Microwave-assisted synthesis and in vitro and in silico studies of pyrano[3,2-c]quinoline-3-carboxylates as dual acting anti-cancer and anti-microbial agents and potential topoisomerase II and DNA-gyrase inhibitors

Abstract

A microwave-assisted method was utilized to synthesize novel pyranoquinolone derivatives as dual acting topoisomerase II/DNA gyrase inhibitors with apoptosis induction ability for halting lung cancer and staphylococcal infection. Herein, the designed rationale was directed toward mimicking the structural features of both topoisomerase II and DNA gyrase inhibitors as well as endowing them with apoptosis induction potential. The absolute configuration of the series was assigned using X-ray diffraction analysis. Cytotoxic activity against NSCLC A549 cells showed that ethyl 2-amino-9-bromo-4-(furan-2-yl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (IC50 ≈ 35 μM) was the most potent derivative in comparison to the positive control Levofloxacin and was selected for further investigation to assess its selectivity (SI = 1.23). Furthermore, in vitro antibacterial screening revealed the potential activity of this bromo derivative against Staphylococcus aureus. Mechanistic studies showed that the aforementioned compound exhibited promising inhibitory activity against topoisomerase II (IC50 = 45.19 μM) and DNA gyrase (IC50 = 40.76 μM) compared to reference standards. In addition, the previous compound induced a A549 cell apoptosis by 38.49-fold and it also increased the total apoptosis by 20.4% compared to a 0.53% increase in the control. Docking simulations postulated its interactions and suggested well fitting into its molecular targets.

Full text

Microwave-assisted synthesis and in vitro and in
silico studies of pyrano[3,2-c]quinoline-3-
carboxylates as dual acting anti-cancer and anti-
microbial agents and potential topoisomerase II
and DNA-gyrase inhibitors†
Ashraf A. Aly, *
a
Hisham A. Abd El-Naby,
a
Essam Kh. Ahmed,
a
Sageda A. Gedamy,
a
Kari Rissanen,
b
Martin Nieger,
c
Alan B. Brown,
d
Michael G. Shehat,
e
Marwa M. Shaaban
f
and Amal Atta
f
A microwave-assisted method was utilized to synthesize novel pyranoquinolone derivatives as dual acting
topoisomerase II/DNA gyrase inhibitors with apoptosis induction ability for halting lung cancer and
staphylococcal infection. Herein, the designed rationale was directed toward mimicking the structural
features of both topoisomerase II and DNA gyrase inhibitors as well as endowing them with apoptosis
induction potential. The absolute configuration of the series was assigned using X-ray diffraction analysis.
Cytotoxic activity against NSCLC A549 cells showed that ethyl 2-amino-9-bromo-4-(furan-2-yl)-5-oxo-
5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (IC
50
z35 mM) was the most potent derivative in
comparison to the positive control Levofloxacin and was selected for further investigation to assess its
selectivity (SI =1.23). Furthermore, in vitro antibacterial screening revealed the potential activity of this
bromo derivative against Staphylococcus aureus. Mechanistic studies showed that the aforementioned
compound exhibited promising inhibitory activity against topoisomerase II (IC
50
=45.19 mM) and DNA
gyrase (IC
50
=40.76 mM) compared to reference standards. In addition, the previous compound induced
a A549 cell apoptosis by 38.49-fold and it also increased the total apoptosis by 20.4% compared to
a 0.53% increase in the control. Docking simulations postulated its interactions and suggested well fitting
into its molecular targets.
1. Introduction
Cancer is a fatal disease characterized by uncontrolled cell
proliferation that invades surrounding tissue and is associated
with a high mortality rate.
1
The WHO's global cancer report
predicts that the percentage of deaths from cancer will double
in the next several years. As a result, many academics are now
considered the development of novel effective anticancer
medications, it has been considered to be an urgent require-
ment.
2
Thus, research on heterocyclic cores possessing anti-
cancer potential has attracted great interest worldwide.
3
Globally, lung cancer is the leading cause of cancer-related
mortality.
4
Adjuvant chemotherapy and antineoplastic agents
are conventional treatments for advanced non-small cell lung
cancer (NSCLC).
5
Since topoisomerase enzymes are crucial for
DNA metabolism, nding enzyme inhibitors is a key goal in the
hunt for novel anticancer medications.
6
They act by inhibiting
topoisomerases from relegating DNA strands aer cleavage,
leading to DNA damage. The majority of anticancer agents are
mainly directed toward DNA topoisomerase inhibition.
7
The
development of novel anticancer medications that specically
target topoisomerase II (Topo II) is a source of interest for
medicinal chemists to overcome resistance and improve
chemotherapeutic outcomes. For instance, etoposide is
a signicant chemotherapeutic drug inhibiting Topo II, which
has been used to treat a variety of human malignancies for over
a
Chemistry Department, Faculty of Science, Minia University, 61519 El-Minia, Egypt.
E-mail: ashrafaly63@yahoo.com; ashraf.shehata@mu.edu.eg; hisham_minia@mu.
edu.eg; essam.mohd@mu.edu.eg; sagedaali332@yahoo.com
b
Department of Chemistry, University of Jyv¨
askyl¨
a, P. O. Box 35, FIN-40014 Jyv¨
askyl¨
a,
Finland. E-mail: kari.t.rissanen@jyu.
c
Department of Chemistry, University of Helsinki, P. O. Box 55, A. I. Virtasen aukio I,
00014 Helsinki, Finland. E-mail: martin.nieger@helsinki.
d
Department of Chemistry and Chemical Engineering, Florida Institute of Technology,
Melbourne, FL 32901, USA. E-mail: abrown@t.edu
e
Department of Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria
21521, Egypt. E-mail: michael.shehat@alexu.edu.eg
f
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria
University, Alexandria 21521, Egypt. E-mail: maraw.mamdouh@alexu.edu.eg; amal.
atta@alexu.edu.eg; missalex_pharma@yahoo.com
†Electronic supplementary information (ESI) available. CCDC 2312660 and
2312661. For ESI and crystallographic data in CIF or other electronic format see
DOI: https://doi.org/10.1039/d4ra06201a
Cite this: RSC Adv.,2025,15, 1941
Received 28th August 2024
Accepted 23rd December 2024
DOI: 10.1039/d4ra06201a
rsc.li/rsc-advances
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1941
RSC Advances
PAPER

20 years and is still among the most oen prescribed anticancer
medications worldwide.
8
Moreover, quinoline and pyran deriv-
atives exhibit a variety of pharmacological activities, making
them important pharmaceutically active heterocyclic
compounds.
9–11
They are important cores for creating new
classes of structural entities used in cancer treatment (Fig. 1).
9,12
The quinoline framework is essential for the development of
anticancer drugs via a variety of mechanisms, including
angiogenesis inhibition, apoptosis induction, growth inhibition
by cell cycle arrest, and cell-migration disruption.
13
In addition,
drugs composed of quinolone and pyran rings targeting
Fig. 1 Reported quinoline-, pyran- and pyranoquinoline-based anticancer and antibacterial derivatives as potential inhibitors of topoisomerase II
and DNA gyrase.
1942 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

