Design, synthesis and spectroscopic and crystallographic characterisation of novel functionalized pyrazole derivatives: biological evaluation for their cytotoxic, angiogenic and antioxidant activities

Article abstract of DOI:10.1007/s11164-018-3445-6

Abstract: The current study presents the synthesis of functionalized pyrazoles (18–29) through 3 + 2 annulation reaction of ethyl 2-(arylidene)-3-oxobutanoates (8–13) with phenylhydrazine hydrochlorides (14–17) in acetic acid under reflux conditions. Stru

Full text of DOI:10.1007/s11164-018-3445-6

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3445-6
Design, synthesis and spectroscopic
and crystallographic characterisation of novel
functionalized pyrazole derivatives: biological
evaluation for their cytotoxic, angiogenic
and antioxidant activities
2
3
•
Achutha Dileep Kumar1 Srinivasan Bharath Rekha N. Dharmappa
•
•
Shivalingegowda Naveen4 Neratur Krishnappagowda Lokanath5
•
•
Kariyappa Ajay Kumar1
Received: 7 February 2018 / Accepted: 18 April 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract The current study presents the synthesis of functionalized pyrazoles (18–29)
through 3 ? 2 annulation reaction of ethyl 2-(arylidene)-3-oxobutanoates (8–13) with
phenylhydrazine hydrochlorides (14–17) in acetic acid under reflux conditions. Struc-
tures of the synthesized new compounds were characterized by spectral and single-
crystal X-ray diffraction studies. Preliminary assessment on their biological activities
showed that compounds 19, 23 and 28 have anticancer and antiangiogenic properties and
compounds 20, 24 and 26 have excellent diphenylpicrylhydrazyl (DPPH) radical scav-
enging activities. Detailed quantitative structure–activity relationship (QSAR) analysis
provided insights into the molecular features that might have contributed towards
increasing potency of inhibition. In summary, we present a study that has successfully
demonstrated the synthesis of novel pyrazole analogues that display anticancer,
antiangiogenic and DPPH free radical scavenging activities, making them lead mole-
cules of choice for further development.
&
Kariyappa Ajay Kumar
ajaykumar@ycm.uni-mysore.ac.in
1
2
3
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysuru 570005, India
Marie Skłodowska-Curie Actions, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal
Department of Biotechnology, JSS College for Arts, Commerce and Science, Ooty Road,
Mysuru, India
4
5
Department of Physics, School of Engineering and Technology, Jain University,
Bangalore 562 112, India
Department of Studies in Physics, Manasagangotri, University of Mysore, Mysuru, India
123

A. Dileep Kumar et al.
Graphical Abstract
Keywords Antiangiogenic Á Antioxidant Á Citotoxic Á Condensation Á Pyrazoles
Introduction
Cancer is a disease characterized by the progressive, persistent, abnormal and
uncontrolled proliferation of cells. It is the second most dreaded cause of mortality
in the world after cardiovascular disorders, and is continuing to be a major health
hazard in developing as well as undeveloped countries [1]. Although there has been
considerable progress to reduce cancer incidence, the number of cancer patients
continues to increase. Chemotherapy is one of the most effective approaches used to
treat cancer patients. However, lack of selectivity and drug resistance reduce the
efficacy of cancer chemotherapy. Therefore, development of effective and safe
anticancer agents with high potency and less toxicity is a major focus for researchers
across the world. The most important aspects in drug design are the affinity of the
small molecule for its target, the specificity of its action, drug metabolism and
bioactivation. Amongst the heterocycles, pyrazoles are the scaffolds of choice in
several medicinal chemistry initiatives. Derivatives of pyrazoles have been shown
to possess varied properties including, but not limited to, being analgesic [2],
antiarrhythmic [3], sPLA2-inhibitory [4], anti-inflammatory [5], antipyretic [6],
anticancer [7], anticonvulsant [8], monoamine oxidase inhibiting [9], antidiabetic
[10], and antibacterial [11]. Pyrazoles have been employed as bifunctional ligands
for metal catalysis, and in various building blocks for medicinal chemistry and
pharmaceutical research. Pyrazoles are synthesized by various conventional routes
[12, 13]. For instance, highly regioselective synthesis of phosphonylpyrazoles by
the reaction of chalcones with an a-diazo-b-ketophosphonate [14], and reaction of
b-enaminoketoesters with phenylhydrazines via a simple and reliable approach have
been successfully demonstrated [15]. The usual acid-catalyzed reaction of a, b-
unsaturated carbonyl compounds with phenylhydrazines to produce pyrazolines is
well established [16]. On the other hand, in the presence of mild bases, reaction of
a, b-unsaturated carbonyl compounds having a benzotriazole group in the a position
with phenylhydrazine produces pyrazoles directly [17].
Angiogenesis is a feature of embryonal development and has prominent roles in
several physiological and pathological conditions, including rheumatoid arthritis,
tumor growth and metastasis, diabetic retinopathy and age-related macular
123

