Biologically active perspective synthesis of heteroannulated 8-nitroquinolines with green chemistry approach

Article abstract of DOI:10.1016/j.bmcl.2017.02.042

A new class of pyrazolo[4,3-c]quinoline (5a-i, 7a-b) and pyrano[3,2-c]quinoline (9a-i) derivatives were designed and synthesized in moderate to good yields by microwave conditions. To enhance the yield of pyrano[3,2-c]quinoline derivatives, multicomponent one-pot synthesis has been developed. The synthesized compounds were identified by spectral and elemental analyses. Compounds 9a and 9i showed good antibacterial activity against Gram-positive and Gram-negative bacterial strains. All of the new compounds exhibited weak to moderate antioxidant activity, compound 9d exerted significant antioxidant power. The cytotoxicity of these compounds were also evaluated against MCF-7 (breast) and A549 (Lung) cancer cell lines. Most of the compounds displayed moderate to good cytotoxic activity against these cell lines. Compound 9i was found to be significantly active in this assay and also induced cell death by apoptosis. Molecular docking studies were carried out using EGFR inhibitor in order to determine the molecular interactions.

Full text of DOI:10.1016/j.bmcl.2017.02.042

Accepted Manuscript
Biologically active perspective synthesis of heteroannulated 8-nitroquinolines
with green chemistry approach
Thangaraj Arasakumar, Sadasivam Mathusalini, Subashini Gopalan, Selvaraj
Shyamsivappan, Athar Ata, Palathurai Subramaniam Mohan
PII:
S0960-894X(17)30176-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.042
BMCL 24714
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 October 2016
8 February 2017
17 February 2017
Please cite this article as: Arasakumar, T., Mathusalini, S., Gopalan, S., Shyamsivappan, S., Ata, A., Mohan, P.S.,
Biologically active perspective synthesis of heteroannulated 8-nitroquinolines with green chemistry approach,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.042
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Biologically active perspective synthesis of heteroannulated 8-nitroquinolines
with green chemistry approach
Thangaraj Arasakumara,b, Sadasivam Mathusalinia, Subashini Gopalana, Selvaraj
Shyamsivappana, Athar Atab,*, Palathurai Subramaniam Mohana,*
a School of Chemical Sciences, Bharathiar University, Coimbatore-641 046, Tamil Nadu, India
b Department of Chemistry, Richardson College for the Environmental Science Complex, The
University of Winnipeg, 599 Portage Avenue, Winnipeg, Manitoba, R3B 2G3, Canada
*Corresponding authors:
Palathurai Subramaniam Mohan,
Email: psmohan59@gmail.com, ps_mohan_in@yahoo.com, Tel +91-422-2428314/ Fax: +91-
422-2459845
Athar Ata,
Email: a.ata@uwinnipeg.ca, Tel 204-786-9389/ Fax: 204-774-2401
Graphical abstract

Abstract
A new class of pyrazolo[4,3-c]quinoline (5a-i, 7a-b) and pyrano[3,2-c]quinoline (9a-i)
derivatives were designed and synthesized in moderate to good yields by microwave conditions.
To enhance the yield of pyrano[3,2-c]quinoline derivatives, multicomponent one-pot synthesis
has been developed. The synthesized compounds were identified by spectral and elemental
analyses. Compounds 9a and 9i showed good antibacterial activity against Gram-positive and
Gram-negative bacterial strains. All of the new compounds exhibited weak to moderate
antioxidant activity, compound 9d exerted significant antioxidant power. The cytotoxicity of
these compounds were also evaluated against MCF-7 (breast) and A549 (Lung) cancer cell lines.
Most of the compounds displayed moderate to good cytotoxic activity against these cell lines.
Compound 9i was found to be significantly active in this assay and also induced cell death by
apoptosis. Molecular docking studies were carried out using EGFR inhibitor in order to
determine the molecular interactions.
Keywords
Pyrazolo[4,3-c]quinoline; Pyrano[3,2-c]quinoline; Antibacterial; Antioxidant; Anticancer;
Molecular docking

