(2023) 17:174
Alzamami et al. BMC Chemistry
https://doi.org/10.1186/s13065-023-01063-5
BMC Chemistry
Open Access
RESEARCH
Novel 8-Methoxycoumarin-3-Carboxamides
with potent anticancer activity against liver
cancer via targeting caspase-3/7 and β-tubulin
polymerization
Ahmad Alzamami1, Eman M. Radwan2, Eman Abo-Elabass3, Mohammed El Behery3,
Hussah Abdullah Alshwyeh4,5, Ebtesam Al-Olayan6, Abdulmalik S. Altamimi7, Nashwah G. M. Attallah8,
Najla Altwaijry9, Mariusz Jaremko10* and Essa M. Saied11,12*
Abstract
In the present study, we explored the potential of coumarin-based compounds, known for their potent anticancer properties, by designing and synthesizing a novel category of 8-methoxycoumarin-3-carboxamides. Our aim
was to investigate their antiproliferative activity against liver cancer cells. Toward this, we developed a versatile
synthetic approach to produce a series of 8-methoxycoumarin-3-carboxamide analogues with meticulous structural
features. Assessment of their antiproliferative activity demonstrated their significant inhibitory effects on the growth
of HepG2 cells, a widely studied liver cancer cell line. Among screened compounds, compound 5 exhibited the most
potent antiproliferative activity among the screened compounds (IC50 = 0.9 µM), outperforming the anticancer drug
staurosporine (IC50 = 8.4 µM), while showing minimal impact on normal cells. The flow cytometric analysis revealed
that compound 5 induces cell cycle arrest during the G1/S phase and triggers apoptosis in HepG2 cells by increasing the percentage of cells arrested in the G2/M and pre-G1 phases. Annexin V-FITC/PI screening further supported
the induction of apoptosis without significant necrosis. Further, compound 5 exhibited the ability to activate caspase3/7 protein and substantially inhibited β-tubulin polymerization activity in HepG2 cells. Finally, molecular modelling analysis further affirmed the high binding affinity of compound 5 toward the active cavity of β-tubulin protein,
suggesting its mechanistic involvement. Collectively, our findings highlight the therapeutic potential of the presented
class of coumarin analogues, especially compound 5, as promising candidates for the development of effective antihepatocellular carcinoma agents.
Keywords Liver cancer, Coumarin analogues, Antiproliferative activity, Cell cycle arrest, Apoptosis, Flow cytometric
analysis, Caspase-3/7, β-tubulin polymerization, Molecular docking
*Correspondence:
Mariusz Jaremko
Mariusz.jaremko@kaust.edu.sa
Essa M. Saied
saiedess@hu-berlin.de
Full list of author information is available at the end of the article
© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
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Alzamami et al. BMC Chemistry
(2023) 17:174
Introduction
Cancer remains a substantial worldwide health concern,
impacting a considerable number of individuals annually
across diverse cancer types [1]. Liver cancer is a notable
malignancy within the spectrum of cancer types, characterized by its elevated incidence rates and unfavorable prognostic outcomes [2]. Hepatocellular carcinoma
(HCC), commonly referred to as liver cancer [3], is an
oncological condition characterized by the initiation of
malignant growth in the hepatic cells, with the potential to metastasize to various anatomical sites [4]. Liver
cancer is a significant global public health concern [5].
Liver cancer ranks as the sixth most frequently diagnosed
cancer [6] and the fourth primary contributor to cancerrelated mortality on a global scale [7], as reported by
the World Health Organization (WHO). Chronic infections of hepatitis B and C viruses are widely acknowledged as the principal risk factors for the development of
liver cancer [8], constituting more than 80% of reported
cases [9]. Furthermore, there are additional risk factors
that should be considered, including diabetes [10], obesity [11], non-alcoholic fatty liver (NAFLD) [12], excessive alcohol consumption [13], exposure to aflatoxins
(toxins produced by certain types of molds) [14], and
specific genetic conditions such as Wilson’s disease [15]
and hemochromatosis [16]. The selection of treatment
modalities for liver cancer is contingent upon the extent
and progression of the ailment [17]. Potential treatment
options for this condition encompass various medical
interventions, including surgical procedures like liver
resection or transplantation, radiation therapy, chemotherapy, targeted therapies, and immunotherapies [18].
The efficacy of treatment is contingent upon the stage of
liver cancer during the point of diagnosis, as well as the
accessibility of resources and expertise required to effectively manage this intricate ailment [19].
Caspases, a class of protease enzymes known as
cysteine proteases, are present in cells as inactive zymogens and are responsible for carrying out the process
of apoptosis [20], also referred to as programmed cell
death. Caspases can be classified into three distinct categories [21]: initiator caspases (including caspase 2, 8, 9,
and 10) [22], executioner caspases (comprising caspase
3, 6, and 7) [23], and inflammatory caspases (encompassing caspase 1, 4, 5, 11, and 12) [24]. These entities
exhibit mutual activation and give rise to a series of biochemical events [25]. The process of cellular dismantling
involves the cleavage of various structural and regulatory
proteins by executioner caspases [26]. It is important to
note that initiator caspases are the first to be activated
in this process. The caspases that play a role in the process of apoptosis can be categorized into two groups
[27]: initiator caspases (such as caspase-9 in mammals)
Page 2 of 22
and effector caspases (such as caspase-3 and caspase-7
in mammals). One of the most intriguing techniques in
cancer treatment is the activation of caspase-3-mediated
induced apoptosis, which leads to cytotoxicity [28]. Many
scientists and researchers have synthesized and studied
bioactive compounds, including quinazoline, coumarin,
thiosemicarbazone, chalcone, and pyrimidines that
induce caspase-3-mediated apoptosis and cytotoxicity
for cancer treatment [29, 30].
Growing empirical evidence indicates that tubulin proteins play a significant role in the metastatic progression
of cancer [31]. Over the course of the past ten years, various studies have identified several tubulin isotypes that
show promise as prognostic markers [32]. These isotypes
have been found to have a correlation with aggressive disease, increased metastatic potential, and a higher likelihood of metastatic relapse in patients [33]. Among these
isotypes, β-tubulin expression has been the primary
focus of investigation [34]. Clinical data has demonstrated a significant association between elevated levels
of β-tubulin protein expression and the manifestation of
aggressive clinical characteristics as well as unfavorable
prognosis in various types of cancer, such as pancreatic,
glioblastoma, gastric, ovarian, breast, colorectal, and
prostate. In both gastric and gliomas cancers, the expression of β-tubulin has been observed to be correlated
with the presence of high-grade malignancy [35]. Previous studies have documented the existence of functional
associations between β-tubulin and metastasis in mouse
models of lung and pancreatic cancer. The depletion of
β-tubulin leads to a reduction in anchorage independent growth, a significant characteristic associated with
the metastatic capacity of non-small cell lung cancer cells
[36]. The suppression of β-tubulin leads to the upregulation of the adhesion-associated tumour suppressor
Maspin. This upregulation inhibits the outgrowth of cell
migration, tumour spheroids, and enhances the sensitivity of non-small cell lung cancer cells to anoikic. Inhibiting the expression of β-tubulin led to diminished growth
of pancreatic cancer cells, along with a decrease in their
capacity to generate tumors and metastasize to distant
organs. In a study conducted by Xiao et al., it was shown
that β-tubulin plays a significant role in imparting brain
metastatic capabilities to breast cancer cells through the
regulation of various crucial signaling molecules that
are involved in cell adhesion and the process of metastasis [37]. The downregulation of β-tubulin resulted
in the modulation of β3-integrin expression, leading
to a decrease in extracellular matrix attachment. The
observed phenomenon was found to be correlated with
a decrease in metastatic capacity, as well as an enhancement in survival rates in a model of brain metastasis [38].
To sum up, these findings indicate that tubulin isotypes,
Alzamami et al. BMC Chemistry
(2023) 17:174
particularly β-tubulin, have a notable impact on enabling
the dissemination of cancer cells and could potentially
act as predictive markers for the advancement of neoplastic diseases and the prognosis of patients. However,
the exact mechanisms by which β-tubulin controls the
metastasis process remain to be fully understood.
In order to enhance the efficacy of drug discovery, a
pragmatic approach involves commencing the process
with natural bioactive substances derived from medicinal
plants or alternative natural origins. Coumarins, which
were initially extracted from melilot flowers and tonka
beans, have been extensively explored for their therapeutical applications, encompassing anti-inflammatory [39],
anticancer [40], antiviral [41], antimicrobial [42], antioxidant [43], and anticoagulant [44]. Warfarin is widely
recognized as a prominent pharmaceutical agent derived
from coumarin [45]. This medication is classified as an
original anticoagulant that has received approval for
its efficacy in reducing the likelihood of blood clot formation [46] and preventing strokes in individuals with
atrial fibrillation and/or those who have undergone cardiac valve replacement surgery [47]. The introduction of
structural alterations to the coumarin scaffold not only
results in the development of novel anticoagulants, but
also induces a shift in the biological properties of newly
synthesized coumarin analogs towards antispasmodic,
vasodilating, antiproliferative, antibiotic, and chemoprotective activities [48]. The anticancer potentials of derivatives of coumarins have been the subject of investigation.
Page 3 of 22
For instance, the efficacy of coumarins containing the
hydrazide-hydrazone moiety has been assessed in relation to their activity against drug-resistant pancreatic
carcinoma cells and various other types of cancer cells
[49]. Molecular hybridization is a valuable drug design
strategy that can enhance the inhibitory potency of
coumarins, while also offering the potential to improve
their pharmacodynamic and pharmacokinetic properties [50]. The process entails the amalgamation of two
or more pharmacophores, with or without the inclusion
of any connecting group(s). Previous studies have demonstrated that the incorporation of supplementary pharmacophores into coumarin has resulted in enhanced
activity, effectiveness, toxicity profiles and oral bioavailability [51].
