Microwave-assisted synthesis of new 2-aryl and 2-alkylimidazolones and evaluation of their in vitro anticancer activity and their in vivo toxicity on zebrafish embryos

Article abstract of DOI:10.1007/s11696-020-01502-w

Herein we describe the synthesis of five new 2-aryl and 2-alkylimidazolone derivatives via an effective one-pot synthetic strategy assisted by microwave irradiations which allowed us to access the desired product in a reduced time reaction compared to the

Full text of DOI:10.1007/s11696-020-01502-w

Chemical Papers (2021) 75:2549–2560
https://doi.org/10.1007/s11696-020-01502-w
ORIGINAL PAPER
Microwave‑assisted synthesis of new 2‑aryl and 2‑alkylimidazolones
and evaluation of their in vitro anticancer activity and their in vivo
toxicity on zebraꢀsh embryos
Samar Bou Zeid1,2 · Aline Hamade · Fadia Najjar · Francois Carreaux · Samar Eid
1
1
2
1
Received: 4 September 2020 / Accepted: 30 December 2020 / Published online: 19 January 2021
©
Institute of Chemistry, Slovak Academy of Sciences 2021
Abstract
Herein we describe the synthesis of ꢀve new 2-aryl and 2-alkylimidazolone derivatives via an eꢁective one-pot synthetic
strategy assisted by microwave irradiations which allowed us to access the desired product in a reduced time reaction com-
pared to the thermal heating and a slightly better yield (48% compared to 45%). The new imidazolone derivatives were
evaluated for their anticancer activity in vitro against MCF-7, MDA-MB-231 and HepG2 cell lines. The results showed good
cytotoxic eꢁects for some of these derivatives on both MCF-7 and HepG2 cell lines in the range of 5.7–11.3 µM. Among the
synthesized derivatives, 2ab and 2b showed the strongest activity with IC values of 7 and 5.7 µM (MCF-7) and 6.2 and
5
0
8
.6 µM (HepG2), respectively. The cytotoxic activities of these derivatives were moderate compared to those of doxorubicin.
However, this product showed higher toxicity in vivo on the development of zebraꢀsh embryos than the synthesized imida-
zolones. These derivatives at high concentrations exhibited some morphological abnormalities on the embryos.
Keywords Imidazolone derivatives · One-pot synthesis · Microwave irradiations · Anticancer activity · Zebraꢀsh embryos
Introduction
2017). Hepatocellular carcinoma (HCC) is considered the
sixth most frequently diagnosed cancer and the fourth lead-
ing cause of death from cancer in the world (Bray et al.
2018). Breast tumors are very heterogeneous and can be
classiꢀed as tumors expressing estrogen and progesterone
receptors (ER+, PR+), tumors expressing ampliꢀed human
epidermal receptors (HER2+) and tumors not expressing
these receptors, called triple-negative breast (ER−, PR−,
HER2−) (Dai et al. 2017). As for the HCC, studies have
shown an overexpression of HER2 protein in several HCC
cell lines (Shi et al. 2019). Considerable eꢁorts have been
invested in establishing eꢂcient treatment strategies of
which the most commonly used are surgery, radiotherapy
and drug treatments (You et al. 2007; Luqmani 2005). In
spite of being eꢁective in the treatment of several types
of cancer, especially in the case of primary tumors, these
strategies can induce tumor heterogeneity resulting in the
reduction of the therapeutic agents eꢂciency and leading
to treatment failure and tumor progression (Foo and Michor
Cancer is a major public health problem worldwide; it
is considered the second leading cause of death globally
(
Wild et al. 2020). In particular, breast cancer is rated as
the second most commonly diagnosed cancer and the lead-
ing cause of cancer death among females (DeSantis et al.
Supplementary Information The online version of this article
(
https://doi.org/10.1007/s11696-020-01502-w) contains
supplementary material, which is available to authorized users.
*
*
*
Fadia Najjar
fnajjar@ul.edu.lb
Francois Carreaux
francois.carreaux@univ-rennes1.fr
Samar Eid
s.eid@ul.edu.lb
1
2
Departments of Biology, Chemistry and Biochemistry,
Laboratoire D’Innovation Thérapeutique, Faculty
of Sciences II, Lebanese University, Fanar, Lebanon
2
014; Luqmani 2005). Thus, it is necessary to develop new
targeted therapies aiming speciꢀc cellular mechanisms.
Many natural products classiꢀed as leads to potential drugs,
contribute signiꢀcantly in the drug discovery and develop-
ment process, especially in cancer treatment (Newman and
Université de Rennes 1, CNRS, ISCR, UMR 6226,
2
3
63 avenue du Général Leclerc, Campus de Beaulieu,
5000 Rennes, France
Vol.:(0
1
123456789)
3

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Chemical Papers (2021) 75:2549–2560
Cragg 2012). As a matter of fact, marine natural products
isolated from marine sponges and organisms, with 1490
new products extracted and identiꢀed in 2017 (Carroll et al.
imidate salts that required the passage of a continuous ꢃow of
hydrogen chloride gas over a solution of the nitriles and alco-
hol in ether or benzene. A new method for synthesizing these
imidate salts by generating hydrogen chloride in situ (Yadav
and Babu 2005), made the Kidawi’s one-pot approach much
more interesting. In light of the above evidence and inspired by
the biological activity of imidazolone derivatives, an eꢁective
one-pot microwave-assisted synthesis was applied to access a
library of new 2-aryl and 2-alkylimidazolone derivatives as
potential anticancer agents (Scheme 1). This non-conventional
heating has emerged as an eꢂcient green method to enhance
products yield and to reduce reaction time (Kappe and Dal-
linger 2009; Strauss and Varma 2006).
