Synthesis, Characterization, Molecular Docking Studies and Anticancer Activity of Schiff Bases Derived from 3-(Substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde and 2-Aminophenol

Article abstract of DOI:10.1007/s10870-018-0727-1

Abstract: A series of new Schiff bases were synthesized by microwave assisted reactions of substituted 1-phenyl-1H-pyrazole-4-carbaldehyde and 2-aminophenol in ethanol and characterized by elemental analysis and spectroscopic (IR, ¹H NMR and MS) data. The crystal structures of four compounds were studied using single-crystal XRD data. Molecular docking studies of all synthesized compounds were performed into the binding site of a protein 3GCW to gain comprehensive understanding into possible binding modes. These compounds were also screened for anticancer activity against the liver (HEP-G2) cell line using the sulphorhodamine-B assay method. Adriamycin i.e. doxorubicin was used as reference standard. One of the compounds shows anticancer activity close to the famous anticancer agent doxorubicin, which was used as control in this study. It is observed that all molecules show activity close to the standard in high concentrations only. Graphical Abstract: Present study describes the anticancer activity and crystal structure study of Schiff bases of substituted pyrazole-4-carbaldehyde with 2-aminophenol. Docking study of all compounds against human hepatoma cell line, HEP-G2 correlates with in vitro activity. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s10870-018-0727-1

Journal of Chemical Crystallography
https://doi.org/10.1007/s10870-018-0727-1
ORIGINAL PAPER
Synthesis, Characterization, Molecular Docking Studies and Anticancer
Activity of Schiff Bases Derived from 3-(Substituted phenyl)-1-phenyl-
1H-pyrazole-4-carbaldehyde and 2-Aminophenol
Sachin S. Wazalwar1 · Anita R. Banpurkar1 · Franc Perdih2
Received: 13 April 2017 / Accepted: 13 August 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of new Schiff bases were synthesized by microwave assisted reactions of substituted 1-phenyl-1H-pyrazole-4-car-
baldehyde and 2-aminophenol in ethanol and characterized by elemental analysis and spectroscopic (IR, 1H NMR and MS)
data. The crystal structures of four compounds were studied using single-crystal XRD data. Molecular docking studies of
all synthesized compounds were performed into the binding site of a protein 3GCW to gain comprehensive understanding
into possible binding modes. These compounds were also screened for anticancer activity against the liver (HEP-G2) cell
line using the sulphorhodamine-B assay method. Adriamycin i.e. doxorubicin was used as reference standard. One of the
compounds shows anticancer activity close to the famous anticancer agent doxorubicin, which was used as control in this
study. It is observed that all molecules show activity close to the standard in high concentrations only.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10870-018-0727-1) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Journal of Chemical Crystallography
Graphical Abstract
Present study describes the anticancer activity and crystal structure study of Schiff bases of substituted pyrazole-4-car-
baldehyde with 2-aminophenol. Docking study of all compounds against human hepatoma cell line, HEP-G2 correlates
with in vitro activity.
Keywords Schiff base · Pyrazole · Single crystal XRD · Docking study · In vitro anticancer study · 3GCW · HEP-G2
the presence of a lone pair of electrons in a sp2 hybridized
Introduction
orbital of the nitrogen atom of the azomethine group is of
considerable chemical and biological importance. Schiff-
base compounds containing an imino group (–C=N–) are
potential anticancer drugs. Many symmetric and asymmet-
ric Schiff bases are derived from salicylaldehyde and other
suitable amines to mimic biological systems [14]. Due to
the straight forward preparation, synthetic flexibility and
the special property of the C=N group, Schiff bases are
generally excellent chelating agents [15–21], especially
when a functional group like–OH is present close to the
azomethine group. Schiff bases have also been reported
to show biological properties, such as antibacterial and
antifungal activities [22–24].
Heterocyclic compounds including pyrazole moieties
receive considerable attention owing to their diverse chemo-
therapeutic actions [1–4]. Drugs based on heterocyclic com-
pounds bearing 1,2-diarylsubstituted pyrazole are among
the list of best-selling pharmaceutical products. Important
pyrazole-based drugs available in the market are apixaban,
celecoxib, fipronil, remogliflazone etabonate, lonazolac, tol-
piprazole, deracoxib, and many more. Crizotinib and ruxoli-
tinib are important pyrazole tethered anticancer drugs [5].
Pyrazole derivatives show anticancer activity due to their
inhibition of various targets such as topoisomeraseII [6–8].
Hence, incorporation of the pyrazole moiety is an important
synthetic strategy in the rational anticancer drug develop-
ment process (Figs. 1, 2).
This article reports the efficient synthesis of the men-
tioned nitrogen containing heterocycles using ethanol as
a solvent by a microwave-assisted synthetic route. Along
with routine spectroscopic studies to ascertain the struc-
ture, an attempt to obtain single-crystals of as many com-
pounds as possible in order to understand structural fea-
tures, bond length, bond angles and atomic spacing are
presented. Molecular docking studies were carried out to
Schiff bases are condensation products of primary
amines with carbonyl compounds. A common structural
feature of these compounds is azomethine group with the
general formula RHC=N–R1, where R and R1 are alkyl,
aryl, cycloalkyl or heterocyclic groups, which may be vari-
ously substituted [9]. Several studies [10–13] showed that
1 3

Journal of Chemical Crystallography
F
Cl
F3C
F
O
N
N
N
N
O
OH
N
N
O
S
O
O
S
O
NH2
(a)
NH2
(b)
(c)
NH2
F
O
O
F
Cl
N
F
N
NH2
N
N
HN
O
N
O
N
N
S
N
F
Cl
N
F
F
O
N
(d)
(e)
(f)
Fig. 1 Pyrazole based drugs. a Deracoxib, b lonazolac, c celecoxib, d apixaban, e tolpiprazole, f fipronil
Fig. 2 Target compounds
N
N
R
N
N
N
R
OH
OHC
3a–i
4a–i
identify the potential binding affinities and the mode of
interaction of the compounds against protein 3GCW, and
anticancer studies were performed using the liver (HEP-
G2) cell line.
1 3

