In vitro anticancer activity of pyrano[3, 2-c]chromene derivatives with both cell cycle arrest and apoptosis induction

Article abstract of DOI:10.1007/s00044-019-02494-3

A series of 2-amino-4-aryl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (4a–m) were synthesized via a one-pot three component condensation reaction between 4-hydroxy-2H-chromen-2-one, various aryl aldehydes and malononitrile in the presence of pi

Full text of DOI:10.1007/s00044-019-02494-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02494-3
ORIGINAL RESEARCH
In vitro anticancer activity of pyrano[3, 2-c]chromene derivatives
with both cell cycle arrest and apoptosis induction
1 ●
2 ●
2 ●
1 ●
2,3 ●
Ahmed M. El-Agrody
Ahmed M. Fouda Mohammed A. Assiri Ahmed Mora Tarik E. Ali
2 ●
4
Mohammed M. Alam Mohammad Y. Alfaifi
Received: 14 August 2019 / Accepted: 21 December 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of 2-amino-4-aryl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (4a–m) were synthesized via a one-pot
three component condensation reaction between 4-hydroxy-2H-chromen-2-one, various aryl aldehydes and malononitrile in
the presence of piperidine as a catalyst in ethanol under microwave irradiation conditions, with good to excellent yields. The
1
13
structure elucidations of all the synthesized compounds were accomplished by spectral data, IR, H NMR, C NMR, MS,
and elemental analyses. The targeted compounds were assessed for their in vitro anticancer activity against mammary gland
breast cancer cell line (MCF-7), human colon cancer (HCT-116), and liver cancer (HepG-2) by using sulphorhodamine B
assay (SRB) method, while doxorubicin, was utilized as standard reference drug. The cancer cells were treated with the
synthesized compounds at differentiable dosages, and cell viability was determined. Compounds 4e, 4f, and 4m exhibited
excellent antitumor activity versus all cancer cell lines with IC50 values ranging from 0.2 to 1.7 μM. The cell cycle arrest
behavior of compounds 4e, 4f, and 4m was investigated. The results illustrated that the potent cytotoxic compounds 4e, 4f,
and 4m induce cell cycle arrest at the G2/M phases and trigger apoptosis in the different tested cancer cells. Finally, the
structure activity relationship (SAR) survey highlighted the antitumor activity of the new molecules that was remarkably
influenced by the hydrophilicity of certain substituents at certain positions.
●
●
●
●
Keywords Microwave irradiation Pyranochromene Antitumor activity Cell cycle SAR
Introduction
therefore, decreases the toxic side effects on normal cells
and also increasing their bioavailability (Kircheis et al.
2002; Hofmeister et al. 2006). Nevertheless, there is still a
necessity for an extensive scientific and commercial interest
in the search of newer, safer, and novel anticancer ther-
apeutic drugs from natural products (Kinghorn et al. 2003,
Ramalhoa et al. 2013).
In recent years, tumor-targeting therapies have shown pro-
mising results for the treatment of cancer, which depends on
certain molecular targets to destroy the tumor cells,
Over the past several years there has been considerable
interest in the development and pharmacology of hetero-
cyclic compounds due to their biological activities in the
human body (Kidwai et al. 2002; Asif 2017; Nikaljea and
Bahetia 2017; Cavalli et al. 2009; Welsch et al. 2010).
Heterocyclic molecules play a vital role in drug discovery;
more than 70% of therapeutic drug molecules are presently
available in the literature. Among these widespread het-
erocyclic compounds, heterocycles containing oxygen
occupy a distinct position because of their wide natural
abundance and broad biological as well as pharmaceutical
significance. In these particular classes of O-heterocycles,
“chromene” heterocyclic scaffolds represent significant
biological activities.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02494-3) contains supplementary
material, which is available to authorized users.
*
Ahmed M. El-Agrody
elagrody_am@yahoo.com
1
2
3
4
Chemistry Department, Faculty of Science, Al-Azhar University,
Nasr City 11884 Cairo, Egypt
Chemistry Department, Faculty of Science, King Khalid
University, Abha 61413, Saudi Arabia
Department of Chemistry, Faculty of Education, Ain Shams
University, Roxy, Cairo, Egypt
Bioloogy Department, Faculty of Science, King Khalid University,
Abha 61413, Saudi Arabia

Medicinal Chemistry Research
Dihydropyrano[3,2-c]chromenes and their derivatives
have recently attracted the attention of synthetic and
medicinal chemists because of their wide range of appli-
cations in various fields of chemistry, biology, and phar-
macology. Some of these compounds exhibit antioxidant
recovery, very expensive reagents, low yields, and tedious
workup.
In continuation of our efforts to discover novel antitumor
agents (Alblewi et al. 2019a, 2019b; Fouda 2017, 2016), we
designed and synthesized novel 4-aryl-2-amino-3-cyano-5-
oxo-4,5-dihydropyrano[3,2-c]chromene via one-pot three
component condensation reaction of 4-hydroxy-2H-chro-
men-2-one with various aryl aldehydes and malononitrile in
the presence of piperidine as a catalyst in ethanol under
microwave irradiation conditions. The identity of all the
new compounds was verified, according to their spectral
(
Kiyani and Tazari 2017), inhibition of cell proliferation,
and induction of apoptosis in K562 human leukemia cells
(
(
Rahimi et al. 2015) diuretic, analgesic, myorelaxant
Bonsignore et al. 1993), anticoagulant (Cingolani et al.
