Novel 1,4-dihydropyrano[2,3-c]pyrazole derivatives: Synthesis, characterization, biological evaluation and in silico study

Article abstract of DOI:10.1016/j.molstruc.2018.12.053

In the present study, a series of novel and biologically potent 6-amino-1-(2,4-dinitrophenyl)-4-phenyl-1,4-dihydropyrano [2,3-c]pyrazole-5-carbonitrile derivatives (5a-5u) have been synthesized through multicomponent reaction between various substituted a

Full text of DOI:10.1016/j.molstruc.2018.12.053

Accepted Manuscript
Novel 1,4-dihydropyrano[2,3-c]pyrazole derivatives: Synthesis, characterization,
biological evaluation and in silico study
Mahesh S. Vasava, Manoj N. Bhoi, Sanjay K. Rathwa, Shilpa S. Shetty, Rikin D.
Patel, Dhanji P. Rajani, Smita D. Rajani, Alpesh Patel, Himanshu A. Pandya, Hitesh
D. Patel
PII:
S0022-2860(18)31480-7
DOI:
https://doi.org/10.1016/j.molstruc.2018.12.053
MOLSTR 25990
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 September 2018
Revised Date: 11 December 2018
Accepted Date: 11 December 2018
Please cite this article as: M.S. Vasava, M.N. Bhoi, S.K. Rathwa, S.S. Shetty, R.D. Patel, D.P. Rajani,
S.D. Rajani, A. Patel, H.A. Pandya, H.D. Patel, Novel 1,4-dihydropyrano[2,3-c]pyrazole derivatives:
Synthesis, characterization, biological evaluation and in silico study, Journal of Molecular Structure
(2019), doi: https://doi.org/10.1016/j.molstruc.2018.12.053.
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ACCEPTED MANUSCRIPT
Novel 1,4-dihydropyrano[2,3-c]pyrazole derivatives: Synthesis,
characterization, biological evaluation and in silico study
a
b
a
c
c
Mahesh S. Vasava , Manoj N. Bhoi , Sanjay K. Rathwa , Shilpa S. Shetty , Rikin D. Patel ,
d
d
e
c
Dhanji P. Rajani , Smita D. Rajani , Alpesh Patel , Himanshu A. Pandya and Hitesh D.
a
Patel*
[a]
Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, Gujarat India.
E-mail: drhiteshpatel1@gmail.com
[b]
Piramal Enterprise Ltd, Plot No.-18, Pharmez, Matoda Village, Ahmedabad.
[c]
Department of Botany, Bioinformatics and Climate Change impacts Management, School of Sciences, Gujarat
University, Ahmedabad, Gujarat, India.
[d]
Microcare Laboratory & Tuberculosis Research Centre, Surat, Gujarat, India,
[e]
Genxplore Diagnostic and Research Centre Pvt. Ltd., Ahmedabad, Gujarat, India.

ACCEPTED MANUSCRIPT
Abstract
In the present study, a series of novel and biologically potent 6-amino-1-(2,4-dinitrophenyl)-
-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile derivatives (5a-5u) have been
4
synthesized through multicomponent reaction between various substituted aromatic aldehyde
derivative (4a-4u), 2, 4-dinitrophenyl hydrazine (1), ethyl acetoacetate (2) and malononitrile
(
3) in the presence of SnCl as a prompt catalyst using both microwave irradiation method as
2
well as conventional method. The structure of synthesized compounds were confirmed by
1
13
various spectroscopic methods such as H-NMR, C-NMR, IR, Mass analysis and elemental
analysis. All the synthesized compounds were subjected in vitro antibacterial, anti-
tuberculosis screening and cytotoxicity MTT assay. In vitro biological study revealed that the
synthesized compound 5a, 7a and 8a are showing good anti-bacterial and anti-tuberculosis
activity. The in silico study of ADME pharmacokinetic properties were also predicted for
synthesized compounds for checking their bioavailability. Furthermore, molecular docking
study of synthesized compounds with enoyl-ACP reductase (oxidoreductase) was carry out to
find out the binding affinity of compounds. Docking study demonstrated that compound 7b
and 7a possessed superior binding affinity with target enzyme by strong hydrogen bonding.
We have also carried out molecular dynamics simulation to check the stability of docked
complex, conformational changes and primary molecular interaction.
Keywords: anti-tuberculosis activity; anti-bacterial activity; molecular docking; molecular
dynamics; one-pot synthesis; pyrano[2,3-c]pyrazole;

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1
. Introduction
The treatment of infectious diseases that are predominantly endemic into the
developing countries, it requires simple medications that can be produced in large quantities
at low cost. Tuberculosis (TB) is most dangerous infectious disease which causes maximum
fatality by subverting the immune system of the human host due to its long and wide
association with humans and is often regarded as a successful pathogen [1]. According to the
WHO report 2017, one-third of the world population is potentially infected with TB and
millions of new cases occur worldwide every year [2].
There are many drugs available for TB in market, among them, Isoniazid (INH),
Rifampicin, Pyrazinamide, and Ethambutol are four milestones in the treatment of TB for
more than 50 years. INH is pro-drug which activate by the mycobacterial catalyse peroxidase
(KatG), which inhibit the enoyl-ACP reductase of the mycobacterial fatty-II type acid. It
involved in the biosynthesis of mycolic acid eventually leading to cell death [3-4]. Studies
have confirmed that the front-line anti-TB drugs such as INH and ethionamide is primary
targeting inhA gene [3]. Freundlich et al. revealed that potent triclosan derivatives that inhibit
InhA in the nanomolar range with minimum inhibitory concentrations of 5–10 µg/mL [5].
Additionally, several compounds have been reported, such as arylamides derivatives [6],
pyrrolidine carboxamide derivatives [7], pyrazole derivatives and indole-5-amides derivatives
[
8] which targeting inhA enzyme. However, the increasing prevalence of multi-drug-resistant
TB (MDR-TB) and extensively drug-resistant TB decrease the effectiveness of these drugs
against tuberculosis. Hence, to stop this infection, there is a burning requirement to develop
inhibitors targeting InhA directly without prerequisite for activation [9].
Owing to the vast research on anti-tubercular activity, many synthesized heterocyclic
compounds have efficiently displayed anti-tubercular activity. Current literature describing
that the 4H-pyrans and pyran-annulated heterocyclic scaffolds have drawn considerable

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interest in medicinal chemistry from the last several years [10-16]. Figure 1 represents some
of the bioactive pyran-annulated heterocyclic compounds which are good antibacterial
agents. Various studies have indicated 4H-pyran derivatives display a potent activity against
mycobacterium [17-29] as well as anticancer [20], cytotoxic [21], anti-inflammatory [22],
anti-HIV [23-24], antimalarial [25], anti-hyperglycemic, and anti-dyslipidemic [26], anti-
neurodegenerative disorders like Alzheimer’s, Parkinson disease, Huntington’s disease [27],
and many more [28-39]. Furthermore, substituted 4H-pyran derivatives have encouraged
increasing roles in synthetic methodologies to promising compounds in the field of medicinal
[
30], pigment industries [31], agrochemical and cosmetics [32]. Keeping this knowledge,
synthetic chemists develop useful synthetic routes to synthesize these heterocyclic
compounds. A lot of synthetic methodologies are already reported.
Nowadays, the most straightforward method for the synthesis of various heterocyclic
compounds involves a three-component reaction or multi-component reaction to avoid the
setbacks such as time-consuming reaction, low yields, toxicity of the chemicals, harmful
organic solvents, dynamic reaction conditions and strenuous work-up. Additionally,
Microwave-assisted synthesis is one of the most prominent method increasing its importance
in the field of organic chemistry [33]. As compared to conventional heating, microwave
irradiation method is much faster and provide higher practical yield in many organic
synthesis [34]. Due to this advantage, this technique is widely used in the organic synthetic
chemistry. In context to the already existing literature [35-45], we decided to use a simple,
rapid, and effective one-pot MCR methodology to synthesize the targeted compounds.
In this current study, our aim was to synthesize new structural moieties under
microwave irradiation and conventional heating. The 1,4-dihydropyrano[2,3-c]pyrazole
derivatives synthesized by the reaction between substituted aldehyde derivatives, ethyl
acetoacetate, malononitrile and 2,4-dinitrophenyl hydrazine using SnCl as a rapid catalyst
2

