Novel pyrazolo[3,4-d]pyrimidine derivatives inhibit human cancer cell proliferation and induce apoptosis by ROS generation

Article abstract of DOI:10.1002/ardp.201900296

The paucity of effective anticancer drugs for successful treatment is a major concern, indicating the strong need for novel therapeutic compounds. In the quest of new molecules, the present study aimed to explore the potential of pyrazolo[3,4-d]pyrimidine derivatives as antiproliferative agents. In vitro anticancer screening of selected compounds was done by the National Cancer Institute's Developmental Therapeutics Programme against a panel of 60 cancer cell lines. The lead compound PP-31d considerably inhibited the growth of cancer cells, such as NCI-H460 (non-small-cell lung cancer), OVCAR-4 (ovarian cancer), 786-0 (renal cancer), A549 (non-small-cell lung cancer), and ACHN (renal cancer), showing strong anticancer potential, among other derivatives. Kinetic studies of PP-31d on NCI-H460 cells revealed a dose-dependent effect with an IC₅₀ of 2 μM. The observed inhibition by PP-31d is attributed to the generation of reactive oxygen species and the subsequent induction of cellular apoptosis, as evidenced by the increase in the hypodiploid (subG1) population, the early apoptotic cell population, and caspase-3/7 activity, the loss of the mitochondrial membrane potential, and the degradation of nuclear DNA. Collectively, our results demonstrated that pyrazolo[3,4-d]pyrimidine derivatives inhibit cancer cell proliferation by inducing apoptosis and, thus, have the potential to be further explored for anticancer properties.

Full text of DOI:10.1002/ardp.201900296

Received: 11 October 2019
Revised: 28 January 2020
Accepted: 3 February 2020
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DOI: 10.1002/ardp.201900296
F U L L P A P E R
Novel pyrazolo[3,4‐d]pyrimidine derivatives inhibit human
cancer cell proliferation and induce apoptosis by ROS
generation
Supreet Gaonkar1 | Mohammed Azharuddin Savanur2
| AfraQuasar A. Nadaf1
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Mahesh S. Najare1 | Shivaraj Mantur1 | Manjunatha Garbhagudi1
Sikandar I. Mulla3 | Imtiyaz Ahmed M. Khazi1
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1Department of Studies in Chemistry,
Karnatak University, Dharwad, Karnataka,
Abstract
India
The paucity of effective anticancer drugs for successful treatment is a major con-
cern, indicating the strong need for novel therapeutic compounds. In the quest of
new molecules, the present study aimed to explore the potential of pyrazolo[3,4‐d]
pyrimidine derivatives as antiproliferative agents. In vitro anticancer screening of
selected compounds was done by the National Cancer Institute's Developmental
Therapeutics Programme against a panel of 60 cancer cell lines. The lead compound
PP‐31d considerably inhibited the growth of cancer cells, such as NCI‐H460
(non‐small‐cell lung cancer), OVCAR‐4 (ovarian cancer), 786‐0 (renal cancer), A549
(non‐small‐cell lung cancer), and ACHN (renal cancer), showing strong anticancer
potential, among other derivatives. Kinetic studies of PP‐31d on NCI‐H460 cells
revealed a dose‐dependent effect with an IC50 of 2 µM. The observed inhibition by
PP‐31d is attributed to the generation of reactive oxygen species and the sub-
sequent induction of cellular apoptosis, as evidenced by the increase in the hypo-
diploid (subG1) population, the early apoptotic cell population, and caspase‐3/7
activity, the loss of the mitochondrial membrane potential, and the degradation of
nuclear DNA. Collectively, our results demonstrated that pyrazolo[3,4‐d]pyrimidine
derivatives inhibit cancer cell proliferation by inducing apoptosis and, thus, have the
potential to be further explored for anticancer properties.
2Department of Biochemistry, Indian Institute
of Science, Bangalore, Karnataka, India
3Department of Biochemistry, School of
Applied Sciences, REVA University, Bangalore,
Karnataka, India
Correspondence
Mohammed Azharuddin Savanur, Department
of Biochemistry, Indian Institute of Science,
Bangalore 560012, India.
Email: azharuddinsavanur1985@gmail.com
Imtiyaz Ahmed M. Khazi, Department of
Studies in Chemistry, Karnatak University,
Dharwad, Karnataka 580003, India.
Email: drimkorgchem@gmail.com
K E Y W O R D S
antiproliferative, apoptosis, cytotoxic, pyrazolo[3,4‐d]pyrimidine
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1
INTRODUCTION
conjunction with surgery and/or radiotherapy with proper diagnosis, is a
prerequisite for adequate and effective cancer treatment. Developing
Chemotherapy is a systemic treatment to combat uncontrolled pro-
liferation and invasion of cells using bioactive small molecules. It is one
of the major therapeutic strategies adopted worldwide for the treat-
ment of different types of cancer. Chemotherapy, either alone or in
improved combinations of different cytotoxic agents has resulted in
clinically relevant molecules helpful in curing cancer patients. However,
despite all innovations to increase chemotherapy efficacy, there are
limitations associated, such as side effects, multidrug resistance of the
These authors contributed equally to this work.
Supreet Gaonkar and Mohammed A. Savanur contributed equally to this study.
