Discovery of 4-alkoxy-2-aryl-6,7-dimethoxyquinolines as a new class of topoisomerase I inhibitors endowed with potent in vitro anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2021.113261

In our attempt to develop potential anticancer agents targeting Topoisomerase I (TOP1), two novel series of 4-alkoxy-2-arylquinolines 14a-p and 19a-c were designed and synthesized based on structure activity relationships of the reported TOP1 inhibitors and structural features required for stabilization of TOP1-DNA cleavage complexes (TOP1ccs). The in vitro anticancer activity of these two series of compounds was evaluated at one dose level using NCI-60 cancer cell lines panel. Compounds 14e-h and 14m-p, with p-substituted phenyl at C2 and propyl linker at C4, were the most potent and were selected for assay at five doses level in which they exhibited potent anticancer activity at sub-micromolar level against diverse cancer cell lines. Compound 14m was the most potent with full panel GI₅₀ MG-MID 1.26 μM and the most sensitive cancers were colon cancer, leukemia and melanoma with GI₅₀ MG-MID 0.875, 0.904 and 0.926 μM, respectively. Melanoma (LOX IMVI) was the most sensitive cell line to all tested compounds displaying GI₅₀ from 0.116 to 0.227 μM, TGI from 0.275 to 0.592 μM and LC₅₀ at sub-micromolar concentration against almost of the tested compounds. Compounds 14e-h and 14m-p were assayed using TOP1-mediated DNA cleavage assay to evaluate their ability to stabilize TOP1ccs resulting in cancer cell death. The morpholino analogs 14h and 14p exhibited moderate TOP1 inhibitory activity compared to 1 μM camptothecin suggesting their use as lead compounds that can be optimized for the development of more potent anticancer agents with potential TOP1 inhibitory activity. Finally, Swiss ADME online web tool predicted that compounds 14h and 14p possessed good oral bioavailability and druglikeness characteristics.

Full text of DOI:10.1016/j.ejmech.2021.113261

European Journal of Medicinal Chemistry 215 (2021) 113261
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of 4-alkoxy-2-aryl-6,7-dimethoxyquinolines as a new class
of topoisomerase I inhibitors endowed with potent in vitro anticancer
activity
, b,
Mostafa M. Elbadawi a *, Wagdy M. Eldehna b, Wenjie Wang c, Keli K. Agama c,
,
**
Yves Pommier c, Manabu Abe a
a Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt
c Developmental Therapeutics Branch & Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD,
USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In our attempt to develop potential anticancer agents targeting Topoisomerase I (TOP1), two novel series
of 4-alkoxy-2-arylquinolines 14a-p and 19a-c were designed and synthesized based on structure activity
relationships of the reported TOP1 inhibitors and structural features required for stabilization of TOP1-
DNA cleavage complexes (TOP1ccs). The in vitro anticancer activity of these two series of compounds was
evaluated at one dose level using NCI-60 cancer cell lines panel. Compounds 14e-h and 14m-p, with p-
substituted phenyl at C2 and propyl linker at C4, were the most potent and were selected for assay at five
doses level in which they exhibited potent anticancer activity at sub-micromolar level against diverse
Received 10 October 2020
Received in revised form
11 January 2021
Accepted 28 January 2021
Available online 9 February 2021
Keywords:
Regioselectivity
Synthesis
Quinoline
Anticancer
cancer cell lines. Compound 14m was the most potent with full panel GI50 MG-MID 1.26
sensitive cancers were colon cancer, leukemia and melanoma with GI50 MG-MID 0.875, 0.904 and
0.926 M, respectively. Melanoma (LOX IMVI) was the most sensitive cell line to all tested compounds
displaying GI50 from 0.116 to 0.227 M, TGI from 0.275 to 0.592 M and LC50 at sub-micromolar con-
mM and the most
m
m
m
Topoisomerase I
centration against almost of the tested compounds. Compounds 14e-h and 14m-p were assayed using
TOP1-mediated DNA cleavage assay to evaluate their ability to stabilize TOP1ccs resulting in cancer cell
death. The morpholino analogs 14h and 14p exhibited moderate TOP1 inhibitory activity compared to
1 mM camptothecin suggesting their use as lead compounds that can be optimized for the development
of more potent anticancer agents with potential TOP1 inhibitory activity. Finally, Swiss ADME online web
tool predicted that compounds 14h and 14p possessed good oral bioavailability and druglikeness
characteristics.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer statistics, 18.1 million new cases and 9.8 million cancer
deaths were estimated. Lung cancer is the most common cancer
Worldwide, cancer is considered as one of the leading health
complications with remarkable increases in the number of patients
annually, which is expected to rise to 21.6 million cases in 2023
compared with 14.1 million in 2012 [1,2]. Based on 2018 global
diagnosed representing 11.6% of all cases and is the leading cause of
cancer death with a mortality rate of 18.4%, followed by breast
cancer with incidence 11.6% and mortality 6.6%, then colorectal
cancer with incidence and mortality 10.2% and 9.2%, respectively.
The incidence and mortality of cancer are fast growing and cancer is
expected to be the leading cause of death in the 21st century [3].
Consequently, tremendous efforts are being accomplished to
develop potent anticancer agents. Among these efforts, the syn-
thesis of compounds with different scaffolds can inhibit potential
chemotherapeutic targets resulting in cancer cell death [4e6].
Quinoline is one of the most privileged scaffolds in drug
* Corresponding author. Department of Chemistry, Graduate School of Science,
Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-
8526, Japan.
** Corresponding author.
E-mail addresses: mostafa_elbadawi@pharm.kfs.edu.eg (M.M. Elbadawi),
mabe@hiroshima-u.ac.jp (M. Abe).
https://doi.org/10.1016/j.ejmech.2021.113261
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
discovery playing a prominent role in medicinal chemistry and is
considered as the most ubiquitous heterocyclic motif with versatile
medicinal applications [7]. Quinoline derivatives were synthesized
and investigated in different applications including antimalarial,
antibacterial and anticancer activity [8e11]. Strikingly, quinoline
scaffold plays a crucial role in anticancer drug discovery and its
derivatives have demonstrated exquisite results as anticancer
candidates through numerous mechanisms of action including DNA
intercalation, inhibition of tubulin polymerization, DNA repair and
angiogenesis, in addition to inhibition of different enzymes that are
critically involved in the process of cancer growth [8,12e14].
Within these prominent enzymes, quinoline derivatives were
found to be inhibitors of EGFR [15e17], VEGFR [12], carbonic
anhydrase enzyme [18], Pim-1 kinase [19], histone deacetylase [20],
c-Met factor [21] and preferentially topoisomerase I enzyme
[22e24].
Topoisomerase I (TOP1) is a ubiquitous nuclear enzyme and is
essential in different cellular processes like replication and tran-
scription. TOP1 relaxes the supercoiled DNA by nicking a single
DNA strand through transesterification reaction using Tyr723 in the
TOP1 active site which attacks DNA at 30-phosphodiester end
forming 30-phosphotyrosine covalent linkage allowing the cleaved
strand to freely rotate around the intact one solving all topological
problems of DNA resulting from replication and transcription. After
DNA relaxation, the 50-hydroxyl end of the broken DNA strand will
reverse the 30-phosphotyrosyl linkage resulting in DNA rejoining
and liberation of TOP1. Normally, the binary TOP1-DNA cleavage
complexes (TOP1ccs) are transient and not detectable, since the
rejoining process is favored [25e30]. Rapidly dividing cancer cells
have overexpressed levels of TOP1 and compromised DNA repair
which demonstrate the increased sensitivity of cancer cells to TOP1
inhibitors. So, TOP1 is considered an effective and validated
chemotherapeutic target for cancer treatment [31e34].
TOP1 inhibitors reversibly bind at the interface of TOP1ccs by
stacking hydrophobic interactions with the base pairs flanking the
TOP1-mediated cleavage site, besides the hydrogen bonding with
TOP1 residues. As a result, TOP1 inhibitors slow down the rejoining
process of the scissile DNA, causing replication fork and tran-
scription complex collision with ternary TOP1-drug-DNA com-
plexes resulting in irreversible TOP1ccs which lead to double strand
breaks and cell death. Hence, TOP1 inhibitors that stabilize TOP1ccs
are known as TOP1 poisons [27,35]. TOP1 poisons are among the
most commonly used and effective anticancer agents which are
eligible as targeted therapies due to their exquisite selectivity [28].
Camptothecin 1 and its FDA approved derivatives; topotecan 2
and irinotecan 3 (Fig. 1) are TOP1 poisons [33,36]. Unfortunately,
the clinical use of topotecan 2 and irinotecan 3 as anticancer drugs
has been hampered due to several drawbacks including metabolic
instability due to hydrolysis of the essential lactone ring at physi-
ological pH, dose-limiting toxicity with myelosuppression and se-
vere diarrhea, poor water solubility and drug efflux-induced
clinical resistance. Therefore, enormous efforts have been estab-
lished to develop better TOP1 poisons [5,37,38]. Meanwhile, it has
been challenging to overcome camptothecins (CPTs) metabolic
instability by simple semisynthetic modifications as the lactone
ring is essential for TOP1ccs binding interactions. Subsequently,
indenoisoquinolines were developed as non-CPT TOP1 poisons by
NCI through screening of drug database for compounds with
cytotoxicity profiles correlated with CPTs. These indenoisoquino-
lines offer diverse advantages over CPTs including chemical sta-
bility, more stable ternary TOP1ccs, undetectable drug efflux-
mediated resistance and targeting TOP1 at different genomic sites
[28]. Interestingly, some of indenoisoquinolines succeeded to enter
phase I clinical trials for treatment of lymphomas and solid tumors
like indotecan (LMP400) 4, indimitecan (LMP776) 5, LMP744 6.
Moreover, dibenzonaphthyridinones Genz-644282 7 and ARC-1118
have good TOP1-targeting activity as the indenoisoquinolines and
are well tolerated in phase I clinical trials (Fig. 1) [28,32,38,39] and
in PDX models [40]. There are some evidences that TOP1 inhibitors
can target specific cancer cell genes more selectively than others.
Thus, extensive efforts have been made to develop novel TOP1 in-
hibitors with different scaffolds based on the structural re-
quirements of the reported TOP1 poisons aiming to discover
promising anticancer agents with better activity and reduced side
effects [24,38,40e44].
Based on those previous findings, we herein report the rational
design and regioselective synthesis of two series of 4-alkoxy-2-
arylquinolines 14a-p and 19a-c as potential anticancer agents
that can trap TOP1ccs. These lead compounds can be optimized to
develop a novel class of potent TOP1 poisons. The design strategy is
depicted in Fig. 2. It is based on the structural features of the re-
ported CPTs and non-CPT TOP1 poisons 1e8 (Fig. 1). From the
crystal structures of TOP1 poisons ternary complexes, it was
revealed that the polycyclic flat planner ring structure is essential to
intercalate and stack on the base pairs at the TOP1-induced
cleavage site, stabilizing the ternary complex and hindering DNA
rejoining. In addition, a hydrogen bond acceptor to project into the
minor groove and form hydrogen bonding with the sole amino acid
residue in this site; Arg364 [26]. For our design, we selected
quinoline core mimicking CPTs 1e3, Genz-644282 7 and ARC-111 8.
For substitutions, we anticipated that the presence of aryl substit-
uent at C2 of quinoline would impart the required polycyclic
structure suitable for the intercalation. Furthermore, N1 or O at C4
may be positioned to form a hydrogen bond with Arg364. Also, it
was found that the alkyl amino side chain with heteroatom found
in indenoisoquinolines 4e6 and dibenzonaphthyridinones 7, 8
serve as hydrogen bond acceptor with Asn352 or Glu356 located in
the major groove of the ternary complex [24]. Hence, we assumed
that the alkyl amino incorporated at C4 position of 2-arylquinoline
will project towards the residues in the major groove acting as a
hydrogen bond acceptor. Moreover, we conserved the two methoxy
groups at C6 and C7 like TOP1 poisons 4e6, which are reported to
hydrogen bond with Asn722 in one of the binding pockets of the
major groove [27]. In order to study the impact of substitution
pattern on the anticancer activity and TOP1 inhibitory activity, we
introduced alkyl chain with different length and different hetero-
cyclic amines at C4, in addition to different aryl substituents at C2
position.
The designed compounds were synthesized and evaluated for
in vitro anticancer activity according to NCI-60 cancer cell lines
screening protocol, then the most promising anticancer agents
were evaluated for TOP1 inhibitory activity using TOP1-mediated
DNA cleavage assays. From this study, we obtained several com-
pounds with promising anticancer activity 14e-h and 14m-p and
some of them 14h and 14p stabilized TOP1ccs. To the best of our
knowledge, this is the first study that reports on 4-alkoxy-2-
arylquinolines as topoisomerase I inhibitors. Thus, in our future
study we will optimize these TOP1 poisons lead compounds to
afford more potent candidates.
2. Results and discussion
2.1. Chemistry
The synthetic routes used for preparation of the two series of
target 4-alkoxy-2-arylquinolines 14a-p and 19a-c are depicted in
Schemes 1 and 2, respectively. The 2-aminoacetophenone deriva-
tive 9 was converted to the benzoyl derivatives 11a, b in excellent
yields (81e85%) by reaction with the appropriate benzoyl chloride
10a, b in the presence of Et3N as a base in dry THF at 0 ꢀC followed
2