Journal of Chemical Crystallography
respectively, represent Ar–OH protons related to OH sub-
stituted acetophenone. Singlets at 8.45–8.86 ppm represent
the proton in Schiff base (CH=N). 4d shows a singlet at
2.36 ppm for Ar–CH3. A singlet at 3.80 ppm represents the
–OCH3 group in 4e. Singlets at 3.85–3.88 ppm represent
two –OCH3 in 4f. Furthermore, the mass spectra and ele-
mental analyses support the structures of compound 4a–i.
Results and Discussion
Chemistry
The synthesis of intermediate and target compounds is
outlined in Scheme 1. Pyrazole containing Schiff bases
4a–i were synthesized from corresponding substituted
1,3-diphenyl-1H-pyrazole-4-carbaldehyde (3a–i) and
2-aminophenol. 3a–i were initially synthesized by the
well-known Vilsmeier-Haack reaction from phenyl hydra-
zones (2a–i) obtained from substituted acetophenone and
phenyl hydrazine Schiff bases (1a–i). Progress of the reac-
tion was monitored by TLC. All synthesized compounds
Single Crystal X‑ray Study
Table 1 describes crystallographic data for 3d, 3f, 4a and
4b (Fig. 3).
The compound 3d crystallizes in the triclinic space group
P–1 (Table 1) with four unique molecules in the asymmet-
ric unit, and compound 3f crystallizes in the monoclinic
space group P21/c with a single unique molecule in the
asymmetric unit. All bond lengths of compounds 3d and
3f are within normal ranges (Table 2). In both compounds
the 1-phenyl-1H-pyrazole moieties are almost planar with
dihedral angles between phenyl and pyrazole rings in the
range of 4.41(10)°–12.92(10)° in 3d and 10.77(8)° in 3 f.
The p-tolyl rings in 3d are inclined to the pyrazole rings
with dihedral angles in the range of 26.13(9)°–31.45(9)°. In
3f a dimethoxyphenyl group is attached to the central pyra-
zole ring at position three with the dihedral angle between
both rings of 34.10(7)°. The dihedral angles between the
carbaldehyde groups and pyrazole rings in the four unique
molecules of 3d are in the range of 7.3(3)°–12.3(3)° and
in 3f this angle is 7.6(3)°. In the crystal structures of 3d
and 3f, molecules are linked via C–H⋯O hydrogen bonding
1
were characterized by H NMR, IR spectroscopy, mass
spectrometry, and elemental analysis.
The IR spectra of compounds 3a–i showed characteristic
υ(CH=O) absorption bands in the region 1660–1677 cm−1
and υ(C=N) bands in the 1513–1522 cm−1 region. The
1HNMR spectra of compounds 3a–i in CDCl3 exhibit
characteristic singlets at 9.95–9.99 ppm attributed to
–CH=O protons, and singlets at 8.46–9.28 ppm attrib-
uted to pyrazolic –CH protons. The IR spectra of com-
pounds 4a–i showed characteristic υ(Ar–OH) absorp-
tion bands in the 3261–3644 cm−1 region υ(CH=N) in
the region 1609–1665 cm−1, and υ(N–N) in the region
1519–1542 cm−1. The 1H NMR spectra of compounds 4a–i
in CDCl3 exhibit characteristic signals at 9.75–10.66 ppm
attributed to Ar–OH protons from aminophenols. Whereas
for compound 4g and 4h singlets at 9.28 and 9.98 ppm,
Scheme 1 General procedure
for synthesis of substituted
pyrazole carbaldehyde and
its Schiff bases with 2-amino
phenol
1 3

Journal of Chemical Crystallography
Table 1 Crystallographic data
for 3d, 3f, 4a and 4b
Compound code
3d
3f
4a
4b
CCDC number
Empirical formula
Formula weight
Color
1534085
C17H14N2O
262.30
Yellow
Plate
1534084
C18H16N2O3
308.33
Yellow
Block
1534087
C22H17N3O
339.38
Colorless
Needle
1534512
C44H34Br2N6O3
854.59
Yellow
Block
Crystal morphology
Temperature (K)
Crystal system
Space group
293(2)
Triclinic
P–1
293(2)
Monoclinic
P 21/c
293(2)
Monoclinic
P 21/c
150(2)
Monoclinic
Cc
Unit cell dimensions (Å)
a
b
c
9.8493(3)
16.1937(6)
17.9391(9)
83.026(4)
74.225(3)
89.748(3)
7.9168(2)
14.5524(3)
13.5459(3)
90
95.213(2)
90
9.8869(2)
21.6405(4)
33.3631(5)
90
96.8060(10)
90
7088.0(2), 16
1.272
0.635
35.003(2)
9.6628(3)
26.5471(10)
90
114.083(4)
90
8197.4(7), 8
1.382
2.882
3456
9734
1013
1.027
α
β
γ
Volume (Å3), Z
2731.87(19), 8 1554.15(6), 4
Calculated density (g cm−3
)
1.276
0.641
1104.0
20,537
726
1.318
0.743
648.0
11,002
211
Absorption coefficient, µ (mm−1
F(000)
)
2848
13,409
941
Reflections collected
Parameters
Goodness-of-fit on F2
R_obs, wR2_obs
1.033
1.015
1.002
0.0508, 0.1602 0.0400, 0.1240 0.0530, 0.1485 0.0951, 0.2785
−0.214, 0.222 −0.158, 0.168 −0.216, 0.184 −1.267, 1.428
Δρmin, Δρmax (eÅ−3
)
Table 2 Important bond distances of 3d and 3f (assigned for each
asymmetric unit in a crystal for all molecules)
Bond distance (Å)
NPy–CAr
3d
3f
N
N1–C5: 1.423(2)
N3–C22: 1.419(2)
N5–C39: 1.4256(19)
N7–C56: 1.423(2)
N1–C4: 1.4278(19)
R
N
O
CPy–CCHO
CPy–CAr
HC=O
C2–C4: 1.450(2)
C2–C18: 1.447(2)
C1–C10: 1.4712(18)
C18–O1: 1.215(2)
C19–C21: 1.443(2)
C36–C38: 1.444(2)
C53–C55: 1.444(2)
Fig. 3 Important bond distances (Å) observed in 3d and 3f
C1–C11: 1.4792(19)
C18–C28: 1.4784(19)
C35–C45: 1.473(2)
C52–C62: 1.472(2)
C4–O1: 1.2105(19)
C21–O2: 1.212(2)
C38–O3: 1.2115(17)
C55–O4: 1.2094(18)
C14–C17: 1.509(2)
C31–C34: 1.513(2)
C48–C51: 1.511(2)
C65–C68: 1.510(3)
where the carbaldehyde groups (in 3d and 3f) as well as one
methoxy group (in 3f) act as a hydrogen bond acceptors
(Table 3). The crystal structure of 3d is stabilized by addi-
tional π⋯π interactions between the five membered pyrazole
rings and the six membered phenyl rings of the adjacent
molecules with centroid-to-centroid distances in the range
of 3.8482(11)–3.9675(11) Å, and dihedral angles between
the rings in the range of 6.41(9)°–13.85(10)°, whereas in
3f a π⋯π interaction between adjacent parallel five mem-
bered pyrazole rings with centroid-to-centroid distance of
3.7086(8) Å is present (Table 4). Furthermore, in structure
3d C–H⋯π and C=O⋯π contacts are also present (Table 5)
(Figs. 4, 5, 6).
CAr–R*
C12–O2: 1.3652(16)
C13–O3: 1.3640(16)
*–R is a substituent on phenyl ring of pyrazole-4-carbaldehyde
1 3