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Arch. Pharm. 2020;e1900296.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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tumor, and limited scope of activity of the available anticancer agents,
making it more challenging for the design and synthesis of novel potent
drugs with less side effects and a broad spectrum of activity. Therefore,
the search for alternative new therapeutic drugs is always in demand.
Pyrimidine and its fused analogs have attracted the interest of
medicinal chemists because of their wide range of biological properties.
Among the fused pyrimidines, pyrazolopyrimidine is an important scaffold
present in many biologically active compounds. As pyrazolopyrimidines
have a structural resemblance with purines, they are considered to be
biologically active isomeric purine analogs. Many biologically active
compounds containing the pyrazolopyrimidine core have been approved
by the Food and Drug Administration (Figure 1).
reacting 1,1,1‐triethoxyethane and malononitrile in acetic
anhydride at 110°C for 4 hr. The cyclocondensation of P1
with 4‐bromophenylhydrazinehydrochloride in ethanol in the
presence of triethylamine at room temperature for 2 hr yielded
the compound 5‐amino‐1‐(4‐bromophenyl)‐3‐methyl‐1H‐pyrazole‐
4‐carbonitrile (P‐2). The key intermediates (P‐31–P‐33) were
synthesised by Suzuki cross‐coupling reaction of P‐2 with
4‐(trifluoromethoxy)phenylboronic acid, phenylboronic acid, and
4‐ethylphenylboronic acid, respectively. Reaction of P‐31–P‐33
with formic acid, formamide, benzonitrile, and phenylacetonitrile in
different physical and chemical environment yields the desired
products PP‐31a–PP‐33a, PP‐31b–PP‐33b, PP‐31c–PP‐33c, and PP
‐31d–PP‐33d, respectively. The structures of the synthesised
compounds were characterized by infrared (IR), 1H nuclear mag-
netic resonance (NMR), 13C NMR, single‐crystal X‐ray diffraction
(SC‐XRD), and mass spectral analysis. The IR band due to CN
stretching at 2,218 cm−1 and NH2 stretching at 3,232 and
3,344 cm−1 supports the formation of the structure P‐31. A singlet
at δ 2.32 ppm due to the CH3 group, a broad singlet at δ 4.74 ppm
(parts per million) corresponding to the NH2 group, and four char-
acteristic doublets of biphenyl at δ 7.31, 7.56, 7.60, and 7.68 ppm
confirm the structure of P‐31. Further, the structure of the inter-
mediate compound P‐31 was confirmed by mass spectral analysis.
The structure of one of the intermediate compounds, P‐33 was
confirmed by single‐crystal X‐ray analysis. A broad singlet at δ
12.35 ppm due to amide NH and a characteristic singlet at δ
8.12 ppm corresponding to C6–H of pyrimidine support the for-
mation of the final compound PP‐31a, which was further confirmed
by the liquid chromatography‐mass spectrometry (LCMS) data. The
1H NMR spectrum of compound PP‐31d shows a characteristic
singlet at δ 4.06 ppm for two protons corresponding to the bridging
CH2 group, which support the formation of compound. Further, the
structure of PP‐31d was confirmed by 13C NMR and DEPT‐135
spectral analysis.
Pyrazolo[3,4‐d]pyrimidines are important pharmacophores that
have shown a broad range of biological potentials, such as anti-
microbial,[1] anti‐inflammatory,[2] antiviral,[3] anticancer,[4] and anti-
tubercular activities,[5], and so forth. In recent years, the pyrazolo[3,4
‐d]pyrimidine ring system has been an important pharmacophore in
anticancer drug discovery.[6–8] There are numerous reports on
pyrazolo[3,4‐d]pyrimidines as selective and potent inhibitors of many
kinases, which play a vital role in cancer cell proliferation.[9–13]
In the present study, we report the synthesis and anti-
proliferative activity of pyrazolo[3,4‐d]pyrimidine derivatives against
a panel of cancer cell lines. This report also explains the mechanism
of induced cell death by generation of reactive oxygen species (ROS)
and subsequent induction of cellular apoptosis.
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RESULTS AND DISCUSSION
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2.1
Chemistry
The synthesis of pyrazolo[3,4‐d]pyrimidine derivatives (PP‐31a–PP‐31d,
PP‐32a–PP‐32d, and PP‐33a–PP‐33d) is outlined in Scheme 1.
2‐(1‐Ethoxyethylidene)malononitrile (P1) was synthesised by
FIGURE 1 Clinically approved drugs
containing the pyrazolopyrimidine ring

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SCHEME 1 Synthesis of the pyrazolo[3,4‐d]
pyrimidine derivatives
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2.2
Single‐crystal X‐ray analysis
crystal structure of a strong bond CN…N (3.088 Å), short contact
interaction between NH…N (2.504 Å), NH…N (2.261 Å), and also
CH… π (2.893 Å) make the molecule more stable (Figure 3). Crystal
data are listed in Table 1.