Design, synthesis and spectroscopic and crystallographic…
degeneration [18]. Angiogenesis-dependent diseases are controlled by using
chemotherapy, immunotherapy and radiation therapy by stimulating the inhibiting
factors [19]. In view of the important roles played by angiogenesis and the fact that
aberrant angiogenesis can sometimes result in pathological and diseased conditions,
there is additional need to synthesize small molecules regulating angiogenesis.
Additionally, deleterious effects arising due to oxidative stress resulting from an
imbalance between free radical generation and their quenching on human health has
huge economic burden. Given the high economic costs of healthcare vis a vis
treatment of conditions such as carcinogenesis, aberrant angiogenesis and free
radical-mediated damage [20], in this paper, we report for the first time the direct
synthesis of functionalized pyrazoles and the results of their cytotoxic, antiangio-
genic and diphenylpicrylhydrazyl (DPPH) radical scavenging activities.
Experimental
Materials and methods
Melting points were determined by an open capillary tube method and are
1
uncorrected. H Nuclear Magnetic Resonance (NMR) and 13C Nuclear Magnetic
Resonance (NMR) spectra were recorded on Agilent 400-MHz and 100-MHz
spectrometers, respectively. The chemical shifts are expressed in d ppm. Mass
spectra were obtained on Electrospray (ESI)/Atmospheric Pressure Chemical
Ionization (APCI)-Hybrid Quadrupole, Synapt G2 High Definition Mass Spectrom-
etry (HDMS) Acquity Ultimate Performance Liquid Chromatography (UPLC)
model spectrometer. Elemental analysis was obtained on a Thermo Finnigan Flash
EA 1112 Elemental analyzer.
Synthesis of benzylidine oxobutanoates, 8–13
To a solution of aromatic aldehydes 1–6 (10 mol) and ethyl acetoacetate 7 (10 mol)
in dichloromethane, a catalytic amount of piperidine (1 mL) and trifluoroacetic acid
(2 mL) were added. Then the solution mixture was refluxed in a water bath for
4–5 h. The progress of the reaction was monitored by thin layer chromatography
(TLVC) and after completion, the reaction mixture was cooled to room temperature
and poured into ice-cold water. The products were extracted into diethyl ether
(50 mL), washed successively with saturated sodium bicarbonate solution, brine
solution and finally with ice-cold water. The organic layer was dried over anhydrous
sodium sulphate and the solvent was evaporated in vacuo. The crude solids were
recrystallized from methyl alcohol to obtain the compounds 8–13.
Synthesis of ethyl 1,5-diaryl-3-methyl-4,5-dihydro-1H-pyrazole-4
carboxylates, 18–29
To a solution of ethyl 2-(4-aryl)-3-oxobutanoate, 8–13 (10 mol) and phenylhy-
drazine hydrochloride, 14–17 (10 mol) in acetic acid (40%) was refluxed for 5–6 h.
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A. Dileep Kumar et al.
The progress of the reaction was monitored by thin layer chromatography (TLC).
After the completion, the mixture was cooled and poured into crushed ice. The
separated solids were filtered and washed successively with 5% NaHCO3 and water.
The crude solids were recrystallized from ethyl alcohol to get target molecules
18–29 in moderate to good yields.
X-ray crystallography
Single crystals of suitable dimensions were chosen carefully for X-ray diffraction
studies. The X-ray intensity data were collected at a temperature of 293(2) K on a
Bruker Proteum2 Charge Couple Device (CCD) diffractometer equipped with an
X-ray generator operating at 45 kV and 10 mA, using CuKa radiation of wavelength
˚
1.54178 A. Data were collected for 24 frames per set with different settings of u (0°
and 90°), keeping the scan width of 0.5°, exposure time of 2 s, the sample-to-
detector distance of 45.10 mm and 2h value at 46.6°. The crystal data and details
concerning data collection and structure refinement are given in Table 1. Parameters
in Crystallography Information File (CIF) format are available as an electronic
supplementary publication from Cambridge Crystallographic Data Centre (CCDC).
Cell culture and in vitro compounds treatment
Michigan Cancer Foundation-7 (MCF-7) breast cancer cell lines were procured
from the National Centre for Cell Sciences (NCCS), Pune, India. The cells were
routinely maintained in Dulbecco’s Modified Eagle Medium (Sigma, St. Louis, MO,
USA) supplemented with 10% heat-inactivated fetal calf serum (HiMedia, Mumbai,
India). Ehrlich ascites tumour (EAT) cells were collected from JSS College of
Pharmacy, Mysore.
Trypan blue dye exclusion assay
The effects of compounds 18–29 on cell viability of MCF-7 breast cancer cell lines
were determined by trypan blue dye exclusion assay. MCF-7 breast cancer cells
treated with or without compounds were harvested and resuspended in 0.4% trypan
blue and the viable cells were counted using haemocytometer. IC50 values were
estimated after 48 h of treatment.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The effect of compounds (18–29) on cell proliferation of MCF-7 breast cancer cells
was determined by MTT assay according to reported procedure. Cells treated with
or without compounds were incubated for 48 h. MTT reagent (5 mg/mL) was added
and the color change due to proliferating cells was estimated.
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Design, synthesis and spectroscopic and crystallographic…
Table 1 Crystal data and structure refinement details
Compound
19
25
CCDC number
Empirical formula
Formula weight
Temperature
1,530,834
C19H17FN2O2
324.35
1,530,833
C19H17BrN2O2
385.26
296(2) K
296(2) K
˚
1.54178 A
˚
1.54178 A
Wavelength
Reflections for cell determination
h range for above
Crystal system
2003
1700
5.45°–64.61°
Triclinic
P - 1
5.45°–62.37°
Monoclinic
P 21/c
Space group
˚
a = 8.6121(5) A
˚
a = 10.597(2) A
Cell dimensions
˚
b = 9.8732(6) A
˚
b = 19.861(4) A
˚
˚
c = 8.907(2) A
c = 10.3398(6) A
a = 75.274(4)°
b = 73.343(4)°
c = 76.295(4)°
801.75(8)°A3
a = 90.00°
b = 109.263(15)°
c = 90.00°
1769.75(7)°A3
4
Volume
Z
2
Density (calculated)
Absorption coefficient
F000
1.344 Mgm-3
0.791 mm-1
1.446 Mgm-3
3.271 mm-1
340
784
Crystal size
0.27 9 0.25 9 0.22 mm
0.29 9 0.26 9 0.22 mm
h range for data collection
Index ranges
5.45°–64.61°
4.45°–62.37°
-10 B h B 9
-11 B k B 11
-12 B l B 11
6890
-12 B h B 12
-22 B k B 21
-9 B l B 10
Reflections collected
Independent reflections
Absorption correction
Refinement method
9119
2630 [Rint = 0.0551]
2736 [Rint = 0.1150]
Multi-scan
Full matrix least-squares on F2
2630/0/219
Multi-scan
Full matrix least-squares on F2
2736/0/219
Data/restraints/parameters
Goodness-of-fit on F2
Final [I [ 2r(I)]
1.055
0.962
R1 = 0.0595, wR2 = 0.1685
R1 = 0.0735, wR2 = 0.1729
R indices (all data)
R1 = 0.0763, wR2 = 0.1938
-3
R1 = 0.1395, wR2 = 0.2181
-3
˚
0.325 and -0.270 e A
˚
0.537 and -0.895 e A
Largest diff. peak and hole
Chorioallantoic membrane assay
The designed series of pyrazoles (18–29) were screened for their antiangiogenic
activity by the chorioallantoic membrane (CAM) assay according to the reported
procedure. Briefly, fertilized hen eggs were surface sterilized using 70% alcohol.
The eggs were incubated in a fan-assisted humidified incubator at 37 °C. On the 4th
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A. Dileep Kumar et al.
day, the eggs were cracked into thin films of the hammock within a laminar flow
cabinet and were further incubated. On the 5th day, when blood vessels were seen
proliferating from the centre of the eggs within the hammock, filter paper discs
loaded with 100 lg of synthesized pyrazoles were placed over the proliferating
blood vessels and the eggs were returned to the incubator. Results for antiangio-
genic effect of the compounds were observed after 24 h.
Giemsa and ethidium bromide/acridine orange staining
EAT cell lines were obtained from the JSS College of Pharmacy, Mysuru. The cells
were centrifuged at 3000 rpm for 5 min and the packed cells were diluted 1:6 times
with phosphate buffer saline. Two milliliters of the diluted cells were treated with
the synthesized compounds 19, 23 and 28 and incubated for 4 h at 37 °C. Untreated
EAT cells served as control. At the end of 4 h, the samples were centrifuged, smears
were made from the cell pellet obtained and fixed with methanol/acetic acid (3:1),
and the morphological features of the cells were observed using different stains.
Batches of both test and control smears were stained with Giemsa’s stain and
acridine orange/ethidium bromide stain that highlights the apoptotic morphology of
the cells when observed under bright field microscope and fluorescent microscope,
respectively.
DPPH radical scavenging activity
Freshly prepared DPPH solution (1 mL, 0.1 mM in 95% methanol) was mixed with
different aliquots of test samples (20, 40, 60, 80 and 100 lg/mL) in methanol.
Ascorbic acid (AA) was used as positive control. The mixture was shaken
vigorously and allowed to stand for 20 min at room temperature. The absorbance
was read against a blank at 517 nm in an ELICO SL 159 UV visible
spectrophotometer. The free radical scavenging potential was calculated as a
percentage (I %) of DPPH discoloration using the Eq. (1);
I% of scavenging ¼ ðA0 À A1=A0Þ Â 100;
ð1Þ
where A0 is the absorbance of the control reaction mixture excluding the test
compounds, and A1 is the absorbance of the test compounds.
Quantitative structure–activity relationship analysis
Quantitative structure–activity relationship (QSAR) analysis was carried out to
determine the variables (molecular parameters of the small molecules) that
demonstrate a correlation with the potency of antioxidant activity displayed. The
physicochemical properties of the small molecules synthesized in this study were
computed from ChemMine tools employing OpenBabel and Joelib descriptors and
ChemBioDraw 14.0. Pearson correlation coefficients were computed individually
on each parameter against the obtained % radical scavenging activity and the
equations were derived by linear regression with GraphPad Prism, version 4.0. The
123