Journal of Chemical Crystallography
Table 3 Hydrogen bond and
C–H⋯π geometry of 3d, 3f, 4a
and 4b
D–H⋯A
D–H (Å)
H⋯A (Å)
D⋯A (Å)
D–H⋯A (°)
Symmetry code
3d
C3–H3⋯O2
C10–H10···O2
C20–H20···O1
C27–H27···O1
C37–H37···O4
C40–H40···O4
C54–H54···O3
C57–H57···O3
C13–H13···Cg6
C47–H47···Cg12
C63 –H63···Cg9
3f
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
0.93
2.40
2.48
2.41
2.51
2.36
2.50
2.42
2.50
2.90
2.95
2.97
3.3161(19)
3.400(2)
3.3233(19)
3.409(2)
3.278(2)
3.386(2)
3.3409(19)
3.434(2)
3.548(2)
3.588(2)
3.682(2)
169
168
168
163
169
159
170
176
128
127
134
1−x, 1−y, 1−z
1−x, 1−y, 1−z
1−x, 1−y, 1−z
1−x, 1−y, 1−z
1−x, 1−y, −z
1−x, 1−y, −z
1−x, 1−y, −z
1−x, 1−y, −z
1−x, −y, 1−z
1−x, −y, −z
1+x, y, z
C5–H5···O3
C8–H8···O1
0.93
0.93
2.50
2.45
3.3254(19)
3.268(2)
148
147
2−x, ½+y, 1½−z
1−x, ½ + y, ½−z
4a
O1–H1A···O3
O1–H1A···N1
O2–H2A···O4
O2–H2A···N4
O3–H3A···O2
O3–H3A···N7
O4–H4A···O1
O4–H4A···N10
C30–H30···O4
C52–H52···O2
C74–H74···O1
C38–H38···Cg3
4b
0.82
0.82
0.82
0.82
0.82
0.82
0.82
0.82
0.93
0.93
0.93
0.93
2.44
2.20
2.44
2.21
2.37
2.21
2.28
2.21
2.48
2.52
2.48
2.86
3.079(3)
2.662(2)
3.112(3)
2.668(3)
3.046(3)
2.670(3)
2.960(3)
2.677(2)
3.383(3)
3.389(3)
3.330(3)
3.648(3)
135
116
140
116
140
116
141
116
165
156
152
143
1+x, y, z
x, y, z
1+x, y, z
x, y, z
x, y, z
x, y, z
−1+x, y, z
x, y, z
1+x, y, z
x, y, z
−1+x, y, z
x, y, z
O1–H1···N1
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.84
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
0.95
2.23
2.48
2.21
2.43
2.21
2.50
2.20
2.18
2.43
2.80
2.50
2.91
2.45
2.47
2.87
2.87
2.89
2.81
2.90
2.93
2.683(14)
3.070(16)
2.678(14)
3.022(15)
2.654(14)
3.259(16)
2.665(14)
2.916(14)
3.267(16)
3.635(14)
3.328(16)
3.729(16)
3.266(18)
3.296(15)
3.569(11)
3.651(11)
3.675(12)
3.543(12)
3.461(13)
3.740(13)
114
128
115
129
113
151
115
147
147
147
146
145
144
145
131
141
141
134
119
144
x, y, z
O1–H1···O4
O2–H2···N4
O2–H2···O3
−½ + x, ½−y, −½ + z
x, y, z
−½ + x, −½−y, −½ + z
x, y, z
½ + x, ½−y, ½ + z
x, y, z
½ + x, ½−y, ½ + z
−½ + x, ½−y, −½ + z
x, y, z
−½ + x, −½−y, −½ + z
x, y, z
½ + x, ½−y, ½ + z
½ + x, ½−y, ½ + z
x, y, z
x, −1+y, z
x, −1+y, z
O3–H3A···N7
O3–H3A···O1
O4–H4A···N10
O4–H4A···O2
C8–H8···O4
C25–H25···Br3
C30–H30···O3
C47–H47···Br2
C52–H52···O1
C74–H74···O2
C34–H34···Cg3
C37–H37···Cg3
C56–H56···Cg15
C59–H59···Cg15
C65–H65···Cg16
C72–H72···Cg8
x, y, z
x, y, z
½ + x, −½−y, ½ + z
Ring centroids for 3d: Cg6 (C28–C33), Cg9 (C45–C50), and Cg12 (C62–C67); for 4a–b: Cg3 (C11–C16),
Cg8 (C39–C44), Cg15 (C77–C82), and Cg16 (C83–C88)
1 3

Journal of Chemical Crystallography
Table 4 π···π interactions in 3d, 3f and 4a
Cg···Cg
CgI···CgJ (Å)
α (°)
β (°)
CgI-Perp (Å)
Slippage (Å)
Symmetry code
3d
Cg1···Cg5
Cg2···Cg4
Cg7···Cg11
Cg8···Cg10
3f
3.9675(11)
3.8482(11)
3.9614(11)
3.8668(11)
13.85(10)
6.41(9)
6.64(10)
10.16(10)
29.90
24.82
29.63
20.64
−3.7700(7)
3.2991(8)
3.6102(7)
–
–
–
–
1+x, y, z
x, y, z
−1+x, y, z
x, y, z
−3.3396(8)
Cg1···Cg1
4a
Cg1···Cg8
3.7086(8)
0
18.03
25.97
−3.5265(6)
3.0961(9)
1.148
–
1−x, 1−y, 1−z
4.2699(14)
17.65(12)
x, y, z
CgI···CgJ, α, β and CgI-Perp are, respectively, the centroid-to-centroid distance between rings I and J, the inter-ring dihedral angle, slip angle,
and the perpendicular distance of CgI from ring J. Ring centroids for 3d–f: Cg1 (N1/N2/C1–C3), Cg2 (C5–C10), Cg4 (N3/N4/C18–C20), Cg5
(C22–C27), Cg7 (N5/N6/C35–C37), Cg8 (C39–C44), Cg10 (N7/N8/C52–C54), Cg11 (C56–C61), for 4a: Cg1 (N2/N3/C8–C10), Cg8 (C39–
C44)
R
N
N
N
OH
Fig. 4 ORTEP plot for 3d
Fig. 6 Important bond distances (Å) observed in 4a and 4b
solvate molecules are present in 4b. All bond lengths of
compounds 4a and 4b are within normal ranges (Tables 5,
6). In both compounds the phenol and pyrazole rings
connected via the azomethine group are nearly copla-
nar; the dihedral angles between both rings are in the
range of 2.45(13)°–8.28(13)° in 4a and 6.8(7)°–8.4(6)°
in 4b. The phenyl rings attached to the pyrazole at posi-
tion 1 are inclined to the pyrazole rings with the dihedral
angles in the range of 14.22(13)°–22.76(13)° in 4a and
13.2(7)°–16.4(6)° in 4b. The aromatic ring attached to
pyrazole at position 3 is twisted out of the pyrazole plane
by 32.87(12)°–39.12(13)° in 4a and 27.7(7)°–30.9(6)° in
Fig. 5 ORTEP plot for 3f
4b. In both crystal structures intramolecular O–H⋯N and
intermolecular O–H⋯O hydrogen bonding is present as
well as C–H⋯O hydrogen bonding (Table 3). Further-
more, C–H⋯π interactions are observed, and C–H⋯Br
Compounds 4a and 4b crystallize in the monoclinic
space groups P21/c and Cc, respectively,with four unique
molecules in the asymmetric unit. Additionally, water
1 3