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 1. TOP1 inhibitors; camptothecins (1e3) and in clinical trials (4e8).
by stirring at room temperature [45,46]. Then, the benzoyl de-
rivatives 11a, b were cyclized under nitrogen using aldol conden-
sation reaction in refluxing dry dioxane and sodium hydroxide to
afford the quinolones 12a, b in 91% and 90% yields, respectively
[47]. The O-alkylated regioisomers 13a-d were prepared by stirring
of quinolones 12a or b with excess KOH and catalytic amount of KI
in dry DMF for 2 h, then the produced nucleophile was reacted with
Finally, the key intermediates 13a-d were converted to the
target 4-alkoxy-2-arylquinolines 14a-p in good to excellent yields
(63e92%) by nucleophilic substitution reaction with different
alicyclic amines in dry DMF in the presence of anhydrous K2CO3 as a
base at 90 ꢀC [48]. Similarly, syntheses of the second series of the
target 4-alkoxy-2-arylquinolines 19a-c (Scheme 2) were accom-
plished using the same procedures for the first series. The struc-
tures of all synthesized compounds were confirmed using 1D and
2D NMR (see supplementary data for 2D NMR), FTIR spectroscopy,
high resolution mass spectrometry (HRMS) and single crystal X-ray
crystallography for one of the final compounds 14b.
the
respective
1-bromo-2-chloroethane
or
1-bromo-3-
chloropropane for 24 h at room temperature to obtain the key in-
termediates 13a-d in good to excellent yields (78e89%) [48].
The quinolones 12a, b are ambident anions and O- versus N-
regioselectivity differs within the same scaffold based on temper-
ature, substituents, solvent and nature of the electrophiles, so we
had to confirm O-regioselectivity of the synthesized key in-
termediates 13a-d to be more confident in understanding the
structure activity relationships of the final products especially, it
was reported that regio-inaccurate structures are present in com-
pound collections even for starting materials available to purchase
from vendors [49e51]. One dimensional (1D) NMR alone is not
enough for this confirmation, so we utilized FTIR spectroscopy
besides the reported NMR-based strategy which depends on using
two dimensional (2D) NMR: 1He13C HSQC, 1He13C HMBC and
1He1H NOESY [49]. 2D NMR spectra for the representative key
intermediate 13a are illustrated in Fig. 3. Regarding FTIR spectrum,
carbonyl stretching band appeared at 1683 cmꢁ1 for the quinolone
starting material 12a, while this band disappeared in 13a after
alkylation which confirmed O-alkylation. Furthermore, HSQC
demonstrated H9/C9 crosspeak that provided the chemical shifts
for C9 and H9 neighboring to the alkylation site, in addition H9/C4
correlation was observed in HMBC spectrum which concluded that
H9 must be within three atoms of C4 and this correlation pattern
impossible in N-alkyl analog. Moreover, the data from NOESY dis-
played a crosspeak between H3 and H9 which means that H3 is
close to H9. Consequently, we can confirm that the synthesized key
intermediates 13a-d are O-regioisomers.
2.2. Single crystal X-ray crystallography
For more certainty of structure elucidation, a single crystal X-ray
crystallography for one of the target 4-alkoxy-2-arylquinolines 14b
was conducted, Fig. 4. Crystals of compound 14b were obtained by
slow evaporation of its solution in chloroform. The crystals were
measured using Bruker APEX-II D8 diffractometer with mono-
chromatic Mo K
a
diffraction radiation type (
l
¼ 0.71073 Å) at 90 K
as ambient temperature. Structure refinement was conducted by
SHELXL. Crystallographic data for compound 14b have been
deposited at Cambridge Crystallographic Data Center and given the
deposition number: CCDC 2023795. The crystallographic data and
refinement are available in the supplementary data. Regarding the
X-ray structure of compound 14b, we can assert the O-regiose-
lectivity of the target 4-alkoxy-2-arylquinolines under the condi-
tions were utilized, thus we can attain more confident study of
structure activity relationships.
2.3. In vitro anticancer activity
The structures for the synthesized 4-alkoxy-2-arylquinolines
14a-p and 19a-c were submitted to the Developmental Therapeu-
tics Program (DTP) of the National Cancer Institute (NCI) (www.dtp.
3

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 2. Rational design for the target compounds generated from the reported TOP1 inhibitors.
nci.nih.gov). All compounds were selected for NCI-60 human can-
cer cell lines screening for their in vitro anticancer activity [52,53].
Interestingly, 4-alkoxy-2-arylquinolines with propyl linker at C4
and p-chlorophenyl at C2 14e-h exhibited significant improvement
in the growth inhibitory activity with GI% ¼ 62e65% compared to
14a-d with ethyl linker. Similarly, the replacement of chloro in
compounds 14e-h with CF3 substituent in 14m-p enhanced the
anticancer activity affording the most potent counterparts in these
series. Conversely, the biosteric replacement of p-substituted
phenyl at C2 with 2-furyl, 2-thienyl and 3-thienyl in 19a-c signifi-
cantly reduced the anticancer activity demonstrating GI% ¼ 46, 20
and 32%, respectively. Consequently, utilizing a propyl linker at C4
and appending a p-substituted phenyl moiety at C2 are suggested
to be crucial elements for the anticancer activity for the herein
reported 4-alkoxy-2-arylquinolines.
2.3.1. Preliminary cytotoxicity screening at 10
Firstly, the selected 4-alkoxy-2-arylquinolines 14a-p and 19a-c
were screened for anticancer activity at a dose of 10 M against a
mM concentration
m
panel of 59 cancer cell lines representing nine cancer types: leu-
kemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and
breast cancers. The sulforhodamine B (SRB) assay was used to
determine the growth and cell viability [54]. The mean % growth
inhibition values (GI%) for compounds 14a-p and 19a-c against NCI-
59 cancer cell lines full panel are depicted in Fig. 5.
Concerning the cytotoxicity screening at 10
mM, compounds
14a-d bearing ethyl linker at C4 position and p-chlorophenyl at C2
position demonstrated low anticancer activity with GI% ¼ 3e17%.
The replacement of chloro in 14a-d with CF3 substituent in 14i-l
slightly enhanced the anticancer activity displaying GI% ¼ 13e34%.
2.3.2. In vitro cytotoxicity evaluation against NCI-59 cancer cell
lines panel at five doses level
Based on the preliminary screening at 10
mM, propyl linker-
4

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Scheme 1. Synthesis of target 4-alkoxy-2-arylquinolines 14a-p; Reagents and conditions: (i) Et3N, THF, 0 ꢀC then rt, overnight; (ii) NaOH, dry dioxane, 110 ꢀC under N2, 4 h; (iii)
KOH, KI, alkyl halide, dry DMF, rt, 24 h; (iv) KI, anhydrous K2CO3, amine, dry DMF, reflux, 12 h.
Scheme 2. Synthesis of target 4-alkoxy-2-arylquinolines 19a-c; Reagents and conditions: (i) Et3N, THF, 0 ꢀC then rt, overnight; (ii) NaOH, dry dioxane, 110 ꢀC under N2, 4 h; (iii)
KOH, KI, 1-bromo-3-chloropropane, dry DMF, rt, 24 h; (iv) KI, anhydrous K2CO3, 4-hydroxypiperidine, dry DMF, reflux, 12 h.
5

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 3. 2D NMR spectra of compound 13a: 1He13C HSQC, 1He13C HMBC and 1He1H NOESY.
Fig. 4. The ORTEP diagram of the final X-ray structure of compound 14b.
bearing 2-arylquinolines 14e-h and 14m-p exhibited significant
growth inhibition against various cancer cell lines representing
different cancer types. Therefore, these compounds were selected
lines. In this regard, the most sensitive cell lines were leukemia
(SR), non-small cell lung cancer (NCIeH226), colon cancer
(COLO205), CNS cancer (SF-295), melanoma (LOX IMVI, SK-MEL-5),
ovarian cancer (NCI/ADR-RES), renal cancer (CAKI-1) and breast
cancer (T-47D) with GI50 values ¼ 0.133, 0.343, 0.401, 0.328, 0.116,
by NCI for further examination at five doses level (0.01e100
Three dose response parameters were calculated: GI50, TGI and
LC50
mM).
.
0.247, 0.458, 0.188 and 0.472 mM, respectively (Table 1). Concerning
Regarding the whole panel of cancer cell lines, compound 14m
displayed the best potent anticancer activity against the entire
the sensitivity against all cancer cell lines, compound 14m
possessed significant antiproliferative activity with full panel GI50
panel of cancer cell lines with GI50 values range 0.116e6.77
mM
MG-MID 1.26
mM and cancer subpanels GI50 MG-MID range
exhibiting GI50 at sub-micromolar concentration over 28 tumor cell
0.875e1.70 M possessing superior activity at sub-micromolar level
m
6

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 5. Mean % growth inhibition of compounds 14a-p and 19a-c against NCI 59 cancer cell line panel.
against colon cancer, then leukemia and melanoma cells with
subpanel GI50 MG-MID values 0.875, 0.904 and 0.926 M, respec-
0.303
mM, respectively (Table 1). In the context of activity over the
m
entire panel cell lines, compound 14e exerted remarkable anti-
tively (Table 2). In addition, compound 14m displayed significant
cancer potential with full panel GI50 MG-MID 1.65
m
M and cancer
cytostatic effect over different cell lines with full panel TGI MG-
subpanels GI50 MG-MID ranged from 1.36 to 2.14
mM with values
MID ¼ 11.61
cell lines, among them melanoma (LOX IMVI and SK-MEL-5) with
TGI 0.275 and 0.645 M, respectively (see supplementary data for
details of TGI values). Besides, it was found that 14m proved to have
a lethal effect against melanoma (LOX IMVI) with LC50 0.656
(see supplementary data for details of LC50 values) and melanoma
LC50 MG-MID 23.7 M (Table 3).
m
M (Table 3) and TGI values ꢂ 5
m
M against 18 cancer
1.36 and 1.37 mM for the most susceptible breast and colon cancers,
respectively (Table 2). Furthermore, compound 14e established
comparable cytostatic influence for various cell lines with full panel
m
TGI MG-MID ¼ 8.71
cancer cell lines, preferably melanoma (LOX IMVI) with
TGI ¼ 0.446 M (supplementary data). Moreover, compound 14e
exerted lethal impact against melanoma (LOX IMVI) with
mM (Table 3) and TGI values ꢂ 5
mM over 19
mM
m
m
Similarly, compound 14o exhibited significant anticancer po-
tential in the second order showing GI50 range from 0.162 to
LC50 ¼ 1.12
m
M (supplementary data) and melanoma LC50 MG-MID
equals 14.6 mM (Table 3).
8.70
m
M and values at sub-micromolar concentration against 24
In a similar fashion, compounds 14f, g and h have exerted good
cancer cell lines. From these cell lines, the most sensitive were
leukemia (SR), non-small cell lung cancer (NCIeH226), colon can-
cer (HCT-15 and COLO205), melanoma (LOX IMVI, SK-MEL-5 and
M14), renal cancer (CAKI-1) and breast cancer (T-47D) demon-
strating GI50 values ¼ 0.193, 0.370, 0.399, 0.406, 0.162, 0.325, 0.327,
anticancer impact over the entire panel of cancer cells with GI50
ranges 0.227e4.27, 0.189e3.85 and 0.217e3.87
mM, respectively,
showing GI50 values at sub-micromolar level for 11, 13 and 11
cancer cell lines, respectively. For compound 14f, leukemia (SR),
melanoma (LOX IMVI) and renal cancer (CAKI-1) were the most
0.272 and 0.454
mM, respectively (Table 1). In regard of the influ-
sensitive with GI50 ¼ 0.373, 0.227 and 0.416
the same sensitivity has been noticed for compounds 14g and 14h
exhibiting GI50 values 0.373, 0.189 and 0.287 M for 14g, while
GI50 ¼ 0.414, 0.217 and 0.305 M for 14h, respectively. In addition,
melanoma (M14) and colon cancer (HT29) showed preferred
M, respectively
mM, respectively, and
ence on the whole panel of cancer cell lines, compound 14o showed
considerable anti-proliferative activity displaying full panel GI50
m
MG-MID 1.57
2.13 M. Breast cancer and melanoma cells were the most affected
ones with GI50 MG-MID 1.13 and 1.19 M, respectively (Table 2). In
the concern of cytostatic potential, compound 14o has established
advantageous effect with full panel TGI MG-MID ¼ 11.58
(Table 3) and TGI values ꢂ 5 M against 14 tumor cell lines and the
most sensitive ones were melanoma (LOX IMVI and SK-MEL-5)
with TGI 0.355 and 1.20 M, respectively (supplementary data).
Also, compound 14o had good lethal activity on melanoma (LOX
IMVI) expressed as LC50 ¼ 0.779 M (supplementary data) and
melanoma LC50 MG-MID ¼ 19.9 M (Table 3).
In a similar manner, compound 14e demonstrated excellent
anticancer effect in the third order with GI50 range 0.191e3.36
mM with subpanels GI50 MG-MID from 1.13 to
m
m
m
sensitivity to 14g with GI50 ¼ 0.412 and 0.492
m
(Table 1). Concerning the sensitivity against all cancer cell lines,
mM
compounds 14f, g and h had favorable anticancer effectiveness
m
with full panel GI50 MG-MID ¼ 1.99, 1.83 and > 3.29
subpanels GI50 MG-MID ranges 1.50e2.51, 1.54e2.30 and 1.40 e
>13.90 M, respectively (Table 2). Melanoma and colon cancer
were the most sensitive to 14f and 14g with GI50 MG-MID 1.50 and
1.65 M for 14f and 1.54 and 1.57 M for 14g, respectively, while
melanoma and renal cancer were the most susceptible for 14h with
GI50 MG-MID 1.40 and 1.66 M, respectively (Table 2). Moreover,
compounds 14f, g and h have exerted good cytostatic action against
diverse cancer cell lines with TGI ꢂ5 M over 18, 16 and 7 cancer
cell lines, respectively, demonstrating melanoma (LOX IMVI) as the
M,
mM and tumor
m
m
m
m
m
m
m
m
M
against the whole panel of tumor cell lines showing GI50 values at
sub-micromolar level over 16 different cell lines. It was established
that leukemia (SR), colon cancer (HT29, COLO205), melanoma (LOX
IMVI and M14) and renal cancer (CAKI-1) were the most susceptible
cell lines with GI50 equal 0.251, 0.454, 0.459.0.191, 0.402 and
m
most affected cell line with TGI ¼ 0.589, 0.435 and 0.592
m
respectively (supplementary data). While, Compounds 14f, h dis-
played lethal impact on melanoma (LOX IMVI) with LC50 ¼ 2.20,
7