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under solvent-free condition with good practical yields. All the synthesised compounds were
screened for their anti-bacterial, anti-tuberculosis activity against various bacterial strain and
H Rv respectively and cytotoxic activity against HeLa cell line.
3
7
In addition, the ADME pharmacokinetic properties of synthesized compounds have
studied for gaining preliminary information concerning their possibly drug-like profile.
Moreover, based on the favourable in vitro antimicrobial results and by considering NADH-
dependent enoyl-Acyl Carrier Protein reductase (enoyl-ACP reductase) as the target receptor,
Molecular docking and molecular dynamics studies was performed against the active site of
the NADH-dependent enoyl-Acyl Carrier Protein reductase (enoyl-ACP reductase) encoded
by the Mycobacterium gene inhA Mycobacterium tuberculosis oxidoreductase enzyme.
2
. Methods and Materials
2
.1 Chemistry
All the chemicals and solvents were purchased from commercial source and were
used in the reaction without further purification. Synthesis of the desired product was carried
out using conventional and microwave irradiation (CEM Discover microwave system, Model
No. 908010; Make up CEM Matthews, Inc, USA) methods. Melting points were determined
by automated melting point system (MPA- Optimelt) and were uncorrected. The completion
of the reactions was checked by thin layer chromatography (TLC). It is silica gel coated
aluminium sheets (silica gel G60 F , Merck) which was visualized by UV radiation and
2
54
various spray reagent. Elemental analysis were performed on vario MICRO cube, elemental
CHNS analyser (Serial Number: 15084053). The IR spectra (in KBr pellets) were recorded
-1
1
13
on a Perkin-Elmer 377 spectrophotometer with absorption in cm . H and C NMR spectra
were run on Bruker 400 and 100 MHz NMR spectrometer respectively, using DMSO-d6 as
solvents. The chemical shifts were expressed in parts per million (δ ppm) with TMS as
internal reference. J values are given in hertz (Hz). Mass spectra was recorded on Advion

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Expression CMS, USA, with electro-spray ionization (ESI) was used as an ion source and
ethanol: formic acid: water was used as a mobile phase. Column Chromatography with 60-
1
20 mesh size of silica gel was used to purify the synthesized compounds.
.1.1 Synthesis of 4-(piperidin-1-yl) benzaldehyde derivatives (6a-6b):
To a stirred solution of 4-fluoro aldehyde (5g) (1.0 eq.) in Methanol was added
2
piperidine (1.0 eq.) and anhydrous Potassium carbonate (10 mol %) at room temperature. The
resulting reaction mixture was heated at 130 °C for 20h. The progress of reaction was
monitored by TLC plate. The reaction mixture was cooled at room temperature and then
poured it into ice cold water and kept for 24h. The solid product was precipitated out, filtered
it using vacuum filtration and washed with hot water. The crude material was crystallized
with Methanol to get pure compounds 4-(piperidin-1-yl) benzaldehyde derivatives 6a-6b with
good practical yield.
2
.1.2 General procedure for the synthesis of 6-amino-1-(2,4-dinitrophenyl)-4-phenyl-1,4
dihydropyrano[2,3-c]pyrazole-5-carbonitrile derivatives (5a-5u):
2
.1.2.1. Microwave irradiation method (Method A)
An equimolar mixture of 2,4-dinitrophenyl hydrazine (1) (1.0 eq.), ethyl acetoacetate
(2) (1.0 eq.), malononitrile (3) (1.0 eq.), aldehyde derivatives (4) (1.0 eq.) in the presence of
SnCl (10 mol %) as a catalyst was irradiated in microwave (CEM Discover microwave) at
2
1
80 W for 10-20 min. The progress of the reaction was continuously observed by TLC
analysis. After the consumption of the starting material, the reaction mixture was cooled at
room temperature and poured into ice cold water and extracted with ethyl acetate (2 x 30
mL). The organic layer was dried with anhydrous Na SO , and the solvent was evaporated
2
4
under reduced pressure. The crude material was purified through column chromatography
using 20% Ethyl acetate in Hexane as an eluent to get desired compound 6-amino-1-(2,4-

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dinitrophenyl)-4-phenyl-1,4dihydropyrano[2,3-c]pyrazole-5-carbonitrile derivatives (5a-5u)
with good practical yield (61-89%).
2
.1.2.2. Conventional Method (Method B)
All the reaction mixtures were taken same as mention in Method A in 50 ml FBF (Flat
Bottomed Flask) in the presence of SnCl (10 mol %) as a catalyst. The reaction mixture was
2
stirred at 80 °C for appropriate time mentioned in Table 1. The progress of the reaction was
observed by TLC analysis. After the completion of reaction, the reaction mixture allowed
cool at room temperature and poured into ice cold water and extracted with ethyl acetate (2 x
3
0 mL). The organic layer was dried with anhydrous Na SO , and the solvent was evaporated
2 4
under reduced pressure. The crude product (5a-5u) were then dried and were further purified
in to column chromatography using 20 % (v/v) Ethyl acetate: Hexane mixture as an eluent
which resulted in 54-81 % yield of the final product.
2
.1.2.3. 6-amino-1-(2,4-dinitrophenyl)-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-
carbonitrile (5a).
Brownish red solid, m.p. 240-243 °C.; Anal. Calc. for C H N O : C, 56.44; H, 2.99; N,
1
9
12
6
5
-1
2
0.78; O, 19.78%. Found- C, 55.85; H, 2.34; N, 20.16%.; IR vmax (KBr) cm : 3323 (N-H ),
str
1
3
106 (C-HAromatic,str), 1613 (C=C ), 2249 (-CN), 1023 (C-O ), 1514, 1346 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.642 (s, 1H), 8.936-8.921 (d, J = 6 Hz, 1H), 8.471 (s, 2H),
;
8
7
1
1
.150-8.142 (d, J = 3.2 Hz, 1H), 7.900 (s, 1H), 7.572-7.567 (d, J = 2 Hz, 2H), 7.358 (s, 1H),
1
3
.317-7.313 (d, J = 1.6 Hz, 2H), 4.656 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 174.91.
51.30, 145.86, 142.19, 139.87, 137.22, 135.31, 129.72, 129.46, 128.23, 128.10, 127.35,
25.29, 125.08, 119.78, 117.92, 116.89, 60.18, 28.14; ESI-MS: m/z Calculated 404.34, found
+
[
m/z] [M+H] 405.1.

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2
5
.1.2.4. (E)-6-amino-1-(2,4-dinitrophenyl)-4-styryl-1,4-dihydropyrano[2,3-c]pyrazole-
carbonitrile (5b).
Yellow solid, m.p. 248-251 °C.; Anal. Calc. for C H N O : C, 58.61; H, 3.28; N, 19.53; O,
2
1
14
6
5
-1
1
8.59%. Found- C, 57.69; H, 3.10; N, 18.83%.; IR vmax (KBr) cm : 3417 (N-H ), 2955 (C-
str
1
HAromatic,str), 1686 (C=C ), 2349 (-CN), 1270 (C-O ), 1596, 1349 (N-O ).; H NMR: (400
str
str
str
MHz, DMSO): δ 9.637 (s, 1H), 8756 - 8.739 (s, J = 6.8 Hz1H), 8.644 (s, 2H), 8.573 - 8.562
;
(d, J = 4.4 Hz, 1H), 7.958 (s, 1H), 7.635-7.620 (d, J = 6 Hz, 2H), 7.511-7.499 (d, J = 4.8 Hz,
1
H), 7.480-7.469 (d, J = 4.4 Hz, 2H), 6.379-6.361 (s, J = 7.2 Hz, 1H), 6.035-6.021 (d, J = 5.6
1
3
Hz, 1H), 4.357 (s, 1H) .; C NMR: (100 MHz, DMSO): δ; 174.82, 152.01, 147.86, 142.97,
1
1
40.35, 136.24, 135.87, 131.02, 128.51, 128.48, 128.37, 128.04, 127.35, 127.02, 123.61,
27.02, 120.34, 119.76, 119.07, 118.65, 60.04, 28.17; ESI-MS: m/z Calculated 430.37, found
+
[
m/z ] [M+H] 431.3.
2
.1.2.5. 6-amino-4-(3-chlorophenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5c).
Light yellow solid, m.p. 260-264 °C.; Anal. Calc. for C H ClN O : C, 52.01; H, 2.53; Cl,
1
9
11
6
5
-1
8
.08; N, 19.15; O, 18.23%. Found- C, 52.34; H, 2.24; N, 19.67%.; IR vmax (KBr) cm : 3282
(
N-H ), 3093 (C-H
), 1612 (C=C ), 2192 (-CN), 1059 (C-O ), 1512, 1344 (N-
Aromatic,str str str
str
1
O ).; H NMR: (400 MHz, DMSO): δ 10.108 (s, 1H), 9.235-9.226 (d, J = 3.6 Hz, 1H), 8.982
str
;
(s, 2H), 8.834-8.821 (d, J = 5.2 Hz, 1H), 7.990 (s, 1H), 7.701-7.692 (d, J = 3.6 Hz, 2H),
1
3
7
1
1
.508-7.490 (d, J = 7.2 Hz, 2H), 4.887 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 174.62.
51.17, 146.43, 142.30, 140.79, 137.60, 137.21, 134.68, 129.73, 128.23, 127.88, 127.37,
25.62, 124.39, 120.03, 118.40, 117.79, 58.42, 26.54; ESI-MS: m/z Calculated 438.78 found,
+
[
m/z] 438.2 [M+H] 439.2.

