A proficient microwave synthesis with structure elucidation and the exploitation of the biological behavior of the newly halogenated 3-amino-1H-benzo[f]chromene molecules, targeting dual inhibition of topoisomerase II and microtubules

Article abstract of DOI:10.1016/j.bioorg.2019.103549

In our endeavors to develop novel and powerful agents with antiproliferative activities, a series of β-enamionitriles, linked to the 8-bromo-1H-benzo[f]chromene moieties (4a-m), was designed and synthesized under microwave irradiation conditions. The stru

Full text of DOI:10.1016/j.bioorg.2019.103549

BioorganicChemistry95(2020)103549
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A proficient microwave synthesis with structure elucidation and the
exploitation of the biological behavior of the newly halogenated 3-amino-
1H-benzo[f]chromene molecules, targeting dual inhibition of topoisomerase
II and microtubules
Ahmed M. Foudaa, Rawda M. Okashab, Fawzia F. Alblewib, Ahmed Morac, Tarek H Afifib,
⁎
Ahmed M. El-Agrodyc,
a Chemistry Department, Faculty of Science, King Khalid University, Abha 61413, Saudi Arabia
b Chemistry Department, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah 30002, Saudi Arabia
c Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City 11884, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
In our endeavors to develop novel and powerful agents with antiproliferative activities, a series of β-enam-
ionitriles, linked to the 8-bromo-1H-benzo[f]chromene moieties (4a-m), was designed and synthesized under
microwave irradiation conditions. The structures of the target compounds were established on the basis of their
spectral data: IR, 1H NMR, 13C NMR, 13C NMR-DEPT/APT, 19F NMR and MS. Furthermore, the antiproliferative
properties were evaluated against the human cancer cell lines MCF-7, HCT-116, and HepG-2 in comparison to
the positive controls Vinblastine and Doxorubicin, employing the viability assay. The obtained results confirmed
that most of the tested molecules revealed strong and selective cytotoxic activities against the three cancer cell
lines. The most potent cytotoxic compounds 4b, 4d, 4e, 4i, and 4k were elected for further examination, such as
the cell cycle analysis, the apoptosis assay, the Caspase production, and the DNA fragmentation. This study also
revealed that the desired compounds stimulate cell cycle arrest at the G2/M phases, increase the production of
Caspases 3, 8, and 9, and finally cause intrinsic and extrinsic apoptotic cell death. Moreover, these compounds
suppress the action of the topoisomerase II enzyme and also disrupt the microtubule functions. The SAR study of
the synthesized compounds verified that the substitution on the phenyl ring of the 1H-benzo[f]chromene nu-
cleus, accompanied with the presence of the bromine atom at the 8-position, increases the ability of these
molecules against different cell lines.
Benzochromenes
Antitumor activity
Cell cycle analysis
Caspases
Topoisomerase
DNA fragmentation
SAR study
1. Introduction
carcinoma (HepG-2), cervical cancer (HeLa), and lung adenocarcinoma
(A549) [18]. The 7-(dimethylamino/amino/hydroxy)-4-(5/4-ni-
Chromene and benzochromene derivatives perform vital roles in
many biological processes, such as antimicrobial [1–4] anti-influenza
virus [5] antiproliferative [6–8] anticancer [9] anti-inflammatory
[10,11] antitubercular [12,13] Alzheimer disease [14] and anti-obesity
[15] effects and agents. A large number of the chromene derivatives
have been prepared with interesting anticancer properties, and several
examples have exhibited cytotoxic effects on cancer related cell lines in
the micro/nano-molar concentration range. For example, the 6-aryl-4H-
chromene derivatives (A) acted as stable and ROS-free antagonistic,
antiapoptotic Bcl-2 proteins [16,17]; the C4-N,N-dialkyl-aniline-sub-
stituted 4-aryl-4H-chromene derivatives (B) contain antiproliferative
properties against the tumor cell lines, namely: hepatocellular
trothiophen-2-yl)-4H-chromene and
the 2,7,8-triamino-4-(5-ni-
trothiophen-2-yl)-4H-chromene derivatives (C) operated as the in-
ducers of apoptosis [19]; the ethyl 2-amino-6-bromo-4-(1-cyano-2-
ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA 14-1) (D) per-
formed as an inhibitor of the Bcl-2 proteins [20], which also demon-
strated selective cytotoxicity against drug-resistant cancer cells that
overexpress the antiapoptotic Bcl-2 family proteins [21,22]. The ethyl
4-((ethoxycarbonyl)methyl)-2-amino-6-phenyl-4H-chromene-3-carbox-
ylate (sHA 14-1) (E) displayed synergism and selective toxicity against
the Bcl-2-overexpressing drug-resistant cancer cells [23], and ethyl 2-
amino-4-(2-morpholino-2-oxoethyl)-6-phenyl-4H-chromene-3-carbox-
ylate (isHA 14-1) (F) acted as an inhibitor of the Bcl-2 protein [17] as
⁎ Corresponding author.
E-mail addresses: elagrody_am@yahoo.com, ahm1954@hotmail.com, elagrody_am47@gmail.com (A.M. El-Agrody).
https://doi.org/10.1016/j.bioorg.2019.103549
Received 10 October 2019; Received in revised form 17 December 2019; Accepted 21 December 2019
Availableonline23December2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
Fig. 1. Chromene drugs (Red highlighted) with different biological activities.
shown in (Fig. 1).
benzo[f]chromene moieties, were synthesized, and their anti-
proliferative activities were surveyed against three cancer cell lines:
MCF-7, HCT-116, and HepG-2. Moreover, the most potent prepared
compounds targeted both the topoisomerease II and microtubules, eli-
citing a cell cycle arrest at the G2/M phases and the induction of the
caspase dependent apoptosis. The structure activity relationships
(SARs) disclose that the introduction of certain lipophilic halogen
substituents on the phenyl ring, attached to the 1-position of the 8-
bromo-1H-benzo[f]chromene moiety, enhanced the antitumor activ-
ities.
Furthermore, the benzochromene derivatives are a very efficacious
choice for the treatment of several human diseases. For instance, the 3-
amino-1-aryl-8-bromo-1H-benzo[f]chromenes (G) and the 3-amino-1-
aryl-8-methoxy-1H-benzo[f]chromenes (H) demonstrated high potency
for the cell invasion assay, the caspase 3/7 assay, and the c-Src kinase
inhibitors [24]. The 3-amino-1-aryl-1H-benzo[f]chromene-2-carboni-
triles (I) exhibited c-Src kinase inhibitory and antiproliferative activities
[25] while the 2-amino-5,6-dihydro-4-aryl-4H-benzo[h]chromene-3-
carbonitriles (J) has good antiproliferative activities against different
cell lines [26]. Additionally, the 2-amino-4-aryl-4H-benzo[h]chromene-
2-carbonitriles (K) revealed c-Src kinase inhibitory and antiproliferative
activities [25] and the 2-amino-4-aryl-8-methoxy-4H-benzo[h]chro-
mene-2-carbonitriles (L) performed as a potent anticancer analog and
targeted the c-Src Kinase enzyme [27] as presented in (Fig. 2).