The therapeutic applications of coumarin derivatives are contingent upon the specific characteristics
and placement of substituents within the fundamental
nucleus (Fig. 1). The impact of incorporating the dihydropyrazole moiety into the coumarin framework was
examined in a study conducted by Hu et al. (Fig. 2) [52].
The authors demonstrated that this particular group of
coumarin analogues exhibits significant hepatoprotective properties through the induction of apoptosis and
the targeting of telomerase activity. The results of this
investigation indicate that coumarin analogues exhibit
promising therapeutic potential for the management
of hepatic disorders. The study conducted by Fayed
et al. demonstrated the diverse anti-cancer properties
Fig. 1 Representative Coumarin-based drugs with various pharmacological effects
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 4 of 22
Fig. 2 Representative sturctures of recently reported coumarin analogues with potent antitumor activity
exhibited by coumarin analogues containing pyridine
hybrids (Fig. 2). These compounds exhibited the ability to induce programmed cell death (apoptosis) and
halt the progression of the cell cycle. Additionally, there
was a notable increase in the activity of caspase-3, an
enzyme involved in apoptosis [53].
The design and synthesis of coumarin hybrids represent a significant and novel approach within the realm
of medicinal chemistry [54]. The coumarin core has
demonstrated the ability to generate a diverse array of
compounds that exhibit therapeutic potential against a
range of diseases, encompassing microbial infections,
cancer, inflammatory conditions, and neurodegenerative disorders. In recent times, a variety of drugs that
are based on coumarin, such as coumadin, acenocoumarol, dicoumarol, phenprocoumon, and novobiocin,
have received approval from the FDA and are currently
being utilized in medical practice [55]. Additionally, a
number of compounds that contain coumarin are currently undergoing clinical trials. The coumarin-based
hybrid compounds were categorized by many research
groups based on their shared biological activities, to
identify their potential therapeutic targets [56]. As
a result of their wide range of applications, coumarin
analogues have emerged as promising research targets
for the pharmaceutical industry [57]. The coumarin
scaffold’s adaptability and simplicity of production
make it a useful starting point for the design of new
compounds with enhanced pharmacological characteristics [58].
In light of the aforementioned and in our ongoing quest
to discover new bioactive chemicals [59–68], we designed
and synthesized a novel series of N-(substituted-phenyl)8-methoxycoumarin-3-carboxamides. The design of
these compounds was mainly based on exploring various
structural features in the 8-methoxycoumarin scaffold
(Fig. 3). The antiproliferative activity of designed compounds was extensively examined against HepG2 cells
by conducting MTT assay and flow cytometric analysis.
We further explored the mode of action of this class of
coumarin analogue by assessing their potency toward
β-tubulin polymerization and Caspase 3/7 proteins.
Finally, we performed the detailed computational analysis
to affirm the binding affinity of this class of compounds
toward the targeted protein(s).
Results and discussion
Synthesis and characterization of novel
8‑methoxycoumarine analogues
The synthesis of 8-methoxycoumarin-3-carboxamides
(4–7) and 8-methoxycoumarin-3-carboxylic acid derivatives (8,9) was successfully accomplished using a multistep synthetic route, as shown in Scheme 1. Starting
with 3-methoxy-2-hydroxybenzaldehyde (1) and diethyl
malonate, ethyl 8-methoxycoumarin-3-carboxylate (2)
was synthesized via a cyclocondensation reaction with
Fig. 3 Illustrative diagram for structural features explored in the presented study
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 5 of 22
Scheme 1 Synthesis of 8-methoxycoumarin-3-carboxamides (4–7) and 8-methoxycoumarin-3-carboxylic acid derivatives (8,9). Reagents
and reaction conditions: a diethylmalonate, piperidine, fusion, 2 h; b Bromine, AcOH, 60 °C, 8 h; c Ammonium acetate, fusion, 6 h; d Acetic
anhydride, reflux, 12 h; e acetic acid, 4N HCl, reflux, 16 h
piperidine as a base catalyst under fusion conditions.
From ethyl 8-methoxycoumarin-3-carboxylate (2), ethyl
5-bromo-8-methoxycoumarin-3-carboxylate (3) was
obtained with good efficiency (71% yield) through halogenation with bromine in glacial acetic acid. By subjecting
ester coumarins 2 and 3 to ammonolysis with ammonia
derived from ammonium acetate under fusion conditions, two key 8-methoxycoumarin-3-carboxamides were
produced. Compound 4, 8-methoxycoumarin-3-carboxamide, was obtained in 53% yield, while compound 5,
5-bromo-8-methoxycoumarin-3-carboxamide, showed
a slightly higher yield of 61%. The confirmation of the
structures of 8-methoxycoumarin-3-carboxamide (4)
and 5-bromo-8-methoxycoumarin-3-carboxamide (5)
was achieved through further transformations. When
subjected to an acetylation reaction, compound 5 was
converted into 5-bromo-8-methoxycoumarin-3-carboxamide (7) with a satisfactory yield of 56%. Similarly,
the acetylation of compound 4 led to the formation of
compound (6), N-(acetyl)8-methoxycoumarin-3-carboxamide, with a yield of 61%. Furthermore, the reaction of 8-methoxycoumarin-3-carboxamide (4) with
4N hydrochloric acid in acetic acid under reflux conditions yielded 56% of 8-methoxycoumarin-3-carboxylic
acid (8). Lastly, the synthesis of 5-bromo-8-methoxycoumarin-3-carboxylic acid (9) involved the halogenation of compound 8 using bromine in glacial acetic acid
at a temperature of 60 °C. Overall, the study successfully demonstrated the synthesis of a diverse range of
8-methoxycoumarin-3-carboxamides and 8-methoxycoumarin-3-carboxylic acid derivatives using ethyl
8-methoxycoumarin-3-carboxylate as the starting material. The described synthetic pathway offers valuable
insights into the potential applications of these compounds in various scientific disciplines, including medicinal chemistry.
The chemical identities and purities of compounds
4–9 were verified through the utilization of several spectroscopic techniques [69], namely proton nuclear magnetic resonance spectroscopy (1H NMR), carbon-13
nuclear magnetic resonance spectroscopy (13C NMR),
and electron ionization mass spectrometry (EI-MS).
These techniques collectively provided confirmation
of the structural compositions of the compounds. The
Alzamami et al. BMC Chemistry
(2023) 17:174
infrared spectra exhibited distinct absorption bands that
corresponded to specific functional groups, including
carbonyl, amide, and bromine [70]. These observations
provided evidence for the existence of these functional
groups within the synthesized compounds. The 1H-NMR
spectra of compounds 4 and 5 exhibited a singlet signal
at δ 3.94 ppm, which can be attributed to the presence
of three protons originating from the methoxy (OCH3)
group. The proton at position 4 of the coumarin ring in
compounds 4 and 5 exhibited singlet signals at chemical
shifts of δ 8.84 and 8.85 ppm, respectively. The protons
associated with the NH2 group in compound 4 exhibit
two singlet signals with chemical shifts of δ 7.95 and
8.09 ppm. Conversely, compound 5 displays a broad singlet signal at δ 8.09 ppm, indicating the presence of the
NH2 group. In compound 4, the aromatic protons exhibit
multiplet signals within the chemical shift range of δ
7.35–7.52 ppm.
In compound 5, two doublet signals are observed at δ
7.38 and 7.67 ppm, indicating the presence of two protons in the aromatic ring. The 13C-NMR spectra of compounds 4 and 5 displayed distinct signals at δ 162.98,
160.52, and 162.44, 159.80, respectively, which can be
attributed to the presence of carbonyl groups in amides
and coumarin rings. Additionally, the carbon signals corresponding to the methoxy groups in compounds 4 and
5 were observed at δ 3.94 ppm. The carbon signals corresponding to the C-O and C-4 positions of the coumarin
ring in compounds 4 and 5 were detected at chemical shifts of δ 148.50, 146.72, 143.80 and 146.70, 141.91,
144.83 ppm, respectively. Additionally, the carbon signals
in the aromatic region and the C-3 position of the pyranone ring were observed within the chemical shift range
of δ 128.84–112.65 ppm. The mass spectra resulting from
electron impact ionization of substances 4 and 5 exhibit
ion peaks at m/z 219 and m/z 297, which correspond to
the molecular formulas C11H9NO4 and C11H8BrNO4,
respectively.
The absence of a proton signal at δ 8.09 ppm in the 1HNMR spectrum of compound 7 indicates the absence of
the amino (NH2) group. Additionally, the presence of two
new singlet signals at δ 11.09 and 2.33 ppm suggests the
presence of one proton in the NH group and three protons in the methyl function of the acetyl (COCH3) group.
The 13C-NMR spectrum of compound 7 revealed the
presence of two distinct carbon signals at chemical shifts
of δ 171.71 and 25.59 ppm. These findings provide evidence for the formation of the acetyl derivative (7) as a
result of the incorporation of the acetyl group (COCH3).
The carbon signals corresponding to the remaining atoms
in 8-methoxy-5-bromocoumarin-3-carboxamide are
detected within the anticipated spectral regions. The verification of the structures of coumarin-3-carboxylic acid
Page 6 of 22
derivatives (8 and 9) was conducted through the analysis
of the signals observed in the 1H-NMR spectra [71]. The
spectrum of 8 demonstrated the lack of two proton signals attributed to the presence of the amino (NH2) group.