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2
.1.2.6. 6-amino-4-(2,5-dimethoxyphenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5d).
Brownish yellow solid, m.p. 286-289 °C.; Anal. Calc. for C H N O : C, 54.31; H, 3.47; N,
2
1
16
6
7
-
1
1
3
8.10; O, 24.12%. Found- C, 54.24; H, 3.31; N, 17.34%.; IR vmax (KBr) cm : 3439 (N-H ),
str
1
076 (C-HAromatic,str), 1626 (C=C ), 2352 (-CN), 1065 (C-O ), 1525, 1381 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 10.929 (s, 1H), 8.914-8.908 (d, J = 2.4 Hz, 1H), 8.589 (s, 2H),
;
8
7
.432-8.420 (s, J = 4.8 Hz, 1H), 7.822 (s, 1H), 7.369 (s, 1H), 7.181-7.159 (d, J = 8.8 Hz, 1H),
13
.066-7.052 (d, J = 5.6 Hz, 1H), 5.005 (s, 1H), 3.778 (s, 3H).; C NMR: (100 MHz, DMSO):
δ; 175.35. 152.08, 151.62, 150.20, 144.78, 142.80, 139.83, 126.71, 124.63, 121.01, 117.90,
1
4
16.86, 115.32, 114.13, 113.67, 113.085, 60.64, 55.80, 54.58, 29.01; ESI-MS: m/z Calculated
+
64.39, found [m/z] [M+H] 465.2.
2
.1.2.7. 6-amino-4-(2-chlorophenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5e).
Brown solid, m.p. 260-264 °C.; Anal. Calc. for C H ClN O : C, 52.01; H, 2.53; Cl, 8.08; N,
1
9
11
6
5
-
1
1
2
9.15; O, 18.23%. Found- C, 52.42; H, 2.69; N, 19.40%.; IR vmax (KBr) cm : 3425 (N-H ),
str
1
979 (C-HAromatic,str), 1595 (C=C ), 2197 (-CN), 1060 (C-O ), 1513, 1343 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ; 9.978 (s, 1H), 9.540-9.534 (d, J=2.4 Hz, 1H), 9.212 (s, 2H),
8
7
1
1
.937-8.925 (s, J = 4.8 Hz, 1H), 7.962 (s, 1H), 7.802 (s, 1H), 7.404-7.398 (d, J = 2.4 Hz, 2H),
1
3
.205-7.192 (d, J = 5.2 Hz, 2H), 4.895 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 175.29,
52.00, 146.02, 143.77, 141.90, 39.68, 136.73, 131.90, 129.11, 128.48, 127.95, 126.64,
26.35, 124.51, 120.03, 117.64, 116.84, 58.97, 24.35; ESI-MS: m/z Calculated 438.78, found
+
[
m/z] [M+H] 439.3.
2
.1.2.8. 6-amino-1-(2,4-dinitrophenyl)-4-(3-nitrophenyl)-1,4-dihydropyrano[2,3-c]pyrazole-
-carbonitrile (5f).
5

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Yellow solid, m.p. 283-286 °C.; Anal. Calc. for C H N O : C, 50.79; H, 2.47; N, 21.82; O,
1
9
11
7
7
-1
2
4.92%. Found- C, 51.24; H, 2.19; N, 21.32%.; IR vmax (KBr) cm : 3316 (N-H ), 3111 (C-
str
1
HAromatic,str), 1619 (C=C ), 2214 (-CN), 1030 (C-O ), 1513, 1334 (N-O ).; H NMR: (400
str
str
str
MHz, DMSO): δ; 10.124 (s, 1H), 9.582-9.571 (d, J=4.4 Hz1H), 8.969 (s, 2H), 8.590-8.578
d, J = 4.8 Hz, 1H), 8.504-8.496 (d, J = 3.2 Hz, 2H), 7.946 (s, 1H), 7.877-7.864 (d, J = 5.2
(
1
3
Hz, 2H), 4.681 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 175.79. 151.14, 146.02, 145.82,
1
1
42.04, 140.60, 137.58, 136.43, 135.29, 133.82, 126.90, 123.73, 121.34, 120.15, 120.06,
+
19.83, 117.01, 59.42, 26.38; ESI-MS: m/z Calculated 449.33, found [m/z] [M+H] 450.1.
2
.1.2.9. 6-amino-1-(2,4-dinitrophenyl)-4-(2-nitrophenyl)-1,4-dihydropyrano[2,3-c]pyrazole-
-carbonitrile (5g).
5
Yellow solid, m.p. 283-286 °C.; Anal. Calc. for C H N O : C, 50.79; H, 2.47; N, 21.82; O,
1
9
11
7
7
-1
2
4.92%. Found- C, 50.19; H, 2.44; N, 21.17%.; IR vmax (KBr) cm : 3313 (N-H ), 2978 (C-
str
1
HAromatic,str), 1597 (C=C ), 2349 (-CN), 1088 (C-O ), 1515, 1345 (N-O ).; H NMR: (400
str
str
str
MHz, DMSO): δ 9.628 (s, 1H), 9.064-9.053 (d, J = 4.4 Hz, 1H), 8.8669 (s, 2H), 8.179-8.165
;
(d, J = 5.6 Hz, 1H), 8.342-8.312 (d, J = 12 Hz, 1H), 7.854-7.833 (d, J = 8.4 Hz, 1H), 7.546
1
3
(
s, 1H), 7.530 (s, 1H), 7.461-7.455(d, J = 2.4 Hz, 1H), 4.982 (s, 1H).; C NMR: (100 MHz,
DMSO): δ; 176.28. 150.84, 150.05, 146.08, 141.62, 140.32, 137.82, 134.57, 133.34, 128.68,
1
27.10, 126.34, 123.93, 123.61, 119.83, 117.01, 116.30, 59.63, 25.67; ESI-MS: m/z
+
Calculated 449.33, found [m/z] [M+H] 450.1.
2
.1.2.10. 6-amino-1-(2,4-dinitrophenyl)-4-(furan-3-yl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carbonitrile (5h).
Dark Red solid, m.p. 263-268 °C.; Anal. Calc. for C H N O : C, 51.78; H, 2.56; N, 21.31;
17
10
6
6
-1
O, 24.35%. Found- C, 50.91; H, 2.39; N, 21.67%.; IR vmax (KBr) cm : 3330 (N-H ), 3121
str
1
(
C-HAromatic,str), 1625 (C=C ), 2211 (-CN), 1180 (C-O ), 1553, 1349 (N-O ).; H NMR:
str
str
str

