Cell cycle arrest and induction of apoptosis of newly synthesized pyranoquinoline derivatives under microwave irradiation

Article abstract of DOI:10.1007/s00044-019-02325-5

A set of 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives were prepared via a one-pot, three-component condensation reaction between the substituted hydroxyquinoline derivatives, some aryl and/or hetaryl aldehydes, and malononitrile in

Full text of DOI:10.1007/s00044-019-02325-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02325-5
ORIGINAL RESEARCH
Cell cycle arrest and induction of apoptosis of newly synthesized
pyranoquinoline derivatives under microwave irradiation
Ahmed M. Fouda1 Ayman M. S. Youssef1,2 Tarek H. Afifi3 Ahmed Mora4 Ahmed M. El-Agrody4
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Received: 22 January 2019 / Accepted: 8 March 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A set of 2-amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives were prepared via a one-pot, three-component
condensation reaction between the substituted hydroxyquinoline derivatives, some aryl and/or hetaryl aldehydes, and
malononitrile in an ethanol/piperidine solution in a microwave irradiation environment. The structure of the prepared
1
compounds was instituted on the foundations of their spectral data: IR, H NMR, 13C NMR, and MS. Four human cancer
cell lines, MCF-7, HCT-116, HepG-2, and A549 were utilized to evaluate the antiproliferative properties of the target
compounds in comparison to the positive controls, Vinblastine and Colchicine using the MTT viability assay. The cell cycle
arrest behavior, detected by propidium iodide as well as the apoptosis induction, which was monitored by the flow
cytometer, using the Annexin V-FITC kits, was investigated. The results illustrated that the potent cytotoxic compounds
induce cell cycle arrest at the G2/M phases and trigger apoptosis in the different tested cancer cells. Finally, the structure
−activity relationship (SAR) study showcases the substitution of some specific groups at the 4-, 6-, and 9-positions in the
prepared 2-amino-4H-pyrano[3,2-h]quinoline derivatives, which indicates that the lipophilicity manipulates the ability of
these moieties against the diverse cell lines.
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Keywords Microwave synthesis 4H-Pyrano[3,2-h]quinoline Antitumor Cell cycle SAR
Introduction
antitubercular (Balamurugan et al. 2010), antiasthmatic
(Doube et al. 1998), antimalarial (Kaur et al. 2009; Spar-
atore et al. 2008), antidiabetic (Edmont et al. 2000), anti-
breast cancer (Shi et al. 2008), immunosuppressive (Batt
et al. 1998), antiviral (Narsinh and Anamik 2001), HIV-1
integrase inhibitory (Sechi et al. 2009), and antiproliferative
(Mol et al. 1984) activities. The fused pyranoquinoline
moiety is an extremely common structural motif, existing in
many naturally occurring or biologically active alkaloids,
such as flindersine, atanine, tabouensinium chloride, zan-
thoxylum simulans, oricine, verprisine, and araliopsis
tabouensis (Wabo et al. 2005; Chen et al. 1997, 1994;
Ramesh et al. 1984). The unique biological activity of the
pyranoquinoline derivatives has made these compounds as
privileged targets in recent medicinal studies. For instance,
compounds that contain a pyranoquinoline nucleus have
been frequently used for bactericidal and bacteriolytic
activities (Siliveri et al. 2017; Magesh et al. 2004), acet-
ylcholinesterase inhibitor (Marco and Martinez-Grau 1997),
antiallergenic (Kamperdick et al. 1999; et al. Cairns et al.
1985), anti-inflammatory (Chen et al. 2007), antimalarial
(Isaka et al. 2001), calcium-signaling inhibition (Koizumi
et al. 2007), platelet aggregation (Chen et al. 1997) and
A heterocyclic-based quinoline nucleus is prevalent in
naturally occurring quinoline alkaloids, which are ther-
apeutics and synthetic analogs with significant biological
activities, such as antimicrobial (Prasad et al. 2017; Has-
sanin et al. 2012; Chang et al. 2010; Thomas et al. 2010),
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02325-5) contains supplementary
material, which is available to authorised users.
* Ahmed M. Fouda
amfouda@hotmail.com
1
Chemistry Department, Faculty of Science, King Khalid
University, Abha 61413, Saudi Arabia
2
Chemistry Department, Faculty of Science, Fayoum University,
Fayoum, Egypt
3
Chemistry Department, Faculty of Science, Taibah University, Al-
Madinah Al-Munawarah 30002, Saudi Arabia
4
Chemistry Department, Faculty of Science, Al-Azhar University,
Nar City, Cairo 11884, Egypt

Medicinal Chemistry Research
antitumor (Fouda 2017; Hammouda et al. 2015; Al-Ghamdi
et al. 2012; El-Agrody et al. 2013; 2012) activities and
agents. Recently, a pyranoquinoline nucleus has been
employed in the pharmacophores with potential medicinal
features against various diseases, like Alzheimer’s (Maalej
et al. 2012), venereal (Wabo et al. 2005) and psychotropic
(Nesterova et al. 1995). Based on these outcomes, we have
designed and synthesized a number of related 2-amino-4-
aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives
and assessed their pharmacological effects. The present
study also examines the mechanisms underlying the cyto-
toxicity of the most potent compounds, utilizing cell cycle
analyses, and the structure−activity relationships (SAR) of
the desired molecules will be highlighted on the effect of
the substituents at the 4-, 6-, and 9-positions towards the
antitumor activity.
irradiation conditions for 2 min at 140 °C, presented in
Scheme 1.
The assignment of the structures of the 4a–h derivatives
1
was established on the basis of their spectral data (IR, H
NMR, 13C NMR and MS) and elemental analyses. For
example, the IR spectrum of compound 4c showed
absorption bands at υ 3481, 3330, and 3219 cm−1 for NH2
in addition to the absorption band of the cyano group at υ
2190 cm−1. On the other hand, the 1H NMR spectrum of 4c
revealed the presence of the NH2 protons at δ 7.30 ppm with
the presence of the aliphatic methine proton at δ 5.42 ppm.
The 13C NMR spectrum of 4c revealed that the carbon
methine signal resonate at δ 30.56 ppm. Furthermore, the
mass spectrum of 4c exhibited (m/z) with a base peak at 335
(M+, 100).
In a similar approach, the reaction of 8-hydroxy-2-
methylquinoline (5) with aryl or hetaryl aldehydes (2a–h)
and malononitrile (3) in an ethanol/piperidine solution
under microwave irradiation conditions yielded the 2-
amino-4-aryl-9-methyl-4H-pyrano[3,2-h]quinoline-3-carbo-
nitrile derivatives (6a–h), as disclosed in Scheme 2.