1
969), antitumor (Emmadi et al. 2012; Perrella et al. 1994;
Baraldi et al. 1992), and antiHIV (Kashman et al. 1992;
Patil et al. 1993). They are also used as cognitive enhan-
cers, for the treatment of neurodegenerative diseases,
including Alzheimer’s disease, amyotrophic lateral
sclerosis, Parkinson’s disease, Huntington’s disease,
AIDS-associated dementia, Down’s syndrome and Schi-
zophrenia and myoclonus (Konkoy et al. 2000), anti-
malarial (Smith et al. 1998), sex-hormonal activity (Moon
et al. 2007), antimicrobial (Kakanejadifard et al. 2016; Li
et al. 2014; Abd-El-Aziz et al. 2004; De Andrade-Neto
et al. 2004; Bedair et al. 2000), anti-viral (Mohr et al.
1
13
data (IR, H NMR, C NMR, mass spectra) and elemental
analyses. All the synthesized compounds were screened for
their antiproliferative activity against MCF-7, HCT-116,
and HepG-2 carcinoma cells and the mechanisms under-
lying the cytotoxicity of the most potent compounds, uti-
lizing cell cycle analyses, were examined. The Structure
activity relationship (SAR) of the target compounds
accentuated the influence of the substituents at different
positions on the antitumor activity.
1
975), anti-inflammatory (Bianchi and Tava 1987), anti-
proliferative (Anderson et al. 2005), TNF-α inhibitor
Abbaspour-Gilandeh et al. 2016), estrogenic (Abd-El-
Results and discussion
(
Aziz et al. 2016), antihelminthic (Costa et al. 2016),
anticonvulsant (Bhat et al. 2008). These derivatives also
constitute in numerous natural products like calanolides,
calanone, calophyllolide etc. (Rueping et al. 2008).
Multicomponent reactions (MCRs) are very important in
organic synthesis due to the formation of carbon–carbon
and carbon-hetero atom bonds in one-pot (Tan et al. 2010;
Yang et al. 2010; Domling 2006 2002; Weber 2002; Ugi
Chemistry
Interaction of 4-hydroxy-2H-chromen-2-one 1 with various
aryl aldehydes 2a–m, malononitrile 3, and piperidine in
EtOH under microwave irradiation conditions for 2 min
afforded the corresponding 2-amino-4-aryl-5-oxo-4,5-dihy-
dropyrano[3,2-c]chromene-3-carbonitrile (4a–m) products
as shown in the (Scheme 1). The maximum power of the
microwave irradiation was optimized by repeating the
reaction at different watt powers (200, 300, 400 W) and
time (1, 1.5, 2 min.). Microwave radiations at 400 W and
reaction time 2 min gave the highest yield. Moreover, the
methine protons (C-4) of compounds 4a–m are chiral
centers.
1
997; Terrett et al. 1995). Simple procedures, high bond
forming efficiency, time and energy saving, and low
expenditures are among the advantages of these reactions
(
Domling and Ugi 2000; Orru and de Greef 2003).
In the literature, few methods have been reported for
the synthesis of 4,5-dihydropyrano[3,2-c]chromenes in
the presence of a variety of catalysts such as K CO under
2
3
microwave irradiation (Kidwai and Saxena 2006), dia-
mmonium hydrogen phosphate, (S)-proline (Abdolmo-
hammadi and Balalaie 2007), H P W O 18H O (Heravi
Spectroscopic data
The assignment of structure 4 was established based on
spectral data. The IR spectra of 4a–m showed the appear-
6
2
18 62
2
et al. 2008), heteropolyacids (HPA) (Heravi et al. 2009),
TBAB (Khurana and Kumar 2009) and DBU (Khurana
et al. 2010), KF-montmorillonite (Shi et al. 2008), nano
ZnO (Paul et al. 2011), alum [KAl(SO ) .12H O] (Karimi
ance of the stretch NH at υ 3462–3323, 3324–3255,
2
−
1
−1
3211–3162 cm , the CN stretch band at υ 2206–2191 cm
−
1
and the CO δ-lactone stretch band at υ 1717–1694 cm .
4
2
2
1
13
and Sedaghatpour 2010), TMGT (Shaabani et al. 2005),
MgO (Seifi and Sheibani 2008), pyridine (Shaker 1996),
The H and C NMR spectra of 4a–m revealed the pre-
sence of 4H signals at δ 5.54–4.48 (s, 1H, H-4) and δ
40.52–33.02 ppm (C-4). Consequently, the mass spectra of
compounds 4a–m and elemental analyses gave further
evidence for the proposed structures (see experimental part
and supplementary materials).
[
bmim]OH (Gong et al. 2009), and Morpholine (Heravi
et al. 2011). However, many of these methods are asso-
ciated with several disadvantages, such as long reaction
time, drastic reaction conditions, difficult catalyst

Medicinal Chemistry Research
Scheme 1 Synthesis of 2-
amino-4-aryl-5-oxo-4,5-
dihydropyrano[3,2-c]chromene-
3
-carbonitrile (4a–m)
Biological evaluation
(4e, 4f, and 4m) was due the inhibitory effect on the cell
cycle process, DNA content was analyzed by flow cyto-
metry using FACS Calibur (Becton Dickinson). Stained
DNA histogram of MFC-7-, HTC-116-, and HepG-2-
treated cells with each derivative at its IC50 values for 24 h
is shown in Fig. 4a. The cell cycle distribution for the
untreated control cells and different reference drugs were
also included in Fig. 4a. The percentage of cells containing
2n (G1 phase), 4n (G2 and M phases), and 4n > 3 > 2n
DNA amount (S phase), assessed via PI staining is shown
in Fig. 4b. All tested compounds have shown significant
increase in cell number of all cancer cells at G2/M phase,
no differences in cell number at S phase, and significant
decrease in G1 compared to control cells. Conversely, only
in the case HTC-116 cells treated with compound 4 m have
showed increased cell arrest at G1 phase and decreased
arrest at G2/M phase. In addition, our tested compounds
showed more or less the same effect on cell cycle arrest
percentage at G2/M compared to Doxorubicin, Colchicine,
and Vinblastine reference drug.