Single crystals of compound P‐33 were developed by slow eva-
poration method using acetonitrile as a solvent. A suitable single
crystal was collected through the polarizing microscope and then
mounted on the Bruker APEX‐II CCD diffractometer system, which
is equipped with a CCD (charge‐coupled device) area detector and
a microfocus sealed tube molybdenum source. The crystal was kept
at 298 K (2) during the data collection with a scan width of 0.5 mm
and a distance of 45 mm from the crystal to the detector. The
structure (Figure 2) was solved using the Olex2 with the XT,[14]
using Intrinsic Phasing, and refined with the ShelXL refinement
package using Least Squares minimization.[15] The presence in the
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2.3
In vitro anticancer activity
To determine the cytotoxic effect of the synthesised compounds,
we deposited the structure of all final compounds in the National
Cancer Institute's Developmental Therapeutics Program (Supple-
mentary Information). Among all, seven compounds (PP‐31a,
PP‐31b, PP‐31c, PP‐31d, PP‐32b, PP‐33a, and PP‐33d) have been

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effect of compounds PP‐31d and PP‐33d on the cellular morphol-
ogy of NCI‐H460 cell was determined. Control cells had a distinct
and well‐defined morphology, whereas cells treated with com-
pound PP‐31d and PP‐33d had indecisive morphology and ap-
peared to be dead (Figure 4b). The cytotoxic effect of PP‐31d on
NCI‐H460 cells was evidenced by the morphological changes in the
phase‐contrast image. The lead compound (PP‐31d) was further
subjected to cell cycle analysis, annexin‐V/PI assay, assessment of
caspase‐3/7 activity, evaluation of mitochondrial membrane po-
tential, and DNA damage studies.
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2.3.1
Compound PP‐31d induces accumulation of
NCI‐H460 cells in the subG1 phase of the cell cycle
Cytotoxic compounds mainly exert their effects by intervening with
cell cycle progression, inhibiting cellular proliferation, and thus, in-
ducing cell death.[16–18] The subG1 phase of the cell cycle represents
the cells that are hypodiploid and undergoing apoptosis.[19,20] NCI‐
H460 cells treated with compound PP‐31d at different concentra-
tions were subjected to cell cycle analysis after propidium iodide (PI)
staining to observe the percentage of cells undergoing apoptosis and
their distribution in different phases of the cell cycle. The results of
the cell cycle analysis of NCI‐H460 cells after treatment with com-
pound PP‐31d are presented in Figure 5a. The DNA content analysis
of NCI‐H460 cells after PP‐31d treatment showed a dose‐dependent
gradual increase in hypodiploid population. Treatment with 10 µM of
compound resulted in 28.6%, which increased to 39.2% at 20 µM
when compared to 1.8% in control groups, suggesting that PP‐31d
promotes cell death through induction of apoptosis. The percentage
distribution of cells in different phases of the cell cycle is represented
in bar diagrams (Figure 5b).
FIGURE 2 ORTEP (Oak Ridge thermal ellipsoid plot) diagram of
compound P‐33 with labeling showing 50% displacement ellipsoids
(crystal structure drew in Mercury 3.7)
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2.3.2
Induction of apoptosis by compound PP‐31d
Loss of plasma membrane asymmetry, such as translocation of phos-
phatidylserine to the outer surface of the plasma membrane, is an
early characteristic of apoptosis,[21,22] which can be detected using
annexin‐V.[23,24] To validate that compound‐induced cell death was as-
sociated with apoptosis, NCI‐H460 cells treated with or without PP‐31d
were analyzed by annexin‐V‐FITC (fluorescein isothiocyanate)/PI
staining, which differentiates viable, early, and late apoptosis or dead
cells. Flow cytometry analysis demonstrated a dose‐dependent increase
in annexin‐V stained cells, representing both the early and late apop-
totic population. As shown in Figure 6a, a substantial increase in the
percentage of early apoptotic cells (49.9%) was observed after PP‐31d
treatment, when compared to the control group (0.5%). Exposure of
NCI‐H460 cells to higher concentrations of the PP‐31d resulted in
shifting to the late apoptotic stage (annexin‐V and PI‐positive), in-
dicating that PP‐31d induces cellular apoptosis in a dose‐dependent
manner. Figure 6b shows the percentage of NCI‐H460 cells in four
different quadrants and presents it in the form of bar graphs.
selected for single‐dose analysis [10 µM] and tested against 60
human cancer cell lines. Compounds PP‐31d and PP‐33d showed
good to moderate inhibition against many cancer cell lines. Initial
screening results revealed that compounds containing a benzyl
group at C‐2 of pyrimidine are more potent and showed enhanced
cytotoxicity. In particular, compound PP‐31d showed strong
growth inhibition (96.6%) of NCI‐H460 cells. Further, we carried
out the dose kinetic study of compound PP‐31d and PP‐33d on NCI
‐H460 cells using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetra-
zolium bromide (MTT) assay. The results showed the strong in-
hibitory activity of compound PP‐31d on NCI‐H460 cells in a dose‐
dependent manner (Figure 4a). In addition, the growth inhibition
effects of these compounds were also tested against human em-
bryonic kidney (HEK293) cells and it was observed that pyrimidine
derivatives have a dose‐dependent effect with a higher IC50
(31.2 µM) value when compared to cancer cells (Figure S2). The

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FIGURE 3 Packing diagram of P‐33 with
CN…N (3.088 Å), NH…N (2.504 Å), NH…N
(2.261 Å) and CH… π (2.893 Å) interactions
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2.3.3
Compound PP‐31d attenuates the
treatment was also assessed by measuring mitochondrial cyto-
chrome c levels and it was observed that the levels of this heme-
protein decreased significantly (Figure S3).