Fig. 2 Lead Topo II inhibitors, apoptosis inducers, DNA gyrase inhibitors and design rationale of the multitarget pyranoquinolones 3a–h.
Scheme 1 Synthesis of ethyl 2-amino-4-(furan-2-yl)-5-oxo-pyrano[3,2-c]quinoline-3- carboxylates 3a–h.
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1943
Paper RSC Advances

topoisomerase are currently widely used as frontline medica-
tions for the treatment of cancer (Fig. 1).
14
Patients with cancer are at an increased risk of bacterial and
antibiotic-resistant infections compared with healthy individ-
uals.
15
Particularly, lung cancer patients undergoing chemo-
therapeutic and surgical treatment are more likely to suffer
from pulmonary complications caused mainly by bacterial
infection. Regarding the bacterial spectrum of lung cancer
patients, Staphylococcus aureus is one of the most frequently
isolated organisms.
16
Moreover, growing evidence indicates that
staphylococcal infection is closely linked to the incidence and
development of several cancer types, signifying that both
inammation and immunity play a certain role in the devel-
opment of cancer; thus, there is an association between Staph-
ylococcus and carcinogenesis.
15,17
Consequently, bacterial
infection may affect the growth of lung cancer by activating
inammatory signaling. Staphylococcus aureus lipoteichoic acid
(LTA) induced a prominent increase in the cellular growth and
proliferation of the NSCLC cell lines A549.
18
Besides, Staphylo-
coccus aureus infection is involved in the regulation of cancer
cell metastasis.
19
Molecular approaches incorporating pyran and quinoline
moieties (Fig. 1) have revealed remarkable antimicrobial activity
against Gram-negative and Gram-positive bacteria as well as
against fungal pathogens.
20–23
Particularly, quinolones are
broad-spectrum antibacterial agents targeting DNA gyrase,
a type II topoisomerase. DNA gyrase plays a vital role in tran-
scription and bacterial DNA replication, making it an essential
therapeutic target in antibacterial drug discovery. However,
adverse effects and emerging drug resistance render the
currently available quinolones less effective.
24
Several quinoline
entities (Fig. 1) with diverse scaffolds have been identied as
DNA gyrase inhibitors, which could serve as good leads for the
development of novel antibacterial agents.
25,26
Additionally,
various synthetic techniques have been utilized to develop new
quinolones or to modify the quinolone scaffold with the aim of
reducing toxicity and overcoming resistance.
27,28
In addition,
spiramycin, a pyran derivative, is a macrolide antibiotic with
activity primarily against Staphylococcus aureus.
29
As a follow-up to our work on the synthesis of biologically
active-fused quinolones, and in light of the aforementioned
activity of pyranes and quinolines, this study aims to combine
the pyrane and quinoline scaffolds into a single molecule in
addition to modifying the nature of the linked quinoline ring by
diversifying the substitution, allowing us to assess the SAR
among the investigated series in the quest of promising
compounds regarding activity (Fig. 2). Herein, we report a novel
series of pyrano[3,2-c]quinoline-3-carboxylates combating the
staphylococcal infection-lung cancer interplay by inhibiting
both DNA gyrase and topoisomerase II with apoptosis induction
ability.
2. Results and discussion
2.1. Chemistry
Pyrano[3,2-c]quinoline-3-carboxylate derivatives 3a–hwere ob-
tained via the reaction of 4-hydroxy-2-oxo-1,2-dihydroquinoline
Table 1 Time and yield of 2-amino-4-(furan-2-yl)-5-oxo-pyrano[3,2-
c]quinoline-3-carboxylates 3a–husing Methods I and II
Compounds
Time (min or h) Yield (%)
Method I (h) Method II (min) Method I Method II
3a 12 3 75 90
3b 14 4 65 92
3c 15 4 77 94
3d 18 7 80 92
3e 20 6 78 88
3f 26 8 64 80
3g 20 9 65 78
3h 24 10 82 85
Fig. 3 Distinctive carbons of compound 3a.
Table 2 NMR spectral data of compound 3a
1
H NMR (DMSO-d
6
)
1
H–
1
H COSY Assignment
7.96 (d, J=8.0 Hz; 1H) 7.58, 7.30 H-10
7.77 (b; 2H) NH
2
7.58 (dd, J=8.1, 7.4 Hz; 1H) 7.96, 7.35, 7.30 H-8
7.37 (d, J=0.8 Hz; 1H) H-50
7.35 (d, J=8.3 Hz; 1H) 7.58 H-7
7.30 (dd, J=7.8, 7.4 Hz; 1H) 7.96, 7.58 H-9
6.28 (d, J=1.8 Hz; 1H) 6.07 H-30
6.07 (dd, J=2.0, 1.0 Hz; 1H) 6.28 H-40
5.03 (s; 1H) H-4
4.11 (ABX
3
,J
AB
=14.2, J
AX
=7.1 Hz; 1H) 1.21 H-3b
4.08 (ABX
3
,J
AB
=14.2, J
BX
=7.1 Hz; 1H) 1.21 H-3b
1.21 (ABX
3
,J
AX
=J
BX
=7.1 Hz; 3H) 4.11, 4.08 H-3c
13
C NMR (DMSO-d
6
) Assignment
167.7 C-3a
160.6, 160.1 C-2, C-20
156.4 C-5
151.9 C-10b
141.0 C-50
137.7 C-6a
131.0 C-8
121.9, 121.6 C-9, C-10
115.30 C-7
112.2, 110.2, 109.5 C-30, C-40, C-10a
105.0 C-4a
74.6 C-3
58.8 C-3b
28.2 C-4
14.3 C-3c
1944 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