Cytotoxic activity assay
The antiproliferative activity of all synthesized compounds
4
a–m were evaluated in vitro against the three human cancer
cell lines, mammary gland breast cancer (MCF-7), human
colon cancer (HCT-116), and liver cancer (HepG-2) in
comparison with doxorubicin as reference drug, using the
standard sulphorhodamine B (SRB) assay (Skehan et al.
1
990). The in vitro cytotoxicity evaluation was achieved
using different concentrations, ranging from 0 to 100 μM.
The results were expressed as growth inhibitory concentration
(
IC ) values, where the necessitated concentration produced
50
a 50% inhibition of cell growth after 24 h of incubation,
compared to the untreated cell control. The IC50 values are
summarized in Table 1 and Fig. 1. The relation between the
surviving cells with different concentrations of tested com-
pounds were plotted to get the survival curve for each type of
cancer cell line after 24 h as depicted in Figs. 2 and 3.
The results in Table 1 revealed that the dihydropyrano
[
3,2-c]chromene derivatives 4e, 4f, and 4m have shown high
Induction of apoptosis by the newly synthesized
compounds
cytotoxicity against all the three carcinoma cell lines as fol-
lows; the MCF-7 (IC50 2.7, 0.6, and 0.32 µM), the HCT-116
(
0
IC50 3.1, 0.2, and 1.7 µM), the HepG-2 (IC50 2.2, 0.5, and
.4 µM), respectively. Whereas the other compounds showed
Blocking cell cycle progression, inducing apoptosis, or
combined effect of both have been used successfully by
many anticancer compounds as a new therapeutic approa-
ches. phosphatidylserine (PS) translocation to the mem-
brane surface is as an accepted marker for apoptosis (Fadok
et al. 1992). Therefore, apoptosis induction by the tested
compounds was measured using Annexin V /PI double
staining flow cytometric assay. Representative dot plots of
the double stained cells (MCF-7, HTC-116, and HepG-2)
after treatment with different tested compounds were shown
in Fig. 5a. The percentage of both early (Annexin V posi-
tive, PI negative) and late (Annexin V positive, PI positive)
apoptosis for treated cells are shown in Fig. 5b. Unlike
necrosis which was not observed in all the results, all treated
cells showed from 10 to 30% increase in total apoptosis
compared to the untreated cells. However, compound 4m
gives higher apoptosis levels in the case of MCF-7 and
a good, moderate activity or inactive cytotoxic activity
against MCF-7, HCT-116, and HepG-2 tumor cell lines.
Cell cycle arrest at G2/M phase by the newly synthesized
compounds
Normal cell growth and division is under the control of
four cell cycle stages (G1, S, G2, and M); however,
unscheduled cell divisions of most cancer cells are by the
downregulation of the cell cycles. Therefore new synthe-
sized anticancer inhibiting the cell cycle represents
important therapeutic intervention in treating proliferative
diseases like cancer (Schwartz and Shah 2005; Williams
and Stoeber 2012). To test whether the potent cytotoxic
activity showed by the new synthesized compounds

Medicinal Chemistry Research
Table 1 The IC50 (µM) of the tested compounds against different
tumor cell lines
SAR studies
The SAR study revealed several crucial structural require-
ments, which enhanced the potency of these halogenated
2
3
-amino-4-aryl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-
-carbonitrile derivatives (4a–m). By exemplifying the
variations in substitution pattern on phenyl ring at 4-
position of 4H-pyran moiety, compounds having electron
withdrawing groups first series (monosubstitents), second
series (disubstitents), and third series (polysubstitents) were
prepared and their effects were examined. It is noteworthy
to observe that the halogenated polysubstitents (third series)
Compound
Ar
IC50 (µM)a
MCF-7
HCT-116 HepG-2
like 2,3,4,5,6-F on phenyl ring of compound 4m have
5
significant impact on activities against MCF-7 cell line than
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
4-FC6H4
>100
>100
>100
disubstitents (second series) like 2,4-F on phenyl ring of
2
2-ClC6H4
3-ClC6H4
4-ClC6H4
4-BrC6H4
2,4-F2C6H3
2,6-F2C6H3
2,3-Cl C H
6.1 ± 0.3
3.3 ± 1.1
4.9 ± 0.6
compound 4f and all the monosubstitents (first series) as
compared with doxorubicin, hinting that grafting a lipo-
philic electron withdrawing polysubstituent fluorine atoms
like 2,3,4,5,6-F and disubstitents like 2,4-F on phenyl ring
12.97 ± 0.9 21.2 ± 1.8 15.9 ± 2.9
>100
>100
>100
2.7 ± 0.2
0.6 ± 0.1
>100
3.1 ± 0.4
0.2 ± 0.1
>100
2.2 ± 0.2
0.5 ± 0.1
>100
5
2
of 4H-pyran moiety is more active than the other dis-
ubstituent (second series) and monosubstituent (first series),
respectively, and the fluorine atom is more beneficial than
the other halogen substituents. Concerning activity against
HCT-116 cell line, compounds 4f and 4m were the most
