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Dual PI3K/Akt Inhibitors Bearing Coumarin-Thiazolidine
Pharmacophores as Potential Apoptosis Inducers in
MCF-7 Cells
Rana M. Abdelnaby 1, *, Heba S. Rateb 2 , Omaima Ali 3 , Ahmed S. Saad 4 , Rania I. Nadeem 5 ,
Sahar M. Abou-Seri 6 , Kamilia M. Amin 6 , Nancy S. Younis 7 and Rasha Abdelhady 8
1 Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University, Cairo 11785, Egypt
2 Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Science and Drug Manufacturing,
Misr University for Science and Technology, 6th of October City 12585, Egypt; heba.sayed@must.edu.eg
3 Egyptian Drug Authority, Cairo 12618, Egypt; omaima_salah@hotmail.com
4 Pharmacology and Toxicology Department, Faculty of Pharmacy, Port Said University, Port Said 42511, Egypt;
mosa1200@yahoo.com
5 Pharmacology and Toxicology Department, Faculty of Pharmacy, Heliopolis University, Cairo 11785, Egypt;
rania.ibrahim@hu.edu.eg
6 Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt;
sahar.shaarawy@pharma.cu.edu.eg (S.M.A.-S.); kamilia.amin@pharma.cu.edu.eg (K.M.A.)
7 Pharmaceutical Sciences Department, Faculty of Clinical Pharmacy, King Faisal University,
Al Hofuf 31982, Al-Ahsa, Saudi Arabia; nyounis@kfu.edu.sa
8 Pharmacology and Toxicology Department, Faculty of Pharmacy, Fayoum University, Fayoum 63514, Egypt;
ram14@fayoum.edu.eg
* Correspondence: rana.mohamed@hu.edu.eg; Tel.: +20-1270551779

Citation: Abdelnaby, R.M.; Rateb,
H.S.; Ali, O.; Saad, A.S.; Nadeem, R.I.;
Abstract: Breast cancer is the most common malignancy worldwide; therefore, the development of
Abou-Seri, S.M.; Amin, K.M.; Younis, new anticancer agents is essential for improved tumor control. By adopting the pharmacophore hy-
N.S.; Abdelhady, R. Dual PI3K/Akt bridization approach, two series of 7-hydroxyl-4-methylcoumarin hybridized with thiosemicarbazone
Inhibitors Bearing Coumarin- (V–VI) and thiazolidin-4-one moieties (VII–VIII) were prepared. The in vitro anticancer activity was
Thiazolidine Pharmacophores as assessed against MCF-7 cells adopting the MTT assay. Nine compounds showed significant cytotoxic-
Potential Apoptosis Inducers in ity. The most promising compound, VIIb, induced remarkable cytotoxicity (IC50 of 1.03 + 0.05 µM).
MCF-7 Cells. Pharmaceuticals 2022, 15, Further investigations were conducted to explore its pro-apoptotic activity demonstrating S-phase
428. https://doi.org/10.3390/ cell cycle arrest. Apoptosis rates following VIIb treatment revealed a 5-fold and 100-fold increase in
ph15040428
early and late apoptotic cells, correspondingly. Moreover, our results showed caspase-9 dependent
Academic Editor: Marialuigia apoptosis induction as manifested by an 8-fold increase in caspase-9 level following VIIb treatment.
Fantacuzzi Mechanistically, VIIb was found to target the PI3K-α/Akt-1 axis, as evidenced by enzyme inhibition
assay results reporting significant inhibition of examined enzymes. These findings were confirmed
Received: 18 January 2022
by Western blot results indicating the ability of VIIb to repress levels of Cyclin D1, p-PI3K, and p-Akt.
Accepted: 29 March 2022
Published: 31 March 2022
Furthermore, docking studies showed that VIIb has a binding affinity with the PI3K binding site
higher than the original ligands X6K. Our results suggest that VIIb has pharmacological potential as
Publisher’s Note: MDPI stays neutral
a promising anti-cancer compound by the inhibition of the PI3K/Akt axis.
with regard to jurisdictional claims in
published maps and institutional affil-
Keywords: PI3K/Akt pathway; MCF-7; 7-hydroxycoumarin; thiazolidin-4-ones; apoptosis; anti-
iations.
cancer activity
Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article Cancer is a multifactorial disease that ranks as the second leading cause of death
distributed under the terms and globally, causing approximately 10 million deaths in 2020. Notably, female breast cancer
conditions of the Creative Commons was reported as the most frequently diagnosed cancer type, surpassing lung cancer [1,2].
Attribution (CC BY) license (https:// The incidence of breast cancer varies globally, it affects 1 out of 20 females worldwide
creativecommons.org/licenses/by/
and 1 out of 8 in developing countries [3]. Major risk factors for breast cancer occurrence
4.0/).
Pharmaceuticals 2022, 15, 428. https://doi.org/10.3390/ph15040428 https://www.mdpi.com/journal/pharmaceuticals

The incidence of breast cancer varies globally, it affects 1 out of 20 females worldwide an
1 out of 8 in developing countries [3]. Major risk factors for breast cancer occurrence in
clude both modifiable factors, such as lifestyle, diet, or hormone replacement therapy, an
Pharmaceuticals 2022, 15, 428 non-modifiable factors, such as age, sex, race, and genetic makeup [4]. 2 of 20
Although surgery and chemotherapy are the mainstream therapeutic strategies fo
breast cancer, still the development of novel targeted cancer therapies sparing toxicitie
to off-cancer
include cells is anfactors,
both modifiable urgentsuch need, mainly due
as lifestyle, diet, to
or the majorreplacement
hormone limitations therapy,
of conventiona
and non-modifiable factors,
chemotherapeutic agents,such as age, sex,
including race, and
systemic genetic
toxicity andmakeup [4]. resistance [3,5].
multidrug
Although
It has beensurgery
notedand that chemotherapy
several signalingare the mainstream
pathways aretherapeutic
dysregulated strategies for
and subsequentl
breast cancer, still the development of novel targeted cancer therapies
have been implicated in the pathogenesis of breast cancer. Remarkably, the signalin sparing toxicities
to off-cancer cells is an urgent need, mainly due to the major limitations of conventional
pathway defined by the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) axi
chemotherapeutic agents, including systemic toxicity and multidrug resistance [3,5].
is a Itchief controller
has been of a several
noted that myriadsignaling
of cellular functions,
pathways including cell
are dysregulated andgrowth and prolifera
subsequently
tion.been
have Moreover,
implicatedaberrations in this molecular
in the pathogenesis of breast pathway
cancer. are critical inthe
Remarkably, breast tumor initia
signaling
pathway defined by the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) axispathway
tion, survival, and angiogenesis [5–8]. Notably, oncogenic activation of this is i
abreast cancer isofmainly
chief controller a myriad attributed
of cellularto the mutation
functions, includingof cell
genes encoding
growth PI3K subunits, in
and proliferation.
Moreover,
cluding p110α aberrations
(PIK3CA) in thisand molecular
p110β pathway
(PIK3CB), arewhere
criticalPIK3CA
in breastmutations
tumor initiation,
were reporte
survival, and angiogenesis [5–8].
in 30–40% of breast cancer patients [9,10]. Notably, oncogenic activation of this pathway in breast
cancerFurthermore,
is mainly attributed
the role toofthe mutation
the PI3K/Akt of genes encoding
signaling network PI3Kinsubunits, including
cancer cells immunomod
p110α (PIK3CA) and p110β (PIK3CB), where PIK3CA mutations were reported in 30–40%
ulation has been clearly highlighted, since Akt hyperactivation was associated with th
of breast cancer patients [9,10].
escape of cancerthe
Furthermore, cells
rolefrom
of theimmune
PI3K/Akt recognition [11]. Consequently,
signaling network recent studies docu
in cancer cells immunomod-
ulation has been clearly highlighted, since Akt hyperactivation was associated with the by in
mented that inhibition of the PI3K/Akt axis enhances tumor immunosurveillance
hibiting
escape of the activation
cancer cells from of immune
immunosuppressive
recognition [11]. pathways [5,6,12].
Consequently, Moreover,
recent studiesin breast can
doc-
cer, dysregulation
umented that inhibition of this
of the signaling
PI3K/Aktaxis axisplays a principal
enhances role in resistance by
tumor immunosurveillance to antineo
inhibiting the activation of immunosuppressive
plastic chemotherapeutic drugs, hormonalpathways therapy,[5,6,12]. Moreover,
and targeted in breast
therapy can- Lately
[5,7,13].
cer, dysregulation of this signaling axis plays a principal role in resistance
the pivotal role of the PI3K/Akt axis in breast carcinogenesis has been characterized to antineoplastic
chemotherapeutic drugs, hormonal
prompting the development of therapy, and targetedstrategies
recent therapeutic therapy [5,7,13]. Lately,inhibit
that could the piv- this path
otal role of the PI3K/Akt axis in breast carcinogenesis has been characterized, prompting
way aiming at both limiting tumor proliferation and/or survival as well as reviving tumo
the development of recent therapeutic strategies that could inhibit this pathway aiming
atfunctional
both limiting immunosurveillance.
tumor proliferation and/or Many clinical
survival studies
as well as have proved
reviving tumorthatfunctional
inhibitors actin
immunosurveillance. Many clinical studies have proved that inhibitors acting on different agent
on different enzymes of this pathway (Figure 1) are very successful therapeutic
with benefits
enzymes of thisagainst
pathwaythe emergence
(Figure of resistance
1) are very successful and with better
therapeutic agents disease prognosis [14
with benefits
16]. the emergence of resistance and with better disease prognosis [14–16].
against
Figure1.1.Reported
Figure Reported PI3K
PI3K andand
AktAkt inhibitors.
inhibitors. Structures
Structures 1 and12and 2 are adapted
are adapted from
from [17], [17], [18],
3 from 3 from [18],
and4 4from
and from [19].
[19].
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 3 of 20
Pharmaceuticals 2022, 15, 428 3 of 20
In the war on cancer, natural products and their derivatives have played a crucial
In developing
role in the war on cancer,
effectivenatural products andagents
chemotherapeutic their derivatives
such as vinca have played aand
alkaloids crucial role
taxols,
in developing effective chemotherapeutic agents such as vinca alkaloids
which inspired our research team to develop new chemotherapeutic agents adopting nat- and taxols, which
inspired our research
urally found scaffolds,team
such toasdevelop
a coumarin newringchemotherapeutic
that was reported agents
over adopting
the years naturally
to have
found scaffolds, such as a coumarin ring that was reported
potent anticancer activity through manipulation of many cellular mechanisms over the years to have
(Figure potent
2)
anticancer
[20–24]. activity through manipulation of many cellular mechanisms (Figure 2) [20–24].
The molecular hybridization
The molecular hybridizationstrategy
strategyhas hasemerged
emergedasasaanovelnovelapproach
approachthat thatinvolves
involves
combining
combining two two oror more
more pharmacophores
pharmacophoresin inone
onemolecule
moleculewith withthe thebenefits
benefitsofofhavinghavinga a
better
better pharmacological profilein
pharmacological profile ineither
eitheradditive
additive(acting
(actingon onthe
thesame
samebiological
biologicaltarget)
target)oror
synergistic
synergistic (modulating
(modulating different
different targets)
targets)manner
mannerfor forthe
theparent
parentmolecules
moleculesand andless
lesslikely
likelyto
develop drug resistance. That attracted large groups of researchers
to develop drug resistance. That attracted large groups of researchers to investigate dif-to investigate different
scaffolds in chemotherapeutic
ferent scaffolds in chemotherapeuticagentsagents
separately and their
separately andmerging
their mergingin oneinmolecule
one molecule [25,26].
Literature survey revealed the potent anticancer effect of several
[25,26]. Literature survey revealed the potent anticancer effect of several coumarin deriv-coumarin derivatives
(Figure 2) owing
atives (Figure to theirtomulti-targeting
2) owing mechanism
their multi-targeting of action
mechanism of in cell biology,
action for example,
in cell biology, for
apoptosis inductioninduction
example, apoptosis and PI3K/AKT inhibition,
and PI3K/AKT which which
inhibition, all finally stop cell
all finally stopproliferation
cell prolif-
and survival
eration processes
and survival [20–24].[20–24].
processes Also, compounds
Also, compounds featuring thiosemicarbazone
featuring thiosemicarbazone linker
and its cyclic analog thaizolidin-4-one ring showed promising effectiveness
linker and its cyclic analog thaizolidin-4-one ring showed promising effectiveness against against many
cancer types as result of ribonucleotide reductase, and carbonic
many cancer types as result of ribonucleotide reductase, and carbonic anhydrase inhibi- anhydrase inhibition,
(Figure 2) [27–30].
tion, (Figure Hence,
2) [27–30] the target
Hence, compounds
the target compounds (V–VIII) were
(V–VIII) weredesigned
designed adopting
adopting the
pharmacophore hybridization technique to have a coumarin scaffold
the pharmacophore hybridization technique to have a coumarin scaffold as the main nu- as the main nucleus
merged with with
cleus merged thiosemicarbazone
thiosemicarbazone linker or or
linker thaizolidin-4-one
thaizolidin-4-onering ringat at position
position C8 C8ofofthe the
coumarin ring, as represented in
coumarin ring, as represented in Figure 3.Figure 3.
Figure 2.
Figure 2. Reported
Reported coumarin
coumarin and
and thiazolidine
thiazolidinecontaining
containinganticancer
anticanceragents.
agents.Structures 5 to
Structures 1010
5 to are
are
adapted, respectively, from [20,24,27,28,31,32].
adapted, respectively, from [20,24,27,28,31,32].
This research work aimed at evaluating the potential cytotoxicity of the novel deriva-
tives in breast cancer cell line MCF-7. Further studies were conducted for the most promis-
ing compound, exploring the enzyme inhibition assay, examining PI3K-α, PI3K-γ, and
Akt-1 isoforms. Secondly, the proapoptotic activity was assessed via investigating cell cycle
distribution alongside apoptosis percentage. In addition, the downstream proteins of the
signaling pathway under investigation were evaluated by Western blot. Eventually, exami-
nation of the binding interactions between the promising derivatives and the nominated
enzymes was conducted by molecular modeling.
Pharmaceuticals
Pharmaceuticals 15,15,
2022,
2022, 428 PEER REVIEW
x FOR 4 of
4 20
of 20
Reference Pharmacophoric Features