The spectroscopic data (IR, 1H NMR, 13C NMR, and MS)
and the elemental analyses of compounds 6a−h were con-
sistent with the predicted structure. The IR spectrum of deri-
vative 6e revealed that the NH2 and cyano groups’ absorption
bands appeared at υ 3409, 3342, 3197 cm−1, and 2208 cm−1,
Results and discussion
Chemistry
Microwave heating is renowned for accelerating organic
reactions, shortening the reaction time, producing high
yields, having a solvent-free state, or being in ethanol
(Mekheimer and Sadek 2009; Surpur et al. 2009; Wu and
Larhed 2005; Kidwai et al. 2005; Kappe 2004; Shi et al.
2004), and aiding in generating products not attainable
through the classical thermal methods (Fouda 2017; Ham-
mouda et al. 2015; Al-Ghamdi et al. 2012; El-Agrody and
Al-Ghamdi 2011), which require a prolonged reaction time,
stoichiometric reagents, and toxic solvents and generate
only moderate yields of the product. Our methodology,
concerning the targeted heterocyclic-based pyranoquinoline
compounds, was initiated through the synthesis of 2-amino-
4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives
(4a–h) via a one-pot, three-component condensation
between the equimolar amounts of 8-hydroxyquinoline (1),
various aryl or hetaryl aldehydes (2a–h), and malononitrile
(3) in ethanol, catalyzed by piperidine under microwave
1
respectively. Additionally, their H NMR spectrum displayed
three signals at δ 7.28, δ 5.9, and δ 2.71 ppm, due to the NH2,
CH, and CH3 groups, respectively. The 13C NMR spectrum of
6e displayed two signals, resonating at δ 31.03 and δ 23.43
ppm, which can be attributed to the CH (pyrano) and CH3
carbons, respectively. Also, the mass spectrum showed (m/z),
403 (M+, 96.79) with a base peak at 40 (100).
Following the same methodology, the interaction of the
5-chloro-8-hydroxyquinoline (7) with several aryl or hetaryl
aldehydes (2a–i) and malononitrile (3) in an ethanol/
piperidine solution in a microwave irradiation environment
delivered the 2-amino-4-aryl-6-chloro-4H-pyrano[3,2-h]
quinoline-3-carbonitrile derivatives (8a–i), Scheme 3. The
newly formed products 8a−i were characterized by their IR,
Scheme 1 2-Amino-4-aryl-4H-pyrano[3,2-h]quinoline-3-carbonitrile
derivatives (4a–h)
Scheme
carbonitrile derivatives (6a–h)
2 2-Amino-4-aryl-9-methyl-4H-pyrano[3,2-h]quinoline-3-

Medicinal Chemistry Research
1H, and 13C NMR spectra. Supporting evidence for the
suggested structures arises from the infrared absorption
bands of compound 8g at υ 3443, 3344, 3238 cm−1, and
2213 cm−1 for the NH2 and cyano groups. On the contrary,
cytotoxic effects of the 2-amino-4-aryl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile derivatives (4, 6, 8) against the
selected human cancer cell lines, namely: mammary gland
breast cancer (MCF-7), human colon cancer (HCT-116),
human hepatocellular carcinoma (HepG-2), and lung car-
cinoma (A549), using Vinblastine and Colchicine as refer-
ence compounds. The in vitro cytotoxicity evaluation was
achieved under various concentrations, ranging from 0 to
50 μg/ml. The results were expressed as growth inhibitory
concentration (IC50) values, where the necessitated con-
centration produced a 50% inhibition of cell growth after
24 h of incubation, compared to the untreated controls. The
IC50 values are summarized in Fig. 1 and Table 1.
From the obtained results, it was elucidated that most of
the prepared compounds displayed excellent to modest or
weak growth inhibitory activity against the tested cancer
cell lines. Investigations of the cytotoxic activity against
MCF-7, HCT-116, HepG-2, and A549 tumor cell lines
indicated that the MCF-7 was the most sensitive cell line to
the influence of the new derivatives. Overall, compounds
4a, d (IC50 = 1.42 0.3, 1.35 0.2, 1.87 0.2, 1.23 0.1
and 1.17 0.11, 1.3 0.12, 1.41 0.18, 38 0.03 µg/ml,
respectively), 6a, d (IC50 = 5.79 0.01, 3.01 0.11 and
1.24 0.3, 1.83 0.2 µg/ml) and 8a, b, d, e (IC50 = 0.9
0.07, 3.04 0.2, 0.7 0.06, 2.67 0.07; 0.99 0.01, 5.81
0.11, 1.23 0.03, 3.1 0.01; 2.47 0.13, 7.94 0.05, 1.48
0.11, 15.6 0.13 and 1.18 0.14, 3.01 0.22, 0.87
0.12, 1.79 0.14 µg/ml, respectively) were found to be the
most potent derivatives of all the tested compounds against
the MCF-7, HCT-116, HepG-2, and A549 tumor cell lines.
In general, the other compounds demonstrated moderate to
fair cytotoxic activities against the four tumor cell types.
1
the H NMR spectrum of 8g revealed the presence of the
NH2 protons at δ 7.48 ppm and the aliphatic methine proton
at δ 5.58 ppm. Further confirmation resulted from the 13C
NMR spectrum of 8g that exhibited signal, resonating at δ
35.98 ppm for the CH carbon. Moreover, the mass spectrum
of 8g revealed (m/z), 341 (M++2, 32.08) with a base peak
at 339 (M+, 100).
The maximum power of the microwave irradiation was
optimized by repeating all the reactions at different watt
powers and periods of times. The optimum condition was
obtained by using microwave irradiations at 400 W and a
reaction time of 2 min, which gave the highest yield of the
desired compounds. It is also important to note that the 4-
position of compounds 4a–h, 6a–h, and 8a–i are chiral
centers, and all the reactions were controlled, using the thin
layer chromatography (TLC) technique.
Biological activity
In vitro cytotoxic activity
The MTT assay (Demirci and Bas¸er 2002; Rahman et al.