One of the enduring threats to longevity of human life is cancer and the affected cases are
expected to be doubled by 2030 from 21 million.1 Lung and breast cancers are most common in
the developing and under developed countries. Though several chemotherapeutic agents
available to date, a major challenge for discovery of small molecules as cancer drugs with
improved efficacy and reduced side effects still remains unsolved.2 Nitrogen bridged
heterocyclic compounds represent a distinct type of anticancer drug candidates, which strongly
induce cell apoptosis3 and in the past decade, many quinoline containing compounds have been
reported as antitumor agents.4 For example, anticancer alkaloid camptothecin contains a
quinoline moiety, which was isolated from Camptotheca acuminate5,6 and its derivatives such as
topotecan and irinotecan are prototypical topoisomerase I inhibitors. In order to improve the
efficacy of quinoline drugs, many pyran, pyrazolo ring appended quinoline derivatives were
developed. It is broadly represented by pyranoquinoline alkaloids and several pyrazoloquinoline
synthetic analogues (Fig. 1), which exhibit a wide range of biological activities.7 Moreover,
several of these alkaloids show higher antiproliferative activity. For example, Huajiaosimuline is
active against ZR-75-1 breast cancer cells, while Zanthosimuline is active against multidrug
resistant KB-VI cancer cells.8
Usually, oxidative stress is caused by reactive oxygen and nitrogen species leading to
variety of disorders including cancer,9 atherosclerosis,10 neuro degeneration and ageing.11
Further, it is responsible for oxidative damage to DNA, proteins, membrane lipids and enhances
tumor cell proliferation, metastasis and angiogenesis.12 Antioxidants are known to scavenge free
radicals and their therapeutic utility can be effective in preventing or supressing several types of
cancer.13 Recently, considerable attention has been paid on finding synthetic antioxidants that
target various signaling pathways that are limited in cancer. On the other hand, treatment of
bacterial infection remains an important and challenging therapeutic problem, because of its
emerging resistance to existing antibiotics.14 Therefore, there is a need for the development of
new chemotherapeutic agents in treating the pathogenic microorganisms and cancer causing
cells.
3-Arylidene-2,3-dihydro-8-nitro-4-quinolones were earlier used as starting material
because of wide spectrum of pharmacological activities.15 In particular, nitro substituted
quinolines have shown promising anticancer effects16,17 against epidermal growth factor receptor

(EGFR)-overexpressing tumor cell lines.18 They are also found to act as selective
antileishmanial,19 neuropeptide Y1 (NPY) receptor antagonists,20 poly(ADP-ribosyl) transferase
(PARP),21 cyclooxygenase-2 (COX-2) inhibitors22 and induce necrotic death of leukaemic cells
HL-6023 in addition to acting as important biosensors.24 In recent years, various substituted
pyrazole and 4H-pyran-annulated heterocyclic scaffolds have been reported to demonstrate
diverse biological activities including anticancer,25 antimicrobial,26 antimalarial,27 anti-HIV28 and
anti-inflammatory29 activities. Particularly, 2-amino-3-cyano-4H-pyran containing heterocycles
showed encouraging anticancer and antibacterial activities.30 Hence, it would be an attractive
idea to incorporate nitroquinoline with pyran and pyrazolo moieties, because it might lead to a
new dimension of structural diversity to the molecules. In our endeavour to adopt green
chemistry approach, we report herein a microwave assisted synthesis of new series of
pyrano[3,2-c]quinoline and pyrazolo[4,3-c]quinoline derivatives and their in vitro antibacterial,
antioxidant and anticancer activities.
CH3
N
O
N
N
CH3
S
N
N
O
O
N
F
O
Br
O
H3C
N
O
N
N
O
N
CH3
N
F
CH3
H
CH3
Zanthosimuline
Huajiaosimuline
Pyrazolo[4,3-c]quinolin-3-ones
Fig. 1. Structure of anticancer alkaloids based on pyranoquinolines and synthetically derived
pyrazoloquinolines.
Our starting compounds, 3-arylidene-2,3-dihydro-8-nitro-4-quinolones (3) were
synthesized by reacting 2,3-dihydro-8-nitro-4-quinolones (1) with substituted aromatic aldehydes
(2) by pyrrolidine base catalysed condensation reaction (Scheme 1)15 and were treated with
hydrazine derivatives (4&6) in ethanol under microwave irradiation (MW) condition, which
afforded the pyrazolo[4,3-c]quinoline (5a-i, 7a-b) derivatives in excellent yields (Table 2).31 To
extend our experiment, 3a was made to react with malononitrile 8 in ethanol (5 mL) in the
presence of pyridine (1 equiv) under MW condition (110 W at 80 °C), which afforded
pyrano[3,2-c]quinoline 9a (55 %, Table 1, entry 5) along with starting 2,3-dihydro-8-nitro-4-