Design, synthesis and spectroscopic and crystallographic…
Pearson correlation coefficients were assessed by assuming a two-tailed distribution
and 95% confidence interval.
Results and discussion
Synthesis and characterization of compounds 8–13 and 18–29
Synthesis of the target polysubstituted pyrazoles (18–29) was accomplished in a
two-step synthetic sequence. Initially, ethyl 2-(arylidene)-3-oxobutanoates (8–13)
were synthesized by the reaction of aromatic aldehydes (1–6) with ethyl
acetoacetate 7 and 3–4 drops of piperidine and trifluoroacetic acid in dichlor-
omethane under reflux conditions as is outlined in Scheme 1. The structures of 8–13
were confirmed by spectral and crystallographic studies. Earlier, we reported the
crystal structures of 9 [22], 10 [23] and 11 [24] which confirms the Z conformation
around the olefinic bond. The characterization data of compounds ethyl 2-benzyli-
dene-3-oxobutanoate 8 [21], ethyl 2-(4-bromobenzylidene)-3-oxobutanoate 12 [25],
and ethyl 2-(2,3-dichlorobenzylidene)-3-oxobutanoate 13 [26] were in agreement
with the literature.
Then, the reaction of ethyl 2-(arylidene)-3-oxobutanoates (8–13) with phenyl-
hydrazine hydrochlorides (14–17) in acetic acid under reflux conditions produced
target substituted pyrazoles (18–29) in good yields (Scheme 2). The structures were
confirmed by spectral and crystallographic studies. For instance, compound 19
shows signals at d 1.16 (J = 7.0 Hz), 2.57, 4.16 (J = 7.2 Hz), 7.14–7.18 and
7.25–7.29 ppm for ester CH3, CH3, OCH2 and aromatic protons, respectively. The
signals correspond to the newly formed pyrazole ring carbons viz. C-4 at d 114.9,
C-5 at d 139.1 and C-3 at d 151.7 ppm. Signals due to two CH3, OCH2 and ester
C=O carbons appeared at d 14.0, 14.2, 59.8 and 163.6 ppm, respectively. It showed
a base peak at m/z 325.10 corresponding to the expected molecular mass. Further,
the structures of 21 [25], 19 and 25 were confirmed by crystallographic studies.
Compound 19 (CCDC: 1530834) crystallized as pale-yellow crystals suitable for
single crystal X-ray diffraction (crystal size 0.27 9 0.25 9 0.22 mm) with the
˚
˚
˚
crystallographic parameters: a = 8.6121(5) A, b = 9.8732(6) A, c = 10.3398(6) A,
a = 75.274(4)°, b = 73.343(4)°, c = 76.295(4)°, V = 801.75(8)°A3, Z = 2. The
Scheme 1 Synthesis of ethyl 2-(arylidene)-3-oxobutanoates (8–13)
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A. Dileep Kumar et al.
Scheme 2 Synthesis of functionalized pyrazoles (18–29)
crystal structure is triclinic with a space group of P - 1 which contains pyrazole
ring as a central core. Compound 25 (CCDC: 1530833) crystallized as pale-yellow
crystals suitable for single crystal X-ray diffraction (crystal size
˚
0.29 9 0.26 9 0.22 mm) with the crystallographic parameters: a = 10.597(2) A,
˚
˚
b = 19.861(4) A, c = 8.907(2) A, a = 90.00°, b = 109.263(15)°, c = 90.00°,
V = 801.75(8)°A3, Z = 2. The crystal structure is monoclinic with a space group
of P 21/c which contains pyrazole ring as the central core. The Oak Ridge Thermal-
Ellipsoid Plot Program (ORTEP) diagram of molecules 19 and 25 with thermal
ellipsoids drawn at 50% probability are shown in Figs. 1 and 2, respectively.
Analytical data of compounds 18–29
Ethyl 3-methyl-1,5-diphenyl-1H-pyrazole-4-carboxylate (18) The characteriza-
tion data was in agreement with the literature [27].
Ethyl 5-(4-fluorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (19) Yield
65%; m.p. 110–113 °C. 1H NMR (CDCl3, d ppm): 1.16 (t, 3H, J = 7.0 Hz, CH3), 2.57
(s, 3H, CH3), 4.16 (q, 2H, J = 7.2 Hz, OCH2), 7.14–7.18 (m, 4H, Ar–H)¸ 7.25–7.29
(m, 5H, Ar–H); 13C NMR (CDCl3; d ppm): 14.0 (1C, CH3), 14.2 (1C, CH3), 59.8 (1C,
OCH2), 114.9 (1C, C-4), 125.3 (2C), 126.7 (1C), 127.4 (2C), 128.5 (2C), 128.6 (2C),
130.2 (1C), 138.7 (1C), 132.4 (1C), 139.1 (1C, C-5), 151.7 (1C, C-3), 163.6 (1C,
C=O). MS (m/z): 325.10 (MH?, 100); anal. calcd. for C19H17FN2O2 (%): C, 70.36; H,
5.28; N, 8.64; found: C, 70.31; H, 5.25; N, 8.61.
Ethyl 1-(3-chlorophenyl)-3-methyl-5-phenyl-1H-pyrazole-4-carboxylate (20) Yield
85%; m.p. 95–96 °C. 1H NMR (CDCl3, d ppm): 1.15 (t, 3H, J = 7.0 Hz, CH3), 2.57
(s, 3H, CH3), 4.16 (q, 2H, J = 7.2 Hz, OCH2), 6.97–7.02 (m, 2H, Ar–H), 7.13–7.16
(m, 2H, Ar–H), 7.20–7.27 (m, 5H, Ar–H); 13C NMR (CDCl3; d ppm): 14.1 (1C,
123