CH3
N N N
S
HN
R1 N
O O R1
OR N
S H O
Coumarin ring thiosemicarbazone thiazolidine ring
Pharmacophore Hybridization
Target Compounds
CH3 CH3
Cyclization
RO O R1O O O
O
NH NH N S
N CH3 N
CH3 R1 N
S R1
O
Va, b & VIa-f VIIa, b & VIIIa-f
Figure 3. Design of the new compounds (V–VIII).
Figure 3. Design of the new compounds (V–VIII).
This research
2. Results work aimed at evaluating the potential cytotoxicity of the novel deriv-
and Discussion
2.1. Chemistry
atives in breast cancer cell line MCF-7. Further studies were conducted for the most prom-
ising compound,
As depictedexploring
in Schemes the1 enzyme
and 2, the inhibition
designedassay, examining
compounds werePI3K-α, PI3K-γ,
prepared. Subse-and
Akt-1 isoforms.
quently, Secondly,
structure the proapoptotic
confirmation was done using activity was assessed
spectral data and via investigating
elemental analysiscell
de-cy-
cle distribution
scribed alongside
in the materials andapoptosis percentage.
methods section. In addition,
The starting the downstream
compounds, proteins of
I–IV, were prepared
as signaling
the reported by our research
pathway underteam in a previous
investigation study
were [33]. Coumarin-thiosemicarbazones
evaluated by Western blot. Eventually,
examination of the binding interactions between the promisingintermediates
(Va, Vb, and VIa–f) resulted from the reaction of the hydrazone b with
IVa, the
derivatives and nom-
different isothiocyanates in good yield; FT-IR
inated enzymes was conducted by molecular modeling. exhibited the disappearance of primary amine
peak and only showed two secondary amine peaks at 3200–3400 cm−1 [34]. Moreover,
1 H-NMR showed extra aromatic protons at 7.3–7.6 ppm for Va and Vb analogs, while for
2. Results and Discussion
the VIa-f, there were the corresponding peaks for the aliphatic substitution at 1.33 and
2.1.
2.4Chemistry
ppm for VIa and VIb, 4–6 ppm for the allyl group in VIc, and the extra aromatic protons
As depicted
at 7.0–7.7 ppm for inVIIf,
Schemes 1 and 2, theCoumarin-thiazolidine-4-ones
g, h derivatives. designed compounds were (VII–VIII)
prepared. then
Subse-
quently, structure via
were synthesized confirmation wasreaction
the cyclization done using
with spectral dataacid;
chloroacetic andthe
elemental analysis
FT-IR showed thede-
scribed in the materials and methods section. The starting compounds, I–IV, were−pre-
disappearance of amino peaks and the appearance of a second carbonyl peak at 1700 cm 1,
whileasthe 1 H-NMR
pared reported bypresented
our researcha peak
teamatin
δ= 2.85–3.86 study
a previous ppm of[33].
the Coumarin-thiosemicarba-
methylene group in the
thiazolidine
zones (Va, Vb,ring
and [35].
VIa–f) resulted from the reaction of the hydrazone intermediates IVa,
b with different isothiocyanates in good yield; FT-IR exhibited the disappearance of pri-
mary amine peak and only showed two secondary amine peaks at 3200–3400 cm−1 [34].
Moreover, 1H-NMR showed extra aromatic protons at 7.3–7.6 ppm for Va and Vb analogs,
while for the VIa-f, there were the corresponding peaks for the aliphatic substitution at
1.33 and 2.4 ppm for VIa and VIb, 4–6 ppm for the allyl group in VIc, and the extra aro-
matic protons at 7.0–7.7 ppm for VIIf, g, h derivatives. Coumarin-thiazolidine-4-ones
(VII–VIII) then were synthesized via the cyclization reaction with chloroacetic acid; the
FT-IR showed the disappearance of amino peaks and the appearance of a second carbonyl
peak
Pharmaceuticals 2022,at
15,1700
x FOR cm −1,REVIEW
PEER while the 1H-NMR
presented a peak at δ = 2.85–3.86 ppm of the meth- 5 of 20
ylene group in the thiazolidine ring [35].
peak at 1700 cm−1, while the 1H-NMR presented a peak at δ = 2.85–3.86 ppm of the meth-
ylene group in the thiazolidine ring [35].
Scheme 1. ReagentsScheme 1. Reagents

Reagents
and conditions: and
(a) Acconditions:
and 2conditions:
O; (b) AlCl (a)(a)
3,Ac 2O;
Ac (b)(b)
2 O;
fusion; AlCl
(c) 3, fusion;
AlCl
ethyl (c) (c)
3 , bromide,
fusion; ethyl bromide,
ethyl
K 2CO K2CO
3bromide, /dry3 /dry
K23CO
/dry ace- ace-
tone; (d) N2H4·H2O,tone; (d) (d)
acetone;
ethanol; 2H
N(e) ·H
2 H24O,
·Hethanol;
N4isothiocyanate (e)derivatives,
2 O, ethanol; isothiocyanate
(e) isothiocyanate derivatives,
ethanol. derivatives,ethanol.
ethanol.
Scheme 2. Reagents and conditions: (f) chloroacetic acid, NaAc/ethanol.
Scheme 2. ReagentsScheme
and conditions: (f)and
2. Reagents chloroacetic
conditions:acid, NaAc/ethanol.
(f) chloroacetic acid, NaAc/ethanol.
2.2. Antitumor Activity
2.2.1. Cytotoxicity Assay
2.2.1.The
Cytotoxicity Assay
in vitro anticancer activity of the designed coumarin-thiosemicarbazones and
2.2.1. Cytotoxicitycoumarin-thiazolidine-4-one
Assay hybrids against MCF-7 cells was tested using the MTT assay,
The in vitro anticancer activity of the designed coumarin-thiosemicarbazones and
The in vitro coumarin-thiazolidine-4-one
anticancer activity of the hybrids
designed coumarin-thiosemicarbazones
against andMTT assay,
MCF-7 cells was tested using the
coumarin-thiazolidine-4-one hybrids
and 5-fluorouracil against
(5-FU) MCF-7
was used cells
as the was tested
reference drug.using the MTT
The tested assay, were used
compounds
in different concentrations, and cell survival was determined after incubation for 48 h as
reported [36,37]. The cytotoxic activity is represented in Table 1 as IC50 (µM) values.
Table 1. The values of IC50 (µM) of the tested compounds in MCF-7 cells.
Compound No. R R1 IC50 (µM)

5-FU - - 27.81 ± 1.41
Va H Ph-CH2 5.13 ± 0.28 ***
Vb H Ph-C=O 47.32 ± 2.47
VIa C2 H5 CH3 11.13 ± 0.58 ***
VIb C2 H5 C2 H5 38.70 ± 2.09
VIc C2 H5 CH2 =CH-CH2 2.72 ± 0.13 ***
VId C2 H5 Ph-CH2 2.61 ± 0.14 ***
VIe C2 H5 Ph-C=O 43.05 ± 2.25
VIf C2 H5 4-OCH3 -Ph 1.21 ± 0.06 ***
VIIa H Ph-CH2 54.80 ± 2.86
VIIb H Ph-C=O 1.03 ± 0.05 ***
VIIIa C2 H5 CH3 20.27 ± 1.06 **
VIIIb C2 H5 C2 H5 57.28 ± 2.99
VIIIc C2 H5 CH2=CH-CH2 4.95 ± 0.26 ***
VIIId C2 H5 Ph-CH2 79.93 ± 4.18
VIIIe C2 H5 Ph-C=O 26.41 ± 1.38 ns
VIIIf C2 H5 4-OCH3 -Ph 11.80 ± 0.62 ***
IC50 values are means ± sd; n = 3. p-value using independent t-test for tested compounds vs. Compound
5-Fluorouracil (5-FU). *** p ≤ 0.001, ** p ≤ 0.01. ns Non-statistically significant p > 0.05.
The cytotoxicity assay results highlighted that both series (open and cyclized analogs)
are promising candidates as antitumor agents ranging from highly active to moderate
activity with IC50 of 1.03–79.90 µM.
In the coumarin-thiosemicarbazone series (Va,b, and VIa–f), the different aliphatic
and aromatic substitutions showed promising cytotoxicity, where some compounds demon-
strated IC50 values statistically significantly lower than that of the reference compound,
5-FU (IC50 = 27.81 + 1.41 µM). Notably, when R = H, the benzyl analog (Va) gave promising
inhibition with IC50 = 5.13 + 0.28 µM, while the benzoyl derivative showed lower activity
with IC50 = 47.32 µM.
Moreover, when R = C2 H5 , the reported results varied between the aliphatic and
aromatic substitutions. The benzyl derivative (VId) showed better activity than its 7-
hydroxyl analog (Va), while for the benzoyl derivative (VIe), the activity did not get any
better but the methoxy derivative showed highly potent inhibition with IC50 = 1.21 µM. For
the aliphatic substitutions, the methyl (VIa) and ethyl (VIb) derivatives showed good to
moderate activities with IC50 of 11.13 and 38.70 µM, while the allyl derivative VIc showed
highly potent activity with IC50 = 2.72 µM.
By looking at the cyclic analogs, thiazolidin-4-one series, the 7-hydroxy coumarin
analogs (VIIa, b), the cyclization led to decreased activity for the benzyl derivative VIIa
(IC50 = 54.80 µM), while the benzoyl derivatives VIIb (IC50 = 1.03 µM) gave the best activity
compared to the open analog Vb (IC50 = 47.32 µM).
In the 7-ethoxy series (VIIIa-f), the compounds had lower inhibitory activity than
the open analogs. The derivatives that displayed higher cytotoxicity than the reference
compound, 5-FU, were VIIIa, VIIIc, and VIIIf, showing IC50 values of 20.27, 4.95, and
11.80 µM, respectively, while VIIIe (IC50 = 26.04 µM) gave comparable activity to 5-FU but
better than the open analog that resulted in IC50 = 43.05 µM.
However, IC50 values recorded for Compounds Vb, VIb, VIe, VIIa, VIIIb, and VIIId
were 47.32, 38.70, 43.05, 54.8, 57.28, and 79.93 µM, correspondingly, which were higher than
the reported value for the reference compound 5-FU.
Statistical significance was tested using an independent t-test comparing the recorded
IC50 values (µM) for the compounds that showed higher cytotoxicity than the reference com-
pound to that of 5-FU where it revealed statistically significant differences for Compounds
Va, VIa, VIc, VId, VIf, VIIb, VIIIc, and VIIIf (p-values < 0.001) as well as Compound
VIIIa, (p-value < 0.01) vs. the reference compound 5-FU. Moreover, the IC50 of Compound
VIIIe was non-significantly different than that of 5-FU (p-value = 0.27).
The metabolic viability of the most potent compounds, VIf and VIIb, in non-cancerous
epithelial cells (MCF 10), was examined using an MTT assay calculating the selectivity
index (SI) [38]. The data provided in Table 2 illustrated that both VIf (IC50 = 20.11 ± 1.05,
SI = 16.61) and VIIb (IC50 = 9.52 ± 0.60, SI = 9.24) have a promising safety profile compared
to 5-FU (IC50 = 36.22 ± 1.89, SI = 1.30). Herein, such results suggested that at the selected
doses, VIf and VIIb potentially will not cause deleterious effects to neighboring non-
cancer cells.
Table 2. IC50 values (µM) of the most cytotoxic compounds (VIf and VIIb) against MCF-10A cell line
and their selectivity indices.
IC50 (µM) IC50 (µM) Selectivity Index

Compound
MCF-10 a MCF-7 MCF-10/MCF-7
VIf 20.11 ± 1.05 1.21 ± 0.06 16.61
VIIb 9.52 ± 0.60 1.03 ± 0.05 9.24
5-FU 36.22 ± 1.89 27.81 ± 1.41 1.30
a IC50 values are means ± sd; n = 3.
Furthermore, the findings of the MTT test proved that the hybridization technique
adopted between coumarin and thiosemicarbazone or thiazolidine-4-one gave highly active
compounds, rendering them very promising candidates for further investigations. The
current study focused on investigating the potential anticancer mechanism for the novel
compound VIIb in MCF-7 cells, whilst the mechanistic details for compound VIf will be
explored in our future work to ponder its anticancer effects in a different cancer cell line.
2.2.2. Enzyme Inhibition Assay