quinolone 1a (30 % yield). In order to improve the yield of the product, this reaction was
subjected to a variety of conditions and the results are presented in Table 1. The table reveals
that, when strong bases like pyrrolidine, Et3N (1 equiv) were used, only trace amount of product
9a is formed and the hydrolysis product 1a was predominantly formed (Table 1, entry 1-2),
while the use of weak bases such as pyridine, piperidine has significantly increased the yield of
product 9a (Table 1, entries 3, 5). This might be due to the basicity and selectivity of bases in the
formation of product. At the same time, in the presence of K2CO3, NH4OAc, KOH in ethanol,
the reactions did not proceed well. Different solvents were also tested in the presence of pyridine
catalyst but unfortunately they had resulted with low yields (Table 1, entries 9, 10). The yield of
9a did not improve with altering concentration of the catalyst (Table 1, entries 11, 12).
Therefore, we tried multicomponent reaction32 (MCR) as another option by taking 2,3-dihydro-
8-nitro-4-quinolones (1), aromatic aldehydes (2), malononitrile (8) and pyridine in ethanol under
MW condition, which afforded exclusively products (9a-i) in higher yields (75-88 %) (Scheme
1, Table 2). Structures of the products (5a-i, 7a-b, and 9a-i) were determined by spectral data.
Table 1 Reaction of 3a with 8 under different conditions
NH2
CN
O
O
O
Catalyast
Experimental conditions
CN
CN
Time (min)
N
H
N
H
F
N
H
F
NO2
1a
NO2
NO2
8
3a
9a
Entry
Catalyst (equiv)
Solvent
Temp (°C) Time (min)a
Yield (%)b
9a 1a
1
2
3
4
5
6
7
Et3N (1.0)
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
80
80
80
80
80
80
80
5
5
6
5
4
5
6
c
c
85
85
45
75
30
d
Pyrrolidine (1.0)
Piperidine (1.0)
45
10
55
d
Morpholine (1.0)
Pyridine (1.0)
K2CO3 (1.0)
NH4OAc (1.0)
d
d

8
KOH (1.0)
Pyridine (1.0)
Pyridine (1.0)
Pyridine (0.5)
Pyridine (2)
EtOH
CH3CN
CH3OH
EtOH
80
80
80
80
80
5
7
6
6
5
d
d
9
8
40
45
34
42
10
11
12
48
44
50
EtOH
aMicrowave irradiation at 110 W. bIsolated Yield. cTrace amount. dNo reaction.
O
R
Ar-CHO
N
H
R= H, Cl
NO2
2
Ar= Aryl/heteroaryl
1
R1
NH2
HN
Pyrrolidine
NH
N
N
O
N
R1
NH2NH2.H2O
6
R
Ar
R
R
Ar
4
Ar
EtOH
EtOH
N
N
H
N
H
H
NO2
7a-b
NO2
R1= H, OCH3
NO2
5a-i
3
CN
Pyridine
CN
8
NH2
CN
Ar
O
O
R
R
N
H
N
H
NO2
NO2
9a-i
1
Scheme 1 Synthesis of pyrazolo, pyranoquinolines