derivatives 1a–hwith ethyl (E)-2-cyano-3-(furan-2-yl)acrylate (2)
(Scheme 1). The reaction was performed under different
conditions, such as ethanol/K
2
CO
3
at reux (Method I) and
ethanol/K
2
CO
3
/microwave (Method II) (Scheme 1).
Interestingly, when the reaction was carried out by Method II,
the yields of products 3a–jwere found to be excellent for some
derivatives (75–94%) and also took a shorter time (Table 1).
Method II showed that the reaction between 1a–hand 2was
completed faster and gave excellent yields (80–94%) compared
with the conventional method (Method I).
Under microwave irradiation, compounds 3a,3b and 3c were
formed completely in a short time (>5 min) and afforded yields
of 90%, 92% and 94%, respectively. The best yield was obtained
in the case of 3c (94%), and the reaction was completed in 4 min
(Table 1). The
1
H NMR spectrum of 3a showed the ethyl protons
as a triplet for CH
3
at d
H
=1.21 (J=7.1 Hz) and the CH
2
-ester at
d
H
=4.11 (ABX
3
,J
AB
=14.2, J
AX
=7.1 Hz; 1H) and d
H
=4.08 ppm
(ABX
3
,J
AB
=14.2, J
BX
=7.1 Hz; 1H). The NH
2
protons resonated
as a broad singlet at d
H
=7.77 ppm, while the H-pyran appeared
as a singlet at d
H
=5.03 ppm. The three protons of furan
appeared as a doublet at d
H
=6.28 (J=1.8 Hz), a double-doublet
at d
H
=6.07 (J=2.0, 1.0 Hz) and a doublet at d
H
=7.37 ppm (J=
0.8 Hz) for H-30, H-40and H-50, respectively. The
13
C NMR
spectrum conrmed the
1
H NMR spectral data. As
13
C NMR
spectrum revealed the ethyl-ester carbon signals at d
C
=14.3
(CH
3
) and 58.8 (CH
2
), while the CH-pyran appeared as a singlet
Fig. 4 Molecular X-ray structure of compound 3a (displacement parameters are drawn at a 50% probability level).
Fig. 5 Distinctive carbons of compound 3b.
Table 3 Spectral data of NMR spectra of compound 3b
1
H NMR (DMSO-d
6
)
1
H–
1
H COSY Assignment
8.07 (dd, J=7.9, 1.0 Hz; 1H) 7.71, 7.39, 6.07 H-10
7.79 (b; 2H) NH-2a
7.71 (ddd, J=8.5, 7.2, 1.3 Hz; 1H) 8.07, 7.58, 7.39 H-8
7.58 (d, J=8.5 Hz; 1H) 7.71, 7.39 H-7
7.39 (dd, J=7.7, 7.4 Hz; 1H) 8.07, 7.71, 7.58 H-9
7.36 (d, J=0.8 Hz; 1H) 6.27 H-50
6.27 (dd, J=3.0, 1.9 Hz; 1H) 7.36, 6.07 H-30
6.07 (d, J=3.0 Hz; 1H) 8.07, 6.27 H-40
5.05 (s; 1H) H-4
4.12 (ABX
3
,J
AB
=10.7, J
AX
=7.1 Hz; 1H) 4.06, 1.21 H-3b
4.06 (ABX
3
,J
AB
=10.7, J
BX
=7.1 Hz; 1H) 4.12, 1.21 H-3b
3.63 (s; 3H) H-6b
1.21 (ABX
3
,J
AX
=J
BX
=7.1 Hz; 3H) 4.12, 4.06 H-3c
13
C NMR (DMSO-d
6
) Assignment
167.6 C-3a
160.0, 159.9 C-2, C-20
156.4 C-5
150.9 C-10b
141.0 C-50
138.5 C-6a
131.5 C-8
122.0 C-9, C-10
114.9 C-7
112.8, 110.2, 108.9 C-30, C-40, C-10a
105.1 C-4a
74.6 C-3
58.8 C-3b
29.3 C-6b
28.9 C-4
14.3 C-3c
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1945
Paper RSC Advances

at d
C
=28.2 ppm. The two carbonyl carbon signals (C-3a and C-
5) for the ester and quinolyl groups resonated at d
C
=167.7 and
156.4 ppm, respectively. The carbon signal of pyran-2-C (C-2) in
the
13
C NMR spectrum appeared at d
C
=160.6 ppm. Distinctive
carbons are shown in Fig. 3. The NMR spectral data for
compound 3a are shown in Table 2.
X-ray structure analysis proved the structure of 3a (Fig. 4) and
was identied as ethyl 2-amino-4-(furan-2-yl)-5-oxo-5,6-dihydro-
4H-pyrano[3,2-c]quinoline-3-carboxylate.
In compound 3b (Fig. 5), the
1
H NMR spectrum revealed the
CH
3
-ester as an ABX
3
,(J
AX
=J
BX
=7.1 Hz) at d
H
=1.21. The CH
2
protons of the ester group appeared as non-equivalent two
protons at d
H
=4.12 (ABX
3
,J
AB
=10.7, J
AX
=7.1 Hz) and at d
H
=
4.06 ppm (ABX
3
,J
AB
=10.7, J
AX
=7.1 Hz). They have a COSY
relationship with each other and with the CH
3
protons of the
ester group. The methyl (N–CH
3
) and CH-pyran (H-4) protons
resonated at d
H
=3.63 and 5.05 ppm. The three protons of furan
appeared as a doublet at d
H
=6.07 (J=3.0 Hz) for H-40and
a double-doublet at d
H
=6.27 ppm (J=3.0, 1.9 Hz) for H-30. H-50
of the furan molecule resonated as a doublet at d
H
=7.36 (J=
0.8 Hz). The amino protons appeared in the
1
H NMR spectrum
at d
H
=7.79. Table 3 illustrates the assigned dvalues of the NMR
spectral data for compound 3b.
X-ray structure analysis proved the structure of 3b (Fig. 6)
and was identied as ethyl 2-amino-4-(furan-2-yl)-6-methyl-5-
oxo-5,6-dihydro-4H-pyrano [3,2-c]quinoline-3-carboxylate.
Fig. 6 Molecular structure of the 1st crystallographic independent molecule of compound 3b (displacement parameters are drawn at a 50%
probability level).
Scheme 2 Mechanism describing the formation of compounds 3a–h.
1946 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

Table 4 IC
50
of compounds 3a–h
Compound Structures
A549 A549
IC
50
(mgmL
−1
)IC
50
(mM)
3a 122.9 3.6 348.8009
3b 127.5 1.1 440.8112
3c 131.55 3.2 417.9811
3d 47.465 3.06 116.194
3e 53.285 0.815 139.3153
3f 30.96 1.47 78.56977
3g 45.97 0.87 125.6143
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1947
Paper RSC Advances

It is noteworthy that compounds 3a and 3b crystallized in
a centrosymmetric space group (P
1 (no. 2)) and the relative
conguration was determined. Therefore, both enantiomers (R
and Sat C4 and C24, respectively) were present in a ratio of 1 : 1.
The mechanism proposed for the formation of compounds
3a–hbegins with a nucleophilic attack of the active CH-3 from
1a–hto the electrophilic carbon in 2via Michael addition to
produce intermediates 4a–h(Scheme 2). Further nucleophilic
attack of the hydroxyl-lone pair in 4a–hthen occurs to the
electrophilic carbon in the nitrile group, forming intermediate
5a–h(Scheme 2). Finally, the aromatization of 5a–hgives the
nal products 3a–h(Scheme 2).
2.2. Biological screening
2.2.1. Cytotoxic activity. The newly synthesized compounds
were evaluated owing to their in vitro antiproliferative activity
against lung epithelial cancer cells A549 using the MTT viability
assay with Levooxacin as the standard drug (Table 4). Interest-
ingly, compound 3h showed the highest antiproliferative activity
with MIC equal to 35.10 mM, which is even greater than the
positive control Levooxacin 410.10 mM. Compounds 3d–g
showed moderate activity among the series but were still more
potent than the reference drug Levooxacin. Compounds 3a–c
exhibited the least potency among the designed compounds. SAR
studies indicated that the halo-pyrano[3,2-c]quinoline-3-
carboxylates clubbed compounds 3f–hwere more potent inhibi-
tors than the other non-halogenated derivatives 3a–e.Moreover,
the pyrano quinolone derivative incorporating the bromo
substituent (compound 3h) exhibited the highest antiproliferative
activity against lung epithelial cancer cells A549. These results
conrm that the incorporation of a halogen, especially a bromine
atom, at the pyrano quinolone ring greatly enhanced the anti-
proliferative activity against lung epithelial cancer cells.
Besides considering cytotoxicity against A549 cells, testing
safety on normal cells WI38 and selectivity to cancer cells is the
main evaluation factor of the studied pyranoquinolones.
Interestingly, the most active derivative 3h exhibited lower
cytotoxic activity against normal cells (IC
50
=43.28 mM) with
a selectivity index of 1.23.
2.2.2. Topoisomerase II inhibition. The in vitro topoisom-
erase II inhibitory prole of the most potent derivative 3h was
studied in reference to the standard etoposide. Compound 3h
exhibited promising inhibitory activity with a micromolar IC
50
value (IC
50
=45.19 mM), and it was slightly less potent than
etoposide (IC
50
=34.99 mM) (Table 5).
2.2.3. Antibacterial activity. Biological evaluation for all the
target compounds was initially performed using the agar
diffusion method as a preliminary step to screen for the most
potent compounds against Staphylococcus aureus ATCC6538
(Table 6). Aer screening, four compounds were selected
because they demonstrated the highest antibacterial activity
(higher inhibition zone diameter) towards the Staphylococcus
Table 4 (Contd. )
Compound Structures
A549 A549
IC
50
(mgmL
−1
)IC
50
(mM)
3h 15.64 1.02 35.10806
Levooxacin —148.2 1.42 410.10
Table 5 Inhibitory profile of 3h against topoisomerase II
Compound Structure IC
50
(mM)
3h 45.19 3.37
Etoposide —34.99 2.58
Table 6 Inhibition zone diameter of compounds 3a–h
Compound
Inhibition zone diameter
(mm) DMSO control (8 mm)
3a 8
3b 8
3c 8
3d 10 0.5
3e 90.3
3f 17.5 0.85
3g 18 1
3h 19 1.1
1948 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