Journal of Chemical Crystallography
Table 5 C=O···π geometry of 3d
as a drug and the second is the drug’s biological target such
as a protein receptor [25]. In calculating the docking score,
the small organic molecule or ligand is considered as a
rigid structure and its interactions with protein receptors are
recorded and expressed in terms of energy associated with
such interactions. In calculations of the Glide energy value,
the conformational, orientational, and positional space of
the docked ligand are taken into consideration [26]. Thus,
the Glide energy value is obtained with torsionally flexible
energy optimization where the ligand is a flexible entity
and all its possible geometrical orientations are considered.
Hence, the Glide energy value is a more reliable, enhanced,
and realistic approach to investigate the docking process.
Both docking score or docking and Glide energy are free
energy values and expressed in kcal mol−1. The docking
studies were carried out using Swissdock, an online web-
server provided by the Molecular Modeling group of the
Swiss Institute of Bioinformatics. Output files were studied
using Chimera 1.1 software by University of California, San
Francisco. Molecular docking of all synthesized compounds
into the binding site of 3GCW protein was performed to gain
a comprehensive understanding into possible binding modes.
Our study and synthetic strategy aims towards synthesis of
a potential anticancer molecule for liver cancer. 3GCW is
the unique name of the PDB file for PCSK9, which is a
secreted protein that degrades low density lipoprotein recep-
tors (LDRs) in the liver by biding to the epidermal growth
factor [27]. It is a part of Hep G2, which is a human liver
cancer cell line. All the synthesized compounds exhibited
good Glide energy and docking scores towards the recep-
tor active pocket, and bind to the active site of the amino
D–H···A
O···Cg (Å)
C=O··· Cg (°)
Symmetry code
3d
C4=O1···Cg4
C21=O2···Cg1
3.4806(16)
3.5294(16)
75.74(11)
81.79(11)
73.59(10)
82.56(12)
1+x, y, z
x, y, z
−1+x, y, z
x, y, z
C38=O3···Cg10 3.4620(15)
C55=O4···Cg7 3.6294(17)
Ring centroids for 3d: Cg1 (N1/N2/C1–C3), Cg4 (N3/N4/C18–C20),
Cg7 (N5/N6/C35–C37), and Cg10 (N7/N8/C52–C54)
interactions are present in 4b. The crystal structure of 4a
is stabilized by additional π⋯π interaction between pyra-
zole rings and phenyl rings of adjacent molecules with
centroid-to-centroid distances of 4.2699(14) Å and a dihe-
dral angle between the rings of 17.65(12)°. No significant
π⋯π interactions are present in 4b (Figs. 7, 8).
Molecular Docking Studies
Molecular docking is a tool used in computer aided structure
based rational drug design. It is designed to predict how
a small molecule binds to a target protein with known 3D
structure, and binding energies determined. Also the posi-
tion of the ligand in the hosts binding site can be visual-
ized. It is helpful for developing better drug candidates
with improved activity. The docking score is the measure of
strength of the non-covalent interaction or binding affinity
between two molecules after they have been docked. Usu-
ally one of the molecules is a small organic compound such
Table 6 Important bond
Bond type
4a
4b
distances (Å) in 4a and 4b
Azomethine C=N
N1–C7: 1.266(3)
N4–C29: 1.270(3)
N7–C51: 1.261(3)
N10–C73: 1.272(3)
N1–C7: 1.292(16)
N4–C29: 1.295(16)
N7–C51: 1.281(16)
N10–C73: 1.280(16)
CAr–NAmine bond distance
C–N in pyrazole ring
N1–C2: 1.407(3)
N4–C24: 1.411(3)
N7–C46: 1.407(3)
N10–C68: 1.407(3)
N1–C2: 1.402(15)
N4–C24: 1.381(15)
N7–C46: 1.423(16)
N10–C68: 1.441(16)
N2–C10: 1.336(3), N3–C8: 1.344(3)
N2–C8: 1.361(15)
N5–C32: 1.332(3), N6–C30: 1.344(3) N3–C10: 1.293(19)
N8–C54: 1.333(3), N9–C52: 1.347(3) N5–C30: 1.369(15),
N11–C76: 1.326(3), N12–C74:
1.349(3)
N6–C32: 1.310(18)
N8–C52: 1.344(17),
N9–C54: 1.354(19)
N11–C74: 1.333(16),
N12–C76:
1.342(19)
N–N in pyrazole ring
N2–N3: 1.363(2)
N5–N6: 1.368(2)
N8–N9: 1.360(2)
N11–N12: 1.363(3)
N2–N3: 1.379(17)
N5–N6: 1.359(17)
N8–N9: 1.349(17)
N11–N12: 1.349(17)
1 3

Journal of Chemical Crystallography
Table 7 Docking interaction with 3GCW protein
Compound
Docking score Glide energy Hydrogen bonding
(kcal mol−1
−7.62
)
(kcal mol−1
−48.65
)
4a
4b
VAL650, ASP422,
GLU426
−7.52
−41.18
LYS243, TYR166,
MET398
4c
4d
4e
4f
4g
4h
−7.67
−7.41
−8.11
−8.48
−8.17
−8.24
−45.58
−44.45
−60.39
−66.49
−57.22
−61.74
LYS243, MET398
LEU135, PHE318
LYS243
LSN207, LYS243
ALA420, GLU159
MET398, ALA242,
TYR166
Fig. 7 Wireframe representation of the crystal structure of 4a show-
ing four molecules in asymmetric unit
4i
−8.15
Doxorubicin −8.40
−61.28
−57.58
GLN172
ALA242
acid residue with H-bonding very close to the standard drug
doxorubicin (Table 7). Among them, the conformations with
lowest binding energies and those ligands exhibiting well
established hydrogen bonds with available oxygen and nitro-
gen in the closest range of 2–4 Å with one or more amino
acids in the receptor active pockets were selected as best
docked ligands. Compound 4f showed the highest dock-
ing score and a Glide energy lower than that of standard
doxorubicin. Its hydrogen bonding distances to amino acids
ASN207and LYS243 are comparatively shorter compared to
those found in doxorubicin. Compound 4f can be considered
to be the best docked ligand with the highest docking score
and lowest Glide energy. The compounds 4e, 4g, 4h and 4i
showed docking scores very close to the standard drug, but
Glide energies were found to be comparatively lower. Com-
pounds 4a, 4b, 4c, 4d have moderate docking scores and
Glide energies. In this study compound 4c showed the short-
est hydrogen bonding distance of 1.12 Å with the amino acid
likely due to presence of the more electronegative chlorine
atom. It is also observed that the presence of electronegative
atoms like oxygen and nitrogen effectively enhances docking
activity (Figs. 9, 10).
Anticancer Activity
In vitro anticancer screening for the synthesized compounds
and doxorubicin as a reference drug control against the liver
Hep G2 cancer cell line was determined using the sulphro-
damine-B assay method.
The cell lines were grown in a RPMI 1640 medium con-
taining 10% fetal bovine serum and 2 mM l-glutamine. For
the presented screening experiment, cells were inoculated
into 96-well micro-titer plates in 90 µL at 5000 cells per
well. After cell inoculation, the microtiter plates were incu-
bated at 37 °C, 5% CO2, 95% air and 100% relative humid-
ity for 24 h prior to addition of the experimental drugs.
The experimental drugs were solubilized in appropriate
solvent to prepare stock solutions with a 10−2 concentra-
tion. At the time of the experiment, four tenfold serial dilu-
tions were made using the complete medium. Aliquots of
10 µL of these different drug dilutions were added to the
appropriate micro-titer wells already containing 90 µL of
medium, resulting in the required final drug concentra-
tions. After compound addition, plates were incubated at
standard conditions for 48 h and assays were terminated
Fig. 8 Wireframe representa-
tion of the crystal structure of
4b showing four molecules in
asymmetric unit
1 3