The absence of these signals served as confirmation of
the formation of an acid derivative. The protons located
on the coumarin ring in compound 8 consist of H-4 of
coumarin, as well as multiplet signals originating from
the aromatic protons. 1H-NMR spectrum of compound
9 exhibited a singlet peak at a chemical shift of 8.45 parts
per million (ppm), corresponding to the H-4 proton of
the coumarin ring. Additionally, two doublet peaks were
observed at chemical shifts of 7.59 and 7.30 ppm, which
can be attributed to the two protons of the aromatic ring.
The mass spectra analysis of compounds 8 and 9 revealed
the presence of ion peaks at m/z 220 and m/z 298, which
can be attributed to the molecular formulas C11H8O5 and
C11H7BrO5, respectively.
Assessment of cytotoxic activity against HepG2 Cells
In our study, we investigated the impact of several newly
synthesized coumarin derivatives (4–9) on the viability
of HepG2 cells, a cell line derived from hepatocellular
carcinoma. To assess the antitumor properties of these
compounds, we utilized the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) colorimetric
assay, a widely used method to evaluate cell viability and
cytotoxicity. As a reference, we included staurosporine,
a known anticancer compound, in our evaluation. The
HepG2 cell line was exposed to various concentrations of
the coumarin derivatives, and the dose-dependent cytotoxicity was assessed (Additional file 1: Table S1). The
obtained IC50 values, which represent the concentration
at which 50% of cell viability is inhibited, are presented
in Table 1 and Fig. 4. Our results demonstrated that the
newly synthesized coumarin derivatives exhibited a
Table 1 Cytotoxic evaluation (IC50, µM) of compounds 4–9,
as compared to staurosporine, against human liver carcinoma
HepG2 cells and WI 38 normal cells
Comp No
4
IC50 value (µM)
HepG2
WI 38
17.2 ± 1.05
NT*
5
0.9 ± 0.05
6
23.6 ± 1.43
NT*
7
2.57 ± 0.16
NT*
8
5.18 ± 0.31
NT*
9
41.6 ± 2.52
NT*
STU
8.4 ± 0.51
24.1 ± 1.46
26.7 ± 1.62
* NTnot tested. The data provided represents the average value with its standard deviation of the mean (SEM),
obtained from at least three separate experiments
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 7 of 22
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
STU
80
70
60
Cell Viability (%)
50
40
30
20
10
-0.41
0.19
0.8
1.4
2
Conc. (LogC, µM)
(A)
(B)
Fig. 4 A The dose-dependent cytotoxic activity of synthesized compounds 4–9 toward HepG2 cells, as compared to staurosporine. The presented
data shows the mean ± standard deviation of the mean obtained from at least three independent experiments. B Summary of presented structure–
activity relationships showing the effect of different substituents
diverse range of antitumor effects, ranging from moderate to highly effective. Among the tested compounds,
the main scaffold of 8-methoxycoumarin-3-carboxamide
(compound 4) showed moderate antiproliferative activity with an IC50 of 17 µM. Interestingly, the introduction
of an acetyl group to the 3-carboxamide of compound
4 (compound 6) resulted in improved cytotoxic activity
with an IC50 of 2.3 µM. This suggests that the newly introduced acetyl group may play a crucial role in the binding
of the compound to its targeted protein(s) by providing
a site for hydrogen bonding acceptors. Moreover, the
bromination of 8-methoxycoumarin-3-carboxamide at
position-5 (compound 5) led to a significant increase in
cytotoxic activity with an IC50 of 0.9 µM. This indicates
that the bromination of the coumarin moiety at position-5 contributes to enhancing the cytotoxicity of the
compound. Conversely, the acetylation of the 3-carboxamide moiety of compound 5 (compound 7) resulted
in a considerable attenuation of cytotoxic activity with
an IC50 of 2.3 µM. This suggests that the presence of
both acetyl and bromo groups in the coumarin scaffold might lead to steric hindrance, affecting the compound’s cytotoxic potential. Furthermore, the hydrolysis
of 8-methoxycoumarin-3-carboxamide (compound 4) to
Alzamami et al. BMC Chemistry
(2023) 17:174
the corresponding 3-carboxylic acid (compound 8) demonstrated a significant improvement in antiproliferative
activity with an IC50 of 5 µM. This indicates the importance of the hydrogen bonding acceptor group (C = O)
at position-3 for the cytotoxicity of this class of compounds. On the other hand, the bromination of 8-methoxycoumarin-3-carboxylic acid scaffold at position-5
(compound 9) led to an almost complete loss of cytotoxicity, with an IC50 of 41 µM. This suggests that the newly
formed methoxycoumarin-3-carboxylic acid has a unique
binding mode that differs from the corresponding 3-carboxamide analogue.
Among screened compounds, compound 5 displayed
the most significant inhibitory effect, with an IC50 value
of 0.9 µM. To put this into perspective, we also compared the activity of compound 5 with that of the anticancer drug staurosporine, which had an IC50 value of
8.4 µM. The substantial difference in IC50 values suggests
that compound 5 possesses higher potency in suppressing the growth of HepG2 cells compared to the standard
drug staurosporine. Encouraged by the promising results
against liver cancer cells, we decided to investigate further and assess the antiproliferative activity of compound
5 against normal lung cells (WI-38 cells). It is essential
to evaluate the selectivity of potential anticancer agents
to ensure minimal harm to healthy cells. Our findings
indicate that compound 5 exhibits a lower cytotoxic
effect on WI38 cells compared to the anticancer drug
staurosporine. The IC50 values for compound 5 against
WI38 cells were 24.1 µM, while staurosporine had IC50
values of 26.7 µM (Additional file 1: Figure S9). This data
demonstrates that compound 5 has a relatively higher
selectivity for cancer cells over normal cells, making it a
more attractive candidate for further investigation as an
anticancer agent. Taken together, our study highlights
the potential of the newly presented class of coumarin
analogues, particularly compound 5, as promising candidates for the development of novel anticancer drugs
targeting liver cancer. The remarkable inhibitory activity of compound 5 against HepG2 cells, coupled with its
reduced cytotoxicity towards normal lung cells, underscores its potential as a lead scaffold for the development
of anticancer agents against liver cancer.
Assessment of cell cycle analysis
Next, our investigations focused on evaluating the effects
of compound 5 on the cell cycle of HepG2 cells utilizing
flow cytometric analysis [72]. The cells were treated with
compound 5 at a concentration corresponding to the IC50
value and the cell cycle distribution was then analyzed,
and the results were compared with untreated control
cells. The findings depicted in Fig. 5 revealed significant
alterations in the cell cycle distribution upon treatment
Page 8 of 22
with compound 5. The treatment led to a reduction in the
cell population in the G0/G1 and S phases, with the percentage of cells in G0/G1 phase decreasing from 49.71%
to 41.09%, and the percentage of cells in the S phase
decreasing from 34.91% to 27.4%. Conversely, there was
a notable increase in the cellular population at the G2/M
and pre-G1 phases. The percentage of cells in the G2/M
phase rose from 15.38% to 31.51%, while the percentage of cells in the pre-G1 phase increased dramatically
from 2.08% to 45.88%. These findings indicate that compound 5 induces a notable arrest in cell growth during
the G1/S phase. The flow cytometric analysis of HepG2
cells treated with compound 5 provided valuable insights
into its effects on the cell cycle distribution. The observed
reduction in the percentage of cells in the G0/G1 and S
phases indicates that compound 5 induces a slowdown in
cell growth during these phases. The G0/G1 phase is the
resting phase, where cells prepare for DNA replication
and cell division, while the S phase is where DNA replication occurs. The decrease in cell population in these
phases suggests that compound 5 interferes with the
normal progression of the cell cycle, possibly by inhibiting key regulatory proteins involved in cell cycle control. Moreover, the considerable increase in the cellular
population at the G2/M phase suggests that compound 5
induces cell cycle arrest at the G2/M checkpoint. This is
a critical control point where the cell ensures that DNA
replication is complete and free from errors before entering mitosis. The observed accumulation of cells in the
G2/M phase indicates that compound 5 halts cell cycle
progression beyond this point, possibly due to its impact
on proteins involved in G2/M transition or mitotic spindle formation. The most remarkable finding was the
dramatic rise in the pre-G1 population upon treatment
with compound 5. The pre-G1 phase is associated with
cells undergoing apoptosis, which is a programmed cell
death process. The significant increase in cells in the preG1 phase suggests that compound 5 effectively induces
apoptosis in HepG2 cells. This implies that the treatment
with compound 5 leads to the activation of apoptotic
pathways, causing the cells to undergo DNA fragmentation and degradation, ultimately leading to cell death.
The ability of compound 5 to induce both G2/M arrest
and apoptosis is intriguing and suggests a multifaceted mechanism of action. G2/M arrest prevents cells
from progressing through the cell cycle, while apoptosis
eliminates the damaged or aberrant cells. These combined effects may contribute to the compound’s potent
anti-cancer activity against HepG2 cells. Taken together
the flow cytometric analysis revealed that compound 5
induces a notable arrest in cell growth during the G1/S
phase and triggers apoptosis in HepG2 cells by increasing
the percentage of cells arrested in the G2/M and pre-G1
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(2023) 17:174
Page 9 of 22
(A)
(B)
HepG2
HepG2+Compound 5
DNA Content (deploid %)
50
40
30
20
10
%
Pr
eG
1
M
%
G
2/
%
S
%
G
0G
1
0
(C)
Fig. 5 Flow cytometric analysis of the cell cycle phases in untreated HepG2 cells (A) and HepG2 cells treated with compound 5 (0.9 µM) (B). C DNA
content in the different cell cycle phases of HepG2 cells
phases. These findings suggest that compound 5 has significant anti-cancer potential and merits further investigation as a potential candidate for cancer therapy.