aureus standard strain. These were further tested to determine
their minimal inhibitory concentration (MIC) using the broth
microdilution method according to the CLSI reference standard
(Table 7) using Spiramycin as the standard drug. Compound 3h
showed the highest activity among the whole series with MIC
90.58 mM. The designed derivative 3h exhibited potency
comparable to the control drug Spiramycin with MIC 37.957
mM. SAR studies revealed that the halogenated pyr-
anoquinolone derivatives 3f–hwere more potent antibacterial
agents than the other non-halogenated derivatives. The bromo-
pyrano quinolone derivative (compound 3h) showed very
promising antibacterial activity and exhibited the highest
antibacterial activity against Staphylococcus aureus. These
results indicate the great importance of the presence of
a halogen, especially the electron withdrawing property of the
bromine atom.
2.2.4. Bacterial DNA gyrase inhibition. DNA gyrase inhibi-
tion assay (Table 8) showed that 3h exhibited signicant
potency against DNA gyrase, and it recorded % inhibition
comparable to the reference Levooxacin. Further investiga-
tions indicated that 3h exhibited micromolar IC
50
(IC
50
=40.76
mM) relative to the reference inhibitor.
2.2.5. Apoptotic investigation and cell cycle analysis. Flow
cytometric analysis of annexin V/PI staining was performed to
determine the apoptotic cell death compared to the necrotic
one to investigate the apoptotic activity of the tested compound
3h (IC50 =35.10 mM), in the untreated and treated lung
epithelial cancer cells A549. As shown in Fig. 7, compound 3h
signicantly stimulated apoptotic lung cancer cell death by
38.49-fold; it increased total apoptosis by 20.4% (8.11% for late
and 12.29% for early) compared to 0.53% (0.19% for late and
0.34% for early) for the control. On the contrary, the compound
stimulated necrotic lung cancer cell death by 2.24-fold; it
induced necrotic cell death by 3.73% compared to 1.66%.
Therefore, the compound treatment favors apoptotic cell death
rather than necrosis.
Cell cycle analysis is a crucial test that investigates the
percentages of the cell population in each cell phase with
cytotoxic substances aer treatment. Lung epithelial cancer
cells A549 were treated with compound 3h. It was subjected to
DNA ow cytometry to determine at which cell cycle the cell
proliferation was arrested. As shown in Fig. 8, the compound
treatment signicantly increased the cell population at the G1
phase by 63.39% compared to the control 57.02%. In compar-
ison, the other phases did not signicantly change.
2.3. Molecular docking
A computational study utilizing MOE 2019.10259 was employed
to predict the binding scores and modes of the investigated
derivatives 3a–hinto the target proteins topoisomerase II (PDB:
Table 7 Minimal inhibitory concentration of compounds 3f–h
Compound MIC (mgmL
−1
) MIC (mM)
3f 78.125 201.983
3g 78.125 210.954
3h 39.062 90.580
Spiramycin
30
32 37.957
Table 8 Inhibitory profile of 3h against DNA gyrase
Compound Structure IC
50
(mM)
3h 40.76 0.64
Levooxacin —31.34 0.45
Fig. 7 Representation of A549 cells treated with 3h and analyzed using flow cytometry after double staining of the cells with annexin-V FITC and PI.
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1949
Paper RSC Advances

Fig. 8 Bar representation of the percentage of cell population at each cell cycle G1, S and G2/M.
Fig. 9 (A) 2D-binding mode of 3h, (B) 3D-interactions of 3h (red sticks), (C) 2D-binding mode of the co-crystallized ligand etoposide, and (D) 3D-
overlay of 3h (red sticks) and etoposide (green sticks) (PDB: 5GWK
31
).
1950 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

5GWK
31
) and DNA gyrase (PDB: 2XCT
32
) compared to co-
crystallized ligands etoposide and ciprooxacin, respectively.
2.3.1. Topoisomerase II. Test compounds tted well in the
co-crystalized ligand binding site with promising binding
scores ranging from −7.55 to −8.19 kcal mol
−1
(ESI Data†),
compared to the redocked ligand etoposide (binding score =
−11.02 at RMSD =0.35 Å). As illustrated in Fig. 9A and B, 3h
interacted with the essential amino acid residue Asp 463
through hydrogen bonds, which can be compared to etoposide.
However, the remaining favorable compounds (ESI Data†) were
able to dock deeply within the enzyme pocket near the DNA,
forming different types of interactions, including hydrogen
bonding and hydrophobic interactions, with both the protein
and DNA.
2.3.2. DNA gyrase. Validation was done by redocking the
co-crystallized ligand ciprooxacin into the enzyme active site
to ensure that its experimental interactions were reproduced
(docking score =−9.92 kcal mol
−1
at RMSD =0.64 Å). Cipro-
oxacin showed a hydrogen bond with Ser1084, and it was also
bonded with DNA via pinteractions with the guanine DG:C9
and adenine bases DA:D13 (Fig. 10). Interestingly, the tested
compounds displayed relatively high docking scores ranging
from −8.03 to −8.63 kcal mol
−1
compared to ciprooxacin.
Additionally, they showed nearly similar binding modes (ESI
Fig. 10 (A) 2D-binding mode of 3h, (B) 3D-interactions of 3h (red sticks), and (C) 2D-binding mode of the co-crystallized ligand ciprofloxacin,
and (D) 3D-overlay of 3h (red sticks) and ciprofloxacin (green sticks) (PDB: 2XCT
32
).
Table 9 ADME analysis of compounds 3a–h
ID M
a
Log P
b
HBD
c
HBA
d
Nrotb
e
TPSA
f
MR
g
Log S
h
F
i
GI absorption BBB
j
Pgp
k
substrate
3a 352.34 2.44 2 5 4 107.55 93.41 −3.47 0.56 High No No
3b 366.37 3.15 1 5 4 96.69 82.02 −2.31 0.56 High No No
3c 380.39 3.38 1 5 5 96.69 103.12 −3.89 0.56 High No No
3d 366.37 3.04 2 5 4 107.55 98.38 −3.77 0.56 High No Yes
3e 382.37 2.75 2 6 5 116.78 99.9 −3.54 0.56 High No Yes
3f 386.79 2.75 2 5 4 107.55 98.42 −4.06 0.56 High No No
3g 370.33 2.81 2 6 4 107.55 93.37 −4.03 0.56 High No No
3h 431.24 3.17 2 5 4 107.55 101.11 −4.38 0.56 High No No
a
M, molecular weight (dalton).
b
ilog P, octanol/water partition coefficient.
c
HBD, hydrogen bond donor.
d
HBA, hydrogen bond acceptor.
e
Nrotb,
# of rotatable bonds.
f
TPSA, total polar surface area.
g
MR, molar refractivity.
h
ilog P, logarithm of compound aqueous solubility.
i
F, Abbott oral
bioavailability score HIA%, human gastrointestinal absorption.
j
BBB permeant, blood–brain barrier penetration.
k
Pgp, permeability glycoprotein.
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1951
Paper RSC Advances