2001; Mosmann 1983) was performed to evaluate the
G2/M phase cell cycle arrest in treated cancer cells
In order to explain the effect of the most potent newly
synthesized compounds (4a, d, 6d, 8a, b, d, e) against the
different cancer cell lines, we have performed a cell cycle
Scheme
3 2-Amino-4-aryl-6-chloro-4H-pyrano[3,2-h]quinoline-3-
carbonitrile derivatives (8a−i)
Fig. 1 IC50 values expressed in
(µg/ml) of 2-amino-4-aryl-4H-
pyrano[3,2-h]quinoline-3-
carbonitrile derivatives against
MCF-7, HCT-116, HepG-2, and
A549 tumor cells

Medicinal Chemistry Research
Table 1 IC50 of the target compounds against MCF-7, HCT-116, HepG-2, and A549 cell lines
Compound
Ar
IC50 (µg/ml)a
MCF-7
HCT-116
HepG-2
A549
4a
3-ClC6H4
1.42 0.3b
34.8 0.15c
1.35 0.2b
43.7 0.13c
1.87 0.2b
NAc
1.23 0.1b
wb
4b
4-BrC6H4
4c
2,6-F2C6H3
2,3-Cl2C6H3
2,3,4,5,6-F5C6
thiophen-2-yl
4-bromothiophen-2-yl
pyridin-3-yl
3-ClC6H4
w
w
w
w
4d
1.17 0.11b
1.3 0.12b
1.41 0.18b
38 0.03b
4e
w
w
w
w
4f
w
w
33.3 0.02
w
52.5 0.06
w
4g
w
w
4h
w
w
w
w
6a
5.79 0.01b
38.9 0.14c
w
21.4 0.1b
22.8 0.13c
w
3.01 0.11b
5.5 0.13c
w
24 0.01b
36.7 0.14b
w
6b
4-BrC6H4
6c
2,6-F2C6H3
3,4-Cl2C6H3
2,3,4,5,6-F5C6
thiophen-2-yl
4-bromothiophen-2-yl
pyridin-3-yl
3-ClC6H4
6d
1.24 0.3b
23.7 0.04b
1.83 0.2b
13.7 0.3b
6e
w
w
w
w
6f
24.4 0.1
45.9 0.2
w
0.9 0.07b
0.99 0.01b
w
2.47 0.13b
1.18 0.14b
w
18.8 0.2
41.54 0.14
36.4 0.13
11.8 0.3
24.8 0.16
21.7 0.1
0.7 0.06b
1.23 0.03b
w
1.48 0.11b
0.87 0.12b
w
15.5 0.2
20.5 0.3
28.9 0.06
2.67 0.07
3.1 0.01b
w
15.6 0.13b
1.79 0.14b
w
6g
6h
b
8a
3.04 0.2
5.81 0.11b
8b
4-BrC6H4
8c
2,6-F2C6H3
2,3-Cl2C6H3
2,4-Cl2C6H3
2,3,4,5,6-F5C6
thiophen-2-yl
4-bromothiophen-2-yl
pyridin-3-yl
—
w
8d
7.94 0.05b
3.01 0.22b
8e
8f
w
8g
w
w
w
w
8h
w
w
w
w
8i
w
w
w
w
Vinblastine
Colchicine
6.1 0.01
17.7 0.03
2.6 0.04
42.8 0.06
4.6 0.11
10.6 0.13
3.78 0.01
21.3 0.03
—
aIC50 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
w = weak activity (IC50 ≥ 50 µg/ml)
bFouda 2017
cEl-Agrody et al. 2013 and NA not active
analysis, using the flow cytometer (Qu et al. 2016; Shen
et al. 2009). The cell cycle distribution for the untreated
control cells, reference drugs, and compound 8b is dis-
played in Fig. 2a, b as the percentage of cells containing 2n
(G1 phase), 4n (G2 and M phases), and 4n > 3n > 2n of
DNA amount (S phase), assessed via PI staining. All the
tested compounds expressed an increased cell number at the
G2/M phases, up to 40% after 24 h, as compared to the 10%
in the control cells, Fig. 2c. These results are accompanied
by a 10% decrease in the cell number at the S phase and a

Medicinal Chemistry Research
Fig. 2 a, b Representative histograms of DNA content distribution of
cell cycle phases of MCF-7 and HCT-116 (a), HepG-2 and A549 (b)
of untreated control cells, reference drugs and compound 8b. DNA
content was stained with P). Other compounds 4a, d, 6d and 8a, d, e
histograms results are shown in supplementary data Fig. 2d. c The
graphical representation of the trend in cell cycle arrest of MCF-7,
HCT-116, HepG-2, and A549 cells after treatment with IC50 con-
centration of tested compounds for 24 h

Medicinal Chemistry Research
Fig. 3 a, b Annexin V/PI assay showing dot plots of MCF-7 and HCT-
116 (a), HepG-2 and A549 (b) of untreated control cells, and treated
cells with reference drugs and compound 8b. Other compounds 4a, d,
6d and 8a, d, e dot plots results are shown in supplementary data Fig.
3d. c The percentage of different cell lines in different stages following
the treatment with IC50 concentration of tested compounds for 24
assessed via Annexin V/PI assay and flow cytometry

Medicinal Chemistry Research
40% decrease in the G1 phase for all cell lines in compar-
ison to the untreated control cells, Fig. 2b. Moreover, the
tested compounds showed higher cell cycle arrest percen-
tages at G2/M than the reference drug Colchicine while they
showed almost the same result as Vinblastine. However, the
compound 4a in the case of the A549 cell line and com-
pound 8a in the case of HepG-2 have presented a slight
increase in the G1 phase cell number and a decrease in the
G2/M phases with no differences in the S phase when
evaluated against the control cells.
group) against all the tumor cells as compared to the stan-
dard drugs Vinblastine and Colchicine (IC50 = 6.1 0.01,
2.6 0.04, 4.6 0.11, 3.78 0.01, and 17.7 0.03, 42.8
0.06, 10.6 0.13, 21.3 0.03 µg/ml, respectively). This
implying that the presence of monochloro and dichloro
atoms of compounds 4a and 4d, respectively, on the phenyl
ring at 4-position of the 4H-pyrano[3,2-h]quinoline was
indispensable regarding the activities of the four cancer cells
as compared to the other substituents and/or hetero moiety.