mitochondrial membrane potential
Increased ROS levels usher disruption of mitochondrial membrane
and degeneracy of the electrochemical gradient (loss in mi-
tochondrial membrane potential).[25–27] As PP‐31d‐treated cells
showed increased ROS levels, it was important to analyze whether
elevated ROS had an impact on mitochondria. Therefore, changes
in the mitochondrial membrane potential were analyzed to de-
termine the deleterious effects of ROS on mitochondria. JC‐1, a
lipophilic cationic dye, was used to determine the effect of PP‐31d
on the mitochondrial membrane potential. JC‐1 emits red fluor-
escence by accumulating in intact mitochondria and is also
membrane‐potential dependent, which helps in measuring changes
in mitochondrial polarization. During apoptosis, mitochondrion
depolarizes and mitochondrial membrane potential collapses,
leading to accumulation of JC‐1 in cytoplasm and emitting green
fluorescence.[28] Flow cytometric analysis demonstrated that
treatment of NCI‐H460 cells with different concentrations of PP‐
31d resulted in depolarization of mitochondrial membrane, as
evidenced by the increase in the number of cells positive for green
fluorescence (Figure 7a). A remarkable reduction in mitochondrial
membrane potential (58.9%) was observed in cells exposed to
20 µM of PP‐31d, suggesting that the compound‐induced cell
death involves mitochondrial depolarization‐associated apoptosis.
Figure 7b shows the percentage of NCI‐H460 cells with high and
low mitochondrial membrane potential after treatment with and
without PP‐31d. Depolarization of mitochondria after PP‐31d
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2.3.4
ROS generation by compound PP‐31d
Oxidative damage to biomolecules caused by enhanced ROS levels
leads to apoptosis and cell death.[29,30] Thus, cytotoxic compounds
that have the ability to enhance ROS levels in cancer cells are po-
tential anticancer agents.[31–33] The ROS‐generating ability of PP‐31d
in NCI‐H460 cells was determined by 2,7‐dichlorodihydro fluorescein
diacetate (DCFDA‐H2) staining (a nonfluorescent dye emits green
fluorescence upon oxidation by ROS) followed by microscopic ana-
lysis. Figure 8a shows enhanced green fluorescence, which is a con-
sequence of DCFDA‐H2 oxidation due to increased ROS levels upon
treatment of cells with PP‐31d. These results indicate that compound
PP‐31d promotes ROS generation and induces apoptosis in cancer
cells.
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2.3.5
Compound PP‐31d induces activation of the
effector caspase‐3/7
The elevated ROS causes oxidative stress and activation of caspases,
leading to the induction of apoptosis.[34,35] Activation of effector
caspase‐3/7 results in DNA condensation and fragmentation, which
are characteristic changes associated with the induction of apoptosis.

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TABLE 1 Crystal data and structure refinement of compound P‐33
DNA. Activation of specific endonucleases that cause DNA frag-
mentation is a known mechanism by which anticancer drugs induce
apoptosis in cancer cells.[36] The ability of PP‐31d to induce changes
in nuclear morphology was assessed by determining the condensa-
tion of chromatin and DNA fragmentation. Staining of NCI‐H460
cells nuclei with 4′,6‐diamidino‐2‐phenylindole (DAPI) after PP‐31d
treatment showed significant chromatin condensation (Figure 8d),
indicating the nuclear changes induced by PP‐31d. Fragmentation of
DNA in cells exposed to compound PP‐31d was determined by
agarose gel electrophoresis. Figure 8c shows fragmented DNA and
ladder formation after PP‐31d treatment in a dose‐dependent man-
ner, further supporting the evidence that these compounds induced
cell death via induction of apoptosis.
Identification code
1
Empirical formula
C19H18N4
Formula weight
302.38
Temperature/K
293(2)
Crystal system
monoclinic
P21/n
Space group
a/Å
11.0142(4)
10.0360(3)
14.9346(4)
90
b/Å
c/Å
α/°
β/°
97.0360(10)
90
γ/°
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3
CONCLUSION
Volume/Å3
1,638.42(9)
4
In summary, a series of 12 pyrazolo[3,4‐d]pyrimidine derivatives
were synthesised and the structure of the compounds was char-
acterized by IR, 1H NMR, 13C NMR, SC‐XRD, and mass spectral
analysis. Among all, seven compounds (PP‐31a, PP‐31b, PP‐31c,
PP‐31d, PP‐32b, PP‐33a, and PP‐33d) were selected for single‐
dose analysis against human cancer cell lines. The structure and
activity relationship indicates that compounds comprising a ben-
zyl group at C‐2 of the pyrimidine are more potent and showed
enhanced cytotoxicity. The cell cycle analysis, DNA damage stu-
dies, annexin‐V/PI assay, evaluation of mitochondrial membrane
potential, and assessment of caspase‐3/7 activity were carried out
to determine the mechanistic details of compound PP‐31d‐
induced cancer cell death. These results indicate that the ob-
served anticancer activity of compound PP‐31d on NCI‐H460
cells is due to the induction of apoptosis. Finally, we conclude that
pyrazolo[3,4‐d]pyrimidine derivatives consisting of a benzyl group
can be carefully modified further to get more potent anticancer
compounds.