Data†). Compound 3h showed the best binding score
(−8.63 kcal mol
−1
) and resided well into the active site through
pinteraction with the DNA adenine base DA:D13 in addition to
hydrogen bonds with Arg458 and Glu477 (Fig. 10). These
observations assumed that screened compounds can be
potential antibacterial agents targeting DNA gyrase as the
docking results are generally consistent with the in vitro anti-
bacterial activities, where 3h was superior to the remaining
compounds.
2.4. In silico drug likeness and molecular property
prediction
Molecular property prediction is becoming a useful tool in
the generation of molecules with the correct parameters to be
useful drug candidates. About 40% of oral drugs fail in
clinical trials because of their poor pharmacokinetic prop-
erties. Drug development involves the assessment of
absorption, distribution, metabolism and excretion (ADME)
increasingly earlier in the discovery process, at a stage when
the considered compounds are numerous, but access to
physical samples is limited. Thus, we applied computational
methods to predict physical and molecular properties to
assess their availability as useful drug candidates. Here, we
present a new SwissADME web tool that provides free access
to a pool of fast yet robust predictive models for physico-
chemical properties, pharmacokinetics, drug-likeness and
medicinal chemistry friendliness, including in-house pro-
cient methods such as iLOGP and Bioavailability Radar
(Table 9).
All the compounds had high GI absorption and obeyed the
Lipinski rule of ve. The overall results showed that the tested
compound showed a very good drug-likeness pharmacokinetic
and pharmacodynamic prole.
3. Conclusion
A series of new pyrano[3,2-c]quinoline-3-carboxylate derivatives
3a–hwere designed and synthesized efficiently using a micro-
wave. The structure of the products was conrmed using
a combination of spectral techniques, including infra-red (IR),
nuclear magnetic resonance (NMR), mass spectrometry (MS)
and elemental analyses, in addition to X-ray structure analysis.
The newly synthesized compounds were evaluated for their in
vitro antiproliferative activity against lung epithelial cancer cells
A549 using an MTT viability assay. Compound 3h showed the
highest activity with an MIC of 15.14 mgml
−1
compared to the
positive control Levooxacin. The cell cycle arrest behavior
detected by propidium iodide and the apoptosis induction was
investigated. In addition, the newly synthesized compounds
were evaluated for their in vitro antibacterial activity against the
standard Staphylococcus aureus strain. Compound 3h showed
the highest antibacterial activity with an MIC of 39.062 mgml
−1
using Levooxacin as the standard reference drug. An in silico
study was performed, including docking of the newly synthe-
sized compounds into topoisomerase II and DNA-gyrase
binding pockets, in addition to predicting their
physicochemical and pharmacokinetic properties. In silico
studies showed the ability of the synthesized members to bind
to the topoisomerase II and DNA-gyrase. Our study revealed that
compound 3h is a promising dual acting anticancer and anti-
bacterial agent targeting topoisomerase II and DNA-gyrase with
acceptable oral bioavailability and physicochemical and phar-
macokinetic properties.
4. Experimental
4.1. Chemistry
Melting points were measured in open capillaries using
a Gallenkamp melting point apparatus (Weiss-Gallenkamp,
Loughborough, UK) and were uncorrected. The IR spectra
were recorded by applying the ATR technique (ATR =Attenu-
ated Total Reection) with an FT device (FT-IR Bruker IFS 88),
Institute of Organic Chemistry, Karlsruhe University, Karls-
ruhe, Germany. The NMR spectra were measured in DMSO-d
6
using a Bruker AV-400 spectrometer, 400 MHz for
1
H, and 100
MHz for
13
C. The chemical shis are expressed in d(ppm)
versus internal tetramethylsilane (TMS) =0for
1
Hand
13
C. The
description of signals includes s =singlet, d =doublet,
dd =doublet of doublet, t =triplet, q =quartet, and
m=multiplet. The following abbreviations were used to
distinguish between signals: Ar-H =aromatic-CH. Signals of
the
13
C NMR spectra were assigned with the help of DEPT90
and DEPT135 and were specied in the following way:
+=primary or tertiary carbon atoms (positive DEPT signal),
−=secondary carbon atoms (negative DEPT signal), and
C
q
=quaternary carbon atoms (no DEPT signal). Correlations
were established using
1
H–
1
HCOSY,
1
H–
13
CHSQCandHMBC
experiments. Mass spectra were recorded using a FAB (fas-
t atom bombardment) Thermo Finnigan Mat 95 (70 eV). The
HRSM was recorded using LC/Q-TOF, 6530 (Aligent Technol-
ogies, Santa Clara, CA, USA) at the Natural Product Research
lab, Faculty of Pharmacy, Fayoum University, Egypt. Elemental
analyses were carried out at the Microanalytical Center, Cairo
University, Egypt. TLC was performed on analytical
Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf254
indicator; TLCs were viewed at l
max
=254 nm.
4.1.1. Starting materials. 1,6-Disubstituted-quinoline-2,4-
(1H,3H)-diones 1a–hwere prepared according to the litera-
ture.
33,34
Ethyl (E)-2-cyano-3-(furan-2-yl)acrylate (2) was
purchased from Aldrich (St. Louis, MO, USA).
4.1.2. Reactions of 1a–h with 2; synthesis of compounds
3a–h
4.1.2.1. Method I. A mixture of 1a–h(1 mmol) and 2
(1 mmol, 0.138 g) in 40 ml absolute ethanol and anhydrous
K
2
CO
3
(1.5 mmol, 0.192 g) was stirred at room temperature for
12–24 h. The reaction was monitored through TLC analysis. The
formed products 3a–hwere ltered offand washed several
times with H
2
O (150 ml) and with ether (30 ml). The obtained
products were recrystallized from the stated solvents to afford
pure compounds 3a–h.
4.1.2.2. Method II. The above method was repeated by
exposing the reaction mixture to MW irradiation for 3–10 min.
The reaction was reuxed in a Milestone Microwave Lab station
1952 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