In the present study, the β-enamionitriles, linked to the 8-bromo-1H-
2. Results and discussion
2.1. Chemistry
The β-enamionitriles (4a-m), attached to the 8-bromo-1H-benzo[f]
Fig. 2. Structure of some 1H-benzo[f]chromene derivatives (blue highlighted) and 4H-benzo[h]chromene derivatives (Green highlighted) with cytotoxic and
apoptotic effects.
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A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
Scheme 1. Synthesis of halogenated 1H-benzo[f]chromene derivatives (4a-m) and 2-(2-fluoro-4-(piperidin-1-yl)benzylidene)malononitrile (5).
chromene groups, were synthesized, as represented in (Scheme 1). The
halogenated phenyl ring at the 1-position of the 3-amino-8-bromo-1H-
benzo[f]chromene-2-carbonitriles (4a-m) was easily acquired in a good
yield by the condensation of 6-bromo-2-naphthol (1) with the appro-
priate aromatic aldehydes (2a-m) and malononitrile (3) in an ethanolic
piperidine solution employing microwave irradiation for 2 min. at
140 °C. In addition, compound 2-(2-fluoro-4-(piperidin-1-yl)benzyli-
dene)malononitrile (5) was unintentionally formed as side product via
the exploitation of 2,4-difluorobenzylidenemalononitrile (6). The
maximum power of the microwave irradiation was optimized by re-
peating the reaction at different watt powers and periods of time, which
delivered the ideal condition at 400 W and reaction time 2 min. and
provided the highest yield of the desired compounds. It is also critical to
remark that the 1-position of compounds 4a-m is a chiral center.
The unexpected formation of compound 5 proceeds simply via the
displacement of the fluorine atom at the formed 2,4-di-
fluorobenzylidenemalononitrile (6) by the piperidinyl moiety via a
nucleophilic aromatic substitution reaction at the 4-postion rather than
the 2-postion due to the steric hindrance of the fluorine atom at the 2-
postion, (Scheme 1). Further, the structure of the 2-(2-fluoro-4-(piper-
idin-1-yl)benzylidene)malononitrile (5) was supported by an in-
dependent synthesis of the same compound through the nucleophilic
substitution of the fluorine atom at the 4-postion of 2g with piperidine
to give 2-fluoro-4-piperidin-1-yl-benzaldehyde (7), followed by the
condensation of 7 with malononitrile (3) in an ethanol solution under
reflux conditions, as illustrated in (Scheme 2).
7.18–7.02 ppm, which were representing the amino groups’ protons,
and at δ 6.11–5.35, which were attributable to the methine protons of
the heterocyclic ring. Meanwhile, the 13C NMR spectra of 4a-m showed
the corresponding methine carbons around
δ 37.16–27.96 ppm.
Additionally, the IR spectrum of compound 5 revealed an absorption
band at υ 2212 cm−1 for the CN group, while the 1H NMR and 13C NMR
spectra of 5 presented the signals of the olefinic proton and carbon at δ
7.86 ppm and δ 149.97 ppm, respectively. Furthermore, the MS spectra
of 4a-m and 5, the 13C NMR-DEPT/APT of compound 4a and 5, 19F
NMR of compound 5 and the single crystal X-ray analysis of compound
4k [28] as a representative example, gave an absolute confirmation for
the target structures.
2.2. Biological activity
2.2.1. In vitro cytotoxic activity
Based on the importance of benzochromene (16–24) as antitumor
agents, we designed halogenated 3-amino-1H-benzo[f]chromenes mo-
lecules with various aryl substitutions. The MTT assay [29–31] was
performed to evaluate the cytotoxic effects of these halogenated 3-
amino-1H-benzo[f]chromene derivatives (4a-m) against the selected
human cancer cell lines, namely: mammary gland breast cancer (MCF-
7), human colon cancer (HCT-116), and human hepatocellular carci-
noma (HepG-2), utilizing Doxorubicin and Vinblastine as reference
compounds. The in-vitro cytotoxicity evaluation was achieved under
different concentrations, expressed as growth inhibitory concentration
(IC50) values, (Table 1) and (Fig. 3).
The characterization of the synthesized compounds using the
spectroscopic data (IR, 1H/13C NMR, 19F NMR, 13C NMR-DEPT/APT,
MS, X-ray) and the elemental analyses were consistent with their
structures. The IR spectra of 4a-m exhibited characteristic absorption
bands around υ 3479–3443, 3338–3320, 3290–3189 cm−1, due to the
amino groups while the nitrile groups’ functionality in 4a-m was de-
tected to be in the range of υ 2203–2190 cm.-1 The 1H NMR spectra of
Interestingly, the results revealed that most the synthesized halo-
genated 3-amino-1H-benzo-[f]chromene derivatives (4a-m) exhibited
excellent to good antiproliferative activity as comparison with the
standard drugs (Doxorubicin and Vinblastine). Among these deriva-
tives, 4d, 4b, 4i, 4k, 4g, 4e, 4l, 4m, 4h, and 4a with IC50 values in the
range of 0.5–5.2 µg/mL, respectively, were found to be the most potent
of all the assessed compounds against the MCF-7 in comparison with
compounds 4a-m displayed singlet signals, resonating around
δ
Scheme 2. Synthesis of 2-(2-fluoro-4-(piperidin-1-yl)benzylidene)malononitrile (5) via 2-fluoro-4-piperidin-1-yl-benzaldehyde (7).
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A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
Table 1
normal cell growth and division; however, most cancer cells undergo
unscheduled cell divisions by the down regulation of the cell cycle.
Therefore, the newly synthesized anticancer derivatives, inhibiting the
cell cycle, represent a critical therapeutic intervention in treating pro-
liferative diseases like cancer [32,33]. To determine whether the potent
cytotoxicity experienced by the novel compounds (4b, 4d, 4e, 4i, and
4k) was due to the inhibitory effect on the cell cycle process, the DNA
content was analyzed by the flow cytometry, utilizing the FACS Calibur
(Becton Dickinson). The MCF-7, HCT-116, and HepG-2 cells were re-
medied with each derivative at its IC50 value for 24 h; moreover, a
controlled experiment with no treatment was incorporated as shown in
the representative cell cycle distribution histograms of the stained DNA
in (Fig. 4a). The results on regulating the cell cycle progression de-
monstrated that all the examined compounds induced a significant in-
crease in the cell population arrest at the G2/M phases with a decreased
cell count in the G1 and S phases, as portrayed in (Fig. 4b). The cell
cycle evaluation established that the analyzed derivatives substantially
arrested the cells’ progression by restricting the G2/M phases.