2019), are being explored for their diꢁerent medical applica-
tions (Mayer et al. 2011; Villa and Gerwick 2010). Among
these natural marine products, several molecules based on a
2
-aminoimidazolone core, such as leucettamine B (Boehm
et al. 1993; Chan et al. 1993), have shown interesting bio-
logical properties. Leucettamine B (Fig. 1) and its synthetic
N-functionalized analogs have manifested signiꢀcant inhibi-
tory activities against protein kinases, CLK1 [CDC (cyclin-
dependent kinase)-like kinase], and DYRKs (dual-speciꢀcity
tyrosine phosphorylation-regulated kinases) (Burgy et al.
2
013; Tahtouh et al. 2012; Debdab et al. 2010, 2011). In the
The cytotoxic activity of the new compounds was then
evaluated in vitro on the breast cancer cell lines MCF-7, MDA-
MB-231 and HCC cell line HepG2. Furthermore, we evaluated
their toxic eꢁect in vivo using the zebraꢀsh model, considered
as a versatile animal model due to the simplicity of its natural
habitat, low cost and ease of breeding (Newman et al. 2014).
The ex-utero development of its transparent embryos allowed
a clear visualization of the development process (Meyers 2018;
Link and Megason 2008) and the evaluation of the toxico-
logical eꢁects of the newly synthesized imidazolones on this
process.
last years the imidazolone scaꢁold has attracted attention
as a potential block for the development of new eꢁective
anticancer agents (Hsu et al. 2020; Abdel Gawad et al. 2016;
Ling et al. 2013; Xiao et al. 2013). Moreover, analogs of
type 2-aryl or 2-alkylimidazolones (Fig. 1) have also been
synthesized (Mokale et al. 2014; Voosala et al. 2014; Shi
et al. 2012; Fozooni and Tikdari 2008); however, the evalu-
ation for their anticancer activity is limited.
In fact, some synthesized (Z)-2-aryl-5-arylmethylideneim-
idazolones were reported to have potent cytotoxic eꢁects
against epithelial carcinoma cancer cell line A549 (Beloglaz-
kina et al. 2016). Eldehna et al. (2019) also reported the syn-
thesis and biological evaluation of novel 4-benzylidene-2-phe-
nyl-imidazolone-based benzenesulfonamides that showed
anti-proliferative activity against colorectal cancer HCT-116
and breast cancer MCF-7 cell lines. The synthetic methodolo-
gies adopted to access these imidazolone derivatives have been
established on the transformation of oxazolone nuclei into imi-
dazolone unit according to several operating conditions. An
eꢂcient multi-component strategy was developed by Kidwai
and Devasia (1962), involving the reaction of the ethyl ester
of glycine with imidate esters and aromatic aldehydes. Nev-
ertheless, it was less adopted due to the tedious preparation of
Experimental
Chemistry
Chemicals, reagents and instruments
Solvents and all other chemical reagents were purchased from
Acros, Sigma-Aldrich and Fluka and were used without fur-
ther puriꢀcation. Solvents were dried by reꢃuxing with the
appropriate drying agents and distilled before use. Toluene
was distilled over sodium and stored over molecular sieves
(3 Å), and ethanol was distilled over calcium hydride (CaH ).
2
Melting points were measured on a melting point appara-
1
tus Stuart SMP10 and were not corrected. H NMR spectra
were recorded on BRUKER AC 300 and AC 400 (300 and
13
4
00 MHz) and C NMR spectra on BRUKER AC 300 and
AC 400 (75 and 101 MHz) spectrometer. Chemical shifts are
expressed in parts per million downꢀeld from tetramethylsi-
lane as an internal standard. Data are given in the following
order: δ value, multiplicity (s: singlet; d: doublet; t: triplet; q:
quartet; m: multiplet; br: broad), number of protons, coupling
constant J given in Hertz. All high-resolution mass spectra
Fig. 1 Structure of leucettamine B and targeted 2-aryl and 2-alkylimi-
dazolones
Scheme 1 Microwave-assisted
synthetic strategy to imida-
zolone derivatives
1
3

Chemical Papers (2021) 75:2549–2560
2551
(
HRMS) were recorded on a Bruker Micro-Tof-Q II or on a
DMSO, δ ppm): 7.89 (s, 1H), 7.71 (d, J = 7.7 Hz, 1H),
7.38–7.27 (m, 5H), 7.24–7.15 (m, 1H), 6.97 (dd, J=7.9,
2.7 Hz, 1H), 6.82 (s, 1H), 3.78 (s, 3H), 3.04 (t, J=7.7 Hz, 2H),
Waters Q-Tof 2 at the CRMPO (Centre Régional de Mesures
de Physiques de l’Ouest–Rennes–France) using positive ion
electrospray. Reactions under microwave irradiations were
realized in the apparatus Anton Paar’s Monowave® 300 (Scan-
Mat Platform, Rennes, France). The microwave instrument
consists of a continuous focused microwave power output from
13
2.85 (t, J=7.7 Hz, 2H). C NMR (101 MHz, DMSO, δ ppm):
172.3, 167.1, 159.7, 141.1, 140.7, 135.9, 130.0, 128.8, 128.8,
126.6, 124.9, 124.0, 117.0, 116.1, 55.5, 31.8, 31.3. HRMS
+
(ESI+): m/z calcd for C H N O Na [M+Na] : 329.1260,
19
18
2
2
0
to 850 W. All experiments were performed using stirring
found 329.1264.
