Antiplatelet activity, molecular docking and QSAR study of novel N′-arylmethylidene-3-methyl-1-phenyl-6-p-chlorophenyl-1H-pyrazolo[3,4-b] pyridine-4-carbohydrazides

Article abstract of DOI:10.1007/s00044-017-2053-0

Abstract: A series of novel N′-arylmethylidene-3-methyl-1-phenyl-6-p-chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2a–2t) has been synthesized from hydrazide (1). The structures of newly synthesized compounds were confirmed by FT-IR, EI-MS, ¹H NMR and ¹³C NMR techniques. The title compounds were evaluated for antioxidant and antiplatelet aggregation effect induced by arachidonic acid (AA) and collagen. All the compounds have exhibited high antioxidant potential and antiplatelet activity but (2c, 2e, 2f, 2g, 2i, 2m, 2o and 2q) have revealed superlative antiplatelet activity. The molecular docking against cyclooxygenase-1 and 2 (COX-1 and COX-2) and quantitative structure-activity relationship (QSAR) were performed in describing their antiplatelet potential against AA and collagen along with antioxidant potential determined by ABTS, DPPH and iron chelating methods. The molecular docking study exhibited that compounds (2c, 2e, 2f, 2g, 2i, 2l, 2m, 2o and 2q) were found to be active against COX-1 while 2o compound also showed activity against COX-2. Compounds 2g and 2l were found to have higher energy stabilization values in comparison to Aspirin. Computational evaluations both molecular docking and QSAR are in good agreement with antiplatelet and antioxidant activities of the compounds (2a–2t). All the compounds especially 2g, 2l, 2m might be promising antiplatelet agents and might be helpful in the synthesis of new drugs for the treatment of cardiovascular and anti-inflammatory diseases. Graphical Abstract: [InlineMediaObject not available: see fulltext.].

Full text of DOI:10.1007/s00044-017-2053-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2053-0
ORIGINAL RESEARCH
Antiplatelet activity, molecular docking and QSAR study of novel
N′-arylmethylidene-3-methyl-1-phenyl-6-p-chlorophenyl-1H-
pyrazolo[3,4-b] pyridine-4-carbohydrazides
Ayesha Ramzan1 Sara Siddiqui1 Ahmad Irfan2,3 Abdullah G. Al-Sehemi2,3
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Aftab Ahmad4 Francis Verpoort5 Adeel H. Chughtai5,6 Misbahul A. Khan1,7
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Munawar A. Munawar1 Muhammad Asim Raza Basra1
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Received: 2 May 2017 / Accepted: 28 August 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of novel N′-arylmethylidene-3-methyl-1-
phenyl-6-p-chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-car-
bohydrazide (2a–2t) has been synthesized from hydrazide
(1). The structures of newly synthesized compounds were
confirmed by FT-IR, EI-MS, 1H NMR and 13C NMR
techniques. The title compounds were evaluated for
antioxidant and antiplatelet aggregation effect induced by
arachidonic acid (AA) and collagen. All the compounds
have exhibited high antioxidant potential and antiplatelet
activity but (2c, 2e, 2f, 2g, 2i, 2m, 2o and 2q) have revealed
superlative antiplatelet activity. The molecular docking
against cyclooxygenase-1 and 2 (COX-1 and COX-2) and
quantitative structure-activity relationship (QSAR) were
performed in describing their antiplatelet potential against
AA and collagen along with antioxidant potential deter-
mined by ABTS, DPPH and iron chelating methods. The
molecular docking study exhibited that compounds (2c, 2e,
2f, 2g, 2i, 2l, 2m, 2o and 2q) were found to be active
against COX-1 while 2o compound also showed activity
against COX-2. Compounds 2g and 2l were found to have
higher energy stabilization values in comparison to Aspirin.
Computational evaluations both molecular docking and
QSAR are in good agreement with antiplatelet and anti-
oxidant activities of the compounds (2a–2t). All the com-
pounds especially 2g, 2l, 2m might be promising
antiplatelet agents and might be helpful in the synthesis of
new drugs for the treatment of cardiovascular and anti-
inflammatory diseases.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2053-0) contains supplementary
material, which is available to authorized users.
* Misbahul A. Khan
misbahakhan@gmail.com
* Munawar A. Munawar
mamunawar.chem@pu.edu.pk
munawaralimunawar@yahoo.com
* Muhammad Asim Raza Basra
asimbasra@gmail.com
asimbasra.chem@pu.edu.pk
1
Institute of Chemistry, University of the Punjab, Lahore 54590,
Pakistan
2
Department of Chemistry, Faculty of Science, King Khalid
University, P.O. Box 9004, Abha 61413, Saudi Arabia
3
Research Center for Advanced Materials Science (RCAMS), King
Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia
4
Center of Excellence in Molecular Biology, University of the
Punjab, Lahore, Pakistan
5
Laboratory of Organometallics, Catalysis, and Ordered Materials,
State Key Laboratory of Advanced Technology for Materials
Synthesis and Processing, Wuhan University of Technology,
Wuhan 430070, China
6
Institute of Chemical Sciences, Bahauddin Zakariya University,
Multan 60800, Pakistan
7
Department of Chemistry, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan

Med Chem Res
Graphical Abstract
methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbo-
hydrazide has displayed excellent antiplatelet activity
(Geraldo et al. 2010).
Hydrazones belong to imperative and influential group of
compounds for useful novel and contemporary drug
development. Now a days researchers have prepared a
number of hydrazones and checked their various biological
activities (Singh and Raghav 2011). Computational
approaches have been broadly used to figure out and
anticipate the potential capabilities of enzyme, ligand and
their interactions. The leading computational methods
include pharmacophore, three-dimensional QSAR and SAR
analysis, molecular dynamics, homology modeling studies,
simple rule-based modeling, and molecular docking (Hutter
2009).
In the present study, we have made an attempt toward the
search of new potent COX inhibitors as antiplatelet drug
with improved efficacy, and performed a SAR study. We
synthesized a series of novel N′-benzylidene-3-methyl-1-
phenyl-6-p-chlorophenyl-1H-pyrazolo[3,4b]pyridine-4-car-
bohydrazides (2a–2t) and then evaluated their potential
antioxidant and antiplatelet activities. All the compounds
have exhibited high antioxidant potential and antiplatelet
activity. Docking studies were also performed against
COX-1 and COX-2, and QSAR analysis was used to find
out the binding interaction of synthesized compounds with
the receptor in comparison to the standard drug Aspirin, in
describing the antiplatelet potential of synthesized com-
pounds. The aim of this study was to design, synthesize and
investigate QSAR correlation along with molecular docking
in order to develop new potent and selective platelet anti-
aggregating drugs.
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Keywords Acyl Hydrazone Density Functional Theory
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Electrostatic Potential Physiochemical Properties
Introduction
Thrombotic disorders, e.g., myocardial infarction, periph-
eral vascular disease, stroke and angina are all caused by a
common primary factor, i.e., platelet adhesion and aggre-
gation, which played an important role in clot formation
(Reddy et al. 2011). Keeping in view, research intention has
been diverted toward selective inhibition of AA and col-
lagen by binding with interactive residue of COX pocket.
Many structure transformation of N′-acylhydrazones have
been done by a number of researchers to enhance the
antiplatelet activity (Haj Mohammad Ebrahim Tehrani et al.
2013).
Hydrazones have immense significance by virtue of their
disparate biological applications, such as antimicrobial,
antidiabetic, anticonvulsant, analgesic, antidepressant, anti-
inflammatory, antiplatelet, antimalarial, antimycobacterial,
vasodilator, anticancer, anti-HIV, and antiviral activities
(Abdel-Wahab et al. 2011; Abu-Surrah et al. 2010; Ajani
et al. 2010; Bezerra-Netto et al. 2006; de Aguiar Cordeiro
et al. 2016; Magda et al. 2011; Patole et al. 2003; Salgın-
Gökşen et al. 2007; Seleem et al. 2011; Sriram et al. 2005).
Some of hydrazones are reported as antichagasic agents
such as N′-(4-nitrobenzylidene)-3-methyl-1-phenyl-6-p-
hydroxyphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydra-
zide has exhibited highest antichagasic activity (Dias et al.
2007) while others are known for enzyme inhibition activ-
ities, (Abdelazem et al. 2015) still some are important in
displaying antiplatelet activity as N′-(benzylidene)-3-
Materials and methods
Chemistry
All chemicals were purchased from Sigma-Aldrich and
other well reputed suppliers. The chemicals were used
mostly as such however when required purified by standard
techniques, i.e., distillation and recrystallization. FT-IR
spectra were recorded on Agilent Technologies 41630 and
1H NMR, 13C NMR recorded on ACD/NMR processor
academic edition at 500 and 125 MHz in dimethyl sulfoxide
(DMSO), respectively. The EIMS Data were taken on JEOl
MS. 600H-1 while the completion of reaction was mon-
itored by silica gel 60 F254 TLC plate.
6-(4-chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]
pyridine-4-carbohydrazide (1) was prepared by the already
reported methods (Dias et al. 2007; Geraldo et al. 2010).