ACCEPTED MANUSCRIPT
(400 MHz, DMSO): δ; 9.561 (s, 1H), 8.884-8.875 (d, J = 3.6 Hz, 1H), 8.585 (s, 2H), 8.382-
8
4
1
1
.371 (s, J = 4.4 Hz, 1H), 7.676 (s, 1H), 7.340-7.328 (d, J = 4.8 Hz, 2H), 6.453-6.442 (d, J =
1
3
.4 Hz, 1H), 4.720 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 175.14, 152.24, 148.07,
43.58, 143.29, 141.06, 140.47, 137.01, 127.68, 124.78, 120.09, 119.81, 118.97, 118.00,
+
09.71, 59.47, 22.59; ESI-MS: m/z Calculated 394.30, found [m/z] [M+H] 395.2.
2
.1.2.11. 6-amino-1-(2,4-dinitrophenyl)-4-(pyridin-2-yl)-1,4-dihydropyrano[2,3-c]pyrazole-
-carbonitrile (5i).
5
Brownish Red Solid, m.p. 272-276 °C.; Anal. Calc. for C H N O : C, 53.34; H, 2.74; N,
1
8
11
7
5
-1
2
4.19; O, 19.74%. Found- C, 54.25; H, 2.34; N, 23.52%.; IR vmax (KBr) cm : 3063 (N-H ),
str
1
2
956 (C-HAromatic,str), 1628 (C=C ), 2798 (-CN), 1123 (C-O ), 1628, 1352 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ; 9.534 (s, 1H), 8.970 (s, 1H), 8.813 (d, 2H), 8.452 (s, 1H),
8
6
1
1
.180 (s, 1H), 7.821 (s, 1H), 7.590-7.582 (d, J = 3.2 Hz, 1H), 7.478-7.462 (d, J = 6.4 Hz, 1H),
1
3
.594 (s, 1H), 4.565 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 172.85, 156.40, 150.35,
48.54, 147.03, 141.76, 141.02, 137.81, 136.38, 128.57, 124.78, 123.39, 120.47, 120.02,
+
18.71, 117.50, 59.05, 27.81; ESI-MS: m/z Calculated 405.324, found [m/z] [M+H] 406.1.
2
.1.2.12. 6-amino-1-(2,4-dinitrophenyl)-4-(pyridin-4-yl)-1,4-dihydropyrano[2,3-c]pyrazole-
-carbonitrile (5j).
5
Red Solid, m.p. 272-275 °C.; Anal. Calc. for C H N O : C, 53.34; H, 2.74; N, 24.19; O,
1
8
11
7
5
-1
1
9.74%. Found- C, 53.28; H, 2.65; N, 23.75%.; IR vmax (KBr) cm : 3669 (N-H ), 2777 (C-
str
1
HAromatic,str), 1616 (C=C ), 2354 (-CN), 1031 (C-O ), 1533, 1359 (N-O ).; H NMR: (400
str
str
str
MHz, DMSO): δ; 9.865 (s, 1H), 8.946-8.933 (s, J = 5.2 Hz, 1H), 8.265 (s, 2H), 8.255-8.247
(s, J = 3.2 Hz, 1H), 7.953-7.943 (d, J = 4 Hz, 2H), 7.440-7.426(d, J = 5.6 Hz, 2H), 7.281 (s,
1
3
2
H), 4.968 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 150.35, 149.79, 146.79, 146.12,

ACCEPTED MANUSCRIPT
1
2
42.53, 140.58, 137.14, 127.83, 124.69, 124.27, 123.77, 120.35, 118.94, 118.06, 60.10,
+
8.90; ESI-MS: m/z Calculated 405.32, found [m/z] [M+H] 406.3.
2
.1.3.13. 6-amino-4-(4-(dimethylamino)phenyl)-1-(2,4-dinitrophenyl)-1,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5k).
Brownish green solid, m.p. 275-279 °C.; Anal. Calc. for C H N O : C, 56.38; H, 3.83; N,
2
1
17
7
5
-1
2
1.91; O, 17.88%. Found- C, 56.15; H, 3.11; N, 20.57%.; IR vmax (KBr) cm : 3324 (N-H ),
str
1
2
986 (C-HAromatic,str), 1692 (C=C ), 2208 (-CN), 1187 (C-O ), 1522, 1356 (N-O ). H
str
str
str
NMR: (400 MHz, DMSO): δ 9.513 (s, 1H), 8.834-8.824 (s, J = 4 Hz, 1H), 8.413 (s, 2H),
;
8
.285-8.278 (d, J = 2.8 Hz, 1H), 7.880 (s, 1H), 7.385-7.374 (d, J = 4.4 Hz, 2H), 6.794-6.782
13
(
d, J = 4.8 Hz, 2H), 4.571 (s, 1H) 2.9335 (s, 6H).; C NMR: (100 MHz, DMSO): δ; 175.69.
1
1
4
51.12, 150.06, 145.67, 142.20, 139.92, 138.29, 129.11, 128.76, 127.08, 124.81, 124.51,
20.34, 117.70, 116.64, 112.83, 112.09, 60.49, 42.76, 42.7, 29.38; ESI-MS: m/z Calculated
+
47.40, found [m/z] [M+H] 448.3.
2
.1.2.14. 6-amino-1-(2,4-dinitrophenyl)-4-(3,4,5-trimethoxyphenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5l).
Light orange solid, m.p. 296-298 °C.; Anal. Calc. for C H N O : C, 53.44; H, 3.67; N,
2
2
18
6
8
-1
1
7.00; O, 25.89. Found- C, 53.20; H, 3.27; N, 17.13%.; IR vmax (KBr) cm : 3312 (N-H ),
str
1
3
098 (C-HAromatic,str), 1621 (C=C ), 2312 (-CN), 1135 (C-O ), 1512, 1335 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.328 (s, 1H), 8.871-8.846 (d, J = 10 Hz, 1H), 8.364 (s, 2H),
;
7
1
1
1
4
.952 (s, 1H), 6.748-6.720 (d, J = 11.2 Hz, 1H), 6.671-6.654 (d, J = 6.8 Hz, 1H), 4.483 (s,
1
3
H), 3.589 (s, 9H).; C NMR: (100 MHz, DMSO): δ; 175.19. 151.75, 151.50, 150.18,
43.93, 142.76, 138.92, 125.68, 123.53, 121.13, 117.86, 116.37, 115.40, 114.34, 112.59,
+
12.21, 60.37, 54.76, 53.87, 29.21; ESI-MS: m/z Calculated 494.41, found [m/z] [M+H]
95.2.

ACCEPTED MANUSCRIPT
2
.1.2.15. 6-amino-1-(2,4-dinitrophenyl)-4-(4-fluorophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5m).
Dark Brown solid, m.p. 254-257 °C.; Anal. Calc. for C H N O F : C, 54.03; H, 2.63; F,
1
9
11
6
5
-1
4
.50; N, 19.90; O, 18.94 %; found C, 54.32; H, 2.14; N, 19.02%; IR vmax (KBr) cm : 3325
(
N-H ), 3036-2028 (C-H
), 1615 (C=C ), 2230 (-CN), 1163 (C-O ), 1511, 1377 (N-
Aromatic str str
str
1
O ); H NMR: (400 MHz, DMSO): δ 10.034 (s, 1H), 9.358 - 9.341 (d, J = 6.8 Hz, 1H),
str
9
2
1
1
.039 (s, 2H), 8.649 - 8.635 (d, J = 5.6 Hz, 1H), 7.967 (s, 1H), 7.161 - 7.140 (d, J = 8.4 Hz,
1
3
H), 7.051 - 7.036 (d, J = 6 Hz, 2H), 4.562 (s, 1H); C NMR: (100 MHz, DMSO): δ 169.95,
58.82, 150.74, 145.90, 142.33, 140.39, 138.86, 130.84, 130.31, 129.38, 128.07, 124.19,
21.32, 118.81, 118.04, 116.41, 115.80, 59.95. 27.93; ESI-MS: m/z Calculated 422.33, found
+
[
m/z] [M+H] 423.5.
2
.1.2.16. 6-amino-4-(4-chlorophenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5n).
Brown solid, m.p. 260-265 °C.; Anal. Calc. for C H ClN O : C, 52.01; H, 2.53; Cl, 8.08; N,
1
9
11
6
5
-
1
1
2
9.15; O, 18.23%. Found- C, 52.38; H, 2.26; N, 19.55%.; IR vmax (KBr) cm : 3317 (N-H ),
str
1
921 (C-HAromatic,str), 1615 (C=C ), 2209 (-CN), 1180 (C-O ), 1521, 1331 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 10.089 (s, 1H), 8.986-8.978 (s, J = 3.2 Hz, 1H), 8.692 (s, 2H),
;
8
.495-8.482 (s, J = 5.2 Hz, 1H), 7.989 (s, 1H), 7.692-7.654 (d, J = 15.2 Hz, 2H), 7.492-7.480
1
3
(
d, J = 4.8 Hz, 2H), 4.892 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 170.31, 150.88, 145.89,
1
1
4
42.67, 140.39, 138.30, 132.35, 131.27, 130.65, 130.51, 127.55, 125.81, 125.32, 125.06,
+
20.32, 118.21, 117.73, 59.48, 28.38; ESI-MS: m/z Calculated 438.78, found [m/z] [M+H]
39.4.
2
.1.2.17. 6-amino-4-(4-cyanophenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5o).