option at 600 rpm. The target temperature was reached within
the required time, and the chosen microwave maximal power
was set to 100 W. The reaction temperature is monitored using
calibrated infrared sensor and the reaction time. The imidates
salts 1a (Kumar et al. 2016), 1b (Yadav and Babu 2005), 1c
(Z)‑5‑(4‑methoxybenzylidene)‑2‑(4‑methoxyphenyl)‑3,
5‑dihydro‑4H‑imidazol‑4‑one 2b
General procedure starting from p-methoxybenzaldehyde
(114 mg, 0.84 mmol) and 1b (200 mg, 0.927 mmol). Yield,
(
Yadav and Babu 2005) and 1d (Shi et al. 2018) were prepared
1
as previously described.
70%; yellow solid, mp 262–264 °C. H NMR (300 MHz,
DMSO, δ ppm): 8.28 (d, J=8.9 Hz, 2H), 8.12 (d, J=8.9 Hz,
2H), 7.14 (d, J=8.9 Hz, 2H), 7.06 (d, J=8.8 Hz, 2H), 6.91
General procedure for the synthesis of 5‑ben‑
zylidene‑2‑aryl/2‑alkyl‑3,5‑dihydro‑4H‑imidazol‑4‑one 2
1
3
(s, 1H), 3.87 (s, 3H), 3.84 (s, 3H). C NMR (101 MHz,
DMSO, δ ppm): 172.6, 163.0, 161.0, 159.7, 139.3, 134.2,
129.6, 127.8, 124.3, 120.9, 114.9, 114.8, 56.0, 55.8. HRMS
Glycine methylester hydrochloride (1.5 eq), the correspond-
ing acetimidate hydrochloride (1.1 eq) and sodium bicarbo-
nate (3 eq) were poured in the microwave vial, ꢃushed with
argon, and anhydrous toluene (0.8 mL) was added to the
powders. To the suspension was then added the aldehyde
+
(ESI+): m/z calcd for C H N O Na [M+Na] : 331.1053,
18
16
2
3
found 331.1056.
(Z)‑2‑(4‑chlorophenyl)‑5‑(4‑methoxybenzylidene)‑3,5‑dihy‑
dro‑4H‑imidazol‑4‑one 2c
(1 eq), the vial was then placed in the Anton Paar monowave
reactor, and the reaction was stirred during 15 min at 150 °C
and at 100 W of maximum power. After the reaction com-
pletion, the suspension was allowed to precipitate at room
temperature. The yellow solid obtained was collected by
General procedure starting from p-methoxybenzaldehyde
(112 mg, 0.826 mmol) and 1c (200 mg, 0.91 mmol). Yield,
1
64%; yellow solid, mp 272–274 °C. H NMR (300 MHz,
ꢀ
ltration, washed twice with water, twice with cold ethanol
DMSO, δ ppm): 8.29 (d, J=8.8 Hz, 2H), 8.16 (d, J=8.6 Hz,
2H), 7.67 (d, J=8.6 Hz, 2H), 7.07 (d, J=8.8 Hz, 2H), 7.01
and twice with isopropyl ether to aꢁord the corresponding
imidazolone as a yellow powder.
1
3
(s, 1H), 3.84 (s, 3H). C NMR (75 MHz, DMSO, δ ppm):
1
72.4, 161.4, 159.2, 138.9, 137.4, 134.6, 129.6, 129.4, 127.6,
(Z)‑5‑(4‑methoxybenzylidene)‑2‑phenethyl‑3,5‑dihy‑
127.6, 126.2, 114.9, 55.8. HRMS (ESI +): m/z calcd for
+
dro‑4H‑imidazol‑4‑one 2aa
C H N O ClNa [M+Na] : 335.0557, found 335.0556.
17
13
2
2
General procedure starting from p-methoxybenzaldehyde
(Z)‑2‑(4‑bromophenyl)‑5‑(4‑methoxybenzylidene)‑3,5‑di‑
hydro‑4H‑imidazol‑4‑one 2d
(
115 mg, 0.85 mmol) and 1a (200 mg, 0.936 mmol). Yield,
1
4
8%; yellow solid, mp 181–183 °C. H NMR (400 MHz,
DMSO, δ ppm): 8.16 (d, J=8.9 Hz, 2H), 7.30 (d, J=4.4 Hz,
General procedure starting from p-methoxybenzaldehyde
(93.5 mg, 0.687 mmol) and 1d (200 mg, 0.756 mmol), Yield,
4
1
2
1
1
H), 7.23–7.17 (m, 1H), 7.02 (d, J =8.9 Hz, 2H), 6.83 (s,
1
H), 3.82 (s, 3H), 3.04 (t, J=7.8 Hz, 2H), 2.84 (t, J=7.8 Hz,
79%; yellow solid mp 286–290 °C. H NMR (300 MHz,
1
3
H). C NMR (101 MHz, DMSO, δ ppm): 172.1, 165.2,
61.1, 141.1, 138.6, 134.1, 128.8, 128.8, 127.4, 126.6,
DMSO, δ ppm): 8.29 (d, J=8.8 Hz, 2H), 8.09 (d, J=8.6 Hz,
2H), 7.81 (d, J=8.6 Hz, 2H), 7.07 (d, J=8.9 Hz, 2H), 7.03
1
3
24.5, 114.7, 55.8, 31.9, 31.6. HRMS (ESI+): m/z calcd
(s, 1H), 3.84 (s, 3H). C NMR (75 MHz, DMSO, δ ppm):
172.3, 161.4, 159.2, 138.9, 134.5, 132.5, 129.5, 127.8, 127.5,
+
for C H N O Na [M+Na] : 329.1260, found 329.1259.