Accumulating evidence highlighted that PI3K signaling pathway upregulation is
highly implicated in breast cancer development and disease progression as well as resis-
tance to hormones and cytotoxic therapy. Therefore, it is essential to elucidate the effect
of the novel compound, VIIb, on the PI3K/Akt signaling pathway by using in vitro PI3K
class 1A enzyme inhibition assay conducted on PI3K-α and PI3K-γ isoforms as well as an
Akt-1 enzyme inhibition assay. Notably, PI3K-α is the most mutated isoform in the PI3K
pathway in breast cancer, whilst the PI3K-γ isoform is not commonly mutated in breast
cancer. Furthermore, targeting the PI3K-γ isoform in breast cancer could contribute to
enhancing the anti-tumor immunity [39].
Data shown in Table 3 demonstrated that VIIb treatment displayed a potent inhibitory
activity on PI3K-α and Akt-1 isoforms where the reported IC50 values were statistically
significantly lower than the reference compound, LY294002. The IC50 values were found to
be 3.70 ± 0.19 and 2.93 ± 0.15 µM, for PI3K-α and Akt-1, correspondingly. Furthermore,
VIIb treatment displayed PI3K-γ isoform inhibition with IC50 of 34.70 ± 1.88 µM. However,
the recorded IC50 value was higher than the reported value of the reference drug.
Compounds
PI3K-α Isoform PI3K-γ Isoform Akt-1 Isoform
VIIb 3.70 ± 0.19 *** 34.70 ± 1.88 2.93 ± 0.15 *
LY294002 8.85 ± 0.46 11.5 ± 0.62 3.53 ± 0.18
Values are means ± sd; n = 3. p-value using independent t-test for VIIb vs. LY294002 reference com-
Pharmaceuticals 2022, 15, 428 pound. *** p≤ 0.001, * p ≤ 0.05. 8 of 20
These findings proved that the hybridization technique adopted in this work suc-
Table 3.inIC
ceeded 50 values (µM)
generating for the inhibition
a promising of PI3K-α,for
dual inhibitor PI3K-γ, and Akt-1axis,
the PI3K/Akt enzymes following
which could be
CompoundinVIIb
beneficial treatment.
treating either hormone- or chemo-resistant breast cancers.
IC50 (µM)
2.2.3. Cell Cycle Analysis and Apoptosis Induction
Compounds
PI3K-α Isoform PI3K-γ Isoform Akt-1 Isoform
VIIb-treated cells were subjected to cell cycle phase distribution analysis as well as
VIIb 3.70 ± 0.19 *** 34.70 ± 1.88 2.93 ± 0.15 *
apoptosis rates determination by flow cytometric analysis in MCF-7 cells, as reported
[40,41]. LY294002 8.85 ± 0.46 11.5 ± 0.62 3.53 ± 0.18
Values are means ± sd; n = 3. p-value using independent t-test for VIIb vs. LY294002 reference compound.
The results of cell cycle analysis revealed marked variability between VIIb-exposed
*** p≤ 0.001, * p ≤ 0.05.
cells vs. control untreated MCF-7 cells, as shown in Table 4 and Figure 4, where VIIb-
treated These
cells findings
showed aproved
higherthat the hybridization
S-phase population oftechnique adopted in
46.02% compared this work
to 36.58% suc-
in control
ceeded
cells. in generating a promising dual inhibitor for the PI3K/Akt axis, which could be
beneficial
However,in treating
VIIb either hormone-
treatment or chemo-resistant
suppressed both G0/G1 breast
and cancers.
G2/M proportions from
53.71% and 9.71%, respectively, in control
2.2.3. Cell Cycle Analysis and Apoptosis Induction untreated MCF-7 cells, to 48.39% and 5.59% in
VIIb-exposed cells, whereas pre-G1 cells, representing apoptotic cells, had a low propor-
VIIb-treated cells were subjected to cell cycle phase distribution analysis as well as
tion and reached 1.55% in untreated cells that significantly increased to 35.25% following
apoptosis rates determination by flow cytometric analysis in MCF-7 cells, as reported [40,41].
VIIb treatment. These data suggested that VIIb treatment induced S-phase accumulation
The results of cell cycle analysis revealed marked variability between VIIb-exposed
and thereby S-phase
cells vs. control untreatedarrest and potentially
MCF-7 subsequently
cells, as shown in Table 4 andcellFigure
death.4, Results of the current
where VIIb-treated
study accord well
cells showed withS-phase
a higher earlier population
research that of highlighted
46.02% comparedthe ability of coumarins
to 36.58% in control to induce
cells.
arrest of various cell cycle phases, potentially leading to apoptosis [26].
Table 4. Cell cycle analysis following 48-h treatment with Compound VIIb.
Table 4. Cell cycle analysis following 48-h treatment with Compound VIIb.
DNA Content %
Cells DNA Content %
Cells G0/G1 S G2/M Pre G1
G0/G1 S G2/M Pre G1
VIIb-treated cells 48.39 46.02 5.59 35.25
VIIb-treated cells 48.39 46.02 5.59 35.25
Control untreated cells 53.71 36.58 9.71 1.55
Figure 4. The cell distribution in cell cycle phases following Compound VIIb exposure.
However, VIIb treatment suppressed both G0/G1 and G2/M proportions from 53.71%
and 9.71%, respectively, in control untreated MCF-7 cells, to 48.39% and 5.59% in VIIb-
exposed cells, whereas pre-G1 cells, representing apoptotic cells, had a low proportion
and reached 1.55% in untreated cells that significantly increased to 35.25% following VIIb
treatment. These data suggested that VIIb treatment induced S-phase accumulation and
thereby S-phase arrest and potentially subsequently cell death. Results of the current study
Figure 4. The cell distribution in cell cycle phases following Compound VIIb exposure.
Control and VIIb-treated (IC50 µM, 48 h) MCF-7 cells were harvested and then sub-
jected to cell cycle analysis by flow cytometry.
Remarkably, clinical oncology aims at developing novel targeted cancer therapies
that could induce apoptosis
accord well within neoplastic
earlier researchcells to enhance
that highlighted thetheir
abilityeradication.
of coumarins Since
to induceloss of
arrest
of various
apoptosis is closely cell cycle
related phases, potentially
to cancer cell survival leading
and to apoptosis
abnormal [26].
growth, induction of
apoptotic signalingControlpathways and VIIb-treated
is a crucial(IC 50 µM, 48 h) in
mechanism MCF-7 cells were
targeted harvested
cancer therapy.and then
To fur-sub-
jected to cell cycle analysis by flow cytometry.
ther explore the linkRemarkably,
between apoptosis ratesaims
clinical oncology andatVIIb treatment,
developing the pro-apoptotic
novel targeted cancer therapies activ-
that
ity of this novelcould
compound was investigated
induce apoptosis in neoplasticbycells
flow cytometry
to enhance theirusing both Since
eradication. Annexinloss ofVapop-
(V)
and propidiumtosis iodide (PI) dyes.
is closely relatedA to distinctive feature
cancer cell survival and ofabnormal
early apoptosis is phosphatidyl-
growth, induction of apoptotic
signaling pathways is a crucial mechanism in targeted
serine transfer toward the cellular surface. Thus, phosphatidylserine can be detected cancer therapy. To further explore
by
the link between apoptosis rates and VIIb treatment, the pro-apoptotic activity of this novel
fluorochrome-tagged anticoagulant protein V. Therefore, the viable cells remained un-
compound was investigated by flow cytometry using both Annexin V (V) and propidium
stained (V−/PI−).iodide
Furthermore,
(PI) dyes. early apoptotic
A distinctive cells
feature of were stained with
early apoptosis V but not PI, demon-
is phosphatidylserine transfer
strating (V /PI )toward
+ − staining. However,
the cellular surface. late
Thus,apoptotic cells showed
phosphatidylserine can beVdetected
positive/PI positive
by fluorochrome-
(V+/PI+) staining, tagged anticoagulant
indicating the lossprotein V. Therefore,
of integrity the viable
of the nuclearcellsorremained
plasmaunstained
membrane (V− /PI
[42–− ).
Furthermore, early apoptotic cells were stained with V but not PI, demonstrating (V+ /PI− )
45].
staining. However, late apoptotic cells showed V positive/PI positive (V+ /PI+ ) staining,
Data of theindicating
currentthe study
loss ofhighlighted
integrity of thethat in non-treated
nuclear or plasma membrane MCF-7[42–45].
cells, 0.48% and
0.17% of examinedData cellsof demonstrated V +/PI− and V+/PI+ staining patterns, correspond-
the current study highlighted that in non-treated MCF-7 cells, 0.48% and 0.17%
ingly, as shownofinexamined
Table 5 cells
anddemonstrated V+ /PI− and V+treatment
Figure 5. Interestingly, /PI+ staining ofpatterns,
MCF-7 correspondingly,
cells with VIIb as
shown in Table 5 and Figure 5. Interestingly, treatment of
caused 5-fold and 100-fold increases in early and late apoptotic cells, respectively, with MCF-7 cells with VIIb caused
5-fold and 100-fold increases in early and late apoptotic cells, respectively, with respect to
respect to control cells where 2.51% and 21.05% of the cells exhibited V+/PI− and V+/PI+
control cells where 2.51% and 21.05% of the cells exhibited V+ /PI− and V+ /PI+ staining
staining patterns, correspondingly.
patterns, correspondingly.
Table 5. Percentage of5.apoptotic

Table Percentagecells after Compound
of apoptotic VIIb treatment.
cells after Compound VIIb treatment.
Apoptosis
Apoptosis %%
Cells Cells Necrosis%
Necrosis%
Total Early Late
Total Early Late
cells 35.25 2.51 21.05 11.69
VIIb-treatedVIIb-treated
cells 35.25 2.51 21.05 11.69
Figure 5. Effect ofFigure

Compound VIIb
5. Effect of on apoptosis
Compound VIIb on induction in MCF-7
apoptosis induction cell line.
in MCF-7 cell line.
Control untreated and MCF-7 cells treated with compound VIIb at its IC50 (µM) for
Control untreated and MCF-7 cells treated with compound VIIb at its IC50 (µM) for
48 h were subjected to apoptotic analysis using Annexin V (V) and propidium iodide (PI)
48 h were subjected to apoptotic
fluorescent dyes. analysis using Annexin V (V) and propidium iodide (PI)
fluorescent dyes.
Results of cell cycle analysis alongside apoptosis induction highlighted the pro-apop-
totic activity of the investigated compound (VIIb). Furthermore, the reported increase in
the necrotic cells following VIIb treatment could be assigned to the hydroxyl group in the
Results of cell cycle analysis alongside apoptosis induction highlighted the pro-
apoptotic activity of the investigated compound (VIIb). Furthermore, the reported increase
in the necrotic cells following VIIb treatment could be assigned to the hydroxyl group in
the structure. In addition, this study demonstrated that VIIb exposure induced changes in
the cellular distribution at different cell cycle phases.
Notably, VIIb exposure resulted in S-phase cell cycle arrest accompanied by a reduc-
tion in the percentage of cells in other phases. The observed effect is possibly attributed
to the down-regulation of the G1-S checkpoint gene P21 that could have allowed G1-S
cell cycle transition despite the presence of DNA damage [46,47]. Moreover, the reported
S-phase cell cycle arrest could be mediated through cyclin A2, which is a pivotal regulator
of the cell cycle and crucial for S-phase and mitotic entry [40,41]. These hypotheses will be
investigated in our future work.
2.2.4. Caspase-9 Assay

Apoptosis is a programmed cell death modality that includes an array of steps such
as activation of caspases alongside endonucleases leading to DNA cleavage, eventually
causing the formation of apoptotic bodies. Caspases are classified into effector caspases,
such as caspase-3, -6, and -7, and initiator caspases, including either caspase-2, -8, and -10
(extrinsic pathway) or caspase-9 (intrinsic pathway) [48]. The level of cleaved caspase-9
was investigated in response to VIIb treatment as an indicator of induction of the apoptotic
pathways, where our results revealed that exposure of MCF-7 cells to Compound VIIb, for
48 h, induced an approximately 8-fold increase in the cleaved form of caspase-9 (p < 0.001)
(Table 6). This result supports our initial hypothesis that the potential anticancer mechanism
of VIIb was through induction of apoptotic pathways.
Table 6. Effect of VIIb treatment on caspase-9 level in MCF-7 cell line.
Cells Caspase-9 Level (ng/mL) Fold

VIIb-treated cells 21.61 ± 0.14 *** 7.96
Control untreated cells 2.712 ± 0.09 1
Values are means ± sd; n = 3. p-value using independent t-test for VIIb vs. control untreated cells. *** p ≤ 0.001.
Data of the current study accord well with a previous report that stated the induction
of apoptosis by a novel coumarin–chalcone hybrid via activation of initiator caspase-9 [49].
Moreover, an earlier study previously highlighted that the anticancer activity of coumarin
derivatives was through induction of caspase-dependent apoptotic pathways [24].
2.2.5. Western Blot

