Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

Article abstract of DOI:10.1080/14756366.2021.1915302

As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth of all malignant tumours-related deaths worldwide. This study reports the synthesis and in?vitro biological assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC₅₀ spanning in ranges 1.48–47.02 and 0.49–68.9 μM, respectively. The three most active benzofurans (4b, 15a and 16a) were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2 inhibitory activity with IC₅₀ equal 77.97, 132.5 and 45.4 nM, respectively.

Full text of DOI:10.1080/14756366.2021.1915302

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Development of 3-methyl/3-
(morpholinomethyl)benzofuran derivatives as
novel antitumor agents towards non-small cell
lung cancer cells
Mohammad M. Al-Sanea, Ghada H. Al-Ansary, Zainab M. Elsayed, Raed M.
Maklad, Eslam B. Elkaeed, Mohamed A. Abdelgawad, Syed Nasir Abbas
Bukhari, Marwa M. Abdel-Aziz, Howayda Suliman & Wagdy M. Eldehna
To cite this article: Mohammad M. Al-Sanea, Ghada H. Al-Ansary, Zainab M. Elsayed, Raed
M. Maklad, Eslam B. Elkaeed, Mohamed A. Abdelgawad, Syed Nasir Abbas Bukhari, Marwa
M. Abdel-Aziz, Howayda Suliman & Wagdy M. Eldehna (2021) Development of 3-methyl/3-
(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell
lung cancer cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 987-999, DOI:
10.1080/14756366.2021.1915302
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 38, NO. 1, 987–999
https://doi.org/10.1080/14756366.2021.1915302
RESEARCH PAPER
Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel
antitumor agents towards non-small cell lung cancer cells
Mohammad M. Al-Saneaa, Ghada H. Al-Ansaryb,c, Zainab M. Elsayedd, Raed M. Maklade,f, Eslam B. Elkaeedg,h
Mohamed A. Abdelgawada,i, Syed Nasir Abbas Bukharia, Marwa M. Abdel-Azizj, Howayda Sulimank and
Wagdy M. Eldehnae
,
b
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia; Department of Pharmaceutical
c
Chemistry, Pharmacy Program, Batterejee Medical College, Jeddah, Saudi Arabia; Department of Pharmaceutical Chemistry, Faculty of
d
Pharmacy, Ain Shams University, Cairo, Egypt; Scientific Research and Innovation Support Unit, Faculty of Pharmacy, Kafrelsheikh University,
e
f
Kafrelsheikh, Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; Institute of
g
Drug Discovery and Development, Kafrelsheikh University, Kafrelsheikh, Egypt; Department of Pharmaceutical Sciences, College of Pharmacy,
AlMaarefa University, Ad Diriyah, Riyadh, Saudi Arabia; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar
University, Nasr City, Cairo, Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef,
Egypt; The Regional Center for Mycology & Biotechnology, Al-Azhar University, Cairo, Egypt; Department of Medical Biochemistry, Faculty of
Medicine, Alexandria University, Alexandria, Egypt
h
i
j
k
ABSTRACT
ARTICLE HISTORY
Received 28 February 2021
Revised 2 April 2021
Accepted 6 April 2021
As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth
of all malignant tumours-related deaths worldwide. This study reports the synthesis and in vitro biological
assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)ben-
zofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcin-
oma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based
derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC50 spanning in
ranges 1.48–47.02 and 0.49–68.9 mM, respectively. The three most active benzofurans (4b, 15a and 16a)
were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and
NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2
inhibitory activity with IC50 equal 77.97, 132.5 and 45.4 nM, respectively.
KEYWORDS
Benzofuran-2-carbohydra-
zide; anticancer agents;
lung cancer; VEGFR-
2 inhibitors
1. Introduction
diverse pharmacophoric groups proved to possess multiple
pharmacological activities for the past decades9,10
Among
these pharmacological activities, benzofuran derivatives exhibit
significant inhibitory carbonic anhydrase11,12 antioxidant13,
anti-
.
Worldwide, lung cancer ranks the most commonly diagnosed can-
cer as the number of cases reported for both sexes merged in
2018 exceeds 2 million new cases accounting for 11.6% of total
cases and 1.8 million lung cancer deaths which presents 18.4% of
the total cancer deaths globally1,2. Although lung cancer is more
commonly diagnosed in males due to the higher incidence of
smoking, yet it is reported to be the second cause of death
among females, preceded by breast cancer2. Accordingly, for the
last few decades, it has been the interest and concern of scientists
to explore new molecules that exhibit significant antiproliferative
activity against the invading lung cancer in the aim of combating
such global health problem3. Interestingly, many of the discovered
molecules proved clinical success for the management of lung
cancer4–6. Yet, the urgent need for more effective and selective
novel anticancer agents is an inevitable necessity which provokes
more rationalised scientific research.
,
anti-Alzheimer’s14, anti-inflammatory15, antibacterial15,16
,
tubercular17, as well as anticancer activities18. Benzofurans were
reported to exert their antiproliferative activities through diverse
mechanisms of cellular proliferation inhibition including apoptosis
induction19–23 and VEGFR-2 inhibitory action24–26
.
The tumour growth mostly depends on the angiogenesis pro-
cess that guarantees formation of new blood vessels from an
existing vasculature27. Angiogenesis process is mainly orchestrated
by diverse pro-angiogenic and anti-angiogenic factors formed by
the tumour cells as well as the host cells. Among the pro-angio-
genic growth factors, vascular endothelial growth factors (VEGFs)
and its receptor VEGFR-2 exert a significant impact on the angio-
genesis process27. Over the past decade, it was well-established
that inhibition of VEGFR-2 is a favourable strategy to afford effi-
cient anticancer agents28.
Benzofurans are regarded as privileged scaffolds upon which
many molecules are pursed that proved clinical utility in many
R
Fruquintinib (ElunateV, Figure 1) is a potent, selective and
fields of medical research7,8. Benzofuran derivatives tagged with orally bioavailable benzofuran-based VEGFR-2 inhibitor29.
CONTACT Wagdy M. Eldehna
wagdy2000@gmail.com
mohmah80@gmail.com
ghada.yassin@bmc.edu.sa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh
33516, Egypt; Mohammad M. Al-Sanea
72341, Saudi Arabia; Ghada H. Al-Ansary
Jeddah, Saudi Arabia
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Aljouf
Department of Pharmaceutical Chemistry, Pharmacy Program, Batterejee Medical College,
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

988
M. M. AL-SANEA ET AL.
Fruquintinib has received its first global approval in China, in treatment of advanced gastric cancer and advanced NSCLC31.
2018, for treatment of metastatic colorectal cancer30. Currently,
Furthermore, PF-00337210 (Figure 1), another benzofuran-based
Fruquintinib is being evaluated in phase III clinical trials for
small molecule, is an orally bioavailable potent inhibitor of VEGFR-
2 that is currently tested in the clinical trials32.
As a part of our ongoing tireless research for discovering novel
anticancer molecules, we herein reported six novel series of ben-
zofuran derivatives. Our design strategy proceeds as two sets of
benzofuran series. The first set is the 3-methylbenzofurans; 4a–d,
6a–c, 8a–c and 11 (Figure 1), whereas the second set is the ben-
zofuran series tagged with morpholino group; 3-(morpholinome-
thyl)benzofurans: 15a–c, 16a–b, 17a–b and 18 (Figure 1). All the
synthesised benzofurans were evaluated for their antiproliferative
activities against lung A549 and NCI-H23 cancer cell lines and
their IC50 values were determined. To test their selectivity poten-
tial for tumour cells, three of the most potent benzofuran deriva-
tives (4b, 15a, and 16a) were selected to be evaluated for their
cytotoxic activity against non-tumorigenic human lung WI-38.
Moreover, in the aim of exploring the mechanistic antiproliferative
activity of the tested molecules at the molecular level, the three
most potent molecules were evaluated for their VEGR-2 inhibitory
activity. In addition, the three most potent antiproliferative com-
pounds (4b, 15a, and 16a) were tested for their antitubercular
activity. Finally, cell cycle analysis studies were conducted, includ-
ing apoptosis assay and Annexin V-FITC assay.