1
9
18
2
2
1
26.4, 126.2, 114.9, 55.8. HRMS (ESI+): m/z calcd for
+
(Z)‑5‑(3‑methoxybenzylidene)‑2‑phenethyl‑3,5‑dihy‑
C H N O BrNa [M+Na] : 379.0052, found 379.0050.
17
13
2
2
dro‑4H‑imidazol‑4‑one 2ab
General procedure starting from m-methoxybenzaldehyde
(
115 mg, 0.85 mmol) and 1a (200 mg, 0.936 mmol). Yield,
1
2
6%; yellow solid, mp 172–174 °C. H NMR (300 MHz,
1
3

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Chemical Papers (2021) 75:2549–2560
Biological activity
under constant control. Animals were maintained under a
1
4–10-h day/night photoperiod cycle and fed three times a
Cell lines: culture conditions and viability assays
day with commercial ꢃakes and live brine shrimp. Natural
spawning was induced in the morning by light. Embryos
were then collected within 30 min and maintained in an E3
(1*x) medium in an incubator at 25 °C to minimize stress
and spontaneous deaths when treating.
Doxorubicin hydrochloride was obtained from EBEWE
Pharma Ges.m.b.H. Nfg.KG A-4866 Unterach, Austria.
MCF-7, hormone-dependent human breast cancer, MDA-
MB-231, hormone-independent human breast cancer and
HepG2 hepatocellular carcinoma cell lines were purchased
originally from ATCC (American Type Culture Collection).
MCF-7 and MDA-MB-231 cells were routinely maintained
in RPMI 1640 medium (Sigma R0883) and HepG2 in
DMEM high glucose medium (Sigma D0819), both medium
containing 1% penicillin streptomycin (Biowest MS006W)
and supplemented with 10% Fetal Bovine Serum (Sigma
F9665) and 1% L-Glutamine (Sigma G7513). The cells were
E3 medium (60*x) is the adequate medium for zebraꢀsh
embryos and was prepared as follows: 34.8 g sodium chlo-
ride, 1.6 g potassium chloride, 5.8 g calcium chloride dihy-
drate, 9.78 g magnesium chloride hexahydrate were dis-
solved in water with a ꢀnal volume 2 L to prepare a 60*x
stock. This solution was autoclaved before adjusting the pH
and diluting, after which it becomes compatible to use for
zebraꢀsh embryos.
cultured at 37 °C in a humidiꢀed incubator with 5% CO
Zebraꢀsh embryo toxicity assay: exposure to the imida‑
zolone derivatives
2
and were passaged twice a week using 1% trypsin (Sigma
T4549). The cytotoxic eꢁects of the imidazolone derivatives
were determined using the trypan blue exclusion assay. It is
based on the concept of dye exclusion to diꢁerentiate viable
cells from dead ones. Indeed, cells that possess damaged
membranes are stained blue, whereas viable cells do not
uptake the dye and remain white. MCF-7, MDA-MB-231
and HepG2 cells were seeded into 24-well culture plates
Fertilized eggs of zebraꢀsh between late blastula and early
gastrula [2.5–5.25 hpf (hours post-fertilization)] were
sorted to ensure only eggs of the same quality were used
in the experiments. This was done to minimize spontane-
ous defects and variability between diꢁerent conditions.
Zebraꢀsh embryos were then transferred to 24-well plates
(5–10 embryos per well) and were maintained in 0.5–1 mL
of E3 medium. Embryos were then exposed to diꢁerent
concentrations of imidazolone derivatives (1 µM–120 µM).
Stock solutions of these compounds at 30 mM were pre-
pared in DMSO and then diluted in E3 medium to obtain
the desired ꢀnal concentrations. The highest concentration
of DMSO following dilution was 0.5% of the total volume.
A control group (E3 1*x) and a vehicle group (0.5% DMSO)
were part of the experiment. Fish embryonic development
was observed directly using a stereoscope, and pictures were
taken using the AmScope software. The malformations
observed and the viability rates were noted and calculated
after 24, 48 and 72 h of treatment.