IC50 values of the target compounds against MCF-7, HCT-116 and HepG-2 cell
lines.
IC50 (µg/mL)a
Compound
Ar
MCF-7
5.2
HCT-116
HepG-2
0.8
4a
4b
4c
4d
4e
4f
4-FC6H4
2-ClC6H4
3-ClC6H4
4-ClC6H4
4-BrC6H4
4-IC6H4
0.03b
0.16
0.14
0.23b
0.14b
0.2
1.0
0.12b
0.12b
0.5
1.0
1.46
6.0
0.35 1.1
8.3
0.14
0.11b
0.35b
0.7
5.2
1.1
0.2
0.5
0.7
0.12b
0.02b
0.11
0.12
0.21
0.14
0.03
0.18
0.02
0.13
2.5
0.5
1.8
59.7
1.7
30.5
1.36
0.5
20.0
4g
4h
4i
2,4-F2C6H3
2,6-F2C6H3
2,3-Cl2C6H3
2,4-Cl2C6H3
2,5-Cl2C6H3
2,6-Cl2C6H3
3,5-Br2-2-
OMeC6H2
–
0.2
0.12 2.0
4.8
0.1
0.17
1.1
1.3
0.6
0.7
2.9
1.54
1.0
0.02
2.4
0.15
0.23
0.16
0.01
0.6
4j
32.0
1.0
0.01 0.7
2.2.3. Induction of apoptosis by the newly synthesized compounds
To further assess whether the tumor suppression property of the
tested compounds was due to apoptotic cell death, the Annexin V/PI
double staining flow cytometric assay was applied to determine whe-
ther the phosphatidylserine (PS) translocation appeared on the mem-
brane surface as an accepted marker for apoptosis [34]. The re-
presentative dot plots of the double stained cells (MCF-7, HCT-116, and
HepG-2), after treatment with the investigated compounds, were re-
vealed in (Fig. 5a). Unlike necrosis, which was not detected, all the
remedied cells revealed an increase from 10 to 25% in total apoptosis in
comparison to the untreated cells. However, compounds 4d and 4k
disclosed higher apoptosis percentages in the case of the MCF-7 (24%)
and HCT-116 (25%) cells when assessed against the HepG-2 (14%)
cells, (Fig. 5b, c, and d). When evaluated against the vehicle’s controls,
the treated cells with compound 4e experienced an increase in a stage
of early apoptosis (Annexin V positive, PI negative) by a 4, 11, and 9 for
MCF-7, HCT-116, and HepG-2, respectively while the late apoptotsis
(Annexin V positive, PI positive) was 27, 16, and 53 for MCF-7, HCT-
116, and HepG-2 cells, respectively, Fig. 5e. In another report, the
Annexin V single positive cells emerged only after treatment with early
anticancer drugs such as Etoposide and Cytarabine, but this did not
occur with the late anticancer drugs such as Doxorubicin and Metho-
trexate [35,36]. The results insinuated that the desired molecules,
performing as drug candidates, exhibited more potent cytotoxicity
during the induction of late apoptosis.
4k
4l
0.06
0.6
8.7
2.8
2.8
0.4
4m
3.6
0.15
Vinblastine
Doxorubicin
6.1
0.4
0.03
0.01
2.6
0.5
0.08
4.6
0.01
0.04
–
0.015 0.9
a
IC50 values expressed in µg/mL as the mean values of triplicate wells from
at least three experiments and are reported as the mean
standard error.
b
[24].
Vinblastine (IC50 = 6.1 µg/mL). Compound 4d (IC50 = 0.5 µg/mL) was
almost equipotent as Doxorubicin (IC50 = 0.4 µg/mL) and emerged as
the most potent counterpart against MCF-7 in this study. Additionally,
compounds 4h, 4e, 4k, 4d, 4j, 4a, 4g, 4b, and 4i (IC50 = 0.5–2.4 µg/
mL) possessed excellent antiproliferative activities against the HCT-116
cells, which proved to excel further than the employed reference drug
Vinblastine (IC50 = 2.6 µg/mL), and compounds 4h, 4e, 4k, 4d, and 4j
displayed less anti-proliferative activities than or equipotent as
Doxorubicin with IC50 values, ranging from 0.5 to 0.7 µg/mL. The de-
rivatives 4j, 4k, 4a, 4h, 4b, 4d, 4i, 4m, 4e, 4g, and 4l demonstrated
superior activities against the HepG-2 cell line with IC50 of 0.6–2.9 µg/
mL as compared to Vinblastine (IC50 = 4.6 µg/mL) while compounds
4j, 4k, and 4a (IC50 = 0.6–0.8 µg/mL) were discovered to be the most
potent counterpart in evaluation with Doxorubicin (IC50 = 0.9 µg/mL).
Lastly, derivatives 4h, 4b, and 4i (IC50 = 1.1–1.3 µg/mL) were almost
equipotent as Doxorubicin.
2.2.4. Increased protein levels of Caspases 3, 8, and 9
2.2.2. Cell cycle distribution at G2/M phase
The Caspases’ members of the cysteine proteases family trigger
apoptosis through an extrinsic pathway, associated with the activation of
The four cell cycle stages (G1, S, G2, and M) ensure a controlled
Fig. 3. IC50 values expressed in (µg/mL) of halogenated 1H-benzo[f]chromene derivatives 4a-m against MCF-7, HCT-116 and HepG-2 tumor cells.
4

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BioorganicChemistry95(2020)103549
the initiator Caspase-8, or an intrinsic pathway, allied with the activation
of the initiator Caspase-9, which finally stems the activation of the ex-
ecutioner Caspases such as Caspase-3 [37,38]. Furthermore, the protein
levels of Caspase-3, Caspase-8, and Caspase-9 were measured, exploiting
the enzyme-linked immunosorbent assay in the different remedied tumor
cells (Fig. 6a, b, and c), to further verify the impact of the analyzed
derivatives on the apoptotic pathway. Fig. 6d illustrates the fold change
for each of the Caspases after their therapy with the examined cancer
cells and compound 4e for 24h in assessment with the untreated control
cells. The up regulation of Caspase-8 and Caspase-9 suggested that the
newly synthesized compounds stimulated apoptosis via the extrinsic/
death receptors and the intrinsic/mitochondrial pathway. Our results are
Fig. 4. The induced cell cycle arrest at the G2/M phase. (a) The representative flow cytometry histograms of the cell cycle distribution. The DNA content was stained
with (PI). (b) the percentage of MCF-7, HCT-116 and HepG-2 cells in the G1, S, and G2/M phases after incubation with compounds 4b, 4d, 4e, 4i, and 4k (IC50
values) for 24 h.
