Design, synthesis and antiproliferative activities of oxidative stress inducers based on 2-Styryl-3,5-dihydro-4H-imidazol-4-one scaffold

Article abstract of DOI:10.1248/cpb.c18-00398

The 2-styryl-3,5-dihydro-4H-imidazol-4-one might be considered as a system with isosteric properties similar to trans-cinnamaldehyde (styrylaldehyde), a safe natural compound that exhibited interesting activities against various cancers. We synthesized a series of compounds that differ structurally in having different alkyl, aryl and heterocyclic substituents at the N3 position of the 2-styryl-4-imidaolone pharmacophore. The compounds were assayed for their cytotoxicity against both cancer and normal cell lines. In addition, their cellular mechanism of action as reactive oxygen species (ROS) inducers were investigated. Many of the synthesized compounds showed higher activities on colon, breast and hepatic cancer cell lines than the parent trans-cinnamaldehyde. Compounds 3a and 3e showed selective antiproliferative activity against cancer cell lines at low micromolar to sub-micromolar IC₅₀ value. Compounds were extremely less toxic on normal cell lines baby hamster kidney fibroblasts (BHK) and human lung tissue fibroblast (WI-38). Similar to transcinnamaldehyde, the colon cancer cell cycle analysis indicated cell cycle changes consistent with increased oxidative stress leading to apoptosis. Compound 3e caused elevation of all cell oxidative indicators of ROS such as a decrease in reduced glutathione, increased malondialdehyde and suppression of catalase and superoxide dismutase activities. Dihydroethidium staining, nuclear fragmentation and increased caspase-3 further confirmed extensive apoptotic induction due to ROS accumulation upon treatment of human colon adenocarcinoma (HCT116) cells with compounds 3a and 3e. Changes in human breast adenocarcinoma (MCF7) cells were less revealing for ROS induction and increased oxidative stress. Conclusion: The compounds represent an example of efficient rescaffolding of a natural compound to a highly potent drug-like analogues.

Full text of DOI:10.1248/cpb.c18-00398

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00398
Advance Publication
July 25, 2018
Design, Synthesis and Antiproliferative Activities of Oxidative
Stress Inducers Based on 2-styryl-3,5-dihydro-4H-imidazol-4-one
Scaffold
Abdelsattar M. Omar1,2*, Tamer M. Abdelghany3, Mohamed S. Abdel-Bakky3,4,
Abdulrahman M. Alahdal1, Mohamed F. Radwan1,5 and Moustafa E. El-Araby1,6*
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah
21589, Saudi Arabia; 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar
University, Cairo 11884, Egypt; 3Department of Pharmacology, Faculty of Pharmacy, Al-Azhar
4
University, Cairo 11884, Egypt; Department of Pharmacology, Faculty of Pharmacy, Aljouf University,
5
Aljouf, Saudi Arabia; Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University,
6
Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University,
Cairo 11790, Egypt.
*
Corresponding Authors that contributed equally to this work
Address correspondence to this author at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz
University, Jeddah, Saudi Arabia; E-mails: asmansour@kau.edu.sa; madaoud@kau.edu.sa.
Ⓒ 2018 The Pharmaceutical Society of Japan

Abstract: The 2-styryl-3,5-dihydro-4H-imidazol-4-one might be considered as a system with isosteric
properties similar to trans-Cinnamaldehyde (styrylaldehyde), a safe natural compounds that exhibited
interesting activities against various cancers. We synthesized a series of compounds that differ
structurally in having different alkyl, aryl and heterocyclic substituents at the N3 position of the 2-styryl-
4-imidaolone pharmacophore. The compounds were assayed for their cytotoxicity against both cancer
and normal cell lines. In addition, their cellular mechanism of action as ROS inducers were investigated.
Many of the synthesized compounds showed higher activities on colon, breast and hepatic cancer cell
lines than the parent trans-cinnamaldehyde. Compounds 3a and 3e showed selective antiproliferative
activity against cancer cell lines at low micromolar to sub-micromolar IC50 value. Compounds were
extremely less toxic on normal cell lines BHK and WI-38. Similar to trans-Cinnamaldehyde, the colon
cancer cell cycle analysis indicated cell cycle changes consistent with increased oxidative stress leading
to apoptosis. Compound 3e caused elevation of all cell oxidative indicators of reactive oxygen species
such as a decrease in reduced glutathione, increased malondialdehyde and suppression of catalase and
superoxide dismutase activities. Dihydroethidium staining, nuclear fragmentation and increased
caspase-3 further confirmed extensive apoptotic induction due to ROS accumulation upon treatment of
HCT116 cells with compounds 3a and 3e. Changes in MCF7 cells were less revealing for ROS induction
and increased oxidative stress. Conclusion: The compounds represent an example of efficient
rescaffolding of a natural compound to a highly potent drug-like analogues.
Keywords: Selective antiproliferative; reactive oxygen species; redox dysregulation; oxidative stress
markers; imidazolone; colon cancer
Chemical and Pharmaceutical Bulletin Advance Publication