2. Results and discussion
2.1. Chemistry
Preparation of target 3-methylbenzofurans (4a–d, 6a–c, 8a–c and
11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b
and 18) are depicted in Schemes 1–4.
Figure 1. Chemical structures of benzofurans Fruquintinib and PF-00337210, as
well as structures of target 2-methylbenzofurans (4a–d, 6a–c and 8a–c) and 3-
(morpholinomethyl)benzofurans (15a–c, 16a–b and 17a–b).
Scheme 1. Synthesis of target 2-methylbenzofurans 4a–d and 6a–c; reagents and conditions: (i) NH2NH2.H2O/isopropyl alcohol/reflux 2 h and (ii) dry toluene/reflux 7 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
989
Scheme 2. Synthesis of target 2-methylbenzofurans 8a–c and 11; reagents and conditions: (i) Ethanol/Cat. Acetic acid/reflux 3 h, (ii) ethanol/cat. Acetic acid/reflux 7 h.
Scheme 3. Synthesis of key intermediate 3-(morpholinomethyl)benzofuran-2-carbohydrazide 14; Reagents and conditions: (i) NBS/carbon tetrachloride/dibenzoyl perox-
ide/reflux 16 h, (ii) Morpholine/Acetonitrile/K2CO3/KI/reflux 8 h, and (iii) NH2NH2.H2O/isopropyl alcohol/reflux 2 h.
First, 3-methylbenzofuran-2-carbohydrazide 2 was prepared
On the other hand, N-bromosuccinimide (NBS) reagent was uti-
from its precursor ethyl 3-methylbenzofuran-2-carboxylate 1 by lised to selectively brominate the reactive benzofuran-3-ylmethyl
nucleophilic substitution reaction using hydrazine hydrate as a group, in a mild bromination step, affording ethyl 3-(bromome-
nucleophile. Then, upon reacting hydrazide 2 with isothiocyanates thyl)benzofuran-2-carboxylate 12. Thereafter, a nucleophilic substi-
3a–d and isocyanates 5a–c in anhydrous toluene, only the more tution reaction at the bromomethyl carbon of compound 12 was
nucleophilic N2 adds to the C¼N double bond of either 3a–d or carried out by morpholine and was triggered by nucleophilic
5a–c to afford the desired 1-acylatedthiosemicarbazides (4a–d) assistance with iodide ion to furnish ethyl 3-(morpholinomethyl)-
and 1-acylatedsemicarbazides (6a–c), respectively (Scheme 1).
benzofuran-2-carboxylate 13. The soft iodide nucleophile would
Furthermore, the key intermediate 3-methylbenzofuran-2-car- selectively attack the soft electrophilic bromomethyl carbon rather
bohydrazide 2 underwent a nucleophilic addition–elimination than the harder one (i.e. the ester in this case) according to the
reaction with the carbonyl group of aldehydes 7a–b as well as HSAB theory33. Such nucleophilic assistance exerted by iodide ion
ketones 9 and 10 to afford aldohydrazones 8a–b and ketohydra- enhances the reactivity of the 3-methylene group via enhance-
zones 8c and 11, respectively. This condensation reaction was car- ment of the leaving group ability by replacing a quite good leav-
ried out in refluxing ethanol with presence of catalytic drops of ing group (i.e. bromide) with a better one (iodide). The next step
acetic acid (Scheme 2).
was quite similar to the preparation of hydrazide 2, in this step,

990
M. M. AL-SANEA ET AL.
Scheme 4. Synthesis of target 3-(morpholinomethyl)benzofurans 15–18; reagents and conditions: (i) dry toluene/reflux 7 h and (ii) ethanol/cat. Acetic acid/reflux 3 h.
the 3-(morpholinomethyl)benzofuran-2-carboxylate ester 13 was
treated with hydrazine hydrate in refluxing ethanol to afford the
desired hydrazide 14 (Scheme 3).
Finally, target 1-acylatedthiosemicarbazides (15a–c), 1-acylated-
semicarbazides (16a,b) and aldohydrazones 17a–b and
ketohydrazones 18 were prepared through reaction of 3-(morpho-
linomethyl)benzofuran-2-carbohydrazide 14 with different isothio- 16a
cyanatobenzene 3a–d, isocyanatobenzene 5a–c and carbonyl
moieties 7a,c and 10, Scheme 4.
Table 1. Cytotoxic impact against non-tumorigenic human lung WI-38 cell line,
as well as mean tumour selectivity index (S.I.) (WI-38/A549 and NCI-H23).
IC50 (mM)
Mean tumour
Compounds
WI-38
A549
NCI-H23
selectivity
4b
15a
19.51 1.18
32.81 1.98
12.86 0.78
1.48 0.08
5.27 0.29
1.50 0.08
5.90 0.29
2.52 0.13
0.49 0.02
5.3
8.4
12.9
methoxy group substituted on the phenyl ring, exhibited antiproli-
ferative activity which is equivalent to that recorded by stauro-
sporine (IC50 ¼ 1.52 mM and 1.50 mM, respectively).
2.2. Biological evaluation
Alternatively, evaluating the antiproliferative activity of the syn-
thesised compounds against NCI-H23 cancer cell line reveals that
all the compounds showed lower antiproliferative activity evident
by their higher IC50 values. The IC50 values of the 3-methylbenzo-
furan series (4a–d, 6a–c, 8a–c and 11) range from 67.22 mM down
to 5.90 mM which is much higher than those values recorded
against A549 cancer cell line (47.02 mM–1.48 mM).
2.2.1. Anti-proliferative activity towards non-small cell lung carcin-
oma A549 and NCI-H23 cell lines
The antiproliferative activity of the synthesised derivatives of the
3-methylbenzofuran series (4a–d, 6a–c, 8a–c and 11) and the 3-
(morpholinomethyl)benzofuran series (15a–c, 16a–b, 17a–b and
18) was evaluated against non-small cell lung carcinoma (NSCLC)
A549 and NCI-H23 cell lines, following the procedures of the MTT
assay34. The antiproliferative activity of the 3-methylbenzofuran
derivatives (4a–d, 6a–c, 8a–c and 11) against A549 cancer cell
line ranges from 47.02 mM down to 1.48 mM. Superiorly, 3-methyl-
benzofuran derivative 4c, with a para-methoxy group grafted on
the terminal phenyl ring, showed the highest antiproliferative
activity (IC50 ¼ 1.48 mM) which is comparable to staurosporine
(IC50 ¼ 1.52 mM), Table 1. Whereas, the antiproliferative potency of
the 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and
18) showed narrower range of activity against the same cell line
with IC50 values ranging from 18.89 mM down to 1.5 mM, which
verifies that substitution of the benzofuran ring by a methyl mor-
pholino group boosts the cytotoxic activity against cell line lung
Controversially, the IC50 values of 3-(morpholinomethyl)benzo-
furan series (15a–c, 16a–b, 17a–b and 18) demonstrated more
potent antiproliferative activity against NCI-H23 cancer cell line
which ranges from 29.75 mM down to 0.49 mM with the exception
of compound 15b that possessed poor antiproliferative activity
with an IC50 value of 68.9 mM. These results again reinforces the
previously observed antiproliferative activity which supports the
conclusion that substituting the benzofuran ring with a methyl
morpholino group is advantageous for the cytotoxic activity
against both tested lung cancer cell lines; A549 and NCI-H23.
Interestingly, compound 16a with a 3-methoxy group proved to
be the most potent compound with 2.53 fold more potent as
compared to staurosporine. Moreover, 15a (IC50 ¼ 2.52 mM) and
15c (IC50 ¼ 2.218 mM) exhibited excellent antiproliferative activity
carcinoma A549. The most potent compound 16a, bearing a 3- as compared to staurosporine (IC50 ¼ 1.24 mM).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
991
2.2.2. Cell cycle analysis
6.90-fold, respectively, and in the sub-G1 phase by 21.76-, 13.13-
To understand the underlying mechanism of the tumour suppres- and 12.54-fold, respectively, as compared to the control (Figure 2).
sion activity of the tested benzofurans, the three most active com-
pounds in this study, 4b, 15a and 16a were further investigated
for their effects on the cell cycle progression in A549 (for 4b) and
NCI-H23 (for 15a and 16a) cancer cell lines.