Additionally, the IC50 values of the second series 6a−h
decreased for the first members (monosubstituent, 6a, IC50
= 5.79 0.01 and 3.01 0.11 µg/ml) and the second mem-
bers (disubstituent, 6d IC50 = 1.24 0.3 and 1.83 0.2 µg/
ml) with respect to breast cancer and human hepatocellular
carcinoma, while the values increased (moderate to weak
activities) for the third and fourth members (polysubstituents
and hetero group) in comparison to the standard drugs
Vinblastine and Colchicine (IC50 = 6.1 0.01, 4.6 0.11,
and 17.7 0.03, 10.6 0.13 µg/ml, respectively), which
suggests that the replacement of hydrogen atom with
monochloro and dichloro atoms of compounds 6a and 6d,
respectively, together with the methyl group at the 9-postion
was imperative for the activities against breast cancer and
human hepatocellular carcinoma. Further investigation of the
impact of the substitution patterns on the phenyl or hetero
ring at the 4-position with the chloro atom at the 6-postion of
the 4H-pyrano[3,2-h]quinoline moiety has rendered the first
members (monosubstituent, 8a, b IC50 = 0.9 0.07, 3.04
0.2, 0.7 0.06, 2.67 0.07 and 0.99 0.01, 5.81 0.11,
1.23 0.03, 3.1 0.01 µg/ml, respectively) and second
members (disubstituent, 8d, e IC50 = 2.47 0.13, 7.94
0.05, 1.48 0.11, 15.6 0.13 and 1.18 0.14, 3.01 0.22,
0.87 0.12, 1.79 0.14 µg/ml, respectively) with a remark-
able enhancement in their antitumor activities against the
four tumor cell types, intimating that grafting lipophilic
electron-withdrawing substituents like the halogens may be
more advantageous than the other substituents and that the
monosubstituent is more active than the disubstituent.
Finally, we can deduce that the substitution patterns on the
phenyl or hetero ring at the 4-position of the 4H-pyrano[3,2-
h]quinoline moiety is a crucial element for the antitumor
activity. The incorporation of electron-donating groups as
the methyl group (hydrophobic group) at the 9-position is
not favorable for activity while the substitution of bulky
electron-withdrawing groups as the chlorine atom (hydro-
phobic group) at the 6-position has greatly enhanced the
activity.
Apoptosis induction in treated cancer cells
Many anticancer compounds exert their effect by blocking
the cell cycle progression, inducing apoptosis, or the
combined effect of both. To verify the linked relation of
the cell cycle arrest and apoptosis, an Annexin/PI double
staining assay was performed (Van Engeland et al. 1998;
Vermes et al. 1995). Dot plots for the untreated control
cells, reference drugs, and compound 8b are shown in Fig.
3a, b.
The percentage of both the early (Annexin V positive, PI
negative) and late (Annexin V positive, PI positive) apop-
tosis for the treated cells are shown in Fig. 3c. All cell lines
treated with compounds 4a and 8a, b, 8d, and 8e for 24 h
generated high apoptosis percentages more or less equal to
Colchicine and Vinblastine. Furthermore, we notice that the
MCF-7, HepG-2, and A549 cells entered a late apoptotic
stage after their treatment with compound 4a. However,
compounds 4d and 6d displayed the lowest percentage of
apoptosis in the case of HCT-116, HepG-2, and A549 cells.
Necrotic cells were not observed with any of the tested
compounds. The results corroborated that the induction of
cytotoxicity occurs through the mechanisms associated with
apoptosis.
SAR studies
The preliminary SAR study was centered on the effect of the
replacement of the hydrogen atoms on the phenyl or hetero
ring at the 4-position of the 4H-pyrano[3,2-h]quinoline
scaffold by the different halogen atoms and the chloro or
methyl group at the 6- and 9-positions. In a comparison of
the cytotoxic activities of the three series 4a−h, 6a−h, and
8a−i against the MCF-7, HCT-116, HepG-2, and A549
tumor cell types, we discovered that the introduction of this
modification has decreased the IC50 values of the first
members (monosubstituent, 4a, IC50 = 1.42 0.3, 1.35
0.2, 1.87 0.2, and 1.23 0.1 µg/ml, respectively) and the
second members (disubstituents 4d, IC50 = 1.17 0.11, 1.3
0.12, 1.41 0.18, and 38 0.03, respectively) and
increased the IC50 values (moderate to weak activities) for
the third and fourth members (polysubstituents and hetero
Conclusion
The biological study was executed to analyze the outcome
of the substituents at different positions on the antitumor

Medicinal Chemistry Research
behavior. Most of the targeted compounds exhibited good
antitumor activities towards the evaluated tumor cell lines.
Additionally, we found that these potent cytotoxic com-
pounds induce cell cycle arrest at the G2/M phases and
trigger apoptotic cell death for the four different cell lines.
Furthermore, the SAR of the desired molecules highlighted
the effect of the substituents at the 4-, 6-, and 9-positions on
the antitumor activity.
2-Amino-4-(4-bromophenyl)-4H-pyrano[3,2-h]quinoline-3-
carbonitrile (4b)
Yellow needles, yield 81%; m.p. 231−232 οC (Lit. m.p. 230
−231 °C; El-Agrody and Al-Ghamdi 2011).
2-Amino-4-(2,6-difluorophenyl)-4H-pyrano[3,2-h]quinoline-
3-carbonitrile (4c)
Yellow crystals from ethanol; yield 85%; m.p. 292−294 °C;
IR (KBr, υ max cm−1): 3481, 3330, 3219 (NH2), 2190
(CN), 1663 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.95
−7.06 (m, 8H, aromatic), 7.30 (bs, 2H, NH2), 5.42 (s, 1H,
H-4); 13C NMR (125 MHz, DMSO-d6) δ: 161.56 (C-2),
150.29 (C-9), 143.66 (C-10b), 137.23 (C-10a), 135.96 (C-
7), 127.91 (C-5), 126.01 (C-6a), 123.70 (C-4a), 122.19 (C-
8), 120.12 (C-6), 119.98 (CN), 52.78 (C-3), 30.56 (C-4),
160.80, 129.98, 119.73, 111.96 (aromatic); MS (m/z) with a
base peak at 335 (M+, 100); C19H11F2N3O (335.31); calcd;
% C: 68.06, % H: 3.31, % N: 12.53; found; % C: 67.99, %
H: 3.24, % N: 12.46.
Experimental section
Materials and equipments
All chemicals were purchased from Sigma-Aldrich Chemical
Co. (Sigma-Aldrich Corp., St. Louis, MO, USA). All melting
points measured with a Stuart Scientific Co. Ltd. apparatus are
uncorrected. The IR spectra were recorded on a KBr disc on a
Jasco FT/IR 460 plus spectrophotometer. The H/13C NMR
1
(500/125 MHz) spectra were measured on BRUKER AV 500
MHz spectrometer in DMSO-d6 as a solvent, using tetra-
methylsilane (TMS) as an internal standard, and chemical
shifts (δ) are expressed in parts per million (ppm). The
microwave apparatus used is Milestone Sr1, Microsynth. The
mass spectra were determined on a Shimadzu GC/MS-
QP5050A spectrometer. 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 %. Reaction courses and product mixtures
were routinely monitored by TLC on silica gel-precoated F254
Merck plates.