Design, synthesis and spectroscopic and crystallographic…
Fig. 1 ORTEP diagram of ethyl 5-(4-fluorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate 19
with thermal ellipsoids drawn at 50% probability
CH3), 14.2 (1C, CH3), 59.9 (1C, OCH2), 113.8 (1C, C-4), 116.7 (1C), 118.5 (1C),
126.4 (1C), 127.3 (2C), 128.2 (2C), 128.6 (1C), 130.9 (1C), 133.6 (1C), 134.3 (1C),
139.5 (1C, C-5), 142.4 (1C), 151.6 (1C, C-3), 163.8 (1C, C=O). MS (m/z): 343.01
(MH?, 37Cl, 33), 341.01 (MH?, 35Cl, 100); anal. calcd. for C19H17ClN2O2 (%): C,
66.96; H, 5.03; N, 8.22; found: C, 66.91; H, 5.01; N, 8.20.
Ethyl 3-methyl-1-phenyl-5-(p-tolyl)-1H-pyrazole-4-carboxylate (21) [28] Yield
1
75%; m.p. 98 °C. H NMR (CDCl3, d ppm): 1.15 (t, 3H, J = 7.0 Hz, CH3), 2.33
(s, 3H, CH3), 2.56 (s, 3H, CH3), 4.15 (q, 2H, J = 7.2 Hz, OCH2), 7.08–7.13 (m, 4H,
Ar–H), 7.15–7.18 (m, 2H, Ar–H), 7.21–7.26 (m, 3H, Ar–H); 13CNMR (CDCl3, d
ppm): 14.0 (1C, CH3), 14.2 (1C, CH3), 21.3 (1C, Ar–CH3), 59.7 (1C, OCH2), 111.8
(1C, C-4), 125.3 (2C), 126.7 (1C), 127.4 (2C), 128.5 (2C), 128.6 (2C), 130.2 (1C),
138.7 (1C), 139.3 (1C), 146.5 (1C, C-5), 151.6 (1C, C-3), 163.8 (1C, C=O). MS (m/
z): 321.13 (MH?, 100); anal. calcd. for C20H20N2O2 (%): C, 74.98; H, 6.29; N, 8.74;
found: C, 74.92; H, 6.27; N, 8.71.
Ethyl 1-(3-chlorophenyl)-3-methyl-5-(p-tolyl)-1H-pyrazole-4-carboxylate (22) Yield
1
73%; m.p. 135–138 °C. H NMR (CDCl3, d ppm): 1.16 (t, 3H, J = 7.0 Hz, CH3),
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A. Dileep Kumar et al.
Fig. 2 ORTEP diagram of ethyl 5-(4-bromophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate 25
with thermal ellipsoids drawn at 50% probability
2.33 (s, 3H, CH3), 2.57 (s, 3H, CH3), 4.15 (q, 2H, J = 7.2 Hz, OCH2), 7.08–7.10 (m,
2H, Ar–H), 7.11–7.16 (m, 3H, Ar–H), 7.19–7.27 (m, 3H, Ar–H); 13C NMR (CDCl3,
d ppm): 14.1 (1C, CH3), 14.2 (1C, CH3), 21.3 (1C, Ar–CH3), 59.8 (1C, CH2), 111.3
(1C, C-4), 116.4 (1C), 118.6 (1C), 125.3 (2C), 126.1 (1C),128.4 (2C), 130.6 (1C),
131.7 (1C), 132.2 (1C), 133.3 (1C), 140.5 (1C), 146.1 (1C, C-5), 151.4 (1C, C-3),
164.0 (1C, C=O). MS (m/z): 357.10 (MH?, 37Cl, 34), 355.08 (MH?, 35Cl, 100);
anal. calcd. for C20H19ClN2O2 (%): C, 67.70; H, 5.40; N, 7.89; found: C, 67.68; H,
5.39; N, 7.86.
Ethyl 5-(4-chlorophenyl)-3-methyl-1-(m-tolyl)-1H-pyrazole-4-carboxylate (23) Yield
1
68%; m.p. 145–149 °C. H NMR (CDCl3, d ppm): 1.15 (t, 3H, J = 7.0 Hz, CH3),
2.24 (s, 3H, CH3), 2.56 (s, 3H, CH3), 4.16 (q, 2H, J = 7.2 Hz, OCH2), 7.13–7.16 (m,
3H, Ar–H), 7.17–7.19 (m, 2H, Ar–H), 7.21–7.29 (m, 3H, Ar–H); 13C NMR (CDCl3,
d ppm): 14.0 (1C, CH3), 14.6 (1C, CH3), 21.3 (1C), 59.8 (1C, OCH2), 113.1 (1C,
C-4), 116.7 (1C), 121.6 (1C), 126.2 (1C), 127.7 (2C), 128.8 (1C), 129.7 (2C), 131.0
(1C), 134.1 (1C), 138.4 (1C), 139.1 (1C), 146.5 (1C, C-5), 151.6 (1C, C-3), 163.5
(1C, C=O). MS (m/z): 357.06 (MH?, 37Cl, 33), 355.06 (MH?, 35Cl, 100); anal.
calcd. for C20H19ClN2O2 (%): C, 67.70; H, 5.40; N, 7.89; found: C, 67.64; H, 5.39;
N, 7.86.
123