Med Chem Res
Synthesis of N′-arylmethylidene-3-methyl-1-phenyl-6-p-
chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4–carbohy-
drazides (2a–2t)
8.02ap (2 s, 1H, H-5), 7.88(sp + ap) (t, J = 7.5 Hz,1H, H-3′′′),
7.68(sp + ap) (d, J = 8.6 Hz 2H, H-3′′, H-5′′), 7.63(sp + ap) (d,
J = 7.4 Hz, 2H, H-3′, H-5′), 7.61ap, 7.53sp (m, 1H, H-4′′′),
7.41(sp + ap) (m, 1H, H-4′) 2.63(sp + ap) (s, 3H, CH3). Rota-
Targeted compounds (2a–2t) were synthesized by the
reaction of 6-(4-chlorophenyl)-3-methyl-1-phenyl-1H-pyr-
azolo[3,4-b]pyridine-4-carbohydrazide (1) (0.37 g, 1 mmol)
with substituted aromatic aldehydes (1 mmol), and heating
under reflux for an hour in glacial acetic acid (5 mL). The
precipitates formed during the reaction were filtered,
washed with cold ethanol, and finally dried in oven at
100 °C. Recrystallization from absolute ethanol gave the
product as yellow colored solids. Yield 25–98%. This
method provided novel, direct and flexible access to diverse
N′-arylmethylidene-3-methyl-1-phenyl-6-p-chlorophenyl-
1H-pyrazolo[3,4-b]pyridine-4–carbohydrazides (2a–2t).
mers percentage sp(73%): ap(27%).
13C NMR (125 MHz,
DMSO-d6): δ = 168.7ap, 162.3sp (C=O), 155.4 (C-6),151.4
(C-7a), 148.8(C-2′′′), 145.1 (N=CH), 142.7 (C-3), 139.3
(C-4), (138.8 C-1′), 137.0 (C-1′′), 135.6 (C-5′′′), 134.4
(C-4′′), 131.6 (C-4′′′), 129.8 (C-3′′, C-5′′), 129.7 (C-3′,
C-5′), 129.6 (C-6′′′), 129.5 (C-2′′, C-6′′), 128.9 (C-1′′′),
128.7 (C-4′), 126.4 (C-5), 125.3ap, 125.0sp(C-3′′′), 121.2sp,
120.9ap(C-2′, C-6′), 113.9 (C-3a), 15.0sp,13.6ap (CH3). MS
(EI+): m/z 510 (M+, 100%), 512 (M + 2, 40), 463 (47), 362
(31), 346 (90), 318 (95), 277 (15), 242 (34).
N′-(3-Nitrobenzylidene)-3-methyl-1phenyl-6-p-chlorophenyl-
1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide
(2c) Com-
N′-(Benzylidene)-3-methyl-1phenyl-6-p-chlorophenyl-1H-
pyrazolo[3,4-b]pyridine-4-carbohydrazide (2a) Compound
2a was prepared by the reaction of 1 with benzaldehyde. The
yellow product was recrystallized from ethanol. Yield: 86%;
m.p 260–262 °C; IR νmax(cm−1):3173 (N-H); 3062 (CH-
Ar); 2965 (CH-Aliph); 1662 (C=O); 1603; 1573; (C=C);
687 (C-Cl):. 1H NMR (500 MHz, DMSO-d6):
δ = 12.44ap, 12.28sp (2 s, 1H, NH) 8.42 sp, 8.37ap (2s, 1H,
N=CH), 8.35(sp + ap) (d, J = 8.6 Hz, 2H, H-2′′, H-6′′),
8.32(sp + ap) (d, J = 7.8 Hz, 2H, H-2′, H-6′), 8.17sp, 8.04ap (2
s, 1H, H-5), 7.8(sp + ap) (d, J = 7.2 Hz, 2H, H-2′′′, H-6′′′),
7.67(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-5′′), 7.62(sp + ap) (t, J
= 8.0 Hz, 2H, H-3′, H-5′), 7.53–7.36(sp + ap) (m,3H, H-3′′′,
H-5′′′, H-4′), 7.32–7.25(sp + ap) (m, 1H, H-4′′′), 2.62 (s, 3H,
pound 2c was prepared by the reaction of 1 with 3-
nitrobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 77%. m.p 270–272 °C; IR νmax
(cm−1): 3188 (N-H); 3054 (CH-Ar); 2935 (CH-Aliph);
1.670 (C=O); 1603 (C=C); 1536, 1357 (NO2); 687 (C-Cl).
1H NMR (500 MHz, DMSO-d6): δ = 12.69ap, 12.55sp (2 s,
1H, NH), 8.63ap, 8.54sp (2 s, 1H, N=CH), 8.36–8.35(sp + ap)
(m, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 7.7 Hz, 2H, H-2′,
H-6′), 8.25sp, 8.07ap (d, J = 7.8 Hz 1H, H-6′′′), 8.18sp,
8.07ap (2 s, 1H, H-5), 7.82sp, 7.74ap (t, J = 8.9 Hz, 1H,
H-5′′′), 7.67(sp + ap) (d, J = 8.6 Hz 2H, H-3′′, H-5′′) 7.62(sp +
ap) (d, J = 7 Hz, 2H, H-3′, H-5′), 7.61(sp + ap) (s, 1H, H-2′′′),
7.57(sp + ap) (t, J = 7.8 Hz, 1H, H-4′′′), 7.38(sp + ap) (m,1H,
H-4′), 2.63(sp + ap) (s, 3H, CH3). Rotamers percentage
CH3). Rotamers percentage sp(74%): ap(26%)
.
13C NMR (125
sp(77%): ap(23%).
13C NMR (125 MHz, DMSO-d6): δ =
MHz, DMSO-d6): δ = 168.1ap, 162.0sp (C=O), 155.4 (C-6),
151.4 (C-7a), 149.5 (N=CH), 142.7 (C-3), 142.3 (C-4),
139.4sp, 139.3ap, (C-1′), 137.1 (C-1′′), 135.6 (C-1′′′), 134.4
(C-4′′), 131.0 (C-4′′′), 129.8 (C-3′′, 5′′), 129.7 (C-3′, C-5′),
129.6 (C-2′′′, C-6′′′), 129.4 (C-2′′, 6′′), 127.8 (C-3′′′, C-5′′′),
127.2 (C-4′), 126.4 (C-5), 121.2sp, 120.9ap(C-2′, C-6′), 113
(C-3a), 15.0sp, 13.7ap (CH3). MS (EI+): m/z 465 (M+,
100%), 467 (M + 2, 40), 362 (46), 346 (60), 318 (52), 278
(9), 242 (25), 140 (7), 77 (13).
168.5ap, 165.8sp (C=O), 155.4 (C-6), 151.4 (C-7a), 148.8
(C-3′′′), 147.2 (N=CH), 142.7 (C-3), 139.3 (C-4), 138.9 (C-
1′), 137.0 (C-1′′), 136.3 (C-1′′′), 135.6 (C-6′′′), 134.0 (C-4′
′), 131.1 (C-5′′′), 129.8 (C-3′′, C-5′′), 129.7 (C-3′, C-5′),
129.6(C-4′′′), 121.8 (C-5), 121.2sp, 121.0ap(C-2′, 6′), 113.8,
112.3 (C-3a), 14.9sp, 13.7ap (CH3). MS (EI+): m/z 510 (M
+, 100%), 512 (M + 2, 39), 480 (21), 362 (35), 346 (80),
318 (54), 277 (11), 242 (23).
N′-(4-Nitrobenzylidene)-3-methyl-1phenyl-6-p-chlorophe-
nyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2d)
Compound 2d was prepared by the reaction of 1 with 4-
nitrobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 85%. m.p300 °C; Lit. (Dias et al. 2007)
[296–298 °C]; IR νmax (cm−1): 3213 (N–H); 3043 (CH-Ar);
2921 (CH-Aliph); 1.661 (C=O); 1594 (C=C); 1533, 1348
(NO2); 687 (C–Cl). 1H NMR (500 MHz, DMSO-d6):
δ = 12.73sp, 12.57ap (2 s, 1H, NH), 8.52ap, 8.36sp (2 s, 1H,
N=CH), 8.34(sp + ap) (d, J = 8.7 Hz, 2H, H-2′′, H-6′′),
8.31(sp + ap) (d, J = 7.6 Hz, 2H, H-2′, H-6′), 8.29sp, 8.19ap (2
s, 1H, H-5), 8.09(sp + ap) (d, J = 7.1 Hz, 2H, H-2′′′, H-6′′′,
N′-(2-Nitrobenzylidene)-3-methyl-1phenyl-6-p-chlorophe-
nyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2b)
Compound 2b was prepared by the reaction of 1 with 2-
nitrobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 75%. m.p 272–274 °C; IR νmax (cm−1):
3181 (N-H); 3010 (CH-Ar); 2853 (CH-Aliph); 1655
(C=O); 1566, 1350 (NO2) 1506 (C=C); 687 (C-Cl).1H
NMR (500 MHz, DMSO-d6): δ = 12.79ap, 12.62sp (2s, 1H,
NH), 8.84sp, 8.52ap (2 s, 1H, N=CH), 8.36(sp + ap) (d, J =
8.6 Hz, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 7.8 Hz, 2H, H-
2′, H-6′), 8.22sp, 8.4ap (2d, J = 7.7 Hz, 1H, H-5′′′), 8.19sp,