2-Amino-4-(2,3-dichlorophenyl)-4H-pyrano[3,2-h]quinoline-
3-carbonitrile (4d)
Yellow crystals from benzene; yield 85%; m.p. 280−281 °C
(Lit. m.p. 240−241 °C; Fouda 2017).
2-Amino-4-(2,3,4,5,6-pentafluorophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (4e)
Yellow crystals from benzene; yield 81%; m.p. 224−226 °C;
IR (KBr, υ max cm−1): 3412, 3338, 3186 (NH2), 2194 (CN),
General procedure for synthesis of 2-amino-4-aryl-
4H-pyrano[3,2-h]quinoline-3-carbonitrile derivatives
(4a–h, 6a–h, 8a–i)
1
1661 (C=N); H NMR (500 MHz, DMSO-d6) δ: 8.96−7.21
(m, 5H, aromatic), 7.35 (bs, 2H, NH2), 5.42 (s, 1H, H-4); 13
C
NMR (125 MHz, DMSO-d6) δ: 160.84 (C-2), 150.36 (C-9),
143.66 (C-10b), 136.00 (C-10a), 130.28 (C-7), 128.07 (C-5),
127.41 (C-6a), 123.85 (C-4a), 122.31 (C-8), 121.74 (C-6),
119.92 (CN), 52.36 (C-3), 31.01 (C-4), 142.63, 140.68,
137.21, 117.64 (aromatic); MS (m/z), 389 (M+, 30.38) with a
base peak at 350 (100); C19H8F5N3O (389.28); calcd; % C:
61.98, H, 3.01; N, 11.41; Found; % C: 61.95, % H: 2.99, %
N: 11.39.
A reaction mixture of 8-hydroxyquinoline (1), 8-hydroxy-2-
methylquinoline (5) or 5-chloro-8-hydroxyquinoline (7)
(0.01 mol), with different aryl or hetaryl aldehydes (2a–h,
or 2a–i) (0.01 mol) and malononitrile (3) (0.01 mol) in
ethanol catalyzed by piperidine under microwave irradiation
conditions was heated for 2 min at 140 °C. After completion
of the reaction, the reaction mixture was cooled to room
temperature and the precipitated solid was filtered off,
washed with methanol, and was recrystallized from ethanol
or ethanol/benzene. The physical and spectral data of
compounds 4a–h, 6a–h and 8a–i are as follows.
2-Amino-4-(thiophen-2-yl)-4H-pyrano[3,2-h]quinoline-3-
carbonitrile (4f)
Yellow crystals from benzene; yield 81%; m.p. 261−262 °C;
IR (KBr, υ max cm−1): 3447, 3328, 3211 (NH2), 2212 (CN),
1626 (C=N); H NMR (500 MHz, DMSO-d6) δ: 8.97−7.17
2-Amino-4-(3-chlorophenyl)-4H-pyrano[3,2-h]quinoline-3-
carbonitrile (4a)
1
(m, 8H, aromatic and thiophene), 7.56 (s, 1H, thiophene H-5),
7.46 (bs, 2H, NH2), 7.18 (s, 1H, thiophene H-3), 7.37 (bs, 2H,
NH2), 5.39 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ:
Yellow crystals from ethanol; yield 84%; m.p. 241−242 °C
(Lit. m.p. 240−241 °C; Fouda 2017).

Medicinal Chemistry Research
160.67 (C-2), 152.33 (C-9), 150.37 (C-10b), 137.39 (C-10a),
136.02 (C-7), 127.98 (C-5), 126.86 (C-6a), 123.97 (C-4a),
122.38 (C-8), 120.86 (C-6), 120.02 (CN), 55.22 (C-3), 36.30
(C-4), 142.57, 126.57, 123.37 (thiophene); MS (m/z), 305
(M+, 30.38) with a base peak at 222 (100); calcd; For
C17H11N3OS (305.35); % C: 66.87, % H: 3.63, N, 13.76;
Found; % C: 66.95, % H: 3.69, % N: 13.83.
2-Amino-4-(2,6-difluorophenyl)-9-methyl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6c)
Yellow crystals from ethanol; yield 81%; m.p. > 300 °C; IR
(KBr, υ max cm−1): 3454, 3341, 3223 (NH2), 2194 (CN),
1655 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.22−7.05
(m, 7H, aromatic), 7.17 (bs, 2H, NH2), 5.40 (s, 1H, H-4),
2.71 (s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ:
161.48 (C-2), 159.13 (C-9), 143.24 (C-10b), 136.23 (C-
10a), 138.08 (C-7), 126.29 (C-5), 125.02 (C-6a), 123.53 (C-
4a), 122.92 (C-8), 120.09 (C-6), 119.98 (CN), 52.88 (C-3),
30.60 (C-4), 24.97 (CH3), 160.83, 129.95, 119.09, 111.96
(aromatic); MS (m/z), 349 (M+, 100); C20H13F2N3O
(349.33); calcd; % C: 68.76, % H: 3.75, % N: 12.03, found;
% C: 68.70, % H: 3.71, % N: 11.96.
2-Amino-4-(4-bromothiophen-2-yl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (4g)
Yellow crystals from benzene; yield 80%; m.p. 258−260 °
C; IR (KBr, υ max cm−1): 3387, 3317, 3193 (NH2), 2202
(CN), 1661 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.96
−7.43 (m, 5H, aromatic), 7.55 (s, 1H, thiophene H-5), 7.37
(bs, 2H, NH2), 7.18 (s, 1H, thiophene H-3), 5.38 (s, 1H, H-
4); 13C NMR (125 MHz, DMSO-d6) δ: 160.62 (C-2),
152.38 (C-9), 150.37 (C-10b), 137.39 (C-10a), 136.07 (C-
7), 127.98 (C-5), 126.89 (C-6a), 123.90 (C-4a), 123.33 (C-
8), 120.86 (C-6), 120.02 (CN), 55.23 (C-3), 36.30 (C-4),
142.74, 126.57, 122.36, 108.16 (thiophene); MS (m/z), 385
(M++2, 96.19), 383 (M+, 100); calcd; For C17H10BrN3OS
(384.25); % C: 53.14, % H: 2.62, N, 10.94; Found; % C:
53.09, % H: 2.57, % N: 10.90.
2-Amino-4-(3,4-dichlorophenyl)-9-methyl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6d)
Yellow crystals from benzene; yield 88%; m.p. 241−242 °
C (Lit. m.p. 240−241 °C; Fouda 2017).