Z
ρ
calc/g/cm3
1.230
μ/mm
0.075
F(000)
644.0
Crystal size/mm3
Radiation
0.23 × 0.24 × 0.25
MoKα (λ = 0.71073)
4.35 to 49.994
2Θ range for data
collection/°
Index ranges
−13 ≤ h ≤ 13, −11 ≤ k ≤ 11,
−17 ≤ l ≤ 17
Reflections collected
56,974
Independent reflections
Data/restraints/parameters
Goodness‐of‐fit on F2
Final R indexes [I ≥ 2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole/e Å−3
CCDC number
2,853 [Rint = 0.0256, Rsigma = 0.0087]
2853/0/211
1.238
R1 = 0.0451, wR2 = 0.1489
R1 = 0.0589, wR2 = 0.1729
0.47/−0.52
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4
EXPERIMENTAL
1526438
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4.1
Chemistry
Thus, the ability of PP‐31d to activate effector caspases was de-
termined using appropriate substrates provided in the caspases‐3/7
Glo assay kit. Treatment of NCI‐H460 cells with PP‐31d showed a
significant increase in caspase‐3/7 activity (2.5‐fold) as compared to
the untreated control, signifying that cell death caused by this
compound is linked to the induction of cell apoptosis (Figure 8b).
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4.1.1
General
All commercially available solvents and reagents were procured
from either Sigma‐Aldrich, S D Fine, or Avra Synthesis Ltd. Thin
layer chromatographs (TLCs) were run on Merck silica gel 60 F254
TLC plates and spots were visualised using ultraviolet light
(254 nm) or permanganate staining. Column chromatography was
carried out with commercially available silica gel and solvents. All
melting points are uncorrected and determined by an open capil-
lary method. NMR spectra were recorded on a 400 MHz Bruker AV
400 spectrometer. Chemical shifts are expressed in ppm relative to
the solvent peak. NMR spin multiplicities are assigned as s (singlet),
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2.3.6
Compound PP‐31d causes chromatin
condensation and DNA fragmentation
Cellular apoptosis is characterized by changes in nuclear morphology,
such as nuclear shrinkage, chromatin condensation, and fragmented

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FIGURE 4 Antiproliferative activity of
compounds PP‐31d and PP‐33d on NCI‐H460
cancer cells. Dose‐dependent effect of
compounds PP‐31d and PP‐33d on human lung
cancer cell (NCI‐H460) proliferation was
determined by MTT (3‐(4,5‐dimethylthiazol‐
2‐yl)‐2,5‐diphenyl tetrazolium bromide) assay.
(a) Graphical representation of percent cell
viability at different concentrations (0–20 µM)
of PP‐31d and PP‐33d, scanning electron
microscopy of triplicates from three different
experiments is represented by error bars,
***p > 0.001. (b) NCI‐H460 cell morphology
after treatment with or without PP‐31d
and PP‐33d compounds was analyzed and
photographed using a phase‐contrast
microscope
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General procedure for the synthesis
of P‐31–P‐33
d (doublet), t (triplet), q (quartet), bs (broad singlet), dd (doublet of
doublet), and m (multiplet, for unresolved lines). Coupling con-
stants (J) were expressed in hertz (Hz). Mass spectra were recorded
using Shimadzu GCMS‐QP2010S.
4.1.2
To
a mixture of P‐2 (2 g, 7.246 mmol), anhydrous potassium
The InChI codes of the investigated compounds together with
carbonate (2 g, 14.493 mmol) and [1,1′‐bis(diphenylphosphino)ferrocene]
some biological activity data are provided as Supporting Information.
dichloropalladium(II) dichloromethane complex (0.295 g 0.3623 mmol)
FIGURE 5 Cell cycle analysis of PP‐31d treated cancer cells. (a) Histograms represent effect of PP‐31d on distribution of NCI‐H460 cells in
different phases of cell cycle at different concentrations (0–20 µM). (b) Percentage of NCI‐H460 cell population in different phases subG1, G1,
S, and G2–M of the cell cycle after treatment with PP‐31d are represented as a bar graph

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FIGURE 6 Induction of cellular apoptosis by compound PP‐31d. (a) Histograms represent effect of PP‐31d on distribution of NCI‐H460 cells
in different phases Q1 (necrotic/dead cells), Q2 (late apoptosis cells), Q3 (live cells), and Q4 (early apoptosis cells) at different concentrations
(0–20 µM). (b) Percentage of NCI‐H460 cell population in Q1, Q2, Q3, and Q4 phases after PP‐31d compound treatment are represented as a
bar graph
FIGURE 7 PP‐31d induces depolarization of mitochondria and loss of membrane potential. (a) Density plot depicts effect of PP‐31d on
JC‐1‐stained NCI‐H460 cells at different concentrations (0–20 µM). Cells emitting green fluorescence and with low mitochondrial membrane
potential are represented as P1. (b) Percentage of cells emitting red fluorescent and with high mitochondrial membrane potential (P2) against
cells emitting green fluorescence and low mitochondrial membrane potential (P1) are represented as a bar graph

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FIGURE 8 Evaluation of PP‐31d induced apoptotic mechanism: Analysis of ROS production, activation of caspases, chromatin condensation,
and DNA fragmentation. (a) Effect of PP‐31d on intracellular ROS production was determined by fluorescence microscopy using ROS‐specific dye
DCFDA‐H2. (b) Effect of PP‐31d on activation of caspase‐3/7 in NCI‐H460 cells was determined by measuring caspase‐3/7 activity using caspase‐3/7
Glo assay and represented as fold increase in activity, ***p > 0.001. (c) Effects of different concentrations (0–20 µM) of PP‐31d on NCI‐H460 cellular
DNA are represented by agarose gel profiles. (d) DAPI‐staining showing the chromatin‐condensed nuclei in apoptotic cells following PP‐31d treatment.