Cyclin D1 activity is essential for the G1 to S phase transition. Moreover, the PI3K/Akt
axis is crucial for cell growth and apoptosis. The effects of VIIb on Cyclin D1 and the
PI3K/Akt pathway in MCF-7 cells was investigated adopting reported procedures [50,51].
As shown in Figure 6, following compound VIIb exposure, the levels of Cyclin D1, p-PI3K,
and p-Akt in MCF-7 cells were effectively suppressed with respect to control untreated cells.
Herein, Western blot results confirmed our previous findings, highlighting the ability of
the novel compound, VIIb, to inhibit the PI3K/Akt signaling pathway. Our results accord
with an earlier study demonstrating that coumarin compounds suppressed Cyclin D1 and
p-Akt protein levels [51]. Consequently, the current work demonstrated that VIIb exposure
induced inhibition of Cyclin D1, which could be attributed to the reported PI3K inhibition
by VIIb, as previously stated [52,53]. Cyclin D1 is a proto-oncogene and a major regulator
of cell cycle progression. In addition, Cyclin D1 overexpression has been linked to both
breast tumorigenesis and tumor progression as well as to the development of endocrine
resistance in breast cancer cells [54,55]. Thus, the illustrated downregulation of Cyclin D1
following VIIb treatment suggests that the novel compound, VIIb, has pharmacological
potential as a therapeutic agent capable of targeting human breast cancer.
Pharmaceuticals2022,
15, x FOR PEER REVIEW
428 11 ofof2020
11
.
Figure 6. Effect of VIIb on Cyclin D1, p-Akt, and p-PI3K protein levels.
Figure 6. Effect of VIIb on Cyclin D1, p-Akt, and p-PI3K protein levels.
Whole-cell protein lysates obtained from both control as well as VIIb-treated (IC50
Whole-cell protein lysates obtained from both control as well as VIIb-treated (IC50
(µM), for 48 h) MCF-7 cells were resolved by SDS-PAGE, and then immunoblotting was con-
(µM), for 48antibodies
ducted with h) MCF-7against
cells were resolved
cyclin by and
D, p-Akt, SDS-PAGE, and then immunoblotting
p-PI3K compared was
to the housekeeping
conducted
protein with antibodies against cyclin D, p-Akt, and p-PI3K compared to the house-
β-Actin.
keeping protein β-Actin.
2.2.6. In Silico Molecular Simulations
2.2.6. In integration
The Silico Molecular Simulations
between experimental and computational methods is a highly at-
tractive The integration between
methodology experimental
in the design and computational
and optimization methodsdrug
field of different is a highly attrac-
candidates.
tive methodology
Accordingly, in the
molecular designwas
docking andconducted
optimization field of the
to illustrate different
bindingdrug candidates.
interactions Ac-
of the
cordingly, molecular docking was conducted to illustrate the binding interactions
novel derivatives VIIb and VIf inside the active site of PI3K-α and Akt-1. It was reported of the
novel
that bothderivatives
Lys 802 andVIIbTyrand
836VIf inside
have the active
a pivotal role insite
theofbinding
PI3K-α ofand Akt-1.
PI3K (PDB It was reported
ID. 4L23) to
that
its both Lys
inhibitors 802whereas
[56], and Tyrthe 836most
haveimportant
a pivotal amino
role in acid
the binding
residuesofinPI3K
Akt-1(PDB
(PDBID.ID.4L23)
3O96)to
its Asn54,
are inhibitors [56],
Trp80, whereas
Ser205, the most
Glu267, important
Lys268, Asn269, amino
and Arg273acid residues
[57]. The in Akt-1 revealed
docking (PDB ID.
3O96)
that bothare Asn54,
PI3K and Trp80, Ser205, Glu267,
Akt-1 enzymes Lys268,
have good Asn269,
docking and
scores andArg273
binding[57]. The docking
affinities with
revealed
both testedthat both PI3K and
compounds, Akt-1 in
as shown enzymes
Tables have
7 andgood8 and docking
Figuresscores
7 andand8. binding affin-
The binding
pattern
ities with both tested compounds, as shown in Tables 7 and 8 and Figures 7 and 8. the
of the two active derivatives VIIb and VIf is similar to the binding pattern of The
reference ligands of
binding pattern X6K
theand
twoIQO.
active derivatives VIIb and VIf is similar to the binding pattern
of the reference ligands X6K and IQO.
Table 7. The docking scores and the main H-bonds observed of the most active compounds against
Table
PI3K 7. The
active docking scores and the main H-bonds observed of the most active compounds against
site.
PI3K active site.
Compound S-Score Interaction Types and Residues
1 X6K −2.2807 H-bonding, Lys802
1 X6K −2.2807 H-bonding, Lys802
2 VIIb −3.7521 H-bonding, Tyr836
2 VIIb −3.7521 H-bonding, Tyr836
3 VIf −3.0518 H-bonding, Lys802
3 VIf −3.0518 H-bonding, Lys802
Table
Table 8. 8.
TheThe docking
docking scores
scores and and the main
the main interactions
interactions observed
observed of the
of the most mostcompounds
active active compounds
against
against Akt active site.
Akt active site.
1 IQO −5.0984 Hydrophobic aromatic, Trp80, Arg273; H-bonding, Asn54.
Hydrophobic aromatic, Trp80,
2 VIIb1 IQO −2.6564 −5.0984 H-bonding, Trp80
Arg273; H-bonding, Asn54.
3 VIf −4.3739 H-bonding, Trp80
2 VIIb −2.6564 H-bonding, Trp80
3 VIf −4.3739 H-bonding, Trp80
Pharmaceuticals
2022,
2022, 428 PEER REVIEW
x FOR 12 of 20
12 of 20
Figure7.
Figure The 2D
7. The 2D binding
bindingpattern
patternofofcompounds:
compounds:(a)(a)
X6K reference
X6K ligand;
reference (b) VIIb;
ligand; (c) VIf
(b) VIIb; (c)inVIf
the in the
active site of PI3K.
active site of PI3K.
Pharmaceuticals2022, 15,428
x FOR PEER REVIEW 13 of
of 20
20
Figure 8. The 2D binding patterns of compounds: (a) IQO reference ligand; (b) VIIb; (c) VIf in the
Figure 8. The 2D binding patterns of compounds: (a) IQO reference ligand; (b) VIIb; (c) VIf in the
active site of Akt.
active site of Akt.
3.3.Materials
Materialsand
andMethods
Methods
3.1.
3.1.Chemistry
Chemistry
All
Allthe
thechemical
chemicalreagents
reagentswere
wereavailable
availablefrom
fromSigma-Aldrich
Sigma-Aldrich(St. (St. Louis,
Louis, MO
MO USA).
USA).
FT-IR spectral analyses (KBr discs) were conducted on a Shimadzu IR Affinity-1
FT-IR spectral analyses (KBr discs) were conducted on a Shimadzu IR Affinity-1 spectro-spectropho-
tometer. 1 H-NMR spectra were conducted on on
a JEOL ECA 300,
photometer. 1H-NMR spectra were conducted a JEOL ECA 300,500
500MHz
MHzspectrometer
spectrometer
using
usingCDCl
CDCl33as asstated.
stated.
The
Thecoumarin
coumarinanalogs
analogsVa,
Va,Vb,
Vb,VIf, VIIa,
VIf, VIIb,
VIIa, and
VIIb, VIIIf
and were
VIIIf werepreviously reported
previously by
reported
our research
by our group,
research and thiosemicarbazone
group, derivatives
and thiosemicarbazone followed
VIa-f VIa-f
derivatives the same
followed the reported
same re-
procedures [33].
ported procedures [33].
General
GeneralProcedures
Proceduresfor
forThiosemicarbazones
ThiosemicarbazonesVVand andVIVISynthesis
Synthesis
The appropriate isothiocyanates (0.05 mol) were added
The appropriate isothiocyanates (0.05 mol) were added to the hydrazone
to the intermediates
hydrazone intermedi-
(0.05 mol), and the reaction mixture was dissolved in ethanol/dimethylformamide and
ates (0.05 mol), and the reaction mixture was dissolved in ethanol/dimethylformamide
reflux continued for 8 h. The solvent was removed under vacuum, and the formed solid
and reflux continued for 8 h. The solvent was removed under vacuum, and the formed
was collected and recrystallized from ethanol.
solid was collected and recrystallized from ethanol.
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-methyl-thiosemicarbazone (VIa):
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-methyl-thiosemicarbazone
◦ C; FT-IR ((ṽ max, cm
Yield
Yield == 85.06%;
85.06%; m.p.
m.p. == 202–204
202–204 °C; cm− −11
):):3286
3286and
and3417
3417 (2NH),
(2NH), 3080
3080
(VIa):
(CH,
(CH, Ar),
Ar), 2981
2981 (CH, aliphatic), 1732 (C=O),
(C=O), 1627
1627 (C=N,
(C=N, imine),
imine), 1597
1597 (C=C,
(C=C, Ar),
Ar), and
and 1188
1188
(C-O, ether); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1.41 (t, j = 9 Hz, 3H, CH3-CH2-O), 2.22
(s, 3H, N=C-CH3), 2.27 (s, 3H, C4-CH3), 2.41 (d, j = 6 Hz, 3H, NH-CH3), 4.16–4.20 (m, 2H,
CH3-CH2-O), 6.17 (s, 1H, C3-Hof coumarin), 6.92 (d, j = 9 Hz, 1H, C6-Harom), 7.62 (d, j = 9
Hz, 1H, C5-Harom), 2.8 and 8.17 (s, 2H, 2NH; exchangeable with D2O); 13C-NMR (400 MHz,
CDCl3) δ (ppm) = 14.55 (CH3-CH2-), 18.47 (CH3-), 23.53 (C=N-CH3), 31.08 (CH3-NH-), 65.01
(C-O, ether); 1 H-NMR (300 MHz, CDCl3 ) δ (ppm): 1.41 (t, j = 9 Hz, 3H, CH3 -CH2 -O), 2.22
(s, 3H, N=C-CH3 ), 2.27 (s, 3H, C4-CH3 ), 2.41 (d, j = 6 Hz, 3H, NH-CH3 ), 4.16–4.20 (m,
2H, CH3 -CH2 -O), 6.17 (s, 1H, C3-Hof coumarin), 6.92 (d, j = 9 Hz, 1H, C6-Harom ), 7.62
(d, j = 9 Hz, 1H, C5-Harom ), 2.8 and 8.17 (s, 2H, 2NH; exchangeable with D2 O); 13 C-NMR
(400 MHz, CDCl3 ) δ (ppm) = 14.55 (CH3 -CH2 -), 18.47 (CH3 -), 23.53 (C=N-CH3 ), 31.08
(CH3 -NH-), 65.01 (CH3 -CH2 -), 108.47–114.44 (3C of coumarin), 127.37 (C5 of coumarin),
142.81 (C10 of coumarin), 151.08 (C4-CH3 of coumarin), 152.17 (C-O-C2 H5 ), 157.61 (-C=O
of coumarin), 159.91 (-C=N-), 178.36 (-C=S);
M+(m/z): 333; Anal calcd: C, 57.64; H, 5.74; N, 12.60; found: C, 57.82; H, 5.79; N, 12.88.
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone (VIb):
YieldYield = 85.06%; = 83.95%; m.p. m.p. = 202–204 = 183 °C; ◦ C; FT-IRFT-IR ((ṽ max, cm cm− −11):):3286
3441and and 3417 3292 (2NH), (2NH), 3080 (CH,
(CH, Ar),Ar), 2981 (aliphatic (CH, aliphatic), CH), 1732 1732 (C=O), 1627 1598 (C=N, (C=C, imine), Ar), and 1597 1184 (C=C, (C-O, Ar), and 1188
ether); 1 H-NMR
(C-O,(400 ether); MHz, 1 H-NMR CDCl3(300 ) δ (ppm): MHz, CDCl 1.33 (t, 3) δ3H, (ppm): j = 81.41 Hz,(t, CH j =3 -CH
9 Hz, 2 -NH),
3H, CH 1.42–1.44
3-CH2-O), (t, 2.22
j = 8 Hz,
(s, 3H, 3H, CH3 -CH
N=C-CH -O), (s,
3), 22.27 2.22 3H, (s,C4-CH 3H, N=C-CH 3), 2.41 3(d, ), 2.30j = 6(s, Hz, 3H, 3H, C4-CH NH-CH 3 ), 3.71–3.77
3), 4.16–4.20 (m,(m, j = 42H, Hz, 2H,
CH3-CH NH-CH 2-O), 26.17 -CH(s, 3 ), 1H,
4.15–4.17 C3-Hof (m, j = 4 Hz, 2H,
coumarin), 6.92CH (d,3j-CH = 9 2Hz, -O),1H, 6.19C6-H (s, 1H, aromC3-H ), 7.62of (d,coumarin),
j=9
Hz, 1H, 6.93C5-H (d, j arom = 8),Hz, 2.8 1H,andC6-H 8.17 (s, arom 2H,), 7.642NH; (d,exchangeable
j = 8 Hz, 1H, C5-H with arom D2O); ,), 138.10
C-NMR and 8.64 (400(s, MHz,2H, 2NH;
exchangeable with D O); M+(m/z):
CDCl3) δ (ppm) = 14.55 (CH23-CH2-), 18.47 (CH3-), 23.53 (C=N-CH3), 31.08 (CH3-NH-), 65.01 347; Anal calcd: C, 58.77; H, 6.09; N, 12.09; found:
C, 58.99; H, 6.18; N, 12.41.
(CH3-CH2-), 108.47–114.44 (3C of coumarin), 127.37 (C5 of coumarin), 142.81 (C10 of cou-
marin), 151.08 (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone
(C4-CH3 of coumarin), 152.17 (C-O-C2H5), 157.61 (-C=O of coumarin), (VIc):
Yield
Yield=178.36 =85.06%;
97.7%; m.p. = 202–204
m.p. 100–102 °C; ◦ C; FT-IR ((ṽ max, cm ):):3286 −
−1 1 3381and and3417 3429(2NH), (2NH),3080 3080
159.91 (-C=N-), (-C=S);
(CH,
(CH,Ar), Ar),2981 (CH, aliphatic), (C=O), 1627 (C=N, imine), 1597H, 1 H-NMR
(C=C,
M+(m/z): 2980Anal
333; (aliphatic calcd: CH),
C, 57.64; 1732H,(C=O), 5.74; N, and 12.60;1597 (C=C,
found: C,Ar, allyl);
57.82; 5.79;Ar), N, 12.88.and MHz,
(300 1188
CDClether);
(C-O, 3 ) δ (ppm):
1H-NMR 1.40(300 (t, j MHz, = 6 Hz,
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone CDCl3H, 3CH 3 -CH2 -O),
) δ (ppm): 1.411.74 (t, j (s,
= 93H, Hz,N=C-CH 3H, CH3-CH 3 ), 2.292-O), (s,2.22 3H,
(VIb): 4-CH
(s, 3H, 3 ), 4.18
N=C-CH (q, 3j =
), 6
2.27 Hz, (s, 2H,3H, CH C4-CH3 -CH 3 ),
2 -O),
2.41 4.3(d, (d,j =j =
6 9
Hz,Hz, 3H,2H, NH-CH
NH-CH 3 ),
2 -CH=CH
4.16–4.20 2 ),
(m,5.22 2H, (d,
jYield
CH = 39-CH Hz, 2H,
-O),
= 83.95%; m.p.
2 CH
6.17 2 -CH=CH
(s, 1H,
= 183 °C; C3-Hof 2 ), 6.00 (m,
coumarin),
FT-IR (ṽ max, cm 1H, CH 6.92 -CH=CH
(d, j = 9 ),
Hz, 6.16
2 ): 3441 2and 3292 (2NH), 3080
−1 1H, (s, 1H,
C6-H C3-H
arom ), 7.62 coumarin),
of (CH, (d, j = 9
Ar), 6.93
Hz,
29811H, (d, j =
C5-Harom
(aliphatic 9 Hz, CH), 1H,
), 2.81732 C6-H
and(C=O), 8.17 ),
arom(s,1598 7.63
2H, 2NH;(d, j = 9
(C=C,exchangeableHz, 1H,
Ar), and 1184with C5-H), 8.20
(C-O,Dether); (s, 1H,
2O); C-NMR 13 NH;
1H-NMR exchangeable
(400 MHz,
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 14 (400
of 20
MHz, with
CDClCDCl 3D
) δ32)O);
(ppm) M+(m/z):
δ (ppm): = 14.551.33 359; (CH
(t, 3H, 3Anal
-CH j =2-),8calcd:
18.47
Hz, CHC,(CH 60.15;
3-CH 3-), H, 5.89;
23.53
2-NH),
N, 11.69;
(C=N-CH
1.42–1.44 ), found:
3(t, 31.08
j = 8 Hz, (CH C,3H,360.34;
-NH-), CHH, - 5.91;
365.01
CH2-O), N, 11.88.
(CH -CH(s,
32.22 2-),3H, 108.47–114.44
N=C-CH3),(3C 2.30of(s,coumarin),
3H, C4-CH127.37 3), 3.71–3.77 (C5 of (m, coumarin),
j = 4 Hz,142.81 2H, NH-CH (C10 of2-cou-
marin), (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzyl-thiosemicarbazone
151.08
CH3), 4.15–4.17 (m, j = 4 Hz, 2H, CH3-CH (C4-CH 3 of coumarin), 152.17 (C-O-C
2-O), 6.19 (s, 1H, C3-H of coumarin), 6.93 (d,
2 H 5 ), 157.61 (-C=O of j = 8(VId):
coumarin),
Yield =
Yield 85.06%;
= 88%; m.p.
m.p. = = 202–204
140–143 ◦ C; FT-IR
°C; FT-IR (ṽ
( max,
max, cm
cm −−11):
): 3286
3358 and
(2NH), 3417 3100 (2NH),(CH, 3080
Ar), 2980
159.91 (-C=N-), 178.36 (-C=S);
Hz, 1H, C6-Harom), 7.64 (d, j = 8 Hz, 1H, C5-Harom,), 8.10 and 8.64 (s, 2H, 2NH;1 exchangeable
(CH,
with D2O); Ar),
(aliphatic 2981
M+(m/z):
M+(m/z): (CH,
CH), 333; aliphatic),
1730
347; Anal (C=O),
Anal calcd: 1732
calcd:1598 (C=O),
C, 57.64; (C=C,
C, 58.77; 1627
Ar),
H, 5.74;H, 6.09;(C=N,
and 1184
N, 12.60; imine),
N, 12.09; (C-O,
found: 1597ether);
found: (C=C,
C, 57.82; Ar),
H-NMR
C, 58.99; H, 5.79; and 1188
(300
N, 12.88.
H, 6.18; MHz,
(C-O, CDCl
ether); ) δ (ppm):
1H-NMR 1.41
(300 (t,
MHz, j = 6 Hz,
CDCl 3H,) δ CH
(ppm): -CH 1.41 -O), (t, 2.25
j = 9 (s,
Hz, 3H, 3H, -N=C-CH
CH -CH ),
-O),2.40 2.22 (s, 3H,
N, 12.41. (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone
3 3 3 2 3 32
(s, 3H, C-4-CH