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Table 1
GI50 values of NCI in vitro cytotoxicity testing of compounds 14e-h and 14m-p at five doses level.a,b
.
Panel/cancer cell lines
Compounds
14e
14f
14g
14h
14m
14n
14o
14p
GI50 (mM)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
SR
2.14
2.72
1.06
1.50
0.251
2.65
4.27
1.63
1.80
0.373
2.79
NT
1.22
2.04
0.373
3.35
NT
2.71
2.85
0.414
1.08
1.84
0.475
0.994
0.133
3.26
13.9
1.79
3.27
0.274
1.83
2.91
0.578
1.70
0.193
2.79
9.58
1.18
2.51
0.242
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCIeH226
NCIeH23
NCIeH322 M
NCIeH460
NCIeH522
Colon Cancer
COLO205
HCC-2998
HCT-116
HCT-15
1.52
1.21
1.38
0.850
0.898
2.45
3.36
0.823
2.36
2.20
1.33
1.98
1.25
1.39
2.83
3.22
0.955
2.19
1.90
1.47
2.37
1.02
1.12
2.95
4.42
0.842
3.28
2.02
1.73
1.98
1.36
0.997
NT
>100
0.958
2.17
0.653
0.982
1.03
0.460
0.343
1.91
6.77
0.528
1.74
1.65
1.03
1.80
1.68
1.59
6.16
10.2
0.835
4.15
0.722
0.803
0.947
0.678
0.370
1.91
8.70
0.595
2.44
1.04
1.61
1.67
1.00
0.406
2.36
10.5
0.639
2.52
0.459
1.86
0.699
0.567
0.454
2.46
0.532
1.95
1.44
0.763
0.700
3.13
0.582
2.34
0.828
0.604
0.492
3.07
0.620
1.97
1.59
0.754
0.610
3.87
0.401
1.45
0.456
0.416
0.446
1.34
0.456
4.07
0.671
0.667
0.486
6.87
0.406
1.28
0.429
0.399
0.415
2.92
0.529
1.41
0.583
0.520
0.540
3.42
HT29
KM12
SW-620
3.12
3.05
3.09
3.57
1.62
3.53
2.91
3.51
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
2.53
1.22
2.44
3.14
1.73
1.79
3.36
1.66
2.28
3.19
1.76
2.81
3.20
1.21
2.57
2.98
1.98
1.89
NT
2.39
0.328
1.87
2.77
1.85
1.05
5.76
1.61
5.75
10.5
2.58
2.12
3.76
0.530
2.08
2.82
1.96
1.10
5.7
1.08
2.99
NT
2.06
1.92
0.349
3.18
2.91
2.46
1.40
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786e0
0.191
1.68
0.402
2.72
2.16
1.48
0.961
1.96
2.03
0.227
1.89
0.591
2.05
2.07
1.49
1.27
2.06
1.90
0.189
1.74
0.412
2.44
2.75
1.45
1.10
1.92
1.94
0.217
2.01
0.545
1.90
NT
1.52
0.832
1.92
2.26
0.116
1.01
0.336
0.709
1.93
1.20
0.247
1.41
0.190
2.65
0.535
2.76
3.85
2.38
1.43
2.28
2.61
0.162
1.26
0.327
1.23
2.64
1.26
0.325
1.70
1.82
0.141
1.38
0.355
1.20
3.32
1.44
0.288
1.67
0.628
1.38
0.881
2.71
3.00
1.03
1.19
0.683
2.42
0.923
3.24
4.34
1.16
2.00
0.925
3.17
0.838
3.23
3.85
1.16
1.77
0.898
NT
0.990
NT
NT
1.58
2.15
1.15
3.00
0.592
2.15
3.71
0.803
0.806
0.458
2.55
0.900
6.10
9.14
3.14
1.66
0.847
10.6
0.651
2.44
2.91
0.710
0.734
0.522
2.30
0.830
3.73
10.3
1.92
1.95
0.640
5.04
2.78
2.19
1.93
0.303
1.17
0.914
2.33
1.72
3.31
1.52
2.43
0.416
1.22
1.26
2.14
1.72
2.62
1.40
2.45
0.287
1.08
0.946
3.73
1.87
1.94
1.59
3.64
0.305
1.12
1.30
NT
2.13
1.58
4.72
2.37
3.18
0.376
1.34
1.44
11.5
2.71
2.17
1.85
1.20
0.272
1.09
0.668
7.80
2.00
1.76
1.51
2.01
0.167
0.706
0.675
13.9
A498
ACHN
CAKI-1
RXF 393
SN 12C
TK-10
UO-31
0.812
0.188
1.13
0.673
3.24
1.78
2.02
1.77
Prostate Cancer
PC-3
DU-145
1.76
2.15
2.02
2.78
1.74
2.54
2.63
NT
1.07
1.37
3.66
4.96
1.34
1.84
2.19
10.1
Breast Cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
1.33
0.907
1.89
1.77
1.02
1.28
1.60
0.665
1.91
2.79
2.26
1.56
1.56
0.666
2.11
1.43
1.31
1.39
2.49
1.01
2.40
2.59
1.96
1.83
0.976
0.565
1.74
1.99
0.472
0.885
2.57
0.919
3.75
6.59
2.97
1.63
1.16
0.536
2.15
1.48
0.454
1.03
1.55
0.498
2.25
5.00
0.654
1.24
MDA-MB-468
a
Bold figure indicates GI50 value at sub-micromolar level.
NT ¼ not tested.
b
8

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Table 2
GI50 mean-graph midpoint (MG-MID)a of subpanel cancer cell lines for compounds 14e-h and 14m-p.
Subpanel cancer cell lines
Compounds
14e
14f
14g
14h
GI50 MG-MID (
14m
M)
14n
14o
14p
m
Leukemia
Non-small cell lung cancer
Colon cancer
CNS cancer
Melanoma
1.53
1.65
1.37
2.14
1.50
1.70
1.66
1.95
1.36
1.65
2.14
1.92
1.65
2.51
1.50
2.25
1.75
2.4
1.60
2.15
1.57
2.30
1.54
1.95
1.79
2.14
1.41
1.83
2.33
>13.90
1.85
2.01
1.40
1.77
1.66
2.63
2.04
>3.29
0.904b
1.60
0.875
1.70
0.926
1.58
1.47
1.22
1.10
1.26
4.49
3.23
2.39
4.72
2.07
4.26
3.45
4.31
3.07
3.59
1.44
1.90
1.25
2.04
1.19
1.46
2.13
1.59
1.13
1.57
3.26
2.41
1.50
2.66
1.15
3.48
2.81
6.14
1.86
2.81
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
1.79
1.99
Full panel GI50 MG-MIDc
a
Median value calculated based on the data from NCI in vitro cytotoxicity screening for each cancer type cell lines.
GI50 full panel mean-graph midpoint (MG-MID) is the average of GI50 values against all cancer cell lines.
Bold figure indicates MG-MID value at sub-micromolar level.
b
c
cancer cell lines with TGI ꢂ5
mM against 4 and 13 tumor cell lines,
respectively. Melanoma (LOX IMVI) was the most sensitive cell line
Table 3
TGI and LC50 mean-graph midpoints (MG-MID)a in
mM for the most potent com-
to both 14n and p with TGI ¼ 0.447 and 0.324
mM, respectively
pounds 14e, m and o.
(supplementary data). In addition, compounds 14n and p displayed
Subpanel cancer cell lines
Compounds
14m
lethal effect against melanoma (LOX IMVI) with LC50 2.13 and
0.747 mM, respectively (supplementary data).
Generally, compound 14m bearing pyrrolidine moiety and p-CF3
phenyl ring, was the most potent analog against all cancer cell lines
14e
LC50
32
14o
TGI LC50
TGI
TGI
LC50
Leukemia
4.99
6.17
81.6
10.29 74.8
13.79 50.6
10.29 40.8
11.87 38.8
with full panel GI50 MG-MID 1.26
cers were colon cancer, leukemia and melanoma with GI50 MG-MID
0.875, 0.904 and 0.926 M, respectively (Table 2). It is noteworthy
mM and the most sensitive can-
Non-small cell lung cancer 10.41 39.7
Colon cancer
CNS cancer
12.72 57.9
8.07 50.4
11.71 44.9
8.31
39.1
10.11 33.5
m
that all the tested compounds 14e-h and 14m-p exhibited exquisite
growth inhibitory influence on specific cell lines at sub-micromolar
level; leukemia (SR), non-small cell lung cancer (NCIeH460), colon
cancer (COLO205, HCT-15 and HT29), melanoma (LOX IMVI and
M14), ovarian cancer (IGROV1) and renal cancer (CAKI-1) (Table 1).
Melanoma (LOX IMVI) was the most sensitive cell line to all com-
pounds 14e-h and 14m-p showing GI50 values of 0.191, 0.227, 0.189,
Melanoma
4.26
14.8
7.66
12.05 39.6
5.80
14.6
55.1
28.9
4.08
28.1
10.22 40.1
16.75 89.5
23.7
81
4.14
19.67 65.5
11.03 35
13.85 42.2
9.29
19.9
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Full panel MG-MIDb
29.6
6.80
40.8
43
8.71
34.67 11.61 56.65 11.58 45.62
a
Median value calculated based on the data from NCI in vitro cytotoxicity
screening for each cancer type cell lines.
b
0.217, 0.116, 0.190, 0.162, 0.141 mM, respectively (Table 1). Mean-
while, TGI values were the best for melanoma (LOX IMVI) against all
Full panel mean-graph midpoint (MG-MID) is the average of TGI or LC50 values
against all cancer cell lines.
compounds 14e-h and 14m-p; 0.446.0.589, 0.435, 0.592, 0.275,
0.447, 0.355, 0.324
melanoma (LOX IMVI) was the most susceptible to the lethal effect
of 14e, f, h and 14m-p displaying LC50 values 1.12, 2.20, 4.46, 0.656,
2.13, 0.779, 0.747 mM, respectively (supplementary data).
With regards to the structure activity relationships, except for
14f, grafting a p-CF3 substituent at the 2-phenyl ring resulted in a
significant enhancement of the overall anticancer activity
compared to the p-Cl substituent. For chloro analogs 14e-h, incor-
poration of pyrrolidine moiety was the most advantageous for
anticancer activity, then piperidine, then 4-hydroxypipeidine, then
morpholine. While for CF3 analogs 14m-p, pyrrolidine was better
for anticancer effect, then piperidine, then morpholine, then 4-
hydroxypiperidine (Fig. 6).
mM, respectively (supplementary data). Also,
4.46
m
M, respectively, compound 14g had lethal activity against
melanoma (SK-MEL-5) with LC50 ¼ 4.87
mM (supplementary data).
Regarding the anticancer efficacy of compounds 14n and 14p
against all cancer cell lines, they exhibited a good inhibitory impact
on the cell growth at sub-micromolar level against 12 and 9 cancer
cell lines with GI50 ranges 0.190e13.9 and 0.141e13.9 mM, respec-
tively (Table 1). For compound 14n, it was found that leukemia (SR),
colon cancer (COLO205 and HT29), melanoma (LOX IMVI) and renal
cancer (CAKI-1) were the most sensitive to its anticancer activity
with GI50 ¼ 0.274, 0.456, 0.486, 0.190 and 0.376
mM, respectively.
While for compound 14p, leukemia (SR), non-small cell lung cancer
(NCIeH226), CNS cancer (SF-295), melanoma (LOXIMVI, M14 and
SK-MEL-5), renal cancer (CAKI-1) and breast cancer (MDA-MB-231/
ATCC) were the most sensitive with GI50 ¼ 0.242, 0.406, 0.349,
2.4. Topoisomerase I-mediated DNA cleavage assay
0.141, 0.355, 0.288, 0.167, 0.498
mM, respectively (Table 1).
Furthermore, compound 14n and 14p had full panel GI50 MG-MID
3.59 and 2.81 M with tumor subpanels GI50 MG-MID ranges
2.07e4.72 and 1.13e6.14 M, respectively (Table 2). Notably, mel-
anoma was the most affected cancer to compound 14n with sub-
panel GI50 MG-MID ¼ 2.07 M, whereas, breast cancer and
melanoma were the most sensitive to compound 14p with GI50
MG-MID ¼ 1.13 and 1.19 M, respectively (Table 2). Moreover,
compounds 14n and p had good cytostatic activity against various
TOP1 poisoning activity of the most potent cytotoxic 4-alkoxy-
2-arylquinolines 14e-h and 14m-p was investigated using TOP1-
mediated DNA cleavage assay which scores the TOP1 poisoning
m
m
activity of the tested compounds compared to 1
mM camptothecin
m
[28]. The tested compounds at 0.1, 1, 10 and 100
mM were incubated
with TOP1 enzyme and 30-[32P]-labeled 117-bp DNA oligonucleo-
tide [29]. TOP1 poisons specifically bind to and trap TOP1ccs
resulting in stabilization of TOP1ccs and DNA cleavage. The
m
9

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 6. Summary of structure activity relationships for anticancer activity.
compound-induced trapped TOP1ccs were visualized using gel
electrophoresis displaying specific cleavage patterns for each
compound in which each gel band represents specific TOP1cc
trapped by the drug. Finally, visual comparison of the lanes pro-
duced by the tested compounds and that produced by 1 mM CPT
was utilized to give semiquantitative scores to indicate the activity
druglikeness and pharmacokinetic properties of the most potent 4-
alkoxy-2-arylquinolines 14e-h and 14m-p to verify that they are
promising candidates from the aspect of pharmacokinetics [57].
The submitted compounds 14e-h and 14m-p, except 14o, were
predicted to display suitable physicochemical and pharmacokinetic
properties; logPo/w range 4.2e5.29, moderate water solubility, high
GI absorption. Despite, compound 14o had potent in vitro anti-
cancer activity, it was predicted to have poor pharmacokinetic
properties; logPo/w 5.56, poor water solubility and low GI absorp-
tion. While, the chloro analogs 14e-h were predicted to cross the
blood brain barrier (BBB), the CF3 analogs 14m-p were not
permeable to BBB, so no predicted CNS side effects (see supple-
mentary data for more details) [58].
For high GI absorption and good oral bioavailability for a com-
pound, the physicochemical properties should be in suitable
ranges. The radar charts in Fig. 9 displayed the oral bioavailability of
compounds 14h and 14p as representatives for the compounds in
these series. This chart has six axes for the six parameters required
for oral bioavailability; lipophilicity (LIPO), size (SIZE), polarity
(POLAR), solubility (INSOLU), saturation (INSATU) and flexibility
(FLEX) and the pink area in this plot represents the optimal ranges
for predicted good oral bioavailability [59,60]. In Fig. 9, the red lines
which represent compounds 14h and 14p are fully incorporated in
the pink area predicting good oral bioavailability (see supplemen-
tary data for the radar charts of the remaining compounds).
Moreover, SwissADME revealed that all tested compounds fulfilled
Lipinski’s rule as one of the major druglikeness characteristics
developed by Pfizer pharmaceutical company predicting that these
compounds with promising pharmacokinetic properties (see sup-
plementary data) [61].
of the tested compounds; 0 (no activity), þ (activity between 20
and 50%, the cleavage activity of 1
50 and 75%, the cleavage activity of 1
tween 75 and 100%, the cleavage activity of 1
(activity equipotent to or more potent than 1
m
M CPT), þþ (activity between
m
M CPT), þþþ (activity be-
m
M CPT) and þþþþ
mM CPT) [48,55,56].
From the chloro analogs 14e-h, compounds 14e-g exhibited
weak TOP1 poisoning activity (þ), while compound 14h displayed
moderate activity (þþ), Fig. 7. Regarding, CF3 analogs 14m-p,
compounds 14m, n displayed no activity (0) and compound 14o
revealed weak activity (þ), while compound 14p exhibited mod-
erate TOP1 inhibitory activity (þþ), Fig. 8. From all tested synthe-
sized compounds, compounds 14h, p bearing morpholine ring,
which was found in diverse TOP1 poisons as indotecan (LMP400) 4
in clinical trials, exhibited the best activity with score þþ demon-
strating that morpholine ring is preferred for TOP1 inhibitory
activity.
Compounds 14h, p exhibited good anticancer activity at sub-
micromolar level against various cancer cell lines and displayed
comparable GI50 ranges 0.217e3.87 and 0.141e13.9
mM, respec-
tively, in addition to the moderate TOP1 poisoning activity leading
to DNA cleavage and cancer cell death.
In summary, we studied the structural requirements for potent
anticancer activity and substituents preferred for TOP1 inhibitory
activity resulting in discovery of compounds 14h, p with novel
scaffold able to exhibit good anticancer effect and moderate TOP1
poisoning activity. Therefore, 14h and 14p can be considered as
TOP1 inhibitors lead compounds will be optimized in our future
study to attain more potential TOP1 inhibitors with potent anti-
cancer activity.
3. Conclusion
In summary, novel two series of 4-alkoxy-2-arylquinolines 14a-
p and 19a-c were designed and synthesized as anticancer agents
targeting TOP1 enzyme. The anticancer potential of all compounds
have been evaluated according to the protocol of NCI. Based on
these results, compound 14m was the most potent with full panel
2.5. Prediction of physicochemical and pharmacokinetic properties
SwissADME online web tool developed by the molecular
modeling group of Swiss Institute of Bioinformatics (SIB) has been
used to compute the physicochemical properties and predict the
GI50 MG-MID 1.26 mM affecting colon cancer, leukemia and mela-
noma at sub-micromolar level. Interestingly, compounds 14e-h and
14m-p showed good anticancer activity against nine cell lines of
10