1. INTRODUCTION
4-Imidazolones (3,5-Dihydro-4H-imidazol-4-one) bearing arylidene at C-5 position and
variety of substituents on positions N-3 and C-2 (Figure 1) is a synthetically accessible
heterocyclic scaffold that has been studied as a potential anticancer 1-3 with other therapeutic
benefits. 4-8 Moreover, this nucleus exists in the marine alkaloid rhopaladins A-D which was
found to exert some interesting biological activities.9 The versatility of 4-imidazolones
attracted our attention during our quest to identify compounds with potential anticancer
activities. For instance, we discovered promising antiproliferative activities of some 4-
imidazolone derivatives such as Cur-3 (Figure 1) against colon cancer cell lines during our
reported research on multi-drug resistance modulating agents.3
From another prespective, it was established that natural small molecules constitute a
valuable source of new leads in anticancer drug discovery because they are usually
characterized by low toxicities and intriguing activities.10 For instance, trans-
Cinnamaldehyde (tCA), the major constituent of cinnamon bark extract, has been extensively
investigated for its activities against various cancer cells such as colon, breast, liver,
melanoma and leukemia (see review by Hong et al).11 Induction of oxidative stress via
increased reactive oxygen species (ROS) has been reported as a major mechanism for the
apoptotic effect of tCA and its closely related derivatives such as BCA (Figure 1).12-13 tCA
and its derivatives were found to exert a pro-oxidant effect leading to elevated ROS as they
also decreased the functional levels of the anti-oxidant cellular components such as
glutathione (GSH) and thioredoxin reductase.14 It has been established that heightened ROS
levels generate an oxidative stress on cancer cells and induce apoptosis on cancer cells.15
However, ROS elevation in healthy normal cells may lead to increased oxidative stress
associated with inflammatory and oncogenic disorders.16 Fortunately, the oxidative stress
caused by tCA and its analogues apparently has more influence on cancer than normal cells
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as they were found to activate the antioxidant Nrf2/Keap1-ARE signaling pathway in non-
immortalized primary fetal colon cells.17 Regardless of its ROS-elevating properties, tCA is
recognized by the FDA as a safe food supplement and flavor.18-19 On the downside, tCA is an
aldehyde with a high metabolic conversion rate and poor pharmacokinetics and it is not
suitable for drug development.20-21 In this report, we aimed to synthesize and investigate
cytotoxic properties of 2-styryl-4-imidazolone series of compounds incorporating the styryl
portion of the tCA.
Figure 1. Imidazol-4-one scaffold, its derivatives alkaloid Rhopaladins and Cur-3 (cytotoxic agent). In the
bottom-left side, the structure of trans-cinnamaldehyde and its derivatives that have proven cytotoxic activities
against cancer cell lines.
2. RESULTS
2.1. Design and Chemical Synthesis
Chemically, 4-imidazolones are electron-withdrawing heterocycles because they contains a
conjugated carbonyl system. Therefore, we hypothesized that appending a styryl group C-2 of
Chemical and Pharmaceutical Bulletin Advance Publication

4-imimidazole would possibly retain the antiproliferative cytotoxic activity of tCA acting by
similar cellular mechanisms of tCA. In theory, heterocyclic 4-imidazolone may resist
metabolic conversion more than easily oxidized aldehydes. More importantly, from prior
experience it has been shown that 4-imidazolone derivatives were able to produce in vivo
activities against inflammation and convulsions.4, 22 Since this work is a first step to explore
the potentiality of 2-styryl-4-imidazolne as anticancer surrogates, a systematic approach was
followed for deciding the list of compounds to prepare. Fixing the group at the 4-postion as
benzylidene and variation of amines at the N-5 of the imidazolone ring aimed to achieve the
activity target.
Figure 2. Design of imidazolone derivatives as tCA mimics.
(Color figure can be accessed in the online version.)
The compounds were synthesized starting from cinnamoylglycine 1 which was condensed
with benzaldehyde according to standard Erlenmeyer procedure to give the azlactone 2, a
known intermediate, that was then reacted with different aliphatic, aromatic and heterocyclic
amines to produce the desired imidazolones 3a-m.4
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1. Synthesis of imidazolone derivatives 3a-m. (a) benzaldehyde, acetic anhydride, heat, 15 min, yield
65%; (b) RNH2, acetic acid, sodium acetate, heating 70-80 °C, yield 16-74%.
IC50 ± S.E. (µM)
Cpd
No.
R
HCT116
3.6 ± 2.19
9.8 ± 1.44
2.7 ± 1.5
35.2 ± 1
0.49 ± 3.9
3.2 ± 1.8
12.1 ± 1.7
13.7 ± 1
MCF-7
HepG2
2.93±2
BHK
777±27
>1000
>1000
>1000
524±21
693±25
>1000
WI-38
>1000
>1000
>1000
>1000
363±17
>1000
>1000
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
2
H
n-Pr
Ph
3.15±1.5
24.6±1.5
1.8±1.6
35.16±1.2
3.9±1.8
18.7±1.16
21±1.08
23.2±1.7
37.1±1.5
31.4±1.5
7.8±4
0.7±1.4
3.2±1.9
30.6±5.8
3.7±2.1
0.39±1.9
28.8±5.3
4.2±1
4-F-Ph
4-OH-Ph
3-CN-Ph
3-CF3-Ph
Bn
33.1±1.1 66.5±5.8
3-pyridyl
79.8 ± 1.5
16.3 ± 1.17
29.15 ± 5
28.6 ± 4.6
2.5 ± 2.9
10.5 ± 1.1
12.4 ± 1.2
43.18±1.5
2.2±2.3
11.8±3.5
3.5±1.8
2.2±1.2
14.4±1.7
>100
>1000
749.2±38
>1000
>1000
>1000
>1000
5-methyl-2-thiazolyl
2-(1,3,4-thidiazolyl)
furfuryl
2.6±1.6
65.0 ±1.4 60.9±6.5
53.3±1.12 45.5±2.8
2-(4-morpholinyl) ethyl
2.6±1.37
17.45±1.3
8.8±2
>1000
584±105
40.8±1.5
tCA
63.9±13
Table 1. Cytotoxic effect of imidazolone derivatives on CRC (HCT116), breast (MCF7) and hepatic (HepG2)
cancer cell line. The last two columns are cytotoxic activities on two normal (non-cancerous) highly
proliferative cell lines: Baby hamster kidney fibroblasts (BHK); and diploid human cell lines (WI-38). IC50
values are calculated as the concentration that kills 50% of the cell population employing SRB assay; data are
presented as means± Standard error of the mean.
The stereochemistry of the benzylidene group at position 5 is an established (Z) isomer
according to literature reports on similar cases.9 The compounds were fully characterized and
Chemical and Pharmaceutical Bulletin Advance Publication