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BioorganicChemistry95(2020)103549
Fig. 4. (continued)
in accordance with previous reports, demonstrating that the Bleomycin,
Doxorubicin, and Mitoxantrone anticancer agents are involved in the
activation of both the extrinsic as well as the intrinsic apoptotic path-
ways, which is accompanied by an increased enzymatic activity of Cas-
pases (Caspase-3, Caspase-8, and Caspase-9) [39].
(0–10 μM) of the analyzed compounds were added to the preformed
microtubules suspensions to monitor the decrease in the fluorescence
intensity, measured as a sign for depolymerization. All the assessed
compounds stimulated rapid depolymerization of the previously de-
veloped microtubules in a concentration-dependent fashion, denoted by
the decrease in the fluorescence values when evaluated against the
Colchicine reference drug, (Fig. 9a). The percentages of the micro-
tubules polymerization inhibition at a 10 μM concentration were 47,
55, and 57 for compounds 4e, 4k, and Colchicine, respectively. In ad-
dition, the remaining examined derivatives destabilize the microtubules
formation by 40%, as depicted in (Fig. 9b). Based on the mechanism of
action, the microtubule inhibitors are usually classified as stabilizer
(paclitaxel and docetaxel) or destabilizer (Vinblastine and Colchicine)
agents [48]. In this existing report, our molecules demonstrated the
inhibition of the microtubules polymerization by destabilizing the
preformed tubulin polymer. Furthermore, all the combined outcomes
signify that the cultivated derivatives (4d, 4e, 4i, and 4k) firstly tar-
geted the topoismorease II and microtubules. Consequently, the in-
hibition of the topoisomerase II and microtubules detain cells at the G2/
M phases and, subsequently, activate extrinsic and intrinsic caspase-
dependent apoptosis, which is ensued by DNA fragmentation and the
proliferation inhibition of cancer cells, Fig. 9c.
2.2.5. Production of apoptotic DNA fragmentation
The presence of Caspase-3 is associated with the typical hallmarks
of apoptosis such as DNA fragmentation [40]. Therefore, the relative
quantity of DNA fragments were detected, employing the diphenyla-
mine (DPA) reagent in the different cancer cells, remedied with com-
pounds 4b, 4d, 4e, 4i, 4k, and Doxorubicin 24 h. The percentage of
DNA fragmentation in all tumor cells, nursed with our yielded deriva-
tives, was double the attained percentage by the cells treated with the
reference drug Doxorubicin, (Fig. 7). These results integrated with the
Caspases’ levels are formerly demonstrated.
2.2.6. Inhibition of Topoisomerase II
Recently, the cell cycle checkpoint arrest has been exercised as a
novel approach to amplify the efficacy and specify the conventional
chemotherapy [41]. In the preceding reports, it was established that the
topoisomerse II inhibitors trigger the G2/M phases arrest due to the
absolute failure of the chromosome decatenation procedure [42,43].
However, it was confirmed that all the new desired molecules prompt
cell cycle arrest at the G2/M phase in the dissimilar tumor lines. Thus,
the possible inhibitory effect for each derivative was explored for the
topoisomerase II activity, exploiting the plasmid-based Topoisomerase
II assay. The capability of the topoisomerase II enzymes to decatenate
the supercoiled plasmid DNA into a relaxed form experienced no ac-
tivity with compound 4b, weak activity with compounds 4e and 4i, and
the strongest inhibitory effect in regards to compounds 4d and 4k,
which is equivalent to the Doxorubicin standard drug, (Fig. 8). Ad-
ditionally, the gained outcomes elucidated the role of the assessed
molecules in the inhibition of Topoisomerase II and the interruption of
a vital cell cycle stage, consequently leading to cell death. These results
are in accordance with Magar et al. work, illustrating the effective in-
hibition of the Topoisomerese II by the produced chromene analogues
as selective targeting anticancer agents [44]. Furthermore, a number of
antitumor drugs (Doxorubicin and Etoposide) were scrutinized for their
cytotoxicity, due to their ability to stabilize a covalent Topoisomerase
II-DNA intermediate [45].
2.3. SAR studies
The SAR study revealed several crucial structural requirements,
which enhanced the potency of these halogenated 3-amino-1H-benzo[f]
chromene derivatives (4a-m). By exemplifying the variations in the
substitution pattern on the phenyl ring at the 1-position of the benzo[f]
chromene moiety, the molecules possessing electron-withdrawing and
electron-donating substituents were prepared with their effects being
analyzed. It is noteworthy to observe that the halogenated mono-
substituents (first series) on the phenyl ring of compounds 4a-f have a
significant impact on the activities against the MCF-7 cell line in eva-
luation against Vinblastine and Doxorubicin, where the activities were
decreased in the order of 4-Cl > 2-Cl > 4-Br > 4-F > 3-Cl > 4-I.
Moreover, for the halogenated disubstituents (second series) on the
phenyl ring of the molecules 4g-l, the order of the antiproliferative
activities is widely varied in accordance to the position and the size of
the halogenated disubstituents on the phenyl ring of the benzo[f]
chromene moiety; subsequently, the activities were decreased in the
order 2,3-Cl2 > 2,5-Cl2 > 2,4-F2 > 2,6-Cl2 > 2,6-F2 > 2,4-Cl2.
The trisubstituent (third series) like 3,5-Br2-2-OMe of compound 4m
demonstrated good activity, as compared to Vinblastine, hinting that
the grafting of the lipophilic electron-withdrawing monosubstituents is
more beneficial than that of the disubstituents and trisubstituents.
Additionally, the moderate size of the lipophilic electron-withdrawing
disubstituents, similar to 2,3-Cl2; 2,5-Cl2; and 2,4-F2, displayed more
activity than the other disubstituents and trisubstituents, respectively.
Regarding the activity against the HCT-116 line, molecules 4h, 4e, 4k,
4d, 4j, 4a, 4g, 4b, and 4i were the most effectual analogs through this
2.2.7. Inhibition of microtubules
Microtubules are major components of the eukaryotic cytoskeleton
that result from the interaction of the alpha and beta tubulins polymers.
Microtubules perform sundry functions in cells, including mitosis;
therefore, the disruption of microtubules generates cell cycle arrest in
the G2/M phases and apoptosis, rendering it an attractive target for
new anticancer drugs [46,47]. The possible depolymerization effects of
the most potent cytotoxic molecules were investigated, employing the
preformed microtubules in a free cell system. Enhanced concentrations
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BioorganicChemistry95(2020)103549
Fig. 5. Apoptotic cell death induced by the halogenated 1H-benzo[f]chromene derivatives: (a) The flow cytometry dot plot represents the Annexin-V-FITC staining in
the X-axis and the PI in the Y-axis. The summary of the Annexin V-FITC apoptosis assay results displays the percentages of early apoptosis, late apoptosis, total
apoptosis, and necrosis in the (b) MCF-7, (c) HCT-116, and (d) HepG-2 treated cells with the indicated drugs (IC50 value) for 24 h. (e) The fold change of early and
late apoptotic cells treated with compound 4e over the control.