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 7. (A) TOP1-mediated DNA cleavage induced by compounds 14e-h. From the left: lane 1, DNA only; lane 2, DNA þ TOP1; lane 3, CPT (1
mM); lane 4, LMP744 (1
mM); lanes 5e20,
compounds 14e-h at 0.1, 1, 10, 100
m
M. Arrows and numbers on the left represent the cleavage sites. (B) 30-[32P]-labeled 117-bp DNA oligonucleotide sequence with the positions of
TOP1 cleavage sites.
different cancers at sub-micromolar concentration. Then,
compound-induced TOP1-mediated DNA cleavage has been
assayed for the promising compounds 14e-h and 14m-p to explore
their ability to stabilize TOP1ccs. This study revealed that com-
pounds 14h and 14p moderately stabilized TOP1ccs compared to
4. Experimental
4.1. Chemistry
4.1.1. General
1
m
M CPT as the first 4-alkoxy-2-arylquinolines able to inhibit TOP1.
Melting points were determined using Yanaco melting point
device and were uncorrected. Infrared (IR) spectra were recorded
by Jasco FT/IR-6600 spectrophotometer as KBr disks and were
expressed in wavenumber (cmꢁ1). NMR spectra were measured
using Bruker Advance III HD at 400 MHz for 1H NMR, 100 MHz for
13C NMR and 376 MHz for 19F NMR in deuterated CDCl3, DMSO‑d6 or
MeOH-d4 using tetramethyl silane (TMS) as an internal standard.
Hence, compounds 14h and 14p were considered as lead com-
pounds which can be optimized to develop more potent anticancer
agents with potential TOP1 inhibitory activity. Finally, SwissADME
online web tool predicted that compounds 14h and 14p possessed
promising pharmacokinetics and druglikeness properties.
11

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 8. (A) TOP1-mediated DNA cleavage induced by compounds 14m-p. From the left: lane 1, DNA only; lane 2, DNA þ TOP1; lane 3, CPT (1
mM); lane 4, LMP744 (1
mM); lanes 5e20,
compounds 14m-p at 0.1, 1, 10, 100
m
M. Arrows and numbers on the left represent the cleavage sites. (B) 30-[32P]-labeled 117-bp DNA oligonucleotide sequence with the positions of
TOP1 cleavage sites.
Coupling constant values (J) were determined in Hertz (Hz) and
chemical shifts ( ) were expressed in ppm. High resolution mass
4.1.2. Synthesis of 1-(2-amino-4,5-dimethoxyphenyl)ethenone (9)
The 2-aminoacetophenone derivative 9 was synthesized ac-
cording to the reported experimental procedure [62,63].
Yellow solid, yield 70%, m.p. 100e102 ꢀC; 1H NMR (400 MHz,
d
spectra (HRMS) were measured with Thermo Fisher Scientific LTQ
Orbitrap XL spectrophotometer using electrospray ionization (ESI)
and were expressed as [MþH]þ or [MþNa]þ at Natural Science
Research and Development Center, Hiroshima University, Japan.
The reaction mixtures were monitored by thin layer chromatog-
raphy (TLC) using Merck silica gel 60F254 aluminum sheets. Column
CDCl3)
d (ppm): 2.53 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 6.12 (s, 1H, phenyl CH), 6.27 (br s, 2H, NH2), 7.12 (s, 1H,
phenyl CH).
chromatography was conducted using silica gel 60 N, 63e210 mm
4.1.3. General procedure for synthesis of the benzoyl derivatives
(11a, b)
Compound 9 (0.976 g, 5 mmol) was dissolved in dry THF (8 mL)
and Et3N (2 mL) and the mixture was cooled in ice bath. Then, a
that was purchased from Kanto Chemical Co. Inc. Unless otherwise
mentioned, all solvents and chemical reagents were commercially
available and were used without further purification.
12

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
Fig. 9. Radar charts for prediction of oral bioavailability generated by SwissADME web tool; the pink area represents the ideal values for oral bioavailability and the red lines
represent compounds (A) 14h and (B) 14p.
solution of the appropriate benzoyl chloride 10a, b (5.1 mmol) in
dry THF (2 mL) was added dropwise. After 30 min at 0 ꢀC, the re-
action mixture was stirred overnight at room temperature. After
reaction completion, the reaction mixture was poured into ice
water (50 mL). The precipitate separated was filtered off, washed
several times with water then methanol, dried under vacuum and
crystallized from acetone to afford compounds 11a, b as yellow
crystals.
temperature. Small amount of water and large amount of hexane
were added to the reaction mixture, then the flask was sonicated
for 2 min. The biphasic mixture was neutralized to pH 7 with 1 M
HCl. The separated solid was filtered, washed with water and dried
to afford 12a or b, respectively.
4.1.4.1. 2-(4-Chlorophenyl)-6,7-dimethoxyquinolin-4(1H)-one (12a).
Yellow solid, Yield 91%, m.p. > 250 ꢀC; IR (KBr,
y
cmꢁ1): 3400 (NH),
3088 (CH aromatic), 2963, 2940 (CH aliphatic), 1685 (C]O), 1507,
4.1.3.1. N-(2-Acetyl-4,5-dimethoxyphenyl)-4-chlorobenzamide (11a).
1466 (C]C aromatic); 1H NMR (400 MHz, MeOH-d4)
d
(ppm): 4.03
Yellow solid, yield 81%, m.p. 182e184 ꢀC; IR (KBr,
y
cmꢁ1): 3163
(s, 3H, OCH3), 4.06 (s, 3H, OCH3), 7.02 (s, 1H, vinyl CH), 7.41 (s, 1H,
phenyl CH), 7.62 (s, 1H, phenyl CH), 7.64 (d, 2H, chlorophenyl 2CH,
J ¼ 8.6 Hz), 7.84 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR
(NHeC]O), 3084, 3058 (CH aromatic), 2995, 2966, 2953, 2934 (CH
aliphatic), 1720 (CH3C]O), 1667 (NHeC]O), 1595 (C]C aromatic);
1H NMR (400 MHz, CDCl3)
d
(ppm): 2.66 (s, 3H, CH3), 3.92 (s, 3H,
(100 MHz, MeOH-d4) d (ppm): 55.78, 56.02, 99.02, 102.21, 104.43,
OCH3), 4.02 (s, 3H, OCH3), 7.31 (s, 1H, phenyl CH), 7.48 (d, 2H,
benzoyl 2CH, J ¼ 8.6 Hz), 7.99 (d, 2H, benzoyl 2CH, J ¼ 8.6 Hz), 8.71
(s, 1H, phenyl CH), 13.01 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
116.35, 128.40, 128.99, 129.53, 131.02, 131.35, 137.33, 137.72, 149.65,
150.47, 155.93, 171.88 (C]O); HRESIMS (m/z): [MþH]þ Calcd for
C
17H15ClNO3, 316.07350; found, 316.07397.
d
(ppm): 28.50, 56.37, 56.49, 103.56, 113.89, 114.64, 128.95, 129.17,
133.28, 138.21, 138.40, 143.91, 155, 165.07 (C]O), 201.35 (C]O);
HRESIMS (m/z): [MþNa]þ Calcd for C17H16ClNO4Na, 356.06601;
found, 356.06613.
4.1.4.2. 6,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quinolin-
4(1H)-one (12b). Yellow solid, Yield 90%, m.p. > 250 ꢀC; IR (KBr,
y
cmꢁ1): 3266 (NH), 3090 (CH aromatic), 2960 (CH aliphatic), 1685
(C]O), 1581, 1518 (C]C aromatic); 1H NMR (400 MHz, DMSO‑d6)
4.1.3.2. N-(2-Acetyl-4,5-dimethoxyphenyl)-4-(trifluoromethyl)ben-
d (ppm): 3.90 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.83 (s, 1H, vinyl CH),
zamide (11b). Yellow solid, Yield 85%, m.p. 128e130 ꢀC; IR (KBr,
y
7.44 (s, 1H, phenyl CH), 7.49 (s, 1H, phenyl CH), 7.98 (d, 2H, CF3-
cmꢁ1): 3160 (NHeC]O), 3061, 3002 (CH aromatic), 2973, 2950,
2934, 2908 (CH aliphatic), 1740 (CH3C]O), 1640 (NHeC]O), 1598
phenyl 2CH, J ¼ 8.3 Hz), 8.11 (d, 2H, CF3-phenyl 2CH, J ¼ 8.3 Hz); 13
C
NMR (100 MHz, MeOH-d4)
d (ppm): 56.27, 56.51, 100.86, 102.78,
(C]C aromatic); 1H NMR (400 MHz, CDCl3)
d
(ppm): 2.63 (s, 3H,
105.62, 117.13, 124.38 (CF3, q, J ¼ 272.4 Hz), 126.45 (CH-C-CF3, q,
J ¼ 3.8 Hz), 129.10, 131.16 (CH-C-CF3, q, J ¼ 32.1 Hz), 137.86, 148.93,
149.21, 154.82, 171.58 (C]O); 19F NMR (376.46 MHz, CDCl3)
CH3), 3.89 (s, 3H, OCH3), 4 (s, 3H, OCH3), 7.27 (s, 1H, phenyl CH), 7.75
(d, 2H, benzoyl 2CH, J ¼ 8.2 Hz), 8.13 (d, 2H, benzoyl 2CH,
J ¼ 8.2 Hz), 8.67 (s, 1H, phenyl CH), 13.07 (s, 1H, NH); 13C NMR
d
(ppm): 61.28 (s); HRESIMS (m/z): [MþH]þ Calcd for C18H15F3NO3,
(100 MHz, CDCl3)
d
(ppm): 28.33, 56.24, 56.31, 103.47,113.73,114.60,
350.10058; found, 350.09995.
123.69 (CF3, q, J ¼ 272.6 Hz),125.80 (CH-C-CF3, q, J ¼ 3.6 Hz),127.83,
133.49 (CH-C-CF3, q, J ¼ 32.7 Hz), 137.80, 138.01, 143.95, 154.85,
4.1.5. General procedure for synthesis of the key intermediates
(13a-d)
164.57 (C]O), 201.31(C]O); 19F NMR (376.46 MHz, CDCl3)
d (ppm):
62.98 (s); HRESIMS (m/z): [MþNa]þ Calcd for C18H16F3NO4Na,
A mixture of 12a or b (3 mmol), KOH (1.009 g, 18 mmol) and KI
(0.498 g, 3 mmol) was stirred in dry DMF (30 mL) for 2 h, then the
respective 1-bromo-2-chloroethane or 1-bromo-3-chloropropane
(15 mmol) was added. The reaction mixture was stirred for
24 h at room temperature. After completion of the reaction, reac-
tion mixture was poured into ice water (50 mL) and the precipi-
tated solid was filtered off, washed with water then hexane and
390.09236; found, 390.09268.
4.1.4. General procedures for synthesis of the quinolones (12a, b)
A mixture of 11a or 11b (1 equivalent), sodium hydroxide (3
equivalents) in dry dioxane was refluxed under N2 at 110 ꢀC for 4 h,
then removed from oil bath and allowed to cool to room
13