2-Amino-4-(2,3,4,5,6-pentafluorophenyl)-9-methyl-4H-
pyrano[3,2-h]quinoline-3-carbonitrile (6e)
2-Amino-4-(pyridin-3-yl)-4H-pyrano[3,2-h]quinoline-3-
carbonitrile (4h)
Yellow crystals from benzene; yield 80%; m.p. 295−296 °C;
IR (KBr, υ max cm−1): 3409, 3342, 3197 (NH2), 2208 (CN),
1663 (C=N); H NMR (500 MHz, DMSO-d6) δ: 8.24−7.12
1
Yellow crystals from benzene; yield 80%; m.p. 264−265 °
C; IR (KBr, υ max cm−1): 3463, 3326, 3191 (NH2), 2191
(CN), 1654 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.48
−7.20 (m, 9H, aromatic and pyridine), 7.31 (bs, 2H, NH2),
5.08 (s, 1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ:
160.44 (C-2), 150.31 (C-9), 148.76 (C-10b), 137.46 (C-
10a), 136.00 (C-7), 127.83 (C-5), 126.89 (C-6a), 124.07 (C-
4a), 122.26 (C-8), 120.92 (C-6), 120.192 (CN), 55.19 (C-3),
38.58 (C-4), 148.41, 143.22, 140.87, 135.42, 123.82 (pyr-
idine); MS (m/z), 300 (M+, 32.82) with a base peak at 222
(100); calcd; For C18H12N4O (300.31); % C: 71.99, % H:
4.03, % N: 18.66. Found; % C: 71.93, % H: 3.98, % N:
18.60.
(m, 4H, aromatic), 7.28 (bs, 2H, NH2), 5.9 (s, 1H, H-4), 2.71
(s, 3H, CH3); 13C NMR (125 MHz, DMSO-d6) δ: 160.87
(C-2), 159.21 (C-9), 143.22 (C-10b), 136.76 (C-10a), 130.15
(C-7), 126.63 (C-5), 125.09 (C-6a), 123.67 (C-4a), 123.03
(C-8), 119.96 (C-6), 118.32 (CN), 52.41 (C-3), 31.03 (C-4),
23.43 (CH3), 142.50, 140.66, 136.09, 114.61 (aromatic); MS
(m/z), 403 (M+, 96.79) with a base peak at 40 (100);
C20H10F5N3O (403.3); calcd; % C: 59.56, H, 2.50; N, 10.42.
Found; % C: 59.46, % H: 2.41, % N: 10.38.
2-Amino-4-(thiophen-2-yl)- 9-methyl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6f)
Yellow crystals from benzene; yield 84%; m.p. 268−269 °C;
IR (KBr, υ max cm−1): 3449, 3343, 3193 (NH2), 2194 (CN),
1662 (C=N); H NMR (500 MHz, DMSO-d6) δ: 9.08−7.22
2-Amino-4-(3-chloroophenyl)-9-methyl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6a)
1
(m, 4H, aromatic and thiophene), 7.57 (s, 1H, thiophene H-5),
7.43 (bs, 2H, NH2), 7.33 (s, 1H, thiophene H-4), 7.22 (s, 1H,
thiophene H-3), 5.35 (s, 1H, H-4), 2.71 (s, 3H, CH3); 13C
NMR (125 MHz, DMSO-d6) δ: 160.49 (C-2), 151.75 (C-9),
151.20 (C-10b), 138.08 (C-10a), 132.55 (C-7), 125.33 (C-5),
125.13 (C-6a), 123.49 (C-4a), 123.33 (C-8), 121.42 (C-6),
119.75 (CN), 55.14 (C-3), 36.04 (C-4), 23.42 (CH3), 142.27,
127.18, 126.60 (thiophene); MS (m/z), 319 (M+, 38.38) with
a base peak at 293 (100); calcd; For C18H13N3OS (319.38); %
Yellow crystals from ethanol; yield 87%; m.p. 238−239 °C
(Lit. m.p. 240−241 °C; Fouda 2017).
2-Amino-4-(4-bromophenyl)- 9-methyl-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6b)
Colorless needles, yield 75%; m.p. 279−280 οC (Lit. m.p.
230–231 °C; El-Agrody and Al-Ghamdi 2011).

Medicinal Chemistry Research
C: 67.69, % H: 4.10, N, 13.16. Found; % C: 67.61, % H:
4.02, % N: 13.08.
2-Amino-6-chloro-4-(2,6-difluorophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8c)
2-Amino-4-(4-bromothiophen-2-yl)-9-methyl-4H-pyrano
[3,2-h]quinoline-3-carbonitrile (6g)
Yellow crystals from ethanol; yield 82%; m.p. 298−290 °C;
IR (KBr, υ max cm−1): 3483, 3328, 3228 (NH2), 2192
(CN), 1656 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 8.21
−7.04 (m, 7H, aromatic), 7.18 (bs, 2H, NH2, D2O
exchangeable), 5.39 (s, 1H, H-4); 13C NMR (125 MHz,
DMSO-d6) δ: 161.54 (C-2), 159.51 (C-9), 143.23(C-10b),
136.07 (C-10a), 136.07 (C-7), 129.86 (C-5), 126.29 (C-6),
125.00 (C-6a), 123.52 (C-8), 120.10 (C-4a), 119.97 (CN),
52.88 (C-3), 30.59 (C-4), 160.83, 129.94, 119.85, 112.11
(aromatic); MS (m/z), 371 (M+, 30.62), 369 (M+, 100);
C19H10ClF2N3O (369.75); calcd; % C: 61.72, % H: 2.73,
% N: 11.36; found; % C: 61.79, % H: 2.79, % N: 11.75.
Yellow crystals from benzene; yield 81%; m.p. 265−267
°C; IR (KBr, υ max cm−1): 3465, 3329, 3198 (NH2), 2190
1
(CN), 1652 (C=N); H NMR (500 MHz, DMSO-d6) δ:
9.05−7.65 (m, 4H, aromatic), 7.57 (s, 1H, thiophene H-
5), 7.45 (bs, 2H, NH2), 7.23 (s, 1H, thiophene H-3), 5.36
(s, 1H, H-4), 2.72 (s, 3H, CH3); 13C NMR (125 MHz,
DMSO-d6) δ: 160.43 (C-2), 151.76 (C-9), 151.20 (C-10b),
138.08 (C-10a), 132.52 (C-7), 126.19 (C-5), 125.36
(C-6a), 125.11 (C-4a), 123.49 (C-8), 121.42 (C-6), 119.75
(CN), 55.13 (C-3), 36.03 (C-4), 23.49 (CH3), 142.21,
127.18, 123.60, 108.33 (thiophene); MS (m/z), 399 (M+
+2, 5.95), 397 (M+, 6.05) with a base peak at 371 (100);
calcd; For C18H12BrN3OS (398.28); % C: 54.28, % H:
3.04, N, 10.55. Found; % C: 54.36, % H: 3.11, % N:
10.60.