Med Chem Res
7.67(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-5′′), 7.62(sp + ap) (t,
J = 8.0 Hz, 2H, H-3′, H-5′), 7.38(sp + ap) (t, J = 7.4 Hz, 1H,
H-4′), 2.62(sp + ap) (s, 3H, CH3). Rotamers percentage
sp(77%): ap(23%).13C NMR (125 MHz, DMSO-d6): δ =
168.4ap, 162.4sp (C=O), 155.4 (C-6), 151.4(C-7a), 148.6
(C-4′′′), 147.1 (N=CH), 142.7 (C-3), 140.6 (C-4), 139.3 (C-
1′′′), 138.8 (C-1′), 137.0 (C-1′′), 135.6 (C-4′′), 129.8 (C-3′′,
C-5′′),129.7 (C-3′, C-5′), 129.6 (C-2′′, C-6′′), 128.8(C-4′),
128.1(C-2′′′, C-6′′′),126.4(C-3′′′, C-5′′′′), 124.6 (C-5),
121.2sp,120.9ap(C-2′, C-6′), 113.9 (C-3a), 14.9sp, 13.6ap
(CH3). MS (EI+): m/z 510 (M+, 100%), 512 (M + 2, 41),
480 (10), 362 (13.4), 346 (87.8), 318 (55), 277 (9.5),
242 (22).
7.6(sp + ap) (m, 1H, H-5′′′), 7.51–7.35(m, 2H, H-4′, H-4′′′),
2.62 (s, 3H, CH3). Rotamers percentage sp(82%): ap(18%)
.
13C NMR (125 MHz, DMSO-d6): δ = 168.0ap, 162.2sp
(C=O), 155.4 (C-6), 151.4 (C-7a), 147.8 (N=CH), 142.7
(C-3), 139.3 (C-4), 139.1 (C-1′), 137.0 (C-1′′), 136.9 (C-1′′
′), 135.6 (C-4′′), 133.5sp, 131.6ap (C-4′′′), 129.9 (C-3′′, C-5′
′), 129.8 (C-3′, C-5′), 129.7 (C-2′′′, C-6′′′), 129.6 (C-2′′, C-6′
′), 126.9 (C-4′), 126.4 (C-5), 122.7 (C-3′′′), 121.2 (C-2′, C-
6′), 121.0 (C-5), 113.8 (C-3a), 14.9sp, 13.6ap (CH3). MS
(EI+): m/z 543 (M+, 76%), 545 (M + 2, 100), 547 (M + 4,
27), 362 (43), 346 (92), 318 (61), 277 (9), 242 (19).
N′-(4-Bromobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2g)
Compound 2g was prepared by the reaction of 1 with 4-
brombenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 84%. m.p 285–287 °C; IR νmax
(cm−1): 3182 (N–H); 3053 (CH-Ar); 2960 (CH-Aliph);
N′-(2-Bromobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2e)
Compound 2e was prepared by the reaction of 1 with 2-
bromobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 86%. m.p 210–212 °C; IR νmax
(cm−1): 3182 (N–H); 3066 (CH-Ar); 2925 (CH-Aliph);
1
1656 (C=O); 1560 (C=C); 681 (C–Cl). H NMR (500
MHz, DMSO-d6): δ = 12.50ap, 12.35sp (2 s, 1H, NH),
8.39sp, 8.36ap (2 s, 1H, N=CH), 8.34(sp + ap) (d, J = 8.6 Hz,
2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 8.3 Hz, 2H, H-2′, H-
6′), 8.17sp, 8.03ap (2 s, 1H, H-5), 7.83ap, 7.76sp (d, J = 8.5
Hz, 2H, H-2′′′, H-6′′′) 7.71(sp + ap) (d, J = 8.5 Hz, 2H, H-3′′′,
H-5′′′) 7.67(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-5′′),
7.50–7.23(sp + ap) (m,3H, H-3′, H-5′, H-4′), 2.61 (s, 3H,
1
1676 (C=O); 1597 (C=C); 690 (C–Cl). H NMR (500
MHz, DMSO-d6): δ = 11.63ap, 11.46sp (2 s, 1H, NH),
10.70ap, 10.19sp (2 s, 1H, N=CH), 8.50ap, 8.34sp (2 s, 1H,
H-5), 8.31(sp + ap) (d, J = 7.7 Hz, 2H, H-2′′, H-6′′), 8.28(sp + ap)
(d, J = 8.6 Hz, 2H, H-2′, H-6′), 7.95–7.90(sp + ap) (m, 1H, H′′′),
7.67(sp + ap) (m,4H, H-3′′, H-5′′, H-3′, H-5′), 7.61(sp + ap) (t,
J = 7.9 Hz, 1H, H-4′′′), 7.42-7.45(sp + ap) (m, 1H, H-4′),
7.39–7.31(sp + ap) (m, 2H, H-3′′′, H-5′′′), 2.63(sp + ap) (s, 3H,
CH3). Rotamers percentage sp(72%): ap(28%).
13C NMR (125
MHz, DMSO-d6): δ = 168.0sp,162.1ap (C=O), 155.4 (C-6),
151.4 (C-7a), 148.3 (N=CH), 142.7 (C-3), 139.3 (C-4),
139.1 (C-1′), 137.0 (C-1′′), 135.6 (C-1′′′), 133.7 (C-4′′),
132.4 (C-4′), 129.8 (C-3′′, C-5′′), 129.7 (3′, 5′), 129.6 (C-2′′
′, C-6′′′), 129.1 (C-2′′, C-6′′), 126.4 (C-3′′′, 5′′′), 124.3 (C-4′′
′), 121.2 (C-2′, C-6′), 120.9 (C-5), 113.8 (C-3a), 14.9sp,
13.7ap (CH3). MS (EI+): m/z 543 (M+, 70%), 545 (M + 2,
91), 547 (M + 4, 27), 362 (46), 346 (100), 318 (75), 277
(14), 242 (39).
CH3). Rotamers percentage sp(65%): ap(35%).
13C NMR (125
MHz, DMSO-d6): δ = 168.9, 166.2, 164.9sp, 161.2ap
(C=O), 155.3 (C-6), 151.4 (C-7a), 144.3 (N=CH), 142.9
(C-3), 141.4 (C-4), 139.3 (C-1′), 138.9 (C-1′′), 137.1ap,
135.5sp(C-1′′′),133.6ap,133.5sp (C-4′′), 132.0 (C-3′′′), 131.8
(C-4′′′), 129.7 (C-3′′, C-5′′), 129.7 (C-3′, C-5′), 129.6 (C-2′′,
C-6′′), 128.5 (C-5′′′), 127.6ap, 127.4sp(C-6′′′), 126.3 (C-4′),
123.8 (C-5), 123.6 (C-2′′′), 121.1 (C-2′, C-6′), 113.4(C-3a),
14.9 (CH3). MS (EI+): m/z 543 (M+, 4%), 545 (M + 2, 5),
547 (M + 4, 2), 419 (73), 362 (12), 346 (100), 318 (80), 277
(10), 242 (22).
N′-(2-Flourobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2h)
Compound 2h was prepared by the reaction of 1 with 2-
flourobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 76%. m.p 265–267 °C; IR: νmax
(cm−1): 3295 (N–H); 3065 (CH-Ar); 2966 (CH-Aliph);
N′-(3-Bromobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2f)
Compound 2f was prepared by the reaction of 1 with 3-
bromobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 97%. m.p 245–247 °C; IR νmax
(cm−1): 3173 (N–H); 3047 (CH-Ar); 1656 (C=O); 1594;
1573; 1536 (C=C); 691 (C–Cl). 1H NMR (500 MHz,
DMSO-d6): δ = 12.56ap, 12.4sp (2 s, 1H, NH), 8.38sp (s, 1H,
N=CH), 8.35(sp + ap) (d, J = 3H, N=CHap, H-2′′, H-6 ′′),
8.32(sp + ap) (d, J = 7.9 Hz, 2H, H-2′, H-6′), 8.17sp, 8.14ap (2
s, 1H, H-5), 8.05ap, 8.01sp (2 s, 1H, H-2′′′), 7.81(sp + ap) (d, J
= 7.8 Hz, 1H, H-6′′′), 7.68(sp + ap) (t, J = 8.4 Hz, H,
H-3′′, H-5′′), 7.63(sp + ap) (t, J = 8.3 Hz, 2H, H-3′, H-5′),
1
1662 (C=O); 1594; 1572; 1507 (C=C); 680 (C-Cl). H
NMR (500 MHz, DMSO-d6): δ = 12.54ap, 12.4sp (2 s, 1H,
NH), 8.65sp, 8.38ap (2 s, 1H, N=CH), 8.36(sp + ap) (d, J =
8.4 Hz, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 7.8 Hz, 2H, H-
2′, H-6′), 8.20sp (s, 1H, H-5), 8.03–8.04(sp + ap) (m, 2H, H-
5ap, H-6′′′), 7.68(sp + ap) (d, J = 8.4 Hz, 2H, H-3′′, H-5′′′),
7.61 (t, J = 7.9 Hz, 2H, H-3′, H-5′), 7.59–7.32(sp + ap) (m,
3H, H-3′′′, H-5′′′, H-4′), 7.25–7.00(sp + ap) (m, 1H, H-4′′′)
2.63ap (s, 3H, CH3). Rotamers percentage sp(74%): ap(26%)
.
13C NMR (125 MHz, DMSO-d6): δ = 165.1sp, 162.1ap