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
dried to afford compounds 13a-d that were used without further
purification.
Then, the appropriate amine (10 mmol) was added to the mixture.
The reaction mixture was refluxed at 90 ꢀC for 12 h, then poured
into ice water (50 mL). The precipitated solid was filtered off,
washed with water then hexane, dried and purified by silica gel
column chromatography using DCM/MeOH affording 14a-p in pure
form.
4 .1. 5 .1. 4 - ( 2 - C h l o ro e t h o x y ) - 2 - ( 4 - c h l o ro p h e nyl ) - 6 , 7 -
dimethoxyquinoline (13a). White solid, Yield 89%, m.p. 170e172 ꢀC;
IR (KBr,
(CH aliphatic), 1596, 1582, 1560, 1512 (C]C aromatic); 1H NMR
(400 MHz, CDCl3)
y
cmꢁ1): 3090, 3016 (CH aromatic), 2990, 2967, 2945, 2887
d
(ppm): 3.98 (t, 2H, CH2Cl, J ¼ 5.8 Hz), 4.02 (s, 3H,
4.1.6.1. 2-(4-Chlorophenyl)-6,7-dimethoxy-4-(2-(pyrrolidin-1-yl)
OCH3), 4.03 (s, 3H, OCH3), 4.49 (t, 2H, CH2eO, J ¼ 5.8 Hz), 6.98 (s,1H,
vinyl CH), 7.40 (s, 1H, phenyl CH), 7.41 (s, 1H, phenyl CH), 7.44 (d,
2H, chlorophenyl 2CH, J ¼ 8.6 Hz), 7.98 (d, 2H, chlorophenyl 2CH,
ethoxy)quinoline (14a). White solid, Yield 80%, m.p. 110e112 ꢀC; IR
(KBr,
y
cmꢁ1): 3000 (CH aromatic), 2961, 2936, 2872 (CH aliphatic),
1592, 1559, 1513 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d
(ppm): 41.54, 56.10, 56.13,
d
(ppm): 1.85 (p, 4H, pyrrolidinyl 2CH2, J ¼ 3.3 Hz), 2.75 (s, 4H,
68.08, 97.34, 99.62, 108.18, 114.78, 128.49, 128.87, 135.04, 138.70,
146.32, 149.29, 152.93, 155.37, 160.30; HRESIMS (m/z): [MþH]þ
Calcd for C19H18Cl2NO3, 378.06655; found, 378.06598.
pyrrolidinyl 2CH2eN), 3.12 (t, 2H, CH2eN, J ¼ 6 Hz), 4.02 (s, 3H,
OCH3), 4.04 (s, 3H, OCH3), 4.43 (t, 2H, CH2eO, J ¼ 6 Hz), 7.07 (s, 1H,
vinyl CH), 7.42 (s, 2H, phenyl CH), 7.46 (d, 2H, chlorophenyl 2CH,
J ¼ 8.6 Hz), 8 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR
4.1. 5. 2. 2-(4-Chlorophenyl)-4-(3-chloropropoxy)-6,7-
(100 MHz, CDCl3) d (ppm): 23.60, 54.59, 54.98, 56.02, 56.12, 67.77,
dimethoxyquinoline (13b). White solid, Yield 83%, m.p. 143.145 ꢀC;
97.44, 99.86, 108.21, 114.94, 128.54, 128.86, 134.95, 138.94, 146.23,
IR (KBr,
(CH aliphatic),1592, 1559,1513 (C]C aromatic); 1H NMR (400 MHz,
CDCl3)
y
cmꢁ1): 3079, 3061, 3000 (CH aromatic), 2960, 2936, 2875
149.07, 152.75, 155.63, 160.93; HRESIMS (m/z): [MþH]þ Calcd for
C
23H26ClN2O3, 413.16337; found, 413.16309.
d
(ppm): 2.43 (p, 2H, CH2, J ¼ 6.1 Hz) 3.83 (t, 2H, CH2Cl,
J ¼ 6.2 Hz), 4.02 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.42 (t, 2H,
CH2eO, J ¼ 6 Hz), 7.06 (s, 1H, vinyl CH), 7.35 (s, 1H, phenyl CH), 7.42
(s, 1H, phenyl CH), 7.45 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz), 8 (d,
2H, chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
4.1.6.2. 1-(2-((2-(4-Chlorophenyl)-6,7-dimethoxyquinolin-4-yl)oxy)
ethyl)piperidin-4-ol (14b). White solid, Yield 72%, m.p. 205e207 ꢀC;
IR (KBr,
aliphatic), 1592, 1560 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
(ppm): 1.59e1.68 (m, 3H, piperidinyl 2CHH’ and OH), 1.90e1.96
y
cmꢁ1): 3226 (OH), 3000 (CH aromatic), 2936, 2918 (CH
d
(ppm): 31.97, 41.35, 56.07, 56.13, 64.90, 97.37, 99.54, 108.29, 114.81,
d
128.53, 128.85, 134.98, 138.84, 146.24, 149.17, 152.81, 155.54, 160.74;
HRESIMS (m/z): [MþH]þ Calcd for C20H20Cl2NO3, 392.08148; found,
392.08127.
(m, 2H, piperidinyl 2CHH’), 2.38e2.44 (m, 2H, piperidinyl 2CHH0-
N), 2.95 (p, 2H, piperidinyl 2CHH0-N, J ¼ 5.3 Hz), 3.12 (t, 2H, CH2eN,
J ¼ 6 Hz), 3.71e3.78 (m, 1H, piperidinyl CHeOH), 4.01 (s, 3H, OCH3),
4.03 (s, 3H, OCH3), 4.39 (t, 2H, CH2eO, J ¼ 6 Hz), 7.06 (s, 1H, vinyl
CH), 7.38 (s, 1H, phenyl CH), 7.42 (s, 1H, phenyl CH), 7.45 (d, 2H,
chlorophenyl 2CH, J ¼ 8.6 Hz), 8 (d, 2H, chlorophenyl 2CH,
4.1.5.3. 4-(2-Chloroethoxy)-6,7-dimethoxy-2-(4-(trifluoromethyl)
phenyl)quinoline (13c). White solid, Yield 81%, m.p. 158e160 ꢀC; IR
(KBr,
2887 (CH aliphatic), 1587, 1565, 1509 (C]C aromatic); 1H NMR
(400 MHz, CDCl3)
y
cmꢁ1): 3095, 3061, 3015 (CH aromatic), 2958, 2943, 2909,
J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 34.52, 51.63, 56.01,
56.13, 56.73, 66.87, 67.54, 97.45, 99.76, 108.23, 114.92, 128.53,
128.86, 134.97, 138.91, 146.22, 149.10, 152.77, 155.60, 160.90; HRE-
SIMS (m/z): [MþH]þ Calcd for C24H28ClN2O4, 443.17321; found,
443.17334.
d
(ppm): 4 (t, 2H, CH2Cl, J ¼ 5.8 Hz), 4.04 (s, 3H,
OCH3), 4.05 (s, 3H, OCH3), 4.52 (t, 2H, CH2eO, J ¼ 5.8 Hz), 7.05 (s, 1H,
vinyl CH), 7.43 (s, 1H, phenyl CH), 7.44 (s, 1H, phenyl CH), 7.73 (d,
2H, CF3-phenyl 2CH, J ¼ 8.2 Hz), 8.15 (d, 2H, CF3-phenyl 2CH,
J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
d
(ppm): 41.51, 56.11, 56.15,
4.1.6.3. 2-(4-Chlorophenyl)-6,7-dimethoxy-4-(2-(piperidin-1-yl)
68.16, 97.66, 99.60, 108.27, 115.05, 124.23 (CF3, q, J ¼ 272.1 Hz),
125.63 (CH-C-CF3, q, J ¼ 3.7 Hz), 127.52, 130.72 (CH-C-CF3, q,
J ¼ 32.5 Hz), 143.64, 146.40, 149.57, 153.08, 155.02, 160.40; 19F NMR
ethoxy)quinoline (14c). White solid, Yield 87%, m.p. 142e144 ꢀC; IR
(KBr,
y
cmꢁ1): 3089, 3006 (CH aromatic), 2952, 2928 (CH aliphatic),
1592, 1560, 1513 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
(376.46 MHz, CDCl3)
d
(ppm): 62.49 (s); HRESIMS (m/z): [MþH]þ
d
(ppm): 1.47 (p, 2H, piperidinyl CH2, J ¼ 6.1 Hz), 1.63 (p, 4H,
Calcd for C20H18ClF3NO3, 412.09218; found, 412.09201.
piperidinyl 2CH2, J ¼ 5.5 Hz), 2.60 (t, 4H, piperidinyl 2CH2eN,
J ¼ 4.8 Hz), 2.96 (t, 2H, CH2eN, J ¼ 6.1 Hz), 4.02 (s, 3H, OCH3), 4.03
(s, 3H, OCH3), 4.40 (t, 2H, CH2eO, J ¼ 6.1 Hz), 7.07 (s, 1H, vinyl CH),
7.40 (s, 1H, phenyl CH), 7.42 (s, 1H, phenyl CH), 7.45 (d, 2H, chlor-
4.1.5.4. 4-(3-Chloropropoxy)-6,7-dimethoxy-2-(4-(trifluoromethyl)
phenyl)quinoline (13d). White solid, Yield 78%, m.p. 133e135 ꢀC; IR
(KBr,
(CH aliphatic), 1592, 1564, 1507 (C]C aromatic); 1H NMR
(400 MHz, CDCl3)
y
cmꢁ1): 3079, 3015 (CH aromatic), 2961, 2934, 2912, 2883
ophenyl 2CH, J ¼ 8.6 Hz), 8 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz); 13
C
NMR (100 MHz, CDCl3)
d (ppm): 24.12, 26.10, 55.23, 56.00, 56.11,
d
(ppm): 2.45 (p, 2H, CH2, J ¼ 6.1 Hz), 43.84 (t, 2H,
57.60, 66.81, 97.47, 99.84, 108.22, 114.97, 128.53, 128.84, 134.93,
138.95, 146.21, 149.06, 152.74, 155.60, 165.98; HRESIMS (m/z):
[MþH]þ Calcd for C24H28ClN2O3, 427.17830; found, 427.17825.
CH2Cl, J ¼ 6.2 Hz), 4.03 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 4.45 (t, 2H,
CH2eO, J ¼ 6 Hz), 7.12 (s, 1H, vinyl CH), 7.38 (s, 1H, phenyl CH), 7.45
(s, 1H, phenyl CH), 7.74 (d, 2H, CF3-phenyl 2CH, J ¼ 8.1 Hz), 8.17 (d,
2H, CF3-phenyl 2CH, J ¼ 8.1 Hz); 13C NMR (100 MHz, CDCl3)
d
(ppm):
4.1.6.4. 4-(2-((2-(4-Chlorophenyl)-6,7-dimethoxyquinolin-4-yl)oxy)
31.95, 41.31, 56.09, 56.14, 64.98, 97.69, 99.51, 108.36, 115.07, 124.25
(CF3, q, J ¼ 271.9 Hz), 125.61 (CH-C-CF3, q, J ¼ 3.7 Hz), 127.56, 130.67
(CH-C-CF3, q, J ¼ 32.4 Hz), 143.78, 146.32, 149.45, 152.95, 155.20,
ethyl)morpholine (14d). White solid, Yield 88%, m.p. 131e133 ꢀC; IR
(KBr,
(CH aliphatic),1592,1560,1513 (C]C aromatic); 1H NMR (400 MHz,
CDCl3)
y
cmꢁ1): 3094, 3055, 3019 (CH aromatic), 2950, 2923, 2879
160.84; 19F NMR (376.46 MHz, CDCl3)
d
(ppm): 62.48 (s); HRESIMS
d
(ppm): 2.66 (t, 4H, morpholinyl CH2eN, J ¼ 4.6 Hz), 2.99 (t,
(m/z): [MþH]þ Calcd for C21H20ClF3NO3, 426.10783; found,
2H, CH2eN, J ¼ 5.8 Hz), 3.74 (t, 4H, morpholinyl 2CH2eO,
J ¼ 4.6 Hz), 4.01 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.40 (t, 2H, CH2eO,
J ¼ 5.8 Hz), 7.04 (s, 1H, vinyl CH), 7.37 (s, 1H, phenyl CH), 7.41 (s, 1H,
phenyl CH), 7.45 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz), 7.99 (d, 2H,
426.10803.
4.1.6. General procedure for synthesis of the target 4-alkoxy-2-
arylquinolines (14a-p)
A mixture of 13a-d (1 mmol), KI (0.83 g, 5 mmol) and anhydrous
K2CO3 (1.38 g, 10 mmol) was stirred in dry DMF (20 mL) for 30 min.
chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm):
54.21, 55.99, 56.13, 57.33, 66.57, 67.01, 97.39, 99.68, 108.25, 114.88,
128.51, 128.86,134.98,138.88, 146.22, 149.13, 152.79, 155.55, 160.81;
14