(
20,000/well), in a total volume of 500 μL/well. After over-
night incubation to ensure cell attachment and well-being,
the medium was aspirated and cells were exposed to the
synthesized imidazolones in duplicate at ꢀnal concentrations
ranging from 0.1 to 60 µM. Stock solutions of these com-
pounds at 30 mM were prepared in DMSO (Sigma-Aldrich
4
1,640) and then diluted in culture medium to obtain the
desired ꢀnal concentrations. The highest concentration of
DMSO following dilution was 0.2% of the total volume, an
amount that was not toxic to the cells. Control cells were
grown in culture medium. The vehicle cells were treated
with 0.2% of DMSO. Plates were incubated for 24, 48
and 72 h. At the end of each time point, the medium was
removed; the cells were collected by the action of trypsin
and counted in a hemocytometer using optical microscopy.
The results were presented as a percentage of the control. All
measurements were taken in duplicate, and each experiment
was repeated at least three times independently.
Results and discussion
Chemistry
Animals
The imidate salts used in this approach were synthesized
according to the protocol described by Yadav and Babu
(2005). This method is based on the generation of hydrogen
chloride in situ following the treatment of acyl chloride with
an alcohol (Scheme 2).
Adult wild-type zebraꢀsh (Danio rerio) of AB and Casper
strain of both sexes were purchased from UMS AMAGEN
CNRS INRA (France) and were used after ethical approval.
Animals were divided in 5-L thermostated tanks at 28± 2 °C
The imidate salts were subsequently used in the prepa-
ration of 2-aryl and 2-alkylimidazolones according to the
strategy described ꢀrst in 1962 (Kidwai and Devasia 1962),
while making modiꢀcations to the reaction conditions. It is a
(
15 ꢀsh/tank) in a constant chemical and biological con-
trolled environment according to international standards.
Mechanical water ꢀltration and aeration were also kept
1
3

Chemical Papers (2021) 75:2549–2560
2553
which resulted in a better conversion (99.7%) and better
yield (48%) of isolated product compared to thermal heat-
ing. Finally in an attempt to achieve a greener process, the
reaction was conducted in a solvent-free medium in the same
conditions as before; however, a decrease in the conversion
to 87% was noted. An increase in the heating temperature to
2
00 °C in a solvent-free medium caused a degradation of the
imidate salt and did not yield the desired product. Therefore,
in a ꢀnal attempt, in a solvent-free medium, the tempera-
ture was maintained at 150 °C and the reaction was carried
for 30 min. In these conditions the product was obtained in
Scheme 2 Preparation of imidate salts
9
2% of conversion that was still lower than the conversion
obtained in the presence of solvent. The microwave-assisted
method in a solvent-based medium has proved to be more
eꢂcient and suitable to reduce the reaction time and slightly
enhance the yield.
Subsequently, the one-pot approach was generalized to
several substrates by varying either the aldehyde or the salt
of imidates. The reactions were carried out in the micro-
wave under the same conditions as above (150 °C, 100 W,
1
5 min) leading to a small library of imidazolone derivatives
(
Fig. 2). The desired products precipitated in the toluene
and were puriꢀed by a simple ꢀltration followed by sev-
eral washes in water and ether. It was not possible to purify
them by column chromatography as they were insoluble in
several organic solvents, such as dichloromethane, ethyl
acetate and partially soluble in methanol and ethanol. All
compounds were obtained in a stereospeciꢀc way with reten-
tion of stereochemistry and the geometry of the exocyclic
double bond that was attributed as being Z by the shield-
ing eꢁect of the carbonyl C-4 on the oleꢀnic proton H-5
Scheme 3 Optimization of the one-pot process under microwave con-
ditions, reaction conditions: (A) 72 °C 2 h, (B) µω 150 °C, 100 W,
1
5 min
(
δH-5=6.82–7.05 ppm) (Debdab et al. 2010; Villemin and
three-component one-pot reaction involving an imidate salt,
a glycine ester and an aromatic aldehyde (Scheme 3). At this
stage, two methods of activation of the reaction have been
employed. The process was carried out under conventional
thermal conditions and also under microwave irradiation in
order to compare the two activation modes. In the interest
of optimizing the conditions, the reaction was conducted
using p-methoxybenzaldehyde and compound 1a, chosen as
model compounds, in the presence of sodium hydrogen car-
bonate as base. In conventional thermal conditions (toluene,
Martin 1995).
The results showed that the one-pot reaction gives better
yields by using imidate salts of aryl type (2b, 2c, 2d) com-
pared to those of alkyl type (2aa, 2ab). This observation
can be explained by the stability of the imidazolones formed
from the aryl imidate salts, due to the conjugation of the
cyclic double bond with the aromatic nucleus of these salts
(Kidwai and Devasia 1962). It could also be explained by the
fact that a small quantity of 2aa and 2ab products was lost in
the precipitation puriꢀcation and was observed in the ꢀltrate
after ꢀltration which was not the case of compounds 2b, 2c
and 2d. However, the diꢁerence in reactivity observed within
these imidates of aryl type seems to be linked to the nature
of the substituents on the aromatic ring. An electron–donor
group on the aromatic nucleus promoted the intramolecular
cyclization of the intermediate, obtained following the addi-
tion of glycine to the imidate salt, by activating the attack of
the amine on the ester based on the proposed mechanism in
Fig. 3. A signiꢀcant decrease in yield was noticed by varia-
tion of the aldehyde (2aa compared to 2ab). This decrease
can be attributed to the diꢁerence in stability of the ꢀnal
7
0 °C), the product was isolated with 45% yield (in 97% of
conversion) after 2 h of reaction, whereas the same reaction
conducted in the same conditions without solvent did not
result in the desired product, but rather a degradation of the
starting products was observed. Furthermore, the reaction
was conducted under microwave irradiations and the condi-
tions were optimized by variation of the heating temperature
and the reaction time. In a ꢀrst attempt, the reaction was
carried at 100 °C under 100 W of maximum power and for
1
5 min to yield the desired product in 88% of conversion.