Table 2 Synthesis of heteroannulated derivatives (5a-i, 7a-b & 9a-i)
Entry Analog
R
R1
Ar
Time (min)
TCRb MCRc TCR
10
Yield (%)a
MCR
d
d
1
2
5a
5b
5c
5d
5e
5f
H
H
H
H
H
H
Cl
H
H
Cl
Cl
H
H
H
H
H
H
H
Cl
H
H
C6H5
4-FC6H4
-
87
90
89
89
91
87
85
83
81
72
73
55
52
50
45
40
38
38
48
46
-
H
8
8
-
-
3
H
4-BrC6H4
-
-
-
-
4
H
4-ClC6H4
8
5
H
4-OCH3C6H4
4-CH3C6H4
4-ClC6H4
7
-
-
6
H
8
-
-
7
5g
5h
5i
OCH3
8
-
-
8
H
H
-
3,4-Di OCH3C6H3
2,4-Di OCH2CH3C6H3
4-FC6H4
9
-
-
9
10
10
10
7
-
-
10
11
12
13
14
15
16
17
18
19
20
7a
7b
9a
9b
9c
9d
9e
9f
-
-
-
4-ClC6H4
-
-
-
4-FC6H4
4
4
4
4
4
5
5
4
4
88
82
84
80
82
75
76
82
87
-
4-BrC6H4
7
-
4-ClC6H4
7
-
4-OCH3C6H4
4-CH3C6H4
2,4-Di OCH3C6H3
3,4-Di OCH3C6H3
4-ClC6H4
7
-
8
-
9
9g
9h
9i
-
10
7
-
-
2-thienyl
10
a Isolated yield, b Two component reaction, c Multicomponent reaction, d No reaction
The pyrazolo and pyrano quinoline derivatives were evaluated for their antibacterial
activities against two Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and two
Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) by agar well diffusion
method using ampicillin as a positive control. Strains were obtained from the Microbial Type
Culture Collection (MTCC) and Gene Bank, Chandigarh, India. These strains were maintained

on LB agar plates at 4 °C overnight and the cultures were prepared in LB broth media. DMSO
was used as solvent and as negative control. Antibacterial activity was determined by measuring
zone of inhibition in mm and compared with ampicillin. The zone of inhibition and minimum
inhibitory concentration are given in Table 3.
Antibacterial activity evaluated for analogues 5a-i, 7a-b and 9a-i against several strains
flaunted a diverse range of inhibitory activity. From the bioassay, it was observed that the
compounds 9a-i with substituents on pyran ring were significantly active against all
microorganisms studied than the analogues 5a-i, 7a-b. Introduction of thiophene core33 at C-4
position of pyran ring (9i) and electron withdrawing group34 at C-4 position of phenyl ring (9a)
exhibited highest activity against all the bacterial strains with inhibition zone 20-29 mm at 5-37
µg/ml as compared to standard ampicillin. Compounds 5b, 5i, 9b, 9c and 9d showed good
activity with inhibition zone ranging between 17-24 mm at 7.5-55.5 µg/ml. 4-Fluoro phenyl on
pyran ring (9a) showed better activity compared to 4-fluoro phenyl on pyrazolo ring (5b).
Therefore it is inferred that, presence of fluorine group and pyran nucleus were responsible for
the enhancement of antibacterial activity. Compounds 5c, 5h, 9e and 9h were found to be active
(15-18 mm at 25.5-55.5 µg/ml) against Gram-negative bacteria but least active (12-19 mm at
12.5-55 µg/ml) against Gram-positive bacterial strains. However, the remaining compounds (5a,
5d, 5e, 5f, 5g, 7a, 7b, 9f & 9g) exhibited poor activity against all microorganisms with inhibition
zone 8-17 mm at 15-200 µg/ml. In general, the order of antibacterial activity of the substituents
at the 4th position of the phenyl ring is F>Br>Cl> OCH3>CH3>H. The better activity of 9i is
ascribed due to the presence of sulphur atom on thiophene moiety.
Table 3 In vitro antibacterial activity of pyrazolo, pyrano quinolines (5a-i, 7a-b & 9a-i)
Compound
ZOI (mm) and MIC (μg/mL) Values
Gram-positive bacteria Gram-negative bacteria
S. aureusa B. subtilisb E. colic P. aeruginosad
ZOI MIC ZOI
ZOI
MIC
60
MIC
60
ZOI
14
MIC
150
5a
5b
5c
5d
8
8
60
12.5
12.5
15
12
21
18
17
20
16
17
10
18
19
16
17
18
55.5
55.5
100
15
25.5
34
16
15
13