at 120 °C for 3–10 min. All reactions were monitored by TLC
with 1 : 1 ethyl acetate/petroleum ether as an eluent and were
carried out until the starting materials were completely
consumed. Aer few minutes (Table 1), microwave irradiation
was stopped, and the reaction mixture was analyzed by TLC.
4.1.2.2.1 Ethyl 2-amino-4-(furan-2-yl)-5-oxo-5,6-dihydro-4H-
pyrano[3,2-c]quinoline-3-carboxylate (3a). Compound 3a was
obtained as pale brown crystals, (DMF/EtOH); yield: m.p. 270–
272 °C; IR (KBr): n
max
/cm
−1
=3460–3463 (NH
2
), 3325 (NH), 3100
(CH-Ar), 2975 (CH-Aliph), 1692 (C]O, ester), 1656 (C]O, qui-
nolone), 1578 (C]C), 1437 (CH
2
), 1375 (CH
3
). NMR (see Table
2). MS (FAB, 3-NBA), m/z(%): 352.1 [M
+
] (18), 353.1 [M + 1] (22),
307.1 (100), 289.1 (35). Anal. calcd C
19
H
16
N
2
O
5
(352.35): C,
64.77; H, 4.58; N, 7.95. Found: C, 64.85; H, 4.48; N, 8.10.
4.1.2.2.2 Ethyl 2-amino-4-(furan-2-yl)-6-methyl-5-oxo-5,6-
dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3b). Compound
3b was obtained as pale brown crystals, (DMF/EtOH); yield:
92%; m.p. 260–262 °C. IR (KBr): n
max
/cm
−1
=3275 (NH), 3050
(CH-Ar), 2928 (CH-Aliph), 1682 (C]O, ester), 1654 (C]O, qui-
nolone), 1614 (C]C), 1463 (CH
2
), 1418 (N–CH
3
), 1375 (CH
3
).
NMR (see Table 3). MS (FAB, 3-NBA), m/z(%): 366.1 [M]
+
(35),
367.1 [M + 1] (45). HRSM [M + Na
+
]=calcd: 389.1114; found:
389.1124. Anal. calcd for C
20
H
18
N
2
O
5
(366.37): C, 65.57; H,
4.95; N, 7.65. Found: C, 65.47; H, 4.85; N, 7.70.
4.1.2.2.3 Ethyl 2-amino-6-ethyl-4-(furan-2-yl)-5-oxo-5,6-dihy-
dro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3c). Compound 3c
was obtained as pale brown crystals, (DMF/EtOH), yield: 94%;
m.p. 264–266 °C. IR (KBr): n
max
/cm
−1
=3268 (NH), 3056 (CH-
Ar), 2828 (CH-Aliph), 1767 (C]O, pyranone), 1655 (C]O,
ester), 1541 (C]O, quinolone), 1485 (CH
2
), 1375 (CH
3
).
1
H NMR
(DMSO-d
6
, ppm): d
H
=8.08 (dd, J=7.9, 0.8 Hz, 1H; H-10), 7.76
(b, 2H; NH-2a), 7.64 (ddd, J=8.3, 7.0, 1.00 Hz, 1H; H-8), 7.57 (d,
J=8.4 Hz, 1H; H-7), 7.34 (dd, J=8.3, 7.5 Hz, 1H; H-9), 7.33 (bs;
H-50), 6.26 (dd, J=3.0, 1.9 Hz, 1H; H-30), 6.03 (d, J=3.0 Hz, 1H;
H-40), 5.06 (s, 1H; H-4), 4.34–4.22 (m, 4H, 2×CH
2
), 1.27 (t, J=
7.1 Hz, 3H, H-3c), 1.20 (t, J=7.0 Hz, 3H, H-6c).
13
C NMR (DMSO-
d
6
, ppm): d
C
=167.6 (C-3a), 159.9, 159.9 (C-2, C-20), 156.3 (C-5),
150.9 (C-10b), 141.0 (C-50), 138.6 (C-6a), 131.4 (C-8), 122.1 (C-9,
C-10), 114.9 (C-7), 112.8, 110.3, 108.9 (C-30, C-40, C-10a), 105.1
(C-4a), 74.6 (C-3), 58.9 (C-3b), 38.4 (C-6b), 28.9 (C-4), 14.3 (C-3c)
12.6 (C-6c). MS (FAB, 3-NBA), m/z(%): 379.06 [M
+
−1] (35),
382.54 [M + 2]
+
(18), 267.07 (100). HRSM [M + Na
+
]=calcd:
403.1270; found: 403.1281. Anal. calcd for C
21
H
20
N
2
O
5
(380.40):
C, 66.31; H, 5.30; N, 7.36. Found: C, 66.35; H, 5.28; N, 7.46.
4.1.2.2.4 Ethyl 2-amino-4-(furan-2-yl)-9-methyl-5-oxo-5,6-
dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3d). Compound
3d was obtained as brown crystals (DMF); yield; 92%; m.p. 280–
282 °C. IR (KBr): n
max
/cm
−1
=3482–3435 (NH
2
), 3266 (NH), 3090
(CH-Ar), 2840 (CH-Aliph), 1737 (C]O, pyranone), 1695 (C]O,
ester), 1521 (C]O, quinolone), 1415 (CH
2
), 1325 (CH
3
).
1
H NMR
(DMSO-d
6
, ppm): d
H
=11.73 (s, 1H, NH), 7.96 (s, 1H; H-10), 7.77
(b, 2H, NH
2
), 7.58 (d, J=8.1, 7.3 Hz, 1H, H-8), 7.35 (d, J=0.8 Hz,
1H, H-50), 7.38 (d, J=8.2 Hz, 1H, H-7), 6.25 (d, J=1.8 Hz, 1H, H-
30), 6.02 (d, J=2.0 Hz, 1.0 Hz, 1H, H-40), 5.06 (s, 1H, H-4), 4.09 (q,
J=7.05 Hz, 2H, H-3b), 2.24 (s, 3H; H-9a), 1.20 (t, J=7.06 Hz, 3H,
H-3c).
13
C NMR (DMSO-d
6
, ppm): d
C
=167.6 (C-3a), 160.6, 159.1
(C-2, C-20), 156.3 (C-5), 151.9 (C-10b), 141.1(C-50), 137.6 (C-6a),
131.0 (C-9), 122.6, 122.6 (C-8, C-10), 115.4 (C-7), 112.2, 110.2,
109.5 (C-30, C-40, C-10a), 105.0 (C-4a), 74.7 (C-3), 58.9 (C-3b),
28.22 (C-4), 20.3 (C-9a), 14.3 (C-3c). MS (FAB, 3-NBA), m/z(%):
366.46 [M]
+
(50), 293.25 [M]
+
(100), 253.22 (100), 224.24 (100).
HRMS [M + Na
+
]=calcd: 389.1114; found: 389.1121. Anal. calcd
for C
20
H
18
N
2
O
5
(366.37): C, 65.57; H, 4.95; N, 7.65. Found: C,
64.57; H, 4.85; N, 7.61.
4.1.2.2.5 Ethyl 2-amino-4-(furan-2-yl)-9-methoxy-5-oxo-5,6-
dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3e). Compound
3e was obtained as brown crystals, (DMF/EtOH); yield: 88%;
m.p. 390–292 °C. IR (KBr): n
max
/cm
−1
=3432–3475 (NH
2
), 3268
(NH), 3056 (CH-Ar), 2828 (CH-Aliph), 1767 (C]O, pyranone),
1655 (C]O, ester), 1541 (C]O, quinolone), 1485 (CH
2
), 1375
(CH
3
).
1
H NMR (DMSO-d
6
, ppm): d
H
=12.23 (s, 1H, NH), 7.98 (s,
1H; H-10), 7.79 (b, 2H, NH
2
), 7.61 (d, J=8.2 Hz, 7.4, 1H, H-8),
7.38 (d, J=0.7 Hz, 1H, H-50), 7.41 (d, J=8.0 Hz, 1H, H-7),
6.27 (d, J=1.8 Hz, 1H, H-30), 6.05 (d, J=2.1 Hz, 1.0, 1H, H-
40), 5.04 (s, 1H, H-4), 4.06 (q, J=7.0 Hz, 2H, H-3b), 3.71 (s, 3H;
H-9a), 1.18 (t, J=7.0 Hz, 3H, H-3c).
13
C NMR (DMSO-d
6
, ppm):
d
C
=167.1 (C-3a), 161.6, 161.5 (C-2, C-5), 158.3 (C-9, C-10b),
154.9 (C-20), 145.7 (C-50), 135.0 (C-6a), 124.7 (C-7), 118.0 (C-
10a), 114.9, 112.7, 110.7 (C-8, C-10, C-40), 107.8 (C-30), 103.1
(C-4a), 75.7 (C-3), 61.3 (C-3b), 56.9 (C-9a), 28.2 (C-4), 14.6 (C-3c).
MS (FAB, 3-NBA), m/z(%): 382.15 [M + 1] (30), 175.93 [M]
+
(45).
HRMS [M + H
+
]=calcd: 383.1243; found: 383.1227. Anal. calcd
for C
20
H
18
N
2
O
6
(382.37): C, 62.82; H, 4.75; N, 7.33. Found: C,
62.72; H, 4.55; N, 7.53.
4.1.2.2.6 Ethyl 2-amino-9-chloro-4-(furan-2-yl)-5-oxo-5,6-dihy-
dro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3f). Compound 3f
was obtained as brown crystals, (DMF/CH
3
OH); yield: 80%; m.p.
310–312 °C. IR (KBr): n
max
/cm
−1
=3432–3475 (NH
2
), 3222 (NH),
3010 (CH-Ar), 2812 (CH-Aliph), 1761 (C]O, pyranone), 1650
(C]O, ester), 1540 (C]O, quinolone), 1475 (CH
2
), 1335 (CH
3
).
1
H NMR (DMSO-d
6
, ppm): d
H
=12.42 (s, 1H, NH), 7.98 (d, J=
8.1 Hz, 1H, H-7), 7.81 (b, 2H, NH
2
), 7.41 (s, 1H, H-10), 7.37 (d, J=
0.9 Hz, 1H, H-50), 7.32 (d, J=8.1 Hz, 1H, H-8), 6.25 (d, J=1.7 Hz,
1H, H-30), 6.01 (d, J=2.0 Hz, 1.0, 1H, H-40), 5.02 (s, 1H, H-4),
4.02 (q, J=7.04 Hz, 2H, H-3b), 1.16 (t, J=7.03 Hz, 3H, H-3c).
13
C NMR (DMSO-d
6
, ppm): d
C
=167.9 (C-3a), 160.5, 160.3 (C-
2, C-20), 158.4(C-5), 155.0 (C-10b), 142.7 (C-50), 135.2 (C-6a),
131.2 (C-9), 128.9, 126.6 (C-8, C-10), 122.6 (C-7), 114.2, 110.7,
108.7 (C-10a, C-40, C-30), 100.6(C-4a), 75.6 (C-3), 61.3 (C-3b),
28.5(C-4), 14.1 (C-3c). MS (FAB, 3-NBA), m/z(%): 386.07 [M +
1] (10), 366.57 [M]
+
(15). HRMS [M + H
+
]=calcd: 387.0747;
found: 387.0617. Anal. calcd for C
19
H
15
ClN
2
O
5
(386.79): C,
59.00; H, 3.91; Cl, 9.17; N, 7.24. Found: C, 59.08; H, 3.95; Cl,
9.21; N, 7.30.
4.1.2.2.7 Ethyl 2-amino-9-uoro-4-(furan-2-yl)-5-oxo-5,6-dihy-
dro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3g). Compound 3g
was obtained as pale brown crystals, (DMF/EtOH); yield: 78%;
© 2025 The Author(s). Published by the Royal Society of Chemistry RSC Adv.,2025,15,1941–1956 | 1953
Paper RSC Advances