In the next assay the temperature was increased to 150 °C
1
3

2554
Chemical Papers (2021) 75:2549–2560
Fig. 2 Yields of the 2-aryl and
2
-alkylimidazolones synthe-
sized via one-pot approach; α:
conversion percentage
products obtained. Indeed, p-methoxybenzaldehyde induces
a stability of the ꢀnal product 2aa via the conjugation of the
exocyclic double bond with the aromatic nucleus, reinforced
by the presence of the methoxy group in the para position.
This eꢁect is absent in the case of a methoxy group in the
meta position (2ab). Furthermore, the absence of an activat-
ing group on the aromatic nucleus of the aldehyde did not
lead to total conversion. Benzaldehyde, used with 1a under
the same conditions as before, resulted in a 78% conversion
to the ꢀnal product. However, attempts to purify this deriva-
tive have not been successful.
with trypan blue against human breast cancer cell lines
MCF-7 (Fig. 4) and MDA-MB-231 (Fig. 5) and against HCC
cell lines HepG2 (Fig. 6). Doxorubicin was used as positive
control. The IC (concentration of the compound which
5
0
caused 50% of cell death) for each cell line was determined
based on dose–response curves, concentrations as a function
of the viability percentages obtained 72 h post-treatment.
The results are summarized in Table 1. It should be noted
that the increased lipophilicity of compounds 2c and 2d (log
p values of 3.13–3.4) caused problems in assessing their
cytotoxic eꢁect. These compounds started to precipitate in
the culture medium even at low concentrations (limit con-
centration 10 µM); therefore, it was not possible to deter-
mine the exact IC values.
Cell viability
5
0
The anticancer eꢁect of the various synthesized imidazolone
derivatives was assessed by means of the cell exclusion test
On breast cancer cell lines, the 2-alkyl and 2-arylimi-
dazolone derivatives 2aa, 2ab and 2b (log p values of
Fig. 3 Proposed reaction
mechanism
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3

Chemical Papers (2021) 75:2549–2560
2555
Fig. 4 Percentage of cell viability of the MCF-7 cell line compared
to the control following treatment with the diꢁerent concentrations of
compounds 2aa (a), 2ab (b), 2b (c), 2c (d) and 2d (e) after 24, 48
and 72 h. Each experiment was carried out in duplicate. *, **, ***,
**** indicate a signiꢀcant diꢁerence compared to the vehicle with
p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, according to
the ANOVA test
Fig. 5 Percentage of cell viability of the MDA-MB-231 cell line
compared to the control following treatment with the diꢁerent con-
centrations of compounds 2aa (a), 2ab (b), 2b (c), 2c (d) and 2d (e)
after 24, 48 and 72 h. Each experiment was carried out in duplicate.
*, **, ***, **** indicate a signiꢀcant diꢁerence compared to the
vehicle with p<0.05, p<0.01, p<0.001 and p<0.0001, respectively,
according to the ANOVA test
1
3

2556
Chemical Papers (2021) 75:2549–2560
Fig. 6 Percentage of cell viability of the HepG2 cell line compared
to the control following treatment with the diꢁerent concentrations of
compounds 2aa (a), 2ab (b), 2b (c), 2c (d) and 2d (e) after 24, 48
and 72 h. Each experiment was carried out in duplicate. *, **, ***,
**** indicate a signiꢀcant diꢁerence compared to the vehicle with
p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, according to
the ANOVA test
Table 1 IC50 values of compounds against diꢁerent cancer cell lines
When tested on the hepatocellular carcinoma cell line
HepG2, the 2-phenethylimidazolone derivative 2ab was
found possessing the most cytotoxic activity with an IC of
Product
IC * (µM)
50
5
0
MDA-MB-231
MCF-7
HepG2
6
.2± 1.15 µM. The displacement of the methoxy substitu-
tion from the meta position (2ab) to the para position (2aa),
reduced the cytotoxic activity by half. The 2-arylimida-
zolone compound 2b also showed an interesting anticancer
2
2
2
2
2
aa
ab
b
c
d
>60
>30
11.3± 2.9
6.2± 1.15
8.6± 0.83
>10
>10
0.04± 0.0096
22± 3.99
25± 3.61
>10
>10
0.01± 0.0008
7± 2.39
5.7± 2.3
>10
>10
0.022± 0.002
eꢁect against this cell line with an IC of 8.6 ± 0.83 µM.
50
Overall, the 5-benzylidene-2-alkyl and 2-arylimidazolones
showed noticeable cytotoxicity against MCF-7 and HepG2
cell lines. In the literature, some 5-benzylidene-2-arylim-
idazolone derivatives have also shown anticancer activity
against epithelial carcinoma cancer cell line A549 with IC
Doxorubicin
*
IC values were derived from single dose–response curves gener-
50
ated from duplicate data points obtained after 72 h of treatment
5
0
values of 0.44 to 12 µM (Beloglazkina et al. 2016).