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Fig. 5. (continued)
Fig. 6. Increased Caspase-3, -8, and -9 levels. The (a) Caspase-3, (b) Caspase-8, and (c) Caspase-9 concentration levels (ng/mL) in the different cell lysates were
determined, using the enzyme-linked immunosorbent assay kit after the treatment of the tumor cells with the indicated compounds (IC50 value) for 24 h. (d) The fold
change for Caspase-3, -8, and -9 in the treated cancer cells with compound 4e was calculated relative to the non-treated control cells.
study in assessment with Vinblastine, intimating that the halogenated
disubstituents possessed excellent antiproliferative activities than the
monosubstitents and trisubstituents. Furthermore, derivatives 4j, 4k,
4a, 4h, 4b, 4d, 4i, 4m, 4e, 4g, and 4l portrayed superior potency
against the HepG-2 line in evaluation with Vinblastine, as compared to
the monosubstitents members and the trisubstituent. Lastly, some of the
selected derivatives of the assessed series demonstrated almost
equipotent to most potent antiproliferative activity in comparison to
Doxorubicin in the treatment of the elected cell lines: the almost
equipotent activity of compound 4d against MCF-7; the equipotent
activity of compounds 4h, 4e and the almost equipotent activity of
compounds 4k, 4d against Hct-116; and the most potent activity of
compounds 4j, 4k, 4a and the equipotent activity of compounds 4b, 4h
against HepG-2.
8

A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
uncorrected. The IR spectra were recorded on a KBr disc on a Jasco FT/
IR 460 plus spectrophotometer. The 1H (500 MHz), 13C (125 MHz), and
19F (377 MHz) NMR spectra were measured on a BRUKER AV 500 MHz
spectrometer in DMSO‑d6, a solvent, using the tetramethylsilane (TMS)
as an internal standard. The 13C NMR spectra were obtained, employing
the distortion-free enhancement by polarization transfer (DEPT) and
the attached proton test (APT). The chemical shifts (δ) are expressed in
parts per million (ppm). The Microwave apparatus utilized is Milestone
Sr1, Microsynth. The mass spectra were determined on a Shimadzu GC/
MS-QP5050A spectrometer. The elemental analysis was carried out at
the Regional Centre for Mycology and Biotechnology (RCMP), Al-Azhar
University, Cairo, Egypt, and the results were within 0.25%. The
reaction courses and product mixtures were routinely monitored by the
thin layer chromatography (TLC) on silica gel precoated F254 Merck
plates.
Fig. 7. Significant increased DNA fragmentation percentage in all treated
cancer cells. The quantitative estimation of DNA fragmentation was discerned,
using the diphenylamine (DPA) reagent. The percentage of DNA fragments in
the cells treated with the indicated compounds was evaluated against the re-
ference drug Doxorubicin.
4.2. General procedure for synthesis of 1H-benzo[f]chromene derivatives
(4a-4m) and 2-(2-Fluoro-4-(piperidin-1-yl)benzylidene)malononitrile (5)
A reaction mixture of 6-bromo-2-naphthol (1) (0.01 mol), different
aromatic aldehydes (2a-m) (0.01 mol), malononitrile (3) (0.01 mol),
and piperidine (0.5 mL) in an ethanol solution (30 mL) was heated
under microwave irradiation conditions for 2 min. at 140 °C. After the
reaction reached completion, the reaction mixture was cooled to room
temperature, and the precipitated solid was filtered off, washed with
methanol, and recrystallised from ethanol or ethanol/benzene. The
physical and spectral data of compounds 4a-m and 5 are as follows:
4.2.1. 3-Amino-8-bromo-1-(4-fluorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4a)
Prepared as previously described [24].
4.2.2. 3-Amino-8-bromo-1-(2-chlorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4b)
Fig. 8. The inhibition of the Topoisomerase II decatenation activity by the
target compounds. The human topoisomerase II-mediated decatenation activity
of kDNA was done, using the topoisomerase II inhibition gel assay. The (-)
kDNA controls with no Topoisomerase II and the (+) decatenated kDNA con-
trols with topoisomerase II. (0) No, (+1) low, (+2) moderate, and (+3) high
inhibition effects.
Colorless needles from ethanol; yield 87%; m.p. 255–256 οC; IR
(KBr) υ (cm−1): 3448, 3328, 3213 (NH2), 2198 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.24–7.01 (m, 9H, aromatic), 7.02 (bs, 2H,
NH2), 5.71 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.7 (C-3),
142.2 (C-4a), 132.1 (C-10a), 130.5 (C-7), 130.3 (C-9), 128.8 (C-6),
128.6 (C-6a), 124.9 (C-10), 119.7 (C-10b), 118.2 (CN), 118.1 (C-5),
115.0 (C-8), 56.1 (C-2), 35.2 (C-1), 147.5, 131.1, 130.1, 129.7, 129.2,
128.2 (aromatic); MS m/z (%): 414 (M++4, 3.11), 412 (M++2, 8.65),
410 (M+, 6.31) with a base peak at 299 (1 0 0); Anal. Calcd for
3. Conclusion
This report portrays a novel and successful endeavor to develop
halogenated 3-amino-1H-benzo[f]chromene derivatives and explore
their dual inhibitory impact on the Topoisomerase II enzyme and mi-
crotubules with potent anticancer activities against three diverse cancer
cell lines: MCF-7, HCT-116, and HepG-2. The dual inhibition of the
Topoisomerase II/microtubules detains cells at the G2/M phases, acti-
vates extrinsic and intrinsic Caspase-dependent apoptosis, and, lastly,
affords DNA fragmentation. Furthermore, the present study offers an
innovative and efficacious strategy for cancer therapy by the dual tar-
geting of the Topoisomerase II/microtubules to overcome the different
medicinal problems encountered with the employment of the conven-
tional combination of Topoisomerase II and tubulin inhibitors. Such
strategy will also render the process of drug choice, administration, and
potent therapeutic efficacy facilitated as well as simplified.
C
20H12BrClN2O: C, 58.35; H, 2.94; N, 6.80. Found: C, 58.31; H, 2.91; N,
6.76%.
4.2.3. 3-Amino-8-bromo-1-(3-chlorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4c)
Colorless needles from ethanol; yield 83%; m.p. 258–259 οC; IR
(KBr) υ (cm−1): 3449, 3327, 3219 (NH2), 2199 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.20–7.05 (m, 9H, aromatic), 7.03 (bs, 2H,
NH2), 5.69 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.8 (C-3),
142.2 (C-4a), 132.2 (C-10a), 130.4 (C-7), 130.3 (C-9), 129.1 (C-6),
128.7 (C-6a), 124.7 (C-10), 120.1 (C-10b), 118.3 (CN), 118.2 (C-5),
115.1 (C-8), 56.1 (C-2), 35.2 (C-1), 142.4, 135.1, 131.1, 129.6, 127.1
(aromatic); MS m/z (%): 414 (M++4, 3.14), 412 (M++2, 12.25), 410
(M+, 9.39) with a base peak at 299 (1 0 0); Anal. Calcd for
4. Experimental section
C
20H12BrClN2O: C, 58.35; H, 2.94; N, 6.80. Found: C, 58.41; H, 3.00; N,
6.80%.