DAPI, 4′,6‐diamidino‐2‐phenylindole; DCFDA, 2,7‐dichlorodihydro fluorescein diacetate; M, marker; ROS, reactive oxygen species
in ethanol (20 ml), different phenylboronic acids (7.971 mmol) were ad-
ded. The reaction mixture was heated at 80°C for 6–8 hr, and the pro-
gress of the reaction was observed by TLC. The reaction mixture was
cooled, extracted with ethyl acetate, and dried over anhydrous MgSO4.
The obtained organic layer was filtered through celite 545 to remove the
remaining fine catalyst particles. Solvent was removed under reduced
pressure and the crude product obtained was purified by column chro-
matography to obtain the key intermediates (P‐31–P‐33).
1‐[(1,1'‐Biphenyl)‐4‐yl]‐5‐amino‐3‐methyl‐1H‐pyrazole‐
4‐carbonitrile (P‐32)
Light yellow solid (78% yield); mp: 130–134°C; 1H NMR
(400 MHz, CDCl3) δ 7.70 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 8.4 Hz,
2H), 7.54 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.24
(t, J = 7.9 Hz, 1H), 4.70 (bs, 2H), and 2.31 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 152.01, 151.06, 150.29, 149.76, 140.34,
139.08, 138.14, 129.37, 128.65, 127.34, 126.89, 106.38, and
13.01; GCMS: m/z 274.
5‐Amino‐3‐methyl‐1‐[4′‐(trifluoromethoxy)‐(1,1′‐biphenyl)‐4‐yl]‐1H‐
pyrazole‐4‐carbonitrile (P‐31)
Light yellow solid (82% yield); mp: 122–124°C; IR (KBr) ʋ cm−1
5‐Amino‐1‐[4′‐ethyl‐(1,1'‐biphenyl)‐4‐yl]‐3‐methyl‐1H‐pyrazole‐
4‐carbonitrile (P‐33)
:
3,344, 3,232, 2,969, 2,925, 2,218, 1,644, 1,561, 1,535, 1,253, 1,167,
834, and 676; 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 8.6 Hz, 2H),
7.60 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H),
4.74 (bs, 2H), and 2.32 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 13C
NMR (101 MHz, CDCl3) δ 151.27, 150.26, 149.19, 149.17, 140.06,
138.39, 136.55, 129.87, 128.19, 124.42, 121.67, 119.03, 114.51, and
12.93; LCMS: m/z 359.
Light green solid (83% yield); mp: 150–152°C; 1H NMR (400 MHz,
CDCl3) δ 7.66 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.57
(d, J = 8.3 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 4.69 (bs, 2H), 2.62
(q, J = 7.8 Hz, 2H), 2.35 (s, 3H) and 1.26 (t, J = 7.6 Hz, 3H); 13C NMR
(101 MHz, CDCl3) δ 152.70, 151.67, 150.94, 149.58, 141.03, 140.36,
139.41, 130.88, 129.26, 128.85, 129.18, 104.97, 34.39, 14.65, and
12.94; GCMS: m/z 302.

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|
4.1.3
General procedure for the synthesis
1‐[(1,1′‐Biphenyl)‐4‐yl]‐3‐methyl‐1H‐pyrazolo[3,4‐d]pyrimidin‐
of PP‐31a–PP‐33a
4‐amine (PP‐32b)
White solid (72% yield); mp: 180–182°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.34 (d, J = 8.2 Hz, 2H), 8.31 (s, 1H), 7.85 (d, J = 7.8 Hz, 2H), 7.81
(d, J = 7.6 Hz, 2H), 7.63–7.29 (m, 4H), 7.33 (t, J = 7.4 Hz, 1H), and 2.63
(s, 3H); 13C NMR (101 MHz, CDCl3) 159.47, 156.68, 153.93, 145.87,
139.24, 137.68, 135.54, 131.39, 128.18, 127.77, 121.94, 120.82,
101.06, and 14.69; GCMS: m/z 301.
To the intermediates P‐31–P‐33 (1 mmol), formic acid (5 ml) was
added and refluxed for 6–8 hr. The reaction mixture was cooled and
the solid was separated by filtration, washed with excess amount of
water, dried, and recrystallized from formic acid to give compounds
PP‐31a–PP‐33a.