m.p. 302–304 °C. IR (KBr): n
max
/cm
−1
=3442–3455 (NH
2
), 3261
(NH), 3052 (CH-Ar), 2820 (CH-Aliph), 1760 (C]O, pyranone),
1659 (C]O, ester), 1549 (C]O, quinolone), 1445 (CH
2
), 1355
(CH
3
).
1
H NMR (DMSO-d
6
, ppm): d
H
=12.45 (s, 1H, NH), 7.88 (d,
J=8.2 Hz, 1H, H-7), 7.81 (b, 2H, NH
2
), 7.37 (d, J=0.8 Hz, 1H, H-
50), 7.20 (d, J=8.2 Hz, 1H, H-8) 7.02 (s, 1H, H-10), 6.27 (d, J=
1.7 Hz, 1H, H-30), 6.02 (d, J=2.01 Hz, 1.0, 1H, H-40), 5.04 (s, 1H,
H-4), 4.03 (q, J=7.01 Hz, 2H, H-3b), 1.14 (t, J=7.01 Hz, 3H, H-
3c).
13
C NMR (DMSO-d
6
, ppm): d
C
=167.6 (C-3a), 160.6, 160.1
(C-2, C-9), 156.3 (C-5), 152.9, 152.6 (C-10b, C-20), 141.9 (C-50),
135.7 (C-6a), 118.0 (C-10a), 115.7, (C-8), 112.6 (C-7), 111.4, 111.3
(C-10, C-40), 106.0 (C-30), 100.9 (C-4a), 74.7 (C-3), 59.9 (C-3b), 28.3
(C-4), 14.3 (C-3c). MS (FAB, 3-NBA), m/z(%): 372.84 [M + 2] (10),
179.25 [M]
+
(85). HRMS [M + H
+
]=calcd: 371.1044; found:
371.1054. Anal. calcd for C
19
H
15
FN
2
O
5
(370.34): C, 61.62; H,
4.08; F, 5.13; N, 7.56. Found: C, 61.60; H, 4.06; F, 5.12; N, 7.50.
4.1.2.2.8 Ethyl 2-amino-9-bromo-4-(furan-2-yl)-5-oxo-5,6-
dihydro-4H-pyrano[3,2-c]quinoline-3-carboxylate (3h). Compound
3h was obtained as brown crystals, (CHCl
3
/EtOH); yield: 85%;
m.p. 320–322 °C. IR (KBr): n
max
/cm
−1
=3462–3465 (NH
2
), 3330
(NH), 3000 (CH-Ar), 2965 (CH-Aliph), 1690 (C]O, ester), 1652
(C]O, quinolone), 1574 (C]C), 1433 (CH
2
), 1370 (CH
3
).
1
HNMR
(DMSO-d
6
,ppm):d
H
=11.93 (s, 1H, NH), 7.98 (s, 1H, H-10), 7.81
(b, 2H, NH
2
), 7.37 (d, J=0.8 Hz, 1H, H-50), 7.56 (d, J=8.2 Hz, 1H,
H-7), 7.42 (d, J=8.3 Hz, 1H, H-8), 6.18 (d, J=1.8 Hz, 1H, H-30),
6.03 (d, J=2.0, 1.0 Hz, 1H, H-40), 5.01 (s, 1H, H-4), 4.08 (q, J=
7.1 Hz, 2H, H-3b), 1.16 (t, J=7.1 Hz, 3H, H-3c).
13
C NMR (DMSO-
d
6
, ppm): d
C
=167.3 (C-3a), 160.5, 160.5 (C-2, C-20), 158.4 (C-5),
155.0 (C-10b), 142.8 (C-50), 138.2 (C-6a), 132.2 (C-8), 126.7 (C-
10), 121.6, 121.5 (C-7, C-10a), 115.2, 110.7, 106.7 (C-9, C-40,C-
30), 100.5 (C-4a), 75.5 (C-3), 61.3 (C-3b), 28.5 (C-4), 14.1 (C-3c). MS
(FAB, 3-NBA), m/z(%): 431.64 [M]
+
(30), 149.78 [M]
+
(100). Anal.
calcd for C
19
H
15
BrN
2
O
5
(431.24): C, 52.92; H, 3.51; Br, 18.53; N,
6.50. Found: C, 53.02; H, 3.48; Br, 18.55; N, 6.40.
4.2. Biological screening
4.2.1. Cytotoxic activity. The anticancer effect of the various
chemical compounds was assayed using the human lung
adenocarcinoma cell line A549. Cells were obtained from ATCC,
cultured and maintained in DMEM high glucose (Biowest,
France) supplemented with 10% FBS (Biowest, France), 100 U
per ml penicillin and 1% streptomycin. Cancer cells were
seeded at a density of 5 ×10
3
cells per well in sterile 96 well at
bottom tissue culture plates the day before treatment. Aer
allowing the cells to adhere for 24 h, serial dilutions of the
tested compounds were added to the seeded cells, and the
plates were incubated for 72 h at 37 °C in a 5% CO
2
incubator.
Aer incubation, MTT (Biobasic, Canada) dissolved in PBS was
added to each well at a nal concentration of 0.5 mg ml
−1
, and
the plates were then incubated at 37 °C for 3 h in the dark. The
MTT solution was then removed, 100 ml DMSO was added to
dissolve the formed formazan crystals, and the absorbance was
measured using a microplate reader (BioTek, USA). The
percentage viability was calculated for each concentration
relative to the control DMSO-treated cells. Graphpad soware
was used to calculate the IC
50
values of each of the tested
compounds.
4.2.2. Topoisomerase II inhibition. The topoisomerase II
enzyme inhibitory activity was performed using the in vitro
toxicology kit assay, MTT based, Stock No. TOX-1 (catalog no.
M-5655) (Sigma ®), according to the manufacturer's
instructions.
4.2.3. Antibacterial activity. To determine MIC, the broth
microdilution method was employed through Mueller-Hinton
broth, as outlined in the approved standard. The compounds
showing the highest activity (as found by applying the agar
diffusion method) were two-fold serially diluted in a round
bottom 96 well plate to obtain a range of nal concentration in
the well from 1250 mgml
−1
to 1.22 mg ml
−1
. For bacterial
suspension preparation, the standardized inoculum was
prepared from a fresh plate of ATCC 6538 (the standard
Staphylococcus aureus strain). The direct colony suspension
method was used, as described in CLSI.
35
Briey, the bacterial
suspension was prepared in 0.9% normal saline and adjusted
visually to obtain turbidity equivalent to a 0.5 McFarland stan-
dard (1–2×10
8
(CFU) mL
−1
). The obtained bacterial inoculum
was further diluted 1 : 100 in double strength cation adjusted
Mueller–Hinton broth to reach 1-2x 10
6
CFU ml
−1
.Aer that,
100 ml of the prepared bacterial suspension was transferred
with equal volume to the serially diluted compounds (100 ml)
present in the 96 well plates. This leads to a nal bacterial
density in the wells of 5–10 ×10
5
CFU ml
−1
. Additional wells for
broth growth positive and negative controls containing either
only bacterial suspension or uninoculated broth were included
in each test. The plates were then incubated at 37 °C for 18 h.
The plates were read using a microplate reader (Biotek, USA),
and the MIC was determined to be the lowest concentration that
inhibited bacterial growth. For solvent control, the MIC of the
used DMSO was determined using the same experimental
settings, as described above. Each experiment was performed at
least twice.
CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically; Approved Standard—Eleventh
Edition. CLSI document M07. Wayne, PA: Clinical and Laboratory
Standards Institute. 2018.
4.2.4. Bacterial DNA gyrase inhibition. In vitro VEGFR-2
kinase inhibitory activity was evaluated using puried E. coli
DNA Gyrase and Relaxed DNA Kit, Protocol TG2000GKIT,
(TopoGEN, Inc.® Florida 32128, USA), following the manufac-
turer's instructions.
4.2.5. Apoptotic investigation and cell cycle analysis. Lung
epithelial cancer cells A549 were seeded into six-well culture plates
(3–5×105 cells per well) and incubated overnight. The cells were
then treated with the target compounds for 48 h. Next, media
supernatants and cells were collected and rinsed with ice-cold
PBS. The next step was suspending the cells in 100 mlof
annexin binding buffer solution “25 mM CaCl
2
,1.4MNaCl,0.1M
Hepes/NaOH, and pH 7.4”and incubation j5495471INTRNODU
with “Annexin V- FITC solution (1 : 100) and propidium iodide
(PI)”at a concentration of 10 mgml
−1
in the dark for 30 min. The
stained cells were then acquired by applying a Cytoex FACS
machine. Data were analyzed using cytExpert soware.
36
1954 |RSC Adv.,2025,15,1941–1956 © 2025 The Author(s). Published by the Royal Society of Chemistry
RSC Advances Paper