Journal of Chemical Crystallography
Fig. 9 Docking pose of doxorubicin with 3GCW protein
Fig. 10 Docking pose of 4c with 3GCW protein
by the addition of cold TCA. Cells were fixed in situ by
the addition of 50 µL of cold 30% (w/v) TCA (final con-
centration, 10% TCA) and incubated for 60 min at 4 °C.
The supernatant was discarded; the plates were washed
five times with tap water and air dried. Sulforhodamine B
(SRB) solution (50 µL) at 0.4% (w/v) in 1% acetic acid was
added to each of the wells, and plates were incubated for
20 min at room temperature. After staining, unbound dye
was recovered and the residual dye was removed by washing
five times with 1% acetic acid. The plates were air dried.
Bound stain was subsequently eluted with 10 mM trizma
base, and the absorbance was read on an Elisa plate reader
at a wavelength of 540 nm with 690 nm reference wave-
length. Percent growth was calculated on a plate-by-plate
basis for test wells relative to control wells. Percent growth
was expressed as the ratio of average absorbance of the test
well to the average absorbance of the control wells× 100.
Using the six absorbance measurements [time zero (Tz),
control growth (C), and test growth in the presence of
drug at the four concentration levels (Ti)], the percentage
growth was calculated at each of the drug concentration
levels. The dose response parameters were calculated for
1 3

Journal of Chemical Crystallography
Table 9 Molar drug concentration required
each test article. Growth inhibition of 50% (GI50) was cal-
culated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the
drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control
cells during the drug incubation. The drug concentration
resulting in total growth inhibition (TGI) was calculated
from Ti=Tz. The LC50 (concentration of drug resulting in
a 50% reduction in the measured protein at the end of the
drug treatment as compared to that at the beginning) indi-
cating a net loss of cells following treatment is calculated
from [(Ti − Tz)/Tz] × 100 = − 50. Values were calculated
for each of these three parameters if the level of activity
was reached; however, if the effect was not reached or was
exceeded, the values for that parameter were expressed as
greater or less than the maximum or minimum concentra-
tion tested [28, 29].
Hep-G2
LC50
TGI
GI50*
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.035753
1.361952
0.205286
0.129875
0.356036
0.020293
0.004815
13.6133
0.001468
0.016786
0.004279
0.002903
0.006219
0.000773
0.000304
0.101864
0.002516
3.11E−07
6.02E−05
0.000207
8.92E−05
6.49E−05
0.000109
2.94E−05
1.92E−05
0.000762
7.16E−05
2.21E−09
0.088407
4.39E−05
ADR
LC50 =concentration of drug causing 50% cell kill
GI50 =concentration of drug causing 50% inhibition of cell growth
TGI=concentration of drug causing total inhibition of cell growth
ADR=adriamycin, positive control compound
Test growth of human hepatoma cell line Hep-G2 was
calculated experimentally at four different concentration lev-
els of each compound (Table 8). Result shows that the syn-
thesized compounds show activity at higher concentration
of 10−4 M. ADR (Adriamycin, positive control compound)
shows % control growth − 61.8 at 10−4 M concentration.
Conclusion
In this study a series of novel 2-{[3-(substitutedphenyl)-
1-phenyl-1H-pyrazol-4-ylmethylene]amino}phenol (4a–i)
have been synthesized from substituted pyrazole-4-car-
baldehydes by microwave-assisted method, which provided
rapid, environmentally benign synthesis. The structures
of all newly synthesized compounds were characterized
by IR, NMR, mass spectrometry, elemental analysis and
X-ray crystal structure analyses. Molecular docking study
of all synthesized compounds into the binding site of
protein 3GCW were provide understanding into possible
binding modes and showed significant activity. Compound
4f showed highest docking score and Glide energy, even
greater than doxorubicin (standard). Compounds 4e, 4g,
Compound 4g shows % control growth of −17.1 at 10−4
M
concentration close to ADR. The GI50 value of 4g was also
found close to that of ADR. The study shows that the pres-
ence of four-substituted OH group increases the antican-
cer activity. The GI50 (concentration of drug causing 50%
inhibition of cell growth) for 4f and 4g was found to be
2.94E−05 and 1.92E−05, respectively, and close to that of
ADR (2.21E−09, Table 9) (Figs. 11, 12).
150.0
Table 8 Test growth of human hepatoma cell line Hep-G2 at four dif-
4a
ferent concentration levels
4b
100.0
Compound
Average value
10−7
4c
M
10−6
M
10−5
M
10−4
M
4d
50.0
4a
4b
4c
4d
4e
4f
4g
4h
4i
121.9
114.0
116.1
112.9
115.5
110.5
115.8
119.8
115.8
11.2
132.6
122.1
120.4
117.9
118.7
123.1
125.0
133.1
130.3
−14.7
126.4
130.0
118.5
115.4
120.4
103.2
106.8
128.1
116.5
−28.4
3.7
24.1
17.6
12.8
20.1
−0.3
−17.1
43.0
12.6
−61.8
4e
4f
0.0
10-7M
10-6M
10-5M
10-4M
4g
4h
4i
-50.0
-100.0
Fig. 11 Graphical representation of % control growth of 4a–i and
ADR
ADR against molar drug concentration
1 3

Journal of Chemical Crystallography
Fig. 12 Images for in vitro anticancer study of 4f (left) and 4g (right)
4h, 4i showed docking scores very close to doxorubicin
with greater glide energies than doxorubicin. Compound
4c showed the shortest hydrogen bonding distance of
1.12 Å among all compounds. This study reveals that the
presence of more electronegative Cl, N and O atoms in a
compound enhances docking activity. Compounds 4f and
4g show anticancer activity close to doxorubicin, which
was used as control in this study. It is observed that all
molecules show activity close to the standard (control) at
high concentration only.
X-ray Structure Determination
All the crystals were obtained by slow evaporation of clear
solution of the compounds at room temperature. A 50:50
mixture of ethanol and chloroform was used as solvent.
Single-crystal X-ray diffraction data were collected on
Agilent Technologies SuperNova Dual diffractometer with
an Atlas detector using monochromated Cu-Kα radiation
(λ=1.54184 Å) at room temperature or at 150 K. The data
were processed using CrysAlis Pro [30]. The structures were
solved by direct methods using the program SHELX2014
[31] or charged-flipping methods using the program Super-
flip [32] and refined on F2 using full-matrix least-squares
procedures (SHELX2014). All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were readily located
in difference Fourier maps and were subsequently treated
as riding atoms in geometrically idealized positions with
Uiso(H)=kUeq(C, N or O), where k=1.5 for OH and methyl
groups, which were permitted to rotate but not to tilt, and 1.2
for all other H atoms unless otherwise noted. In the crystal
structure 4b water oxygen atoms O5 and O6 as well as O7
and O8 are disordered over two positions with refined ratio
of 0.40:0.60 and 0.65:0.35, respectively, and ISOR restraints
were used for their refinement. Hydrogen atoms on water
oxygen atoms O5–O8 were not found in difference Fourier
maps and were not included in the refinement. Crystallo-
graphic data are listed in Table 1.
Experimental Protocols
General (Materials and Methods)
All starting materials and solvents were purchased from
commercial sources and used without further purification.
A microwave oven of SAMSUNG, 800 W output with digi-
tal timer and clock was used for syntheses. Melting points
were determined in open capillaries using an electro thermal
melting point apparatus and are uncorrected. The progress
of reactions was monitored by TLC. Infrared (IR) spectra
(4000–600 cm−1) of the samples were recorded using a Per-
kin–Elmer Spectrum 100, equipped with a Specac Golden
Gate Diamond ATR as a solid sample support. Elemental
(C, H, N) analyses were obtained using a Perkin–Elmer 2400
1
Series II CHNS/O Elemental Analyzer. H NMR spectra
were recorded with a Bruker Avance III 500 nuclear mag-
netic resonance spectrometer with TMS as internal refer-
ence. MS spectra were recorded with a VG-Analytical Auto-
Spec Q instrument.
General Procedure
Synthesis of 3-(Substituted
phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
A mixture of substituted acetophenone 1a–i (0.01 M), phe-
nyl hydrazine (0.01 M), one drop of conc. HCl and etha-
nol as a solvent was taken in a 100 mL conical flask and
1 3