Design, synthesis and spectroscopic and crystallographic…
Ethyl 1-(3-chlorophenyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-carboxy-
1
late (24) Yield 85%; m.p. 56–58 °C. H NMR (CDCl3, d ppm): 1.15 (t, 3H,
J = 7.0 Hz, CH3), 2.56 (s, 3H, CH3), 4.16 (q, 2H, J = 7.2 Hz, OCH2), 6.97–6.99 (m,
2H, Ar–H), 7.12–7.19 (m, 3H, Ar–H), 7.21–7.26 (m, 3H, Ar–H); 13C NMR (CDCl3,
d ppm): 14.1 (1C, CH3), 14.4 (1C, CH3), 60.1 (1C, OCH2), 113.8 (1C, C-4), 116.7
(1C), 117.3 (2C), 118.8 (1C), 126.4 (1C), 128.3 (1C), 130.5 (2C), 131.1 (1C), 134.2
(1C), 140.1 (1C, C-5), 141.6 (1C), 151.6 (1C, C-3), 162.1 (1C), 163.4 (1C, C=O).
MS (m/z): 361.06 (MH?, 37Cl, 34), 359.04 (MH?, 35Cl, 100); anal. calcd. for
C19H16ClFN2O2 (%): C, 63.60; H, 4.49; N, 7.81; found: C, 63.57; H, 4.48; N, 7.78.
Ethyl 5-(4-bromophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (25) Yield
80%; m.p. 87–88 °C. 1H NMR (CDCl3, d ppm): 1.16 (t, 3H, J = 7.0 Hz, CH3), 2.57
(s, 3H, CH3), 4.15 (q, 2H, J = 7.2 Hz, OCH2), 7.11 (dd, 2H, J = 6.8, J = 1.6 Hz,
Ar–H), 7.14 (m, 2H, J = 6.2, J = 1.2 Hz, Ar–H), 7.24–7.27 (m, 3H, Ar–H),
7.42–7.44 (m, 2H, Ar–H); 13C NMR (CDCl3, d ppm): 14.1 (1C, CH3), 14.5 (1C,
CH3), 60.0 (1C, OCH2), 112.9 (1C, C-4), 123.7 (1C), 124.5 (2C), 125.8 (1C), 128.5
(2C), 129.7 (2C), 131.8 (2C), 132.4 (1C), 138.3 (1C), 141.6 (1C, C-5), 151.2 (1C,
C-3), 163.3 (1C, C=O). MS (m/z): 387.02 (MH?, 81Br, 98), 384.01 (MH?, 79Br,
100); anal. calcd. for C19H17BrN2O2 (%): C, 59.23; H, 4.45; N, 7.27; found: C,
59.20; H, 4.44; N, 7.25.
Ethyl 5-(2,3-dichlorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (26) Yield
65%; m.p. 51–53 °C. 1H NMR (CDCl3, d ppm): 1.14 (t, 3H, J = 7.0 Hz, CH3), 2.54
(s, 3H, CH3), 4.17 (q, 2H, J = 7.2 Hz, OCH2), 6.97–6.99 (m, 2H, Ar–H), 7.00–7.16
(m, 3H, Ar–H), 7.20–7.24 (m, 4H, Ar–H); 13C NMR (CDCl3, d ppm): 14.0 (1C,
CH3), 14.6 (1C, CH3), 60.2 (1C, OCH2), 113.5 (1C, C-4), 124.4 (2C), 126.3 (1C),
127.1 (1C), 127.6 (1C), 129.2 (2C), 130.1 (1C), 131.3 (1C), 131.8 (1C), 132.9 (1C),
139.1 (1C), 141.9 (1C, C-5), 151.7 (1C, C-3), 163.0 (1C, C=O). MS (m/z): 378.01
(M ? 4, 11), 376.02 (M ? 2, 63), 374.05 (M?, 100); anal. calcd. for C19H16Cl2-
N2O2 (%): C, 60.81; H, 4.30; N, 7.47; found: C, 60.76; H, 4.29; N, 7.45.
Ethyl 5-(2,3-dichlorophenyl)-1-(2,4-dimethylphenyl)-3-methyl-1H-pyrazole-4-
1
carboxylate (27) Yield 65%; m.p. 45–47 °C. H NMR (CDCl3, d ppm): 1.16 (t,
3H, J = 7.0 Hz, CH3), 1.97 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.54 (s, 3H, CH3), 4.18
(q, 2H, J = 7.2 Hz, OCH2), 7.15–7.20 (m, 3H, Ar–H), 7.24–7.29 (m, 3H, Ar–H);
13C NMR (CDCl3, d ppm): 14.0 (1C, CH3), 14.3 (1C, CH3), 40.7 (2C, NCH3), 60.2
(1C, OCH2), 113.4 (1C, C-4), 120.1 (1C), 123.8 (1C), 125.8 (1C), 126.1 (1C), 127.3
(1C), 130.1 (1C), 131.1 (1C), 132.0 (1C), 133.1 (1C), 135.0 (2C), 139.6 (1C), 141.5
(1C, C-5), 151.6 (1C, C-3), 163.3 (1C, C=O). MS (m/z): 406.09 (M ? 4, 13), 404.08
(M ? 2, 64), 402.10 (M?, 100); anal. calcd. for C21H20Cl2N2O2 (%): C, 62.54; H,
5.00; N, 6.95; found: C, 62.51; H, 4.98; N, 6.93.
Ethyl 5-(4-chlorophenyl)-1-(2,4-dimethylphenyl)-3-methyl-1H-pyrazole-4-car-
1
boxylate (28) Semi-solid, yield 68%. H NMR (CDCl3, d ppm): 1.19 (t, 3H,
J = 7.0 Hz, CH3), 1.94 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.53 (s, 3H, CH3), 4.19 (q,
2H, J = 7.2 Hz, OCH2), 7.28–7.39 (m, 3H, Ar–H), 7.56–7.59 (m, 4H, Ar–H); 13C
NMR (CDCl3, d ppm): 14.1 (1C, CH3), 14.3 (1C, CH3), 15.5 (1C, CH3), 20.7 (1C,
123