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
HRESIMS (m/z): [MþH]þ Calcd for C23H26ClN2O4, 429.15756; found,
4.1.6.9. 6,7-Dimethoxy-4-(2-(pyrrolidin-1-yl)ethoxy)-2-(4-(tri-
429.15778.
fluoromethyl)phenyl)quinoline (14i). White solid, Yield 86%, m.p.
116e118 ꢀC; IR (KBr, cmꢁ1): 3096, 3019 (CH aromatic), 2954, 2878
y
4.1.6.5. 2-(4-Chlorophenyl)-6,7-dimethoxy-4-(3-(pyrrolidin-1-yl)
(CH aliphatic), 1592, 1560, 1522, 1509 (C]C aromatic); 1H NMR
(400 MHz, CDCl3) (ppm): 1.83 (p, 4H, pyrrolidinyl 2CH2,
propoxy)quinoline (14e). White solid, Yield 92%, m.p. 116e118 ꢀC; IR
d
(KBr,
(CH aliphatic), 1591, 1560, 1512 (C]C aromatic); 1H NMR (400 MHz,
CDCl3)
y
cmꢁ1): 3090, 3061, 3000 (CH aromatic), 2955, 2905, 2876
J ¼ 3.4 Hz), 2.73 (p, 4H, pyrrolidinyl 2CH2eN, J ¼ 3.4 Hz), 3.10 (t, 2H,
CH2eN, J ¼ 6 Hz), 4.02 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 4.43 (t, 2H,
CH2eO, J ¼ 6 Hz), 7.10 (s, 1H, vinyl CH), 7.43 (s, 1H, phenyl CH), 7.44
(s, 1H, phenyl CH), 7.73 (d, 2H, CF3-phenyl 2CH, J ¼ 8.1 Hz), 8.16 (d,
d
(ppm): 1.81 (p, 4H, pyrrolidinyl 2CH2, J ¼ 3.3 Hz), 2.2 (p 2H,
CH2, J ¼ 6.8 Hz), 2.56 (s, 4H, pyrrolidinyl 2CH2eN), 2.72 (t, 2H,
CH2eN, J ¼ 7.3 Hz), 4.02 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.34 (t, 2H,
CH2eO, J ¼ 6.4 Hz), 7.07 (s, 1H, vinyl CH), 7.40 (s, 1H, phenyl CH),
7.41 (s, 1H, phenyl CH) 7.45 (d, 2H, chlorophenyl 2CH, J ¼ 8.5 Hz), 8
(d, 2H, chlorophenyl 2CH, J ¼ 8.5 Hz); 13C NMR (100 MHz, CDCl3)
2H, CF3-phenyl 2CH, J ¼ 8.1 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm):
23.62, 54.62, 55.00, 56.02, 56.12, 67.99, 97.71, 99.85, 108.28, 115.21,
124.25 (CF3, q, J ¼ 272 Hz), 125.60 (CH-C-CF3, q, J ¼ 3.7 Hz), 127.55,
130.62 (CH-C-CF3, q, J ¼ 32 Hz), 143.88, 146.28, 149.32, 152.87,
d
(ppm): 23.49, 28.70, 53.12, 54.34, 56.06, 56.11, 66.83, 97.44, 99.76,
155.23, 161.06; 19F NMR (376.46 MHz, CDCl3)
d (ppm): 62.49 (s);
108.23, 114.97, 128.55, 128.81, 134.88, 139.00, 146.18, 149.04, 152.71,
155.61, 161.15; HRESIMS (m/z): [MþH]þ Calcd for C24H28ClN2O3,
427.17830; found, 427.17853.
HRESIMS (m/z): [MþH]þ Calcd for C24H26F3N2O3, 447.18900; found,
447.18820.
4.1.6.10. 1-(2-((6,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quino-
lin-4-yl)oxy)ethyl)piperidin-4-ol (14j). White solid, Yield 76%, m.p.
4.1.6.6. 1-(3-((2-(4-Chlorophenyl)-6,7-dimethoxyquinolin-4-yl)oxy)
propyl)piperidin-4-ol (14f). White solid, Yield 76%, m.p.155e157 ꢀC;
176e178 ꢀC; IR (KBr, cmꢁ1): 3360 (OH), 3006 (CH aromatic), 2945,
y
IR (KBr,
aliphatic), 1589, 1560, 1513 (C]C aromatic); 1H NMR (400 MHz,
CDCl3) (ppm): 1.56e1.64 (m, 3H, piperidinyl 2CHH’), 1.68 (br s, 1H,
y
cmꢁ1): 3410 (OH), 3009 (CH aromatic), 2924 (CH
2925, 2900 (CH aliphatic), 1591, 1566, 1511 (C]C aromatic); 1H
NMR (400 MHz, CDCl3) (ppm): 1.59e1.67 (m, 2H, piperidinyl
d
d
2CHH’), 1.84 (br s, 1H, OH), 1.89e1.94 (m, 2H, piperidinyl 2CHH’),
2.38e2.44 (m, 2H, piperidinyl 2CHH0-N), 2.95 (p, 2H, piperidinyl
2CHH0-N, J ¼ 5.2 Hz), 2.99 (t, 2H, CH2eN, J ¼ 6 Hz), 3.71e3.76 (m,
1H, piperidinyl CHeOH), 4.01 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.40
(t, 2H, CH2eO, J ¼ 6 Hz), 7.09 (s, 1H, vinyl CH), 7.39 (s, 1H, phenyl
CH), 7.44 (s,1H, phenyl CH), 7.73 (d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz),
8.15 (d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
OH), 1.89e1.93 (m, 2H, piperidinyl 2CHH’), 2.12e2.22 (m, 4H,
piperidinyl 2CHH0-N, CH2)), 2.60 (t, 2H, CH2eN, J ¼ 7.3 Hz), 2.82 (t,
2H, piperidinyl 2CHH0-N, J ¼ 5.7 Hz), 3.70e3.75 (m, 1H, piperidinyl
CHeOH), 4.02 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.31 (t, 2H, CH2eO,
J ¼ 6.4 Hz), 7.04 (s, 1H, vinyl CH), 7.39 (s, 1H, phenyl CH), 7.42 (s, 1H,
phenyl CH), 7.45 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz), 8 (d, 2H,
chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d
(ppm):
d (ppm): 34.41, 51.59, 56.02, 56.13, 56.69, 66.83, 67.38, 97.77, 99.73,
26.91, 34.48, 51.20, 55.01, 56.06, 56.11, 66.74, 67.87, 97.41, 99.72,
108.24,114.95,128.56,128.82,134.92,139.00,146.20,149.08,152.75,
155.63, 161.11; HRESIMS (m/z): [MþH]þ Calcd for C25H30ClN2O4,
457.18886; found, 457.18893.
108.24, 115.16, 124.24 (CF3, q, J ¼ 272.1 Hz), 125.60 (CH-C-CF3, q,
J ¼ 3.7 Hz), 127.57, 130.65 (CH-C-CF3, q, J ¼ 32.4 Hz), 143.81, 146.26,
149.38, 152.91, 155.24, 160.97; 19F NMR (376.46 MHz, CDCl3)
d
(ppm): 62.48 (s); HRESIMS (m/z): [MþH]þ Calcd for C25H27F3N2O4,
477.19957; found, 477.19897.
4.1.6.7. 2-(4-Chlorophenyl)-6,7-dimethoxy-4-(3-(piperidin-1-yl)pro-
poxy)quinoline (14g). White solid, Yield 76%, m.p. 130.132 ꢀC; IR
4.1.6.11. 6,7-Dimethoxy-4-(2-(piperidin-1-yl)ethoxy)-2-(4-(tri-
fluoromethyl)phenyl)quinoline (14k). White solid, Yield 90%, m.p.
(KBr,
y
cmꢁ1): 3095, 3000 (CH aromatic), 2930, 2880 (CH aliphatic),
1591, 1560, 1512 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
138e140 ꢀC; IR (KBr,
y
cmꢁ1): 3096, 3009 (CH aromatic), 2939 (CH
d
(ppm): 1.45 (t, 2H, piperidinyl CH2, J ¼ 5.6 Hz), 1.60 (p, 4H,
aliphatic), 1592, 1560, 1508 (C]C aromatic); 1H NMR (400 MHz,
CDCl3)
piperidinyl 2CH2, J ¼ 5.6 Hz), 2.15 (p, 2H, CH2, J ¼ 6.9 Hz), 2.43 (br s,
4H, piperidinyl 2CH2eN), 2.56 (t, 2H, CH2eN, J ¼ 7.4 Hz), 4.01 (s, 3H,
OCH3), 4.02 (s, 3H, OCH3), 4.29 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.03 (s, 1H,
vinyl CH), 7.38 (s, 1H, phenyl CH), 7.40 (s, 1H, phenyl CH), 7.44 (d,
2H, chlorophenyl 2CH, J ¼ 8.6 Hz), 7.99 (d, 2H, chlorophenyl 2CH,
d
(ppm): 1.47 (p, 2H, piperidinyl CH2, J ¼ 5.4 Hz), 1.63 (p, 4H,
piperidinyl 2CH2, J ¼ 5.6 Hz), 2.61 (t, 4H, piperidinyl 2CH2eN,
J ¼ 5.8 Hz), 2.97 (t, 2H, CH2eN, J ¼ 6.1 Hz), 4.03 (s, 3H, OCH3), 4.04 (s,
3H, OCH3), 4.42 (t, 2H, CH2eO, J ¼ 6.1 Hz), 7.12 (s, 1H, vinyl CH), 7.42
(s, 1H, phenyl CH), 7.44 (s, 1H, phenyl CH), 7.74 (d, 2H, CF3-phenyl
2CH, J ¼ 8.2 Hz), 8.17 (d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz); 13C NMR
J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 24.41, 25.99, 26.68,
54.76, 55.95, 56.04, 56.09, 66.92, 97.39, 99.76,108.21, 114.98,128.55,
128.80, 134.88, 139.00, 146.17, 149.04, 152.71, 155.59, 161.13; HRE-
SIMS (m/z): [MþH]þ Calcd for C25H30ClN2O3, 441.19395; found,
441.19409.
(100 MHz, CDCl3) d (ppm): 24.11, 26.08, 55.23, 56.01, 56.12, 57.59,
66.88, 97.77, 99.81, 108.28, 115.22, 124.26 (CF3, q, J ¼ 272 Hz), 125.59
(CH-C-CF3, q, J ¼ 3.7 Hz), 127.54, 130.62 (CH-C-CF3, q, J ¼ 32.4 Hz),
143.87, 146.27, 149.33, 152.87, 155.21, 161.06; 19F NMR (376.46 MHz,
CDCl3)
d
(ppm): 62.48 (s); HRESIMS (m/z): [MþH]þ Calcd for
4.1.6.8. 4-(3-((2-(4-Chlorophenyl)-6,7-dimethoxyquinolin-4-yl)oxy)
C25H28F3N2O3, 461.20465; found, 461.20395.
propyl)morpholine (14h). White solid, Yield 79%, m.p. 144e146 ꢀC;
IR (KBr,
2883 (CH aliphatic), 1590, 1559, 1511 (C]C aromatic); 1H NMR
(400 MHz, CDCl3)
y
cmꢁ1): 3091, 3064, 3027, 3004 (CH aromatic), 2951, 2903,
4.1.6.12. 4-(2-((6,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quino-
lin-4-yl)oxy)ethyl)morpholine (14l). White solid, Yield 63%, m.p.
d
(ppm): 2.16 (p, 2H, CH2, J ¼ 6.8 Hz), 2.50 (t, 4H,
133e135 ꢀC; IR (KBr, cmꢁ1): 3096, 3021 (CH aromatic), 2959,
y
morpholinyl CH2eN, J ¼ 4.4 Hz), 2.61 (t, 2H, CH2eN, J ¼ 7.2 Hz), 3.72
(t, 4H, morpholinyl 2CH2eO, J ¼ 4.4 Hz), 4.02 (s, 3H, OCH3), 4.03 (s,
3H, OCH3), 4.33 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.04 (s, 1H, vinyl CH), 7.38
(s, 1H, phenyl CH), 7.41 (s, 1H, phenyl CH), 7.45 (d, 2H, chlorophenyl
2CH, J ¼ 8.6 Hz), 8 (d, 2H, chlorophenyl 2CH, J ¼ 8.6 Hz); 13C NMR
2920, 2882 (CH aliphatic), 1591, 1566, 1510 (C]C aromatic); 1H
NMR (400 MHz, CDCl3) (ppm): 2.67 (t, 4H, morpholinyl CH2eN,
d
J ¼ 4.6 Hz), 3 (t, 2H, CH2eN, J ¼ 5.8 Hz), 3.74 (t, 4H, morpholinyl
2CH2eO, J ¼ 4.6 Hz), 4.02 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 4.42 (t,
2H, CH2eO, J ¼ 5.8 Hz), 7.09 (s, 1H, vinyl CH), 7.39 (s, 1H, phenyl CH),
7.44 (s, 1H, phenyl CH), 7.73 (d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz), 8.16
(d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
(100 MHz, CDCl3)
d (ppm):26.26, 53.84, 55.51, 56.05, 56.11, 66.51,
66.97, 97.37, 99.68, 108.27, 114.92, 128.54, 128.83, 134.93, 138.99,
146.21, 149.10, 152.76, 155.60, 161.07; HRESIMS (m/z): [MþH]þ
Calcd for C24H28ClN2O4, 443.17321; found, 443.17340.
d
(ppm): 54.21, 56.00, 56.13, 57.32, 66.65, 66.99, 97.69, 99.67, 108.32,
115.14, 124.25 (CF3, q, J ¼ 271.4 Hz), 125.61 (CH-C-CF3, q, J ¼ 3.7 Hz),
15