2-Amino-6-chloro-4-(2,3-dichlorophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8d)
Pale yellow crystals from benzene; yield 87%; m.p. 300–
301 °C (Lit. m.p. 240−241 °C; Fouda 2017).
2-Amino-4-(pyridin-3-yl)- 9-methyl-4H-pyrano[3,2-h]
2-Amino-6-chloro-4-(2,4-dichlorophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (6h)
quinoline-3-carbonitrile (8e)
Yellow crystals from benzene; yield 82%; m.p. 281−282
°C; IR (KBr, υ max cm−1): 3454, 3341, 3191 (NH2), 2194
(CN), 1655 (C=N); H NMR (500 MHz, DMSO-d6) δ:
Pale yellow crystals from benzene; yield 85%; m.p. 276
−277 °C (Lit. m.p. 240−241 °C; Fouda 2017).
1
8.58−7.12 (m, 8H, aromatic and pyridine), 7.23 (bs, 2H,
NH2), 5.05 (s, 1H, H-4), 2.70 (s, 3H, CH3); 13C NMR
(125 MHz, DMSO-d6) δ: 160.48 (C-2), 159.11 (C-9),
148.80 (C-10b), 137.03 (C-10a), 136.08 (C-7), 126.32
(C-5), 125.66 (C-6a), 124.05 (C-4a), 122.93 (C-8),
120.93 (C-6), 120.21 (CN), 55.34 (C-3), 38.64 (C-4),
24.66 (CH3), 148.37, 141.01, 135.35, 126.18, 123.81
(pyridine); MS (m/z), 314 (M+, 26.46) with a base peak at
236 (100); calcd; For C19H14N4O (314.34); % C: 72.60, %
H: 4.49, % N: 17.82. Found; % C: 72.67, % H: 4.55, % N:
17.88.
2-Amino-6-chloro-4-(2,3,4,5,6-pentafluorophenyl)-4H-
pyrano[3,2-h]quinoline-3-carbonitrile (8f)
Yellow crystals from benzene; yield 81%; m.p. 230−231°C;
IR (KBr, υ max cm−1): 3404, 3335, 3189 (NH2), 2195 (CN),
1
1660 (C=N); H NMR (500 MHz, DMSO-d6) δ: 9.07−7.44
(m, 4H, aromatic), 7.41 (bs, 2H, NH2), 5.42 (s, 1H, H-4); 13
C
NMR (125 MHz, DMSO-d6) δ: 160.51 (C-2), 151.21 (C-9),
143.21 (C-10b), 137.83 (C-10a), 130.35 (C-7), 127.46 (C-5),
125.92 (C-6), 125.44 (C-6a), 123.49 (C-8), 122.93 (C-4a),
119.62 (CN), 52.41 (C-3), 30.75 (C-4), 142.63, 140.67,
132.44, 118.97 (aromatic); MS (m/z), 425 (M++2, 18.15),
423 (M+, 5.91) with a base peak at 40 (100); C19H7ClF5N3O
(423.72); calcd; % C: 53.86, H, 1.67; N, 9.92. Found; % C:
53.80, % H: 1.61, % N: 9.86.
2-Amino-6-chloro-4-(3-chlorophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8a)
Yellow crystals from benzene; yield 87%; m.p. 272−273 °C
(Lit. m.p. 240−241 °C; Fouda 2017).
2-Amino-6-chloro-4-(thiophen-2-yl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8g)
2-Amino-6-chloro-4-(4-bromophenyl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8b)
Yellow crystals from benzene; yield 80%; m.p. 283−282 °
C; IR (KBr, υ max cm−1): 3443, 3344, 3238 (NH2), 2213
(CN), 1667 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.05
−7.22 (m, 7H, aromatic and thiophene), 7.58 (s, 1H,
Yellow crystals from ethanol; yield 88%; m.p. 229−230 °C
(Lit. m.p. 240−241 °C; Fouda 2017).

Medicinal Chemistry Research
thiophene H-5), 7.48 (bs, 2H, NH2), 7.24 (s, 1H, thiophene
H-3), 7.47 (bs, 2H, NH2), 5.38 (s, 1H, H-4); 13C NMR (125
MHz, DMSO-d6) δ: 160.42 (C-2), 151.79 (C-9), 151.25 (C-
10b), 138.06 (C-10a), 132.53 (C-7), 127.43 (C-5), 126.19
(C-6), 125.35 (C-6a), 123.60 (C-8), 121.43 (C-4a), 119.78
(CN), 55.06 (C-3), 35.98 (C-4), 142.23, 127.19, 125.38,
123.38 (thiophene); MS (m/z), 341 (M++2, 32.08) with a
base peak at 339 (M+, 100); calcd; For C17H10ClN3OS
(339.8); % C: 60.09, % H: 2.97, N, 12.37. Found; % C:
60.00, % H: 2.90, % N: 12.30.
Rockville, MD). The cells were grown on RPMI-1640
medium supplemented with 10% inactivated fetal calf
serum and 50 µg/ml gentamycin. The cells were maintained
at 37 °C in a humidified atmosphere with 5% CO2 and were
subcultured 2−3 times a week.
Cytotoxicity evaluation using viability assay
The tumor cell lines were suspended in the medium at a
concentration of 5 × 104 cells/well in Corning® 96-well tissue
culture plates and then incubated for 24 h. The tested com-
pounds with concentrations ranging from 0 to 50 μg/ml were
then added into 96-well plates (six replicates) to achieve
different conc. for each compound. Six vehicle controls with
media or 0.5% DMSO were run for each 96-well plate as a
control. After incubating for 24 h, the numbers of viable cells
were determined by the MTT test. Briefly, the media was
removed from the 96-well plates and replaced with 100 μl of
fresh culture RPMI-1640 medium without phenol red then 10
µl of the 12 mM MTT stock solution (5 mg of MTT in 1 ml of
PBS) to each well including the untreated controls. The 96-
well plates were then incubated at 37 οC and 5% CO2 for 4 h.
An 85-μl aliquot of the media was removed from the wells,
and 50 µl of DMSO was added to each well and mixed
thoroughly with the pipette and incubated at 37 οC for 10 min.
Then, the optical density was measured at 590 nm with the
microplate reader (Sunrise, TECAN, Inc, USA) to determine
the number of viable cells and the percentage of viability was
calculated as [1 − (ODt/ODc)] × 100%, where ODt is the
mean optical density of wells treated with the tested sample
and ODc is the mean optical density of untreated cells. The
relation between surviving cells and drug concentration is
plotted to get the survival curve of each tumor cell line after
treatment with the specified compound. The 50% inhibitory
concentration (IC50), the concentration required to cause toxic
effects in 50% of intact cells, was estimated from graphic
plots of the dose−response curve for each conc. (Demirci and
Bas¸er 2002; Rahman et al. 2001; Mosmann 1983).