5e
15
12
13
19
19
12
13
23
20
21
21
13
14
14
15
23
24
17.5
50
50
12.5
10
50
55
5
17
12
14
19
23
12
10
26
20
24
20
12
15
15
18
29
30
20
50
14
10
12
18
19
12
8
42.5
80.5
80.5
34
10
8
200
200
100
55.5
55.5
200
100
37
5f
5g
50
13
16
18
8
5h
12.5
7.5
50
5i
34
7a
80.5
97.5
17
7b
60
14
20
20
18
21
15
13
13
16
20
22
9a
7.5
12.5
10
22
18
21
20
18
15
12
18
22
24
9b
10
10
10
55
40
40
17.5
5
25.5
17
37
9c
55.5
37
9d
12.5
50
17
9e
25.5
42.5
80.5
25.5
17
55.5
150
150
55.5
37
9f
20
9g
9h
20
17.5
5
9i
Ampicillin
2.5
2.5
8.5
18.5
c
d
a Staphylococcus aureus b Bacillus subtilis Escherichia coli Pseudomonas aeruginosa
The newly synthesized quinoline derivatives were evaluated for their antioxidant potential
by DPPH, ferric reducing antioxidant power and total antioxidant capacity methods.
In this study, we have determined the antioxidant properties of compounds (5a-i, 7a-b &
9a-i) using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity with respect to the
standard ascorbic acid. The samples were prepared at different concentrations and the IC50 values
of tested compounds were shown in figure 2. All the compounds showed weak to moderate
scavenging activity. However, methoxy substituted compounds 5e, 5g, 5h, 9d, 9f and 9g showed
significant scavenging activity (IC50 = 65.42-169.1 µg/ml) compared to the standard used (IC50 =
28.02 µg/ml) and among them, compound 9d showed highest scavenging activity with IC50 value
65.42 µg/ml. This might be due to the presence of the electron releasing methoxy group at the
para position of the phenyl ring, which may enhance the stabilization of the resulting oxygen
radical.35

Fig. 2. Free radical scavenging activity of the synthesized compounds (5a-i, 7a-b & 9a-i).
The reducing antioxidant power of all the compounds were performed and compared to
butylated hydroxytoluene (BHT) with different concentration levels (Fig. 3). From the
experiments, it was observed that compounds 9d and 5g showed significant activity. The above
behaviour might be due to the presence of methoxy group on phenyl ring and the chloro
substituent at the sixth position of quinoline ring. All other compounds have shown weak activity
compared to standard BHT.
Fig. 3. Ferric reducing antioxidant activity of the synthesized compounds (5a-i, 7a-b & 9a-i).

The total antioxidant assay was carried out for all the new analogues. Antioxidant
activities are expressed as the number of equivalents of ascorbic acid. Among the tested
compounds, 9d showed highest total antioxidant capacity, whereas compounds 5a, 5c, 9f showed
mild activity. However, the remaining compounds did not show total antioxidant activities as
effective as the standard. The results are presented in Fig. 4.
Fig. 4. Total antioxidant capacity of the compounds (5a-i, 7a-b & 9a-i).
All the new quinoline derivatives (5a-i, 7a-b and 9a-i) were evaluated for their anticancer
activity against two human cancer cell lines of breast adenocarcinoma (MCF-7) and lung
adenocarcinoma (A549) using MTT assay. Doxorubicin was used as standard and the results are
given in Table 4. The results suggested that compounds 9d, 9h and 9i have displayed excellent
cell growth inhibition against MCF-7 cell line with IC50 <20 µM, whereas compounds 5b, 9a and
9i exhibited significant cell growth inhibition against A549 cell line with IC50 <23.5 µM. This
potency is comparable to the standard. Interestingly, compound 9i was the most active with IC50
value 14.26 µM, 15.41 µM against MCF-7 and A549 cell lines respectively, compared to the
standard doxorubicin, which displayed IC50 of 15.12 µM (MCF-7) and 18.56 µM (A549).
However, the remaining compounds showed only moderate inhibitory effect against both the cell
lines.