Med Chem Res
(C=O), 155 (C-2′′′), 151.7 (C-6), 151.4 (C-7a), 142.6 (C-3),
139.1 (C-4), 138.8 (C-1′), 129.8 (C-1′′), 129.6 (C-4′′), 127.6
(C-4′′′), 126.9 (C-3′′, C-5′′), 126.5 (3′, 5′), 125.6 (C-6′′′),
121.2 (C-2′′, C-6′′), 114.3 (C-5), 113.6 (C-2′, 6′), 112.4 (C-
3a), 15.0sp,13.9ap (CH3). MS (EI+): m/z 483 (M+, 100%),
485 (M + 2, 23), 463 (25), 362 (22), 346 (61), 318 (60), 277
(7), 242 (19).
137.0sp,135.6ap(C-1′′′), 131.3 (C-4′′), 130.1 (C-2′′′, C-6′′′),
129.8(C-3′′, C-5′′, 129.7) (C-3′, C-5′), 129.6 (C-2′′, C-6′′),
126.4 (C-4′), 121.2sp, 120.9ap (C-5), 116.6sp, 116.4ap(C-3′′′,
C-5′′′), 113.8 (C-2′, C-6′), 112.4 (C-3a), 14.9sp, 13.6ap
(CH3). MS (EI+): m/z 483(M+, 100%), 485 (M + 2, 46),
362(49), 346 (94), 318 (89), 277 (14), 242 (32).
N′-(2-Chlorobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2k)
Compound 2k was prepared by the reaction of 1 with 2-
chlorobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 75%. m.p 212–214 °C; IR νmax
(cm−1): 3188(N–H); 3054 (CH-Ar); 2900 (CH-Aliph);
N′-(3-Flourobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2i)
Compound 2i was prepared by the reaction of 1 with 3-
flourobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 97%. m.p 267–270 °C; IR νmax
(cm−1): 3178 (N–H); 3065 (CH-Ar); 2970 (CH-Aliph);
1663 (C=O); 1595; 1575; 1507 (C=C); 1351 (F); 689 (Cl).
1H NMR (500 MHz, DMSO-d6): δ = 12.57sp, 12.39ap (2 s,
1H, NH), 8.42(sp + ap) (s, 1H, N=CH), 8.35(sp + ap) (d, J =
8.6 Hz, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 7.6 Hz, 2H, H-
2′, H-6′), 8.17sp, 8.04ap (2 s, 1H, H-5), 7.67(sp + ap) (d, J =
8.5 Hz, 2H, H-3′′, H-5′′), 7.63(sp + ap) (dd, J = 14.1, 5.8 Hz,
2H, H-3′, H-5′), 7.56(sp + ap) (dd, J = 13.9, 7.9 Hz, 2H,
H-3′′′, H-5′′′), 7.38(sp + ap) (t, J = 7.3 Hz, 1H, H-4′),
7.36–7.31 (m, 1H, H-4′′′), 2.62 (s, 3H, CH3). Rotamers
percentage sp(80%): ap(20%).13C NMR (125 MHz, DMSO-
d6): δ = 163.8sp, 162.1ap (C=O), 162.0 (C-3′′′), 155.4 (C-6),
151.4 (C-7a), 147.9 (N=CH), 142.7 (C-3), 139.3 (C-4),
139.1 (C-1′), 137.0 (C-1′′), 135.6 (C-1′′′), 131.6 (C-4′′),
129.8 (C-3′′, C-5′′), 129.7 (C-3′, C-5′), 129.6 (C-2′′, C-6′′),
126.4 (C-4′), 124.2 (C-6′′′), 121.2 (C-2′, C-6′), 120.9
(C-5′′′), 117.7 (C-4′′′), 113.9 (C-2′′′), 113.8 (C-5), 113.7 (C-
3a), 14.9sp, 13.6ap (CH3). MS (EI+): m/z 483 (M+, 100%),
485 (M + 2, 45), 362 (25), 346 (89), 318 (70), 277 (10), 242
(27).
1
1654 (C=O); 1607, 1593 (C=C); 691 (C–Cl). H NMR
(500 MHz, DMSO-d6): δ = 12.57(sp + ap) (s, 1H, NH),
8.86sp, 8.57ap (2 s, 1H, N=CH) 8.36 (d, J = 8.0 Hz, 2H, H-
2′′, H-6′′), 8.32(sp + ap) (d, J = 8.0 Hz, 2H, H-2′, H-6′),
8.21sp, 8.05ap (2 s, 1H, H-5), 8.11–8.13(sp + ap) (d, J = 7.6
Hz, 1H, H-6′′′) 7.74–7.43(sp + ap) (m, 6H, H-3′, H-5′, H-3′′
H-5′′, H-3′′′, H-4′′′), 7.38sp, 7.32ap (t, J = 7.0 Hz, 1H, H-4′),
2.63(sp + ap) (s, 3H, CH3). Rotamers percentage sp(75%)
:
ap(25%)
.
13C NMR (125 MHz, DMSO-d6): δ = 167.5sp,
162.1ap (C=O), 155.5(C-6), 151.4 (C-7a), 145.5 (N=CH),
142.7 (C-3), 139.3 (C-4), 138.9 (C-1′), 137.2 (C-1′′), 135.6
(C-1′′′), 133.9 (C-2′′′), 132.4 (C-4′′), 132.2sp, 132.1ap,
(C-4′′′), 131.6sp,130.5ap(C-3′′′), 129.8 (C-3′′, C-5′′), 129.7
(C-3′, C-5′), 129.6 (C-6′′′), 129.1 (C-2′′, C-6′′), 128.2
(C-3′′′, C-5′′′), 127.6 (C-4′), 126.4 (C-5), 121.2sp,120.9ap(C-
2′, C-6′),113.9 (C-3a), 15.0ap, 14.4sp (CH3). MS (EI+): m/z
499 (M+, 100%), 501 (M + 2, 64), 503 (M + 4, 13), 362
(37), 346 (93), 318 (69), 277 (8), 242 (27).
N′-(3-Chlorobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2l)
Compound 2l was prepared by the reaction of 1 with 3-
chlorobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 60%. m.p 246–247 °C; IR νmax (cm−1):
3178 (N–H); 3051 (CH-Ar); 2923 (CH-Aliph); 1656 (C=O);
1595; 1572; 1506 (C=C); 683 (C–Cl). 1H NMR (500 MHz,
DMSO-d6): δ = 12.56ap, 12.42 sp (2s, 1H, NH), 8.40(sp + ap)
(s, 1H, N=CH), 8.34(sp + ap) (d, J = 8.6 Hz, 2H, H-2′′, H-6′′),
8.31(sp + ap) (d, J = 7.8 Hz, 2H, H-2′, H-6′), 8.17sp, 8.04ap (2
s, 1H, H-5), 7.86(sp + ap) (s, 1H, H-2′′′), 7.77(sp + ap) (d, J =
6.4 Hz, 1H, H-6′′′), 7.66(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-
N′-(4-Flourobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2j)
Compound 2j was prepared by the reaction of 1 with 4-
flourobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 97%. m.p 269–271 °C; Lit (Dias et al.
2007) [230 °C]. IR νmax (cm−1): 3185(N–H); 3062 (CH-
Ar); 2900 (CH-Aliph); 1.655 (C=O); 1595 (C=C); 691
(C–Cl). 1H NMR (500 MHz, DMSO-d6): δ = 12.45sp,
12.29ap (2 s, 1H, NH), 8.41ap, 8.37sp (2 s, 1H, N=CH),
8.36(sp + ap) (d, J = 4.4 Hz, 2H, H-2′′, H-6′′), 8.33(sp + ap) (d,
J = 12.6, 8.5 Hz, 2H, H-2′, H-6′), 8.16ap, 8.02 sp (2s, 1H,
H-5), 7.87(sp + ap) (d, J = 8.67, 5.7 Hz, 2H, H-2′′′, H-6′′′)
7.67(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-5′′), 7.62(sp + ap)
(t, J = 8.0 Hz, 2H, H-3′, H-5′), 7.35(sp + ap) (m, 2H, H-3′′′,
H-5′′′), 7.12(sp + ap) (t, J = 8.8 Hz, 1H, H-4′), 2.62(sp + ap) (s,
5′′), 7.62
(t, J = 7.5 Hz, 2H, H-3′, H-5′), 7.54(sp + ap)
(sp + ap)
(m,1H, H-4′′′), 7.38(sp + ap) (t, J = 7.3 Hz, 1H, H-5′′′),
7.34–7.23(sp + ap) (m, 1H, H-4′), 2.62(sp + ap) (s, 3H, CH3).
Rotamers percentage sp(78%): ap(22%).
13C NMR (125 MHz,
DMSO-d6): δ = 167.5sp, 162.2ap (C=O), 155.4 (C-6), 151.4
(C-7a), 147.9 (N=CH), 142.7 (C-3), 139.4 (C-4), 139.3 (C-
1′), 139.0 (C-1′′), 137.0 (C-1′′′′), 136.6 (C-3′′′), 135.6 (C-5′′′),
134.2 (C-C-4′′), 130.6 (C-4′′′), 129.8 (C-3′′, C-5′′), 129.7 (C-
6′′′), 129.6 (C-2′′′), 129.6 (C-2′′, C-6′′), 127.1 (C-3′′, C-5′′),
3H, CH3). Rotamers percentage ap(77%): sp(23%).
13C NMR
(125 MHz, DMSO-d6): δ = 165.2 (C-4′′′), 163.0ap, 162.1sp
(C=O), 155.4 (C-6), 151.4 (C-7a), 148.4 (N=CH), 144.8ap,
142.7sp(C-3), 140.4 (C-4), 139.3(C-1′), 139.2(C-1′′),