2.2.3. Apoptosis assay
The apoptotic effect of the three benzofurans (4b, 15a and 16a)
was evaluated using Annexin VFITC/PI (AV/PI) dual staining assay
to validate if their observed cytotoxic effect is due to non-specific
Analysing the results for the three benzofurans on the two
investigated cell lines revealed that they affected the cell cycle
necrosis or physiological apoptosis. The apoptotic effect of 3-
progression in a similar manner, where 4b, 15a and 16a reduced
methylbenzofuran derivative 4b was observed against A549 cell
the G0–G1 phase by 0.67-, 0.56- and 0.47-folds, respectively.
line using a control, where 4b affected 42.05% of apoptosis com-
Moreover, the S phase was also attenuated by 0.46, 0.50 and, 0.51
pared to that triggered by control which was 1.37% (Figure 3).
fold, respectively. This reduction was accompanied by augmenta-
Moreover, the apoptotic effects of 3-(morpholinomethyl)benzo-
tion of the cell population in the G2/M phase by 2.67-, 5.33- and
furan derivatives 15a and 16a were evaluated in NCI-H23 cancer
Figure 2. Effect of 3-methylbenzofuran derivative 4b on the phases of cell cycle of A549 cells, and effect of 3-(morpholinomethyl)benzofuran derivatives 15a and 16a
on the phases of cell cycle of NCI-H23 cells.
Figure 3. Effect of 3-methylbenzofuran derivative 4b on the percentage of annexin V-FITC-positive staining in Non-small cell lung cancer A549 cells.
Figure 4. Effect of 3-(morpholinomethyl)benzofuran derivatives 15a and 16a on the percentage of annexin V-FITC-positive staining in Non-small cell lung cancer NCI-
H23 cells.

992
M. M. AL-SANEA ET AL.
cell line. Results revealed that benzofuran 15a produced 34.59% indicative parameter for the relative safety of the three tested
of total apoptosis, as well as, benzofuran 16a caused 36.81% of
total apoptosis compared to the control which produced only
2.09% of total apoptosis (Figure 4). Based on the previous results,
compounds 4b, 15a and 16a proved to induce apoptosis signifi-
cantly in both cancer cell lines; A-549 and NCI-H23.
compounds is the mean tumour selectivity value which was calcu-
lated for the three compounds to be 5.3, 8.4 and 12.9, respect-
ively. These values prove that the three tested benzofurans
exhibited selective antiproliferative activity against tumour cells
while sparing the normal cells.
2.2.4. Cytotoxic activity against non-tumorigenic human lung WI-
38 cell line
2.2.5. VEGFR-2 inhibitory assay
Antiproliferative agents are designed to inhibit the cellular growth
of tumour cells selectively, while sparing the normal cells.
Accordingly, the three most potent benzofurans; 4b, 15a and 16a
were evaluated for their cytotoxic activity against non-tumorigenic
human lung WI-38 cell line and their IC50 values were compared
to those detected against the lung cancer cell lines A549 and NCI-
H23 (Table 2).
Scrutinising the results revealed that the three tested benzofur-
ans are barely toxic to the non-tumerigenic human lung WI-38
cell line with high IC50 values of 19.51 mM, 32.81 mM and 12.86 mM,
respectively (Table 2). On the other hand, they exhibited marked
antiproliferative activity against the lung cancer cell lines A549
and NCI-H23 with much lower IC50 values of 1.48 mM, 5.27 mM and
1.5 mM for A549 cancer cell line, and 5.90 mM, 2.52 mM and 0.49 mM
Surveying the literature revealed that VEGFR-2 inhibition is one of
the well-known reported antiproliferative mechanisms of benzo-
furan-based small molecules24–26,29–32. Accordingly, the potential
VEGFR-2 inhibitory activity of the three most potent molecules;
4b, 15a and 16a were evaluated in the aim of exploring the cellu-
lar proliferation inhibitory potential of the tested compounds at
the molecular level. Interestingly, results revealed that all of the
three tested benzofurans (4b, 15a and 16a) exhibited excellent
inhibition of VEGFR-2 at nanomolar range with IC50 values of
77.97 nM, 132.5 nM and 45.4 nM, although none of them was
superior to sorafenib (IC50¼ 34.68 nM) (Table 3). This suggests that
the herein reported benzofurans exert their antiproliferative activ-
ity by inhibition of VEGFR-2 resulting in inhibition of angiogenesis
for NCI-H23 cancer cell line, respectively (Table 2). A much more and ultimately cell death.
Table 2. In vitro anti-proliferative activity of target 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b
and 18) against lung A549 and NCI-H23 cancer cell lines.
IC50 (mM)a
Compounds
X
R
A549
NCI-H23
4a
4b
4c
4d
6a
6b
6c
8a
8b
8c
11
15a
15b
15c
16a
16b
S
S
S
3-OCH3
4-OCH3
3-CF3
4-F
3-OCH3
4-OCH3
4-Cl-3-CF3
3-OCH3
4-OCH3
4-OCH3
–
3-OCH3
4-OCH3
3-CF3
3-OCH3
4-OCH3
H
10.09 0.55
1.48 0.08
36.49 0.2
12.79 0.7
15.11 0.83
12.5 0.68
47.02 2.57
44.1 2.41
9.204 0.5
16.35 0.89
30.19 1.65
5.273 0.29
5.7 0.31
6.303 0.34
1.50 0.08
18.89 1.03
6.01 0.03
10.83 0.59
3.697 0.2
1.52 0.05
34.18 1.71
5.90 0.29
24.36 0.12
67.22 3.36
56.66 2.83
9.24 0.46
24.54 1.23
14.89 0.74
6.148 0.31
13.95 0.7
12.05 0.6
2.525 0.13
68.9 3.44
2.218 0.11
0.49 0.02
5.089 0.25
22.97 1.15
3.043 0.15
29.75 1.49
1.24 0.02
S
O
O
O
H
H
CH3
–
S
S
S
O
O
H
H
–
17a
17b
18
4-OCH3
–
Staurosporine
–
–
aIC50 values are the mean SD of three separate experiments.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
993
Table 3. Inhibitory activity of target benzofurans 4b,
15a and 16a against VEGFR-2.
moderate to weak anti-tubercular activity. The 3-(morpholinome-
thyl)benzofuran derivatives 15a and 16a revealed moderate activ-
ity with MIC values equal 62.5 and 125 mg/ml, respectively,
whereas the 3-methylbenzofuran derivative 4b showed high MIC
value equals 250 mg/ml (Table 4).
IC50 (nM)
Compounds
VEGFR-2
4b
15a
16a
Sorafenib
77.97 4.6
132.5 7.8
45.4 2.7
34.68 2.6
2.4. Docking studies
This section aimed to predict the plausible binding mode of target
benzofuran-based small molecules herein reported with VEGFR-2
binding site, as well as correlating the retrieved biological activ-
ities with binding poses to establish guidance for future optimisa-
tion of the lead compounds. As it is an error prone technique,
molecular docking is to be validated by comparison to an experi-
mental reference; accordingly the co-crystalized Sorafenib was re-
docked to its VEGFR-2 active site (PDB: 4ASD37) where the calcu-
lated RMSD values between the co-crystalized Sorafenib and
docked poses were 0.71 Å (Supporting materials, Figure S1). Also,
the re-docking validation perfectly reproduced all original interac-
tions between the co-crystallized Sorafenib and the VEGFR-2
active site.
The examined benzofuran derivatives (4b, 15a and 16a)
achieved favourable interaction pattern and good binding affin-
ities (S ¼ ꢀ9.9, ꢀ10.5 and ꢀ10.4 kcal/mole, Table 5) within their
VEGFR-2, in comparison to the reference Sorafenib that achieved a
score of ꢀ11.1 kcal/mole. In details, benzofuran derivatives 4b,
15a and 16a were successfully engaged in many types of hydro-
phobic and hydrogen bonding interactions. Of particular import-
ance, the three benzofurans were engaged in two hydrogen
bonding interactions through their NH and carbonyl oxygen
(C¼O) functionalities of the hydrazide linker with the key GLU-885
and ASP-1046 amino acids residues, respectively (Figures 5–7), In
addition, the oxygen of the grafted methoxy group on the ter-
minal phenyl moieties acted as HBA and achieved a third hydro-
gen bond interaction with CYS-1045, Figures 5–7.