In general, structure activity relationship (SAR) studies for these compounds reveal that
quinoline fused 2-amino-3-cyanopyran36 skeleton showed better activity when compared to
pyrazolo skeleton. This might be due to the presence of 4H-pyran ring37 and strong electron
withdrawing cyano group.38 The C-4 substituents on 2-amino-3-cyanopyran ring could alter the
cytotoxicity against both the cell lines. The excellent activity of compound 9i might be due to
presence of thiopene moiety36e,39 which induces anticancer activity. The next most promising
compounds 9c and 9h having chlorine substituent showed cytotoxicity against MCF-7 and A549
cell lines. Moreover, compounds 5b, 5c, 5d, 5g, 9a, 9b, 9c and 9h having electron withdrawing
groups (F, Cl, Br) on C-4 position of the phenyl ring exhibited better cytotoxicity effects and this
might be due to electromeric effect of halogen groups.40 But at the same time, compounds 5e, 5g,
9d having methoxy group at para position of the phenyl ring36c showed better activity against
MCF-7 cell line. No significant difference was observed between dimethoxy (5h, 9f & 9g) and
diethoxy (5i) substituted compounds, which exerted significant inhibitory effects.36a It was
observed that 2-amino-3-cyanopyran skeleton along with the hetero aryl ring and electron rich
aryl groups are responsible for improved cytotoxicity.
Table 4 Cytotoxicity and IC50 (µM) of synthesized compounds (5a-i, 7a-b & 9a-i)
Compound
MCF-7a
53.38
45.98
34.72
41.35
31.31
72.80
25.76
58.54
65.73
98.74
A549b
59.78
23.21
29.57
28.66
40.17
45.79
31.98
46.36
50.91
63.59
Compound
MCF-7a
78.48
35.67
24.72
22.73
16.78
34.12
51.23
56.34
18.02
14.26
15.12
A549b
95.13
22.14
34.75
28.12
35.57
56.27
44.61
50.65
25.48
15.41
18.56
5a
5b
5c
5d
5e
5f
7b
9a
9b
9c
9d
9e
5g
5h
5i
9f
9g
9h
9i
7a
Doxorubicin
a Human breast adenocarcinoma b Human lung adenocarcinoma

Apoptosis is one of the major pathways that leads to the process of cell death and
morphological changes. These changes include blebbing, nuclear fragmentation, cell shrinkage
and chromatin condensation. Cancer cells often have irregular apoptotic pathways and induction
of cancer cell apoptosis by natural or synthetic analogues is considered an effective therapy for
cancer.41 Therefore it was of interest to investigate the apoptosis inducing effect of 9i on MCF-7,
A549 cell lines, which was observed under a fluorescence microscope after being stained by
acridine orange / ethidium bromide (AO/EB) and DAPI. Compound 9i with treated cells showed
above mentioned characteristic morphological features, whereas these distinctive morphological
features are not observed in the control cells (Fig. 5).
Apoptotic, necrotic and live cells can be differentiated by using acridine orange/ethidium
bromide (AO/EB) fluorescent staining assay.42 Acridine orange permeates the live as well as
dead cells and emits green fluorescence, whereas ethidium bromide can stain only dead cells
with loss in membrane integrity and emits orange fluorescence after intercalation into DNA. The
compound 9i treated and untreated MCF-7, A549 cell lines were stained with AO/EB dye and
analysed under fluorescence microscopy, which is shown in Fig. 5. The control cells appeared as
green coloured nucleus with normal morphology. The cells, after treatment with 9i at its 50%
inhibitory concentration showed colour and morphological changes. The morphological changes
of treated cells when compared with that of the untreated cells revealed apoptotic cell death.
Apoptotic cells were observed as orange coloured bodies, whereas the necrotic cells appeared red
in colour. These morphological findings indicated that compound 9i inhibits cell viability and
induces apoptosis in MCF-7 and A549 cells.
DAPI (4′, 6-diamidino-2-phenylindole) is a fluorescent dye that forms complexes with
double stranded DNA and it is useful to detect the chromatin condensation or nuclear damage.
The DAPI stains the apoptotic cells as bright coloured due to the condensed nucleus, which is a
typical apoptotic characteristic. Therefore it was considered of interest to detect the effect of
compound 9i in MCF-7, A549 cell lines by using DAPI staining method. Fig. 5 clearly shows
that the nuclear structure of control cells was intact, whereas compound 9i treated cells exhibited
condensed nuclei.