(VIb): N=C-CH 3 ), 4.16–4.21
3 ), 2.27 (s,(m, 3H, 2H, CH
C4-CH 3 -CH3 ), 2.41
2 -O), 4.91
(d, j = (d,
6 j
Hz, = 6 Hz,
3H, 2H,
NH-CH CH 3
2 -Ph),
), 4.16–4.206.18 (s, 1H,
(m, C3-Hof
2H,
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone (VIc):
CH3Yieldcoumarin),
-CH -O), 6.17 6.92 (s, (s,
1H, j= 9 Hz,
C3-Hof 1H, C6-H
coumarin), arom6.92 ), 7.03–7.04
(d, j = 9 (m,
Hz, 5H,1H, phenyl),
C6-H 7.63
), (d, j(d,
7.62 = 9j =Hz, 9 1H,
Pharmaceuticals 2022, 15, x FOR PEER REVIEW= 97.7%; m.p. = 100–102 °C; FT-IR (ṽ max, cm ): 3381 and 34293292
2Yield = 83.95%; m.p. = 183 °C; FT-IR (ṽ max, cm ): 3441 and (2NH),
−1 −1 arom
(2NH), 30803080 (CH,
14 of (CH, 20
Hz,Ar), C5-H
1H, C5-H
2981 ), 7.84
arom(aliphatic ), 2.8 andand CH),8.228.17 (s,(s,2H,
1732 2H,
(C=O),2NH;2NH; 1598exchangeable
exchangeable
(C=C,Ar, Ar),allyl);with
andwith DD
1184 2 O);
2O); M+(m/z):
(C-O, 13 C-NMRether); 409;
(400
1H-NMR Anal
MHz, calcd:
3) (400
arom
Ar), 2980 (aliphatic CH), 1732 (C=O), and 1597 (C=C, 1H-NMR (300 MHz, CDCl
C,3) 64.53; H, 5.66; N, 10.26; found: C,
j = 64.81; H,23.53 5.74; N, 10.44. 3), 31.08 (CH
δCDCl MHz,
(ppm): δ1.40(ppm)
CDCl (t, j3=)=14.55 Hz,(CH
δ6(ppm): 3H,1.333-CH
CH(t, 2-),
3-CH3H, 18.47
2-O), 8(CHHz,3-),
1.74 CH
(s, 3H,3-CH (C=N-CH
2-NH), 31.42–1.44
N=C-CH ), 2.29 (s, (t, 3H,j =34-CH -NH-),
8 Hz,3),3H, 65.01
4.18CH3-
(CH -CH (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone
-), 108.47–114.44 (3C of coumarin), 127.37 (C5 of coumarin), 142.81 (C10 of cou- (VIe):
(q, j =CH
3 2-O),2H,
6 Hz, 2 2.22CH (s,3-CH
3H, 2N=C-CH -O), 4.3 (d, 3), 2.30
j = 9 ◦Hz, (s, 3H, 2H,C4-CH NH-CH 3), 23.71–3.77
-CH=CH −): 2
1 ):3286
(m,
), 5.22 j = 4
(d, Hz, j = 92H, Hz, NH-CH
2H, 2-
marin), YieldYield
151.08
CH3), 4.15–4.17 = 85.06%;
= 88%;
(C4-CH m.p.
m.p. of == 202–204
220–223
coumarin), °C;C;
152.17 FT-IR (C-O-C(ṽ
( max, H cm
), −1
157.61 3468 (-C=Oand and 3417
of3414 (2NH),
(2NH),
coumarin), 3080
(d, j3059
2), 6.00 (m, j = 4 CH Hz,2-CH=CH 2H, CH3-CH 2-O), (s,6.191H, (s, 1H,of C3-H of coumarin), 6.93 (d, j6.93 =8
3 2 5
CH2-CH=CH (m, 1H, 2), 6.16 C3-H coumarin), = 9 1Hz,
(CH,
159.91 (CH,
Hz, Ar),
(-C=N-),
1H, Ar), 2981
C6-H 2981
178.36(CH,
(aliphatic
), aliphatic),
(-C=S);
7.64 (d, CH),
j = 8 1732
1724
Hz, 1H, (C=O),
(C=O), C5-H 1627
1597 ,), (C=N,
(C=C,
8.10 and imine),
Ar), and
8.64 1597
1174
(s, 2H, (C=C,
(C-O,
2NH; Ar),
ether); and
exchangeable 1188
H-NMR
1H, C6-Harom), 7.63 arom (d, j = 9 Hz, 1H, C5-H), 8.20 (s, 1H, NH; exchangeable with D2O); arom
(400
(C-O,
M+(m/z): MHz,
ether); CDCl
333;1H-NMR
Anal 3 ) δ347; (ppm):
(300 MHz, 1.41 (t,H,jC,
CDCl =3) 8, 3H,
δ (ppm): CH1.41 3 -CH (t, 2 -O,),
j 12.09;
= C,
9 Hz,2.45 (s,H,3H,
3H, CH 3N=CCH
-CH H,3 ),2.22
-O), 2.53
with
M+(m/z): D
359;2O); M+(m/z):
Anal calcd:calcd: C, 60.15; C, 57.64;
Anal H,calcd:5.89; 5.74;
N,58.77; N, 12.60;
11.69; H,found:6.09; found:N,
C, 60.34; 57.82;
H,found:5.91;5.79; C,
N, 58.99; N,212.88.
11.88. 6.18;
(s, (s,
3H, 3H, C4-CH ), 4.21 (q, j = 8 Hz, 2H,
N=C-CH33 2.27 (s, 3H, C4-CH3), 2.413(d, j 2= 6 Hz, 3H, NH-CH3), 4.16–4.20 (m, 2H,
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone CH -CH -O), 6.18 (s, 1H, C3-H of coumarin), 7.01
N, 12.41.
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzyl-thiosemicarbazone
CH
(VIb): (d,3-CHj = 82-O), Hz,6.17 1H, (s, C6-H),1H, C3-Hof 7.45 (t, coumarin),
t, j = 8, 2H,6.92 3-H(d, and j =5-H 9 Hz, arom 1H,), 7.56C6-H (t,arom j =), 87.62 Hz,(d, 1H, j =4-H 9
(VId): (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone (VIc):
Hz, of
Yieldphenyl,),
1H, =C5-H83.95%; 7.69
arom ), (d,and
2.8
m.p. j==183 88.17 Hz,
°C; (s, 1H,
2H,
FT-IR C5-H),
2NH;(ṽ max, 7.92
exchangeable
cm (d,
−1): 2H, 3441 C2-H
with
and D and
2O);C6-H
3292 13C-NMR
(2NH), of3080 phenyl),
(400 (CH, MHz, 8.93
Yield Yield
= 88%; = 97.7%; =m.p. = 100–102 °C; FT-IR (ṽ max, ):cm −1 ): 3381 and 3429(CH, (2NH), Ar),)3080 (CH,
Ar),CDCland312.96
2981 )(aliphatic
δ (ppm) (s,m.p.
2H,=CH), 2NH;
14.55
140–143
(CH
1732 exchangeable°C; FT-IR
3-CH2-),
(C=O), 18.47
1598
(ṽ
with max,
(CH
(C=C, D3Ar), cm−113
2 O);
-), 23.53
and
3358
C-NMR
(C=N-CH
1184
(2NH), (400
(C-O,
3100
MHz,
3),ether);
31.08 (CH 1CDCl
H-NMR 3-NH-),
2980
3 δ (400 (ppm)
65.013)=
Ar),
(aliphatic 2980 CH), (aliphatic
1730 -), (C=O), CH), 1732
1598 (C=O),
(C=C, and
Ar), 1597 (C=C,
and 1184), (C-O, Ar, allyl);
ether); 1H-NMR1 H-NMR (300 MHz,
(300 MHz,(4C of CDCl
(CH 14.603-CH (CH 32)-),δ3 -CH
108.47–114.44 18.76(t, (CH(3C -),
3of 24.10
j =3coumarin), (C=N-CH 127.37 64.81
(C5 of (CH 3 -CH
coumarin), 2 -), 111.19–114.27
j3=),142.81 (C10 of3),cou-
MHz,
CDCl )CDCl
δ 3(ppm):
δ (ppm): 1.40 (t, 2j (t,
(ppm):
1.41 = 61.33j Hz,
= 6 3H,
Hz, 3H, CH
3H, CH8 Hz,
-CH 2-O),
3-CH
CH 3-CH
1.74
2-O),
23-NH),
(s,
2.25 3H,(s, 1.42–1.44
N=C-CH
3H, -N=C-CH 3),(t,2.29 82.40
(s, Hz,
3H, (s,3H,
4-CH
3H, CH C-3-4.18
CH coumarin),
marin),
-O), 2.22 151.08(s, 127.41–133.48
3H, (C4-CH
N=C-CH 3 of ),(7C,
2.30 Ar),
coumarin), (s, 150.86
3H, 152.17
C4-CH (C10 ),of
(C-O-C coumarin),
3.71–3.77 2H5), (m, 157.61151.98
j = 4 (-C=O
Hz, (-C=O 2H, of of coumarin),
coumarin),
NH-CH
2(q,
3), j4.16–4.21
4-CH154.55 = 6 Hz, 2H, (m, CH 2H, 3-CH 3-O),
CH2157.42 -CH4.3
3
2-O), (d,4.91 j = 9(d, Hz, 2H,
j =159.80 3
6 Hz, NH-CH 2H, CH 2-CH=CH
2-Ph), 6.18 2), 5.22 (s, 1H, = 9 Hz,2-2H,
(d, jC3-Hof
CH 159.91
), 4.15–4.17 (C4 of
(-C=N-), (m, coumarin),
178.36
j = 4 Hz,(-C=S); 2H, CH (C -CH2 H52-O-C),
-O), 6.19 (s, 1H,(-C=N-),
C3-H 166.30
of coumarin),(C=O-Ph), 6.93 176.43
(d, jj ==(-C=S);
3CH
coumarin), 2-CH=CH 6.92 423; (s,2),j 6.00
= 9 Hz, (m, 1H, 1H, C6-H CH2-CH=CH3
arom), 7.03–7.04 2), 6.16 (m, (s, 1H, 5H, C3-H
phenyl), of coumarin),
7.63 H, (d,5.18; j6.93
= 9 N, (d,
Hz,1H, 98Hz,
Hz, M+(m/z):
1H, M+(m/z):
C6-H ),333;Anal
7.64 Anal(d, calcd:
j calcd:
= 8 Hz,C, C,62.40;
1H,57.64; C5-HH, H, 5.00;
5.74;
,), N, N,
8.109.92;12.60;
and found: found:
8.64 (s,C, 2H,62.57;
C, 57.82;
2NH; H, 5.79;
exchangeable 10.04.
N, 12.88.
C5-H1H, arom), C6-H 7.84arom
General
arom
and ), 7.63
8.22 (s,
procedures
(d,2H, j = 2NH; 9 Hz, exchangeable
for the C,
1H, C5-H), 8.20
synthesis
arom
with
of6.09;
(s,D1H, 2O); NH;
thiazolidine-4-ones M+(m/z): exchangeable 409; and
(VII Analwith calcd:
VIII):
D2O);
as re-
with D 2(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone
M+(m/z):O); M+(m/z):
359; Anal 347; calcd:Anal C, calcd:
60.15; H, 58.77;
5.89; H,
N, 11.69; N,found:12.09; C, found:
60.34; C,
H, 58.99;
5.91; N,H, 6.18;
11.88.
C, 64.53; H,
ported [33]. 5.66; N, 10.26; found: C, 64.81; H, 5.74; N, 10.44.
N, (VIb):
12.41. (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzyl-thiosemicarbazone
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone
YieldThe =intermediates
83.95%; m.p. V = 183 or VI °C;(0.005 FT-IRmol) (ṽ max, andcm chloroacetic
−1): 3441 and acid 3292 (0.00505
(2NH),mol, 3080 0.618
(CH,g)
(VIe): (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone
(VId): (VIc):
Ar),in sodium
2981 acetate
(aliphatic (0.00505
CH), mol, 0.414 g) and 15 mL absolute ethanol were reacted for 8h
= 1732 (C=O), 1598 (C=C, Ar), and 1184 and(C-O, ether); H-NMR (400
1
Yield
Yield ==reflux.
Yield 97.7%;
88%; = 88%;m.p.m.p. m.p. = 100–102
= 220–223 140–143 °C; °C; °C;
FT-IRFT-IR FT-IR (ṽmax,
(ṽiced max,
(ṽcoldmax,
cm cm −1cm
−1):
): 34683381
−1 ):separated
3358
and 3429(2NH),
(2NH),
3414 (2NH),
3100 3080(CH,
(CH,
3059 (CH,
Ar), 2980
under After that, washing with water the crystalline solid that
Ar),MHz,
Ar), (aliphatic
2981
CDCl
2980(aliphatic
(aliphatic 3) δ (ppm):
CH), CH),
1730
CH),
1.33(C=O),
1732
(C=O),
1724
(t,1598
(C=O),
3H,and j(C=C,
1597
= 81597 Hz,
(C=C,Ar), CHand
(C=C, 3-CH2-NH),
Ar), Ar, 1184
and allyl);
1174 (C-O, 1.42–1.44
1H-NMR (300
(C-O, ether);ether);
(t,1H-NMR
j 1=MHz,8 Hz,(300
H-NMR
3H, MHz,
CDCl(4003)
CH3-
CH was -O), collected
2.22 (s, and
3H, recrystallized
N=C-CH ), 2.30 from (s, ethanol.
3H, C4-CH ), 3.71–3.77 (m, j = 4 Hz, 2H, NH-CH 2-
δ (ppm):
MHz, CDCl 2
CDCl 1.40
3) δ δ(t,(ppm):
3) (ppm):
j = 6 1.41 Hz, 1.41 3H,
(t, (t,j =CH 33-CH
j6=Hz, 8, 3H, 2-O),
3H, CHCH 1.743-CH
3-CH
(s,2-O,),
23H,
-O), N=C-CH
3
2.25 (s,
2.45 (s, 3H, 3), 2.29
3H, -N=C-CH
N=CCH (s, 3H, 3),3),
4-CH
2.532.40(s, ), 3H,
3(s, 4.18
3H, C-
CH
(q, j4-CH ),
= 63),Hz, 4.15–4.17
2H, (m, j = 4 Hz, 2H, CH -CH -O), 6.19 (s, 1H, C3-H of coumarin), 6.93 (d, j = 8
(q,CH j = 38-CH Hz,2-O), 2H,4.3 CH(d, j =2-O),9 Hz,6.18 2H,(d, NH-CH 2-CH=CH 2), 5.22 (d, j =(s,9(d,Hz,j C3-Hof
=2H,
3 3 2
C4-CH 3), 4.16–4.21
4.21 (m, 2H, CH 3-CH
3-CH 2-O),4.91 (s, j1H,
= 6 Hz, C3-H 2H, of CH 2-Ph),
coumarin), 6.18 7.01 1H, 8
CHHz,
Hz, -CH=CH1H, C6-H
2coumarin),
1H, C6-H), 2), 6.92arom),
6.00
7.45 (m,
(s,
(t,
7.64
t,j =1H,
(d,
j =9 8, CH
Hz, j = 21H, 8 Hz,C6-H
2H,-CH=CH
1H, C5-H
3-H and2arom ),5-H
6.16 (s,,),1H,
arom
), 7.03–7.04
arom), 7.56(m,
8.10 C3-Handof8.64
(t, j5H,
(s, 2H, 2NH;
= coumarin),
phenyl),
8 Hz, 1H, 4-H 6.93of
7.63 exchangeable
(d,
(d, jj == 99 Hz,
phenyl,), Hz,1H,
1H, withC6-H
C5-H D 2O);),M+(m/z):
arom ), 7.63
7.84 and (d, j 347;
8.22 = 9 Anal
(s, Hz, 2H, calcd:
1H,2NH; C5-H),C, 58.77;
8.20
exchangeable H,(s, 6.09;
1H,with N,
NH; D 12.09; found:
exchangeable
O); M+(m/z): C, 58.99;
with
409; Anal DH, 2O);6.18;
calcd:
7.69 (d, j = 8 Hz, 1H, C5-H), 7.92 (d, 2H, C2-H and C6-H of phenyl), 8.93 and 12.96 (s, 2H,
arom 2
N,
M+(m/z): 12.41. 359; H,Anal calcd:
2NH; C,exchangeable
64.53; 5.66;with N, 10.26; DC, 60.15;
2O);found: 13C-NMR H,C, 5.89;
64.81;
(400 N, MHz,
11.69;
H, 5.74; found:
CDClN, 10.44. C, 60.34; H, 5.91; N, 11.88.
3) δ (ppm) = 14.60 (CH3-CH2-),
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone (VIc):
18.76 (CH3-), 24.10 (C=N-CH3), 64.81 (CH3-CH2-), 111.19–114.27 (4C of coumarin), 127.41–
(VId): Yield = 97.7%; m.p. = 100–102 °C; FT-IR (ṽ max, cm −1): 3381 and 3429 (2NH), 3080 (CH,
(Z/E)-2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-methyl-thiazolidin-
4-one (VIIIa):
Yield Yield = 85.06%;
= 89%; m.p. m.p. == 202–204 245–247 °C; ◦ C; FT-IR ((ṽ max, cm cm− −11):):3286
3040and (CH,3417 Ar),(2NH),2985 (aliphatic 3080
(CH,CH), Ar),1735 2981and1722 (CH, aliphatic), (2C=O), 1732 1624 (C=N, (C=O), imine), 1627 (C=N, and 1600 imine), (C=C, 1597Ar); (C=C, 1 H-NMRAr), and (400 1188 MHz,
CDCl
(C-O, ether);
3 ) δ (ppm):
1H-NMR 1.44(300 (t, j
MHz,= 8 Hz, CDCl 3H, 3 ) CH
δ -CH
(ppm):
3 2 -O),
1.41 2.39
(t, j =(s,9 3H,
Hz, 4-CH
3H, CH 3 ), 32.42
-CH 2(s,
-O),3H, 2.22 N=C-
CH ), 3.36
(s, 3H, 3N=C-CH3), 2.27 (s, 3H,(s, 3H, 4-N-CH ), 3.76 (s, 2H, S-CH -CO),
3 C4-CH3), 2.41 (d, 2j = 6 Hz, 3H, NH-CH3), 4.16–4.20 4.17 (q, j = 8 Hz, 2H, CH 3 -CH (m, 2 2H,6.16
-O),
CH(s,3-CH 1H, C3-Hof
2-O), 6.17 coumarin),
(s, 1H, C3-Hof 6.91 coumarin),
(d, j = 8 Hz,6.92 1H, (d, C6-H j =arom9 Hz, ), 7.55
1H,(d, C6-H j = arom
8 Hz, 1H,C5-H
), 7.62 (d, j =arom 9 ,);
Hz,M+ 1H,(m/z):C5-Harom 373; Anal
), 2.8 and calcd:8.17 C, (s, 57.89;
2H, 2NH; H, 5.13; N, 11.25; found:
exchangeable with DC, 2O); 58.04;
13C-NMR H, 5.19; (400 N,MHz,11.39.
CDCl3) δ(Z/E)-2-{[1-(7-Ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-3-ethyl-thiazolidin-4-
(ppm) = 14.55 (CH3-CH2-), 18.47 (CH3-), 23.53 (C=N-CH3), 31.08 (CH3-NH-), 65.01
(CHone 3-CH (VIIIb):
2-), 108.47–114.44 (3C of coumarin), 127.37 (C5 of coumarin), 142.81 (C10 of cou-
Yield = 85.06%; 89%; m.p. == 202–204
177–179 °C; ◦ C; FT-IR ((ṽ max, cm −−11
marin), Yield
151.08 = (C4-CH m.p. 3 of coumarin), 152.17 (C-O-C2cm H5), ):): 3286
3086and
157.61 (CH,
(-C=O 3417Ar),of(2NH),
2978
coumarin), 3080
(aliphatic
(CH,CH), Ar), 17302981 (CH,
and1720 aliphatic),
(2C=O), 1732
and (C=O),
1624 (C=N, 1627 (C=N,
imine), imine),
1600 (C=C, 1597 (C=C,
Ar); 1 H-NMRAr), and (400 1188 MHz,
159.91 (-C=N-), 178.36 (-C=S);
CDCl
(C-O, ether);
M+(m/z): 3 ) δ 1(ppm):
H-NMR 1.36
(300 (t,MHz, j =
333; Anal calcd: C, 57.64; H, 5.74; 3N, 12.60;8 Hz,
CDCl 3H,
3 ) δ CH
(ppm): -CH 1.412 -N-thiazolidine),
found: C, 57.82; H, 5.79; N, 12.88.3H,
(t, j = 9 Hz, 3H, 1.44
CH 3 (t,
-CH j 2 =
-O),8 Hz,
2.22
CH
(s, 3H, -CH2 -O),3),2.38
3N=C-CH 2.27(s, (s,3H, 3H,4-CH C4-CH
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone 3 ), 32.41
), 2.41 (s, (d, 3H,j = N=C-CH
6 Hz, 3H, 3 ), NH-CH
3.74 (s, 2H, S-CH2 -CO),
3), 4.16–4.20 (m, 3.94 2H, (q,
CHj3-CH
(VIb): = 8 Hz, 2-O), 2H, 6.17 4-N-CH
(s, 1H,2 -CH3), C3-Hof4.11–4.18 coumarin), (m, 6.92 2H, CH (d, 3j-CH = 9 2Hz, -O),1H, 6.15C6-H (s, 1H, arom C3-Hof
), 7.62 (d, coumarin),
j=9
Hz,6.871H,
Yield (d,
C5-H =j =83.95%;
8 Hz, ), 2.8 1H, and
m.p. C6-H =8.17183 (s,°C;),2H,7.55
FT-IR2NH; (d,(ṽj exchangeable
=max,8 Hz,cm 1H,−1):C5-H with
3441 andD2);O); M+(m/z):
3292 13C-NMR
(2NH), 387;(400
3080 AnalMHz, calcd:
14 (CH,
arom arom arom
Pharmaceuticals 2022, 15, x FOR PEER REVIEW of 20
CDClC, 58.90;
) δ (ppm) H, 5.46;
= 14.55 N, 10.85;
(CH -CHfound, -), C,
18.47
Ar), 2981 (aliphatic CH), 1732 (C=O), 1598 (C=C, Ar), and 1184 (C-O, ether); H-NMR (400
3 3 2 59.07;
(CH H,
3-), 5.54;
23.53 N, 11.02.
(C=N-CH 3 ), 31.08 (CH 1 3-NH-), 65.01
(CH
MHz, 3-CH CDCl(Z/E)-3-Allyl-2-{[1-(7-ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-4-
2-),3108.47–114.44
) δ (ppm): 1.33(3C (t, 3H, of coumarin),
j = 8 Hz, CH 127.37
3-CH2(C5 -NH), of coumarin),
1.42–1.44 (t,142.81 j = 8 Hz, (C10 3H, ofCH cou- 3-
CH one
marin), (VIIIc):
2-O), 151.082.22 (s, (C4-CH 3H, N=C-CH 3 of coumarin), 3), 2.30 (s, 3H, 152.17 C4-CH (C-O-C 2H5), 157.61
3), 3.71–3.77 (m, j (-C=O
= 4 Hz, of
2H, coumarin),
NH-CH 2-
◦ C; FT-IR ((ṽ max, cm cm− −11
CH3),Yield
159.91 Yield
(-C=N-),
4.15–4.17 = 85.06%; 92%;j =m.p.
= 178.36
(m, m.p.
4(-C=S);
Hz, == 202–204
222–224
2H, CH3-CH °C; ):):3286
2-O), 6.19 (s, 1H, C3-H of coumarin), 6.93 (d, j = 8
3061and (CH,3417 Ar),(2NH),2981 (aliphatic 3080
1 H-NMR
(CH,
Hz,CH), Ar),
M+(m/z):
1H, C6-H 2981
1737 (CH,
and1726
333;
arom Anal
), 7.64 aliphatic),
(2C=O),
(d, calcd:
j = 8 Hz, C, 1732
1612
57.64;
1H, (C=O),
(C=N,H, 5.74;
C5-H 1627
imine),
arom,), N,(C=N, and
12.60;
8.10 and imine),
1604
8.64(C=C,
found: 1597
C,
(s, 57.82;
2H, (C=C,
Ar2NH; H, Ar),