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
127.53, 130.66 (CH-C-CF3, q, J ¼ 32.3 Hz), 143.81, 146.30, 149.42,
J ¼ 7.2 Hz), 3.73 (t, 4H, morpholinyl 2CH2eO, J ¼ 4.4 Hz), 4.03 (s, 3H,
OCH3), 4.04 (s, 3H, OCH3), 4.35 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.10 (s, 1H,
vinyl CH), 7.40 (s,1H, phenyl CH), 7.44 (s,1H, phenyl CH), 7.74 (d, 2H,
CF3-phenyl 2CH, J ¼ 8.2 Hz), 8.16 (d, 2H, CF3-phenyl 2CH,
152.94, 155.18, 160.91; 19F NMR (376.46 MHz, CDCl3)
d (ppm): 62.49
(s); HRESIMS (m/z): [MþH]þ Calcd for C24H26F3N2O4, 463.18392;
found, 463.18341.
J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 26.25, 53.84, 55.49,
4.1.6.13. 6,7-Dimethoxy-4-(3-(pyrrolidin-1-yl)propoxy)-2-(4-(tri-
56.07, 56.13, 66.59, 66.96, 97.69, 99.65, 108.33, 115.17, 124.25 (CF3, q,
fluoromethyl)phenyl)quinoline (14m). White solid, Yield 88%, m.p.
J ¼ 272 Hz), 125.59 (CH-C-CF3, q, J ¼ 3.7 Hz), 127.57, 130.64 (CH-C-
126e128 ꢀC; IR (KBr,
y
cmꢁ1): 3084, 3067 (CH aromatic), 2994,
CF3, q, J ¼ 32.4 Hz), 143.92, 146.28, 149.36, 152.89, 155.25, 161.16; 19
F
2967, 2944, 2874 (CH aliphatic), 1593, 1561, 1508 (C]C aromatic);
NMR (376.46 MHz, CDCl3) d (ppm): 62.48 (s); HRESIMS (m/z):
1H NMR (400 MHz, CDCl3)
d
(ppm): 1.81 (p, 4H, pyrrolidinyl 2CH2,
[MþH]þ Calcd for C25H28F3N2O4,477.19957; found, 477.19943.
J ¼ 3.3 Hz), 0.2.20 (p, 2H, CH2, J ¼ 6.9 Hz), 2.56 (p, 4H, pyrrolidinyl
2CH2eN, J ¼ 3.3 Hz), 2.72 (t, 2H, CH2eN, J ¼ 7.3 Hz), 4.02 (s, 3H,
OCH3), 4.03 (s, 3H, OCH3), 4.35 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.11 (s, 1H,
vinyl CH), 7.41 (s,1H, phenyl CH), 7.43 (s,1H, phenyl CH), 7.73 (d, 2H,
CF3-phenyl 2CH, J ¼ 8.1 Hz), 8.16 (d, 2H, CF3-phenyl 2CH, J ¼ 8.1 Hz);
4.1.7. General procedure for synthesis of the aroyl derivatives (16a-
c)
The aroyl derivatives 16a-c were synthesized by reaction of the
2-aminoacetophenone derivative 9 with the appropriate aroyl
chloride 15a-c using the same procedure as 11a, b.
13C NMR (100 MHz, CDCl3)
d (ppm): 23.48, 28.68, 53.08, 54.32,
56.07, 56.11, 66.90, 97.74, 99.74, 108.29, 115.22, 124.26 (CF3, q,
J ¼ 272.1 Hz), 125.56 (CH-C-CF3, q, J ¼ 3.5 Hz), 127.56, 130.58 (CH-C-
4.1.7.1. N-(2-Acetyl-4,5-dimethoxyphenyl)furan-2-carboxamide
CF3, q, J ¼ 32.4 Hz), 143.93, 146.24, 149.31, 152.84, 155.23, 161.24; 19
F
(16a). Yellow solid, yield 72%, m.p. 162e164 ꢀC; IR (KBr, cmꢁ1):
y
NMR (376.46 MHz, CDCl3)
d
(ppm): 62.47 (s); HRESIMS (m/z):
3138 (NHeC]O), 3009, 3001 (CH aromatic), 2968, 2939, 2856 (CH
aliphatic), 1670 (CH3C]O), 1635 (NHeC]O), 1562, 1521 (C]C ar-
[MþH]þ Calcd for C25H28F3N2O3, 461.20465; found, 461.20425.
omatic); 1H NMR (400 MHz, CDCl3)
d (ppm): 2.64 (s, 3H, CH3), 3.90
4.1.6.14. 1-(3-((6,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quino-
lin-4-yl)oxy)propyl)piperidin-4-ol (14n). White solid, Yield 88%,
(s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.54 (dd, 1H, furyl CH, J ¼ 3.5,
1.7 Hz), 7.21 (dd, 1H, furyl CH, J ¼ 3.5, 0.8 Hz), 7.29 (s, 1H, phenyl
CH), 7.60 (dd, 1H, furyl CH, J ¼ 1.7, 0.8 Hz), 8.65 (s, 1H, phenyl CH),
m.p. 173e175 ꢀC; IR (KBr,
y
cmꢁ1): 3392 (OH), 3000 (CH aromatic),
2935 (CH aliphatic), 1590, 1560, 1508 (C]C aromatic); 1H NMR
(400 MHz, CDCl3) (ppm): 1.56e1.64 (m, 2H, piperidinyl 2CHH’),
12.92 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
d (ppm): 28.31, 56.20,
d
56.38, 103.59, 112.29, 113.74, 114.63, 115.21, 137.52, 143.74, 145.01,
148.28, 154.64, 157.19 (C]O), 200.81 (C]O); HRESIMS (m/z):
[MþNa]þ Calcd for C15H15NO5Na, 312.08424; found, 312.08429.
1.74 (br s, 1H, OH), 1.88e1.92 (m, 2H, piperidinyl 2CHH’), 2.13e2.22
(m, 4H, piperidinyl 2CHH0-N and CH2), 2.61 (t, 2H, CH2eN,
J ¼ 7.3 Hz), 2.82 (t, 2H, piperidinyl 2CHH0-N, J ¼ 5.8 Hz), 3.69e3.75
(m, 1H, piperidinyl CHeOH), 4.03 (s, 3H, OCH3), 4.04 (s, 3H, OCH3),
4.33 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.09 (s, 1H, vinyl CH), 7.40 (s, 1H,
phenyl CH), 7.44 (s, 1H, phenyl CH), 7.73 (d, 2H, CF3-phenyl 2CH,
J ¼ 8.2 Hz), 8.16 (d, 2H, CF3-phenyl 2CH, J ¼ 8.2 Hz); 13C NMR
4.1.7.2. N-(2-Acetyl-4,5-dimethoxyphenyl)thiophene-2-carboxamide
(16b). Yellow solid, yield 72%, m.p. 163e165 ꢀC; IR (KBr,
y
cmꢁ1):
3115 (NHeC]O), 3082, 3003, 3000 (CH aromatic), 2938, 2916,
2852 (CH aliphatic), 1654 (CH3C]O), 1635 (NHeC]O), 1596, 1522
(100 MHz, CDCl3)
d
(ppm): 26.90, 34.47, 51.20, 54.99, 56.08, 56.12,
(C]C aromatic); 1H NMR (400 MHz, CDCl3)
d (ppm): 2.64 (s, 3H,
66.81, 67.85, 97.72, 99.69, 108.30, 115.20, 124.27 (CF3, q,
CH3), 3.90 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.14 (dd, 1H, thiophene
CH, J ¼ 5, 3.8 Hz), 7.29 (s, 1H, phenyl CH), 7.56 (dd, 1H, thiophene
CH, J ¼ 5, 1.1 Hz), 7.81 (dd, 1H, thiophene CH, J ¼ 3.8, 1.1 Hz), 8.63 (s,
1H, phenyl CH), 12.98 (s, 1H, NH); 13C NMR (100 MHz, CDCl3)
J ¼ 272.7 Hz), 125.59 (CH-C-CF3, q, J ¼ 3.8 Hz), 127.59, 130.62 (CH-C-
CF3, q, J ¼ 32.7 Hz),143.93, 146.26, 149.34, 152.87, 155.28, 161.20; 19
F
NMR (376.46 MHz, CDCl3)
d (ppm): 62.48 (s); HRESIMS (m/z):
[MþH]þ Calcd for C26H30F3N2O4, 491.21522; found, 491.21472.
d (ppm): 28.37, 56.24, 56.40, 103.33, 113.77, 114.22, 128.01, 128.65,
131.27, 138.10, 140.45, 143.67, 154.89, 160.90 (C]O), 201.13 (C]O);
HRESIMS (m/z): [MþNa]þ Calcd for C15H15NO4SNa, 328.06140;
found, 328.06149.
4.1.6.15. 6,7-Dimethoxy-4-(3-(piperidin-1-yl)propoxy)-2-(4-(tri-
fluoromethyl)phenyl)quinoline (14o). White solid, Yield 83%, m.p.
109e111 ꢀC; IR (KBr, cmꢁ1): 3094, 3006 (CH aromatic), 2939, 2857
y
(CH aliphatic),1590,1561,1509 (C]C aromatic); 1H NMR (400 MHz,
CDCl3)
4.1.7.3. N-(2-Acetyl-4,5-dimethoxyphenyl)thiophene-3-carboxamide
d
(ppm): 1.46 (t, 2H, piperidinyl CH2, J ¼ 5.6 Hz), 1.60 (p, 4H,
(16c). Yellow solid, yield 65%, m.p. 178e180 ꢀC; IR (KBr, cmꢁ1):
y
piperidinyl 2CH2, J ¼ 5.6 Hz), 2.16 (p, 2H, CH2, J ¼ 6.9 Hz), 2.43 (br s,
4H, piperidinyl 2CH2eN), 2.57 (t, 2H, CH2eN, J ¼ 7.4 Hz), 4.02 (s, 3H,
OCH3), 4.03 (s, 3H, OCH3), 4.31 (t, 2H, CH2eO, J ¼ 6.4 Hz), 7.08 (s, 1H,
vinyl CH), 7.40 (s, 1H, phenyl CH), 7.43 (s, 1H, phenyl CH), 7.72 (d,
2H, CF3-phenyl 2CH, J ¼ 8.2 Hz), 8.16 (d, 2H, CF3-phenyl 2CH,
3198 (NHeC]O), 3099, 3089, 3033, 3006 (CH aromatic), 2953,
2932, 2910, 2850 (CH aliphatic), 1655 (CH3C]O), 1633 (NHeC]O),
1594, 1519 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
d (ppm):
2.65 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 7.30 (s, 1H,
phenyl CH), 7.39 (dd, 1H, thiophene CH, J ¼ 5.1, 3 Hz), 7.67 (dd, 1H,
thiophene CH, J ¼ 5.1, 1.3 Hz), 8.13 (dd, 1H, thiophene CH, J ¼ 3,
1.3 Hz), 8.69 (s, 1H, phenyl CH), 12.87 (s, 1H, NH); 13C NMR
J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 24.40, 25.99, 26.67,
54.76, 55.92, 56.06, 56.10, 66.99, 97.70, 99.72, 108.28, 115.23, 124.26
(CF3, q, J ¼ 272.1 Hz), 125.56 (CH-C-CF3, q, J ¼ 3.8 Hz), 127.57, 130.58
(CH-C-CF3, q, J ¼ 32.4 Hz), 143.95, 146.24, 149.31, 152.84, 155.23,
(100 MHz, CDCl3)
d (ppm): 28.41, 56.24, 56.42, 103.35, 113.83,
114.29, 126.38, 126.68, 129.44, 138.33, 138.36, 143.62, 154.91, 161.72
161.23; 19F NMR (376.46 MHz, CDCl3)
d
(ppm): 62.47 (s); HRESIMS
(C]O), 201.10 (C]O); HRESIMS (m/z): [MþNa]þ Calcd for
(m/z): [MþH]þ Calcd for C26H30F3N2O3, 475.22030; found,
C15H15NO4SNa, 328.06140; found, 328.06122.
475.22006.
4.1.8. General procedure for synthesis of the quinolones (17a-c)
The quinolones 17a-c were prepared using the same procedures
as 12a, b.
4.1.6.16. 4-(3-((6,7-Dimethoxy-2-(4-(trifluoromethyl)phenyl)quino-
lin-4-yl)oxy)propyl)morpholine (14p). White solid, Yield 79%, m.p.
105e107 ꢀC; IR (KBr,
y
cmꢁ1): 3096, 3000 (CH aromatic), 2958,
2939, 2868, 2853 (CH aliphatic), 1591, 1565, 1508 (C]C aromatic);
1H NMR (400 MHz, CDCl3)
(ppm): 2.17 (p, 2H, CH2, J ¼ 6.8 Hz), 2.50
(t, 4H, morpholinyl CH2eN, J ¼ 4.4 Hz), 2.62 (t, 2H, CH2eN,
4.1.8.1. 2-(Furan-2-yl)-6,7-dimethoxyquinolin-4(1H)-one
Yellow solid, yield 61%, m.p. > 250 ꢀC; IR (KBr, cmꢁ1): 3403 (NH),
3099, 3077, 3039, 3000 (CH aromatic), 2946, 2913 (CH aliphatic),
(17a).
d
y
16