Table 4. Anti-tubercular activity of the target com-
pounds 4b, 15a and 16a.
Compounds
MIC (mg/ml)
4b
500
62.5
125
0.24
15a
16a
INH
Table 5. The detailed bonding interactions and binding scores for the benzofur-
ans 4b, 15a and 16a.
Score
Distance
(Å)
Compounds
kcal\mole
Bonding interaction
4b
ꢀ9.9
Hydrogen bond with GLU885
Hydrogen bond with CYS1045
Hydrogen bond with ASP1046
Pi-alkyl with LEU889
2.66
2.38
3.05
5.37
4.88
2.67
5.41
4.17
4.91
2.68
2.88
2.47
2.58
5.33
5.13
4.57
5.04
5.46
5.34
4.33
4.66
2.29
2.87
2.47
2.93
5.33
5.25
5.18
4.54
5.04
5.40
5.48
4.07
4.79
2.29
Pi-anion with ASP1046
Pi-donor with ASP1046
Pi-alkyl with LYS868
Pi-alkyl with CYS1045
Pi–Pi interaction with PHE1047
Pi-lone pair with PHE1047
Hydrogen bond with GLU885
Hydrogen bond with CYS1045
Hydrogen bond with ASP1046
Pi-alkyl with LEU889
15a
ꢀ10.4
Alkyl–alkyl with VAL898
Alkyl–alkyl with LEU1019
Alkyl–alkyl with ILE1044
Pi-alkyl with HIS1026
Pi-alkyl with LYS868
Pi-alkyl with CYS1045
Furthermore, the appended morpholine moiety in benzofurans
15a and 16a was able to form various hydrophobic interactions
with non-polar residues such as VAL-898, LEU-1019 and ILE-1044,
which should explain the higher energy score achieved by com-
pounds 15a and 16a than compound 4b that lacks the morpho-
Pi–Pi interaction with PHE1047
Pi-lone pair with PHE1047
Hydrogen bond with GLU885
Hydrogen bond with CYS1045
Hydrogen bond with ASP1046
Pi-alkyl with LEU889
16a
ꢀ10.5
line moiety, Figures 5–7. Table
5 summarises the bonding
interactions between benzofurans 4b, 15a and 16a and VEGFR-2
binding site.
Pi-alkyl with ILE888
Alkyl–alkyl with VAL898
Alkyl–alkyl with LEU1019
Alkyl–alkyl with ILE1044
Pi-alkyl with HIS1026
Pi-alkyl with LYS868
Pi-alkyl with CYS1045
3. Conclusions
The current study presented the synthesis and in vitro biological
assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c
and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b,
17a–b and 18) as potential anticancer agents towards non-small
cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2
inhibitory activity. The 3-methylbenzofuran counterparts 4c, with a
Pi–Pi interaction with PHE1047
Pi-lone pair with PHE1047
2.2. Anti-tubercular activity
Lung infections, headed by tuberculosis have been implicated as para-methoxy group grafted on the terminal phenyl ring, exerted
potentially contributing to lung cancer35. In this study, we decided the best antiproliferative activity against A549 cell line (IC50
¼
to explore the potential anti-tubercular activity of target benzo- 1.48 mM) which is comparable to staurosporine (IC50 ¼ 1.52 mM),
furan derivatives17. The three most promising benzofuran-based whereas the 3-(morpholinomethyl)benzofurans 15a, 15c and 16a
derivatives herein reported (4b, 15a and 16a) were assessed for displayed excellent activity against NCI-H23 cell line (IC50 ¼ 2.52,
their potential anti-tubercular activity towards M. tuberculosis fol- 2.21 and 0.49 mM). Benzofurans (4b, 15a and 16a) were further
lowing the procedures of the Microplate Alamar Blue Assay36, and investigated for their effects on the cell cycle progression and
using INH as a reference anti-TB drug. The results are expressed apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell
as minimum inhibitory concentration and presented in Table 4.
lines. The examined compounds significantly affected the cell
The results of the Microplate Alamar Blue assay highlighted cycle progression and provoked apoptosis within the tested
that the tested benzofuran derivatives (4b, 15a and 16a) exerted cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed

994
M. M. AL-SANEA ET AL.
Figure 5. 2D (A) and 3D (B) interactions of 3-methylbenzofuran derivative 4b within VEGFR-2 binding site (PDB: 4ASD).
Figure 6. 2D (A) and 3D (B) interactions of 3-(morpholinomethyl)benzofuran derivative 15a within VEGFR-2 binding site (PDB: 4ASD).
Figure 7. 2D (A) and 3D (B) interactions of 3-(morpholinomethyl)benzofuran derivative 16a within VEGFR-2 binding site (PDB: 4ASD).
good VEGFR-2 inhibitory activity with an IC50 value equal to 77.97, 5.3, 8.4 and 12.9, respectively. The conducted molecular docking
132.5 and 45.4 nM, respectively. In addition, the cytotoxic impact study highlighted that benzofuran derivatives 4b, 15a and 16a
of the three benzofurans was assessed towards normal lung WI-38 were successfully engaged in many types of hydrophobic and
cells, where they elicited a mean tumour selectivity values equal hydrogen bonding interactions. Of particular importance, they

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
995
were engaged in two hydrogen bonding interactions through 112.06, 66.63 (O(CH2)2), 53.25 (N(CH2)2), 51.01 (CH2). Anal. Calcd.
their NH and carbonyl oxygen (C¼O) functionalities of the hydra- for C14H17N3O3 (275.31): C, 61.08; H, 6.22; N, 15.26; Found: C,
zide linker with the key GLU-885 and ASP-1046 amino acids resi- 60.84; H, 6.25; N, 15.33.
dues, respectively, whereas, the oxygen of the grafted methoxy
group achieved a third hydrogen bond interaction with CYS-1045.
4.1.4. General method for the synthesis of thiosemicarbazides
4a–d, 15a–c and semicarbazides 6a–c, 16a,b
4. Experimental
The proper isocyanate or isothiocyanate derivative (0.7 mmol) was
dissolved in 5 ml of stirred dry toluene. Similarly, a solution of the
proper hydrazide 2 or 14 (0.6 mmol) in 3 ml of hot stirred toluene/
dioxane solvent mixture (5:1, respectively) was prepared. To this
latter solution was dropwise added the isocyanate or isothiocyan-
ate solution in toluene, and then the reaction mixture was
refluxed for 7 h. The absence of the TLC spot of the starting
hydrazide was safely considered a sign of the reaction coming to
an end. After that, the precipitate formed upon cooling was fil-
tered off, dried by suction, washed with toluene (3 ꢂ 4 ml), dried
again in hot air oven for 5 h at 90 ꢁC. Further recrystallization from
dioxane-toluene mixture affords the desired thiosemicarbazides
4a–d, 15a–c and semicarbazides 6a–c, 16a,b in good yield.
4.1. Chemistry
Solvents used were of HPLC grade and purchased from Sigma-
Aldrich (Chicago, IL). Reaction follow up was carried out using pre-
coated F254 Merck TLC plates (Kenilworth, NJ). Shimadzu FT-IR
spectrometer (Shimadzu, Kyoto, Japan) was used for functional
group analysis of the synthesised compounds. NMR spectrometric
analyses were carried out using Bruker-Avance 400 NMR spec-
trometer (100 MHz for 13C- and 400 MHz for H-NMR experiments,
1
respectively). Chemical shifts were recorded in ppm after setting
standard solvent signals (DMSO-d6 at d: 2.54 ppm for 1H- and d:
40.45 ppm for 13C NMR spectra). Multiplicities were reported with
their first-order J coupling constants (Hz) for doublets (d); triplets
(t); quartettes (q) as well as second-order J constants (whenever
possible) for the second-order splitting (e.g. doublet of triplets dt).
Stuart apparatus was used for determination of melting points.