Journal of Chemical Crystallography
subjected to microwave irradiation for 1–2 min at 450 W
at an interval of 5 s each. The resulting solid products 2a–i
were dried, dissolved in excess of DMF, and 0.015 M POCl3
was added dropwise under cold conditions. The reaction
mixture is allowed to attain room temperature and refluxed
at about 80 °C for 1 h conventionally, allowed to cool and
then hydrolyzed by water to yield a white solid. The solid
was washed with ample water to remove excess HCl, then
filtered, dried and recrystallised using ethanol. Recrystallisa-
tion proceeded from a saturated solution in a mixture of eth-
anol and chloroform in a dark chamber at low temperature.
1H, Pyrazole-CH); 7.71–7.73 (d, 4H, Ar–H); 7.42–7.45
(t, 2H, Ar–H); 7.30–7.33 (t, 1H, Ar–H); 6.94–6.97 (d, 2H,
Ar–H), 3.80 (s, 3H, OCH3).
3 - ( 3 , 4 - D i m e t h o x y p h e n y l ) - 1 - p h e n y l - 1 H- p y r a -
zole-4-carbaldehyde(3f) Brown, yield 65%, melting point
80 °C. IR (υmax/cm−1): 3010 (Ar–CH), 2835 (C–H in
1
CHO), 1660 (C=O), 1522 (C=N), 1442 (C=C). H NMR
(500 MHz, CDCl3, δ/ppm): 9.99 (s, 1H, –CHO); 8.45 (s,
1H, Pyrazole-CH); 7.72–7.73 (d, 2H, Ar–H); 7.43–7.46 (t,
2H, Ar–H); 7.31–7.38 (m, 3H, Ar–H); 6.91–6.93 (d, 1H,
Ar–H); 3.88 (s, 3H, OCH3); 3.91 (s, 3H, OCH3).
Spectral Data
3 - ( 4 - H y d r ox y p h e ny l ) - 1 - p h e ny l - 1 H- p y r a z o l e - 4 -
carbaldehde(3g) Off white, yield 76%, melting point
150 °C. IR (υmax/cm−1): 3337 (Ar–OH), 3110 (Ar–CH),
2815 (C–H in CHO), 1680 (C=O), 1513 (C=N), 1445
1,3-Diphenyl-1H-pyrazole-4-carbaldehde(3a) Off
white
needle like crystals, yield 89%, Melting point 135 °C. IR
(υmax/cm−1): 3121 (Ar–CH), 2828 (C–H in CHO), 1677
1
1
(C=O), 1519 (C=N), 1443 (C=C). H NMR (500 MHz,
(C=C); H NMR (500 MHz, CDCl3, δ/ppm): 9.95 (s, 1H,
CDCl3, δ/ppm): 9.96 (s, 1H, –CHO); 8.46 (s, 1H, Pyrazole-
CH); 7.68–7.72 (t, 4H, Ar–H); 7.55–7.57 (d, 2H, Ar–H);
7.43–7.46 (t, 2H, Ar–H); 7.32–7.35 (t, 1H, Ar–H).
–CHO); 9.79 (s, 1H, Ar–OH); 9.28 (s, 1H, Pyrazole-CH);
7.97–7.98 (d, 2H, Ar–H); 7.77–7.79 (d, 2H, Ar–H); 7.55–
7.58 (t, 2H, Ar–H); 7.40–7.43 (t, 1H, Ar–H); 6.87–6.89 (d,
2H, Ar–H).
3 - ( 4 - B r o m o p h e n y l ) - 1 - p h e n y l - 1 H- p y r a z o l e - 4 -
carbaldehyde(3b) Off white needle like crystals, yield 88%,
melting point 130 °C. IR (υmax/cm−1): 3124 (Ar–CH), 2828
(C–H in CHO), 1677 (C=O), 1519 (C=N), 1443 (C=C). 1H
NMR (500 MHz, CDCl3, δ/ppm): 9.99 (s, 1H, –CHO); 8.48
(s, 1H, Pyrazole-CH); 7.72–7.76 (m, 4H, Ar–H); 7.43–7.46
(t, 4H, Ar–H); 7.31–7.34 (t, 1H, Ar–H).
3 - ( 3 - H y d r ox y p h e ny l ) - 1 - p h e ny l - 1 H- p y r a z o l e - 4 -
carbaldehde(3h) Off white, yield 72%, melting point
90 °C. IR (υmax/cm−1): 3443 (Ar–OH), 3124 (Ar–CH), 2896
(C–H in CHO), 1660 (C=O), 1519 (C=N), 1443 (C=C);
1H NMR (500 MHz, CDCl3, δ/ppm): 9.98 (s, 1H, –CHO);
9.66 (s, 1H, Ar–OH); 9.29 (s, 1H, Pyrazole-CH); 7.98–7.99
(d, 2H, Ar–H); 7.56–7.59 (t, 2H, Ar–H); 7.42–7.45 (t, 1H,
Ar–H); 7.29–7.35 (m, 2H, Ar–H); 6.88–6.89 (d, 1H, Ar–H).
3 - ( 4 - C h l o r o p h e n y l ) - 1 - p h e n y l - 1 H- p y r a z o l e - 4 -
carbaldehyde(3c) Off white needle like crystals, yield 87%,
melting point 122 °C. IR (υmax/cm−1): 3124 (Ar–CH), 2835
(C–H in CHO), 1667 (C=O), 1513 (C=N), 1449 (C=C),
3 - ( 3 - N i t r o p h e n y l ) - 1 - p h e n y l - 1 H- p y r a z o l e - 4 - c
arbaldehyde(3i) Greenish yellow, yield 87%, melting
point 140 °C. IR (υmax/cm−1): 3131 (Ar–CH), 2835 (C–H
in CHO), 1680 (C=O), 1596 (C=N), 1525 (C–NO2) 1425
1
678 (C–Cl). H NMR (500 MHz, CDCl3, δ/ppm): 9.97 (s,
1H, –CHO); 8.46 (s, 1H, Pyrazole-CH); 7.75–7.77 (d, 2H,
Ar–H); 7.71–7.72 (d, 2H, Ar–H); 7.44–7.47 (t, 2H, Ar–H);
7.40–7.42 (d, 2H, Ar–H); 7.32–7.35 (t, 1H, Ar–H).
1
(C=C); H NMR (500 MHz, CDCl3, δ/ppm): 10.02 (s,
1H, –CHO); 8.51 (s, 1H, Pyrazole-CH); 8.24–8.27 (t, 2H,
Ar–H); 7.74–7.75 (d, 2H, Ar–H); 7.59–7.63 (t, 1H, Ar–H);
7.46–7.50 (t, 2H, Ar–H); 7.35–7.38 (t, 1H, Ar–H).
1-Phenyl-3-p-tolyl-1H-pyrazole-4-carbaldehyde(3d) Off
white, yield 89%, melting point 100 °C. IR (υmax/cm−1):
3125 (Ar–CH), 2838 (C–H in CHO), 1673 (C=O), 1518
(C=N), 1450 (C=C). 1H NMR (500 MHz, CDCl3, δ/ppm):
9.98 (s, 1H, –CHO); 8.46 (s, 1H, Pyrazole-CH); 7.71–7.73
(d, 2H, Ar–H); 7.62–7.63 (d, 2H, Ar–H); 7.42–7.45 (t, 2H,
Ar–H); 7.30–7.33 (t, 1H, Ar–H); 7.23–7.25 (d, 2H, Ar–H);
2.36 (s, 3H, Ar–CH3).
Synthesis of 2-[(3-(Substituted
phenyl)-1-phenyl-1H-pyrazole 4-ylmethylene)amino]
phenol
A series of Schiff bases 4a–i was synthesized according to
Scheme 1. A mixture of synthesized 3-(substituted phenyl)-
1-phenyl-1H-pyrazole-4-carbaldehyde (0.01 M) 3a–i, 2-ami-
nophenol (0.01 M), one drop of conc. HCl and ethanol as a
solvent was subjected to microwave irradiation for 2–3 min
at 450 W power at an interval of 5 s each, which results in
the formation of yellow solid on cooling. The products were
washed with cold water followed by washing with saturated
3 - ( 4 - M e t h ox y p h e ny l ) - 1 - p h e ny l - 1 H- p y r a z o l e - 4 -
carbaldehyde(3e) Off white, yield 88%, melting point
110 °C. IR (υmax/cm−1): 3123 (Ar–CH), 2834 (C–H in
1
CHO), 1667 (C=O), 1515 (C=N), 1455 (C=C). H NMR
(500 MHz, CDCl3, δ/ppm): 9.97 (s, 1H, –CHO); 8.45 (s,
1 3