A. Dileep Kumar et al.
CH3), 60.3 (1C, OCH2), 112.5 (1C, C-4), 118.6 (1C), 123.2 (1C), 125.6 (1C), 128.8
(2C), 129.3 (2C), 130.0 (1C), 133.9 (1C), 134.7 (1C), 136.3 (1C), 140.1 (1C), 141.8
(1C, C-5), 151.2 (1C, C-3), 163.1 (1C, C=O). MS (m/z): 370.09 (M?, 37Cl, 34),
368.10 (M?,35Cl, 100); anal. calcd. for C21H21ClN2O2 (%): C, 68.38; H, 5.74; N,
7.59; found: C, 68.31; H, 5.73; N, 7.57.
Ethyl 1-(2,4-dimethylphenyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-car-
1
boxylate (29) Semi-solid, yield 70%. H NMR (CDCl3, d ppm): 1.21 (t, 3H,
J = 7.0 Hz, CH3), 1.95 (s, 3H, CH3), 2.31 (s, 3H, CH3), 2.54 (s, 3H, CH3), 4.21 (q, 2H,
J = 7.2 Hz, OCH2), 7.29–7.37 (m, 3H, Ar–H), 7.57–7.60 (m, 4H, Ar–H); 13C NMR
(CDCl3, d ppm): 14.2 (1C, CH3), 14.3 (1C, CH3), 15.7 (1C, CH3), 20.9 (1C, CH3),
60.3 (1C, OCH2), 112.4 (1C, C-4), 117.8 (2C), 118.5 (1C), 122.10 (1C), 125.7 (1C),
128.2 (1C), 129.8 (2C), 134.7 (1C), 135.3 (1C), 140.1 (1C), 141.6 (1C, C-5), 151.3
(1C, C-3), 159.4 (1C), 163.2 (1C, C=O). MS (m/z): 352.11 (M?, 100); anal. calcd. for
C21H21FN2O2 (%): C, 71.57; H, 6.01; N, 7.95; found: C, 71.54; H, 5.99; N, 7.93.
Anti-angiogenic activity of compounds 18–29
In order to evaluate the anti-angiogenic activity of the pyrazole derivatives 18–29,
we first assessed their in vitro cytotoxic effect on MCF-7 breast cancer cell lines
following 48 h of exposure using trypan blue and (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) [29]. This in vitro screening is performed to
identify the compounds amongst the series that exhibit cytotoxic effects. Tamoxifen
(TMX) was used as a standard, and DMSO as negative control; the results of the
screening are summarized in Table 2. In the trypan blue assay, compounds 19, 23
and 28 showed excellent cell growth inhibition with IC50 values of 7.9, 10.6 and
8.2 lM, respectively, in comparison with the standard TMX’s 11.4 lM. The effect
of compounds on cell proliferation tested using MTT assay reveals that, compounds
19, 23 and 28 have maximum cytotoxic effect with IC50 values of 7.6, 10.4 and
8.5 lM, respectively. These results were in agreement with those reported with
trypan blue (Table 2).
Obviously, from the results, compound 19 (7.9 lM) with fluoro substitution at
the para position of the phenyl ring exhibited the highest activity against MCF-7
cells in trypan blue with MTT assay IC50 values of 7.9 and 7.6 lM when compared
with the standard. The results reveal that compounds 26 and 27 have no cytotoxic
effect and compounds 18, 20, 24 and 29 possess moderate cytotoxic effects on
MCF-7 cells. Compounds 19, 23 and 28 were considered as lead compounds for
further investigation. The cytotoxic effects of synthesized pyrazole derivatives
18–29 on MCF-7 cell lines were well within the range exhibited by similar and
structurally related pyrazoles [29].
To study the morphological features of the cells, we subjected the cells to Giemsa
and ethidium bromide/acridine orange staining [30]. In this, experimental batches of
both test and control smears were stained with Giemsa’s stain and acridine orange/
ethidium bromide stain that highlights the apoptotic morphology of the cells when
observed under florescent microscope and as photographed employing a Nikon
123