Fig. 5. Fluorescence microscopy image of 9i on MCF-7 and A549 cell lines. (a & c) Control
cells of MCF-7 and A549; (b & d) Cells treated with IC50 value of 9i. Respective control cells
stained with AO/EB appear in green colour; apoptotic cells appear in orange colour; necrotic
cells appear in red colour. DAPI staining of 9i-treated cells (b & d) show nuclear apoptotic cells.
In order to theoretically calculate the binding mode of 8-nitroquinoline derivatives with the
active center of epidermal growth factor receptor (EGFR) (PDB code: 1M17),43 molecular
docking of the selected compounds 5b, 5g, 9a, 9d, 9h and 9i into the ATP binding domain of
EGFR were performed by Autodock software.44 The binding energies of the docked compounds
5b, 5g, 9a, 9d, 9h and 9i were -6.08, -4.98, -4.76, -6.19, -5.97, 5.93 kcal/mol, respectively.
Among them, compounds 5b, 9d, 9h and 9i show high binding energy with the EGF receptor.
These results match well with the observed anticancer activity of the synthesised compounds. In
particular, compound 9d binds very well with EGF receptor (-6.19 kcal/mol) which shows two
crucial hydrogen bonding interaction with Glu738 (2.30 Å) and Arg817 (2.60 Å) through –NH2
substituent of pyran ring and quinoline ring, respectively (Fig. 6). Compound 5a has formed two
hydrogen bonds with active site of amino acids. Oxygen atom of –NO2 group in the quinoline
ring acts as H-bond acceptor forming one H-bond with OG1 atom of Thr766 with bond length of
2.10 Å. The H-atom of –NH group in the quinoline ring has formed another hydrogen bond with
OG1 atom of Thr766 with bond length of 2.20 Å. The compounds 9h and 9i showed each two
hydrogen bonding interactions with Asp813, Asn818 and Lys721, Met769 amino acids,

respectively. The compounds 5g and 9a show low binding energy and each formed one hydrogen
bonding interaction with Thr766 and Asp818, respectively.
Fig. 6. Docking studies on compounds 5b, 5g, 9a, 9d, 9h and 9i with EGF receptor.
In short, we have synthesized new biologically active pyrazolo, 2-amino-3-cyanopyran
ring fused 8-nitroquinoline derivatives. The yield of 2-amino-3-cyanopyran quinoline analogues

were improved via multicomponent reaction under microwave condition. The compounds 5a-i,
7a-b & 9a-i were studied for their antibacterial, antioxidant and antibacterial activities. The
synthetic analogues with thiophene moiety (9i) and 4-fluorophenyl (9a) substituents at the C-4
position of the pyran ring showed good antibacterial activity compared to all other compounds.
The antioxidant activity of the compounds were screened by three methods. Among them,
compound 9d possessed significant antioxidant potential and showed more cytotoxicity to cancer
cell lines. This might be due to the presence of methoxy group at para position of the phenyl
ring. Further, compound 9d displayed wide range of free radical scavenging activities.
Cytotoxicity studies were carried out by MTT assay method against MCF-7 and A549 cell lines.
In particular, the anticancer activity of compound 9i was quite comparable with that of the
standard drug doxorubicin against both the cell lines. The methoxy substituted analogue 9d
exhibited almost equipotent efficacy with standard drug against breast cancer cell line MCF-7.
However, fluorine substituted analogues 5b, 9a displayed promising inhibitory effects against
lung cancer cell line A549. The AO/EB and DAPI nuclear staining studies suggested that
compound 9i induced apoptosis in MCF-7 and A549 cell lines. As exemplified by our SAR
studies, the compounds containing 2-amino-3-cyanopyran skeleton along with thiophene moiety
and halogenated aryl group at C-4 position exhibited good anticancer and antibacterial activities
compared to the compounds containing pyrazolo skeleton.
Acknowledgement
The research at The University of Winnipeg was supported by Natural Sciences and Engineering
Research Council of Canada.
References and notes
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31. General procedure for the synthesis of pyrazolo[4,3-c]quinoline derivatives (5a-i, 7a-b): 3-
Arylidene-2,3-dihydro-8-nitro-4-quinolone (3) (1 mmol) and phenyl hydrazine hydrochloride (4)
/ hydrazine (6) (2 mmol) were mixed with 5 ml of ethanol sealed and subjected to microwave
irradiation, programmed at 80 °C and 110 W for 7-10 min. After completion of the reaction, the
mixture was brought into room temperature, the solid separated by filtration, dried and
recrystallized from ethanol to get the title compounds which were found to be in good purity
(TLC) and yield.
3-(4-Chloro-phenyl)-6-nitro-2-phenyl-3,3a,4,5-tetrahydro-2H-pyrazolo[4,3-c]quinoline (5d)
1
Dark red solid, mp 165-167 °C; IR (KBr) υmax 3363, 1587, 1522 cm-1; H NMR (400 MHz,
CDCl3): δ 8.18-8.12 (m, 3H, Ar-H), 7.42-7.37 (m, 4H, Ar-H), 7.21 (t, 2H, J = 8.4 Hz, Ar-H),
7.06 (dd, 2H, J = 1.2, 7.6 Hz), 6.90 (t, 1H, J = 8.0 Hz, Ar-H), 6.78 (t, 1H, J = 7.6 Hz, Ar-H), 4.89
(d, 1H, J = 11.2 Hz, CH), 4.03-3.97 (m, 1H, N-CH2), 3.62 (t, 1H, J = 12.4 Hz, N-CH2), 3.55-3.48
13
(m, 1H, CH); C NMR (100 MHz, DMSO): δ 43.82, 51.29, 69.21, 114.24, 115.39, 117.22,
119.97, 127.32, 128.12, 128.74, 129.17,130.81, 131.96, 132.27, 140.65, 142.32, 145.74, 145.79;
HRMS (TOF-ES+) m/z [M+H]+ calcd for C22H18ClN4O2 405.1040, found 405.1038.
32. Synthesis of pyrano[3,2-c]quinoline derivatives (9a-i)