FLASH 2000 CHNS/O analyser was utilised for performing elemen-
4.1.4.1. N-(3-Methoxyphenyl)-2–(3-methylbenzofuran-2-carbonyl)-
hydrazine-1-carbothioamide (4a). White crystals, mp
¼
172–174 ꢁC, yield 80%; IR (cmꢀ1): 3484 (br, NH groups), 1653 (C¼O
hydrazide), 1276 (C¼S thiosemicarbazide). 1H NMR (400 MHz,
DMSO-d6) d: 2.61 (s, CH3 C-3 of benzofuran, 3H), 3.77 (s, OCH3,
3H), 6.77 (d, H-4 of 3-CH3OC6H4-, J ¼ 8.1 Hz, 1H), 7.11 (d, H-6 of 3-
CH3OC6H4-, J ¼ 7.9 Hz, 1H), 7.22 (s, H-2 of 3-CH3OC6H4-, 1H), 7.26
(t, H-5 of 3-CH3OC6H4-, J ¼ 8.1 Hz, 1H), 7.41 (t, H-5 of benzofuran,
J ¼ 7.4 Hz, 1H), 7.55 (t, H-6 of benzofuran, J ¼ 7.7 Hz, 1H), 7.66 (d,
H-7 of benzofuran, J ¼ 8.3 Hz, 1H), 7.82 (d, H-4 of benzofuran,
J ¼ 7.8 Hz, 1H), 9.79 (s, NH, 2H), 10.65 (s, NH, 1H). 13C NMR
(100 MHz, DMSO-d6) 181.29 (C¼S), 159.76 (C¼O), 153.3, 142.54,
140.82, 129.35, 129.14, 128.02, 123.8, 122.85, 121.66, 118.19,
112.13, 111.77, 110.83, 55.56 (OCH3), 9.27 (CH3). Anal. Calcd. for
C18H17N3O3S (355.41): C, 60.83; H, 4.82; N, 11.82; O, 13.50; S, 9.02.
Found: C, 61.11; H, 4.85; N, 11.89.
tal analysis. Compounds
ously reported.
1
and 223, and 1238 were previ-
4.1.2. Preparation of ethyl 3-(morpholinomethyl)benzofuran-2-
carboxylate (13)
Compound 13 was prepared by refluxing ethyl 3-(bromomethyl)-
benzofuran-2-carboxylate 12 (1.98 g, 7 mmol) in acetonitrile with
an excess of morpholine (1.31 g, 15 mmol) using anhydrous potas-
sium carbonate as a base. The reaction completion was followed
up by TLC. After consumption of the ester 12, the inorganic salts
were filtered off. Then, the solvent was evaporated and the resi-
due recrystallized from ether-DCM (3:1) giving the desired product
as white powder; yield (1.56 g 77%). This product was used in the
next step for synthesis of hydrazide 14 without further
purification.
4.1.4.2. N-(4-Methoxyphenyl)-2-(3-methylbenzofuran-2-carbonyl)-
hydrazine-1-carbothioamide (4b). White crystals, mp
¼
190–193 ꢁC, yield 74%; IR: ѵ cmꢀ1: 3324, 3228, 3166 (NH groups),
1676 (C¼O hydrazide), 1248 (C¼S thiosemicarbazide). 1H NMR
(400 MHz, DMSO-d6) d 10.63 (s, 1H), 9.75 (s, 1H), 9.68 (s, 1H), 7.81
(d, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 8.3 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H),
7.40 (t, J ¼ 7.5 Hz, 1H), 7.34 (d, J ¼ 8.1 Hz, 2H), 6.93 (d, J ¼ 8.5 Hz,
2H), 3.78 (s, OCH3, 3H), 2.61 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
181.71 (C¼S), 159.72 (C¼O), 157.16, 153.29, 142.61, 132.55, 129.36,
128.69, 127.98, 127.78, 123.77, 122.75, 121.63, 113.64, 112.11,
55.66 (OCH3), 9.28 (CH3). Anal. Calcd. for C18H17N3O3S (355.41): C,
60.83; H, 4.82; N, 11.82; O, 13.50; S, 9.02. Found: C, 61.07; H, 4.86;
N, 11.91.
4.1.3. General method for preparation of hydrazides 2 and 14
The appropriate ester 1 or 13 (4 mmol) was dissolved in absolute
isopropyl alcohol (12 ml). Then, (1 g, 20 mmol) of 99% hydrazine
hydrate was drop-wise added before the whole mixture was
refluxed for 2 h. Solvent and most reagents were then distilled off,
and the residue washed several times with water. Crystallisation
from ethanol 85% afforded the final crystalline hydrazide.
4.1.3.1. 3-Methylbenzofuran-2-carbohydrazide (2). White crystals,
mp ¼ 138–140 ꢁC (reported mp ¼ 135 ꢁC38), yield ¼ 75%.
4.1.4.3. 2-(3-Methylbenzofuran-2-carbonyl)-N-(3-(trifluoromethyl)-
4.1.3.2. 3-(Morpholinomethyl)benzofuran-2-carbohydrazide (14). phenyl)hydrazine-1-carbothioamide (4c). White crystals, mp ¼
White crystals, mp ¼ 207–209 ꢁC, yield 70%; 1H NMR (400 MHz, 170–172 ꢁC, yield 75%; IR: 3242 (br, NH groups), 1676 (C¼O hydra-
DMSO-d6) d: 10.45 (s, NH, 1H), 7.90 (d, H-4 of benzofuran, zide), 1256 (C¼S thiosemicarbazide). 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.0 Hz, 1H), 7.60 (d, H-7 of benzofuran, J ¼ 8.0 Hz, 1H), 7.43 (t, d 10.74 (s, 1H), 10.05 (s, 1H), 10.02 (s, 1H), 7.91 (d, J ¼ 6.9 Hz, 2H),
H-6 of benzofuran, J ¼ 8.0 Hz, 1H), 7.32 (t, H-5 of benzofuran, 7.83 (d, J ¼ 7.8 Hz, 1H), 7.67 (d, J ¼ 8.3 Hz, 1H), 7.59 (d, J ¼ 9.3 Hz,
J ¼ 7.2 Hz, 1H), 4.62 (s, 2H, NH2), 3.97 (s, 2H, –CH2-morpholine), 1H), 7.54 (t, J ¼ 7.1 Hz, 2H), 7.41 (t, J ¼ 7.5 Hz, 1H), 2.62 (s, 3H). 13C
3.58 (s, 4H, CH2OCH2 of morpholinyl moiety), 2.51 (s, 4H, CH2NCH2 NMR (100 MHz, DMSO-d6) 181.44 (C¼S), 159.76 (C¼O), 153.32,
of morpholinyl moiety). 13 C NMR (100 MHz, DMSO-d6) 158.84 142.42, 140.55, 129.76, 129.33, 128.91, 128.11, 125.92, 123.84,
(C¼O), 153.44, 145.47, 128.88, 127.19, 123.79, 122.26, 119.59, 123.21, 123.21, 122.14, 121.70, 120.51, 112.13, 9.28 (CH3). Anal.

996
M. M. AL-SANEA ET AL.
Calcd. for C18H14F3N3O2S (393.38): C, 54.96; H, 3.59; F, 14.49; N, NMR (100 MHz, DMSO-d6) 180.98 (C¼S), 159.50 (C¼O), 153.61,
10.68; O, 8.13; S, 8.15. Found: C, 55.21; H, 3.61; N, 10.61.
145.29, 140.77, 137.83, 129.38, 128.74, 128.69, 127.80, 125.80,
124.07, 122.28, 120.90, 118.09, 112.26, 110.82, 66.83 (O(CH2)2),
55.56 (OCH3), 52.95 (N(CH2)2), 50.70 (CH2). Anal. Calcd. for
C22H24N4O4S (440.52): C, 59.98; H, 5.49; N, 12.72; O, 14.53; S, 7.28.
Found: C, 60.24; H, 5.55; N, 12.79.