2
.44–2.93) were found to be inactive against the prolifera-
The diꢁerence of anticancer activity noted between ER+
cell line MCF-7 and HER2+ cell line HepG2 on the one
hand and triple-negative cell line MDA-MB-231 on the other
hand could be attributed to the diꢁerent kinases expressions
in these cell lines. In fact, it has been reported that CLK2 is
ampliꢀed and overexpressed in HER2+ and ER+ cell lines
and that downregulation of CLK2 inhibited breast cancer
growth in cell culture (Yoshida et al. 2015), whereas inhibi-
tion of hyperactive CDKs in triple-negative breast cancer
resulted in decreased cell migration and invasion (Tarase-
wicz et al. 2014). These ꢀndings could suggest possible
selective interactions of the synthesized imidazolone deriva-
tives with speciꢀc kinases.
tion of the hormone-independent breast cancer line MDA-
MB-231. As for the hormone-dependent breast cancer line
MCF-7, the results show that the highest anticancer activ-
ity was observed for the 2-arylimidazolone compound 2b
(
IC = 5.7 ± 2.3 µM) having a methoxy group in the para
50
position on each of the aromatic nuclei. As for the 2-pheneth-
ylimidazolone derivatives, compound 2aa, having a methoxy
substitution in the para position of the benzylidene moiety,
did not show any interesting cytotoxic eꢁect (IC >30 µM).
50
However, an enhanced cytotoxic activity was noted, when
the methoxy group was displaced from para to meta position
in compound 2ab, showing an IC50 of 7± 2.39 µM.
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Chemical Papers (2021) 75:2549–2560
2557
All the imidazolone derivatives were less potent than the
doxorubicin drug used as positive control. However, the latter
can induce acute and chronic cardiotoxicity that may occur at
low doses (Octavia et al. 2012). It also showed toxicity on car-
diogenesis, heart formation and survival of zebraꢀsh embryos
Table 2 Percentage of mortality and types of morphological malfor-
mation caused by each analog in zebraꢀsh embryos at 72 hpf
Product % of mor-
tality (at
Morphological malformations at 72 hpf
Cardiac Yolk sac Curved
Hatching
7
2 hpf)
edema edema
tail/Short
tail
retarda-
tion
(Ma et al. 2018; Chang et al. 2014).
2
2
2
aa
1
Zebrafsh embryo toxicity assay
0
0
6
10
12
50
50
−
−
−
+
+
−
−
−
−
−
+
+
+
+
−
−
+
+
+
+
+
−
−
−
−
−
+
+
3
The newly synthesized imidazolone derivatives and doxoru-
bicin drug have been tested for their toxic eꢁect in vivo on
the development of zebraꢀsh embryos exposed to increasing
concentrations (1–120 µM) of these compounds. Morphologi-
cal abnormalities and mortality of the embryos were studied.
The summary of mortality and observed malformations of
zebraꢀsh embryos at 72 hpf with the diꢁerent compounds are
presented in Table 2.
1
3
6
9
1
0
0
0
0
20
ab
1
0
0
5
5
13
15
13
−
−
−
−
+
−
−
−
−
−
−
+
+
+
−
−
−
−
+
+
+
−
−
−
−
−
−
−
3
0
Below concentrations of 10 µM, no incidence of mortal-
ity and malformation was observed with all the tested com-
pounds. The mortality rate slightly increased after exposure to
1
3
6
9
1
b
1
3
1
3
6
9
1
d
1
3
1
c
0
0
0
20
2ab and 2b at concentrations higher than 60 µM. Furthermore,
treatment with 2aa starting from 60 µM caused a signiꢀcantly
increased mortality at 48 and 72 hpf. At 72–96 hpf, there were
signiꢀcant diꢁerences in the malformation phenotypes in the
zebraꢀsh embryos that were exposed to the various com-
pounds. The predominant eꢁects observed after 2ab (Fig. 7)
and 2aa (Fig. 8) exposure were pericardial edema, yolk sac
edema, short and contorted tail. For 2ab, starting from 30 µM
and at 72 hpf, the length of the embryos became much shorter,
whereas cardiac edema, yolk sac edema and short yolk sac
extension were well noted at 60 µM. In addition, a signiꢀcant
contraction of the tail has been observed at 60 µM (Fig. 7).
However, 2aa compound treatment induced tail contraction
at 10 µM and yolk sac and pericardial edema starting from
0
0
0
10
10
15
15
−
−
−
−
−
+
+
−
−
−
−
+
+
+
−
−
−
−
+
+
+
−
−
−
−
−
−
−
0
0
0
0
20
2
2
0
0
0
−
−
−
−
−
−
−
−
−
−
−
−
0
0
3
0 µM (Fig. 8). Furthermore, a delay in zebraꢀsh hatching
was observed only for 2aa compound at high concentrations.
It was not possible to determine the eꢁect of 2c and 2d
compounds at high concentrations due to their precipitation
in the E3 medium. After 72 h of exposure to 2c and 2d, there
was no signiꢀcant mortality, neither eꢁects on hatching delay
nor malformation in embryonic development, when com-
pared with control group with all concentrations used below
1
3
1
0
0
0
−
−
−
−
−
−
−
−
−
−
−
−
Doxorubicin
1
3
1
3
6
9
1
0
0
20
50
100
100
100
−
−
−
+
+
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
−
−
+
+
+
+
0
0
0
0
1
0 µM. Overall, the 5-benzylidene-2-alkyl and 2-arylimida-
zolones induced signiꢀcant mortality and abnormalities only
at high concentrations. These compounds were less toxic on
the development of zebraꢀsh embryos than doxorubicin drug
that caused 50% of lethality at 30 µM.