purities were confirmed using spectral analyses (NMR, LC/MS) in addition to elemental
analyses.
The amines selected for the final step in the chart varied from aliphatic (3b, 3m), to phenyl
(3b), substituted phenyl (3c, 3d, 3e, 3f), heterocyclic (3i, 3j, 3k, 3l, 3m) or arylalkyl (3h, 3l),
in addition to the unsubstituted N-5 analogue (3a). Moreover, the compound set covered a
range of polar to non-polar substituents as well as variable electron densities on the aromatic
rings according to the varied N-3 substituents.
Our first objective in this research was to identify cytotoxic compounds with an acceptable
level of potency (< 5µM). Once this step was accomplished, we aimed to study cellular
mechanisms, aiming to confirm apoptosis induction via ROS elevation in a similar way to the
natural lead tCA23 and its derivatives.12, 24 In addition, potency against not only cancer cell
lines, but also against normal, highly proliferative mammalian cells were sought as an
indicator of electivity and preliminary toxicity.25
2.2. Biological Screening:
2.2.1. Cytotoxicity and Cell Cycle Arrest
All compounds were screened for their cytotoxic activity using SRB26 assay against three
different cancer cell lines: human colon adenocarcinoma (HCT116), human breast
adenocarcinoma (MCF7) and human hepatocellular carcinoma (HepG2). These cell lines
represent the most common spread of malignant diseases. The majority of our compounds
exhibited significant growth inhibition of cancer cells against all three cancer cell lines (Table
1). For example, compounds 3a, 3c, 3e and 3m showed universal cytotoxic activities ranged
from 0.5 to 4.0 µM IC50 values. Generally, many of the tested compounds had higher
cytotoxic activities than the lead tCA. Some compounds showed selective inhibition against a
certain cell line. In this regard, compounds 3b, 3h and 3j showed more potent activities
Chemical and Pharmaceutical Bulletin Advance Publication

against HepG2 than HCT116 or MCF7. The 3-(3-cyanophenyl) imidazolone derivative (3f)
was clearly a selective inhibitor of HCT116. The anti-proliferative activities of many
imidazolones 3 were higher than the isosteric oxazolone 2.
The synthesized compounds demonstrated differential activity towards cancer cell lines over
normal (non-cancerous cell lines). The selectivity test was performed by inhibition assay
against two types of normal highly proliferative cell lines: Baby Hamster Kidney fibroblasts
(BHK) and Diploid Human WI-38 cells. For instance, the potent anticancer compound 3a
killed cancer cells of colon, breast and liver at IC50 values at 3.6, 3.15 and 2.96 respectively.
This compound was remarkably safe to the normal cells as illustrated in Figure 3.
Compounds 3a and 3e had no appreciable toxic activity on both BHK and WI-38 cell lines as
their selectivity index against cancer cells was found to be more than 100 times over normal
cells. Some of the screened compounds (3h, 3l and 3m) showed lower selectivity as they
inhibited BHK and WI-38 cell lines with IC50 values less than 100 µM.
Figure 3. Dose-Response Curves of cytotoxic effect of compound 3a on normal cell lines: BHK (Blue) and WI-
38 (Black); In addition to cancer cell lines: HCT-116 (Red), HepG-2 (purple) and MCF-7(Green).
(Color figure can be accessed in the online version.)
Chemical and Pharmaceutical Bulletin Advance Publication

The changes in the cell cycle profile were found to be time dependent as the distribution of
the percentage of cells population in each stage varied considerably from zero h (control) to
24 h to 48 h (Figure 4). In HCT116, the cytotoxic compounds 3a and 3e caused non-
significant changes on the Pre-G phase cell population compared to control after 24 h. On the
other hand, the 48 h treatment resulted in a dramatic increase in the percentage of cells at Pre-
G by 4 and 5 folds respectively compared to control. Moreover, both compounds induced a
significant increase in the S-phase population with a concomitant decrease in the G0/G1
population, indicating S-phase arrest.
The changes in the Pre-G phase were less clear in MCF7 cells for all three tested compounds
3a, 3c and 3m. Furthermore, longer time incubation of 3c did not result in an increase in Pre-
G, while all three compounds increased the population of cells at G0/G1 phase. This may
indicate that these compounds might act through a different mechanism on MCF7 than
HCT116 cell lines.
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Figure 4. Effect of compounds 3a, 3e, 3c, and 3m on cell cycle distribution of HCT116 and MCF7 cell lines
(Color figure can be accessed in the online version.)
2.2.2. Mechanisms of Cytotoxicity in HCT116 Cells
In order to further investigate the mechanism of cytotoxicity of the universally potent
compounds, we conducted different experiments on the compounds 3a and 3e. The two
compounds were expected to induce apoptosis of cancer cells by oxidative stress and
downstream consequences similar to tCA due to their above mentioned design.
To study the effect of 3a and 3e on free radical generation and oxidative stress induction,
HCT116 cells were incubated with the IC50 of compounds 3a and 3e for 48 h. At the end of
the incubation period, the cell lysates were assayed for the content of redox regulators. Both
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compounds lead to an increase
in oxidati
ve stress ma
rkers by va
riable degrees (figure 5). The
phenolic derivative 3e caused sharp elevation of MDA to almost twice of its concentration in
untreated cells, indicating large increase in the oxidative stress. In addition, 3e caused
extensive depression (about 10 fold) of the ROS scavengers CAT and SOD. The
unsubstituted 3a gave much weaker effects on all oxidative parameters. Furthermore, a test
.-
for superoxide anion radical (O2 ) confirmed the above results of oxidative stress induction
.-
due to ROS accumulation. The amount of O2 produced upon treatment of HCT116 cells
with 3a and 3e was elevated (figure 6).
3a
3e
250
200
150
100
50
0
GSH
CAT
SOD
MDA
Figure 5. Effect of compounds 3a and 3e on oxidative stress parameters (MDA, GSH, SOD, and CAT)
in HCT116 cells. The values are expressed as percentage of controls and calculated from the means of three
independent experiments.
(Color figure can be accessed in the online version.)
Chemical and Pharmaceutical Bulletin Advance Publication