Annexin V‑FITC/ PI screening
Next, we have conducted Annexin V-FITC/PI screening
to further investigate the impact of compound 5 on the
growth of HepG2 cells and comprehensively analyze programmed cell death. The Annexin V-FITC/PI assay is a
widely used method to differentiate between apoptotic
and necrotic cells based on their membrane integrity
and phospholipid exposure [73]. As shown in Fig. 6, the
results indicated a substantial increase in the total percentage of programmed cell death in HepG2 cells upon
treatment with compound 5. Specifically, the percentage
of programmed cell death rose from 2.08% in untreated
cells to 45.88% after treatment with compound 5, representing a remarkable 22-fold increase. Furthermore, the
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 10 of 22
(A)
(B)
HepG2
HepG2+Compound 5
Programed cell death (%)
50
40
30
20
10
0
ta
To
l
is
os
t
op
rly
Ea
t
op
ap
te
ap
os
is
is
os
r
ec
N
La
(C)
Fig. 6 A: The apoptosis effect on human HepG2 cell line catalyzed by compound 5. B: Compound 5 induced apoptosis effect in the human HepG2
cell line
analysis revealed a notable increase in the percentage of
apoptosis during both the early and late phases in compound 5-treated HepG2 cells. The percentage of cells
undergoing early apoptosis increased from 0.46% in the
control group to 19.51% after compound 5 treatment,
indicating a significant 42-fold increase. Similarly, the
percentage of cells in late-stage apoptosis rose from 0.22%
in the control group to 22.16% after compound 5 treatment, representing an impressive 100-fold increase. On
the other hand, compound 5 did not induce a remarkable
effect on the necrosis pathway in HepG2 cells. The percentage of cells undergoing necrosis increased from 1.4%
(2023) 17:174
in untreated cells to 4.21% after compound 5 treatment,
resulting in a threefold increase. The results from the
Annexin V-FITC/PI screening provide crucial insights
into the mode of action of compound 5 in inducing cell
death in HepG2 cells. Annexin V-FITC is a marker that
binds specifically to phosphatidylserine (PS), a phospholipid that becomes externalized on the cell membrane
during early apoptosis. Propidium iodide (PI) is a dye that
can penetrate cells with compromised membranes, typically found in late-stage apoptosis or necrosis. The substantial rise in the total percentage of programmed cell
death upon treatment with compound 5 indicates that it
effectively induces cell death in HepG2 cells. The significant increase in both early and late-phase apoptosis suggests that compound 5 triggers the apoptotic pathway at
multiple stages, leading to cell demise. Notably, the lack
of a significant effect on necrosis suggests that compound
5 primarily induces apoptosis rather than necrosis in
HepG2 cells. This is important since apoptosis is a highly
regulated process, and its induction is a favorable outcome for anti-cancer therapies, as opposed to necrosis,
which is often associated with cell death caused by injury
or external damage. Overall, these findings from the
Annexin V-FITC/PI screening support the ability of compound 5 to induce apoptosis in HepG2 cells and its lack
of significant induction of necrosis reinforce its potential
as a promising candidate for anti-cancer therapy.
Assessment of Caspase‑3/7 activity
Caspase-3 is a cysteine-aspartic protease protein that
facilitates the enzymatic cleavage of specific target proteins. It consists of two subunits, specifically a 12-kDa
subunit and a 17-kDa subunit, which are characterized by
the presence of three and five thiol functions, respectively
[20, 74]. Caspases are initially produced as inactive zymogens, known as procaspases [75], which subsequently
activated in response to specific internal and/or external signals, such as the increased production of reactive
oxygen species (ROS). The crucial involvement of the terminal caspase-3/7 activation is observed in the initiation
of apoptosis and subsequent cell demise in neoplastic
cells [28]. To gain more insight into the apoptotic mode
of action for compound 5, we assessed the active caspase-3/7 levels in HepG2 cells utilizing a green flow cytometric assay. The results demonstrated that compound
5 treatment led to a substantial increase in the levels of
active Caspase-3/7 in HepG2 cells. Compared to the control group of untreated HepG2 cells, there was a remarkable 5.6-fold rise in the levels of active Caspase-3/7 upon
treatment with compound 5. This noteworthy increase
in Caspase-3/7 activity indicates that compound 5 has
the ability to promote apoptosis by triggering the activation of these key apoptotic proteins in HepG2 cells
Page 11 of 22
(Fig. 7). These findings provide valuable insights into the
potential mechanism of action of compound 5 as an anticancer agent. Activation of Caspase-3/7 is a pivotal step
in the apoptotic pathway, leading to the programmed
destruction of cancer cells. The significant increase in
active Caspase-3/7 levels observed in HepG2 cells treated
with compound 5 indicates that this compound has a
potent ability to induce apoptosis in cancer cells. The
observed rise in Caspase-3/7 activity complements our
results, which showed compound 5’s inhibitory effect
on β-tubulin polymerization. By disrupting microtubule
dynamics and simultaneously promoting Caspase-3/7
activation, compound 5 likely triggers multiple pathways
that contribute to the inhibition of cancer cell growth
and the induction of apoptosis. These results support
the hypothesis that compound 5’s anti-cancer activity in
HepG2 cells could be attributed, at least in part, to its
capacity to activate the apoptotic pathway.
Assessment of β‑tubulin polymerization activity
Microtubules are widely recognized as a significant target for anti-cancer therapy due to their pivotal involvement in cellular division and the preservation of cellular
morphology [76]. The impact of numerous anti-cancer
agents is achieved through their ability to disrupt microtubule dynamics [77]. This disruption ultimately results
in the deregulation of mitotic spindles, leading to cell
cycle arrest in cancer cells and subsequently inducing apoptosis [78]. Toward this end, we further aimed
to assess investigated the impact of compound 5 on
β-tubulin polymerization activity in HepG2 cells. Thus,
HepG2 cells were treated with compound 5 at (0.9 µM)
for a duration of 24 h and the activity of β-tubulin
20000
Casp 3/7 Activity (FLU)
Alzamami et al. BMC Chemistry
15000
10000
5000
0
Co
nt
r
H
ol
ep
G2
un
d
5
po
m
Co
Fig. 7 Effect of compound 5 on the level of caspase 3/7 in HepG2
cells
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 12 of 22
polymerization was explored using spectrophotometry at
450 nm. The findings indicate that compound 5 exhibits a
significant inhibitory effect toward β-tubulin polymerization as evidenced by a threefold reduction in the concentration of β-tubulin polymerization, as compared to the
untreated HepG2 cells (Fig. 8). These findings indicate
that compound 5 interferes with the process of microtubule assembly, thereby disrupting the proper formation
of mitotic spindles. Microtubules are essential structures
for cell division, and their dynamic behavior is critical for
the accurate segregation of chromosomes during mitosis.
By disrupting microtubule dynamics, compounds like 5
can induce mitotic defects and lead to cell cycle arrest in
cancer cells, ultimately triggering programmed cell death
(apoptosis). The observed inhibitory effect of compound
5 on β-tubulin polymerization strengthens the rationale
for considering it as a candidate for anti-cancer therapy.
By targeting the microtubule network, compound 5 may
hinder the uncontrolled proliferation of cancer cells and
potentially offer a more selective approach to cancer
treatment. Overall, these results suggest that compound
5’s anti-cancer activity may be attributed, at least in part,
to its disruption of microtubule dynamics, leading to
potential cell cycle arrest and apoptosis.
In silico molecular modeling study
Finally, we aimed to affirm the inhibitory activity of compound 5 toward β-tubulin activity by assessing its binding affinity toward the active site utilizing molecular
modelling analysis. The application of molecular docking
simulation has been widely utilized and demonstrated
to be efficacious in examining the interaction between
β-tubulin polymerization (ng/mL)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Co
nt
r
H
ol
ep
G2
d
5
un
po
m
Co
Fig. 8 Inhibitory activity of compound 5 toward β-tubulin
polymerization activity in HepG2 cells
a bioactive ligand and the active site of a particular protein, as well as in assessing its binding score [79]. For this
purpose, we retrieved the crystal structure of tubulin
(PDB code: 4yh2) along with its co-crystallized ligand,
C2-ligand, from the Protein Data Bank (PDB). Prior to
molecular modeling, unnecessary chains, water molecules, and other irrelevant entities were removed to
ensure the accuracy of the docking process. The docking methodology was carefully adjusted to achieve low
RMSD values, ensuring a reliable representation of
the interactions as predicted by the crystal structure.
The analysis revealed a series of interactions, including hydrogen bonding and hydrophobic interactions,
between the C2-ligand and multiple amino acid residues
within the binding pocket. The binding score obtained
for the interaction between the C2-ligand and tubulin
was -12.46 kcal/mol, indicating a strong binding affinity.
This negative binding score suggests a favorable interaction, signifying that the C2-ligand has the potential to
bind effectively to the active site of the tubulin protein.
The formation of hydrogen bonds and hydrophobic interactions with specific amino acid residues within the binding pocket further supports the credibility of the docking
results. The network of hydrophobic interactions involving residues like LEU255, ALA316, ALA354, and TYR202
demonstrates the involvement of specific amino acids in
reinforcing the binding between the C2-ligand and tubulin. In addition to the hydrogen bonding interactions,
a network of hydrophobic interactions was identified
between the C2-ligand and specific amino acid residues
in the binding pocket. The residues involved in these
hydrophobic interactions included LEU255, ALA316,
ALA354, and TYR202, with varying distances between
the ligand and the amino acids (Fig. 9, Table 2).