and allyl);
5.79;
exchangeable and 1188
N, 12.88.
(400
(C-O,
with MHz,H-NMR
Dether);
1 CDCl ) (300 δ (ppm): MHz,1.44 CDCl
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone
2O); M+(m/z):3 347; Anal calcd: C, 58.77; H, 6.09; N,
(t, 3j)=δ 8(ppm): Hz, 3H, 1.41 CH (t,3 -CH
j12.09;
= 92Hz, -O),found: 2.35CH
3H, (s, 358.99;
C, 3H,
-CH4-CH 2-O), 2.22
H, 6.18;3 ), 2.42
(s, (s, 3H,
3H,
(VIb): N=C-CH N=C-CH 3), 2.273 ), (s,(s, 2H,3H, S-CHC4-CH 2 -CO),
3), 2.41 4.13 (d, (d, j =j 6= Hz,
8 Hz, 3H, 2H, NH-CHallylic3), CH ), 4.19 (q,
4.16–4.20
2 (m, j= 2H,8 Hz,
N, 12.41.
CH2H,3-CH
Yield CH2 -O), -CH 6.17 -O),
(s, 5.41
1H, (d,
C3-Hof j = 8 Hz,
coumarin), 2H,
= 83.95%; m.p. = 183 °C; FT-IR (ṽ max, cm ): 3441 and 3292 (2NH), 3080(VIc):
3 2
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone
CH 6.92
2 -CH=CH(d, j
−1 = 92 ),
Hz, 5.96 1H, (m, C6-H1H, CH
arom ),
2 -CH=CH
7.62 (d, (CH,96.16
j 2=),
Hz, (s,1H, 1H, C5-H C3-Hof ), 2.8 and
coumarin), 8.17 (s,
6.91 2H, (d, 2NH;
j = 8 exchangeable
Hz, 1H, C6-H), with 7.56 D (d,O); j = C-NMR
8 Hz, (400
1H,C5-H); MHz, 13
Ar), 2981
Yield(aliphatic arom
= 97.7%; m.p. CH),=1732 100–102 (C=O), °C; 1598 FT-IR(C=C, (ṽ max, Ar),cm and−1): 1184
3381 (C-O, 2
and 3429
13
ether);(2NH), 1 H-NMR 3080 (CH, (400 C-
CDCl
MHz,
Ar),NMR 3)
2980 CDCl (400
δ (ppm) 3) δMHz,
(aliphatic = 14.55
(ppm): CH),CDCl (CH
1.33
1732 )-CH
33(t, (ppm)
δ(C=O),
3H, 2-),j 18.47
=and 8 =Hz, 14.67
(CH
1597 CH (CH
3-),
3-CH
(C=C, 32-CH
23.53 -NH),
Ar, 2 -),1.42–1.44
(C=N-CH
allyl); 18.71 3), (CH
1H-NMR 31.08 -),
= 823.30
(CH
(t,3j(300 3-NH-),
Hz,
MHz, (C=N-CH
3H, CDClCH33-) 3 ),
65.01
32.52 (CH -Thiazolidine), 45.04 (CH 2 , Allyl),
(CH
CH 3-CH2.22
2-O),
δ (ppm): 2-), 108.47–114.44
1.40 (s,
2(t, 3H,
j = 6N=C-CH
Hz, 3H, (3C 3),
CH of2.30
coumarin),
3-CH (s,2-O),3H, C4-CH
1.74 127.37(s,64.62
3),(C5
3H, (-CH
3.71–3.77
N=C-CH 2 of
of coumarin), (m,ethyl),
3), 2.29 j = 4142.81
(s, 107.96–116.26
Hz, 3H, 2H,(C10
4-CH NH-CH of
3), 4.18
(4C
cou- 2- of
coumarin),
marin),
CH 151.08 118.19
(C4-CH (-CH2 of of allyl),
coumarin), 125.29 152.17 (C5 of coumarin),
(C-O-C H ), 130.26
157.61 (CH
(-C=O of allyl),
of 150.33
coumarin), = (C10
(q, j3),
= 64.15–4.17
Hz, 2H, (m, CH3j-CH = 4 Hz,
3
2-O), 2H,4.3CH (d, 3j-CH = 9 Hz, 2-O),2H, 6.19NH-CH (s, 1H, 2C3-H
2 5
-CH=CH of coumarin),
2), 5.22 (d, j6.93 = 9 Hz, (d, j2H, 8
of
159.91
Hz,21H, coumarin),
(-C=N-),
C6-H2arom 152.25
178.36
), 7.64 (C4-CH
(-C=S); of coumarin), 157.46 (C-O, ethyl), 159.28 (C=N, thiazolidine),
CH -CH=CH ), 6.00 (m,(d, 1H, j =CH 8 Hz, 3
2-CH=CH1H, C5-H arom,),
2), 6.16 (s,8.10
1H,and C3-H 8.64 (s, 2H, 2NH;
of coumarin), 6.93exchangeable
(d, j = 9 Hz,
159.52
M+(m/z): (C=N), 160.57 (-C=O of coumarin), 171.50 (-C=O, thiazolidine); M+(m/z): 399; Anal
with
Pharmaceuticals 2022, 15, x FOR PEER1H,
REVIEW D2O);arom
C6-H ), 333;
M+(m/z):7.63Anal 347;
(d, j calcd:
=Anal 9 Hz, C, 57.64;
calcd:
1H, C, H,58.77;
C5-H), 5.74; 8.20 N, 6.09;
H, 12.60;
(s, 1H, N,found:
12.09;
NH; C,found:
57.82; C,
exchangeable H, 5.79;
58.99; with N,H, 12.88.
14D6.18;
2O);
of 20
calcd: C, 60.13; H, 5.30; N, 10.52;
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone
N, 12.41. found, C, 6.28; H, 5.34; N, 10.66.
M+(m/z): 359; Anal calcd: C, 60.15; H, 5.89; N, 11.69; found: C, 60.34; H, 5.91; N, 11.88.
(VIb): (Z/E)-3-Benzyl-2-{[1-(7-ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone (VIc):
4-one
Yield (VIIId):
= 83.95%; m.p. = 183
Yield = 97.7%; m.p. = 100–102 °C;◦FT-IR (ṽ max, cm ): 3381 °C; FT-IR (ṽ max, cm −1−1): 3441 and 3292 (2NH),
and 3429 (2NH), 3080 (CH, 3080 (CH,
(VId): −−11
Ar), Yield
2981
Ar), 2980 Yield = 85.06%;
(aliphatic = 86%; m.p.
m.p.
CH), 1732== 202–204
171–172
(C=O), °C;C;
1598 FT-IR
(C=C, ((ṽ max,
Ar), and cm
cm 1184 ):): 3286
3040
(C-O, and
(CH, 3417
ether); Ar), (2NH),
2980
1H-NMR 3080
(aliphatic
(400
Yield(aliphatic
= 88%; m.p. CH), 1732 (C=O),
= 140–143 °C; FT-IR and 1597 (ṽ max, (C=C,cm Ar, −1):allyl);
3358 (2NH),1 H-NMR
1 H-NMR 3100(300(CH, MHz, Ar),CDCl 2980 3)
(CH,
MHz,
δ CH), Ar),
CDCl
(ppm): 1.40 17242981 3) (CH,
(C=O),
δ (ppm):
(t, 1730
j = 6 Hz, aliphatic),
1618 1.33 (C=N,
3H, 1598(t, 3H,
CH3-CH 1732
imine),
j = (C=O),
8 Hz,
2-O),
and CH
1.7416271597
3 -CH (C=N,
(s, 1184(C=C,
-NH),
3H, N=C-CH
2 imine),
Ar);1.42–1.44 1597
3), 2.29
(C=C,
(t, j (400
= 8
(s, 3H, 4-CHAr),
MHz,
Hz, and
3H, 3),
1188
CDCl
CH4.18 3-3 ) δ
(aliphatic CH), (C=O), (C=C, Ar), and (C-O, ether); 1H-NMR (300 MHz,
(ppm):
(C-O, 1.44 (t, j =N=C-CH
8 Hz,
(3003H, ),CH 3 -CH 32)-O), 2.39 (s, ), 3H, (t,4-CH ), 2.43 (s, 3H,32H, N=C-CH 3 ),2-3.77
= 36ether);
1H-NMR MHz, CDCl δ C4-CH
(ppm):
CH j2-O), 31.41 j = 93(m, Hz, 3H, CH -CH 92-O), 2.22
(q,
CDCl δ2.22
) Hz, (ppm): (s,
2H, 3H,
CH 1.41 3-CH (t, j2-O),
= 6 Hz, 34.3 2.30
3H,j(s,
(d, =CH 93H, Hz,
3-CH 2H,
2-O), NH-CH
2.25 3.71–3.77
(s,2-CH=CH
3H, -N=C-CH 2j),= 5.22
4 Hz, 3),(d,2.40j = (s, NH-CH
Hz,3H,2H, C-
(s,
CH (s,
3H, 2H,N=C-CH
),34.15–4.17 S-CH -C-O),
),
(m, 2.27
j(m,
=2H, 4.56
(s,
4 1H,Hz, 3H,(q,2H, j =
C4-CH
CH8 Hz, ), 2H,
3-CH22-O), 2.41 CH (d,6.19 -CH
j = 6
(s, -O),
Hz,
1H, 5.05
3H,
C3-H (s,
NH-CHof2H, Benzyl
coumarin),), 4.16–4.20CH 6.93 ), 6.12
(m,
(d, =(s,81H,
2H,
CH
4-CH 32-CH=CH
), 4.16–4.21
23
2), 6.00 (m, CH CH 3-CH 2-CH=CH
3
2-O), 4.91 ), 6.16
(d,
3
j (s,
= 6
2
1H,Hz, C3-H
2H, CH
of coumarin), 3
2-Ph), 6.186.93 (s,
2
(d, jC3-Hof
1H, = 9 jHz,
CH
Hz, C3--CH
31H, of coumarin),
2-O),
C6-H 6.17 ), (s,
7.64 6.88C3-Hof
1H, (d,8j Hz, = 8coumarin),
Hz, 1H, C6-H 6.92 (d,
,),arom ),j and
6.89–7.39
= 9 Hz, 1H, (m, C6-H5H, Ar),
arom), 7.55
7.62 (d,
(d,Dj j2=O);
=89Hz,
1H,
1H,
C6-H
coumarin), C5-H);
arom ),arom
6.92 7.63
(s,
M+(m/z): = 9(d,
j (d, j =j =91H,
Hz, 449;
Hz,C6-H
Anal
1H,arom
1H,
calcd:
C5-H
C5-H),), C, arom
7.03–7.048.20
64.13;
8.10
(s,(m,
H,
1H, 5H,
5.16;
8.64
NH; (s,
phenyl),
N, 9.35;
2H,
exchangeable 2NH;
7.63
found: (d,exchangeable
C, j with
=64.25;
9 Hz,1H, H, 5.23;
Hz,
with 1H,
D C5-H
2O); arom), 2.8347;
M+(m/z): and Anal 8.17 (s, 2H,
calcd: 2NH;
C, 58.77; exchangeable
H, 6.09; N,Dwith12.09; D2found:
O); H,C-NMR
13
C, 58.99; (400 H, MHz,
6.18;
Pharmaceuticals 2022, 15, x FOR PEERM+(m/z):
C5-H
REVIEW arom),359; 7.84 Anal
and 8.22 calcd: (s,C,2H, 60.15; 2NH; H, exchangeable
5.89; N, 11.69; found:
with C, 60.34;
2O); M+(m/z): 5.91;
409; N,Anal 11.88.14calcd:
of 20
CDCl
N, N,
12.41. 9.44.
3) δ (ppm) = 14.55 (CH3-CH2-), 18.47 (CH3-), 23.53 (C=N-CH3), 31.08 (CH3-NH-), 65.01
C, 64.53; H, 5.66; N, 10.26; found: C, 64.81; H, 5.74; N, 10.44.
(CH -CH (Z/E)-3-Benzoyl-2-{[1-(7-ethoxy-4-methylcoumarin-8-yl)-ethylidene]-hydrazono}-thiazolidin-
2-), 108.47–114.44 (3C of coumarin), 127.37 (C5 of coumarin), 142.81 (C10 of
3(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone cou-
(VIc):
(VId): (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone
4-one
marin), Yield (VIIIe):
151.08 (C4-CH of coumarin), 152.17 (C-O-C H ), 157.61 (-C=O of coumarin),
Yield == 97.7%;
(VIe):Yield 88%; m.p. m.p.= =140–143
3 100–102°C; °C;FT-IR FT-IR(ṽ(ṽmax,
◦ C; FT-IR
max,cm cm−1):):3358
−12 3381
5
−−11
and 3429
(2NH), 3100 (2NH),(CH,3080 Ar), (CH,2980
159.91
Ar), 2980 Yield
(-C=N-), = 85.06%;
(aliphatic = 178.36
50%; CH),m.p.
m.p.
(-C=S);
1732 == 202–204
242–244
(C=O), °C;
and 1597 ((ṽ max,
(C=C, Ar, cm
cmallyl); ):):3286
13064
H-NMR and
(CH, 3417
(300Ar), (2NH),
MHz,2981CDCl 3080
(aliphatic 3)
(aliphatic
Yield CH),
= 88%; 1730m.p. (C=O),
=(2C=O),
220–223 1598°C; (C=C, FT-IR Ar), and
(ṽimine),
max,(C=N, 1184
cm −1 (C-O,
): 3468(C=C, ether);
and 3414 1 H-NMR(2NH),
1 H-NMR (300
3059 MHz,
(CH,
(CH,
δ CH),
(ppm): Ar),
M+(m/z): 17162981
1.40 and
(t, (CH,
333;j = 1670
Anal
6 aliphatic),
Hz, calcd:
3H, CH C, 1732
1624
57.64;
-CH (C=O),
(C=N, H,
-O), 1627
5.74;
1.74 N,
(s, and
12.60;
3H, imine),
1597
found:
N=C-CH 1597
C,
), 57.82;
2.29 (C=C,
Ar);
(s, H,
3H, Ar),
5.79;
4-CH and
N, (400 1188
12.88.
), 4.18 MHz,
CDCl2981
Ar), 3) δ (ppm):
(aliphatic 1.41 CH),(t, j 1724
= 6 Hz, 3 3H, 1597 CH3-CH
2 2-O), 2.25 (s, 3H, (C-O, -N=C-CH
3 3), 2.40 (s, 3H, 3 C-
j =(C=O), δ(C=C, Ar), and (t,21174 ether); 1H-NMR (400
CDCl
(C-O, 3 ) δ2H,
ether); (ppm):
1H-NMR 1.41(300 (t, MHz, 8 Hz, CDCl
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone 3H,3) CH -CH
(ppm):
32H, 2 -O),
1.41 2.35 j =(s,9 3H,Hz,24-CH 3H, CH 3 ), 32.46
-CH 2(s,
-O),3H, 2.22 N=C-
(q,
4-CH
MHz,j = 36
), Hz,
CDCl4.16–4.21 3) δ
CH (m,
(ppm): 3-CH 2H, 2-O),
1.41 CH (t,4.3
3 -CH
j =(d,8,
2 j
-O), =
3H, 9 Hz,
4.91
CH (d,
3=-CH j NH-CH
= 6
2-O,),Hz, -CH=CH
2H,
2.45 CH
(s, 3H,
2 -Ph), ),
N=CCH5.22
6.18 (d,
(s, j
3),(s,
=
1H,
2.53 9 Hz,
C3-Hof
(s, 2H,
3H,
(s, CH
3H,
(VIb): 3 ),
N=C-CH 3.86 (s,3 ),2H,2.27 S-CH
(s, 3H, -CO), C4-CH 4.17 3 ), (q,
2.41 j (d,
2CH2-CH=CH2), 6.16 (s, 1H, C3-Hof3 coumarin), 8 j Hz,
= 6 2H,
Hz, CH
3H, -CH
NH-CH 2 -O), 3 ), 6.16
4.16–4.20 1H, (m, C3-H2H, of
CH
C4-CH 2-CH=CH
coumarin), 3), 4.21
2), 6.00
6.92 (q, (m,
(s,j =j(d,
=8 9jHz,1H,2H,
Hz, 1H,CH C6-H 3-CH arom ), 7.03–7.04
2-O), 6.18 (s, (m, 5H,
1H, C3-H phenyl),
of coumarin),7.63 6.93 j (d,
(d, 7.01 = 9j(d, = 9jHz,
Hz,1H, = 8
CH coumarin),
3-CH 2-O), 6.93 = 8 Hz, 1H, C6-H ), 7.38–7.62 (m, 3H, C3, C4, C5-H of phenyl),
Yield ),6.17
= 83.95%; (s,(d,1H,j =C3-Hof
m.p. =92H,183 °C;coumarin),
FT-IR (ṽ max,
arom 6.92 (d,
cm j 1H,
=): 93441
Hz, 1H,
and C6-H(2NH),
3292 arom), 7.62 arom 3080 (d,D(CH,
j2O);
=9
−1
1H,
C5-H C6-H ),arom
7.84 7.63 Hz, 1H, C5-H), 8.20 (s, NH; exchangeable with
Hz,
Hz,
Ar),
1H,
7.90
1H,
aromC6-H),
2981 (d,
C5-H j =arom
(aliphatic 8and7.45
Hz,
), 2.8
8.22
(t,and
2H,
CH),
(s,
t,C2,j =8.17
1732
8,
C6-H 2H,2NH;
(s,
(C=O), 2H,3-Hexchangeable
arom ofand
2NH;
1598
5-H
phenyl),
(C=C,
arom
exchangeable Ar),
),with
7.83 (d, D
7.56
and
(t,
1H, 2O);
with
1184
j =C5-H8M+(m/z):
D
(C-O,
Hz,
2O); arom 1H,
13 409; of
4-H
); M+(m/z):
C-NMR
ether);
Anal
1H-NMR (400
calcd:
phenyl,),
463;
MHz,
(400 Anal
M+(m/z):
C,
7.6964.53;
(d, j H,
= 359;
8 5.66;
Hz, Anal N,
1H, calcd:
10.26;
C5-H), C,
found: 60.15;
7.92 C,
(d, H, 5.89;
64.81;
2H, C2-H H,N,5.74; 11.69;
and N,
C6-H found:
10.44.of C, 60.34;8.93
phenyl), H, 5.91; and N,
12.96 11.88. (s, 2H,
CDCl
MHz,calcd:3)CDCl C,362.19;
δ (ppm) = 14.55
) δ (ppm): H, 4.57; (CHN,
1.33 -CH
3(t, 9.07;
3H, 2-),jfound,
18.47
= 8 Hz, C,CH
(CH 62.37;
3-),
3-CH
H,
23.53 4.61;
2-NH),(C=N-CH N, 9.13.
1.42–1.44 3), 31.08 (t, j (CH
= 8 Hz, 3-NH-), 3H, 65.01
CH3-
2NH; (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzyl-thiosemicarbazone
(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone
exchangeable with D(3C 2O); 13C-NMR (400 MHz, CDCl3) δ (ppm) = 14.60 (CH3-CH2-),
(CH
CH
(VId): -CH -), 108.47–114.44 of coumarin),
2-O), 2.22 (s, 3H, N=C-CH3), 2.30 (s, 3H, C4-CH3), 3.71–3.77 (m, j = 4 Hz, 2H, NH-CH
3 2 127.37 (C5 of coumarin), 142.81 (C10 of cou- 2-
(VIe):(CH3-), 24.10 (C=N-CH3), 64.81 (CH3-CH2-), 111.19–114.27 (4C of coumarin), 127.41–
18.76
marin),
CH3),Yield 151.08
4.15–4.17 (C4-CH
(m, j = 4 3Hz, of coumarin),
2H, CH -CH 152.17
-O), 6.19 (C-O-C(s, 1H, 2HC3-H
5), 157.61 of (-C=O
coumarin), of coumarin),
6.93 (d, j = 8
(7C,==Ar), 88%; m.p.
m.p. ==(C10 140–143 °C;
°C; FT-IR FT-IR (ṽ max, cm
cm−1): 3358 (2NH), 3100 (CH, Ar), 2980
3 2 −1
133.48 Yield 88%;150.86 220–223 of coumarin), (ṽ
151.98 max,(-C=O ): of
3468 and 3414
coumarin), (2NH),
154.55 (C4 3059 of (CH,
cou-
159.91
Hz, 1H,
(aliphatic (-C=N-),
C6-H CH),(C arom 178.36
),
1730 7.64 (-C=S);
(d,
(C=O), j = 8
1598 Hz, 1H,
(C=C, C5-HAr), arom ,),
andAr), 8.10
1184 and(C-O, 8.64 (s,
ether); 2H, 1 2NH;
H-NMR exchangeable
(300 MHz,
Ar),
marin),2981157.42 (aliphatic 2HCH),5-O-C), 1724 159.80 (C=O), 1597
(-C=N-), (C=C,
166.30 and 1174176.43 (C-O,(-C=S); ether); 1H-NMR (400
with
CDCl M+(m/z):
D ) O);
2δ (ppm): 333;
M+(m/z): 1.41Anal 347;
(t, jcalcd:
= Anal6 Hz, C, 57.64;
calcd:
3H, C,
CH H,58.77;
5.74;
-CH -O),N, (C=O-Ph),
H, 12.60;
6.09;
2.25 N,found:
(s, 12.09;
3H, C,found:
-N=C-CH57.82; C, H,
),
M+(m/z):
5.79;(s,
58.99;
2.40 N,H,3H,
423;
12.88.
6.18;C-
MHz,calcd:
Anal 3 CDClC, 3) δ (ppm):
62.40; H, 1.41
5.00; (t,
N, j 9.92;
= 8, 3H, found: CHC,
3 3-CH2
62.57; 2-O,), H, 2.45
5.18; (s,N,3H, 10.04. N=CCH3), 2.53 (s, 3H,
3
N,
4-CH (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-ethyl-thiosemicarbazone
12.41. ), 4.16–4.21 (m, 2H, CH -CH -O), 4.91 (d, j = 6 Hz, 2H, CH -Ph), 6.18 (s, 1H, C3-Hof
C4-CH 3 3), 4.21procedures
General (q, j = 8 Hz,for 2H,the 3 CHsynthesis
3-CH2-O),of
2 6.18 (s, 1H, C3-H of coumarin),
thiazolidine-4-ones 2
(VII and VIII): 7.01 (d,asj =re-8
(VIb): (Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-allyl-thiosemicarbazone
coumarin), 6.92 (s, j = 9 Hz, 1H, C6-H ), 7.03–7.04 (m, 5H, phenyl), 7.63 (d, jof= phenyl,),(VIc):
9 Hz,1H,
Hz,
ported 1H, C6-H), 7.45 (t, t, j =
[33].= 83.95%; m.p. = 183 °C; FT-IR (ṽ max, cm−1−1 8, 2H, 3-H and
arom 5-H arom ), 7.56 (t, j = 8 Hz, 1H, 4-H
C5-H Yield
Yield = 97.7%; m.p. = 100–102 °C; FT-IR (ṽ max, cm ): 3441
): 3381 and
and 3292
3429 (2NH),
(2NH), 3080
3080 (CH,
(CH,
7.69 (d, arom j ),= 7.84
8 Hz,and 1H,8.22 C5-H), (s, 2H, 7.922NH; (d, 2H, exchangeable
C2-H and C6-H withof D2phenyl),
O); M+(m/z): 8.93 and 409;12.96 Anal(s, calcd:
2H,
Ar),
Ar), 2981H,
2980 (aliphatic
(aliphatic
5.66; N,CH), CH), 1732 (C=O), 15981597 (C=C, Ar), and 11841(C-O, ether); 1 H-NMR (400
C,
2NH;64.53; exchangeable with1732
10.26; Dfound: (C=O),
2O); 13C-NMR C, 64.81;and H, 5.74;
(400 (C=C,
MHz, N,Ar, allyl);
10.44.
CDCl H-NMR
3) δ (ppm) = 14.60 (CH3-CH2-),
(300 MHz, CDCl 3)
MHz,
δ (ppm): CDCl 1.40 3) (t,
δ (ppm):
j = 6 Hz, 1.333H, (t, CH 3H, -CHj = 8 Hz,
-O), CH
1.74 -CH
3(s, 3H, 2-NH),N=C-CH 1.42–1.44 ), 2.29 (t, j
(s, = 8
3H, Hz,4-CH 3H, CH
), 4.18 3-
18.76(Z/E)-1(1-(7-ethoxy-4-methylcoumarin)ethylidene)-4-benzoyl-thiosemicarbazone
3 2
(CH3-), 24.10 (C=N-CH3), 64.81 (CH3-CH2-), 111.19–114.27 (4C of coumarin), 127.41– 3 3
CH
(q, j2-O),
= 6 Hz, 2.222H, (s, CH 3H,3-CH N=C-CH 2-O), 34.3 ), 2.30(d, j(s, = 93H, Hz,C4-CH 2H, NH-CH 3), 3.71–3.77 2-CH=CH (m, 2j),= 5.22 4 Hz, (d,2H, j = NH-CH
9 Hz, 2H, 2-