ACCEPTED MANUSCRIPT
Light yellow solid, m.p. 266-268 °C.; Anal. Calc. for C H N O : C, 55.95; H, 2.58; N,
2
0
11
7
5
-1
2
2.84; O, 18.63%. Found- C, 53.89; H, 2.68; N, 21.38%.; IR vmax (KBr) cm : 3443 (N-H ),
str
1
3
028 (C-HAromatic,str), 1696 (C=C ), 2324 (-CN), 1124 (C-O ), 1498, 1365 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.724 (s, 1H), 8.954 - 8.942 (d, 1H), 8.571 (s, 2H), 8.336 -
;
8
1
1
1
4
.321 (d, J = 6 Hz, 1H), 7.898 (s, 1H), 7.620-7.612 (d, J = 3.2 Hz, 2H), 7.589-7.560 (d, J =
1
3
1.6 Hz, 2H), 4.632 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 173.02, 152.02, 147.41,
42.30, 140.67, 137.39, 130.11, 129.76, 127.20, 124.17, 120.53, 118.95, 118.84, 117.91,
+
09.42, 109.13, 108.86, 59.40, 28.71; ESI-MS: m/z Calculated, 429.35 found [m/z] [M+H]
30.3.
2
.1.2.18. 6-amino-1-(2,4-dinitrophenyl)-4-(3-hydroxyphenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5p).
Dark brown solid, m.p. 258-260 °C.; Anal. Calc. For C H N O : C, 54.29; H, 2.88; N,
19
12
6
6
-1
1
9.99; O, 22.84%. Found- C, 54.75; H, 2.16; N, 19.34%.; IR vmax (KBr) cm : 3313 (N-H ),
str
1
3
103 (C-HAromatic,str), 1620 (C=C ), 2313 (-CN), 1085 (C-O ), 1514, 1368 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ (ppm) 9.859 (s, 1H), 9.158-9.135 (d, J = 9.2 Hz, 1H), 8.859 (s,
H
2
H), 8.372-8.356 (d, J = 6.4 Hz, 1H), 7.759 (s, 1H), 7.251-7.236 (d, J = 6 Hz, 2H), 7.047 (s,
13
1
H), 6.939-6.921 (d, J = 7.2 Hz, 1H), 5.051 (s, 1H), 4.667 (s, 1H).; C NMR: (100 MHz,
DMSO): δ; 171.83, 155.74, 150.62, 146.71, 142.10, 139.90, 137.52, 136.71, 129.87, 127.35,
1
4
24.17, 122.05, 120.32, 117.58, 113.43, 112.18, 59.52, 28.82; ESI-MS: m/z Calculated
+
20.34, found [m/z] [M+H] 421.1.
2
.1.2.19. 6-amino-4-(4-bromophenyl)-1-(2,4-dinitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5q).
Brown solid, m.p. 273-276 °C.; Anal. Calc. for C H BrN O : C, 47.22; H, 2.29; Br, 16.54;
1
9
11
6
5
-1
N, 17.39; O, 16.55%. Found- C, 48.42; H, 2.64; N, 17.11%.; IR vmax (KBr) cm : 3314 (N-

ACCEPTED MANUSCRIPT
1
H ), 2938 (C-H
str
), 1596 (C=C ), 2350 (-CN), 1126 (C-O ), 1506, 1346 (N-O ).; H
str str str
Aromatic,str
NMR: (400 MHz, DMSO): δ (ppm) 9.694 (s, 1H), 8.869-8.836 (d, J = 13.2 Hz, 1H), 8.574
H
(s, 2H), 8.352-8.330 (d, J = 8.8 Hz, 1H), 7.895 (s, 1H), 7.621-7.594 (d, J 10.8 = , 2H), 7.047
1
3
(
s, 1H), 7.320-7.304 (d, J = 6.4 Hz, 2H), 4.835 (s, 1H). C NMR: (100 MHz, DMSO): δ;
1
1
4
70.56. 151.67, 146.38, 143.16, 140.32, 138.04, 134.09, 132.70, 131.61, 131.27, 130.74,
27.36, 124.15, 120.48, 120.28, 118.43, 117.59, 59.87, 29.09; ESI-MS: m/z Calculated
+
83.23, found [m/z] [M+H] 483.5.
2
.1.2.20. 6-amino-4-(5-bromo-2-hydroxyphenyl)-1-(2,4-dinitrophenyl)-
,4dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5r).
1
Orange solid, m.p. 288-293 °C.; Anal. Calc. for C H BrN O : C, 45.71; H, 2.22; Br, 16.01;
1
9
11
6
6
-1
N, 16.83; O, 19.23%. Found- C, 45.22; H, 2.68; N, 16.20%.; IR vmax (KBr) cm : 3387 (N-
1
H ), 2803 (C-H
str
), 1598 (C=C ), 2214 (-CN), 1127 (C-O ), 1513, 1345 (N-O ).; H
Aromatic,str
str
str
str
NMR: (400 MHz, DMSO): δ 9.361 (s, 1H), 8.670-8.651 (s, J = 7.6 Hz, 1H), 8.576 (s, 2H),
;
8
.281-8.270 (s, J = 4.4 Hz, 1H), 8.148 (s, 1H), 7.873-7.861 (d, J = 4.8 Hz, 1H), 7.472-7.460
1
3
(
d, J = 4.8 Hz, 2H), 6.363 (s, 1H), 5.827 (s, 1H).; C NMR: (100 MHz, DMSO): δ; 172.24,
1
1
56.88, 151.23, 146.64, 142.21, 139.85, 137.22, 136.00, 126.94, 124.61, 122.45, 120.38,
19.55, 118.20, 117.58, 115.68, 114.79, 59.68, 26.76; ESI-MS: m/z Calculated 499.23, found
+
[
m/z] [M+H] 500.6.
2
.1.2.21. 6-amino-1-(2,4-dinitrophenyl)-4-(4-nitrophenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (5s).
Dark orange solid, m.p. 283-285 °C.; Anal. Calc. for C H N O : C, 50.79; H, 2.47; N,
1
9
11
7
7
-1
2
1.82; O, 24.92%. Found- C, 50.33; H, 2.29; N, 21.08%.; IR vmax (KBr) cm : 3281 (N-H ),
str
1
3
110 (C-HAromatic,str), 1612 (C=C ), 2314 (-CN), 1137 (C-O ), 1514, 1346 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ; 9.584 (s, 1H), 8.727-8.703 (d, J = 9.6 Hz, 1H), 8.538 (d, 2H),

ACCEPTED MANUSCRIPT
8
.371-8.359 (d, J = 4.8 Hz, 1H), 8.247 (s, 1H), 7.773-7.762 (d, J = 4.4 Hz, 2H), 7.658-7.629
1
3
(
d, J = 11.6 Hz, 2H), 4.594 (s, 1H).; C NMR: (100 MHz, DMSO): δc (ppm) 27.87, 59.62,
1
1
16.87, 118.03, 120.06, 123.79, 124.04, 124.91, 127.08, 130.22, 130.90, 136.83, 139.13,
40.60, 142.11, 145.95, 146.29, 151.37, 173.20.; ESI-MS: m/z Calculated 449.33, found
+
[
m/z] [M+H] 450.1.
2
.1.2.22. 6-amino-4-(3,4-dimethoxyphenyl)-1-(2,4-dinitrophenyl)-1,4-
dihydropyrano[2,3c]pyrazole-5-carbonitrile (5t).
Black solid, m.p. 286-289 °C.; Anal. Calc. for C H N O : C, 54.31; H, 3.47; N, 18.10; O,
21
16
6
7
-1
2
4.12%. Found- C, 54.62; H, 3.27; N, 17.86%.; IR vmax (KBr) cm : 3315 (N-H ), 2975 (C-
str
1
HAromatic,str), 1615 (C=C ), 2215 (-CN), 1085 (C-O ), 1511, 1379 (N-O ).; H NMR: (400
str
str
str
MHz, DMSO): δ; 9.580 (s, 1H), 8.784-8.772 (s, J = 4.8 Hz, 1H), 8.264 (s, 2H), 7.97-7.963 (s,
J = 2.8 Hz, 1H), 7.579 (s, 1H), 7.242-7.231 (s, J = 4.4 Hz, 1H), 6.817-6.790 (d, J = 10.8 Hz,
1
3
2
1
1
H), 4.861 (s, 1H), 3.871 (s, 6H).; C NMR: (100 MHz, DMSO): δ; 172.22, 151.12, 150.67,
46.24, 145.92, 142.29, 140.40, 136.87, 128.24, 128.07, 124.20, 122.31, 120.42, 117.92,
17.31, 113.12, 112.83, 60.09, 56.67, 55.82, 28.71; ESI-MS: m/z Calculated 464.39, found
+
[
m/z] [M+H] 465.2.
2
.1.2.23. 6-amino-1-(2,4-dinitrophenyl)-4-(4-hydroxy-3-methoxyphenyl)-1,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5u).
Reddish brown solid, m.p. 278-283 °C.; Anal. Calc. for C H N O : C, 53.34; H, 3.13; N,
2
0
14
6
7
-1
1
8.66; O, 24.87% Found- C, 52.74; H, 3.42; N, 18.17%.; IR vmax (KBr) cm : 3313 (N-H ),
str
1
3
075 (C-HAromatic,str), 1622 (C=C ), 2314 (-CN), 1134 (C-O ), 1514, 1366 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.428 (s, 1H), 8.965-8.952 (d, J = 5.2 Hz, 1H), 8.689 (s, 2H),
;
8
.224-8.204 (d, J = 8 Hz, 1H), 7.847 (s, 1H), 7.042-7.037 (d, J = 2 Hz, 1H), 6.748-6.725 (d, J
1
3
=
9.2 Hz, 2H), 4.865 (s, 1H), 4.834 (s, 1H), 3.674 (s, 3H).; C NMR: (100 MHz, DMSO): δ;