4.3. Molecular docking
The docking scores and binding modes of the newly synthesized
compounds with the target proteins (DNA gyrase and topo-
isomerase II) were predicted by employing the MOE 2019.10259
soware.
4.4. Investigated derivative preparation
First, Chemdraw soware was employed to draw the tested
compounds, which were then imported to an optimized data-
base, as previously discussed.
37
4.5. Protein preparation
3D crystal structures of the target proteins (PDB: 5GWK
31
and
PDB: 2XCT
32
) were obtained from the protein data bank web-
site,
38
followed by structure preparation and optimization for
established docking, as described in detail.
35
Subsequently,
docking of the assessed derivatives 3a–hto the target protein
complex was carried out by applying the default docking
protocol. For each docked structure, the pose with prominent
amino acid interactions, the best docking score, and RMSD
were selected and visualized.
Data availability
Data will be made available on request. CCDC 2312660 (3a,
https://www.ccdc.cam.ac.uk/structures/search?
Ccdc=2312660&Author=Nieger&Access=referee) and 2312661
(3b,https://www.ccdc.cam.ac.uk/structures/search?
Ccdc=2312661&Author=Nieger&Access=referee) contain the
supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Author contributions
A. A. Aly: conceptualization, writing, and editing; H. A. Abd El-
Naby, E. K. Ahmed: supervision; S. A. Gedamy: methodology,
writing the dra; M. Nieger, K. Rissanen: X-ray, writing dra;
Alan B. Brown: editing; Marwa M. Shaaban, Amal Atta: biology,
molecular modeling, writing and editing; Michael G. Shehat:
biology.
Conflicts of interest
The authors declare no conict of interest.
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