In contrast to the C2-ligand, compound 5 demonstrated a notable affinity for binding to the active site
of the tubulin protein, as indicated by a binding score
of -11.89 kcal/mol. This score reflects a strong binding
interaction between compound 5 and the tubulin protein,
suggesting its potential as a tubulin inhibitor. The analysis of the binding mode revealed that compound 5 has
the ability to interact with the tubulin protein through
its 3-hydroxyphenyl-acetamide moiety, forming a total
of four hydrogen bonds with various amino acid residues located within the binding pocket (Fig. 10). Hydrogen bonding interactions play a crucial role in stabilizing
ligand–protein complexes and promoting specific binding. Furthermore, the binding affinity between compound 5 and its binding site was reinforced by a network
of hydrophobic interactions involving specific amino acid
residues within the cavity. The residues involved in these
hydrophobic interactions included LEU248, LEU255,
ALA316, ALA354, ILE318, LYS352, and MET259, with
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 13 of 22
Fig. 9 Representative binding modes of C2-ligand inside the active site of tubulin (PDB code: 4yh2) protein through 2D (A), H-binding (B), aromatic
(C), and hydrophobic interactions (D)
Table 2 Binding score and interactions of C2-ligand into the active cavity of β-tubulin protein
Binding score (kcal/mol)
Hydrophobic interactions
−12.46
Hydrophilic interactions (Hydrogen
bonding)
Distance (A)
ASN167
2.95
CYS241
3.47
Q4ED504
3.68
Q4ED504
3.58
LEU255
3.7, 3.75
ALA 316
3.78, 3.77, 4.35
ALA354
3.8
TYR202
5.58
varying distances between the ligand and the amino
acids (Table 3). These identified interactions provide
valuable insights into the potential inhibitory activity of
compound 5 against the tubulin protein. By binding to
the active site of tubulin and forming hydrogen bonds
and hydrophobic interactions, compound 5 may disrupt microtubule dynamics, a critical process in cellular
division and maintaining cellular structure. This disruption could lead to cell cycle arrest and induce apoptosis,
making compound 5 a promising candidate for anti-cancer therapy.
The current findings further support and validate our
previous research concerning the inhibitory effect of
compound 5 on the tubulin protein in an in vitro environment. Moreover, the results of our study suggest that
the compound 8-methoxy-azacoumarin-3-carboxamide,
a part of compound 5’s chemical structure, holds potential as a foundational structure for the development of
Alzamami et al. BMC Chemistry
(2023) 17:174
Page 14 of 22
Fig. 10 Representative binding modes of compound 5 inside the active site of tubulin (PDB code: 4yh2) protein through 2D (A), H-binding (B),
aromatic (C), and hydrophobic interactions (D)
Table 3 Binding score and interactions of compound 5 into the active cavity of β-tubulin protein
Binding score (kcal/mol)
Hydrophobic interactions
−11.89
Hydrophilic interactions (Hydrogen
bonding)
Distance (A)
ALA354
3.23
LEU255
2.36, 2.18
LEU248
3.84, 5.39
LEU255
3.59
ALA 316
4.27, 4.45
ALA354
3.5, 4.9
ILE318
4.62
LYS352
4.49
MET259
5.46
highly effective tubulin inhibitors. This information could
inspire future drug design efforts to optimize compound
5 or its derivatives as potential anti-cancer agents targeting tubulin. Taken together, the molecular docking analysis demonstrates a notable affinity of compound 5 for the
active site of the tubulin protein. The formation of hydrogen bonds and hydrophobic interactions supports its
potential as a tubulin inhibitor. These findings contribute
to our understanding of compound 5’s inhibitory effect
on tubulin and highlight its potential as a candidate for
Alzamami et al. BMC Chemistry
(2023) 17:174
further development in the quest for novel and effective
tubulin inhibitors for cancer treatment.
Material and method
Instrumentation, reagents and analysis
Reputable commercial hubs were the source of all the
chemicals and solvents, used in the current study. During purchase of chemicals high level purity and basic
standards of analytical grade were observed using different quality control protocols to ensure the precision
and reproducibility of the experimental work. Reliable
and accurate results during characterization and analysis of compounds was made sure by proper calibration of
instruments, careful sample preparation and appropriate
standerds used. Bruker avance 400 MHz and 100 MHz
spectrometers were used for 1H and 13C NMRs respectively, while DMSO-d6 having tetra methyl silane solvent
was used as internal standard. δ shifts (parts per million)
is the key indicator while characterization and structural
orientation of compounds while using the NMR. Microanalytical analysis for the determination of C, H, and N
contents were carried out using Perkin-Elmer 2400 series
with CHN analyzer, helping the accurate analysis and
molecular formula determination of the compounds.
EI-MS was performed using an Agilent Technologies
6890N gas chromatograph (GC) equipped with a 5973mass spectrometer selective detector. The EI-MS analysis
confirmed the molecular weights and ionic charges of the
compounds by providing data on their mass-to-charge
ratio. Crystalline compounds’ melting points were determined without any corrections using an electrothermal
melting point equipment. Purity, crystallinity, and identity of a compound is dependent on its melting point and
for the verification of all above these mentioned parameters they were compared with standards. The vibrations
and functional groups of compounds in the IR region
were analyzed by recording their spectra using an infrared (IR) spectrometer (Bruker FT-8000). Infrared spectra
provide information about the chemical bonds and functional groups present in a substance, allowing for more
precise identification and characterization.
Synthetic protocols and analytical assessments
Synthesis of Compound 2
Compound 1 was synthesized through the fusion of a
mixture containing 0.01 mol of 3-methoxy-2-hydroxybenzaldehyde and 0.01 mol of diethylmalonate on a
hot-plate. The reaction was conducted in the presence
of 1 mL of piperidine for a duration of 5 min. Following
that, a volume of 20 mL of ethanol was introduced into
the reaction mixture, which was then subjected to reflux
for a duration of 2 h. Following the completion of the
reaction as judged by TLC analysis, the reaction mixture
Page 15 of 22
was subsequently cooled and carefully transferred into
an ice-water bath with continuous stirring. Subsequently,
the mixture was subjected to neutralization using a solution of (2%, aqueous) dil. hydrochloric acid. The resultant solid was subsequently gathered through the process
of filtration. Ethyl 8-methoxycoumarin-3-carboxylate (2)
was obtained according to above mentioned process, as
white crystals. Yield 81%, m.p. 99 °C. IR (KBr) υmax: 1736,
1715 (C = O), 1605, 1585 (C = C), 1086, 1037 (C-O) cm−1.
1
H-NMR (DMSO-d6, ppm) δ: 1.35 (t, 3H, CH3), 3.94
(s, 3H, OCH3), 4.33 (q, 2H, OCH2), 7.32–7.47 (m, 3H,
Ar–H), 8.73 (s, 1H, H- coumarin). 13C-NMR (DMSOd6, ppm) δ: 164.51, 163.10 (C = O), 157.23, 154.57 (C-O),
149.25 (C-coumarin), 135.03, 130.91, 125.42, 118.82,
118.33, 116.56 (C-coumarin and C-aromatic), 61.69
(OCH2), 56.60 (OCH3), 14.53 (CH3) (in agreement with
previous reports [80, 81]).
Synthesis of brominated compounds 3 and 9
Compounds 1 and 8 (0.01 mol) were dissolved in 20 ml
of glacial acetic acid. Subsequently, a solution of bromine (0.01 mol) in glacial acetic acid was added dropwise to the mixture of compounds 1 and 8 while stirring
at a temperature of 60℃. After a duration of 10 min, the
color of bromine was eliminated, and a yellow solution
persisted. During this stage of the experiment, a solution containing 0.5–1.0 ml of Br2/AcOH was introduced
with continuous agitation at ambient temperature for a
duration of 8 h. Subsequently, the resulting mixture was
carefully poured into water while maintaining a stirring
motion. The solid that was generated underwent separation via filtration, followed by washing with water, drying, and subsequent recrystallization from an appropriate
solvent, resulting in the formation of compounds 3 and 9.
Ethyl 5-bromo-8-methoxycoumarin-3-carboxylate (3) As
pale-yellow crystals, yield 71%, m.p. 155 °C. IR (KBr) υmax:
1745, 1720 (C = O), 1610, 1588 (C = C), 1091, 1036 (C-O)
cm−1. 1H-NMR (DMSO-d6, ppm) δ: 1.36 (t, 3H, CH3), 3.94
(s, 3H, OCH3), 4.34 (q, 2H, OCH2), 7.38 (d, 1H, Ar–H),
7.66 (d, 1H, Ar–H), 8.60 (s, 1H, H-coumarin ring). MS:
m/z (%) = 328 (M+ + 2, 67.03), 326 (M+, 100), 300 (6.81),
299 (6.31), 283 (21.40), 281 (8.52), 255 (11.30), 254 (18.87),
248 (8.14), 247 (1.11), 219 (7.18), 181 (7.60), 156 (6.39), 119
(9.14), 105 (9.30), 104 (25.72). Anal. Calcd for C13H11BrO5
(M. wt. = 326): C, 47.85; H, 3.77. Found: C, 47.51; H, 3.20.