Med Chem Res
126.5 (C-4′), 126.4 (C-5), 121.2 (C-2′, C-6′), 113.8 (C-3a),
14.9ap, 13.7sp (CH3). MS (EI+): m/z 499 (M+, 88%), 501
(M + 2, 77), 503 (M + 4, 13), 362 (48), 346 (100), 318 (58),
277 (20), 242 (31).
recrystallized from ethanol. Yield: 73%. m.p 280–283 °C;
IR νmax (cm−1): 3197 (N–H); 3066 (CH-Ar); 2966 (CH-
Aliph); 1654 (C=O); 1596; 1572; 1507(C=C); 689 (C–Cl).
1H NMR (500 MHz, DMSO-d6): δ = 12.31ap, 12.14sp (2 s,
1H, NH), 8.63ap, 8.37sp(2 s, 1H, N=CH) 8.34(sp + ap) (d, J
= 8.0 Hz, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 8 Hz, 2H, H-
2′, H-6′), 8.15sp, 8.01ap(2 s, 1H, H-5), 7.82ap, 7.75sp (d, J =
8.6 Hz, 2H, H-2′′′, H-6′′′), 7.67(sp + ap) (d, J = 8 Hz, 2H, H-
3′′, H-5′′), 7.62(sp + ap) (t, J = 8.1 Hz, 2H, H-2′, H-5′),
7.38(sp + ap) (t, J = 7.0 Hz, 1H, H-4′), 7.27, 6.84(sp + ap) (m,
2H, H-3′′′, H-5′′′), 3.84sp, 3.68ap (2s, 3H, OCH3), 2.62(sp +
N′-(4-Chlorobenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2m)
Compound 2m was prepared by the reaction of 1 with 4-
chlorobenzaldehyde. The yellow product was recrystallized
from ethanol. Yield: 63%. m.p 285–287 °C; IR νmax (cm−1):
3188 (N–H); 3057 (CH-Ar); 2928 (CH-Aliph); 1656 (C=O);
1595; 1548; 1560 (C=C); 690 (C–Cl). 1H NMR (500 MHz,
DMSO-d6): δ = 12.50ap, 12.31sp (2 s, 1H, NH), 8.41sp, 8.36ap
(2 s, 1H, N=CH), 8.34(sp + ap) (d, J = 8.6 Hz, 2H, H-2′′, H-6′
′), 8.31(sp + ap) (d, J = 8.2 Hz, 2H, H-2′, H-6′), 8.16sp, 8.03ap
(2 s, 1H, H-5) 7.83(sp + ap) (d, J = 8.5 Hz, 2H, H-2′′′,H-6′′′),
7.67(sp + ap) (d, J = 8.6 Hz, 2H, H-3′′, H-5′′), 7.62(sp + ap) (t, J
= 8.4 Hz, 2H, H-3′, H-5′), 7.57(sp + ap) (t, J = 8.5 Hz, 2H, H-
3′′′, H-5′′′), 7.38(sp + ap) (t, J = 7.3 Hz, 1H, H-4′), 2.62(sp + ap)
(s, 3H, CH3). Rotamers percentage sp(75%): ap(25%).
13C
ap)
NMR (125 MHz DMSO-d6): δ = 172ap, 161.8sp (C=O),
161.7 (C-4′′′), 155.5 (C-6), 151.4 (C-7a), 149.4 (N=CH),
142.8 (C-3), 139.5 (C-4), 139.3 (C-4′′), 137.1 (C-1′), 135.6
(C-1′′), 129.8 (C-1′′′), 129.7 (C-3′′, C-5′′), 129.6 (C-3′, C-
5′), 129.5 (C-2′′′, C-6′′′), 128.7 (C-2′′, C-6′′), 127.2 (C-4′),
126.2 (H-5), 121.2 (C-2′, C-6′), 114.9 (C-3′′′, C-5′′′), 113.4
(C-3a), 55.8sp,55.7ap (OCH3), 14.9sp, 13.6ap (CH3). MS
(EI+): m/z 495 (M+, 86%), 497 (M + 2, 38), 464 (4), 362
(100), 346 (26), 318 (59), 277 (10), 242 (22).
(s, 3H, CH3). Rotamers percentage sp(73%): ap(27%).
13C
NMR (125 MHz, DMSO-d6): δ = 168.0ap, 162.1sp (C=O),
155.4 (C-6), 151.4 (C-7a), 148.2 (N=CH), 145.1 (C-3),
142.7 (C-4), 139.3 (C-4′′′), 139.1 (C-1′), 137.0 (C-1′′), 135.6
(C-4′′), 135.5ap, 133.3sp (C-1′′′), 129.8 (C-2′′′, C-6′′′), 129.7
(C-3′′, C-5′′), 129.6 (C-3′, C-5′), 129.5 (C-3′′′′, C-5′′′), 129.5
(C-2′′, C-6′′), 126.4 (C-4′), 121.2 (C-2′, C-6′), 120.9 (C-5),
113.8 (C-3a), 14.9sp, 13.6ap (CH3). MS (EI+): m/z 499 (M+,
100%), 501 (M + 2, 62), 503 (M + 4, 16), 362 (45), 346
(79), 318 (63), 277 (8), 242 (30).
N′-(2-Hydroxy-3-methoxybenzylidene)-6-(4-chlorophenyl)-
3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohy-
drazide (2p) Compound 2p was prepared by the reaction
of 1 with 2-hydroxy-3-methoxybenzaldehyde. The yellow
product was recrystallized from ethanol. Yield: 68%. m.p
215–217 °C; IR νmax (cm−1): 3393 (OH); 3006 (CH-Ar);
2920 (CH-Aliph); 1654 (C=O); 706 (C–Cl). 1H NMR (500
MHz, DMSO-d6): δ = 10.88ap, 10.70sp (2 s, 1H, NH),
10.19sp, 8.99ap (2 s, 1H, N=CH), 8.30(sp + ap) (d, J = 7.9 Hz,
2H, H-2′′, H-6′′), 8.28(sp + ap) (d, J = 8.5 Hz, 2H, H-2′, H-
6′), 7.94sp, 7.33ap (2 s, 1H, H-5), 7.66(sp + ap) (d, J = 8.4 Hz,
2H, H-3′′, H-5′′), 7.61(sp + ap) (t, J = 7.8 Hz, 2H, H-3′, H-5′),
7.40–7.26 (m, 2H, H-4′, H-6′′′), 7.13sp, 7.08ap (d, J = 7.9
Hz, 1H, H-4′′′), 6.91(sp + ap) (t, J = 7.9 Hz, 1H, H - 5′′′),
3.61(sp + ap) (s, 3H, OCH3) 2.61(sp + ap) (s, 3H, CH3). Rota-
N′-(2-Methoxybenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2n)
Compound 2n was prepared by the reaction of 1 with 2-
methoxybenzaldehyde. The yellow product was recrys-
tallized from ethanol. Yield: 77%. m.p 229–231 °C; IR νmax
(cm−1): 3, 156 (N–H); 3006 (CH-Ar); 2940 (CH-Aliph);
1
1648 (C=O); 1598; 1561; 1491 (C=C); 688 (C–Cl). H
NMR (500 MHz, DMSO-d6): δ = 12.38ap, 12.23sp (2 s, 1H,
NH), 8.78sp, 8.50ap (2 s, 1H, N=CH), 8.36(sp + ap) (d, J = 7.8
Hz, 2H, H-2′′, H-6′′), 8.32(sp + ap) (d, J = 7.8 Hz, 2H, H-2′, H-
6′), 8.17sp, 8.02ap (2 s, 1H, H-5), 7.96(sp + ap) (d, J = 7.5 Hz,
1H, H-6′′′), 7.67(sp + ap) (d, J = 8.2 Hz, 2H, H-3′′, H-5′′),
7.62(sp + ap) (t, J = 7.4 Hz, 2H, H-3′, H-5′) 7.52–7.35(sp + ap)
(m, 2H, H-4′′′, H-4′), 7.15–7.08(sp + ap) (m, 2H, H-5′′′, H-3′′′),
6.73ap (t, J = 1H, H-5ap′′′) 3.88sp, 3.82ap (2 s, 3H, OCH3),
mers percentage sp(70%): ap(30%).
13C NMR (125 MHz,
DMSO-d6): δ = 168sp, 164.9ap (C=O), 163.2 (C-2′′′), 155.3
(C-6), 151.4 (C-7a), 149.0 (N=CH), 148.4 (C-3′′′), 142.9
(C-3), 139.3 (C-4), 138.9 (C-1′), 137.1 (C-1′′), 135.5 (C-4′′),
130.5 (C-6′′′), 129.7 (C-3′′, C-5′′), 129.6 (C-3′, -5′), 129.6
(C-2′′, C-6′′), 126.3 (C-4′), 122.5 (C-5), 121.1 (C-2′, C-6′),
120.1 (C-6′′′), 119.8 (C-4′′′), 118.7 (C-5′′′), 113.4 (C-
3a),56.4 (OCH3), 14.9 (CH3). MS (EI+): m/z 511 (M+,
80%), 513 (M + 2,), 419 (80), 362 (26), 346 (100), 318
(81), 277 (12), 242 (24).
2.63(sp + ap) (s, 3H, CH3). Rotamers percentage sp(76%)
:
ap(24%). MS (EI+): m/z 495 (M+, 81%), 497 (M + 2, 29),
464 (6), 362 (100), 346 (24), 318 (53), 277 (10), 242 (24).
N′-(3-Hydroxybenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2q)
Compound 2q was prepared by the reaction of 1 with 3-
hydroxybenzaldehyde. The yellow product was recrys-
tallized from ethanol. Yield: 39%. m.p 290–292 °C; IR νmax
N′-(4-Methoxybenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2o)
Compound 2o was prepared by the reaction of 1 with
4-methoxybenzaldehyde. The yellow product was

Med Chem Res
(cm−1): 3394 (OH); 3043 (CH-Ar); 2963 (CH-Aliph); 1650
(C=O); 689 (Cl). 1H NMR (500 MHz, DMSO-d6): δ =
12.36ap, 12.21sp (2 s, IH, NH), 8.51ap, 8.35sp (2 s, 1H,
N=CH), 8.33(sp + ap) (d, J = 6.2 Hz, 2H, H-2′′, H-6′′),
8.30(sp + ap) (d, J = 10 Hz, 2H, H-2′, H-6′), 8.14sp, 8.06ap (2
s, 1H, H-5), 7.99sp,7.93ap (2 s, 1H, OH), 7.69–7.59(sp + ap)
(m, 5H, H-3′′, H-5′′, H-3′, H-5′, H-2′′′), 7.42-7.26(sp + ap) (m,
3H, H-4′′, H-4′, H-6′′, H-5′′′), 2.61(sp + ap) (s, 3H, CH3).
Rotamers percentage sp(76%):ap(24%).13C NMR (125 MHz,
DMSO-d6): δ = 161.9 (C=O), 155.5 (C-3′′′), 151.7 (C-6),
150.7 (C-7a), 149.5 (N=CH), 146.2 (C-3), 139.3 (C-4),
139.2 (C-4′′) 139.1 (C-1′), 137.0 (C-1′′′), 135.6 (C-1′′),
135.4 (C-4′), 129.8 (C-3′′, C-5′′), 129.7 (C-2′′, C-6′′), 129.6
(C-3′, C-5′), 126.5 (C-5), 121.2 (C-2′, C-6′), 120.1 (C-6′′′),
115.1 (C-2′′′), 113.3 (C-5′′′), 112.9 (C-3a), 112.2 (C-4′′′),
14.8sp, 13.6ap(CH3). MS (EI+): m/z 481 (M+, 78%),
483 (M + 2, 30), 419 (58), 362 (55), 346 (100), 318 (27.7),
242 (33).
151.4 (C-7a), 149.5 (C-1′′′), 146.2 (C-3′′′), 142.7 (C-3),
139.3 (C-4), 139.2 (N=CH), 139.1 (C-1′), 137.0 (C-1′′),
135.6 (C-4′′), 129.8 (C-3′′, C-5′′), 129.7 (C-3′, C-5′), 129.6
(C-2′′, C-6′′), 126.4 (C-4′), 121.1 (C-2′, C-6′), 120.9 (C-5),
115.0 (C-5′′′), 113.7 (C-4′′′), 112.8ap, 112.3sp (C-3a), 14.9sp,
13.6ap (CH3). MS (EI+): m/z 455 (M+, 100%), 457 (M + 2,
40), 346 (75), 318 (67), 277 (14), 242 (26).
Molecular docking studies
Protein (Receptor) structure preparation
In order to understand the insight mechanism of binding
mode of synthesized compound with receptor residues
molecular docking simulations were performed for com-
pounds (2a–2t and Aspirin). Molecular docking studies
were performed by using MOE 2009.10 (Ahad et al. 2015;
Ahmad et al. 2015; Ahmad et al. 2015). The human PGH-2
synthase 3D model of COX-1 (PDB-ID: 1Q4G) and COX-2
(PDB-ID: 3NT1) were retrieved from PDB (source www.
rcsb.org//pdb/). The molecular modeling and computational
research is also widely used in literature for drug designing
(Karmakar and Singh 2017; Li et al. 2014; Srikala et al.
2017).
AMBER99 force field function of MOE was used for 3D
protonation and energy minimization by receptor molecule
optimization. The slope was set at 0.5 and receptor was
reduced till its root mean square slope reached below point
0.05. After 3D protonation of receptor protein and hide
molecule option was used to hydrogen molecule. Again
MOE option of surface and map was used for pinpointing
the pocket residues and docking site. This 3D protonated
and energy minimized receptor molecules were used for
molecular docking analysis.
N′-(4-Hydroxybenzylidene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2r)
Compound 2r was prepared by the reaction of 1 with 4-
hydroxybenzaldehyde. The yellow product was recrys-
tallized from ethanol. Yield: 75%. m.p ›300 °C; Lit. (Dias
et al. 2007) [›310 °C]. IR νmax (cm−1): 3313 (OH); 3199
(CH-Ar); 3097 (CH-Aliph); 1647 (C=O); 692 (C–Cl).
N′-(4-(Dimethylamino)benzylidene)-6-(4-chlorophenyl)-3-
methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohy-
drazide (2s) Compound 2s was prepared by the reaction of
1 with 4-dimethylaminobenzaldehyde. The yellow product
was recrystallized from ethanol. Yield: 98%. m.p 214–215 °C;
Lit. (Geraldo et al. 2010) [212 °C]; IR (ν-cm−1): 3192
(N–H); 1696 (C=O). MS (EI+): m/z 499 (M+, 1%), 501
(M + 2, 34), 419 (84), 377 (21), 362 (13), 346 (100), 318
(74), 277 (12), 242 (20).
Ligands preparation and database construction
N′-(Furan-2-yl-methylene)-6-(4-chlorophenyl)-3-methyl-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide (2t)
Compound 2t was prepared by the reaction of 1 with furan-
2-carbaldehyde. The yellow product was recrystallized from
ethanol. Yield: 60%. m.p 250–252 °C; IR: νmax (cm−1):
3175 (N–H); 3011 (CH-Ar); 2944 (CH-Aliph); 1652; 1600;
1559 (C=O). 1H NMR (500 MHz, DMSO-d6): δ = 12.36ap,
12.22 sp (2s, 1H, NH), 8.51ap, 8.34sp (2 s, 1H, N=CH),
8.32(sp + ap) (d, J = 7 Hz, 2H, H-2′′, H-6′′), 8.30(sp + ap) (d, J
= 6.2 Hz, 2H, H-2′, H-6′), 8.14sp, 8.06ap (2 s, 1H, H-5),
7.99ap, 7.92 sp (2s, 1H, H-3′′′), 7.66(sp + ap) (d, J = 8.6 Hz,
2H, H-3′′, H-5′′), 7.6 (t, J = 7.9 Hz, 2H, H-3′, H-5′),
7.38(sp + ap) (t, J = 7.2 Hz, 1H, H-4′), 7.03sp, 6.77ap (d, J =
3.3 Hz, 2H, H-4"', H-5"'), 2. 62(sp + ap) (s, 3H, CH3). Rota-
The scaffolds of synthesized compounds (2a–2t and
Aspirin) were constructed by using chem3D pro 12.0. Their
3D structures were then saved in .mol file for docking
analysis. The ligands structure prepared by addition
of hydrogen atom and energy minimization at 0.05 gra-
dients was done with the help of MMFF94X force field
option in MOE. These ligands were then added into a
database and were used for docking against the targeted
receptor protein.
Molecular docking analysis
Molecular docking analysis was executed after receptor
protein and ligands molecule preparation. The .mbd format
was used for saving the docking output database file
mers percentage sp(75%): ap(25%)
DMSO-d6): δ = 167.8 161.9sp (C=O), 155.4 (C-6),
.
13C NMR (125 MHz,
ap,