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
1640 (C]O), 1581, 1541, 1507 (C]C aromatic); 1H NMR (400 MHz,
160.42; HRESIMS (m/z): [MþH]þ Calcd for C18H19ClNO3S,
MeOH-d4)
d
(ppm): 4.06 (s, 3H, OCH3), 4.10 (s, 3H, OCH3), 6.84 (dd,
364.07687; found, 364.07706.
1H, furyl CH, J ¼ 3.6,1.7 Hz), 7.20 (s,1H, vinyl CH), 7.49 (s,1H, phenyl
CH), 7.57 (dd, 1H, furyl CH, J ¼ 3.6, 0.6 Hz), 7.64 (s, 1H, phenyl CH),
7.84 (s, 1H, NH), 7.97 (dd, 1H, furyl CH, J ¼ 1.7, 0.6 Hz),; 13C NMR
4.1.9.3. 4-(3-Chloropropoxy)-6,7-dimethoxy-2-(thiophen-3-yl)quin-
oline (18c). Yellow solid, yield 73%, m.p. 133e135 ꢀC; IR (KBr,
y
(100 MHz, MeOH-d4)
d
(ppm): 55.61, 55.88, 98.89, 99.60, 101.76,
cmꢁ1): 3089, 3000 (CH aromatic), 2965, 2940, 2914 (CH aliphatic),
113.21, 114.43, 115.27, 136.89, 141.15, 145.41, 147.10, 150.14, 156.51,
169.23 (C]O); HRESIMS (m/z): [MþH]þ Calcd for C15H14NO4,
272.09173; found, 272.09195.
1590, 1508 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
d
(ppm):
2.43 (p, 2H, CH2, J ¼ 6.1 Hz), 3.83 (t, 2H, CH2Cl, J ¼ 6.2 Hz), 4.01 (s,
3H, OCH3), 4.03 (s, 3H, OCH3), 4.41 (t, 2H, CH2O, J ¼ 6 Hz), 7.02 (s,1H,
vinyl CH), 7.34 (s, 1H, phenyl CH), 7.40 (s, 1H, phenyl CH), 7.41 (dd,
1H, thiophene CH, J ¼ 5, 3 Hz), 7.77 (dd, 1H, thiophene CH, J ¼ 5,
1.2 Hz), 7.93 (dd, 1H, thiophene CH, J ¼ 3, 1.2 Hz); 13C NMR
4.1.8.2. 6,7-Dimethoxy-2-(thiophen-2-yl)quinolin-4(1H)-one (17b).
Yellow solid, yield 91%, m.p. > 250 ꢀC; IR (KBr,
y
cmꢁ1): 3401 (NH),
3076, 3003 (CH aromatic), 2938, 2902, 2866 (CH aliphatic), 1634
(100 MHz, CDCl3) d (ppm): 31.99, 41.37, 56.04, 56.09, 64.85, 97.67,
(C]O), 1595, 1519, 1502 (C]C aromatic); 1H NMR (400 MHz,
99.67, 108.18, 114.72, 123.57, 126.14, 126.72, 143.23, 146.21, 148.90,
MeOH-d4)
d
(ppm): 4.05 (s, 3H, OCH3), 4.10 (s, 3H, OCH3), 7.17 (s, 1H,
152.68, 152.81, 160.53; HRESIMS (m/z): [MþH]þ Calcd for
vinyl CH), 7.37 (dd, 1H, thiophene CH, J ¼ 5, 3.8 Hz), 7.51 (s, 1H,
phenyl CH), 7.61 (s, 1H, phenyl CH), 7.76 (s, 1H, NH), 7.90 (dd, 1H,
thiophene CH, J ¼ 5, 1.1 Hz), 8.04 (dd, 1H, thiophene CH, J ¼ 3.8,
C18H19ClNO3S, 364.07687; found, 364.07751.
4.1.10. General procedures for synthesis of the target 4-alkoxy-2-
arylquinolines (19a-c)
1.1 Hz); 13C NMR (100 MHz, MeOH-d4)
d (ppm): 55.78, 56.06, 98.90,
101.46, 101.86, 114.68, 129.22, 130.06, 131.94, 133.71, 137.34, 146.02,
A mixture of 18a-c (1 mmol), KI (0.83 g, 5 mmol) and anhydrous
K2CO3 (1.38 g, 10 mmol) was stirred in dry DMF (20 mL) for 30 min.
Then, 4-hydroxymorpholine (1.01 g, 10 mmol) was added to the
mixture. The reaction mixture was refluxed at 90 ꢀC for 12 h, then
poured into ice water (50 mL). The precipitated solid (19b, c) was
filtered off, washed with water then hexane. For 19a, the aqueous
layer was extracted with Ethyl acetate (50 mL ꢃ 3), then the organic
layers were collected and washed with water and brine. The
organic layer was dried over anhydrous Na2SO4, then evaporated
under vacuum. The products 19a-c were purified by silica gel col-
umn chromatography using DCM/MeOH.
150.50, 156.91, 168.12 (C]O); HRESIMS (m/z): [MþH]þ Calcd for
C
15H14NO3S, 288.06889; found, 288.06918.
4.1.8.3. 6,7-Dimethoxy-2-(thiophen-3-yl)quinolin-4(1H)-one (17c).
Yellow solid, yield 82%, m.p. > 250 ꢀC; IR (KBr, cmꢁ1): 3406 (NH),
3052 (CH aromatic), 2964, 2930 (CH aliphatic), 1635 (C]O), 1507
(C]C aromatic); 1H NMR (400 MHz, MeOH-d4)
(ppm): 4.01 (s, 3H,
y
d
OCH3), 4.05 (s, 3H, OCH3), 6.89 (s, 1H, vinyl CH), 7.34 (s, 1H, phenyl
CH), 7.63 (s, 1H, phenyl CH), 7.76 (dd, 1H, thiophene CH, J ¼ 5.1,
1.5 Hz), 7.70 (dd, 1H, thiophene CH, J ¼ 5.1, 2.8 Hz), 7.82 (s, 1H, NH),
8.21 (dd, 1H, thiophene CH, J ¼ 2.8, 1.5 Hz); 13C NMR (100 MHz,
MeOH-d4)
d
(ppm): 55.44, 55.64, 98.86, 102.61, 103.98, 116.82,
4.1.10.1. 1-(3-((2-(Furan-2-yl)-6,7-dimethoxyquinolin-4-yl)oxy)pro-
125.57, 126.72, 127.89, 134.26, 136.85, 146.05, 149.07, 155.47, 173.60;
HRESIMS (m/z): [MþH]þ Calcd for C15H14NO3S, 288.06889; found,
288.06915.
pyl)piperidin-4-ol (19a). Yellow solid, yield 75%, m.p. 165e167 ꢀC;
IR (KBr,
aliphatic), 1589, 1560, 1509 (C]C aromatic); 1H NMR (400 MHz,
CDCl3) (ppm): 1.57e1.66 (m, 2H, piperidinyl 2CHH’), 1.76 (br s, 1H,
y
cmꢁ1): 3416 (OH), 3007 (CH aromatic), 2929 (CH
d
4.1.9. General procedure for synthesis of the key intermediates
(18a-c)
The key intermediates 18a-c were synthesized using the same
procedures as 13a-d.
OH), 1.89e1.94 (m, 2H, piperidinyl 2CHH’), 2.12e2.22 (m, 4H,
piperidinyl 2CHH0-N and CH2), 2.60 (t, 2H, CH2eN, J ¼ 7.3 Hz), 2.82
(t, 2H, piperidinyl 2CHH0-N, J ¼ 5.8 Hz), 3.69e3.75 (m, 1H, piper-
idinyl CHeOH), 4 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 4.31 (t, 2H,
CH2eO, J ¼ 6.4 Hz), 6.55 (dd, 1H, furyl CH, J ¼ 3.5, 1.7 Hz), 7.10 (s, 1H,
vinyl CH), 7.12 (dd, 1H, furyl CH, J ¼ 3.5, 0.5 Hz), 7.36 (s, 1H, phenyl
CH), 7.40 (s, 1H, phenyl CH), 7.56 (dd, 1H, furyl CH, J ¼ 1.7, 0.5 Hz);
4.1.9.1. 4-(3-Chloropropoxy)-2-(furan-2-yl)-6,7-dimethoxyquinoline
(18a). Yellow solid, yield 81%, m.p. 139e141 ꢀC; IR (KBr,
y
cmꢁ1):
3092, 3011 (CH aromatic), 2952, 2940, 2879 (CH aliphatic), 1594,
13C NMR (100 MHz, CDCl3)
d (ppm): 26.88, 34.48, 51.20, 55.03,
1565, 1510 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
d
(ppm):
56.03, 56.10, 66.78, 67.93, 95.96, 99.88, 108.09, 108.67, 112.06,
115.02, 143.24, 146.12, 148.49, 148.88, 152.68, 154.21, 160.81; HRE-
SIMS (m/z): [MþH]þ Calcd for C23H29N2O5, 413.20710; found,
413.20676.
2.43 (p, 2H, CH2, J ¼ 6.1 Hz), 3.82 (t, 2H, CH2Cl, J ¼ 6.3 Hz), 4 (s, 3H,
OCH3), 4.02 (s, 3H, OCH3), 4.42 (t, 2H, CH2O, J ¼ 5.9 Hz), 6.56 (dd,1H,
furyl CH, J ¼ 3.4, 1.7 Hz), 7.11 (s, 1H, furyl CH), 7.12 (s, 1H, vinyl CH),
7.33 (s, 1H, phenyl CH), 7.41 (s, 1H, phenyl CH), 7.57 (dd, 1H, furyl
CH, J ¼ 1.7, 0.7 Hz); 13C NMR (100 MHz, CDCl3)
d
(ppm): 31.97, 41.34,
4.1.10.2. 1-(3-((6,7-Dimethoxy-2-(thiophen-2-yl)quinolin-4-yl)oxy)
56.04, 56.12, 64.98, 95.96, 99.72, 108.13, 108.77, 112.08, 114.86,
143.31, 146.16, 148.44, 148.98, 152.76, 154.09, 160.44; HRESIMS (m/
z): [MþH]þ Calcd for C18H19ClNO4, 348.09971; found, 348.09988.
propyl)piperidin-4-ol (19b). Yellow solid, yield 86%, m.p. 88e90 ꢀC;
IR (KBr,
aliphatic), 1589, 1507 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
(ppm): 1.56e1.65 (m, 2H, piperidinyl 2CHH’), 1.73 (br s, 1H, OH),
y
cmꢁ1): 3400 (OH), 3091, 3000 (CH aromatic), 2935 (CH
d
4.1.9.2. 4-(3-Chloropropoxy)-6,7-dimethoxy-2-(thiophen-2-yl)quin-
1.88e1.94 (m, 2H, piperidinyl 2CHH’), 2.12e2.22 (m, 4H, piperidinyl
2CHH0-N and CH2), 2.60 (t, 2H, CH2eN, J ¼ 7.3 Hz), 2.82 (t, 2H,
piperidinyl 2CHH0-N, J ¼ 5.8 Hz), 3.69e3.75 (m, 1H, piperidinyl
CHeOH), 4 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 4.30 (t, 2H, CH2eO,
J ¼ 6.4 Hz), 7.04 (s, 1H, vinyl CH), 7.12 (dd, 1H, thiophene CH, J ¼ 5,
3.7 Hz), 7.35 (s, 1H, phenyl CH), 7.36 (s, 1H, phenyl CH), 7.40 (dd, 1H,
thiophene CH, J ¼ 5, 1.1 Hz), 7.63 (dd, 1H, thiophene CH, J ¼ 3.7,
oline (18b). Yellow solid, yield 76%, m.p. 168e170 ꢀC; IR (KBr,
y
cmꢁ1): 3071, 3005 (CH aromatic), 2934, 2880 (CH aliphatic), 1591,
1511 (C]C aromatic); 1H NMR (400 MHz, CDCl3)
d
(ppm): 2.42 (p,
2H, CH2, J ¼ 6.1 Hz), 3.83 (t, 2H, CH2Cl, J ¼ 6.2 Hz), 4 (s, 3H, OCH3),
4.03 (s, 3H, OCH3), 4.40 (t, 2H, CH2O, J ¼ 6 Hz), 7.05 (s, 1H, vinyl CH),
7.12 (dd, 1H, thiophene CH, J ¼ 5, 3.7 Hz), 7.31 (s, 1H, phenyl CH),
7.37 (s, 1H, phenyl CH), 7.41 (dd, 1H, thiophene CH, J ¼ 5, 1.1 Hz),
7.63 (dd, 1H, thiophene CH, J ¼ 3.7, 1.1 Hz); 13C NMR (100 MHz,
1.1 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 26.89, 34.48, 51.21,
55.03, 56.02, 56.11, 66.72, 67,89, 96.23, 99.91, 108.06, 115.04, 124.62,
127.48, 127.80, 146.01, 146.05148.81, 151.65, 152.64, 160.79; HRE-
SIMS (m/z): [MþH]þ Calcd for C23H29N2O4S, 429.18425; found,
CDCl3) d (ppm): 31.97, 41.36, 56.03, 56.12, 64.91, 96.21, 99.76, 108.10,
114.89, 124.68, 127.57, 127.84, 145.94, 146.06, 148.91, 151.61, 152.71,
17

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
429.18414.
plates were air dried. Bound stain was subsequently solubilized
with 10 mM trizma base, and the absorbance was read on an
automated plate reader at a wavelength of 515 nm. For suspension
cells, the methodology was the same except that the assay was
terminated by fixing settled cells at the bottom of the wells by
4.1.10.3. 1-(3-((6,7-Dimethoxy-2-(thiophen-3-yl)quinolin-4-yl)oxy)
propyl)piperidin-4-ol (19c). Yellow solid, yield 65%, m.p.
158e160 ꢀC; IR (KBr, cmꢁ1): 3401 (OH), 3100, 3000 (CH aromatic),
y
2931 (CH aliphatic), 1591, 1509 (C]C aromatic); 1H NMR (400 MHz,
CDCl3) (ppm): 1.56e1.65 (m, 2H, piperidinyl 2CHH’), 1.72 (br s, 1H,
gently adding 50 mL of 80% TCA (final concentration, 16% TCA).
d
Using the seven absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth was calculated at
each of the drug concentrations levels. Percentage growth inhibi-
tion was calculated as:
OH), 1.89e1.93 (m, 2H, piperidinyl 2CHH’), 2.12e2.22 (m, 4H,
piperidinyl 2CHH0-N and CH2), 2.60 (t, 2H, CH2eN, J ¼ 7.3 Hz), 2.82
(t, 2H, piperidinyl 2CHH0-N, J ¼ 5.8 Hz), 3.69e3.75 (m, 1H, piper-
idinyl CHeOH), 4.01 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 4.30 (t, 2H,
CH2eO, J ¼ 6.4 Hz), 7 (s, 1H, vinyl CH), 7.37 (s, 1H, phenyl CH), 7.39
(s, 1H, phenyl CH), 7.41 (dd, 1H, thiophene CH, J ¼ 5, 3 Hz), 7.76 (dd,
1H, thiophene CH, J ¼ 5, 1.2 Hz), 7.92 (dd, 1H, thiophene CH, J ¼ 3,
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/ ¼ Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti < Tz
1.2 Hz); 13C NMR (100 MHz, CDCl3)
d (ppm): 26.91, 34.48, 51.21,
55.04, 56.03, 56.07, 66.67, 67.88, 97.69, 99.83, 108.13, 114.86, 123.50,
126.08, 126.76, 143.35, 146.15, 148.80, 152.60, 152.87, 160.89; HRE-
SIMS (m/z): [MþH]þ Calcd for C23H29N2O4S, 429.18425; found,
429.18414.
Three dose response parameters were calculated for each
experimental agent. Growth inhibition of 50% (GI50) was calculated
from [(Ti-Tz)/(C-Tz)] x 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
(TGI) was calculated from Ti ¼ Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end of
the drug treatment as compared to that at the beginning) indicating
a net loss of cells following treatment was calculated from [(Ti-Tz)/
Tz] x 100 ¼ ꢁ50.
4.2. In vitro anticancer activity
The cytotoxicity in vitro assay was conducted at National Cancer
Institute (NCI), Bethesda, USA against 59 cancer cell lines, as re-
ported earlier [64,65]. The one-dose data were reported as a mean
graph of the percent growth of treated cells. The number reported
for the one-dose assay was growth relative to the no-drug control
and relative to the time zero number of cells. This allowed detection
of both growth inhibition (values between 0 and 100) and lethality
(values less than 0). For example, a value of 100 means no growth
inhibition. A value of 40 would mean 60% growth inhibition. A value
of 0 means no net growth over the course of the experiment. A
value of ꢁ40 would mean 40% lethality. A value of ꢁ100 means all
cells were dead.
4.3. Topoisomerase I-mediated DNA cleavage assay
A 30-[32P]-labeled 117-bp DNA substrate oligonucleotide was
prepared as described previously [55]. Radiolabeled DNA was
incubated with recombinant human TOP1 in 20
(10 mmol/L Tris-HCl, pH 7.5, 50 mmol/L KCl, 5 mmol/L MgCl2,
0.1 mmol/L EDTA, and 15
g/mL BSA) at 30 ꢀC for 20 min in the
mL reaction buffer
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
m
presence of the indicated drug concentrations. Reactions were
terminated by adding SDS (0.5% final concentration) followed by
the addition of two volumes of loading dye (80% formamide,
10 mmol/L sodium hydroxide, 1 mmol/L sodium EDTA, 0.1% xylene
cyanol, and 0.1% bromophenol blue). Aliquots of reaction mixtures
were subjected to 16% denaturing PAGE. Gels were dried and
visualized by using PhosphorImager and Image Quant software
(Molecular Dynamics).
2 mM
L
-glutamine. For a typical screening experiment, cells were
inoculated into 96 well microtiter plates in 100
mL at plating den-
sities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 ꢀC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line were fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at
the time of drug addition (Tz). Experimental drugs were solubilized
in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate was thawed and diluted
to twice the desired final maximum test concentration with com-
Declaration of competing interest
The authors have declared no conflict of interest.
plete medium containing 50
fold or ½ log serial dilutions were made to provide a total of five
drug concentrations plus control. Aliquots of 100 L of these
different drug dilutions were added to the appropriate microtiter
wells already containing 100 L of medium, resulting in the
required final drug concentrations. Following drug addition, the
plates were incubated for an additional 48 h at 37 ꢀC, 5% CO2, 95%
air, and 100% relative humidity. For adherent cells, the assay was
terminated by the addition of cold TCA. Cells were fixed in situ by
mg/mL gentamicin. Additional four, 10-
Acknowledgement
m
Mostafa M. Elbadawi was supported by MEXT scholarship pro-
vided by the Ministry of Education, Culture, Sports, Science and
Technology of Japan (scholarship no. 182582). Yves Pommier and
Keli K. Agama are supported by the Center for Cancer Research, the
Intramural Program of the National Cancer Institute (Z01-BC-
006161). The authors are thankful to Developmental Therapeutics
Program (DTP) of the National Cancer Institute (NCI) for in vitro
anticancer screening of the newly synthesized compounds. Also,
the authors are grateful to Dr. Sayaka Hatano for help in X-ray
crystallography. Moreover, we would like to thank Professor Nor-
imatsu Morioka and Dr. Yoki Nakamura (Department of Pharma-
cology, Graduate School of Biomedical and Health Sciences,
Hiroshima University, Japan) for their informative discussions.
m
the gentle addition of 50 mL of cold 50% (w/v) TCA (final concen-
tration, 10% TCA) and incubated for 60 min at 4 ꢀC. The supernatant
was discarded, and the plates were washed five times with tap
water and air dried. Sulforhodamine B (SRB) solution (100 mL) at
0.4% (w/v) in 1% acetic acid was added to each well, and plates were
incubated for 10 min at room temperature. After staining, unbound
dye was removed by washing five times with 1% acetic acid and the
18

M.M. Elbadawi, W.M. Eldehna, W. Wang et al.
European Journal of Medicinal Chemistry 215 (2021) 113261
isoforms I and II inhibitors, J. Enzym. Inhib. Med. Chem. 34 (2019) 1457e1464,
https://doi.org/10.1080/14756366.2019.1652282.
Appendix A. Supplementary data
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[20] S. Li, L. Hu, J. Li, J. Zhu, F. Zeng, Q. Huang, L. Qiu, R. Du, R. Cao, Design, synthesis,
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