2-Amino-6-chloro-4-(4-bromothiophen-2-yl)-4H-pyrano[3,2-
h]quinoline-3-carbonitrile (8h)
Yellow crystals from benzene; yield 80%; m.p. 279−280 °
C; IR (KBr, υ max cm−1): 3387, 3297, 3240 (NH2), 2204
(CN), 1660 (C=N); 1H NMR (500 MHz, DMSO-d6) δ: 9.05
−7.65 (m, 4H, aromatic), 7.58 (s, 1H, thiophene H-5), 7.48
(bs, 2H, NH2), 7.23 (s, 1H, thiophene H-3), 5.37 (s, 1H, H-
4); 13C NMR (125 MHz, DMSO-d6) δ: 160.41 (C-2),
151.78 (C-9), 151.21 (C-10b), 138.06 (C-10a), 132.53 (C-
7), 127.49 (C-5), 126.19 (C-6), 125.34 (C-6a), 123.61 (C-
8), 121.41 (C-4a), 119.79 (CN), 55.05 (C-3), 35.99 (C-4),
142.18, 127.18, 123.50, 108.34 (thiophene); MS (m/z), 420
(M++4, 20.08), 418 (M++2, 99.08), 416 (M+, 59.41) with
a base peak at 354 (100); calcd; For C17H9BrClN3OS
(418.69); % C: 48.77, % H: 2.17, N, 10.04. Found; % C:
48.85, % H: 2.24, % N: 10.11.
2-Amino-6-chloro-4-(pyridin-3-yl)-4H-pyrano[3,2-h]
quinoline-3-carbonitrile (8i)
Yellow crystals from benzene; yield 80%; m.p. 273−274 °C;
IR (KBr, υ max cm−1): 3358, 3328, 3192 (NH2), 2190 (CN),
1
1660 (C=N); H NMR (500 MHz, DMSO-d6) δ: 9.02−7.36
(m, 8H, aromatic and pyridine), 7.35 (bs, 2H, NH2), 5.07 (s,
1H, H-4); 13C NMR (125 MHz, DMSO-d6) δ: 160.22 (C-2),
151.07 (C-9), 148.81 (C-10b), 138.11 (C-10a), 132.37 (C-7),
126.25 (C-5), 125.18 (C-6), 125.03 (C-6a), 123.31 (C-4a),
121.42 (C-8), 119.92 (CN), 55.10 (C-3), 38.36 (C-4), 148.61,
142.70, 140.36, 135.47, 124.13 (pyridine); MS (m/z), 336 (M+
+2, 3.88) 334 (M+, 12.07) with a base peak at 112 (100);
calcd; For C18H11ClN4O (334.76); % C: 64.58, % H: 3.31, %
N: 16.74. Found; % C: 64.51, % H: 3.24, % N: 16.67.
Cell cycle analysis
Cell cycle analysis was carried out using a Propidium
Iodide Flow Cytometry Kit (Abcam, UK) (Qu et al. 2016;
Shen et al. 2009). Different cell lines were seeded in a six-
well plate at a concentration of 5×104 per well, then incu-
bated for 24 h. Cells were cultured for an additional 24 h in
the absence (control) or presence of tested newly synthe-
sized compounds with a concentration equal to the IC50
value. After that cells were harvested and fixed using 66%
ethanol on ice, and stored at 4 °C for at least 2 h. After
rewashing with PBS, cells were resuspended in 200 μl of 1×
propidium iodide + RNAse staining solution for 30 min at
room temperature in the dark. The DNA content in each cell
Biological screening
Cell culture
The tumor cell lines breast adenocarcinoma (MCF-7),
human colon carcinoma (HCT-116), hepatocellular carci-
noma (HepG-2) and lung carcinoma (A549) were obtained
from the American Type Culture Collection (ATCC,

Medicinal Chemistry Research
nucleus was determined by a FACS Calibur flow cytometer
(BD Biosciences, Franklin Lakes, NJ, USA). Finally, cell
cycle phase distribution was analyzed using Cell Quest Pro
software (BD Biosciences) showing collected propidium
iodide fluorescence intensity on FL2.
regioselective Friedländer synthesis and antitubercular evaluation
of 2,9-diaryl-2,3-dihydrothieno[3,2-b]quinolones. Eur
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Quantification of apoptosis by flow cytometry
Annexin V-FITC apoptosis assay was performed by using
Annexin V-FITC/PI double staining detection kit (BD
Pharmingen, USA) (Van Engeland et al. 1998; Vermes
et al. 1995). Briefly, cells were cultured at a density of
5×104 per well in six-well plates, and treated with IC50
concentration of the different tested compounds for 24 h.
The staining procedure was performed following the man-
ufacturer’s instruction. Flow cytometric analysis was per-
formed on FACS Calibur flow cytometer (BD Biosciences,
Franklin Lakes, NJ, USA). Annexin V-FITC was detected
through (FL1) channel, while PI was detected through the
(FL2) channel. Finally, a minimum of 10,000 cells per
sample were acquired and analyzed using Cell Quest Pro
software (BD Biosciences).
Statistics
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All data were expressed as the means standard deviation
(SD), from at least three independent experiments with
similar results. Statistical analysis and figures were per-
formed by GraphPad Prism 5.01 (GraphPad software, San
Diego, CA, USA).
Acknowledgements The authors extend their appreciation to the
Deanship of Science Research at King Khalid University for funding
this work through General Research Project under Grant Number (G.
R.P-168-39). In addition, the authors deeply thank the Regional Center
for Mycology & Biotechnology (RCMP), Al-Azhar University, Cairo,
Egypt, for carrying out the antitumor study and also, Mr. Ali Y. A.
1
Alshahrani for drawing the H NMR and 13C NMR spectra.
Compliance with ethical standards
Fouda AM (2017) Halogenated 2-amino-4H-pyrano[3,2-h]quinoline-3-
carbonitriles as antitumor agents and structure-activity relation-
ships of the 4-, 6-, and 9-positions. Med Chem Res 26:302–313
Hammouda MAA, EL-Hag FA-AA, El-Serwy WS, El-Manawaty MA
(2015) Synthesis and characterization of new fused 4H-
Pyranquinoline carbonitrile derivatives with anticipated anti-
tumor biological activity. RJPBCS 6:200–208
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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