active analogs through this study as compared with dox-
orubicin, suggesting that the halogenated disubstitents
g
h
i
5.9 ± 0.6
10.9 ± 4.1
>100
5.1 ± 0.6
4.5 ± 0.2
2
6
3
2,4-Cl2C6H3
2,5-Cl2C6H3
2,6-Cl2C6H3
3,4-Cl2C6H3
15.1 ± 6.1 1.6 ± 0.4
j
>100
>100
>100
>100
k
l
>100
>100
>100
>100
m
2,3,4,5,6-F5C6 0.32 ± 0.04 1.7 ± 0.3
0.4 ± 0.5
(second series) like 2,4-F2 and polysubstituent like
Doxorubicin
–
0.7 ± 0.09 0.6 ± 0.03 0.9 ± 0.1
2
,3,4,5,6-F possessed excellent antiproliferative activities
5
aIC50 values expressed in µM as the mean values of triplicate wells
from at least three experiments and are reported as the mean ±
standard error
than the other disubstituent (second series) and mono-
substitents (first series), implying that grafting a lipophilic
electron withdrawing disubstituent like 2,4-F and poly-
2
substituent like 2,3,4,5,6-F is more beneficial than the other
5
substituents. In addition, compounds 4m and 4f were dis-
played the more potent activities against HepG-2 cell line as
compared with doxorubicin, hinting that the polysubstituent
like 2,3,4,5,6-F5 and disubstituent like 2,4-F2 exhibited
superior potency than the other disubstituent (second series)
and monosubstitents (first series). The other compounds
showed a good, moderate activity or inactive cytotoxic
activity against MCF-7, HepG2, and HCT-116 tumor
cell lines.
Conclusion
Fig. 1 IC50 of the tested compounds on MCF-7, HCT-116, and HepG-
2
, cells with different concentrations for 24 h
In summary, we have demonstrated an elegant protocol for
the synthesis of the halogenated 2-amino-4-aryl-5-oxo-4,5-
dihydropyrano[3,2-c]chromene-3-carbonitriles (4a–m) and
characterized successfully. All the synthesized compounds
were tested as antitumor agents against three human
cancer cell lines: liver (HepG-2), colon (HCT-116), and
breast (MCF-7). The results have shown that some of the
compounds exhibited significant activity against all
HTC-116 while compounds 4e and 4f give higher levels in
the case of HepG-2 cells. The percentage of apoptosis in all
tested cells treated with different reference drugs (Doxor-
ubicin, Colchicine, and Vinblastine) was comparable with
our new synthesized tested compounds.

Medicinal Chemistry Research
Fig. 2 Dose response curve of compounds 4a–f with different concentrations for 24 h. against MCF-7, HepG-2, and HCT-116 cell lines
cancer cell lines. Among them, compounds 4e, 4f, and 4m
were the most potent cytotoxic agents all tumor cell lines
compared with doxorubicin as a reference drug. In addi-
tion, the results showed that the potent cytotoxic effect
observed occurs through the mechanisms associated with
cell cycle arrest at G2/M and induction of apoptosis.
Therefore, the present studies reveal that the 3,4-dihy-
dropyrano[3,2-c]chromene derivatives provide a valuable
new therapeutic intervention for the treatment of cancer
diseases, and the 4e, 4f, and 4m are the effective lead
compounds for anticancer drug discovery and further
research.

Medicinal Chemistry Research
Fig. 3 Dose response curve of compounds 4g–m and Doxorubicin with different concentrations for 24 h. against MCF-7, HepG-2, and HCT-116
cell lines

Medicinal Chemistry Research
Fig. 4 Induced cell cycle arrest
at G2/M phase. a Representative
flow cytometry histograms of
cell cycle distribution. DNA
content was stained with (PI).
b Percentage of MCF-7, HCT-
1
16, and HepG-2 cells in the G1,
S, and G2/M phases after
incubation with compounds 4e,
f, and m (IC50 value) for 24 h

Medicinal Chemistry Research
Fig. 5 Apoptotic cell death
induced by pyrano[3,2-c]
chromene derivatives: a Flow
cytometry dot plot represents
Annexin-V-FITC staining in
X-axis and PI in Y-axis.
b Summary of the Annexin
V-FITC Apoptosis assay results
showing the percentages of early
apoptosis, late apoptosis, total
apoptosis, and necrosis treated
cells with the indicated drugs
(
IC50 value) for 24 h

Medicinal Chemistry Research
Experimental section
Materials and methods
1
3
C NMR (DMSO-d , 125 MHz,) δ (ppm) = 160.0 (C, CO),
6
1
58.7 (C, C-10b), 154.6 (C, C-2), 152.8 (C, C-6a), 140.8
(C, C-1′), 133.6 (C, C-2′), 133.0 (C, C-3′), 131.2 (C, C-8),
30.1 (C, C-4′), 129.4 (C, C-5′), 128.3 (C, C-6′), 125.3 (C,
1
All chemicals used were of commercial grade and were
used without any further purification. Melting points
were determined using a Stuart Scientific melting point
apparatus SMP 10 and are uncorrected. Thin Layer
Chromatography (TLC) was carried out using aluminum
sheets pre-coated with silica gel F254 purchased from
Sigma Aldrich. IR spectra (KBr) were recorded on a
Thermo Fisher Scientific Nicolet iS50 FT-IR spectro-
meter. H NMR (500 MHz) and C NMR (125 MHz)
spectra were recorded on a Bruker Avance 500 MHz
instrument in CDCl3 or DMSO-d6 with TMS as an
internal standard.