Figure 6. Superoxide free radical detection. Cells were grown on a cover slip in a 12 well plate for 24 hour,
then exposed to the IC50 of compounds 3a and 3e for 24 hours. Cells then were incubated with 10μM
dihydroethidium (DHE), at 37ºC for 30 min and1μM DAPI was used as counter stain. The cover slips were then
mounted and visualized by Leica fluorescence microscope. Red fluorescence represents DHE staining in the top
row and blue stain for DAPI in the middle row.
(Color figure can be accessed in the online version.)
Immunofluorescence assays were performed to investigate the effect of 3a and 3e on the
expression of cleaved-caspase 3. Treatment of HCT116 cancer cells with the IC50 of 3a and
3e significantly increased the expression and nuclear translocation of cleaved-caspase 3 (red)
compared with non-treated cells (Figure 7), indicating that our compounds halted the
HCT116 growth and induced cellular apoptosis. These findings were further confirmed by
the presence of typical morphological features of apoptotic nuclei including, 1- Chromatin
condensation (increase in cell membrane permeability and uptake of the fluorescent stain
4',6'-diamidino-2-phenylindole (DAPI), leaving a stronger blue fluorescence), 2- Pyknosis, 3-
The nuclear envelope becomes discontinuous and the DNA inside it is fragmented
(karyorrhexis) which are the hallmark of apoptotic cells (Figure 8).
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Figure 7. Effects of 3a and 3e on the cellular expression and nuclear translocation of cleaved-caspase 3 in
human colon cancer cells (HCT116). Immunofluorescence staining of cleaved-caspase 3 expressions in human
HCT116 cells treated for 24 h with 3a, and 3e. The cells were stained with DAPI to visualize nuclei (blue) and
with Cy3-coupled secondary antibodies to visualize the distribution of caspase-3 (red). Scale bar, 50µm.
(Color figure can be accessed in the online version.)
Figure 8. Effects of 3a and 3e treatment on the nuclear structures of HCT116 cells. HCT116 cells were grown
on cover slips and treated with 3a or 3e. Cells were incubated for 24 h and fixed in methanol / 0.02% EDTA.
The cells were stained with DAPI to visualize the nuclei. Treated cells show nuclear condensation and
fragmentation along with the condensed blue fluorescence of DAPI.
(Color figure can be accessed in the online version.)
3. DISCUSSION
The results of both cytotoxicity and cell cycle analysis indicated that the 2-styryl-4-
imidazolone scaffold (styryl group conjugated to an electron-withdrawing moiety) was a
promising antiproliferative pharmacophore. In 39 cytotoxicity tests, 19 times the IC50 was at a
low micromolar (<5 µM) range. The advantage of the new compounds was highlighted by
being safe on normal cells (Compounds 3a and 3e) with IC50 values higher than 100 µM. It is
worthy to mention that the 2-phenyl analogue of 3e (5-benzylidene-3-(4-hydroxyphenyl)-2-
Chemical and Pharmaceutical Bulletin Advance Publication

phenyl-3,5-dihydro-4H-imidazol-4-one) was reported to be inactive against MCF7 cancer cell
line2 while our 2-styryl derivative 3e exhibited good cytotoxicity (IC50=3.9 µM) against the
same cell line. This underscores the importance of the 2-styryl group for the cytotoxic
activities.
Even though some compounds (3h, 3l and 3m) showed proliferation inhibition activities
(under 100 µM) against BHK and WI-38 cell lines, their selectivity index were still high
against cancer cells over normal cells. This was interpreted based on selectivity that ranged
from 8-25 times provided that IC50 values against cancer cell lines are <10 µM. The
structure-activity relationships (SAR) revealed that the antiproliferative potencies seemed
less affected by the type of N3-substitution. The less clear SAR is usually complicated by
pharmacokinetic factors in cell-based phenotypic screening.27
The cell cycle analysis of HCT116 cancer cells, but not MCF7, confirmed the similarity of
cellular behavior of our compounds with that of BCA, a cinnamaldehyde analogue (Figure
1).24 BCA and our compounds 3a and 3e caused accumulation of cells in the pre-G phase.
This cell cycle effect was confirmed by the presence of a pre-G peak in the cell cycle profile
analysis and that may have resulted from degradation or fragmentation of genetic material,
indicating a possible role for apoptosis in their cell growth inhibition activities. Moreover,
compound 3a induced a significant increase in the S-phase population with a concomitant
decrease in the G0/G1 population, indicating S-phase arrest. Comparing the cell cycle effects
of our compounds on HCT116 colon cancer cells and MCF7 breast cancer cells indicated that
the mechanism of cytotoxic activities differ between the two cell lines. Therefore, we
postulated that MCF7 cells might be killed by another mechanism other than ROS induction
and could not pursue the investigation on ROS elevation markers on MCF7. We focused our
subcellular studies on HCT116 cells and conducted several confirmatory tests to measure the
Chemical and Pharmaceutical Bulletin Advance Publication