5-Bromo-8-methoxycoumarin-3-carboxylic acid (9) As
colorless crystals, yield 53%, m.p. 210 °C. IR (KBr) υmax:
3360–2650 (br. OH), 1726–1718 (br. C = O), 1605, 1586
(C = C), 1081, 1031 (C-O) cm−1. 1H-NMR (DMSO-d6,
ppm) δ: 3.94 (s, 3H, OCH3), 7.29–7.31 (d, 1H, Ar–H),
7.58–7.60 (d, 1H, Ar–H), 8.45 (s, 1H, H-coumarin). 13C-
Alzamami et al. BMC Chemistry
(2023) 17:174
NMR (DMSO-d6, ppm) δ: 164.78, 156.72 (C = O), 146.61,
144.93 (C-O), 144.61 (C-coumarin), 128.40, 122.97,
118.34, 116.84, 112.24 (C-aromatic and C-3 of pyranone
ring), 56.85 (OCH3). MS: m/z (%) = 300 (M+ + 2, 21.03),
299 (M+ + 1, 6.31), 298 (M+, 21.54), 297 (M+-1, 3.43), 256
(32.18), 255 (12.29), 254 (37.81), 228 (1.36), 227 (1.82), 226
(4.58), 213 (14.63), 212 (3.46), 211 (18.34), 204 (1.15), 203
(4.88), 185 (13.91), 183 (16.48), 175 (9.17), 157 (15.38),
156 (6.01), 155 (10.24), 141 (43.53), 140 (52.06), 139 (100),
120 (6.36), 119 (12.34), 113 (9.87), 111 (39.95), 104 (4.65),
103 (12.80), 96 (24.29), 95 (2.08), 94 (15.40), 93 (13.93),
91 (8.70), 89 (6.31), 76 (9.03), 75 (24.00), 74 (16.02). Anal.
Calcd for C11H7BrNO5 (M. wt. = 298): C, 44.29; H, 2.35.
Found: C, 44.04; H, 2.13.
Synthesis of 5‑substituted
8‑methoxycoumarin‑3‑carboxamides (4 and 5)
A blend comprising ester coumarin 2 or 3 (0.01 mol) and
ammonium acetate (5 mol.) was subjected to fusion on a
hot plate at a temperature range of 140℃ for a duration
of 6 h. Subsequently, the mixture was cooled and introduced into water while being agitated. The precipitated
powder was separated through the process of filtration,
followed by washing with water, subsequent drying, and
recrystallization using an appropriate solvent, resulting
in the formation of compounds 4 and 5.
Page 16 of 22
δ: 162.44, 159.80 (C = O), 146.81, 145.91 (C-O), 144.79
(C-coumarin), 128.84, 121.16, 118.63, 117.33, 112.65
(C-aromatic and C-3 of pyranone ring), 56.94 (OCH3).
MS analysis: m/z (%) = 299 (M+ + 2, 74.90), 298 (M+ + 1,
27.75), 297 (M+, 100), 233 (8.13), 231 (14.27), 220 (4.09),
219 (53.00), 218 (76.14), 217 (2.89), 213 (13.40), 211 (18.13),
203 (19.43), 202 (19.56), 191 (1.09), 190 (49.77), 189 (3.06),
185 (6.91), 184 (6.25), 183 (10.63), 175 (7.45), 174 (12.27),
173 (4.95), 169 (3.59), 167 (2.57), 162 (2.12), 158 (7.69),
157 (12.25), 148 (6.07), 147 (9.90), 146 (2.79), 145 (2.76),
133 (8.56), 132 (6.32), 119 (22.87), 118 (16.41), 117 (14.48),
105 (12.75), 104 (14.84), 103 (44.53), 91 (20.07), 90 (15.95),
89 (25.89), 88 (9.37), 87 (13.04), 77 (12.09), 76 (29.94), 75
(46.82), 74 (20.96), 63 (7.39), 62 (2.34), 50 (3.37). Anal.
Calcd for C11H8BrNO4 (M. wt. = 297): C, 44.48; H, 2.70;
N, 4.70. Found: C, 44.18; H, 2.32, N, 4.47.
Synthesis of acetyl derivatives 6, 7
A mixture containing compound 4 or 5 (0.01 mol) dissolved in acetic anhydride (25 mL) was subjected to
reflux for a duration of 12 h. Subsequently, the mixture
was cooled and gradually added to a stirred solution of
ice-water. The reaction mixture was allowed to stand for
a duration of 24 h, following which the resultant product
was obtained through the process of filtration. Subsequently, the product was subjected to a water wash and
subsequently dried. Ultimately, the formation of compounds 6 and 7 was achieved through the crystallization
of the solid product using an appropriate solvent.
8-Methoxycoumarin-3-carboxamide (4) The entitled
compound afforded as pale-yellow crystals. Yield 55%,
m.p. 253 °C. IR (KBr) υmax: 3315–3187 (NH2), 1726–1690
(br. C = O), 1611, 1590 (C = C), 1083, 1045 (C-O) cm−1.
1
H-NMR (DMSO-d6, ppm) δ: 3.94 (s, 3H, OCH3), 7.35–
7.52 (m, 3H, Ar–H), 7.95 (s, 1H, NH2), 8.09 (s, 1H, NH2),
8.87 (s, 1H, H-coumarin). 13C-NMR (DMSO-d6, ppm)
δ: 163.02, 160.62 (C = O), 148.49, 146.68, 143.79 (C-coumarin), 125.47, 121.58, 119.83, 119.51, 116.49 (C-aromatic and C-3 of pyranone ring), 56.71 (OCH3). MS: m/z
(%) = 220 (M+ + 1, 14.50), 219 (M+, 100), 203 (35.33), 176
(11.18), 175 (10.51), 174 (6.21), 173 (5.06), 133 (32.07), 131
(3.02), 120 (7.96), 119 (14.36), 118 (12.96), 117 (3.92), 105
(29.26), 104 (9.60), 102 (5.94), 91 (12.96), 90 (11.88), 89
(31.65), 77 (45.22), 76 (35.40), 75 (16.45), 74 (16.85), 65
(10.09), 63 (22.72), 62 (17.43), 51 (23.99), 50 (18.49). Anal.
Calcd for C11H9NO4 (M. wt. = 219): C, 60.27; H, 4.11; N,
6.39. Found: C, 60.12; H, 3.97, N, 6.11.
N-acetyl 8-methoxycoumarin-3-carboxamide (6) As colorless crystals, yield 67%, m.p. 285 °C. IR (KBr) υmax: 3227
(NH), 1725–1689 (br. C = O), 1605, 1582 (C = C), 1078, 1042
(C-O) cm−1. NMR spectra: no data because compound 6
insoluble in solvent. MS: m/z (%) = 262 (M+ + 1, 1.13), 261
(M+, 15.73), 220 (15.83), 219 (100), 204 (6.61), 203 (74.69),
202 (1.41), 191 (15.14), 190 (1.22), 176 (17.88), 175 (11.90),
174 (8.38), 173 (5.83), 161 (4.91), 148 (17.51), 147 (0.05), 146
(4.84), 133 (31.19), 132 (3.29), 120 (13.04), 119 (18.49), 118
(17.53), 117 (10.14), 105 (41.74), 104 (12.11), 103 (12.00), 102
(9.11), 92 (7.73), 91 (18.86), 90 (15.39), 89 (40.25), 88 (12.16),
87 (4.46), 78 (3.66), 77 (32.87), 76 (29.71), 75 (11.40), 74
(7.93), 73 (3.90), 65 (6.82), 63 (11.19), 62 (6.64), 51 (15.30), 50
(9.45). Anal. Calcd for C13H11NO5 (M. wt. = 261): C, 59.77;
H, 4.21; N, 5.36. Found: C, 59.59; H, 4.01, N, 5.11.
5-Bromo-8-methoxycoumarin-3-carboxamide
(5) As
pale yellow crystals, yield 61%, m.p. 285 °C. IR (KBr) υmax:
3336, 3189 (NH2), 1723–1691 (br. C = O), 1610, 1588
(C = C), 1067, 1035 (C-O) cm−1. 1H-NMR ((DMSO-d6,
ppm) δ: 3.94 (s, 3H, OCH3), 7.37–7.39 (d, 1H, Ar–H),
7.67–7.69 (d, 1H, Ar–H), 8.09 (br. s, 2H, NH2), 8.76 (s,
1H, H-4 of coumarin) ppm. 13C-NMR (DMSO-d6, ppm)
N-acetyl 5-bromo-8-methoxycoumarin-3-carboxamide
(7) As pale yellow crystals, yield 56%, m.p. 220 °C. IR
(KBr) υmax: 3225 (NH), 1723–1696 (br. C = O), 1612,
1590 (C = C), 1083, 1062 (C-O) cm−1. 1H-NMR (DMSOd6, ppm) δ: 2.33 (s, 3H, COCH3), 3.94 (s, 3H, OCH3),
7.36–7.71 (m, 2H, Ar–H), 8.53 (s, 1H, H-4 of coumarin
ring), 11.09 (br. s, 1H, NH). 13C-NMR ((DMSO-d6, ppm)
Alzamami et al. BMC Chemistry
(2023) 17:174
δ: 171.71, 162.80, 162.41, 162.29, 159.78, 158.78 (C = O
of more than two isomer), 146.77, 146.69, 145.90 (C-O),
144.81, 144.67, 144.60 (C-4 of pyranone ring), 129.13,
128.84, 122.96, 121.12, 118.61, 118.39, 117.69, 117.32,
112.65, 112.59 (C-aromatic and C-3 of pyranone ring),
57.00, 56.93 (OCH3 of two isomer), 25.59 (CH3). MS analysis: m/z (%) = 341 (M+ + 2, 27.20), 340 (M+ + 1, 9.59), 339
(M+, 39.51), 338 (M+-1, 20.28), 300 (10.38), 299 (45.60),
298 (15.98), 297 (39.20), 283 (49.54), 282 (48.53), 281
(54.54), 280 (55.45), 279 (8.65), 271 (23.90), 270 (10.78),
269 (27.81), 268 (28.07), 261 (6.48), 260 (100), 256 (21.67),
255 (9.86), 254 (26.83), 219 (16.45), 218 (46.59), 217
(71.35), 212 (20.94), 211 (15.71), 210 (25.44), 203 (21.80),
202 (36.00), 199 (27.55), 198 (19.40), 197 (24.21), 196
(22.98), 190 (41.75), 189 (19.40), 175 (24.61), 174 (15.69),
173 (15.51), 167 (15.07), 166 (19.41), 158 (16.49), 157
(24.22), 156 (14.27), 155 (28.27), 141 (39.93), 140 (25.11),
139 (83.88), 138 (82.81), 119 (19.60), 118 (16.56), 117
(12.17), 104 (6.87), 103 (35.59), 102 (15.79), 91 (8.05), 90
(7.08), 89 (11.82), 76 (6.27), 75 (22.37), 74 (11.19). Anal.