C-9), 123.1 (C, C-10), 119.4 (C, C-7), 117.2 (C, CN), 113.4
(C, C-10a), 103.5 (C, C-4a), 57.1 (C, C-3), 39.5 (CH, C-4);
EIMS m/z 334 [M+2] 352 (33.25), [M] 350 (100).
+
+
2-Amino-4-(3-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]
chromene-3-carbonitrile (4c) Colorless crystals, mp
271–272 °C (Lit. mp 272−274 °C; Kiyani and Tazari
2017; m.p. 275–277° C; Hazeri et al. 2014); yield 81%; IR
1
13
(KBr) υ
3323, 3255, 3194 (NH ), 2206 (CN),
max
1
2
1696 cm− (CO δ-lactone);
1
H
NMR (DMSO-d6,
500 MHz,) δ (ppm) = 7.90–7.32 (10H, m, Ar-H, NH ),
2
1
3
4.51 (1H, s, CH-pyran); C NMR (DMSO-d , 125 MHz,)
6
δ (ppm) = 160.1 (C, CO), 158.6 (C, C-10b), 154.3 (C, C-
2), 152.8 (C, C-6a), 146.4 (C, C-1′), 144.6 (C, C-3′),
133.6 (C, C-5′), 133.0 (C, C-2′), 131.7 (C, C-8), 130.9 (C,
C-4′), 128.2 (C, C-6′), 125.2 (C, C-9), 123.5 (C, C-10),
119.6 (C, C-7), 117.1 (C, CN), 113.6 (C, C-10a), 103.8
(C, C-4a), 58.0 (C, C-3), 39.9 (CH, C-4); EIMS m/z 352
General procedure for the synthesis of 2-amino-4-aryl-5-oxo-
4
,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (4a–m)
A mixture of 4-hydroxy-2H-chromen-2-one 1 (0.01 mol),
various aromatic aldehyde 2a–m (0.01 mol), malononitrile 3
+
+
(0.01 mol), and 0.5 ml of piperidine in ethanol (30 mL) in a
[M+2] (33.05), [M] 350 (100).
100 mL round bottom flask was heated under microwave
irradiation conditions for 2 min. After successful completion
of the reaction, monitored by TLC, the reaction mixture was
cooled to room temperature, the obtained solid product was
separated by filtration and washed with methanol and purified
by recrystallization from ethanol to afford the pure products
2-Amino-4-(4-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]
chromene-3-carbonitrile (4d) Colorless crystals, mp
263–264 °C (Lit. mp 261 °C; El-Agrody et al. 2000); yield
84%; IR (KBr) υ
3381, 3311, 3189 (NH ), 2192 (CN),
max
2
1713 cm− (CO δ-lactone);
1
1
H
NMR (DMSO-d6,
4
a–m. The physical and spectral data of compounds 4a–m
500 MHz,) δ (ppm) = 7.92–7.30 (10H, m, Ar-H, NH ), 4.50
2
1
3
are as follows:
(1H, s, CH-pyran); C NMR (DMSO-d , 125 MHz,) δ
6
(ppm) = 159.5 (C, CO), 157.9 (C, C-10b), 153.5 (C, C-2),
2
-Amino-4-(4-fluorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]
152.2 (C, C-6a), 145.9 (C, C-1′), 142.3 (C, C-4′), 133.0 (C,
C-2′),131.7 (C, C-6′), 130.1 (C, C-3′), 129.6 (C, C-5′),
128.4 (C, C-8), 124.6 (C, C-9), 122.5 (C, C-10), 119.0 (C,
C-7), 116.5 (C, CN), 112.9 (C, C-10a), 103.5 (C, C-4a),
chromene-3-carbonitrile (4a) Pale yellow crystals, mp
2
2
2
5
64–265 °C (Lit. mp 260−262 °C, Mehrabi and Abusaidi
010); yield 83%; IR (KBr) υ
194 (CN), 1716 cm (CO δ-lactone); H NMR (DMSO-d ,
3379, 3292, 3191 (NH ),
max
2
−1
1
+
57.5 (C, C-2), 39.4 (CH, C-4); EIMS m/z 352 [M+2]
6
+
00 MHz,) δ (ppm) = 7.90–7.13 (10H, m, Ar-H, NH ), 4.4
(10.65), [M] 350 (33.71), 239 (100).
2
(
(
1H, s, CH-pyran); 13C NMR (DMSO-d , 125 MHz,) δ
6
ppm) = 163.0 (C, CO), 160.6 (C, C-10b), 158.5 (C, C-4′),
2-Amino-4-(4-bromophenyl)-5-oxo-4,5-dihydropyrano[3,2-
c]chromene-3-carbonitrile (4e) Colorless crystals, mp
259–260 °C (Lit. mp 254 °C; El-Agrody et al. 2000); yield
153.8 (C, C-2), 152.7 (C, C-6a), 140.1 (C, C-1′), 133.5 (C, C-
2′, C-6′), 130.3 (C, C-8), 125.2 (C, C-9), 123.1 (C, C-10),
119.7 (C, C-7), 117.1 (C, CN), 115.9 (C, C-3′, C-5′), 113.5
82%; IR (KBr) υ
3385, 3291, 3187 (NH ), 2191 (CN),
2
max
1709 cm− (CO δ-lactone);
1
1
H
NMR (DMSO-d6,
(C, C-10a), 104.4 (C, C-4a), 58.4 (C, C-3), 39.9 (CH, C-4);
+
EIMS m/z 334 [M] (100).