pro-oxidant activities of our compounds on this type of cancer cells. In addition, the potent
anticancer compounds 3h, 3l and 3m that showed some cytotoxic activities against normal
cell lines (BHK and WI-38) were also excluded from mechanistic investigations.
In tracking the pro-oxidant effect of 2-styryl-4-imidazolone derivatives (3a and 3e), we
followed models from previous studies that focused on cellular markers of oxidative stress
rather than isolated target screening as several pathways might be involved in producing
higher oxidative stress inside the cancer cells.28-29 Four cellular markers were selected here,
the non-enzymatic glutathione (GSH, reduced marker) and malondialdehyde (MDA, oxidized
marker) as well as redox enzymes catalase and superoxide dismutase (SOD). The effect of
the two potent compounds 3a and 3e on the ROS markers were significantly different. The N-
(4-hydroxyphenyl) analogue 3e consistently decreased the cellular activities associated with
CAT and SOD. Moreover, it decreased the cellular level of GSH with concomitant increase
in the lipid peroxidation product MDA. This indicates universal interruption in the cellular
redox system, which is a hallmark of apoptosis.30 The NH analogue 3a induced only a slight
increase in ROS parameters indicating a possible different cytotoxic mechanism than 3e.
Nonetheless, we found strong evidence for apoptotic cell death upon treatment of HCT116
cells by both compounds. Our observation of cleaved caspase 3 over expression and nuclear
translocation after treatment by 3a and 3e within HCT116 cell offered another evidence of
apoptosis induction via ROS generation. Cleaved caspase 3 induction of cell death is
consistent with studies on tCA, BCA (Figure 1).21, 24
The morphological changes of HCT116 cell nuclei further confirmed the pro-apoptotic
activity of compounds 3a and 3e (Figure 7). The nuclear morphology shows chromatin
condensation (evidenced by an increase in cell membrane permeability and uptake of DAPI),
leaving a stronger blue fluorescence, pyknosis and karyorrhexis. All these features are
Chemical and Pharmaceutical Bulletin Advance Publication

considered as the hallmark of apoptotic cells.31 All above mechanistic studies confirmed that
the cytotoxic activities of 3a and 3e are similar but not identical between the two compounds.
Compound 3e clearly induces apoptotic changes via ROS induction. However, compound 3a
also induced cell apoptosis but it was not clear that ROS induction is the dominant factor in
cell death.
4. CONCLUSION
In this work, we developed 4-imidazolone derivatives bearing 2-styryl group with fixed
benzylidene sustituent on C-5 and variable substituents on N-3 as potent cytotoxic agents
against colon, breast and hepatic cancer cell lines. Two compounds (3a and 3e) could
selectively inhibit cancer cell growth by the induction of oxidative stress similar to the styryl
analogue 2`-benzyloxycinnamaldehyde (BCA) as revealed by several experiments on ROS
markers, apoptotic markers and cell-cycle changes. This research is preceded by only few
reports about the cancer cell inhibition profiles of 4-imidazolone derivatives especially in the
area of ROS elevation. In the future, we are looking forward to further investigate the
pharmacokinetics and establish a stronger pharmacological profile for the discussed
compounds, including in vivo toxicity and efficacy studies. New variations could be
introduced in the future to investigate the structure-activity relationship of substitutions on
the phenyl rings of the benzylidene and styryl moieties. Finally, the highly selective
anticancer of potent compound with virtually no toxicity against normal cell lines is indeed
encouraging to pursue these 4-imidazolone analogues for drug development.
5. EXPERIMENTAL
5.1. General
All melting points were uncorrected and measured using the capillary melting point
instrument BI 9100 (Barnstead Electrothermal, UK). Infrared spectra were recorded on a
Chemical and Pharmaceutical Bulletin Advance Publication

Thermo Scientific Nicolet iS10 FT-IR Spectrometer (King Fahd Center for Medical Research,
King Abdulaziz University, Jeddah, Saudi Arabia). In this report, we only listed the important
IR stretching bands, including NH, OH, CH, C=O, C=N and/or C=C. In FT-IR, all samples
were measured neatly. 1H NMR spectra were determined on an AVANCE-III 600 MHz and
AVANCE-III HD 850 MHz spectrometers (Bruker, Germany), and chemical shifts were
expressed as ppm against TMS as an internal reference (King Fahd Center for Medical
Research and Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia). LC/MS
analysis were performed on an Agilent 6320 Ion Trap HPLC–ESI-MS/DAD (Santa Clara,
CA, USA) with the following settings: The analytes were separated using an Macherey-Nagel
Nucleodur-C18 column (150 mm length × 4.6 mm i.d., 5 μm) (Macherey-Nagel GMBH &
Co. KG, Duren, Germany). Mobile phase; isocratic elution using a mixture of isopropanol
and 0.01M ammonium acetate in water (65 : 35, v/v). The flow rate was 0.4 mL/min.; total
run time = 20 min. Purities were reported according to percentage of Peak Areas at
wavelength 280 nm. Microanalyses were operated using Vario, an Elmentar apparatus
(Shimadzu, Japan), Organic Microanalysis Unit (Cairo University, Giza, Egypt) . Column
chromatography was performed on a silica gel 60 (particle size 0.06 mm - 0.20 mm).
5.2. Chemical Synthesis
The previously reported compounds cinnamoylglycine 1,32 azlactone 233 and imidazolone
derivatives 3a,34 3b,35 and 3c33 were prepared according to procedures reported below, and
their physical and spectral properties were confirmed. Purity of compounds were first
assessed qualitatively using Thin Layer Chromatography (TLC), 1H NMR and quantitatively
using LC/MS (UV detection at 254 nM). A compound was screened only if purity was
confirmed to be above 95%.
(4Z)-Benzylidene-1-(4-fluorophenyl)-(2E)-styryl-5(4H)-imidazolone (3d).
Chemical and Pharmaceutical Bulletin Advance Publication

