instructions.[43] Approximately 5.0 × 103 cells in 100 µL were placed in 96-well plates as
triplicates and treated with IC50 concentrations of test compounds at 37 °C with 5% CO2 for 24
h. LDH activity was determined using absorbance values at 492 – 630 nm using a microplate
reader. The cytotoxicity assay results were given as the percent cytotoxicity according to the
following formula: % Cytotoxicity = [(Experimental Value - Low Control /High Control - Low
Control) × 100].
Cell imaging
Cells were plated in 96-well plates at a density of 5.000 cells per well and were incubated for
24 h. Afterwards, 15, 20, 25, 37.5, and 50 µg/mL of the test compounds were administered and
morphology alterations of the cells were screened by phase contrast microscopy for every 6 h
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10.1002/cbdv.202000139
Chemistry & Biodiversity
during 24 h. Images of control and test compound treated cells were taken at the end of the
process using a digital camera attached to an inverted microscope.
DNA binding studies
UV spectroscopy was used to determine the interaction of the compounds with CT-DNA and
to calculate the binding constants (퐾푏). A CT-DNA solution was prepared by dissolving 2.5
mg CT-DNA in 10.0 mL Tris–HCl buffer (20 mM Tris–HCl, 20 mM NaCl at pH 7.4) which
was then stored in the refrigerator. The concentration of CT-DNA was determined
spectrophotometrically using the known Ɛ value of 6600 M−1 cm−1 at 260 nm. After dissolving
the CT-DNA fibers in Tris–HCl buffer, the purity of this solution was checked using the
absorbance ratio A260/A280. The CT-DNA solution in the buffer displayed an A260/A280 ratio of
1.89, indicating that the DNA was sufficiently pure. These compounds were dissolved in
DMSO and diluted with Tris-HCl buffer to obtain 50 µM concentrations. Test compounds in
the solutions were incubated at 37 °C for about 30 min before the measurements. The UV
absorption titrations were conducted by keeping the concentration of these compounds fixed
while varying the CT-DNA concentrations (50-250 μM). Absorption spectra were recorded
using 1-cm-path quartz cuvettes at room temperature.
Calculation of IC50 and % inhibition
IC50 value is a concentration that inhibits half of the cells in vitro. The half maximal inhibitory
concentrations (IC50) of the test and control compounds were calculated using XLfit5 or an
excel spreadsheet and represent µM at 95% confidence intervals. The proliferation assay results
were expressed as percent inhibition according to the following formula: % Inhibition = [1 -
(Absorbance of Treatments / Absorbance of DMSO) × 100].
Docking study
Docking studies were performed by the optimization of ligands and obtaining proteins
corresponding to the specific cancer cell line from the protein database. The molecular drawings
of studied ligands were prepared by using GausView 5.0.8 and the mechanic optimizations
were calculated with Gaussian 09W.[44,45] Molecular docking studies were performed between
HEX 8.0.0 program for the complexes studied and the target protein.[46]
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10.1002/cbdv.202000139
Chemistry & Biodiversity
Acknowledgements
The authors are indebted to the Gaziosmanpasa University, Scientific Research Projects
Commission (Project No: BAP-2015/91) and TÜBİTAK (Scientific and Technological
Research Council of Turkey; Project No: 114Z696) for financial supports.
Author Contribution Statement
M. B. G., B. Y., F. E., O. Ö., Y. B. and M. C. designed and performed the experiments, analyzed the
data and M. B. G. and M. C. wrote the paper. A. A. performed the antiproliferative activity experiments,
analyzed the data of compounds. S. E. carried out molecular docking study of compounds.
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