3‐Methyl‐1‐[4′‐(trifluoromethoxy)‐(1,1′‐biphenyl)‐4‐yl]‐1,5‐
dihydro‐4H‐pyrazolo[3,4‐d]pyrimidin‐4‐one (PP‐31a)
1‐[4′‐Ethyl‐(1,1′‐biphenyl)‐4‐yl]‐3‐methyl‐6‐phenyl‐1H‐pyrazolo[3,4‐
d]pyrimidin‐4‐amine (PP‐33b)
White solid (85% yield); mp: >250°C; 1H NMR (400 MHz, dimethyl
sulfoxide [DMSO]‐d6) δ 12.35 (s, 1H), 8.14 (s, 1H), 8.13 (d, J = 5.8 Hz,
2H), 7.83–7.80 (m, 4H), 7.44 (d, J = 8.3 Hz, 2H), and 2.51 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ 159.53, 155.19, 151.65, 148.24, 139.36,
137.89, 136.19, 128.41, 127.68, 126.70, 126.06. 124.55, 117.03,
106.39, and 13.36; GCMS: m/z 386.
White solid (68% yield); mp: 197–199°C; 1H NMR (400 MHz, DMSO‐d6)
δ 8.35 (d, J = 8.4 Hz, 2H), 8.32 (s, 1H), 7.83 (d, J = 7.8 Hz, 2H), 7.78 (d,
J = 8.2 Hz, 2H), 7.66–7.32 (m, 4H), 2.73 (q, J = 7.3 Hz, 2H), 2.61 (s, 3H),
and 1.27 (t, J = 7.5 Hz, 3H); 13C NMR (101 MHz, CDCl3) 160.08, 157.38,
152.76, 144.88, 139.56, 138.23, 136.19, 132.06, 128.79, 127.34,
122.68, 121.41, 100.83, 35.49, 14.58, and 12.75; GCMS: m/z 329.
1‐[(1,1′‐Biphenyl)‐4‐yl]‐3‐methyl‐1,5‐dihydro‐4H‐pyrazolo[3,4‐d]pyr-
imidin‐4‐one (PP‐32a)
|
4.1.5
General procedure for the synthesis
White solid (81% yield); mp: >250°C; 1H NMR (400 MHz, DMSO‐d6)
δ 12.38 (s, 1H), 8.17 (s, 1H), 8.15 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 8.7 Hz,
2H), 7.72 (d, J = 7.3 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.3 Hz,
1H), and 2.55 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 157.91, 152.28,
148.91, 146.04, 139.16, 138.11, 137.58, 128.95, 127.58, 127.24,
126.56, 121.45, 105.81, and 13.33; GCMS: m/z 302.
of PP‐31c–PP‐33c and PP‐31d–PP‐33d
To a suspension of P‐31–P‐33 (1 mmol), potassium tert‐butoxide
(0.2 equiv) in tert‐butanol (5 ml), benzonitrile, or phenylacetonitrile
(1.2 equiv) was added and heated at 80°C for 4–6 hr. The reaction
mixture was concentrated under vacuum, solid was dissolved in water
and neutralized with 1 N HCl. The solid separated from filtration was
dried and recrystallized from ethanol to give the desired products PP‐
31c–PP‐33c and PP‐31d–PP‐33d.
1‐[4'‐Ethyl‐(1,1′‐biphenyl)‐4‐yl]‐3‐methyl‐1,5‐dihydro‐4H‐
pyrazolo[3,4‐d]pyrimidin‐4‐one (PP‐33a)
White solid (87% yield); mp: >250°C; 1H NMR (400 MHz, DMSO‐d6)
δ 12.35 (s, 1H), 8.16 (s, 1H), 8.13 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.7 Hz,
2H), 7.61 (d, J = 7.3 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 2.65 (q, J = 7.6 Hz,
2H), 2.53 (s, 3H), and 1.25 (t, J = 7.8 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 158.15, 152.39, 148.82, 147.06, 139.54, 138.11, 137.58,
130.12, 128.75, 126.45, 125.73, 121.97, 106.11, 33.08, 14.94, and
13.31; GCMS: m/z 330.
3‐Methyl‐6‐phenyl‐1‐[4′‐(trifluoromethoxy)‐(1,1′‐biphenyl)‐4‐yl]‐1H‐
pyrazolo[3,4‐d]pyrimidin‐4‐amine (PP‐31c)
White solid (72% yield); mp: 215–218°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.49 (dd, J = 6.7, 3.1 Hz, 2H), 8.46 (d, J = 8.7 Hz, 2H), 7.91–7.87
(m, 4H), 7.66–7.41 (m, 7H), and 2.69 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 161.74, 158.57, 155.64, 147.77, 143.20, 138.83, 138.67,
137.90, 135.35, 133.29, 132.18, 130.39, 129.45, 128.30, 127.44,
121.41, 120.29, 99.29, and 14.49; GCMS: m/z 461.
|
4.1.4
General procedure for the synthesis
of PP‐31b–PP‐33b
1‐[(1,1′‐Biphenyl)‐4‐yl]‐3‐methyl‐6‐phenyl‐1H‐pyrazolo[3,4‐d]pyr-
imidin‐4‐amine (PP‐32c)
The intermediates P‐31–P‐33 (1 mmol) were refluxed in formamide
for 4–6 hr. The reaction mixture was cooled and the precipitate was
filtered, dried, and recrystallized from ethanol to afford the desired
products PP‐31b–PP‐33b.