Journal of Chemical Crystallography
solutions of sodium bisulphate to remove excess of unre-
acted aldehyde, filtered, dried and recrystallised using hot
ethanol. Further recrystallisation was performed in a mixture
of ethanol and chloroform (1:1 v/v) by slow evaporation in
a dark chamber at low temperature.
Found: C, 77.59%; H, 5.19%; N, 11.60%. MS (ESI+)
m/z354 (MH+). HRMS calcd. for C29H20N3O: 354.1606.
Found: 354.1604. IR (υmax/cm−1): 3372 (Ar–OH), 3138
1
(Ar–CH), 1624 (HC=N), 1595 (C=C), 1537 (N–N). H
NMR (500 MHz, CDCl3, δ/ppm): 9.97 (s, 1H, Ar–OH);
8.64 (s, 1H, HC=N); 8.45 (s, 1H, Ar–H); 7.54–7.55 (d,
2H, Ar–H); 7.43–7.45 (t, 2H, Ar–H); 7.31 (t, 1H, Ar–H);
7.23–7.24 (d, 2H, Ar–H); 7.18 (s, 1H, Ar–H); 7.08–7.14
(m, 2H, Ar–H); 6.93–6.94 (d, 1H, Ar–H); 6.78–6.81 (t,
1H, Ar–H); 2.36 (s, 3H, Ar–CH3).
2-[(1,3-Diphenyl-1H-pyrazol-4-ylmethylene)amino]
phenol(4a) Pale yellow needle like crystals, yield 84%,
melting point 142 °C. Anal. Calcd. for C22H17N3O (339.14
g mol−1): C, 77.86%; H, 5.05%; N, 12.38%. Found: C,
77.76%; H, 5.15%; N, 12.32%. MS (ESI+) m/z 340 (MH+).
HRMS calcd. for C22H18N3O: 340.1450. Found: 340.1455.
IR (υmax/cm−1): 3426 (Ar–OH), 3141 (Ar–CH), 1613
2-{[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-ylmeth-
ylene]amino}phenol(4e) Dark yellow needle like crys-
tals, yield 82%, melting point 140 °C. MS (ESI+) m/z 370
(MH+). HRMS calcd. for C23H20N3O2: 370.1550. Found:
370.1549. IR (υmax/cm−1): 3676 (Ar–OH), 3127 (Ar–CH),
1665 (HC=N), 1595 (C=C), 1519 (N–N), 2980 (OCH3).
1H NMR (500 MHz, CDCl3, δ/ppm): 9.97 (s, 1H, Ar–
OH); 8.44 (s, 1H, HC=N); 8.45 (s, 1H, Ar–H); 7.71–7.73
(d, 4H, Ar–H); 7.42–7.45 (t, 2H, Ar–H); 7.30–7.33 (t, 1H,
Ar–H); 7.18 (s, 1H, Ar–H); 6.95–6.97 (d, 2H, Ar–H); 3.80
(s, 3H, Ar–OCH3).
1
(HC=N), 1596 (C=C), 1542 (N–N). H NMR (500 MHz,
CDCl3, δ/ppm): 9.99 (s, 1H, Ar–OH); 8.48 (s, 1H, HC=N);
8.56 (s, 1H, Ar–H); 7.84–7.85 (d, 4H, Ar-H); 7.59–7.63(d,
4H, Ar–H); 6.95–7.02 (m, 3H, Ar–H); 6.80–6.85 (t, 3H,
Ar–H).
2-{[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-ylmeth-
ylene]amino}phenol(4b) Pale yellow needle like crys-
tals, yield 88%, melting point 158 °C. Anal. Calcd. for
C22H16BrN3O·½H2O (427.26 g mol−1): C, 61.87%;
H, 4.01%; N, 9.83%. Found: C, 61.37%; H, 3.58%; N,
9.57%. MS (ESI+) m/z 418 (MH+). HRMS calcd. for
2-{[3-(3,4-Dimethoxyphenyl)-1-phenyl-1H-pyrazol-4-yl-
methylene]amino}phenol(4f) Dark brown needle like
crystals, yield 89%, melting point 135 °C. Anal. Calcd. for
C24H21N3O3·H2O (417.42 g mol−1): C, 69.05%; H, 5.55%;
C22H17BrN3O: 418.0555. Found: 418.0546. IR(υmax
/
cm−1): 3390 (Ar–OH), 3132 (Ar–CH), 1624 (HC=N), 1595
1
(C=C), 1537 (N–N), 686 (Ar–Br); H NMR (500 MHz,
N, 10.06%. Found: C, 68.87%; H, 5.20%; N, 9.31%. IR (υmax/
CDCl3, δppm): 9.96 (s, 1H, Ar–OH); 8.61 (s, 1H, HC=N);
8.46 (s, 1H, Ar–H); 7.76–7.77 (d, 2H, Ar–H); 7.54–7.59
(m, 3H, Ar–H); 7.44–7.47 (t, 2H, Ar–H); 7.31–7.34 (t,
1H, Ar–H); 7.19 (s, 1H, Ar–H); 7.10–7.15 (m, 2H, Ar–H);
6.80–6.83 (t, 1H, Ar–H); 6.94–6.96 (d, 2H, Ar–H).
cm−1): 3644 (Ar–OH), 3127 (Ar–CH), 1665 (HC=N), 1595
1
(C=C), 1519 (N–N), 2980 (OCH3). H NMR (500 MHz,
CDCl3, δ/ppm): 9.99 (s, 1H, Ar–OH); 8.65 (s, 1H, HC=N);
8.45 (s, 1H, Ar–H); 7.86–7.87 (d, 2H, Ar–H); 7.72–7.73
(d, 2H, Ar–H); 7.07–7.08 (d, 2H, Ar–H);6.90–6.92 (d, 2H,
Ar–H); 7.18 (s, 1H, Ar–H); 6.78–6.81 (t, 1H, Ar–H); 3.88
(s, 4H, Ar–OCH3); 3.85 (s, 2H, Ar–OCH3).
2-{[3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-ylmeth-
ylene]amino}phenol(4c) Dark yellow needle like crys-
tals, yield 87%, melting point 145 °C. Anal. Calcd. for
C22H16ClN3O (373.83 g mol−1): C, 70.68%; H, 4.31%;
N, 11.24%. Found: C, 70.31%; H, 4.12%; N, 10.87%. MS
(ESI+) m/z 374 (MH+). HRMS calcd. for C22H17ClN3O:
374.1060. Found: 374.1059. IR (υmax/cm−1): 3390 (Ar–
OH), 3137 (Ar–CH), 1619 (HC=N), 1595 (C=C), 1537
2-{[3-(4-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-ylmeth-
ylene]amino}phenol(4g) Pale yellow solid, yield 81%,
melting point 135 °C. Anal. Calcd. For C22H17N3O3
(355.39 g mol−1): C, 74.35%; H, 4.82%; N, 11.82%. Found:
C, 73.08%; H, 4.75%; N, 11.32%. MS (ESI+) m/z 356
(MH+). HRMS calcd. for C22H17N3O3: 356.1399. Found:
356.1399. IR (υmax/cm−1): 3425 (Ar–OH), 3150 (Ar–
1
(N–N), 835 (Ar–Cl). H NMR (500 MHz, CDCl3, δ/
1
ppm): 9.97 (s, 1H, Ar–OH); 8.61 (s, 1H, HC=N); 8.46
(s, 1H, Ar–H); 7.76–7.78 (d, 2H, Ar–H); 7.60–7.61 (d,
1H, Ar–H); 7.47–7.41 (m, 3H, Ar–H); 7.32–7.34 (t, 1H,
Ar–H); 7.19 (s, 2H, Ar–H); 7.15–7.10 (m, 2H, Ar–H);
6.95 (d, 1H, Ar–H); 6.83–6.80 (t, 1H, Ar–H).
CH), 1609 (HC=N), 1554 (C=C), 1540 (N–N). H NMR
(500 MHz, CDCl3, δ/ppm): 9.75 (s, 1H, Ar–OH); 9.28 (s,
1H, Ar–OH); 8.83 (s, 1H, HC=N); 8.66 (s, 1H, Ar–H);
7.97–7.98 (d, 2H, Ar–H); 7.63–7.64 (d, 2H, Ar–H); 7.55–
7.57 (t, 2H, Ar–H); 7.38–7.39 (t, 2H, Ar–H); 7.19–7.20 (d,
1H, Ar–H); 7.04–7.06 (t, 1H, Ar–H); 6.90 (t, 3H, Ar–H);
6.80–6.91 (t, 1H, Ar–H).
2-[(1-Phenyl-3-p-tolyl-1H-pyrazol-4-ylmethylene)amino]
phenol(4d) Dark brown needle like crystals, yield
82%, melting point 120 °C. Anal. Calcd. for C29H19N3O
(353.42 g mol−1): C, 78.16%; H, 5.42%; N, 11.86%.
2-{[3-(3-Hydroxyphenyl)-1-phenyl-1H-pyrazol-4-ylmethyl-
ene]amino}phenol(4h) Greenish yellow solid, yield 78%,
1 3