4.1. Materials and Equipment’s
All chemicals were purchased from Sigma-Aldrich Chemical Co.
(Sigma-Aldrich Corp., St. Louis, MO, USA). All the melting points were
4.2.4. 3-Amino-8-bromo-1-(4-chlorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4d)
measured with
a
Stuart Scientific Co. Ltd apparatus, which are
Prepared as previously described [24].
9

A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
Fig. 9. The destabilization of the preformed microtubules by the target compounds in the cell-free system. (a) The dose response curves for the effect of the
synthesized compounds 4b, 4d, 4e, 4i, and 4k at four different concentrations (µM) on the depolymerization of the preformed microtubules, as monitored by the
decrease in the fluorescence intensity. The excitation and emission wavelengths were 360 and 420 nm. (b) The graphical representation of the microtubules
inhibition percentage of the different examined derivatives in comparison with Colchicine. (c) A schematic presentation of the possible mechanisms of action of the
investigated compounds.
4.2.5. 3-Amino-8-bromo-1-(4-bromophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4e)
4.2.8. 3-Amino-8-bromo-1-(2,6-difluorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4h)
Prepared as previously described [24].
Colorless crystals from ethanol; yield 80%; m.p. 298–299 °C; IR
(KBr) υ (cm−1): 3453, 3338, 3224 (NH2), 2190 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.24–7.05 (m, 8H, aromatic), 7.16 (bs, 2H,
NH2), 5.68 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 161.1 (C-3),
159.1 (C-4a), 147.5 (C-10a), 131.9 (C-7), 130.6 (C-9), 129.8 (C-6),
129.8 (C-6a), 129.5 (C-10), 128.7 (C-10b), 124.0 (C-5), 119.9 (C-8),
119.6 (CN), 53.6 (C-2), 28.0 (C-1), 160.3, 159.1, 130.4, 118.1, 113.1,
112.3 (aromatic); MS m/z (%): 414 (M++2, 13.66), 412 (M+, 14.79)
with a base peak at 300 (1 0 0); Anal. Calcd for C20H11BrF2N2O: C,
58.13; H, 2.68; N, 6.78. Found: C, 58.08; H, 2.63; N, 6.72%.
4.2.6. 3-Amino-8-bromo-1-(4-iodophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4f)
Colorless crystals from ethanol/benzene; yield 87%; m.p.
227–228 °C; IR (KBr) υ (cm -1): 3448, 3318, 3181 (NH2), 2201 (CN); 1H
NMR (DMSO‑d6, 500 MHz) δ: 8.22–6.98 (m, 9H, aromatic), 7.05 (bs,
2H, NH2), 5.32 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.5 (C-
3), 147.1 (C-4a), 132.1 (C-10a), 130.3 (C-7), 130.0 (C-9), 129.0 (C-6),
128.8 (C-6a), 125.9 (C-10), 120.1 (C-10b), 118.2 (C-5), 118.1 (CN),
115.4 (C-8), 57.3 (C-2), 37.4 (C-1), 145.2, 137.5, 129.3, 92.6 (aro-
4.2.9. 3-Amino-8-bromo-1-(2,3-dichlorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4i)
matic); MS m/z (%): 504 (M++2, 31.16) with a base peak at 502 (M+
,
100); Anal. Calcd for C20H12BrIN2O: C, 47.74; H, 2.40; N, 5.57. Found:
C, 47.89; H, 2.54; N, 5.71%.
Colorless crystals from ethanol; yield 84%; m.p. 295–296 °C; IR
(KBr) υ (cm -1): 3448, 3329, 3213 (NH2), 2193 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.24–7.00 (m, 8H, aromatic), 7.18 (bs, 2H,
NH2), 5.78 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.8 (C-3),
144.8 (C-4a), 132.1 (C-10a), 130.5 (C-7), 130.4 (C-9), 128.8 (C-6),
128.7 (C-6a), 124.8 (C-10), 119.6 (C-10b), 118.3 (CN), 118.1 (C-5),
114.5 (C-8), 55.5 (C-2), 36.1 (C-1), 147.5, 132.1, 129.4, 129.2, 129.0,
124.8 (aromatic); MS m/z (%): 450 (M++6, 0.35), 448 (M++4, 2.54),
446 (M++2, 5.54), 444 (M+, 3.36) with a base peak at 300 (1 0 0);
Anal. Calcd for C20H11BrCl2N2O: C, 53.84; H, 2.49; N, 6.28. Found: C,
53.90; H, 2.61; N, 6.40%.
4.2.7. 3-Amino-8-bromo-1-(2,4-difluorophenyl)-1H-benzo[f]chromene-2-
carbonitrile (4g)
Colorless crystals from ethanol; yield 66%; m.p. 300–301 °C; IR
(KBr) υ (cm−1): 3479, 3337, 3290 (NH2), 2200 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.63–7.05 (m, 8H, aromatic), 7.14 (bs, 2H,
NH2), 5.78 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 161.3 (C-3),
159.5 (C-4a), 147.1 (C-10a), 130.8 (C-7), 130.7 (C-9), 129.9 (C-6),
129.7 (C-6a), 129.5 (C-10), 128.6 (C-10b), 123.4 (C-5), 119.8 (C-8),
119.7 (CN), 53.7 (C-2), 28.6 (C-1), 160.1, 159.1, 131.1, 129.6, 117.2,
112.1 (aromatic); MS m/z (%): 414 (M++2, 16.63), 412 (M+, 17.73)
with a base peak at 300 (1 0 0); Anal. Calcd for C20H11BrF2N2O: C,
58.13; H, 2.68; N, 6.78. Found: C, 58.18; H, 2.72; N, 6.82%.
4.2.10. 3-Amino-8-bromo-1-(2,4-dichlorophenyl)-1H-benzo[f]chromene-
2-carbonitrile (4j)
Colorless crystals from ethanol; yield 82%; m.p. 297–298 °C; IR
10

A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
(KBr) υ (cm -1): 3448, 3330, 3210 (NH2), 2193 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.23–7.02 (m, 8H, aromatic), 7.14 (bs, 2H,
NH2), 5.67 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.7 (C-3),
141.3 (C-4a), 132.1 (C-10a), 130.3 (C-7), 129.3 (C-9), 128.6 (C-6),
128.5 (C-6a), 124.8 (C-10), 119.5 (C-10b), 118.3 (CN), 118.1 (C-5),
114.4 (C-8), 55.6 (C-2), 34.8 (C-1), 147.5, 132.2, 132.0, 131.5, 130.5,
129.0 (aromatic); MS m/z (%): 450 (M++6, 0.48), 448 (M++6, 3.61),
446 (M++2, 7.69), 444 (M+, 4.95) with a base peak at 300 (1 0 0);
Anal. Calcd for C20H11BrCl2N2O: C, 53.84; H, 2.49; N, 6.28. Found: C,
53.95; H, 2.59; N, 6.39%.