Design, synthesis and spectroscopic and crystallographic…
Table 2 IC50 values of designed series of compounds 18–29 on trypan blue and MTT assay at 48 h in
MCF-7 cells
Compounds
Trypan blue assay IC50 value (lM)
MTT assay IC50 value (lM)
Negative control
–
–
18
19 0.27
7.9 – 0.07
17 0.17
44 0.12
71 0.19
10.6 – 0.07
14 0.11
84 0.25
[ 100
21 0.18
7.6 – 0.08
16 0.22
49 0.16
65 0.13
10.4 – 0.08
13 0.12
87 0.19
[ 100
19
20
21
22
23
24
25
26
27
[ 100
[ 100
28
8.2 – 0.13
19 0.15
11.4 0.07
8.5 – 0.16
21 0.20
11.4 0.09
29
Positive control
Based on the IC50 values, compounds 19, 23 and 28 were chosen as lead compounds as indicated by IC50
values in bold entries
Fig. 3 Cytotoxic and antiangiogenic effect of compounds 19, 23 and 28. (A) Giemsa staining of normal
cells and cells treated with compounds 19, 23 and 28 highlighting widespread cell death. (B) Ethidium
bromide/acridine orange staining of normal cells and cells treated with compounds 19, 23 and 28
highlighting cell death. (C) CAM photos illustrates the formation of blood vessels in normal cells with
regression of angiogenesis in cells treated with compounds 19, 23 and 28
123

A. Dileep Kumar et al.
D3200 camera (Fig. 3a, b). The apoptotic morphology clearly indicated the
potential cytotoxic effect of compounds 19, 23 and 28.
The anti-angiogenic activity of the designed series of pyrazoles 18–29 was
evaluated in the shell less CAM assay [30], which is the most widely used assay to
study angiogenesis in vivo. In this model, compounds 18–29 induced moderate to
good aviculture zone formation in the developing embryos. Amongst the tested
compounds, compound 19, 23 and 28 clearly show regression of newly formed
micro-vessels which were found around the area of disc implantation compared to
the other compounds in the series (Fig. 3c). This regression of micro-vessel density
in the CAM model is highly evident of inhibition of angiogenesis and it correlates
with our in vitro results more accurately.
DPPH radical scavenging activity of compounds 18–29
As an additional investigation, we also performed the DPPH radical scavenging
assay of the synthesized compounds 18–29 according to Blois method [31, 32]. The
results were expressed as I % standard deviations (SD; n = 3). The results of the
findings (Table 3) clearly showed that the newly synthesized compounds exhibited
good activities. Preliminary investigation results reveal that compounds 20, 24 and
26 with chlorophenyl substitutions of the pyrazole showed stronger DPPH
scavenging activity in comparison with the standard ascorbic acid. Compounds
19, 23 and 28 showed scavenging abilities similar to that displayed by the standard.
However, the rest of the compounds showed lesser activities. From these results, it
Table 3 DPPH radical scavenging activity of the synthesized pyrazoles 18–29
Compounds
% Radical scavenging activitya
20 (lg/mL)
40 (lg/mL)
60 (lg/mL)
80 (lg/mL)
100 (lg/mL)
18
19
20
21
22
23
24
25
26
27
28
29
AAb
15.21 0.65
18.45 0.11
23.54 0.21
14.12 0.87
15.61 0.21
16.98 0.28
19.03 0.87
17.22 0.32
23.54 0.21
14.01 0.32
17.35 0.26
13.98 0.40
15.08 0.89
16.12 0.87
20.16 0.98
24.56 0.76
13.99 0.43
17.34 0.76
18.97 0.32
20.98 0.10
17.96 0.89
24.56 0.76
16.21 0.97
18.90 0.55
15.30 0.40
16.87 0.89
21.12 0.41
23.67 0.11
29.21 0.41
19.76 0.32
23.45 0.51
23.76 0.58
24.84 0.32
20.54 0.76
29.21 0.41
21.03 1.01
22.34 0.35
20.16 0.86
21.98 0.31
24.22 0.32
25.65 0.76
31.54 0.32
22.54 0.12
24.98 0.54
25.75 0.42
27.76 0.11
22.05 0.32
31.54 0.32
23.11 0.50
25.24 0.50
23.08 0.75
24.25 0.22
26.08 0.41
28.94 0.20
34.65 0.41
23.95 0.31
27.95 0.56
28.07 0.71
29.87 0.32
25.76 0.26
34.65 0.41
26.05 0.63
28.75 0.65
25.35 0.82
28.65 0.98
aValues are mean SD of three replicates (n = 3)
bAscorbic acid = positive control
123