A mixture of the oxazolone 2 (2 mmol, 0.554 g) and 4-fluoroaniline (2 mmol, 0.222 g) in
acetic acid (10 mL) containing freshly fused sodium acetate (0.04 g, 0.5 mmol) was heated at
90°C with constant stirring for 6 h. Upon completion of the reaction, the excess acid was
made alkaline with 10% NHCO3 (30 mL) solution and extracted with EtOAc (2 x 25 mL).
The combined organic extract was washed with water, then 1 M HCl (25 mL), followed by
water and brine and then dried with Na2SO4. After solvent removal to dryness under reduced
pressure, the residue was subjected to flash column chromatography (petroleum ether / DCM
/ MeOH. The product was isolated as an orange solid (0.622 g, 74%), mp 200°C. 1H NMR
(600 MHz, CDCl3) δH ppm 8.32 (2H, d, J = 7.15 Hz), 8.09 (1H, d, J = 15.81 Hz), 7.51-7.55
(3H, m), 7.50 (1H, s), 7.46 (1H, d, J = 7.53 Hz), 7.38-7.43 (3H, m) 7.35 (2H, dd, J = 9.03,
4.89 Hz), 7.25-7.30 (3H, m), 6.60 (1H, d, J = 16.19 Hz); IR (FT-IR, cm–1): 3065.7, 1708.7,
1622.6, 1508.4; LC-MS (ESI) RT = 11.5 min, m/z 369.3[M + H]+.
(4Z)-Benzylidene-1-(4-hydroxyphenyl)-(2E)-styryl-5(4H)-imidazolone (3e).
This compound was prepared according to the procedure described for the synthesis of 3d
starting from oxazolone 2 (2 mmol, 0.554 g) and 4-aminophenol (2 mmol, 0.218 g) as a
greenish yellow solid (0.49 g, 65%), mp 286-289°C. 1H NMR (600 MHz, DMSO-d6) δH ppm
8.38 (2H, d, J = 7.5 Hz), 7.97 (1H, d, J = 15.81 Hz), 7.64 (2H, m), 7.55-7.50 (2H, m), 7.48
(1H, m), 7.44-7.40 (3H, m), 7.21 (2H, m), 7.15 (1H, s), 6.94 (2H, m), 6.63 (1H, d, J = 16.1
Hz); IR (FT-IR, cm–1): 3322.3, 3027.7, 1691.3, 1617.6, 1506.19. LC-MS (ESI) RT = 6.2 min,
m/z 367.2 [M + H]+. Anal. Calcd for (C24H18N2O2) C, 78.67; H, 4.95; N, 7.65; O, 8.73; found
C, 78.98; H, 4.85; N, 7.48.
(4Z)-Benzylidene-1-(3-cyanophenyl)-(2E)-styryl-5(4H)-imidazolone (3f).
This compound was prepared according to the procedure described for the synthesis of 3d
starting from a mixture of the oxazolone 2 (2 mmol, 0.554 g) and the 3-aminobenzonitrile (2
Chemical and Pharmaceutical Bulletin Advance Publication

mmol, 0.236 g) as a crystalline yellow solid (0.352 g, 47%), mp 227-228°C. 1H NMR (600
MHz, CDCl3) δH ppm 8.32 (2H, d, J = 7.5 Hz), 8.13 (1H, d, J = 15.81 Hz), 7.81 (1H, dt, J =
7.81 and 1.18 Hz), 7.73 (2H, m), 7.63 (1H, m), 7.57-7.51 (4H, m), 7.58 (1H, m), 7.44-7.41
(3H, m), 7.31 (1H, s), 6.57 (1H, d, J = 15.8 Hz); IR (FT-IR, cm–1): 3082.4, 2231.0, 1718.4,
1625.4, 1508.54. LC-MS (ESI) RT = 9.0 min, m/z 376.3[M + H]+.
(4Z)-Benzylidene-(2E)-styryl-1-(3-trifluoromethylphenyl)-5(4H)-imidazolone (3g).
This compound was prepared according to the procedure described for the synthesis of 3d,
starting from oxazolone 2 (2 mmol, 0.554 g) and 3-trifluoromethylaniline (2 mmol, 0.322 g).
The product 3g was isolated as a brownish red crystalline solid (0.622 g, 74%), mp 202-
204°C. 1H NMR (600 MHz, CDCl3) δH ppm 8.30 (2H, d, J = 7.5 Hz), 8.12 (1H, d, J = 15.81
Hz), 7.78 (1H, d, J = 7.91 Hz), 7.73 (1H, t, J = 7.72 Hz), 7.67 (1H, s), 7.58 (1H, d, J = 7.53
Hz), 7.53 (4H, m), 7.47 (1H, m), 7.43-7.39 (3H, m), 7.31 (1H, s), 6.60 (1H, d, J = 15.81 Hz);
IR (FT-IR, cm–1): 3060.4, 1710.3, 1625.4, 1510.1.
(4Z)-Benzylidene-1-(3-pyridyl)-(2E)-styryl-5(4H)-imidazolone (3i).
This compound was prepared according to the procedure described for the synthesis of 3d
starting from a mixture of the oxazolone 2 (2 mmol, 0.554 g) and the 3-aminopyridine (2
mmol, 0.188 g). The product 3i was collected yellow solid (0.315, 44%), mp 222-223 °C.
The 1H NMR (600 MHz, CDCl3) results were as follows: δH ppm 8.76 (1H, d, J = 4.14 Hz),
8.67 (1H, s), 8.32 (2H, d, J = 8.66 Hz), 8.14 (1H, d, J = 15.81 Hz), 7.77 (1H, dd, J = 10.54,
1.51 Hz), 7.57-7.51 (5H, m), 7.48 (1H, d, J = 7.53 Hz), 7.41 (3H, d, J = 4.14 Hz), 7.32 (1H,
s), 6.61 (1H, d, J = 15.81 Hz); IR (FT-IR, cm-1) 3062.5, 1714.4, 1624.9, 1479.9; LC-MS
(ESI), RT = 6.5 min, m/z 352.3 [M + H]+.
(4Z)-Benzylidene-1-(5-methylthiazol-2-yl)-(2E)-styryl-5(4H)-imidazolone (3j).
Chemical and Pharmaceutical Bulletin Advance Publication