Med Chem Res
containing receptor ligand complexes. In order to evaluate
the ligands interactions with receptor protein active sites
residue, complexes with minimum S values were taken.
MOE ligX option was used for analysis of best docked pose
having highest negative binding energy value (S value),
highest hydrogen bonding, and π–π interactions.
formula.
Percent scavenging activity
¼ ½1 À ðAbs of test compound=Abs of controlފ  100
ABTS antioxidant activity
Biological activities
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
antioxidant activity was also performed in the same way as
for DPPH. Whereas, ABTS (7 mM) solution was prepared
by mixing it with potassium persulfate (2.45 M, 2.6 mL)
solution. The resulting solution was incubated for 12–16 h
in dark. Greenish blue solution obtained was diluted with
ethanol to give the 0.7 absorbance at 734 nm. All the
samples were run in triplicates. Trolox was used as standard
drug.
Antiplatelet activity
Antiplatelet activity against AA and collagen was deter-
mined on human whole blood as previously described
method along with slight modifications (Storey et al. 1998).
Blood (5 mL) was drawn from healthy volunteer by careful
venipuncture (after getting their consents), poured into tri-
sodium citrate (0.5 mL; 3.8%) containing falcon tube. The
tube with blood was then incubated at 37 °C for 30 min. The
aliquots (460 μL) of this pre-incubated blood was mixed
with 600 μL fixing solution (0.5% formaldehyde in PBS) to
take the baseline platelet count at t = 0 min. In a separate
tube, 20 μL of antagonist, i.e., DMSO/Aspirin/compounds
(2a–2t) were dispensed in falcon containing 460 μL whole
blood and stirred for 2 min at 37 °C. Platelet count was
recorded after 6 min on platelet counter (Sysmex XT-1800i)
after adding and mixing 20 μL of agonist, i.e., AA/collagen.
Aspirin was used as a control while percentage inhibition
was calculated by the following formula. All assays were
completed within 90 min of venipuncture.
Iron (Fe2+) chelating activity
Iron (Fe2+) chelating activity was performed by the pre-
viously reported method with slight modifications (Narsin-
ghani et al. 2013). A volume of 40 µL of sample was
incubated with 40 µL ferric chloride (200 µM) and 20 µL of
o-phenanthroline solution (0.05%) at room temperature for
10 min. Absorbance was measured at 510 nm and trolox
was taken as standard.
Percentage inhibition = (Agonist platelet count/Baseline
platelet count) × 100
Results and discussion
Percentage aggregation = [(Baseline platelet count −
Agonist platelet count)/Baseline platelet count] × 100
(Where baseline platelet count is the first blood draw
count at t = 0 min).
1
Characterization: H and 13C NMR spectra
The synthesis of new N′-benzylidene-3-methyl-1-phenyl-6-
p-chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazide
(2a–2t) was carried out in five steps as shown in Scheme 1.
Condensation of 3-aminocrotonitrile with phenyl hydrazine
gave 5-amino-3-methyl-1-phenyl-1H-pyrazole which on
reaction with p-chlorobenzaldehyde followed by ethyl
pyruvate afforded ethyl 6-p-chlorophenyl-3-methyl-1-phe-
nyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylate. The ester
treated with hydrazine monohydrate yielded 6-p-chlor-
ophenyl-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-
carbohydrazide (1) and finally N′-(arylmethylidene)-3-
methyl-1-phenyl-6-p-chlorophenyl-1H-pyrazolo[3,4-b]pyr-
idine-4-carbohydrazides (2a–2t) were prepared from the
condensation of 1 with various aromatic aldehydes in gla-
cial acetic acid in 25–98% yield (Supporting Supplementary
Table S1).
Antioxidant activity
2,2-Diphenyl-1-picrylhydrazyl (DPPH) anti-oxidant activity
Free radical scavenging effect of synthesized compounds
was determined with DPPH assay (Baylac and Racine
2003). Different concentrations (1 and 8 mM, 10 μL) of
compounds in respective solvents were mixed with DPPH
(100 μM, 90 μL) with total volume of 100 µL in 96-well
plates. This reaction mixture was incubated at 37 °C for 30
min. Microplate reader (BIO RAD Model 680) was used to
record the absorbance of resulting reaction mixture at 517
nm. Experiments were performed in triplicate
while trolox was used as standard antioxidant. The
decrease in absorbance showed increased level of radical
scavenging activity which was determined by following
Structure elucidation of synthesized compounds (2a–2t)
was determined using different spectroscopic techniques
such as FT-IR, H NMR, 13C NMR and EI-MS spectral
1

Med Chem Res
Scheme 1 Synthesis of the
target N′-benzylidene-3-methyl-
1-phenyl-6-p-chlorophenyl-1H-
pyrazolo[3,4-b]pyridine-4-
carbohydrazides (2a-2t)
data. The reaction was checked by TLC and purity of
compounds was entrenched from sharp melting points.
Assignment of specific characteristic IR bands furnished the
important indications for formation of (2a–2t). FT-IR
spectra of synthesized compounds displayed their char-
acteristics stretching vibration in the range 3150–3300 cm−1
for NH and 1648–1680 cm−1 for C=O. 1H NMR spectra of
synthesized compounds showed characteristics signals in
the expected regions; 2.62–2.63 ppm for CH3 and 12–13
ppm for NH along the aromatic proton in the respective
region. The 13C NMR clearly showed a CH3 region in
13–14 ppm.
Steric factor also minimized the possibility of Z(>C=N)
geometric isomers (III–IV, Fig. 1). It has been claimed by
the researcher on the basis of X-Ray data that acyl hydrazone
exist only in E(>C=N) geometrical isomers (i.e., I and II,
Fig. 1), their further study confirmed that the duplicate sig-
nals in acyl hydrazones are due to amide conformers [sp
(EC=N, EN–N) (I) and ap(EC=N, E N–N) (II)(Fig. 1) (Glidewell
et al. 2004a, 2004b; E Wyrzykiewicz and Prukala 1998).
Similar conclusions have also been drawn by many
authors utilizing the different NMR techniques (Nakka et al.
2010; Patorski et al. 2012; Syakaev et al. 2006). The
duplicate appearance of signals have been observed in the
NMR spectra of N′-(arylmethylidene)-6-p-chlorophenyl-3-
methyl-1-phenyl-1H-pyrazol[3,4-b]pyridine-4-carbohy-
drazide (2a–2p, 2t), the intensities of duplicate signal
confirmed the existence of syn-periplanar (sp) conformers
as major component as compared to anti-pariplanar (ap)
conformers due to less steric hindrance of attached moieties
in case of sp conformers (Supporting Supplementary Fig.
S1) (Gerardo Palla et al. 1986). The assignment of the
signals for sp and ap conformers is indicated in Supporting
Supplementary Tables S2, S3.
In 1H NMR of compounds (2a–2p, 2t), duplicate
appearance of signals were observed for NH, N=CH, H-5,
CH3 and aryl protons. It has already been reported in lit-
erature that acyl hydrazones normally exist as isomeric
mixture (Koop et al. 2001; G Palla et al. 1982). Depending
on the arrangement of the group around C=N as well as
around N–N and C(O)–N bond, different conformers may
exist (Fig. 1) (Fraser et al. 1981; Uwe Himmelreich et al.
1990; U Himmelreich et al. 1993; Gerardo Palla et al. 1986;
Elzbieta Wyrzykiewicz et al. 2000). Z(N–N) (V–VIII, Fig.
1) conformations could not be realized due to steric repul-
sion between aroyl group and benzylidene moieties.
The NH proton in ap conformer of acylhydrazones (2a–
2p, 2t) appeared downfield to that of NH proton in sp