Calcd for C13H10BrNO5 (M. wt. = 339): C, 46.02; H, 2.95;
N, 4.13. Found: C, 45.87; H, 2.73, N, 4.02.
Synthesis of 8‑methoxycoumarin‑3‑carboxylic acid (8)
A solution of coumarin-3-carboxamide (4, 0.01 mol)
was dissolved in acetic acid (15 ml), then 15 ml 4N HCl
was added. The reaction mixture was refluxed for 16 h,
and then the contents of the reaction were poured into
crushed ice. The resulting mixture was left at room temperature overnight, and the solid that formed was filtered
off. The solid was then washed with water and dried.
Afterward, recrystallization from ethanol was carried
out to further purify the product. Colorless crystals.
Yield 78%, m.p. 186 °C, IR (KBr) υmax: 3350–2851 (br.
OH), 1725–1706 (br. C = O), 1606, 1591 (C = C), 1036,
1021 (C-O) cm−1. 1H-NMR (DMSO-d6, ppm) δ: 3.93 (s,
3H, OCH3), 7.31–7.46 (m, 3H, Ar–H), 8.73 (s, 1H, H-4
of coumarin). 13C-NMR (DMSO-d6, ppm) δ: 164.49,
156.90 (C = O), 148.95, 146.67 (C-O), 144.24 (C-4 of
coumarin ring), 125.18, 121.52, 119.07, 118.96, 116.61
(C-aromatic and C-3 of pyranone), 56.60 (OCH3). MS
analysis: m/z (%) = 221 (M+ + 1, 16.00), 220 (M+, 96.36),
203 (22.34), 177 (8.93), 176 (100.00), 161 (15.58), 149
(7.22), 148 (38.72), 147 (20.17), 146 (6.35), 141 (7.33), 139
(34.97), 133 (61.14), 131 (4.08), 120 (17.92), 119 (12.91),
118 (18.20), 117 (2.41), 111 (7.98), 105 (52.55), 104 (7.43),
103 (12.87), 102 (10.13), 91 (17.12), 90 (12.72), 89 (24.94),
88 (5.49), 77 (33.45), 76 (16.34), 75 (7.56), 65 (7.99), 63
(5.95), 62 (1.90), 51 (13.66). Anal. Calcd for C11H8O5 (M.
wt. = 220): C, 60.00; H, 3.64. Found: C, 59.83; H, 3.33.
Page 17 of 22
In vitro assessment of cytotoxic activity against HepG2
and WI 38 cells
The antitumor efficacy of the recently developed coumarin compounds 4–9 was evaluated against the HepG2
and WI 38 cell lines (Sigma-Aldrich, USA, St. Louis,
product number 85011430, and 90020107, respectively)
utilizing the MTT assay technique. The cells were seeded
in 96-well plates at a density of 1 × 104 and incubated at
37 °C for 48 h in the presence of 5% CO2. Following the
incubation period, the cells were subjected to various
concentrations of the prepared molecules and subsequently incubated for a duration of 24 h. The MTT dye
was introduced after a 24 h period of drug treatment and
subsequently incubated for a duration of 4 h at a temperature of 37 °C. Subsequently, a volume of 100 μL of
dimethyl sulfoxide (DMSO) was introduced into each
well in order to facilitate the dissolution of the purple
formazan that had been generated. The quantification
of the color intensity of the formazan product, which
serves as an indicator of the cellular growth condition, is
performed by employing an ELISA plate reader set at a
wavelength of 570 nm. The experimental conditions were
implemented with a minimum of three replicates, and
the experiments were conducted on at least three separate occasions.
Cell cycle analysis
The quantification of DNA content in the cell cycle analysis was performed using a FACS Calibur flow cytometer
at a wavelength of 488 nm, following the guidelines provided by the manufacturer. In this study, a total of 2 × 105
cells per well were subjected to treatment with a specific
compound, referred to as molecule 5, at concentrations
corresponding to its IC50 for a duration of 24 h. Following
the completion of the treatment, the cells underwent two
rounds of washing and were subsequently resuspended in
phosphate-buffered saline (PBS). Following the washing
step, a volume of 0.7 ml of absolute ethanol was subsequently introduced, and the mixture was incubated at a
temperature of -20 °C for a duration of 20 min. Following the washing step, a volume of 500 μl of RNase was
introduced, and subsequently, the mixture was incubated
for a duration of 30 min. Next, the addition of PI was carried out, followed by a 30-min incubation period, during
which exposure to light was carefully avoided.
Annexin V‑FITC/ PI screening
Apoptosis detection in HepG-2 cells was conducted utilizing the BioVision® annexin‐V‐FITC apoptosis detection kit in accordance with the instructions provided by
Alzamami et al. BMC Chemistry
(2023) 17:174
the manufacturer. The quantification of apoptosis was
performed using a FACS Calibur flow cytometer, with
a wavelength of 488 nm. In a concise manner, a total of
1–5 × 105 cells were obtained through the process of
centrifugation. The cells were subsequently exposed to
compound 5 at concentrations corresponding to its IC50
value for a duration of 24 h. Following this treatment, the
cells were resuspended in a binding buffer solution with
a volume of 500 μl. The addition of Annexin-V-FITC and
PI was performed. Subsequently, the specimens were
subjected to incubation at ambient temperature for a
duration of 5 min, while being shielded from light. The
analysis of Annexin-V-FITC binding was conducted utilizing a dedicated signal detector.
Assessment of caspase‑3/7 activity
The HepG2 cell line was subjected to treatment with
compound 5, and subsequently, an analysis was conducted using the Cell Event caspase-3/7 green flow assay.
The Cytometry Assay Kit, identified by Catalog Number C10427 (Cayman, USA), is utilized in the field of
academic research. The cell suspensions were subjected
to centrifugation at 100 g for a duration of 7 min at a
temperature of 37 °C, following which the supernatants
were carefully extracted. The pellets were reconstituted
in a 500 μl solution of phosphate buffer solution (PBS).
Subsequently, a volume of 0.5 μL of the cell Event Caspase-3/7 Green detection Reagent was introduced to all
cellular samples. The cells were subjected to incubation
at a temperature of 37 °C for a duration of 30 min. Subsequently, the cells were assessed utilizing the BD FACS
CALIBER flow cytometer.
Assessment of β‑tubulin polymerization activity
Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen/Life Technologies) containing 10% fetal bovine
serum (FBS) (Hyclone), 10 µg/mL of insulin (Sigma),
and 1% penicillin–streptomycin was used for culturing
the HepG2 cell line. The cancer cell suspension and the
tested compound 5 were dispensed in a 96-well plate at
a volume of 100 μL each per well. After incubating the
plate for 18–24 h, the enzyme assay for tubulin was performed. The microtiter plate used in this study was precoated with an antibody specific to TUB β. Following
that, standards or samples were added to their respective
wells on the microtiter plate, along with a biotin-conjugated antibody that specifically binds to TUB β. Subsequently, the addition of Avidin-Horseradish Peroxidase
(HRP) conjugate to individual microplate wells was followed by an incubation period. Upon addition of the
TMB substrate solution, only the wells that contain TUB
β, biotin-conjugated antibody, and enzyme-conjugated
Avidin will manifest a discernible alteration in coloration.
Page 18 of 22
To conclude the enzymatic reaction between the enzyme
and substrate, a solution of sulfuric acid was introduced,
leading to a color change. This color change was then
quantitatively measured using spectrophotometry at
a specific wavelength of 450 nm ± 10 nm. To determine
the concentration of TUB β in the samples, the optical
density (O.D.) of the samples was compared to a standard curve. The experiments were conducted in triplicate,
repeating the process three times for each sample, to
ensure the accuracy and consistency of the results.
In silico computational studies
The binding affinity of this particular chemical class to
the active site of the tubulin protein was assessed through
a thorough computational analysis using MOE software.
The application of molecular docking enables the assessment of the binding affinity between small molecules and
the binding site of a particular protein, thereby providing
valuable insights into the mechanism of action of pharmacological compounds. Compound 5 was selected to
investigate the affinity of the developed N-(substitutedphenyl)-8-methoxycoumarin-3-carboxamide analogues
for the functional pocket of the tubulin protein. Using
Chem.Draw, we were able to determine compound 5’s
2-dimensional structure, which paved the way for additional computer investigation of the molecule. Tubulin
protein crystallographic structures are abundant in the
Protein Data Bank (PDB), which has greatly aided our
research. We zeroed focused on the tubulin protein crystal structure (PDB code: 4yh2) in particular. These structures allow for in-depth investigation of the proteins’
structural characteristics and probable binding sites by
providing information about their three-dimensional
organization. Multiple experimental techniques were
employed to prepare the 3D structures of the tubulin protein for subsequent docking simulations. To account for
the ionization states of amino acid residues at a specific
pH, the structures were initially subjected to protonation.