500 MHz,) δ (ppm) = 7.91–7.25 (10H, m, Ar-H, NH ),
2
1
3
4
.48 (1H, s, CH-pyran); C NMR (DMSO-d , 125 MHz,)
6
2
-Amino-4-(2-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]
δ (ppm) = 159.5 (C, CO), 157.9 (C, C-10b), 153.5 (C, C-2),
152.2 (C, C-6a), 142.7 (C, C-1′), 133.0 (C, C-3′), 131.6 (C,
C-5′), 131.3 (C, C-2′), 130.0 (C, C-6′), 129.6 (C, C-8),
124.6 (C, C-9), 122.5 (C, C-10), 120.2 (C, C-4′) 119.0 (C,
C-7), 116.5 (C, CN), 112.9 (C, C-10a), 103.4 (C, C-4a),
chromene-3-carbonitrile (4b) Colorless crystals, mp 278-
2
2
3
79 °C (Lit. m.p. 272−274 °C; Kiyani and Tazari 2017; mp
75–277 °C; Hazeri et al. 2014); yield 82%; IR (KBr) υ
max
−
1
398, 3284, 3179 (NH ), 2199 (CN), 1708 cm (CO δ-
2
1
+
lactone); H NMR (DMSO-d , 500 MHz,) δ (ppm) =
7
57.5 (C, C-3), 36.5 (CH, C-4); EIMS m/z 396 [M+2]
6
+
.92–7.27 (10H, m, Ar-H, NH ), 4.98 (1H, s, CH-pyran);
(90.65), [M] 394 (100).
2

Medicinal Chemistry Research
1
2
[
2
-Amino-4-(2,4-difluorophenyl)-5-oxo-4,5-dihydropyrano
3,2-c]chromene-3-carbonitrile (4f) Yellow crystals, mp
74–275 °C; yield 80%; IR (KBr) υ 3379, 3295, 3192
NH ), 4.98 (s, 1H, CH-pyran); 3C NMR (DMSO-d6,
2
125 MHz,) δ (ppm) = 159.4 (C, CO), 158.1 (C, C-10b),
154.1 (C, C-2), 152.2 (C, C-6a), 139.4 (C, C-1′), 133.4 (C,
C-2′), 133.1 (C, C-4′), 132.4 (C, C-6′), 132.1 (C, C-3′),
128.8 (C, C-8), 127.8 (C, C-5′), 124.7 (C, C-9), 122.5 (C,
C-10), 118.6 (C, C-7), 116.6 (C, CN), 112.8 (C, C-10a),
102.5 (C, C-4a), 56.0 (C, C-3), 33.9 (CH, C-4); EIMS m/z
[M+4]+ 388 (6.15), [M+2]+ 386 (38.65), [M]+ 384
(60.31), 349 (100).
max
−
1
1
(
NH ), 2192 (CN), 1712 cm (CO δ-lactone H NMR
2
(
DMSO-d , 500 MHz,) δ (ppm) = 7.91–7.00 (m, 9H, Ar-H,
6
13
NH ), 4.73 (s, 1H, CH-pyran) C NMR (DMSO-d6,
1
1
2
25 MHz,) δ (ppm) = 162.4 (C, CO), 161.3 (C, C-10b),
60.4 (C, C-2′), 159.4 (C, C-2), 158.2 (C, C-4′), 152.2 (C,
C-6a), 133.0 (C, C-6′), 131.5 (C, C-8), 126.4 (C, C-9),
24.7 (C, C-10), 122.4 (C, C-1′), 118.9 (C, C-7), 116.6 (C,
CN), 112.9 (C, C-10a), 111.7 (C, C-5′), 104.1 (C, C-4a),
02.4 (C, C-3′), 56.3 (C, C-3), 30.9 (CH, C-4); EIMS m/z
1
2-Amino-4-(2,5-dichlorophenyl)-5-oxo-4,5-dihydropyrano
[3,2-c]chromene-3-carbonitrile (4j) Pale brown crystals,
1
+
[
M] 352 (100); Anal. Calcd for C H F N O (352.07):
mp 270–271 °C; yield 79%; IR (KBr) υ
3415, 3289,
19
10
2
2
3
max
−
1
1
C, 64.78; H, 2.86; N, 7.95. Found: C, 64.84; H, 2.90;
N, 8.00.