Design, synthesis and spectroscopic and crystallographic…
is anticipated that the presence of a monochloro-substituted phenyl ring at the 1
position of the pyrazole ring influences the increasing DPPH radical scavenging
abilities of the designed series of pyrazoles.
Quantitative structure–activity relationship analysis
To understand the potential molecular features that were contributing towards the
potency of the observed antioxidant activity by some of the compounds, a
systematic QSAR analysis was carried out. A pairwise analysis on a total of 38
molecular parameters was assessed for possible correlation with the potency of
observed antioxidant activity for the 12 compounds reported (18–29; Fig. 4). The
molecular parameters included, but are not limited to, variables such as the
molecular weight, LogP, presence/absence of various halogenated substituents,
hydrogen bonding donors/acceptors, presence and number of single and double
Fig. 4 Pairwise correlation analysis for four different physicochemical parameters computed for
compounds from series 18–29 against the DPPH and hydroxyl radical scavenging activity (HRS). In the
plots, the y-axis represents the % radical scavenging activity and the x-axis indicates the various
physicochemical parameters computed for compounds employing Joelib and OpenBabel. The plots were
generated in GraphPad Prism, version 4.0, and the linear regression and correlation analysis were carried
out using the functions employed by the software. ‘‘Pearson’s r indicates the correlation coefficient
123

A. Dileep Kumar et al.
bonds, molecular connectivity parameters like Zagreb group index, shape, etc.
Before discussing the obtained results, it would be appropriate to point out that the
12 molecules designed and synthesized in this study share a highly conserved
invariable core with minor aliphatic and halogenated substitutions. Hence, the minor
variability in the microenvironment of the small molecules translating into potency
of antioxidant activity presents an appropriate exercise in modeling an accurate
QSAR model.
The pairwise comparison shows that, among other parameters, the potency of the
antioxidant activity doesn’t correlate with parameters like LogP and the molecular
weight of the synthesized compounds (Fig. 4a, b). However, the hydrogen bonding
acceptor 1 parameter correlated negatively with the potency of antioxidant activity
having a reasonably good Pearson’s correlation coefficient with a P value less than
0.04, indicating reasonable significance (Fig. 4c). This indicates that in the current
series of compounds, the lesser the hydrogen bonding acceptance potential of a
small molecule, the greater the possibility that it will have potent antioxidant
activity. The parameter that showed positive correlation with the potency of
antioxidant activity was the content of the halide chlorine (Fig. 4d). However, the
statistics of the correlation was not very strong, indicating that chloride substitu-
tion’s effect on potency is highly regional. For instance, compound 27, in spite of
having two chlorine substitutions, showed relatively less potent antioxidant activity.
Future studies would attempt the synthesis of further compounds in this series for
the sake of statistical rigor of the analysis. Two-dimensional QSAR analysis
employing both multiple linear regressions (MLR) and partial least square
regression (PLR) would be attempted. As an additional measure, depending on
the availability of crystal structure information for all the synthesized compounds in
the series, three-dimensional QSAR strategies using comparative molecular field
analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)
would also be attempted. However, it would have to be pointed out that the success
of QSAR predictions, to a large extent, depend on the training set and it would be
important to implement rigorous training subroutines employing either genetic
algorithms or far more traditional approaches.
Conclusions
To sum up, in the present work, we report a procedure for the direct synthesis of
pyrazoles from various ethyl 2-(4-aryl)-3-oxobutanoates. The ethyl ester function in
a substituted pyrazole ring is posited to be the key feature for the biological potency
of the synthesized compounds. From the results of the biological evaluation, its
evident that amongst the designed series, compounds 19, 24 and 28 exhibit
promising cytotoxic and angiogenic properties; compounds, 20, 24 and 26 possesses
good antioxidant properties. The demonstrated synthesis paves the way for future
efforts at synthesizing derivatives of pyrazoles that could find widespread
applications in medicinal chemistry and efforts aimed at alleviating the dreadful
outcomes that oxidative stress imposes.
123

Design, synthesis and spectroscopic and crystallographic…
Acknowledgements The authors are grateful to the IOE Instrumentation Facility, Vijnana Bhavana,
University of Mysore, for recording spectra and X-ray diffraction studies, and JSS College of Pharmacy,
Mysuru, for providing cell lines.
Compliance with ethical standards
Conflict of interest All authors declare no conflict of interest including financial, personal or other
relationships with other people or organizations for this article.
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