ACCEPTED MANUSCRIPT
1
1
71.22, 152.14, 148.05, 146.18, 145.23, 142.20, 140.00, 136.71, 129.04, 127.10, 124.26,
22.19, 120.21, 118.09, 117.66, 114.79, 112.82, 59.48, 55.91, 28.59; ESI-MS: m/z
+
Calculated 450.36, found [m/z] [M+H] 451.6.
2
.1.3. Synthesis of 6-amino-1-(2,4-dinitrophenyl)-4-(4-(piperidin-1-yl)phenyl)-1,4-
dihydropyrano[2,3-c]pyrazole-5-carbonitrile derivatives (7a-7b):
An equimolar mixture of 2, 4-dinitrophenyl hydrazine (1) (1.0 eq.), ethyl acetoacetate
(2) (1.0 eq.), malononitrile (3) (1.0 eq.) and aldehyde derivatives (6a-6b) (1.0 eq.) were taken
into 50 ml FBF (Flat Bottomed Flask) in the presence of SnCl (10 mol %) as a catalyst. The
2
reaction mixture was stirred at 80 °C for appropriate time mentioned in Table 1 and also
heated at 180 W for 10-20 min under microwave irradiation. The isolation of yield performed
same as 5a-5u to get compound 7a-7b.
2
.1.3.1. 6-amino-1-(2,4-dinitrophenyl)-4-(4-morpholinophenyl)-1,4-dihydropyrano[2,3-c]
pyrazole-5-carbonitrile (7a).
Dark brown solid, m.p. 302-304 °C.; Anal. Calc. for C H N O : C, 56.44; H, 3.91; N,
2
3
19
7
6
-
1
2
2
0.03; O, 19.61%. Found- C, 55.76; H, 3.82; N, 20.34%.; IR vmax (KBr) cm : 3284 (N-H ),
str
1
804 (C-HAromatic,str), 1599 (C=C ), 2216 (-CN), 1060 (C-O ), 1469, 1381 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.921 (s, 1H), 8.854 (s, 2H), 8.630 (s, 1H), 7.834-7.811 (d, J =
;
9
.2 Hz, 1H), 7.756-7.738 (d, J = 7.2 Hz, 1H), 7.223-7.201 (d, J = 8.8 Hz, 2H), 7.051-7.031
(d, J = 8 Hz, 2H), 4.131 (s, 1H), 3.818-3.806 (t, J = 4.8 Hz, 4H), 3.402-3.364 (t, J = 15.2 Hz,
1
3
4
1
6
H).; C NMR: (100 MHz, DMSO): δ; 169.08. 151.80, 149.47, 144.49, 144.41, 141.54,
37.11, 129.41, 129.07, 128.11, 124.27, 123.80, 121.92, 116.63, 115.90, 112.19, 111.94,
5.87, 64.90, 60.64, 54.34, 53.20, 29.01; ESI-MS: m/z Calculated 489.44, found [m/z]
+
[
M+H] 490.3.

ACCEPTED MANUSCRIPT
2
.1.3.2. 6-amino-1-(2,4-dinitrophenyl)-4-(4-(piperidin-1-yl)phenyl)-1,4-dihydropyrano[2,3-
c]pyrazole-5-carbonitrile (7b).
Brownish orange solid, m.p. 298-301 °C.; Anal. Calc. for C H N O : C, 59.13; H, 4.34; N,
2
4
21
7
5
-
1
2
2
0.11; O, 16.41%. Found- C, 59.31; H, 4.08; N, 20.65%.; IR vmax (KBr) cm : 3140 (N-H ),
str
1
989 (C-HAromatic,str), 1641 (C=C ), 2316 (-CN), 1174 (C-O ), 1544, 1383 (N-O ).; H
str
str
str
NMR: (400 MHz, DMSO): δ 9.454 (s, 1H), 8.821-8.794 (d, J = 10.8 Hz, 1H), 8.554 (s, 2H),
;
8
.046-8.022 (d, J = 9.6 Hz, 1H), 7.590 (s, 1H), 7.069-7.046 (d, J = 9.2 Hz, 2H), 7.000-6.978
1
3
(
d, J = 8.8 Hz, 2H), 4.153 (s, 1H), 3.321-3.308 (t, J = 5.2 Hz, 4H), 1.494-1.395(m, 6H).; C
NMR: (100 MHz, DMSO): δ; 174.59, 153.09, 151.64, 148.05, 143.29, 138.75, 135.83,
1
5
4
30.16, 129.85, 128.07, 124.15, 123.81, 121.01, 119.13, 116.89, 113.00, 112.87, 60.24,
+
4.06, 53.64, 25.07, 24.91, 23.90; ESI-MS: m/z Calculated 487.47, found [m/z ] [M+H]
88.4.
2
5
.1.4. Synthesis of 6-amino-1-isonicotinoyl-4-phenyl-1, 4-dihydropyrano [2, 3-c] pyrazole-
-carbonitrile derivative (8a):
An equimolar mixture of isonicotinohydrazide (1a) (1.0 eq.), ethyl acetoacetate (2)
(1.0 eq.), malononitrile (3) (1.0 eq.) and aldehyde derivatives (4a) (1.0 eq.) were taken in 50
ml FBF (Flat Bottomed Flask) in the presence of SnCl2 (10 mol %) as a catalyst. The
isolation method of the product yield is same as 5a-5u to obtain compound 6-amino-1-
isonicotinoyl-4-phenyl-1, 4-dihydropyrano [2, 3-c] pyrazole-5-carbonitrile 8a.
2
.1.4.1.
6-amino-1-isonicotinoyl-4-(4-nitrophenyl)-1,4-dihydropyrano[2,3-c]pyrazole-5-
carbonitrile (8a).
Light yellow solid, m.p. 268-270 °C.; Anal. Calc. for C H N O : C, 58.76; H, 3.11; N,
1
9
12
6
4
-1
2
1.64; O, 16.48%. Found- C, 58.25; H, 3.07; N, 21.38%.; IR vmax (KBr) cm : 3186 (N-H ),
str
1
3
001 (C-HAromatic,str), 1685 (C=C ), 2205 (-CN), 1142 (C-O ), 1560 (N-O ).; H NMR:
str
str
str

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(400 MHz, DMSO): δ; 8.815-8.797 (d, 2H), 7.957 (s, 2H), 7.650-7.638 (d, J = 4.8 Hz, 2H),
7
1
1
2
.479-7.458 (d, J = 8.4 Hz, 2H), 6.534-6.512 (d, J = 8.8 Hz, 2H), 6.492 (s, 1H), 4.526 (s,
1
3
H).; C NMR: (100 MHz, DMSO): δ; 174.82. 167.02, 157.34, 152.01, 150.67, 142.25,
41.03, 131.68, 130.09, 129.57, 124.02, 123.83, 123.04, 122.34, 117.86, 117.02, 60.62,
+
9.18; ESI-MS: m/z Calculated 388.34, found [m/z] [M+H] 389.2.
2
.2 Biological assay
2
.2.1 In vitro anti-bacterial activity
The in vitro anti-bacterial activity of synthesized compounds were performed against
four different bacterial strains (E. coli MTCC 443, P. aeruginosa MTCC 1688, S. aureus
MTCC 96, S. pyogenus MTCC 442) and compered with standard drugs Ampicillin,
Chloramphenicol and Ciprofloxacin. This activity was performed in accordance with method
in Patel et al. and Mungra et al. The highest dilution showing at least 99% inhibition has
taken as MIC value [53-62].
2
.2.2 In vitro anti-tuberculosis activity and MDR-TB study
The anti-tuberculosis screening of the synthesized compounds was carried out using
method describe by Patel et al. and Mungra et al. [53-62]. The MIC of test compound was
taken as < 20 colonies or no development of colonies occurred at the concentration. The
M. tuberculosis H Rv strain was tested with standard drug isoniazid for the comparison
3
7
intense. The multi-drug resistance tuberculosis activity of the synthesized compounds
against M. tuberculosis H Rv. (MDR-TB strain was collected from National Institute for
3
7
Research in Tuberculosis, Chennai, Tamil Nadu, and India). (The strain was identified by
phenotype and genotype).
2
.2.3 Cytotoxicity assay protocol