4.1.4.4. N-(4-Fluorophenyl)-2-(3-methylbenzofuran-2-carbonyl)hy-
drazine-1-carbothioamide (4d). White crystals, mp ¼ 206–210 ꢁC,
yield 83%; IR: 3305, 3186 (br), (NH groups), 1652 (C¼O hydrazide),
1224 (C¼S thiosemicarbazide). 1H NMR (400 MHz, DMSO-d6) d
10.69 (s, 1H), 9.87 (s, 1H), 9.83 (s, 1H), 7.82 (d, J ¼ 7.7 Hz, 1H), 7.66
(d, J ¼ 8.3 Hz, 1H), 7.58–7.52 (m, 1H), 7.52–7.44 (m, 2H), 7.40 (t,
J ¼ 7.3 Hz, 1H), 7.20 (t, J ¼ 8.8 Hz, 2H), 2.61 (s, 3H). Anal. Calcd. for
C17H14FN3O2S (343.38): C, 59.46; H, 4.11; F, 5.53; N, 12.24; O, 9.32;
S, 9.34. Found: C, 59.67; H, 4.08; N, 12.35.
4.1.4.9. N-(4-Methoxyphenyl)-2-(3-(morpholinomethyl)benzofuran-
2-carbonyl)hydrazine-1-carbothioamide (15b). White crystals, mp
¼ 198–200 ꢁC, yield 79%; IR: 3273 (br), 3230 (NH groups), 1643
(C¼O hydrazide), 1239 (C¼S thiosemicarbazide). 1H NMR
(400 MHz, DMSO-d6) d 11.94 (s, 1H), 9.80 (s, 1H), 9.73 (s, 1H), 7.99
(d, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H),
7.41 (t, J ¼ 7.4 Hz, 1H), 7.34 (d, J ¼ 6.7 Hz, 2H), 6.94 (d, J ¼ 8.2 Hz,
2H), 4.07 (s, 2H), 3.78 (s, OCH3, 3H), 3.63 (s, 4H, CH2OCH2 of mor-
pholinyl moiety), 2.57 (s, 4H, CH2NCH2 of morpholinyl moiety). 13C
NMR (100 MHz, DMSO-d6) 181.60 (C¼S), 159.13 (C¼O), 157.22,
153.59, 145.29, 132.44, 129.38, 128.76, 128.69, 127.77, 124.04,
122.28, 120.85, 113.81, 112.24, 66.83 (O(CH2)2), 55.68 (OCH3), 52.97
(N(CH2)2), 50.74 (CH2). Anal. Calcd. for C22H24N4O4S (440.52): C,
59.98; H, 5.49; N, 12.72; O, 14.53; S, 7.28. Found: C, 60.18; H, 5.47;
N, 12.75.
4.1.4.5. N-(3-Methoxyphenyl)-2-(3-methylbenzofuran-2-carbonyl)-
hydrazine-1-carboxamide (6a). White crystals, mp ¼ 162–164 ꢁC,
yield 72%; IR: 3354, 3299, 3276 (NH groups), 1711 (C¼O hydra-
zide), 1660 (C¼O semicarbazide). 1H NMR (400 MHz, DMSO-d6) d
10.37 (s, 1H), 8.90 (s, 1H), 8.25 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.66
(d, J ¼ 8.3 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.41 (t, J ¼ 7.4 Hz, 1H),
7.23–7.13 (m, 2H), 7.06 (d, J ¼ 7.5 Hz, 1H), 6.58 (d, J ¼ 8.0 Hz, 1H),
3.75 (s, OCH3, 3H), 2.59 (s, 3H). 13 C NMR (100 MHz, DMSO-d6)
160.15 (C¼O), 155.77 (C¼O of urea), 153.34, 142.48, 141.38,
137.83, 129.89, 129.33, 128.69, 127.97, 125.80, 123.80, 122.69,
121.66, 112.13, 111.3, 55.36 (OCH3), 9.18 (CH3). Anal. Calcd. for
C18H17N3O4 (339.35): C, 63.71; H, 5.05; N, 12.38; O, 18.86. Found: C,
63.49; H, 5.09; N, 14.45.
4.1.4.10. 2-(3-(Morpholinomethyl)benzofuran-2-carbonyl)-N-(3-(tri-
fluoromethyl)phenyl)hydrazine-1-carbothioamide (15c). White crys-
tals, mp ¼ 210–212 ꢁC, yield 74%; IR: 3449 (br), 3256 (NH groups),
1652 (C¼O hydrazide), 1260 (C¼S thiosemicarbazide). 1H NMR
(400 MHz, DMSO-d6) d 11.87 (s, 1H), 10.07 (s, 2H), 8.01 (d,
J ¼ 7.8 Hz, 1H), 7.90 (d, J ¼ 7.7 Hz, 2H), 7.72 (d, J ¼ 8.3 Hz, 1H), 7.61
(t, J ¼ 7.8 Hz, 1H), 7.55 (t, J ¼ 7.6 Hz, 2H), 7.42 (t, J ¼ 7.5 Hz, 1H),
4.12 (s, 2H), 3.63 (s, 4H, CH2OCH2 of morpholinyl moiety), 2.59 (s,
4H, CH2NCH2 of morpholinyl moiety). 13C NMR (100 MHz, DMSO-
d6) 181.10 (C¼S), 159.28 (C¼O), 153.60, 144.97, 140.57, 129.67,
129.38, 128.71, 127.89, 125.91, 124.09, 123.21, 122.42, 121.89,
120.50, 112.24, 66.44 (O(CH2)2), 53.01 (N(CH2)2), 50.80 (CH2). Anal.
Calcd. for C22H21F3N4O3S (478.49): C, 55.22; H, 4.42; F, 11.91; N,
11.71; O, 10.03; S, 6.70. Found: C, 55.39; H, 4.38; N, 11.64.
4.1.4.6. N-(4-Methoxyphenyl)-2-(3-methylbenzofuran-2-carbonyl)-
hydrazine-1-carboxamide (6b). White crystals, mp ¼ 236–240 ꢁC,
yield 78%; IR: 3370, 3324, 3288 (NH groups), 1681 (C¼O hydra-
zide), 1650 (C¼O semicarbazide). 1H NMR (400 MHz, DMSO-d6) d
10.34 (s, 1H), 8.72 (s, 1H), 8.16 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.65
(d, J ¼ 8.3 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.48–7.36 (m, 3H), 6.89
(d, J ¼ 8.6 Hz, 2H), 3.74 (s, OCH3, 3H), 2.59 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) 160.16 (C¼O), 156.11 (C¼O of urea), 154.94,
153.32, 142.57, 133.18, 129.35, 128.68, 127.92, 125.80, 123.77,
122.56, 121.64, 120.83, 114.27, 112.11, 55.6 (OCH3), 9.19 (CH3).
Anal. Calcd. for C18H17N3O4 (339.35): C, 63.71; H, 5.05; N, 12.38; O,
18.86. Found: C, 63.89; H, 5.07; N, 12.46.
4.1.4.11. N-(3-Methoxyphenyl)-2-(3-(morpholinomethyl)benzofuran-
2-carbonyl)hydrazine-1-carboxamide (16a). White crystals, mp ¼
238–240 ꢁC, yield 82%; IR: 3305 (br, NH groups), 1711 (C¼O hydra-
zide), 1640 (C¼O semicarbazide). 1H NMR (400 MHz, DMSO-d6) d
11.49 (s, 1H), 8.93 (s, 1H), 8.43 (s, 1H), 7.96 (d, J ¼ 7.6 Hz, 1H), 7.67
(d, J ¼ 8.4 Hz, 1H), 7.49 (t, J ¼ 8.0 Hz, 1H), 7.37 (t, J ¼ 7.6 Hz, 1H),
7.16–7.19 (m, 2H), 7.02 (d, J ¼ 7.6 Hz, 1H), 6.57 (d, J ¼ 8.0 Hz, 1H),
4.01 (s, 2H), 3.72 (s, OCH3, 3H), 3.60 (s, 4H, CH2OCH2 of morpho-
linyl moiety), 2.51 (s, 4H, CH2NCH2 of morpholinyl moiety). 13C
NMR (100 MHz, DMSO-d6) 160.07 (C¼O), 159.29 (C¼O of urea),
155.40, 153.64, 144.99, 141.28, 129.94, 129.38, 128.74, 128.68,
127.75, 124.03, 122.40, 121.03, 112.24, 111.29, 107.78, 104.79,
66.51 (O(CH2)2), 55.38 (OCH3), 53.12 (N(CH2)2), 50.89 (CH2). Anal.