Method 1: Two component reaction
A mixture of 3-arylidene-2,3-dihydro-8-nitro-4-quinolones (3) (1 mmol), malononitrile (8)
(1 mmol) and pyridine (0.05 mmol) as a catalyst in ethanol (5 ml) was sealed and subjected to
microwave irradiation for 7-10 min. The progress of the reaction was monitored by TLC. After
completion of the reaction, the excess solvent was evaporated. The residue was dissolved in ice
cold water and extracted with ethyl acetate. Combined organic layers were washed with brine
solution and dried over anhydrous sodium sulfate. Then, the crude residue was subjected to
column chromatography using ethyl acetate:petroleum ether mixture (1:4) to obtain the pure
products (9).
Method 2: Multicomponent reaction
A mixture of 2,3-dihydro-8-nitro-4-quinolones (1) (1 mmol), aromatic aldehydes (2) (1
mmol), malononitrile (8) (1 mmol) and pyridine (0.05 mmol) in dry ethanol (5 ml) was sealed
and subjected to microwave irradiation (80 °C at 110 W) for 4-5 min. After completion of the
reaction (indicated by TLC), the excess solvent was evaporated. The residue was dissolved in ice
cold water and extracted with ethyl acetate. Combined organic layers were washed with brine
solution and dried over anhydrous sodium sulfate. Then, crude residue subjected to column
chromatography using ethyl acetate:petroleum ether mixture (1:4) to obtain the pure products
(9) with high yield.
2-Amino-4-(4-fluoro-phenyl)-7-nitro-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (9a)
1
Red solid, mp 211-213 °C; IR (KBr) υmax 3360, 3352, 2192, 1579, 1510 cm-1; H NMR (400
MHz, DMSO-d6): δ 8.38 (s, 1H, Q-NH), 7.89 (d, 1H, J = 8.8 Hz, Ar-H), 7.50 (d, 1H, J = 6.4 Hz,
Ar-H), 7.32 (t, 2H, J = 6.4 Hz, Ar-H), 7.21 (t, 2H, J = 8.8 Hz, Ar-H), 6.98 (s, 2H, NH2), 6.61 (t,
1H, J = 8.0 Hz, Ar-H), 4.31 (d, 1H, J = 17.2 Hz, N-CH2), 4.04 (s, 1H, CH), 3.90 (d, 1H, J = 17.6
Hz, N-CH2); 13C NMR (101 MHz, DMSO-d6): δ 43.81, 47.32, 55.97, 106.64, 114.43, 115.53 (d,
2JCF = 21.5 Hz), 117.28, 119.92, 125.97, 126.57, 129.59, 129.67, 129.70, 133.79, 136.44, 138.38,
138.41, 142.52, 159.31, 161.39, (d, 1JCF = 244.2 Hz); HRMS (TOF-ES+) m/z [M-3H]+ calcd for
C19H10FN4O3 361.0737, found 361.0794.
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Graphical abstract