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Human cervical cancer cell line (HeLa) were used for in vitro cytotoxicity study of
the synthesized compounds 7a, 8a, 5a and 5r. The cytotoxic activity protocol was used
according method used in Desai et al. [63].
2
.3 Computational Study
2
.3.1 ADMET property prediction
A set of ADMET-related properties of the synthesized compounds were predicted by
using Qikprop programs (Schrödinger, LLC, New York, NY, 2015) [64, 65]. LigPrep module
were used to prepare the compounds and utilized for the calculation of pharmacokinetic
parameters by QikProp module. The program QikProp generates physically relevant
descriptors, and uses them to perform ADMET predictions and utilizes the method of
Jorgensen [67] to calculate pharmacokinetic properties and descriptors.
2
.3.2 Molecular Docking
The potential binding mode and the binding interaction of the ligand with enoyl-ACP
reductase (oxidoreductase) have been investigated by using Maestro 10.7 Schrödinger, LLC,
New York, NY, 2012. The 3D structures of all the compounds were obtained using Marvin
suites and saved as SDF file. The 3D crystal structures of enoyl-ACP reductase
(oxidoreductase) from Mycobacterium tuberculosis (PDB ID: 5MTQ; resolution 2.6cÅ) were
retrieved from the protein data bank (www.rcsb.org). The protein (5MTQ) was choose based
on structure similarity of ligands, resolution and expression system. Morevere, The protein
organism is Mycobacterium Tubercolisis (H Rv Strain) which is encourage us to do the
3
7
docking in the active site of this protein. The ligand with the lowest energy, correct Lewis
structure, tautomers and ionization states (pH 7.0±2.0) for each of the ligands were generated
and optimized with default settings were prepared for molecular docking using Ligprep

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module of Schrödinger. The OPLS_2005 [65] force field were used for computing partial
atomic charges. The proteins were prepared for docking using Protein Preparation Wizard
module in Maestro. Bond order and formal charges were assigned and hydrogen atoms were
added to the crystal structure. Further to refine the structure OPLS-2005 force field parameter
was used to alleviate steric clashes. The receptor grid, which was generated using Glide 5.8
(Maestro, Schrödinger, LLC, New York, NY, 2012) with default settings for all parameters.
An approximately large grid size was preferred to include all active site residues involved in
substrate binding. The Glide extra-standard precision (XP) mode was used for docking of the
generated receptor grid file along with all prepared ligand conformers. Default settings were
retained for the scoring and refinement.
2
.3.4 Molecular Dynamics
The natural dynamics on different timescales of docked complex of compound 7b and
protein, thermal average of the molecular properties of complex is carried with the help of
molecular dynamics (MD) stimulation. MD simulations have been conducted with Desmond
[
65-67] program, as implemented Schrödinger Materials Science Suite 2015-4 [68].
OPLS_2005 [65] force field with NPT ensemble class have been used, while pressure and
temperature were set to 1.0325 bar and 300 K, respectively. The system was modelled by
placing one NATPB (5-[(4-nitrophenyl)acetamido]-2-(4-tert-butylphenyl)benzoxazole)
molecule into the cubic box with around 3000 water molecules and the simulation time was
set to 10 ns cut off radius was set to 12 Å, pressure to 1.0325 bar, while temperature was set
to 300 K. In all cases when Desmond was used, input and output files were manipulated by
Maestro graphical user interface application of Schrödinger Materials Science Suite 2015-4.
3
. Results and Discussion
3
.1 Chemistry

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In the present study. We have synthesized a series of novel compounds based on
pyrano[2,3-c]pyrazole (5a-5u, 7a-7b and 8a) which was illustrated in Scheme 1-3. The
synthesis of the series of heterocyclic scaffold performed using MCR methodology following
direct addition of the substituted aldehyde derivative (4a-4u) with the corresponding 2, 4-
dinitrophenyl hydrazine (1), ethyl acetoacetate (2) and malononitrile (3) in the presence of
SnCl using Methanol as solvent under microwave irradiation and conventional method
2
(Scheme 2-3). Targeted compound 6-amino-1-isonicotinoyl-4-phenyl-1, 4-dihydropyrano [2,
3
-c] pyrazole-5-carbonitrile (8a) (Scheme 3) was synthesised using substituted aldehyde
derivative (4a-4u), isonicotinohydrazide (1a), ethyl acetoacetate (2) and malononitrile (3) in
the same condition as a 5a-5u. Whereas, to synthesise compound 7a-7b, a 4- substituted
aldehyde derivatives were initially synthesized (6a-6b) (Scheme 1) via condensation of 4-
fluoro aldehyde, piperidine and morpholine in the presence of anhydrous potassium carbonate
using Methanol as solvent.
From the current literature survey, we concluded that many researcher have reported
desired product formation with lower product yields and longer hours reaction time by using
different solvent and catalyst for the synthesis of 1,4-dihydropyrano[2,3-c]pyrazole
derivatives [35-45, 69]. Despite this challenge, we have optimized the reaction conditions
using conventional and microwave irradiation to find out the superior method for the
synthesis of targeted compound 5a with higher yield and shorter reaction time. In both the
conditions, we first performed a trial reaction with equivalent mole of benzaldehyde,
malononitrile and 2,4-dinitrophenyl hydrazine in the presence of SnCl using water,
2
Methanol, ethanol as solvent and without solvent at various temperature (Table 1). In the
conventional method, Compound 5a with yield of 67% in 4.2 hrs was obtained when the
reaction was performed without catalyst at 80 ºC temperature. While, when the reaction was
performed in Methanol at 80 °C temperature by using SnCl as a catalyst the yield and time
2

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of product 5a could be improved to 80 % in 1.4 hours. However, the targeted product 5a was
synthesized in 25 min with 88 % of yield in microwave irradiation method, where Methanol
was used as a solvent in the presence of SnCl as a catalyst at 180 W (140 °C) temperature.
2
Most of the compounds were synthesized using neat condition in the microwave irradiation
method. However, we have also used neat condition while synthesizing compounds via
conventional method but the yield of the reaction was quite low as compared to in Methanol.
The synthesized compounds are enantiomeric mixture there is no synthetic selectivity. The
yield of compound 5a (80 % by conventional method and 88 % by microwave method)
synthesized by our method was significantly higher, than the previously reported protocols
[
35-45, 69].
All the synthesized compounds 5a-5u, 7a-7b, 8a were characterized by various
1
13
spectroscopic methods such as H NMR, C NMR, IR spectra, Mass analysis and elemental
analysis. We have confirmed the synthesised compound 5a from IR analysis. IR band at 3323
-1
and 3106 cm which confirmed the presence of N-H
and aromatic character (C-Hstretch)
stretch
-1
in the moiety respectively. When -CN group showed vibration peak at 2249 cm , while C-
-1
Ostretch showed peak at band at 1023 cm confirm the formation of pyran ring in molecule.
1
From the H NMR data of compound 5a, The aromatic protons were resonated at 9.642,
8
.936-8.921 δ ppm (Nitro containing aromatic ring) and other benzene (Aromatic) ring
showed peak at 7.572-7.567, 7.358 and 7.317-7.313 δ ppm. The -NH group resonated at
2
8
.471 δ ppm. The chiral carbon containing proton resonated at 4.656 δ ppm which also
13
supported to synthesised compound 5a. Also C-NMR gave full structural conformation of
synthesised compound. The peaks at 27.93, 60.18 and 174.91 δ ppm correspond to cyclised
chiral carbon, –C-CN containing carbon and -NH containing carbon respectively which gave
2
full confirmation of 5a.The mass spectra of compound 5a showed molecular ion peak at m/z
=
404.2 (M+H) which support to the structure of compound 5a.