Med Chem Res
Fig. 1 Eight possible (E and Z)
conformer of acyl-hydrazones
conformers. It can be attributed to the close proximity of
>C=O in ap isomer, which may exert field and anisotropic
effect. The N=CH proton in sp conformers appeared down
field to that of N=CH proton in Eap isomer in most of the
cases probably due to electronic effect (inductive effect,
field effect, and anisotropic effect) of nearby moieties. The
same behavior had already been reported in literature
(Syakaev et al. 2006).
chlorophenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides
(2a–2t). It has been found that all the compounds exhibited
high antioxidant potential except of 2c, 2f, and 2t because
of readily available methyl proton which may scavenge the
ABTS, DPPH, and Fe free radical. Results are tabulated in
Supporting Supplementary Table S4.
The antiplatelet activity has been displayed by all the
compounds but (2c, 2e, 2f, 2g, 2i, 2m, 2o and 2q) have
revealed superlative antiplatelet activity. All the results were
summarized in Table 1.
The signals for H-5 proton in sp was appeared downfield
due to anisotropic effect of >C=O moiety which seems to
be lessen in ap due to distortions in structure planes as a
result of torsional strain of nearby groups. By the similar
reasons, the CH3 group in sp conformers appeared on
downfield as compared to the CH3 in ap (which in most of
the cases is overlapped by residual DMSO signals). The
chemical shift of phenyl protons of benzylidene moiety
have been found to be affected by the existence of sp/ap
conformations. The presence of carbon signals more than
the expected indicate the presence of conformational mix-
ture (Bock and Pedersen 1974). The 1H and 13C NMR
spectra have been recorded in DMSO-d6. Appearance of
double signals, confirmed the formation of sp/ap con-
formations (Supporting Supplementary Fig. S3). Accom-
panying NMR, the compounds (2a–2t) were subjected to
ESI-MS analysis for detecting the molecular mass and
confirmation of synthesized compounds. Results obtained
for structure elucidation of all the synthesized compounds
(2a–2t) provided solid and strong affirmation and endor-
sement for the complete synthesis of target compounds.
Molecular docking studies
Finding interactive residues in COX-1 and COX-2
The main focus of the present work was sorting of most
potent compound for antiplatelet activity from the series
(2a–2t) via Molecular Dockings and computational
screening with targeted protein. The docked compounds (2o
2c, 2e, 2f, 2g, 2l, 2i, 2m, 2o, 2q, 2s and Aspirin) showed the
binding interaction with the pocket residues. Present work
also showed that 11 compounds out of 20 compounds
involved in the hydrogen bond formation with the inter-
acting residues for antiplatelet activity against (COX-1 and
COX-2) (Fig. 2a, b). Thus these synthesized compounds
(2a–2t) can be considered potent antiplatelet drugs and
further be used with diverse approaches including phar-
macological and drug designing (Table 2a, b).
Computational detail
Biological activity
To understand the radical scavenging procedure H-atom
transfer, and one-electron transfer mechanisms are fre-
quently used (Belcastro et al. 2006; Wright et al. 2001).
Usually, H-atom transfer mechanism is used to understand
DPPH, ABTS, iron chelating and antiplatelet activity assays
were used to evaluate the antioxidant and antiplatelet
activity
of
N′-arylmethylidene-3-methyl-1-phenyl-6-

Med Chem Res
Table 1 Antiplatelet activity
results of synthesized
compounds (2a–2t)
Compound no.
R group
AA (100 μM)
Collagen (5 μg/mL)
% Inhibition
% Inhibition
% Aggregation
% Inhibition
2a
2b
2c
H
73.47 2.6
72.60 2.7
86.54 2.1
73.33 3.3
78.94 3.7
70.12 25
70.12 1.5
76.44 2.4
68.59 2.6
71.64 3.4
75.55 2.6
75.30 2.8
74.08 3.7
70.12 2.5
71.34 4.6
80.34 2.3
71.11 1.1
75.55 2.6
68.88 1.9
77.33 3.3
93.2 1.23
26.53 2.8
51.16 1.9
13.45 0.29
26.66 2.7
21.05 2.3
29.87 0.5
29.87 2.5
23.55 2.6
31.40 2.4
28.35 2.6
24.44 2.4
24.69 1.2
25.91 2.3
29.87 0.5
28.65 1.4
20.64 3.0
28.88 1.9
24.44 1.4
31.11 1.1
22.66 2.7
6.81 1.4
73.47 2.6
74.22 2.2
79.81 3.9
78.26 0.7
73.68 2.1
74.26 0.1
75.14 2.1
88.44 1.4
73.68 2.1
78.22 2.2
54.97 0.6
97.07 0.2
80.70 2.5
74.45 0.9
83.62 2.3
79.82 2.6
67.90 0.8
77.77 1.8
83.62 1.3
78.66 2.7
94.41 2.01
26.52 2.4
25.77 1.8
20.18 2.1
21.73 1.3
26.31 1.9
25.73 1.9
24.85 3.1
11.55 1.6
26.31 1.9
21.77 1.8
45.02 2.4
2.923 2.7
19.29 2.5
23.55 1.2
16.37 2.7
20.17 44
32.10 2.1
22.22222
2NO2
3NO2
4NO2
2Br
2d
2e
2f
3Br
2g
2h
2i
4Br
2F
3F
2j
4F
2k
2l
2Cl
3Cl
2m
2n
2o
2p
2q
2r
4Cl
2OCH3
4OCH3
2OH-3OCH3
3OH
4OH
2s
4-N,N(CH3)2
4-Furan-2-yl
16.37 2.7
21.33 3.3
5.59 2.43
2t
Aspirin
the radical scavenging activity for those compounds which
have –OH group. In such compounds, hydrogen atom
transfer would be favorable. While the hydrazone deriva-
tives which have been studied in the present work don’t
have –OH group/groups except 2p–2r. Thus in the present
study, we have shed light on the one-electron transfer
mechanism for the hydrazone derivatives. Recently, density
functional theory (DFT) has been proved as a proficient
method that reproduces the experimental data (Chaudhry
et al. 2014; Chaudhry et al. 2014; Irfan and Al-Sehemi
2014). In the present study, ground state geometries have
been optimized by adopting the B3LYP functional (Irfan
et al. 2016; Irfan et al. 2017) and 6–31G** basis set. The
reactivity descriptors have been computed by DFT
approach from the Eqs. 1–8. Details about methodology can
be found in the reference (Al-Sehemi et al. 2012).
Softness (S) was calculated as:
S ¼ 1=2η
ð4Þ
Electrophilicity index (ωi) was calculated from the fol-
lowing equation:
ωi ¼ m2=2η
ð5Þ
Chemical potential (μ) was calculated from the following
equation:
μ ¼ ÀðHOMO þ LUMO=2Þ
ð6Þ
Ionization potentials (IP) and electron affinities (EA)
have been calculated as under
Mulliken electronegativity (χ) was calculated from as:
IP ¼ ÀEHOMO
EA ¼ ÀELUMO
ð7Þ
ð8Þ
χ ¼ ðEHOMO þ ELUMOÞ=2
ð1Þ
The hardness (η) was computed by using Eq. 2:
All calculations were performed by Gaussian 09 software
(Frisch et al. 2009) except QSAR investigations which have
been done in Spartan ‘14 v1.1.8’ at B3LYP/6-31G* level
which has been proved an efficient and reasonable to
interpret the SAR and other physiochemical properties (Al-
Sehemi et al. 2017; Al-Sehemi et al. 2016).
η ¼ ðELUMO À EHOMOÞ=2
ð2Þ
Electrophilicity (ω) was calculated from the following
Eq. 3:
2
ð3Þ
ω ¼ ðEHOMO þ ELUMO=2Þ =2η

Med Chem Res
Electronic properties
delocalized at the (dimethylaminobenzylidene)-hydrazide
moiety. In 2a, 2e–2m, and 2t, mainly the LUMOs are
localized on the pyrazolo[3,4-b]pyridine moiety (benzyli-
dene and phenyl of chlorophenyl), which are also taking
part in the formation of LUMOs. The intra-molecular
charge transfer (ICT) has been observed from phenyl moi-
ety to the rest of the ring. In 2b–2d, the LUMO is localized
The highest occupied molecular orbitals (HOMOs) and
lowest unoccupied molecular orbitals (LUMOs) have been
displayed in Fig. 3.The HOMOs are delocalized on the 1-
phenyl-1H-pyrazolo[3,4-b]pyridine moiety in all the studied
hydrazone derivatives 2a–2t except 2 s in which HOMO is