This compound was prepared according to the procedure described for the synthesis of 3d
starting from oxazolone 2 (2 mmol, 0.554 g) and 2-amino-5-methylthiazole (2 mmol, 0.228
g). The product 3j was collected as a yellow crystalline solid (0.16 g, 21%), mp 144-145 °C.
1H NMR (600 MHz, CDCl3) δH ppm 8.32 (2H, d, J = 7.15 Hz), 8.24-8.14 (2H, m), 7.71 (2H,
d, J = 6.78 Hz), 7.56-7.50 (2H, m), 7.50-7.41 (4H, m), 7.38 (1H, d, J = 1.13 Hz), 7.32 (1H, s),
2.54 (3H, s); IR (FT-IR, cm–1): 3052.8, 2977.7, 2919.1, 1713.5, 1636.0, 1513.64.
(4Z)-Benzylidene-(2E)-styryl-1-(1,3,4-thiadiazol-2-yl)-5(4H)-imidazolone (5k).
This compound was prepared according to the procedure described for the synthesis of 3d,
starting from oxazolone 2 (2 mmol, 0.554 g) and 3-trifluoromethylaniline (2 mmol, 0.202 g).
The product was isolated as a yellow solid (0.197 g, 27%), mp 219-222°C. 1H NMR (600
MHz, CDCl3) δH ppm 8.73 (1H, d, J = 4.52 Hz), 8.65 (1H, d, J = 1.88 Hz), 8.30 (2H, d, J =
7.15 Hz), 8.12 (1H, d, J = 15.81 Hz), 7.56-7.47 (4H, m), 7.45 (1H, d, J = 7.15 Hz), 7.40-7.36
(2H, m), 7.31 (1H, s), 6.60 (1H, d, J = 15.81 Hz).
(4Z)-Benzylidene-1-(furan-2-ylmethyl)-(2E)-styryl-5(4H)-imidazolone (3l).
This compound was prepared according to the procedure described for the synthesis of 3d,
starting from oxazolone 2 (2 mmol, 0.554 g) and furfurylamine (2 mmol, 0.177 mL). The
product was isolated as a yellow solid (0.118 g, 16%), mp 145-147°C. 1H NMR (600 MHz,
CDCl3) δH ppm 8.29-8.23 (2H, m), 8.15 (1H, d, J = 15.81 Hz), 7.66 (2H, dd, J = 7.91, 1.13
Hz), 7.51-7.38 (7H, m), 7.22 (1H, s), 7.02 (1H, d, J = 15.81 Hz), 6.39-6.34 (2H, m), 4.98 (2H,
s); IR (FT-IR, cm–1): 3065.2, 3030.4, 2955.9, 1705.5, 1625.7, 1508.2; LC-MS (ESI) RT =
19.4 min, m/z 355.2 [M + H]+.
(4Z)-Benzylidene-1-[2-(4-morpholinyl)ethyl]-(2E)-styryl-5(4H)-imidazolone (3m).
Chemical and Pharmaceutical Bulletin Advance Publication

This compound was prepared according to the procedure described for the synthesis of 3d,
starting from oxazolone 2 (2 mmol, 0.554 g) and 2(4-morpholinyl)ethylamine (2 mmol, 0.262
mL). The product was isolated as a yellow solid (0.235 g, 30%), mp 186-187. 1H NMR (600
MHz, CDCl3) δH ppm 8.27 (2H, d, J = 7.53 Hz), 8.19 (1H, d, J = 15.43 Hz), 7.74-7.66 (2H,
m), 7.54-7.40 (6H, m), 7.19 (1H, m), 6.96 (1H, d, J=13.93 Hz), 3.96 (1H, br s), 3.72-3.69 (3H,
m), 2.69-3.62 (4H, br m), 2.12 (4H, s); IR (FT-IR, cm–1): 3032.1, 2926.17, 1689.3, 1626.0,
1512.8.
5.3. Biological Screening
5.3.1. Cell culture:
Human breast adenocarcinoma cell lines (MCF-7), Human colon adenocarcinoma
(HCT-116), Human hepatocellular carcinoma (HepG-2), baby hamster kidney cells (BHK-
21) and human lung tissue fibroblast (WI-38) were originally purchased from American type
culture collection (ATCC, Wesel, Germany) and grown in the tissue culture lab of the
Egyptian company for production of vaccines, sera and drugs (Vacsera, Giza, Egypt). The
cells were transferred to our laboratory and maintained in Dulbecco Modified Eagle's
medium (DMEM) or Roswell Park Memorial Institute medium (RPMI1640) both are
supplemented with 1% of 100 mg/ mL of streptomycin, 100 units/ mL of penicillin and 10%
of heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) in a humidified, 5% (v/v)
CO2 atmosphere at 37 ºC.
5.3.2. Cytotoxicity Assays
The sulforhodamine B (SRB) assays were performed according to Skehan et al.18
Briefly, exponentially growing cells were trypsinized, counted and seeded at the appropriate
densities (5000 cells/100 µL/ well) into 96-well microtiter plates. Cells were incubated in a
Chemical and Pharmaceutical Bulletin Advance Publication