20
*
% of mortality (at 72 hpf) for control and vehicle (0.5% of DMSO)
are zero, and none of the morphological malformations at 72 hpf are
detectable
1
3

2558
Chemical Papers (2021) 75:2549–2560
Fig. 7 Zebraꢀsh embryos treated with the 2ab product for 72 h a
CTRL—control; b embryo treated with 3 μM c embryo treated with
f embryo treated with 120 μM. Scale bar 250 µm. ce: cardial edema;
yse: yolk sac edema; sy: short yolk sac extension; ct: contracted tail
3
0 μM; d embryo treated with 60 μM; e embryo treated with 60 μM;
Fig. 8 Photographs of
encountered malformations,
resulting from the treatment
of Casper zebraꢀsh embryos
with compounds 2aa at 96 hpf.
a CTRL—control; b embryo
treated with 10 μM c embryo
treated with 30 μM; d embryo
treated with 60 μM; scale bar
2
50 µm. ce: cardial edema; yse:
yolk sac edema; ct: contracted
tail
Conclusions
Besides their cytotoxic activity on cancer cell lines, these
compounds showed biological interactions in vivo mani-
fested in morphological abnormalities observed on the
development of zebraꢀsh embryos only at high concentra-
tions. Although a limited number of imidazolone deriva-
tives are presented here, it is obvious that a much larger
diversity can be achieved through this versatile process to
enrich this family of molecules and subsequently conduct
a more detailed SAR. This preliminary study showed that
these compounds may be considered as promising mol-
ecules in the further search of novel active and non-toxic
anticancer agents.
We optimized the synthesis of new 2-aryl and 2-alky-
limidazolone derivatives, in a one-pot procedure under
microwave irradiations. The latter have shown to be more
efficient than thermal heating by providing similar or
slightly better yields of the imidazolones while reducing
the reaction time from 2 h to 15 min. The cytotoxicity of
the imidazolone derivatives was evaluated in vitro against
tumor cell lines. This family of compounds exhibited inter-
esting anticancer activity against ER+ and PR+ cell line
MCF-7 and HER2+ cell line HepG2. Among the synthe-
sized derivatives, compound 2b expressed the best cyto-
toxic eꢁect against MCF-7 cell line with an IC value
Acknowledgements We would like to thank the Lebanese University
and the National Council for Scientiꢀc Research (CNRS)—Leba-
non, for the ꢀnancial support of Samar Bou Zeid during her thesis and
for the ꢀnancial support of this project. We would also like to thank
the Edde Association—Lebanon. The funders had no role in the study
design or during data collection and analysis as well as preparation
of the manuscript. This work was also supported by the University of
Rennes 1 and by the management committee of scientiꢀc research at
the Lebanese University.
5
0
of 5.7 µM, whereas compound 2ab was the most active
against HepG2 cell line with an IC of 6.2 µM. Further
5
0
studies should be conducted to identify the involvement
of protein kinases, overexpressed in these types of tumors,
in the pathways responsible for the observed cytotoxicity.
1
3

Chemical Papers (2021) 75:2549–2560
2559
Author contributions All authors contributed to the study conception
and design. Material preparation, data collection and analysis were
performed by all authors. The ꢀrst draft of the manuscript was written
by Samar Bou Zeid, and all authors commented on previous versions
of the manuscript. All authors read and approved the ꢀnal manuscript.
regulated kinases derived from the marine sponge leucettamine
B: modulation of alternative pre-RNA splicing. J Med Chem
54(12):4172–4186. https://doi.org/10.1021/jm200274d
Debdab M, Renault S, Lozach O, Meijer L, Paquin L, Carreaux F,
Bazureau J-P (2010) Synthesis and preliminary biological evalu-
ation of new derivatives of the marine alkaloid leucettamine B as
kinase inhibitors. Eur J Med Chem 45(2):805–810. https://doi.
org/10.1016/j.ejmech.2009.10.009
Compliance with ethical standards
DeSantis CE, Ma J, Goding Sauer A, Newman LA, Jemal A (2017)
Breast cancer statistics, 2017, racial disparity in mortality by
state. CA Cancer J Clin 67(6):439–448. https://doi.org/10.3322/
caac.21412
Conflict of interest All authors declare that they have no conꢃict of
interest.
Ethics approval Adult zebraꢀsh (Danio rerio) were used after ethical
Eldehna WM, Abdelrahman MA, Nocentini A, Bua S, Al-Rashood ST,
Hassan GS, Bonardi A, Almehizia AA, Alkahtani HM, Alharbi
A, Gratteri P, Supuran CT (2019) Synthesis, biological evalu-
ation and in silico studies with 4-benzylidene-2-phenyl-5(4H)-
imidazolone-based benzenesulfonamides as novel selective car-
bonic anhydrase IX inhibitors endowed with anticancer activity.
Bioorganic Chem 90:103102. https://doi.org/10.1016/j.bioor
g.2019.103102
approval.
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