3.2.1. Cytotoxicity Assay
Cell culture:
MCF-7 breast cancer cell line (ATCC® HTB-22™) plus non-tumorigenic epithelial cell
line (MCF 10, ATCC® CRL-10317™) were supplied from VACSERA (Cairo, Egypt) and
cultured in Dulbecco’s Modified Eagle Medium (Invitrogen-Life Technologies, Carlsbad,
CA, USA) with 1% antibiotic solution (streptomycin–penicillin) plus 10% fetal bovine serum
(Hyclone) in a 5% (v/v) humidified CO2 incubator at 37 ◦ C.
MTT assay:
IC50 of each of the tested compounds was studied by MTT assay where cells were treated
with trypsin, counted, and then plated in sterile microtiter plates (density: 1.2–1.8 × 104 cells/well).
Firstly, cells were kept in a humidified atmosphere (37 ◦ C, 24 h), and then incubated with
serial concentrations of the tested compounds. After 48 h, the medium was aspirated, and
then cells were incubated with 5% MTT solution (M-5655; Sigma Aldrich, St. Louis, MO
USA) (200 µL/well, 2 h), allowing the dye to metabolize into the colored insoluble for-
mazan complex that was then dissolved in the appropriate solubilization solution (M-8910)
(200 µL/well), for 30 min with gentle mixing at room temperature. The UV absorbance
was measured using a microplate reader (570 nm), and cell viability was determined with
respect to untreated control cells. The cytotoxic potencies of synthesized derivatives were
expressed as IC50 value, which represents the concentration of tested compound capable of
inducing 50% inhibition in cell proliferation. The values were means ± sd; n = 3.
3.2.2. PI3K and Akt Enzyme Inhibition Assays