humidified atmosphere at 37°C for 24 h. Then, the cells were exposed to different compounds
at the desired concentrations, (0.01, 0.1, 1, 10, 100 and 1000µM) or to 1% dimethyl sulfoxide
(DMSO) for 72 h. At the end of the treatment period, the media were removed, and the cells
were fixed with 10% trichloroacetic acid at 4ºC for 1 h. The cells then were washed with tap
water four times and incubated with SRB 0.4% for 30 min. Excess dye was removed by
washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye was dissolved in 10
mM Tris base solution for OD determination at 510 nm using a SpectraMax plus Microplate
Reader (Molecular Devices, CA). Cell viability was expressed relative to the untreated
control cells and the IC50s were calculated using Graph pad prism 5 software (GraphPad
Software, Inc., CA, USA).
5.3.3. Cell Cycle Analysis
To analyze the DNA content by flow cytometry, different cell lines were seeded at a density
of 2 x 106 cell / T75 flask for 24 h; then, the cells were exposed to different compounds at
their IC50 concentration for 24 h. The cells were collected by trypsinization, washed with
phosphate-buffered saline (PBS) and fixed in ice-cold absolute alcohol. Thereafter, cells were
stained using CycletestTM Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA)
according to the manufacturer’s instructions. Cell cycle distribution was determined using a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
5.3.4. Effect on Redox Regulator Activities in HCT116 Cells
HCT116 cells were incubated with the IC50 of compounds 3a and 3e for 48 h. At the end of
treatment period, cells were washed thrice with PBS, collected by trypsinization, total
protein content were extracted by sonication in ice cold PBS in the dark, protein amounts
were quantified using Bio-rad DC protein assay kit. Equal amount of protein lysate were used
for determination of reduced glutathione (GSH) content36, the content of lipid peroxidation
Chemical and Pharmaceutical Bulletin Advance Publication

product, malondialdehyde (MDA)37, enzymatic activities of catalase (CAT)38 and superoxide
dismutase(SOD)39.
5.3.5. Effect on Superoxide Anion Radical Generation in HCT116 Cells
.-
The superoxide anion (O2 ) production was tested by dihydrethidium (DHE) staining within
HCT116 cancer cells (Figure 5). HCT116 cells were cultured on sterile cover slips (Harvard
Apparatus, 22 mm2) in a 6-well plate at a density of 2 ×105 cells/well. Cells were allowed to
attach to the cover slips for 24 hours in a humidified atmosphere at 37ºC and 5% CO2. The
cells were then exposed to compounds 3a and 3e for 24 h. At the end of the exposure time,
the growth medium was removed, followed by washing thrice with phosphate buffered saline
.-
(PBS). The cells were then incubated with the O2 probe dihydroethidium (DHE), 10µM, and
the nuclear stain 4', 6-Diamidino-2-phenylindole, dihydrochloride (DAPI), 1µM, for 30 min
at 37°C. After incubation, cells were washed thrice with PBS, a drop of mounting medium
was added to a slide, and the cover slips were inverted on the mounting medium and sealed.
Different fields were captured at 20X magnification power using a fluorescent microscope
(Leica DM 5500B).
5.3.6. Effect on Cleaved- Caspase 3 protein expression in HCT116 Cells
Cleaved caspase-3 rabbit mAb was purchased from Cell Signaling Technology (Cell
Signaling Technology, MA), A Cy3-conjugated goat anti-rabbit antibody was purchased from
Jackson ImmunoResearch (West Grove, PA, USA). 4′,6′-Diamidino-2-phenylindole (DAPI)
was purchased from Sigma–Aldrich (St. Louis, MO, USA).
HCT116 cells were cultured on 22-mm2 sterile coverslips (Harvard Apparatus) in sterile six-
well plates at a density of 2 × 105 cells/well. Cells were exposed to IC50 concentration of
compounds 3a, 3e or 1% DMSO in fresh serum-free medium for 24 h. At the end of the
exposure, cells attached to cover slips were washed with PBS and fixed with ice cold
Chemical and Pharmaceutical Bulletin Advance Publication

methanol / 0.01% ethylenediaminetetraacetic acid (EDTA) for 20 min. After fixation, cells
were blocked for 30 min at room temperature with 0.1% Triton X-100 / PBS containing 5%
bovine serum albumin (BSA), washed with PBS and incubated for 45 min with the primary
antibody (diluted in 5% BSA / PBS / 0.1% Triton X-100) as indicated. Following washing,
the bound antibodies were detected by Alexa 488-conjugated goat anti-rabbit Cy3 secondary
antibodies. To stain the nuclei, cells were incubated with DAPI, and the cells were mounted
in Fluoromount G (Biozol) and analyzed with the Leica DM 5500 B fluorescence microscope
(Leica, Buffalo Grove, IL, USA).
ACKNOWLEDGMENTS
This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz
University, Jeddah, under grant No. (166-015-D1434). The authors, therefore, acknowledge
with thanks DSR technical and financial support. We appreciate the efforts of Mr. Magdy
Ghazy, Mr. Mohamed Elmehyawy (NMR Unit, King Fahd Center for Medical Research,
KAU) and Mr. Ahmed Kammona (Faculty of Pharmacy, KAU) to perform NMR and LC-MS
analyses of new compounds. We also appreciate the efforts of Mr. Amr El-Araby for
reviewing and editing the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

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