(DMSO‑d6, 377 MHz) δ: −109.2 (Ar-F); MS m/z (%): 255 (M+, 12.07)
with a base peak at 172 (1 0 0); Anal. Calcd for: C15H14FN3: C, 70.57; H,
5.53; N, 16.46. Found: C, 70.46; H, 5.45; N, 16.38%.
4.2.15. Preparation
of
2-(2-fluoro-4-(piperidin-1-yl)benzylidene)
malononitrile (5) from 2-fluoro-4-piperidin-1-yl-benzaldehyde (7) and
malononitrile (3)
Prepared as previously described [49].
4.3. Biological screening
4.2.11. 3-Amino-8-bromo-1-(2,5-dichlorophenyl)-1H-benzo[f]chromene-
2-carbonitrile (4k)
4.3.1. Cell culture and cytotoxicity evaluation using viability assay
The derivatives 4a-m was initially evaluated for their in vitro anti-
tumor activities against three different human cell lines: MCF-7, HCT-
116, and HepG-2. The in-vitro cytotoxicity evaluation was performed at
the Regional Center for Mycology & Biotechnology (RCMP), Al-Azhar
University under different concentrations (50, 25, 12.5, 6.25, 3.125,
1.56, and 0 µg/mL); Vinblastine and Doxorubicin were employed as
standard cytotoxic drugs. The measurements of cell growth and the in-
vitro cytotoxicity evaluation were determined, using the viability assay,
as described in literature [29–31] and the results were cited in Table 1
and Fig. 3.
Yellow crystals from ethanol; yield 81%; m.p. 293–294 °C; IR (KBr)
υ (cm -1): 3443, 3321, 3211 (NH2), 2195 (CN); 1H NMR (DMSO‑d6,
500 MHz) δ: 8.21–7.02 (m, 8H, aromatic), 7.17 (bs, 2H, NH2), 5.67 (s,
1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 159.8 (C-3), 144.2 (C-4a),
132.5 (C-10a), 130. 6 (C-7), 129.5 (C-9), 128.6 (C-6), 128.8 (C-6a),
124.7 (C-10), 119.5 (C-10b), 118.3 (CN), 118.1 (C-5), 114.0 (C-8), 55.4
(C-2), 35.5 (C-1), 147.5, 132.0, 131.6, 130.4, 130.0, 128.8 (aromatic);
MS m/z (%): 450 (M++6, 1.35), 448 (M++6, 9.54), 446 (M++2,
22.54), 444 (M+, 13.36) with a base peak at 164 (1 0 0); Anal. Calcd for
C
20H11BrCl2N2O: C, 53.84; H, 2.49; N, 6.28. Found: C, 53.73; H, 2.39;
N, 6.19%.
4.3.2. Cell cycle analysis
4.2.12. 3-Amino-8-bromo-1-(2,6-dichlorophenyl)-1H-benzo[f]chromene-
2-carbonitrile (4l)
The cell cycle arrest distribution was performed, exploiting the
Propidium Iodide Flow Cytometry Kit (ab139418, Abcam) as previously
described [50]. The human cancer cell lines MCF-7, HCT-116, and
HepG-2 at the 5 × 104 cells were cultured in 60-mm dishes in the
presence of various tested compounds with the concentration equal to
the IC50 values for 24 h. The cells were then harvested and fixed in a
100% ice cold ethanol at +4 °C for at least 2 h. After rewashing with
PBS, the cells were incubated with a 200 μL 1X Propidium Iodide
(PI) + RNase Staining Solution for 30 min at room temperature in the
dark. The DNA content in each cell nucleus was decided by a FACS
Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, and USA).
The cell cycle phase distribution was analyzed, using the Cell Quest Pro
software (BD Biosciences), which display the collected propidium io-
dide fluorescence intensity on FL2.
Colorless crystals from ethanol; yield 80%; m.p. 318–319 °C; IR
(KBr) υ (cm -1): 3447, 3326, 3210 (NH2), 2195 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.21–7.28 (m, 8H, aromatic), 7.12 (bs, 2H,
NH2), 6.11 (s, 1H, H-1); 13C NMR (DMSO‑d6, 125 MHz) δ: 160.2 (C-3),
148.2 (C-4a), 134.3 (C-10a), 131.9 (C-7), 130.6 (C-9), 128.9 (C-6),
128.9 (C-6a), 124.6 (C-10), 119.3 (C-10b), 117.9 (CN), 117.8 (C-5),
112.7 (C-8), 52.7 (C-2), 35.0 (C-1), 137.1, 135.0, 131.0, 131.1, 129.8,
129.4 (aromatic); MS m/z (%): 450 (M++6, 0.65), 448 (M++6, 4.01),
446 (M++2, 10.86), 444 (M+, 6.76) with a base peak at 300 (1 0 0);
Anal. Calcd for C20H11BrCl2N2O: C, 53.84; H, 2.49; N, 6.28. Found: C,
53.90; H, 2.58; N, 6.35%.
4.2.13. 3-Amino-8-bromo-1-(3,5-dibromo-2-methoxyphenyl)-1H-benzo[f]
chromene-2-carbonitrile (4m)
4.3.3. Annexin V-FITC apoptosis assay
Colorless crystals from ethanol; yield 86%; m.p. 290–291 °C; IR
(KBr) υ (cm -1): 3443, 3320, 3209 (NH2), 2202 (CN); 1H NMR
(DMSO‑d6, 500 MHz) δ: 8.22–7.19 (m, 7H, aromatic), 7.07 (bs, 2H,
NH2), 5.38 (s, 1H, H-1), 3.77 (s, 3H, OCH3); 13C NMR (DMSO‑d6,
125 MHz) δ: 159.6 (C-3), 144.4 (C-4a), 132.1 (C-10a), 130.3 (C-7),
130.0 (C-9), 128.8 (C-6), 128.7 (C-6a), 125.9 (C-10), 120.1 (C-10b),
118.2 (CN), 118.1 (C-5), 115.5 (C-8), 57.3 (C-2), 55.2 (CH3), 37.2 (C-1),
147.1, 131.3, 128.8, 112.8, 102.9 (aromatic); MS m/z (%): 450
The apoptosis assay was performed with an Annexin V-FITC/PI
double staining apoptosis detection kit (K101, Biovison), exercising a
flow cytometer [51]. The human cells treated with the different newly
synthesized derivatives (IC50 value) were harvested by the trypsiniza-
tion, washed twice with 4 °C PBS, and re-suspended in the binding
buffer. Subsequently, the Annexin V-FITC and Propidium iodide (PI)
solutions were added to stain the cells before the analysis by the flow
cytometry, where a minimum of 10,000 cells per sample were acquired.