Subsequently, the structures underwent refinement procedures aimed at eliminating all components other than
the target protein. This involved the removal of superfluous chains and water molecules, as well as the assignment
of partial charges to the remaining atoms. The process
of energy minimization was employed to determine the
protein conformations that exhibit the highest stability.
The MMFF94X force field was utilized to represent the
internal charge distribution. There were adjustments
made to the docking technique to improve its reliability
and accuracy. In this analysis, we made some minor alterations, such as using the Triangle Matcher placement
method and the London dG score formula. To improve
ligand binding prediction accuracy, a specialized procedure was developed. Careful analysis was performed
Alzamami et al. BMC Chemistry
(2023) 17:174
to evaluate the binding affinity and interaction mode of
the first co-crystallized ligand, and the results were compared to those published in prior research to verify the
effectiveness of the improved methodology. This contrast
was used as a benchmark against which the credibility
of the docking findings could be assessed. After running
the docking simulations, the resulting data was subjected
to a thorough analysis. The selection of binding modes
with high binding affinity was based on their potential to
offer valuable insights into the intended targets, warranting further investigation. Quantitative assessments of the
ligand-receptor interactions were conducted by estimating the docking scores and binding energies, utilizing the
specified binding modes. The dependability and accuracy
of the docking predictions were enhanced by the use of a
thorough docking process that included the positioning
of Triangle Matcher and the use of the London dG scoring function. High-affinity binding modes were identified
after an evaluation of the ligand’s binding interactions,
allowing for a comprehensive analysis of the ligand’s
potential binding affinity and energetics. Fifty different protein conformations were created throughout our
study. The interactions between the ligand (compound 5)
and the amino acid residues were then studied by evaluating these conformations. In addition, the determination of the binding energy for each conformation was
performed in order to gain a deeper understanding of the
chemical’s stability and potential affinity within the binding site. This was achieved by quantifying the strength of
the interactions between the ligand and the protein.
Conclusion
The presented study provides compelling evidence for
the potential therapeutic value of a novel class of 8-methoxycoumarin-3-carboxamides in the treatment of hepatocellular carcinoma (HCC). Liver cancer remains a
significant global health challenge, and existing pharmacological treatments have limitations, making the search
for effective and safe therapeutic agents imperative. The
8-methoxycoumarin-3-carboxamides synthesized in this
study demonstrated remarkable antiproliferative activity against HepG2 cells, a representative model of HCC.
Notably, compound 5 emerged as the most promising candidate, displaying exceptional inhibitory effects
with an IC50 value of 0.9 µM, surpassing the anticancer
drug staurosporine while exhibiting minimal toxicity to
normal cells. The mechanisms underlying the antiproliferative activity of compound 5 were elucidated, showing its ability to induce cell cycle arrest during the G1/S
phase and trigger apoptosis without significant necrosis
in HepG2 cells. Furthermore, compound 5 effectively
activated caspase3/7 proteins and disrupted β-tubulin
polymerization, indicating its potential to interfere with
Page 19 of 22
critical cellular processes involved in cancer cell growth
and survival. The molecular modeling analysis reaffirmed
the strong binding affinity of compound 5 to the active
cavity of β-tubulin protein, further supporting its potential as an effective therapeutic agent targeting HCC. The
presented findings underscore the promising therapeutic
potential of 8-methoxycoumarin-3-carboxamides, especially compound 5, in combatting hepatocellular carcinoma. Although these results are encouraging, further
studies, including in vivo experiments, are warranted
to validate the efficacy of compound 5 and related analogues. Nevertheless, our research lays the foundation
for the discovery of new, potent anti-HCC agents and
provides valuable insights into the mechanisms of action
that can guide future drug development efforts.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13065-023-01063-5.
Additional file 1: Scheme S1. Main fragmentation pattern of compounds
4 and 6. Scheme S1. Main fragmentation pattern of compounds 5 and 7.
Figure S1a. 1H NMR spectra of compound 2. Figure S1b. Mass spectrum
of compound 2. Figure S2a. 1H NMR spectra of compound 3. Figure
S2b. Mass spectrum of compound 3. Figure S3a. 1H NMR and 13C NMR
spectra of compound 4. Figure S3b. Mass spectrum of compound 4.
Figure S4a. 1H NMR and 13C NMR spectra of compound 5. Figure S4b.
Mass spectrum of compound 5. Figure S5. Mass spectrum of compound
6. Figure S6a. 1H NMR and 13C NMR spectra of compound 7. Figure
S6b. Mass spectrum of compound 7. Figure S7a. 1H NMR and 13C NMR
spectra of compound 8. Figure S7b. Mass spectrum of compound 8.
Figure S8a. 1H NMR and 13C NMR spectra of compound 9. Figure S8b.
Mass spectrum of compound 9. Figure S9. The dose-dependent cytotoxic
activity of synthesized compound 5 toward WI 38 cells, as compared to
STU. The presented data shows the mean ± standard deviation of the
mean obtained from at least three independent experiments. Table S1.
Cytotoxic evaluation of compounds 4–9, as compared to STU, against
human liver carcinoma HepG2.
Acknowledgements
The authors would like also to thank the Deanship of Scientific Research at
Shaqra University for supporting this work. The authors also acknowledge the
Princess Nourah bint Abdulrahman University for funding this work through
the Researchers Supporting Project number (PNURSP2023R89), Princess
Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. This study was also
supported by Researchers Supporting Project number (RSP2023R111), King
Saud University, and Riyadh, Saudi Arabia.
Author contributions
Conceptualization, AA, EMR, EA-E, MEB, MJ, and EMS; methodology, AA, EMR,
EA-E, MEB, EAO, ASA, and EMS; software, EMR, EA-E, HAA, EAO, ASA, NGMA, NA,
MJ, and EMS; validation, AA, MEB, EAO, ASA, NGMA, and NA; formal analysis,
AA, EMR, EA-E, MEB, HAA, EAO, ASA, and EMS; investigation, AA, EMR, MEB,
ASA, NGMA, MJ, and EMS; data curation, EMR, EA-E, MEB, HAA, EAO, N.A., and
MJ; writing—original draft preparation, AA, EMR, EA-E, MEB, MJ, and EMS;
writing—review and editing, AA, EMR, EA-E, MEB, HAA, EAO, ASA, NGMA, NA,
MJ, and EMS; visualization, EMR, EA-E, MEB, HAA, EAO, ASA, NGMA, and NA;
supervision, AA, EMR, MEB, EAO, MJ, and EMS; project administration, AA, EMR,
MEB, EAO, MJ, and EMS; funding acquisition, AA, EAO, NA, MJ, and EMS. All
authors have read and agreed to the published version of the manuscript.
Funding
Open Access funding enabled and organized by Projekt DEAL. We acknowledge
also support by the Open Access Publication Fund of Humboldt-Universität zu
Alzamami et al. BMC Chemistry
(2023) 17:174
Berlin. This research was funded by Faculty of Science, and Port-Said University,
and Suez Canal University, Egypt. The authors also extend their appreciation to
Princess Nourah bint Abdulrahman University for funding this work through the
Researchers Supporting Project Number (PNURSP2023R89), Princess Nourah
bint Abdulrahman University, Riyadh, Saudi Arabia. This study was also supported by Researchers Supporting Project Number (RSP2023R111), King Saud
University, and Riyadh, Saudi Arabia. The authors would like also to thank the
Deanship of Scientific Research at Shaqra University for supporting this work.
Availability of data and materials
All data generated or analyzed during this study are included in this article
(and its additional information files). Samples from compounds 4–9 are available from the authors.
Page 20 of 22
7.
8.
9.
10.
11.
Declarations
12.
Ethics approval and consent to participate
Not applicable.
13.
Consent for publication
Not applicable.
14.
Competing interests
The authors declare that they have no competing interests.
15.
Author details
1
Clinical Laboratory Science Department, College of Applied Medical Science, Shaqra University, AlQuwayiyah 11961, Sahqra, Saudi Arabia. 2 Chemistry Department (The Division of Organic Chemistry), Faculty of Science,
Port-Said University, Port-Said, Egypt. 3 Chemistry Department (The Division
of Biochemistry), Faculty of Science, Port-Said University, Port-Said, Egypt.
4
Department of Biology, College of Science, Imam Abdulrahman Bin Faisal
University, 31441 Dammam, Saudi Arabia. 5 Basic & Applied Scientific Research
Centre, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, 31441 Dammam, Saudi Arabia. 6 Department of Zoology, College of Science, King Saud
University, Riyadh, Saudi Arabia. 7 Department of Pharmaceutical Chemistry,
College of Pharmacy, Prince Sattam Bin Abdulaziz University, PO Box 173,
11942 Alkharj, Saudi Arabia. 8 Egyptian Drug Authority EDA Previously NODCAR
, Cairo, Egypt. 9 Department of Pharmaceutical Sciences, Princess Nourah bint
Abdulrahman University, P.O. Box 84428, 11671 Riyadh, Saudi Arabia. 10 Division
of Biological and Environmental Sciences and Engineering, Smart-Health
Initiative and Red Sea Research Center, King Abdullah University of Science
and Technology, P.O. Box 4700, 23955-6900 Thuwal, Saudi Arabia. 11 Chemistry
Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt.
12
Institute for Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2,
12489 Berlin, Germany.
16.
17.
18.
19.
20.
21.
22.
Received: 1 August 2023 Accepted: 23 October 2023
23.
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