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3
.2 Biology
3
.2.1 In vitro anti-bacterial activity
A series of synthesized compounds were evaluated for their anti-bacterial activity
against different bacterial stain such as E. coli, MTCC 443, P. aeruginosa MTCC 1688, S.
aureus MTCC 96, S. pyogenus MTCC 442 as compared to the standard drug. The activity
data were summarized in Table 2. From the bioassay, it can be stated that the compound 8a
which was hybridised molecule of isoniazid with a pyran ring showed significant
antibacterial activity against E. coli and P. aeruginosa at MIC value 12.5 µg/mL. Analogue
5
a with high lipophilicity (C log P = 4.1319) exhibited excellent antibacterial activity against
E. coli and P. aeruginosa at MIC value 12.5 µg/mL and 6.25 µg/mL respectively. In addition,
Compound 5l which contained two methoxy groups showed good activity against E. coli and
P. Aeruginosa at MIC value 12.5 µg/mL and 25 µg/mL correspondingly. Compound 5n (C
log P = 4.7019) and 7a (C log P =3.4449) with higher lipophilicity displayed higher MIC
value 12.5 µg/mL, 12.5 µg/mL, respectively amongst all the active compounds when tested
against S. pyogenus. Moreover, analogue 5b and 7a (MIC value 25 µg/mL) shows better
inhibitory effect against S. aureus. Based on the results, it can be stated that in case on
inhibition of the bacterial strain, higher inhibiting efficiency of compounds comes with
higher lipophilicity than with lower lipophilicity. Remaining compounds exhibited
comparatively superior antibacterial activity against all the tested bacterial strains in
comparison to the standard drugs Ampicillin, Chloramphenicol and Ciprofloxacin. The C log
P values (P is the partition coefficient) of all the synthesised compounds as well as standard
drugs are mentioned in Table 2. Overall, all the results showed that the MIC value less than
that of standard drugs were considered as promising anti-bacterial agent.
3
.2.3 In vitro anti-tuberculosis activity and MDR-TB study

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The promising results obtained from the antibacterial activity encouraged us to go for
further biological screening such as in vitro anti-tubercular activity. In vitro anti-tuberculosis
activity of all the newly synthesized compounds was investigated against Mycobacterium
tuberculosis H Rv strain by Lowenstein-Jensen method and the corresponding results are
3
7
shown in Table 2. The result showed that analogue 8a enhance good activity (25 µg/mL) due
to the presence of isoniazid moiety instead of 2, 4-dinitrophenyl hydrazine presented in rest
molecules. In addition, compound 7a and 5r showed 25 µg/mL of MIC against mycobacterial
strain.
Moreover, Compound 5u showed moderate activity against H Rv strain with MIC
3
7
value 50 µg/mL. All the remaining compounds were found to display reasonable activity at
MIC ranging from 50 to 500 µg/mL. Further, the compounds were screened against MDR-TB
(First-line anti-TB drug resistance) in clinical strains. From the data (Table 2) of activity
against MDR strain it was observed that only compound 7a demonstrate moderate activity at
MIC value 50 µg/mL. The remaining result of tested compounds against MDR-TB strain
were comparatively poor. The lower effectiveness of synthesized compounds against strain
not being adapted to laboratory environments and different media, which may affect their
apparent susceptibility to certain inhibitors and they exhibit poor effect against strain in vitro
resulted in the poor activity.
3
.2.4 Cytotoxicity assay
In vitro cytotoxic activity of synthesized compounds 5a, 5r, 7a and 8a was evaluated
against HeLa cell line (human cervical cancer) by the MTT colorimetric assay [70]. The
result are summarized in table 3. Percentage cell viability of HeLa cell line at various
concentrations for these compounds are shown in figure 2. It was observed that there is no
any remarkable cytotoxic effect on HeLa cell line, Perticulery the tested compound 7a. This

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activity suggest that these compounds have good proficiency for their future in vivo activity
for antimicrobial agents.
3
.3 Computational Study
3
.3.1 ADMET property prediction
In silico, pharmacokinetic properties leading to drug-likeness of the synthesized
compounds were predicted using Qikprop module (Schrodinger, LLC, New York, NY, 2015).
The predicted important pharmacokinetic properties of synthesized compounds along with
their acceptable limits are showed in Table 4 as compared with the reference compounds.
Most of the compounds were found to have good bioavailability except 5s. One of the
important factor to be studied in concern with the absorption of the drug molecule is
intestinal absorption, which was ascertained by Caco-2 cell permeability (QPPCaco)
prediction. The synthesized compound shows moderate results predicting intestinal
absorption in Caco-2 cell permeability. Further, The Qikprop descriptor for blood/brain
partition coefficient QPlogBB showed reliable prediction for the synthesized compounds and
reference drugs.
The predicted results for QPlogkhsa descriptor of Qikprop indicating the predicted
values of human serum albumin binding indicated that molecules were found to in the
+
permissible range (-1.5-1.5). The active molecules were accessed for IC value of HERG K
5
0
channel blockage prediction, which indicated that the predicted values are in the acceptable
range (<-5) as compared to the standard reference entities. The aqueous solubility parameter
(QPlog S) of the synthesized compounds were assessed and the compounds were found to be
falling out of the acceptable range (-6.5-0.5) signifying incomplete solubility of the
synthesized compounds which can be further improved using solubility enhancers.
3
.3.2 Molecular docking

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Due to lack of enzymatic study, we decided to perform in silico computational study
of the synthesized compounds which provided more information that could be applied to
design new molecules with more potent biological activity. Therefore, Molecular docking
analysis was performed to obtain the detailed molecular interactions and to estimate the
binding affinity of the synthesized compounds with enoyl-ACP reductase (oxidoreductase)
(PDB ID: 5MTQ). Synthesized pyrano [2,3-c]-pyrazole derivatives and standard ligand
Isoniazid was docked into the active site of enoyl-ACP reductase (oxidoreductase). The
docking results of the ligands with enoyl-ACP reductase (oxidoreductase) as per the Glide
calculations are given in Table 5. The results of docking simulations demonstrated high
binding affinity with good interactions for critical residues present in enoyl-ACP reductase
(oxidoreductase). Synthesized compound 7b, standard compound isoniazid and isoniazid
+
with co-factor (NAD ) showed the catalytic site with binding energies of -10.19, -6.34 and -
+
6
.23 kcal/mol, respectively. The purpose of docking with INH-NAD is to compare the
+
docking result with INH because most of known INH inhibitors bind next to the NAD co-
factor. The docking pose of INH without co-factor and with co-factor is presented in figure 4.
As per the docking simulations, 7b was docked into enoyl-ACP reductase (oxidoreductase)
active site with -10.19 kcal/mol of binding energy higher than Isoniazid was seen to be
forming conventional hydrogen bond with Gln214 and Arg225, along with carbon hydrogen
bond with Pro156 which also show Pi-Cation interaction and Vai203, Met199, Phe149,
Leu218, Met155 shows alkyl and Pi-Alkyl interaction with amino acids of enoyl-ACP
reductase (oxidoreductase) in Figure 3a. When the docking snapshot of isoniazid in complex
with enoyl-ACP reductase (oxidoreductase) from Mycobacterium tuberculosis was analysed,
it was seen that it docked into the active site of enoyl-ACP reductase (oxidoreductase) with -
6
.34 kcal/mol of energy and found to be forming direct hydrogen bonds with Gly239;
Lys377, and Heme, along with hydrophobic interactions with Ile232; Leu384; Leu230;

ACCEPTED MANUSCRIPT
Leu234; Phe163; Phe226; Val350; Ala264; Ile354; Ala260; Phe291; and Pro241 of enzyme
in figure 3b.
Among 25 compounds analysed, all compounds were successfully docked with a
binding energy range of -10.19 to -2.39 kcal/mol. The compound 7b showed the highest
binding affinity of -10.19 kcal/mol of binding energy. The compound with highest binding
affinity, 7b was further analysed using molecular dynamic simulation to check the stability of
docked complex.
3
.3.3 Molecular Dynamics
Molecular Dynamics (MD) simulations were performed to analyse the stability,
conformational changes and underlying molecular interactions at atomic level of enoyl-ACP
reductase (oxidoreductase) with compound 7b at 10 ns. The MD simulations were carried out
within the given temperature, pressure and volume defined by simulation quality parameters.
The studies were performed in different standard simulation parameters: (i) Root mean square
deviation (RMSD), (ii) Root mean square fluctuations (RMSF), (iii) The Ligand Root Mean
Square Fluctuation, (iv) Protein-Ligand contacts; (v) total energy, and (vi) Ligand Torsion
Profile and properties. The main objective of MD simulation was to understand the dynamics
and stability of enoyl-ACP reductase (oxidoreductase) from Mycobacterium tuberculosis
protein and its interaction with compound 7b. The overall statistical data after the analysis of
the MD trajectories are explained with figure 5.
RMSF of the enoyl-ACP reductase (oxidoreductase) protein in complex with 7b
pattern of residue fluctuations were found to be similar from docked structure of protein-
ligand shown in Figure 3 and 5 (a). Protein depicted quite stable structure without lacking any
major interaction throughout the simulation period. Intra molecular hydrogen bonds were
analysed to understand the underlying forces better in relation to protein structure stability.
Protein rigidity is represented by increased intra molecular hydrogen bonds. This observation