Journal of Chemical Crystallography
melting point 175 °C. MS (ESI+) m/z 356 (MH+). HRMS
calcd. for C22H18N3O3: 356.1399. Found: 356.1394. IR
(υmax/cm−1): 3261 (Ar–OH), 3091 (Ar–CH), 1665 (HC=N),
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1
1595 (C=C), 1529 (N–N). H NMR (500 MHz, CDCl3, δ/
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ppm): 10.66 (s, 1H, Ar–OH); 9.98 (s, 1H, Ar–OH); 9.30 (s,
1H, HC=N); 7.98–7.99 (d, 2H, Ar–H); 7.57–7.59 (t, 2H,
Ar–H); 7.42–7.44 (t, 1H, Ar–H); 7.35–7.28 (m, 4H, Ar–H);
7.20–7.22 (t, 2H, Ar–H); 7.00–7.01 (d, 1H, Ar–H); 6.85–
6.89 (m, 2H, Ar–H).
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2-{[3-(3-Nitrophenyl)-1-pheny1H-pyrazol-4-ylmethylene]
amino}phenol(4i) Greenish yellow solid, yield 76%, melt-
ing point 180 °C. MS (ESI+) m/z 385 (MH+). HRMS calcd.
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for C22H17N4O3: 385.1301. Found: 385.1296. IR (υmax
/
cm−1): 3148 (Ar–OH), 3080 (Ar–CH), 1683 (HC=N), 1585
(C=C), 1519 (N–N), 1525 (Ar–NO2). 1H NMR (500 MHz,
CDCl3, δ/ppm): 10.02 (s, 1H, Ar–OH); 8.79 (s, 1H, HC=N);
8.51 (s, 1H, Ar–H); 8.24–8.27 (t, 2H, Ar–H); 7.75–7.75 (d,
2H, Ar–H); 7.60–7.61 (t, 1H, Ar–H); 7.48 (t, 2H, Ar–H);
7.37(t, 1H, Ar–H); 6.19 (s, 3H, Ar–H); 6.97 (s, 1H, Ar–H).
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Acknowledgements The authors are thankful to the Anti-Cancer Drug
screening facility (ACDSF) at ACTREC, Tata Memorial Centre, Navi
Mumbai for providing the in vitro SRB assay for anti-cancer activity eval-
uation of drugs. Financial support from the Slovenian Research Agency
(ARRS) through project P1-0175 is gratefully acknowledged. We thank
the EN-FIST Centre of Excellence, Ljubljana, Slovenia, for using the
SuperNova diffractometer. The authors acknowledge SwissDock, a free
protein ligand docking web service powered by EADock DSS by the
Molecular Modeling group of the Swiss Institute of Bioinformatics for
providing docking service. Authors are also thankful to The University
of California, San Francisco for use of Chimera 1.1 software to analyze
the results of docking.
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Affiliations
Sachin S. Wazalwar1 · Anita R. Banpurkar1 · Franc Perdih2
2
* Sachin S. Wazalwar
Faculty of Chemistry and Chemical Technology, University
sacheein@gmail.com
of Ljubljana, Večna pot 113, 1000 Ljubljana, Slovenia
1
Department of Applied Chemistry, Rajiv Gandhi College
of Engineering, Research and Technology, Chandrapur,
MS 442403, India
1 3