The Annexin V-FITC binding (FL1) and PI (FL2) were analyzed, using
the Cell Quest Pro software (BD Biosciences).
(M++6, 22.01), 566 (M++4, 6.01), 564 (M++2, 6.14), 562 (M+
,
2.07) with a base peak at 300 (1 0 0); Anal. Calcd for C21H13Br3N2O2: C,
44.64; H, 2.32; N, 4.96. Found: C, 44.71; H, 2.40; N, 5.05%.
4.2.14. 2-(2-Fluoro-4-(piperidin-1-yl)benzylidene)malononitrile (5)
Orange needles from ethanol; yield 32%; m.p. 222–223 °C (Lit. m.p.
224 °C; 32); IR (KBr) υ (cm -1): 2212 (CN); 1H NMR (DMSO‑d6, 500 MHz)
δ: 7.98 (t, 1H, Ph-H3), 7.86 (s, 1H, ]CH), 6.89 (dd, 1H, Ph-H5), 6. 79
(dd, 1H, Ph-H6), 3.52, 3.37 (m, 4H, piperidinyl 2,6-CH2), 1.63–1.56 (m,
6H, piperidinyl 3,4,5-CH2); 13C NMR (DMSO‑d6, 125 MHz) δ: 150.0
(]CH), 116.30 (CN), 71.0 (C-1), 165.9, 156.4, 129.9, 115.59, 110.4,
98.8 (aromatic), 48.1, 25.6, 24.3 (piperidinyl); 13CNMR-APT
(DMSO‑d6, 125 MHz) spectrum CH, CH3 [positive (up)], CH2, Cq [ne-
gative (down)], revealed the following signals at δ: 165.9 (aromatic ↓),
156.4 (aromatic ↓), 150.0 (]CH ↑), 129.9 (aromatic ↑), 116.3 (CN ↓),
115.5 (aromatic ↓), 110.4 (aromatic ↓), 98.8 (aromatic ↑), 71.0 (C-1 ↓),
48.1 (piperidinyl ↓), 25.6 (piperidinyl ↓), 24.3 (piperidinyl ↓); 19F NMR
4.3.4. Determination of Caspase-3, Caspase-8, and Caspase-9 production
Caspase-3, Caspase-8, and Caspase-9 protein levels, the cancer cells
were treated with the investigated compounds (IC50 value) and in-
cubated for 24 h. Then, the Caspase-3, Caspase-8, and Caspase-9 con-
centrations level in the different cell lysates were verified, exploiting
the enzyme-linked immunosorbent assay kit (KHO1091, BMS2024, and
BMS2025; Invitrogen), according to the instructions of the manu-
facturer. The colored product was measured at 450 nm by a plate reader
(ROBONIK P2000 Elisa Reader, Karnataka, India) after the reaction was
terminated by the addition of the stop solution. The concentrations of
the samples were calculated from the standard curve, and the results
were presented as nanogram per milliliter (ng/mL). There was no cross-
reactivity with the other Caspases.
11

A.M. Fouda, et al.
BioorganicChemistry95(2020)103549
4.3.5. DNA fragmentation
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The Topoisomerase II Assay Kit (TG1001-1, TopoGEN) was carried
out according to the manufacture instruction to discern if compounds
(4b, 4d, 4e, 4i, and 4k) inhibited the catalytic decatenation activity of
the topoisomerase II [53]. The decatenated reaction products were se-
parated by the agarose gel electrophoresis prior to the photo doc-
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The altering microtubules polymerization was performed, em-
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(BK011, Cytoskeleton). In accordance with the manufacturer’s in-
structions, the purified tubulin from the porcine brain (2 mg/mL) were
polymerized efficiently in the assay buffer (80 mM PIPES, pH 6.9, 2 mM
MgCl2, 0.5 mM EGTA, 1 mM GTP, and 15% glycerol) at 37 °C to reach a
maximum fluorescence after 30 min. After polymerization, the reaction
mixtures were incubated with the increasing concentrations of the
different tested molecules (0–10 μM) in 96-well microtiter plates. The
fluorescence changes were measured in the Spectramax Gemini fluor-
escence plate reader (Molecular Devices, Sunnyvale, CA) at 37 °C, using
excitation at 360 nm and emission at 420 nm. The destabilizing effect of
our compounds on the preformed microtubules was measured as a
decrease in the fluorescence intensity. The data were assessed against
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4.3.8. Statistics
All data were expressed as the means
standard deviation (SD)
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Declaration of Competing Interest
[18] A. Parthiban, M. Kumaravel, J. Muthukumaran, R. Rukkumani, R. Krishna,
H.S.P. Rao, Synthesis, in vitro and in silico anti-proliferative activity of 4-aryl-4H-
chromene derivatives, Med. Chem. Res. 25 (2016) 1308–1315.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[19] Z. Saffari, H. Aryapour, A. Akbarzadeh, A. Foroumadi, N. Jafari, M.F. Zarabi,
A. Farhangi, In vitro antitumor evaluation of 4H-chromene-3-carbonitrile deriva-
tives as a new series of apoptotic inducers, Tumor. Biol. 35 (2014) 5845–5855.
[20] J.-L. Wang, D. Liu, Z.-J. Zhang, S. Shan, X. Han, S.M. Srinivasula, C.M. Croce,
S. Alnemri, Z. Huang, Structure-based discovery of an organic compound that binds
Bcl-2 protein and induces apoptosis of tumor cells, Proc. Natl. Acad. Sci. USA 97
(2000) 7124–7129.
Acknowledgements
The authors extend their appreciation to the Deanship of Science
Research at King Khalid University, Saudi Arabia for funding this work
through General Research Project under Grant Number (R.G.P.1/111/
40). In addition, the author deeply thanks the Regional Center for
Mycology & Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt,
for carrying out the antitumor study and elemental analyses.
[21] Y. Dai, M. Rahmani, S.J. Corey, P. Dent, S. Grant, A Bcr/Abl-independent, Lyn-
dependent form of imatinib mesylate (STI-571) resistance is associated with altered
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103549.
[24] H.E.A. Ahmed, M.A.A. El-Nassag, A.H. Hassan, R.M. Okasha, S. Ihmaid, A.M. Fouda,
T.H. Afifi, A.A. Aljuhani, A.M. El-Agrody, Introducing novel potent anticancer
agents of 1H-benzo[f]chromene scaffolds, targeting c-Src kinase enzyme with MDA-
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