3211 (NH ), 2196 (CN), 1717 cm (CO δ-lactone); H
2
NMR (DMSO-d , 500 MHz,) δ (ppm) = 7.91–7.45 (9H, m,
Ar-H, NH ), 4.98 (1H, s, CH-pyran); C NMR (DMSO-d6,
6
1
3
2
2
[
2
-Amino-4-(2,6-difluorophenyl)-5-oxo-4,5-dihydropyrano
3,2-c]chromene-3-carbonitrile (4g) Colorless crystals, mp
95–297 °C; yield 79%; IR (KBr) υ 3391, 3282, 3175
125 MHz,) δ (ppm) = 160.0 (C, CO), 158.7 (C, C-10b),
155.0 (C, C-2), 152.8 (C, C-6a), 142.8 (C, C-1′), 133.6 (C,
C-5′), 132.7 (C, C-6′), 131.9 (C, C-3′), 131.8 (C, C-2′),
130.9 (C, C-8), 129.4 (C, C-4′), 125.2 (C, C-9), 123.2 (C,
C-10), 119.2 (C, C-7), 117.2 (C, CN), 113.2 (C, C-10a),
102.6 (C, C-4a), 56.4 (C, C-3), 35.7 (CH, C-4); EIMS m/z
[M+4]+ 388 (46.65), [M+2]+ 386 (72.65), [M]+ 384
(6.31), 349 (100); Anal. Calcd for C H Cl N O (385.20):
max
−
1
1
(
NH ), 2205 (CN), 1705 cm (CO δ-lactone); H NMR
2
(
DMSO-d , 500 MHz,) δ (ppm) = 7.91–7.06 (9H, m, Ar-H,
6
13
NH ), 4.94 (s, 1H, CH-pyran); C NMR (DMSO-d6,
1
1
2
25 MHz,) δ (ppm) = 161.8 (C, CO), 161.7 (C, C-10b),
59.8 (C, C-2′), 158.8 (C, C-6′), 154.0 (C, C-2), 152.1 (C,
1
9
10
2
2
3
C-6a), 133.2 (C, C-4′), 129.9 (C, C-8), 124.8 (C, C-9),
23.6 (C, C-10), 118.9 (C, C-7), 117.9 (C, C-1′), 116.7 (C,
CN), 112.6 (C, C-10a), 112.0 (C, C-3′, C-5′), 101.6 (C, C-
a), 54.3 (C, C-3), 26.4 (CH, C-4); EIMS m/z 352 [M]+
100); Anal. Calcd for C H F N O (352.07): C, 64.78; H,
C, 59.24; H, 2.62; N, 7.27. Found: C, 59.29; H, 2.61;
N, 7.32.
1
4
(
2-Amino-4-(2,6-dichlorophenyl)-5-oxo-4,5-dihydropyrano
[3,2-c]chromene-3-carbonitrile (4k) Colorless crystals, mp
19
10
2
2
3
2
.86; N, 7.95. Found: C, 64.72; H, 2.81; N, 7.90.
296–297 °C; yield 81%; IR (KBr) υ
3418, 3276, 3172
max
−
1
1
(
NH ), 2200 (CN), 1705 cm (CO δ-lactone); H NMR
2
2
-Amino-4-(2,3-dichlorophenyl)-5-oxo-4,5-dihydropyrano
(DMSO-d , 500 MHz,) δ (ppm) = 7.90–7.39 (8H, m, Ar-H,
6
1
3
[
2
3,2-c]chromene-3-carbonitrile (4h) Colorless crystals, mp
93–295 °C; Yield 80%; IR (KBr) υ 3404, 3291, 3180
NH ), 5.54 (1H, s, CH-pyran); C NMR (DMSO-d6,
2
125 MHz,) δ (ppm) = 159.2 (C, CO), 158.9 (C, C-10b),
154.5 (C, C-2), 152.2 (C, C-6a), 136.0 (C, C-1′), 135.1 (C,
C-2′), 134.3 (C, C-6′), 133.2 (C, C-4′), 130.3 (C, C-3′),
129.7 (C, C-5′), 128.7 (C, C-8), 124.8 (C, C-9), 122.4 (C,
C-10), 118.5 (C, C-7), 116.6 (C, CN), 112.5 (C, C-10a),
101.1 (C, C-4a), 53.4 (C, C-3), 33.3 (CH, C-4); EIMS m/z
[M+4]+ 388 (2.65), [M+2]+ 386 (18.55), [M]+ 384
(28.31), 239 (100); Anal. Calcd for C H Cl N O
max
−
1
1
(
(
NH ), 2199 (CN), 1711 cm (CO δ-lactone); H NMR
DMSO-d , 500 MHz,) δ (ppm) = 7.92–7.29 (9H, m, Ar-H,
2
6
13
NH ), 5.08 (s, 1H, CH-pyran); C NMR (DMSO-d6,
1
1
2
25 MHz,) δ (ppm) = 159.4 (C, CO), 158.2 (C, C-10b),
54.1 (C, C-2), 152.2 (C, C-6a), 143.0 (C, C-1′), 133.1 (C,
C-3′), 131.9 (C, C-2′), 130.5 (C, C-5′), 129.3 (C, C-4′),
29.2 (C, C-8), 128.5 (C, C-6′), 124.7 (C, C-9), 122.5 (C,
C-10), 118.6 (C, C-7), 116.6 (C, CN), 112.8 (C, C-10a),
02.7 (C, C-4a), 56.1 (C, C-3), 35.1 (CH, C-4); EIMS m/z
1
1
9
10
2
2
3
(385.20): C, 59.24; H, 2.62; N, 7.27. Found: C, 59.30; H,
2.66; N, 7.31.