Calcd. for C22H24N4O5 (424.46): C, 62.25; H, 5.70; N, 13.20; O, 18.85.
Found: C, 62.47; H, 5.65; N, 13.26.
4.1.4.7. N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-(3-methylbenzo-
furan-2-carbonyl)hydrazine-1-carboxamide (6c). White crystals, mp
¼ 240–242 ꢁC, yield 80%; IR: 3346, 3296 (br), (NH groups), 1713
(C¼O hydrazide), 1662 (C¼O semicarbazide). 1H NMR (400 MHz,
DMSO-d6) d 10.45 (s, 1H), 9.39 (s, 1H), 8.60 (s, 1H), 8.14 (s, 1H),
7.86 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.65 (t, J ¼ 9.9 Hz, 2H), 7.55 (t,
J ¼ 7.7 Hz, 1H), 7.40 (t, J ¼ 7.4 Hz, 1H), 2.60 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) 160.21 (C¼O), 155.87 (C¼O of urea), 153.35,
142.40, 139.88, 137.82, 132.33, 129.31, 128.67, 127.38, 126.85,
125.78, 124.67, 123.82, 122.91, 119.24, 112.13, 9.18 (CH3). Anal.
Calcd. for C18H13ClF3N3O3 (411.77): C, 52.51; H, 3.18; Cl, 8.61; F,
13.84; N, 10.21; O, 11.66. Found: C, 52.37; H, 3.19; N, 10.13.
4.1.4.8. N-(3-Methoxyphenyl)-2-(3-(morpholinomethyl)benzofuran-
2-carbonyl)hydrazine-1-carbothioamide (15a). White crystals, mp
¼ 190–192 ꢁC, yield 84%; IR: 3262 (br, NH groups), 1647 (C¼O
hydrazide), 1277 (C¼S thiosemicarbazide). 1H NMR (400 MHz,
DMSO-d6) d 11.95 (s, 1H), 9.86 (s, 2H), 7.99 (d, J ¼ 7.7 Hz, 1H), 7.72
(d, J ¼ 8.2 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.42 (t, J ¼ 7.4 Hz, 1H),
4.1.4.12. N-(4-Methoxyphenyl)-2-(3-(morpholinomethyl)benzofuran-
2-carbonyl)hydrazine-1-carboxamide (16b). White crystals, mp ¼
242–244 ꢁC, yield 73%; IR: 3423, 3320, 3256 (NH groups), 1706
(C¼O hydrazide), 1639 (C¼O semicarbazide). 1H NMR (400 MHz,
DMSO-d6) d 11.50 (s, 1H), 8.78 (s, 1H), 8.37 (s, 1H), 8.00 (d,
7.32–7.17 (m, 2H), 7.10 (d, J ¼ 7.6 Hz, 1H), 6.77 (d, J ¼ 7.8 Hz, 1H), J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 1H), 7.54 (t, J ¼ 7.7 Hz, 1H),
4.08 (s, 2H), 3.78 (s, OCH3, 3H), 3.63 (s, 4H, CH2OCH2 of morpho- 7.49–7.26 (m, 4H), 6.90 (d, J ¼ 8.6 Hz, 2H), 4.04 (s, 2H), 3.74 (s,
linyl moiety), 2.58 (s, 4H, CH2NCH2 of morpholinyl moiety). 13C OCH3, 3H), 3.63 (s, 4H, CH2OCH2 of morpholinyl moiety), 2.54 (s,

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997
4H, CH2NCH2 of morpholinyl moiety). 13C NMR (100 MHz, DMSO- (C¼N hydrazone). 1H NMR (400 MHz, DMSO-d6) d 10.67 (s, 1H),
d6) 159.31 (C¼O), 155.75 (C¼O of urea), 155.02, 154.79, 153.63, 8.14 (d, 1H), 7.82 (d, J ¼ 7.8 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 1H), 7.55 (t,
145.10, 133.41, 133.05, 128.76, 127.71, 124.02, 122.36, 120.90, J ¼ 7.7 Hz, 1H), 7.41 (t, 1H), 7.36 (d, J ¼ 7.2 Hz, 1H), 7.32 (d,
120.85, 120.38, 114.42, 114.33, 112.23, 66.51 (O(CH2)2), 55.61 J ¼ 7.3 Hz, 1H), 7.27 (d, J ¼ 7.4 Hz, 1H), 2.85 (dt, J ¼ 12.0, 5.9 Hz, 4H),
(OCH3), 53.11 (N(CH2)2), 50.88 (CH2). Anal. Calcd. for C22H24N4O5 2.62 (s, 3H), 1.91 (p, J ¼ 6.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6)
(424.46): C, 62.25; H, 5.70; N, 13.20; O, 18.85. Found: C, 62.01; H, 156.56 (C¼O), 155.98, 153.37, 143.01, 140.79, 132.55, 130.10,
5.73; N, 13.24.
129.46, 129.15, 127.89, 126.76, 125.23, 123.83, 122.60, 121.59,
112.25, 29.35 (CH2 of C-4 of dihydronaphthalen-1(2H)-one), 26.42
(CH2 of C-3 of dihydronaphthalen-1(2H)-one), 21.88 (CH2 of C-2 of
dihydronaphthalen-1(2H)-one), 9.29 (CH3). Anal. Calcd. for
C20H18N2O2 (318.38): C, 75.45; H, 5.70; N, 8.80; O, 10.05. Found: C,
75.21; H, 5.71; N, 8.83.
4.1.5. General method for the synthesis of hydrazones 8a–c, 11,
17a,b and 18
The appropriate hydrazide derivative 2 or 14 (0.6 mmol) was
heated at reflux while stirring with suitable aldehyde or ketone
derivative (0.7 mmol) in ethanol (8 ml) using catalytic amount of
3–4 drops of glacial acetic acid for 3–7 h. Solvent was then dis-
tilled off and the residue was suspended in distilled water while
vigorous stirring for 15 min. After filtration and suction drying, the
powder obtained was recrystallized from dioxan/isopropyl alcohol
mixture to afford the final benzofuran-based hydrazones 8a–c, 11,
17a,b and 18.
4.1.5.5. N’-Benzylidene-3-(morpholinomethyl)benzofuran-2-carbo-
hydrazide (17a). White crystals, mp ¼ 174 ꢁC, yield 85%; 1H NMR
(400 MHz, DMSO-d6) d 12.72 (s, 1H), 8.52 (s, 1H), 8.02 (d, J ¼ 7.8 Hz,
1H), 7.79 (d, J ¼ 6.6 Hz, 2H), 7.72 (d, J ¼ 8.3 Hz, 1H), 7.59–7.46 (m,
4H), 7.42 (t, J ¼ 7.5 Hz, 1H), 4.09 (s, 2H), 3.64 (s, 4H, CH2OCH2 of
morpholinyl moiety), 3.54 (s, 4H, CH2NCH2 of morpholinyl moiety).
13C NMR (100 MHz, DMSO-d6) 156.00 (C¼O), 153.65, 149.06,
144.84, 134.61, 130.81, 129.37, 128.89, 127.90, 127.73, 124.09,
122.64, 121.79, 112.26, 66.62 (O(CH2)2), 53.30 (N(CH2)2), 51.07
(CH2). Anal. Calcd. for C21H21N3O3 (363.42): C, 69.41; H, 5.82; N,
11.56; O, 13.21. Found: C, 69.60; H, 5.89; N, 11.62.