The in vitro inhibition of PI3K and Akt kinase activities by VIIb was assessed using a
PI3Kα (p110α/p85) assay kit (Catalog #79781; BPS Bioscience, Inc, San Diego, CA, USA),
PI3Kγ (p110γ/PIK3R5) assay kit (Catalog #79803; BPS Bioscience, Inc, San Diego, CA,
USA), and Akt Kinase Activity assay kit (ab139436; Abcam, Cambridge, UK) following the
manufacturer’s instructions as described previously [58,59]. The results were expressed
as IC50 values using dose–response curves and linear regression equations. LY294002
compound was taken as the reference compound. The values were means ± sd; n = 3.
3.2.3. Cell Cycle Analysis and Apoptosis Induction

Cell cycle analysis and apoptosis rates following VIIb treatment were investigated
using a Propidium Iodide Flow Cytometry Kit (ab139418; Abcam, Cambridge, UK) [60]
and Annexin V-FITC apoptosis kit (Catalog: K101-25; BioVision Research Products, San
Francisco, CA, USA) [61], respectively, following the manufacturer’s instructions as stated
by a previous study [17].
3.2.4. Determination of the Cleaved Caspase-9 Level

The level of cleaved caspase-9 was assessed in both untreated/control MCF-7 cells and
following VIIb treatment using DRG® Caspase-9 (human) ELISA (EIA-4860), following
the manufacturer’s instructions (DRG International Inc., Springfield, NJ, USA). The data
were means ± sd; n = 3.
3.2.5. Western Blot

Western blot analysis was conducted using the following primary antibodies: anti-p-
Akt (1:5000; ab81283), anti-Cyclin D1 (1:1000; ab226977), anti-p-PI3K (1:1000; ab278545),
and anti-beta-actin (1:1000; ab8227). Firstly, VIIb-treated and untreated cells were rinsed
with PBS, and then cold lysis buffer was added to induce cell lysis. After centrifugation,
the supernatants were collected, and harvested proteins were quantified by Bradford assay
and then resolved on SDS-PAGE followed by electroblotting onto polyvinylidene fluo-
ride membranes. Later, membrane blocking was conducted using 5% skimmed milk in
0.1% Tween-20 in PBS (PBST). Then membranes were incubated with the aforementioned
primary antibodies at 4 ◦ C. Following 12-h incubation, the membranes were soaked in
PBST thrice followed by 1-h incubation with appropriate secondary antibodies. Antibodies
were supplied from Abcam (Cambridge, UK). The signals were visualized with a chemi-
luminescence ECL kit (Perkin Elmer, Waltham, MA, USA) following the manufacturer’s
instructions, and images were obtained using a Biorad Imager.
3.2.6. Molecular Simulation Studies

Molecular docking of the two target compounds into the PI3K protein (PDB code 4L23)
and Akt protein (PDB code 3O96) was performed using the MOE software package. Initially,
the downloaded protein was prepared, and water molecules were removed followed by
the minimization step. The standard settings were kept in the docking steps. The 2D
interaction diagrams for the best 10 poses were studied, and the highest-scoring binding
poses were selected and compared to the reference ligands.
3.3. Statistical Analysis

Unpaired t-tests (GraphPad Prism v7.00) were performed to investigate the signifi-
cance levels between tested Compound VIIb and control samples or reference compounds.
p < 0.05 was considered significant.
4. Conclusions
By adopting the pharmacophore hybridization approach, new series of 7-hydroxyl-4-
methylcoumarin and their 7-ethoxy analogs bearing thiosemicarbazone (V–VI) or thiazolidin-
4-one moiety (VII and VIII) were designed, prepared, and then their in vitro cytotoxicity
against MCF-7 cells examined by MTT assay. Nine compounds, namely Va, VIa, VIc, VId,
VIf, VIIb, VIIIa VIIIc, and VIIIf, demonstrated significant cytotoxicity; thus, they are
considered promising antiproliferative agents against MCF-7 cells. Overall, the present
study exemplified that one of these derivatives, VIIb, induced significant cytotoxicity at a
low concentration of 1.03 ± 0.05 µM. Further investigations were conducted to unravel the
mechanistic details of this observation. Mechanistically, VIIb exerted its effect via dual inhi-
bition of PI3K/Akt kinase activity, as manifested by the results of enzyme inhibition assay,
and was further confirmed by Western blot results. Additionally, VIIb treatment induced
S-phase cell cycle arrest alongside induction of caspase-9 mediated apoptosis. Further,
Western blot results demonstrated potential Compound VIIb modulation of anti-apoptotic
Cyclin D1, as evidenced by its decreased protein expression. Eventually, molecular docking
illustrated the binding patterns of this compound with the targeted enzymes PI3K and
Akt-1. These newly designed and synthesized coumarin hybrids are excellent candidates
for further investigation and optimization targeting signal transduction pathways in the
treatment of cancer. In particular, the currently observed antitumor efficacy of the novel
coumarin derivative VIIb in MCF-7 cells suggests the potential to evolve as a promising
anti-cancer compound via dual inhibition of the PI3K/Akt axis.
Author Contributions: Conceptualization, R.M.A. and K.M.A.; Methodology, S.M.A.-S. and O.A.;
Software, R.M.A., H.S.R.; Validation, H.S.R., R.I.N., O.A., N.S.Y. and A.S.S.; Formal Analysis, R.M.A.,
R.A., H.S.R., R.I.N., N.S.Y., O.A. and A.S.S.; Investigation, R.A.; Resources, R.M.A.; Data Curation,
R.A.; Writing—Original Draft Preparation, R.M.A., O.A., H.S.R. and R.A.; Writing—Review and Edit-
ing, R.M.A., H.S.R., R.A., S.M.A.-S. and K.M.A.; Visualization, R.I.N., N.S.Y. and A.S.S.; Supervision,
K.M.A. and S.M.A.-S.; Project Administration, R.M.A. and R.A.; Funding Acquisition, N.S.Y., O.A.,
R.I.N. and A.S.S. All authors have read and agreed to the published version of the manuscript.
Funding: The APC was funded by the Deanship of Scientific Research, Vice Presidency for Graduate
Studies and Scientific Research, King Faisal University, Saudi Arabia, grant number 216.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: We acknowledge Mohamed M. Hussein (Cairo University) for his significant

contribution to this work, as it would not have been accomplished without his exceptional support.
Conflicts of Interest: The authors declare no conflict of interest.
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Pharmaceuticals 2022, 15, 428. https://doi.org/10.3390/ph15040428 https://www.mdpi.com/journal/pharmaceuticals

Reference Pharmacophoric Features

Scheme 1. ReagentsScheme 1. Reagents

Scheme 2. Reagents and conditions: (f) chloroacetic acid, NaAc/ethanol.

Compound No. R R1 IC50 (µM)

IC50 (µM) IC50 (µM) Selectivity Index

2.2.2. Enzyme Inhibition Assay

Table 5. Percentage of5.apoptotic

Figure 5. Effect ofFigure

2.2.4. Caspase-9 Assay

Table 6. Effect of VIIb treatment on caspase-9 level in MCF-7 cell line.

Cells Caspase-9 Level (ng/mL) Fold

2.2.5. Western Blot

(s, 3H, C-4-CH

Pharmaceuticals 2022, 15, x FOR PEER REVIEW 14 of 20

3.2. Antitumor Activity

3.2.2. PI3K and Akt Enzyme Inhibition Assays

3.2.3. Cell Cycle Analysis and Apoptosis Induction

3.2.4. Determination of the Cleaved Caspase-9 Level

3.2.5. Western Blot

3.2.6. Molecular Simulation Studies

3.3. Statistical Analysis

Acknowledgments: We acknowledge Mohamed M. Hussein (Cairo University) for his significant

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