Med Chem Res
Fig. 2 a Two and three dimensional interaction diagrams of 2e, 2g, 2l,
2o and Aspirin using Naproxen pocket against COX-1 and COX-2.
Interaction diagrams were attained by using ligand interaction analysis
feature of MOE. b Two and three dimensional interaction diagrams of
2c, 2e, 2f, 2i, 2m, 2o, 2q, 2s and Aspirin using selected residues.
Interaction diagrams were attained by using ligand interaction analysis
feature of MOE while Arg120, Arg83, and Tyr355 residue of COX-1
were used
on the nitro-benzylidene unit revealing ICT from 1-phenyl-
1H-pyrazolo[3,4-b]pyridine to nitro-benzylidene moiety. In
2n–2s, the LUMO is localized on the pyrazolo[3,4-b]pyr-
idine. The ICT has been observed from phenyl to the pyr-
azolo[3,4-b]pyridine and chlorophenyl. As in 2b–2d, the
strong electron deactivating group is at ortho, meta, and
para positions, respectively as a result nitro-benzylidene
moiety withdraw electron toward itself while in 2n–2o,
electron activating group methoxy is substituted in corollary
methoxybenzylidene is usually not taking part in the for-
mation of the LUMO. It is expected that strong electron
activating group –N(CH3)2 in 2s is playing significant role
by which HOMO is distributed at (dimethyl amino-benzy-
lidene)-hydrazide unit. In Aspirin, the HOMO is distributed
at phenol moiety and the LUMO at phenol as well as
carboxylic group leading the ICT from phenol to the
anchoring site (carboxylic group). In trolox, the HOMO is
distributed at conjugated ring along with some charge at
–OH and –CH3 groups while the LUMO is localized at
whole of the molecule revealing ICT from left to the right
side of the system.
In Supporting Supplementary Table S5, we have tabu-
lated the HOMO energies (EHOMO), LUMO energies
(ELUMO), HOMO–LUMO energy gaps (Egap), IP, and EA at
the B3LYP/6-31G** level of theory. The EHOMO levels of
indigenously synthesized and studied hydrazone derivatives
are lower than the trolox while higher than Aspirin while
Egap is smaller than the trolox and Aspirin. The largest Egap
has been observed for the 2j, 2n–2o amongst all the
hydrazone derivatives. Moreover, with respect to charge

Med Chem Res
Enzyme selected
Table 2 (A) Docking result
using the selected naproxen
pocket for COX-1 and 2 and (B)
selected residues
(A)
Compound
Van der Waal forces (kcal/mol)
Interacting residues
2e (R = 2Br)
2g (R = 4Br)
2l (R = 3Cl)
Aspirin
−3.08
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg120, Tyr355
COX-1
COX-1
COX-1
COX-1
COX-2
COX-2
−21.57
−17.78
−15.61
−10.72
−11.48
2o (R = 2OCH3)
Aspirin
Arg120, Tyr355
Tyr355
(B)
sp Compound
2c (R = 3NO2)
2e (R = 2Br)
2f (R = 3Br)
2i (R = 3 F)
2m (R = 4Cl)
2o (R = 2OCH3)
2q (R = 3OH)
Van der Waal forces (kcal/mol)
Interacting residues
Enzyme selected
COX-1
−6.58
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
Arg83, Arg120, Tyr355
−3.20
COX-1
−14.54
−16.48
−17.53
−19.35
−12.99
COX-1
COX-1
COX-1
COX-1
COX-1
PDB Code = 1Q4G and 3NT1 for COX-1 and COX-2, respectively from already published articles
transfer ability of the compounds, it is expected that the
higher EA of 2b–2m might lead to enhanced electron
transport aptitude in these contenders than the 2a. The
scavenging of free radical can be evaluated by single
electron donation. IP is an important descriptor by which
the range of electron transfer can be assessed. By removing
electron from HOMO one-electron transfer radical cation
can be gained. It can be seen that 2b, 2e, 2h, 2k, 2n, 2o, and
2t have the smaller IP values as compared to the parent
compound 2a revealing that in these materials electron
transfer mechanism might be more encouraging for the
scavenging of free radicals (Supporting Supplementary-
Table S5).
this MEP surface maps, it is expected that the oxygen of
C=O and nitrogen of pyrazole would be favorable elec-
trophilic reactive sites and benzylidene-hydrazine moiety as
a nucleophilic reactive site. Furthermore, in 2b–2d, the nitro
group would be favorable electrophilic reactive site.
QSAR study
Different QSAR descriptors, e.g., μD = dipole moment,
area, volume, partition coefficient (Log P), hydrogen bond
donor (HBD), hydrogen bond acceptor, polar surface area
(PSA), solvation energy, and polarizability of hydrazone
derivatives have been tabulated in Table 3. Previously, it
has been shown that PSA should not exceed 120 A2 for the
orally active drug which are transported by trans cellular
route (Kelder 1999; van de Waterbeemd et al. 1998) and
<100 A2 for brain penetration (van de Waterbeemd et al.
1998) or <60–70 A2 (Kelder et al. 1999). The PSA of all the
studied hydrazone derivatives is less than 120 A2. More-
over, the PSA of all the studied hydrazone (except 2b–2d)
derivatives is also smaller than 60 A2. On other, lipophili-
city of the studied compounds ranges from 5.56 to 8.26.
The lipophilicity increases by substituting the electron
deactivating groups (–F, –Cl, –Br, and NO2) while the
electron activating group (–OCH3) and furan moiety
decreases lipophilicity than parent compound 2a. From the
previous studies, it is widely accepted that the compounds
having very high/low log P values do not have good bio-
availability as they cannot cross hydrophilic and lipophilic
barricades, respectively. However, these compounds might
be used as lead compounds to synthesize better antiplatelet
Molecular electrostatic potential (MEP)
The 3-D surface maps of the MEP are best tools to know the
molecular interactions as well as the relative reactivity sites
for the nucleophilic and electrophilic attack. In Fig. 4, we
have illustrated the MEP surface maps of hydrazone deri-
vatives from 2a–2t along with reference compounds to
understand the positive and negative electrostatic potential
(ESP) regions. In Fig. 4, red, blue and green colors repre-
sent the negative, positive and zero potential regions. It is
anticipated that the electrophilic and nucleophilic attack
would be favorable on the negative and positive ESP. The
negative ESP can be seen on the oxygen of C=O and
nitrogen of pyrazole in 2a whereas the positive ESP at
benzylidene-hydrazine moiety. Almost analogous tendency
has been observed in all the studied derivatives. In 2b–2d,
the negative ESP can also be seen on the nitro group. From

Med Chem Res
Fig. 3 The distribution pattern of the HOMOs and LUMOs of the studied compounds (2a–2t) at ground states

Med Chem Res
Fig. 4 The molecular electrostatic potential surfaces of the N′-benzylidene derivatives (2a–2t). Red, blue and green color regions represented the
negative, positive and zero electrostatic potential of derivative (2a–2t) (color figure online)
Table 3 Different QSAR
μD (Debye)
Area (A2)
Volume (A3)
Log P HBD HBA Pol.
PSA (A2) S.E. (KJ/mol)
descriptors of (2a–2t) obtained
at B3LYP/6-31 G** level of
theory
2a
2b
2c
2d
2e
2f
4.92
8.77
6.94
4.18
6.02
5.41
3.81
6.18
5.23
4.18
6.24
5.40
3.82
5.24
6.99
7.91
5.31
4.61
8.08
5.83
479.29
502.94
505.38
505.22
491.56
499.8
462.52
484.27
484.15
484.11
480.19
480.69
480.66
466.87
467.14
467.14
476.10
476.25
476.24
489.39
489.62
496.44
469.62
469.60
511.76
440.96
174.86
264.27
7.43
7.46
7.46
7.46
8.26
8.26
8.26
7.59
7.59
7.59
7.99
7.99
7.99
7.30
7.14
6.75
7.04
7.04
7.67
5.56
1.18
4.29
0
1
1
1
1
1
1
1
1
0
1
1
1
0
0
1
1
1
0
1
1
2
6
9
9
9
6
6
6
6
6
6
6
6
6
7
7
8
7
7
7
7
7
2
78.02 47.51
79.97 85.16
79.9 86.70
−28.37
−45.2
−43.31
−34.97
−32.11
−33.27
−31.09
−31.46
−27.05
−24.95
−32.62
−31.8
79.54 86.61
79.47 46.49
79.51 47.69
2g
2h
2i
499.7
79.5
47.62
482.9
78.39 46.05
78.41 47.60
78.39 47.59
79.14 46.96
79.15 47.65
79.14 47.65
80.20 52.60
80.21 54.50
80.76 70.95
78.59 67.22
78.59 67.21
82.09 48.34
76.29 54.91
54.25 51.99
61.50 49.81
485.15
485.12
493.46
495.22
495.2
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
−29.57
−32.44
−35.38
−38.73
−48.77
−49.24
−33.65
−32.89
−33.03
−34.81
506.42
509.10
515.79
488.19
488.15
530.67
460.01
196.35
275.19
Aspirin 2.65
Trolox 2.78
μD dipole moment, HBD hydrogen bond donor, HBA hydrogen bond acceptor, PSA polar surface area, S.E.
solvation energy, Pol. polarizability

Med Chem Res
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agents with good log P value. Similar compounds have
been reported in literature having better biological activities
with high log P value (Dias et al. 2007).
Conclusion
In summary, we evaluated the potential of novel N′-ben-
zylidene-3-methyl-1-phenyl-6-p-chlorophenyl-1H-pyrazolo
[3,4b]pyridine-4carbohydrazides (2a–2t) as new antith-
rombotic agents. These compounds were synthesized
starting from the simple chemicals via functional group
replacement approach and their structures were elucidated
by various spectroscopic techniques. All compounds except
of 2c, 2f, 2t have exhibited high antioxidant potential and
compounds (2c, 2e, 2f, 2g, 2i, 2m, 2o, 2q) have showed
superlative antiplatelet activity, respectively. Docking
results revealed that compounds involved in the hydrogen
bond formation with the interacting residues for antiplatelet
activity against (COX-1 and COX-2) and were found in
accordance with the experimental results. QSAR results are
in good agreement with antiplatelet and antioxidant activ-
ities of the compounds (2a–2t). Our finding suggest that the
2g, 2l and 2m might be promising antiplatelet agents and
will be helpful in the designing of new drugs for the
treatment of cardiovascular, thromboembolic, and anti-
inflammatory diseases.
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Acknowledgements We are thankful to Higher Education Com-
mission of Pakistan for research funding Indigenous 5000 PhD fel-
lowship Batch VII (PIN # 117-6703-PS7-083) and Ex-Supervisor
Professor Dr. Riffat Parveen (Late) for her countless efforts. Professor
Dr. Makshoof Athar, Director, Institute of Chemistry, University of the
Punjab, Lahore for his support and providing facilities to carry on this
research work. We are also thankful to University of Health Sciences,
Lahore for providing the Elisa reader and platelet counter facility.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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