1
+
+
+
[
M+4] 388 (9.25), [M+2] 386 (65.25), [M] 384 (100);
Anal. Calcd for C H Cl N O (385.20): C, 59.24; H, 2.62;
2-Amino-4-(3,4-dichlorophenyl)-5-oxo-4,5-dihydropyrano
1
9
10
2
2
3
N, 7.27. Found: C, 59.20; H, 2.57; N, 7.21
[3,2-c]chromene-3-carbonitrile (4l) Colorless crystals, mp
2
47–249 °C; yield 80%; IR (KBr) υ
3400, 3321, 3193
max
−
1
1
2
[
2
-Amino-4-(2,4-dichlorophenyl)-5-oxo-4,5-dihydropyrano
3,2-c]chromene-3-carbonitrile (4i) Colorless crystals, mp
62–263 °C (Lit. mp 257–259 °C; Abdolmohammadi and
Balalaie 2007); yield 83%; IR (KBr) υ 3462, 3296, 3162
(NH ), 2203 (CN), 1696 cm (CO δ-lactone); H NMR
2
(DMSO-d , 500 MHz,) δ (ppm) = 7.90–7.29 (8H, m, Ar-H,
6
1
3
NH ), 4.54 (1H, s, CH-pyran); C NMR (DMSO-d6,
2
125 MHz,) δ (ppm) = 159.5 (C, CO), 158.0 (C, C-10b),
153.8 (C, C-2), 152.2 (C, C-6a), 144.4 (C, C-1′), 133.0 (C,
C-3′), 131.0 (C, C-4′), 130.6 (C, C-2′), 129.8 (C, C-5′),
max
−
1
1
(
(
NH ), 2200 (CN), 1716 cm (CO δ-lactone); H NMR
DMSO-d , 500 MHz,) δ (ppm) = 7.92–7.34 (8H, m, Ar-H,
2
6

Medicinal Chemistry Research
1
29.8 (C, C-6′), 128.2 (C, C-8), 124.6 (C, C-9), 122.6 (C,
C-10), 118.9 (C, C-7), 116.5 (C, CN), 112.9 (C, C-10a),
02.7 (C, C-4a), 57.0 (C, C-3), 36.2 (CH, C-4); EIMS m/z
temperature in the dark. Finally, cells were stained using
Propidium Iodide Flow Cytometry Kit (Abcam, UK) and
DNA content in each cell nucleus was determined by a
FACS Calibur flow cytometer (BD Biosciences, Franklin
Lakes, NJ, USA). Cell cycle phase distribution was ana-
lyzed using Cell Quest Pro software (BD Biosciences)
showing collected propidium iodide fluorescence intensity
on FL2.
1
+
+
+
[
M+4] 388 (9.65), [M+2] 386 (64.35), [M] 384 (100);
Anal. Calcd for C H Cl N O (385.20): C, 59.24; H, 2.62;
N, 7.27. Found: C, 59.19; H, 2.57; N, 7.21.
1
9
10
2
2
3
2
-Amino-4-(2,3,4,5,6-pentafluorophenyl)-5-oxo-4,5-dihydro-
pyrano[3,2-c]chromene-3-carbonitrile (4m) Colorless
crystals, mp 257–259 °C; yield 65%; IR (KBr) υ 3419,
Annexin V/PE double staining assay
max
−
1
3
324, 3191 (NH ), 2195 (CN), 1694 cm (CO δ-lactone);
2
1
4
H NMR (DMSO-d , 500 MHz,) δ (ppm) = 7.91–7.50 (m,
Cells at a density of 5 × 10 per well were treated with IC
6
50
13
5
H, Ar-H, NH ), 5.06 (s, 1H, CH-pyran); C NMR
concentration of the different tested compounds for 24 h.
The staining procedure was done following the manu-
facturer’s instruction using Annexin V-FITC/PI double
staining detection kit (BD Pharmingen, 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).
2
(
DMSO-d , 125 MHz,) δ (ppm) = 161.2 (C, CO), 160.1 (C,
6
C-10b), 159.1 (C, C-5′), 157.9 (C, C-4′), 153.8 (C, C-2),
52.7 (C, C-6a), 149.7 (C, C-3′), 133.8 (C, C-2′), 130.9 (C,
C-6′), 126.8 (C, C-8), 125.4 (C, C-9), 123.1 (C, C-10),
19.5 (C, C-7), 117.3 (C, CN), 113.3 (C, C-10a), 110.8 (C,
1
1
C-1′), 103.8 (C, C-4a), 57.4 (C, C-3), 33.0 (CH, C-4); EIMS
m/z [M] 406 (100); Anal. Calcd for C H F N O
+
19
7
5
2
3
(
1
406.26): C, 56.17; H, 1.74; N, N, 6.90. Found: C, 56.23; H,
.784; N, N, 6.95.
Statistics
Biological screening
All data were expressed as the means ± standard deviation
(SD), from at least three independent experiments with
Cell culture
similar results. Statistical analysis and figures were per-
formed by GraphPad Prism 5.01 (GraphPad Software, San
Diego, CA. USA).
The tumor cell lines, mammary gland breast cancer cell line
(
MCF-7), human colon carcinoma (HCT-116), and hepa-
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
tocellular carcinoma (HepG-2), were obtained from the
American Type Culture Collection (ATCC, Rockville,
MD). The cells were grown on an RPMI-1640 medium,
supplemented with 10% inactivated fetal calf serum and
(R.G.P.1/111/40). In addition, the authors deeply thank the Regional
Center for Mycology & Biotechnology (RCMP), Al-Azhar University,
Cairo, Egypt, for carrying out the antitumor study, elemental analyses
5
0 µg/mL gentamycin. The cells were maintained at 37 °C
1
and also, for Mr. Ali Y. A. Alshahrani for drawing the H NMR and
in a humidified atmosphere with 5% CO and were sub-
culture two to three times a week.
13
2
C NMR spectra.
Compliance with ethical standards
Cytotoxicity evaluation using viability assay
Conflict of interest The authors declare that they have no conflict of
The cytotoxic activity was appraised, using the standard
sulphorhodamine B (SRB) assay, as reported previously
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
(
Skehan et al. 1990).
Analysis of cellular DNA content
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