4.1.5.1. N’-(3-Methoxybenzylidene)-3-methylbenzofuran-2-carbohy-
drazide (8a). White crystals, mp ¼ 242–244 ꢁC, yield 86%; IR: 3222
(NH hydrazone), 2834 (Csp2-H hydrazone) 1653 (C¼O hydrazone),
1608 (C¼N hydrazone). 1H NMR (400 MHz, DMSO-d6) d 12.12 (s,
1H), 8.57 (s, 1H), 7.84 (d, J ¼ 7.9 Hz, 1H), 7.69 (d, J ¼ 8.1 Hz, 1H),
7.57 (t, J ¼ 7.3 Hz, 1H), 7.47–7.37 (m, 2H), 7.32 (s, 2H), 7.07 (d,
J ¼ 7.4 Hz, 1H), 3.86 (s, OCH3, 3H), 2.63 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) 160.04 (C¼O), 156.37 (C¼O of urea), 153.31, 148.83,
142.52, 136.22, 130.49, 129.48, 128.10, 123.91, 123.41, 121.69,
120.68, 116.85, 112.18, 111.61, 55.67 (OCH3), 9.29 (CH3). Anal.
Calcd. for C18H16N2O3 (308.34): C, 70.12; H, 5.23; N, 9.09; O, 15.57.
Found: C, 69.90; H, 5.18; N, 9.13.
4.1.5.6.
N’-(4-Methoxybenzylidene)-3-(morpholinomethyl)benzo-
furan-2-carbohydrazide (17b). White crystals, mp ¼ 200 ꢁC, yield
79%; IR: 3449 (br, NH hydrazone), 2844 (Csp2-H hydrazone) 1681
(C¼O hydrazone), 1611 (C¼N hydrazone). 1H NMR (400 MHz,
DMSO-d6) d 12.60 (s, 1H), 8.44 (s, 1H), 8.01 (d, J ¼ 7.8 Hz, 1H), 7.73
(d, J ¼ 7.4 Hz, 2H), 7.71 (s, 1H), 7.54 (t, J ¼ 7.7 Hz, 1H), 7.41 (t,
J ¼ 7.4 Hz, 1H), 7.08 (d, J ¼ 8.2 Hz, 2H), 4.08 (s, 2H), 3.85 (s, OCH3,
3H), 3.64 (s, 4H, CH2OCH2 of morpholinyl moiety), 2.54 (s, 4H,
CH2NCH2 of morpholinyl moiety). 13C NMR (100 MHz, DMSO-d6)
161.50 (C¼O), 155.82, 153.61, 148.92, 145.01, 129.38, 128.92,
127.79, 127.14, 124.06, 122.56, 121.42, 114.86, 112.24, 66.61
(O(CH2)2), 55.81 (OCH3), 53.28 (N(CH2)2), 51.05 (CH2). Anal. Calcd.
for C22H23N3O4 (393.44): C, 67.16; H, 5.89; N, 10.68; O, 16.27.
Found: C, 67.03; H, 5.91; N, 16.36.
4.1.5.2. N’-(4-Methoxybenzylidene)-3-methylbenzofuran-2-carbohy-
drazide (8b). White crystals, mp ¼ 236–240 ꢁC, yield 83%; IR: 3195
(NH hydrazone), 2836 (Csp2-H hydrazone) 1645 (C¼O hydrazone),
1606 (C¼N hydrazone). 1H NMR (400 MHz, DMSO-d6) d 11.97 (s,
1H), 8.54 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.77–7.63 (m, 3H), 7.55 (t,
J ¼ 7.7 Hz, 1H), 7.41 (t, J ¼ 7.5 Hz, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 3.85
(s, OCH3, 3H), 2.63 (s, 3H). 13C NMR (100 MHz, DMSO-d6) 161.39
(C¼O), 156.21 (C¼O of urea), 153.27, 148.85, 142.67, 129.52,
129.26, 127.96, 127.33, 123.84, 123.03, 121.60, 114.84, 112.13,
55.78 (OCH3), 9.26 (CH3). Anal. Calcd. for C18H16N2O3 (308.34): C,
70.12; H, 5.23; N, 9.09; O, 15.57. Found: C, 69.87; H, 5.20; N, 9.17.
4.1.5.7. N’-(3,4-Dihydronaphthalen-1(2H)-ylidene)-3-(morpholino-
methyl)benzofuran-2-carbohydrazide (18). White crystals, mp ¼
200 ꢁC, yield 87%; IR: 3449 (br, NH hydrazone), 1685 (C¼O hydra-
zone), 1618 (C¼N hydrazone). 1H NMR (400 MHz, DMSO-d6) d
11.31 (s, 1H), 8.16 (d, J ¼ 7.4 Hz, 1H), 8.02 (d, J ¼ 7.8 Hz, 1H), 7.75
(d, J ¼ 8.2 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H),
7.40–7.30 (m, 2H), 7.27 (d, J ¼ 7.3 Hz, 1H), 4.04 (s, 2H), 3.64 (s, 4H,
CH2OCH2 of morpholinyl moiety), 2.52 (s, 4H, CH2NCH2 of morpho-
linyl moiety), 2.84 (s, 4H), 2.06–1.83 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) 155.97 (C¼O), 154.56, 153.77, 145.67, 140.78, 132.47,
130.10, 129.15, 129.07, 127.78, 126.79, 125.25, 124.10, 122.26,
120.04, 112.34, 66.23 (O(CH2)2), 53.51 (N(CH2)2), 50.94 (CH2), 29.26
(CH2 of C-4 of dihydronaphthalen-1(2H)-one), 26.91 (CH2 of C-3 of
dihydronaphthalen-1(2H)-one), 21.75 (CH2 of C-2 of dihydronaph-
thalen-1(2H)-one). Anal. Calcd. for C24H25N3O3 (403.48): C, 71.44; H,
6.25; N, 10.41; O, 11.90. Found: C, 71.63; H, 6.28; N, 10.43.
4.1.5.3. N’-(1-(4-Methoxyphenyl)ethylidene)-3-methylbenzofuran-2-
carbohydrazide (8c). White crystals, mp ¼ 160 ꢁC, yield 81%; IR:
3393 (NH hydrazone), 1683 (C¼O hydrazone), 1607 (C¼N hydra-
zone). 1H NMR (400 MHz, DMSO-d6) d 10.70 (s, 1H), 7.87 (d,
J ¼ 8.0 Hz, 2H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.70 (d, J ¼ 8.3 Hz, 1H), 7.55
(t, J ¼ 7.7 Hz, 1H), 7.40 (t, J ¼ 7.5 Hz, 1H), 7.04 (d, J ¼ 8.4 Hz, 2H),
3.84 (s, OCH3, 3H), 2.61 (s, 3H), 2.40 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) 161.03 (C¼O), 156.64 (C¼O of urea), 153.35, 143.04,
130.66, 129.47, 129.09, 128.53, 127.83, 123.81, 122.45, 121.55,
114.22, 112.24, 55.72 (OCH3), 14.75 (CH3 of acetophenone), 9.27
(CH3). Anal. Calcd. for C19H18N2O3 (322.36): C, 70.79; H, 5.63; N,
8.69; O, 14.89. Found: C, 70.96; H, 5.67; N, 8.60.
4.2. Biological evaluation
4.1.5.4. N’-(3,4-Dihydronaphthalen-1(2H)-ylidene)-3-methylbenzo-
The procedures of experiments performed for the biological
furan-2-carbohydrazide (11). White crystals, mp ¼ 180–182 ꢁC, evaluations in this study have been provided in the
yield 89%; IR: 3383 (NH hydrazone), 1686 (C¼O hydrazone), 1607 Supporting materials.

998
M. M. AL-SANEA ET AL.
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Acknowledgements
The authors extend their sincere appreciation to the central
laboratory at Jouf University for support this study. M. M. Al-Sanea
also extends his appreciation to the Korea Institute of Science and
Technology (KIST) for funding this work through the Grant name
“2021 KIST School Partnership Project.”
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Disclosure statement
No potential conflict of interest was reported by the author(s).
16. Hiremathad A, Patil MR, Chand K, et al. Benzofuran: an
emerging scaffold for antimicrobial agents. RSC Adv 2015;5:
96809.
Funding
17. Xu Z, Zhao S, Zaosheng L, et al. Benzofuran derivatives and
their anti-tubercular, anti-bacterial activities. Eur J Med
Chem 2019;162:266–76.
The authors extend their appreciation to the Deputyship for
Research & Innovation, Ministry of Education in Saudi Arabia for
funding this work through the project number "375213500."
ꢀ
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