White solid (70% yield); mp: 192–194°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.47 (dd, J = 7.2, 3.1 Hz, 2H), 8.43 (d, J = 8.7 Hz, 2H), 7.89–7.83
(m, 4H), 7.63–7.38 (m, 7H), 7.30 (t, J = 7.8 Hz, 1H), and 2.67 (s, 3H);
13C NMR (101 MHz, CDCl3) δ 161.53, 158.49, 154.83, 144.70,
138.21, 137.84, 137.24, 134.70, 132.93, 132.07, 130.37, 129.58,
129.04, 128.76, 127.13, 120.81, 100.14, and 14.50; GCMS: m/z 377.
3‐Methyl‐1‐[4′‐(trifluoromethoxy)‐(1,1′‐biphenyl)‐4‐yl]‐1H‐
pyrazolo[3,4‐d]pyrimidin‐4‐amine (PP‐31b)
White solid (69% yield); mp: 205–207°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.33 (d, J = 8.8 Hz, 2H), 8.31 (s, 1H), 7.85 (d, J = 8.4 Hz, 4H), 7.65
–7.31 (m, 4H), and 2.67 (s, 3H); 13C NMR (101 MHz, CDCl3) 158.64,
156.60, 154.36, 147.79, 143.15, 138.68, 138.63, 135.54, 128.35,
127.40, 121.45, 120.38, 118.84, 100.39, and 14.47; GCMS: m/z 385.
1‐[4′‐Ethyl‐(1,1′‐biphenyl)‐4‐yl]‐3‐methyl‐6‐phenyl‐1H‐pyrazolo[3,4‐
d]pyrimidin‐4‐amine (PP‐33c)
White solid (69% yield); mp: 197–200°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.46–8.41 (m, 4H), 7.87 (d, J = 8.2, 2H), 7.84 (d, J = 8.1 Hz, 2H),
7.65–7.39 (m, 7H), 2.71 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H), and 1.25

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(t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 162.05, 159.16,
154.77, 144.82, 138.30, 137.89, 137.33, 135.09, 132.52, 132.11,
130.65, 129.21, 128.49, 128.09, 127.69, 122.38, 99.83, 35.91, 14.47,
and 12.89; GCMS: m/z 405.
added and incubated for 4 hr. Cells were lysed using 10% sodium
dodecyl sulfate (100 µl/well) in 0.01 N HCl and absorbance was
measured using Tecan i‐200 microplate reader at 570 nm, with a
reference wavelength of 640 nm. Percent viability was calculated
by considering vehicle control as 100%. In another experiment, NCI‐
H460 cells after treatment with compound PP‐31d and PP‐33d as
mentioned above were photographed using a phase‐contrast micro-
scope to determine changes in cellular morphology.
6‐Benzyl‐3‐methyl‐1‐[4‐(4‐trifluoromethoxyphenyl)phenyl]‐1H‐
pyrazolo[3,4‐d]pyrimidin‐4‐amine (PP‐31d)
White solid (75% yield); mp: 192–194°C; 1H NMR (400 MHz, DMSO‐
d6) 8.35 (d, J = 8.7 Hz, 2H), 7.83 (t, J = 9.3 Hz, 4H), 7.68–7.09 (m, 9H),
4.04 (d, J = 13.2 Hz, 2H), and 2.63 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 167.47, 158.64, 155.43, 147.76, 143.03, 138.88, 138.81, 138.69,
135.31, 128.95, 128.30, 128.16, 127.33, 126.09, 121.41, 120.19,
118.85, 98.73, 45.30, and 14.46; GCMS: m/z 475.
|
4.2.3
Cell cycle analysis
NCI‐H460 cells, after treatment with compound PP‐31d, were sub-
jected to cell cycle analysis, and the distribution of cells in different
phases of the cycle was determined by flow cytometry. Briefly,
0.1 × 106 cells were treated with different concentrations (5, 10, and
20 µM) of compounds for 24 hr. After treatment, the cells were
harvested by trypsinization, washed with phosphate‐buffered saline
(PBS), and fixed in 70% chilled ethanol. Rehydration of cells was
carried out using PBS and treated with 50 µl ribonuclease A (5 mg/ml
in PBS, DNase‐free) for 4 hr at 37°C to hydrolyze RNA. The cells
were then stained with PI (50 µg/ml in PBS) for 30 min at 37°C,
and the DNA content was analyzed on the FL‐2A channel of a flow
cytometer (FACS Calibur, BD) equipped with a 488 nm argon laser.
The data were analyzed by Cell Quest Pro software (BD) for the
distribution of cells in different phases of the cell cycle.
1‐[(1,1′‐Biphenyl)‐4‐yl]‐6‐benzyl‐3‐methyl‐1H‐pyrazolo[3,4‐d]pyr-
imidin‐4‐amine (PP‐32d)
White solid (69% yield); mp: 179–182°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.33 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.0 Hz,
2H), 7.62–7.13 (m, 10H), 4.04 (s, 2H), and 2.60 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ 168.15, 158.65, 156.04, 143.98, 139.13, 138.65,
138.02, 136.11, 130.94, 128.43, 127.84, 127.66, 127.32, 126.71,
123.88, 121.90, 99.08, 45.32, and 14.47; GCMS: m/z 391.
6‐Benzyl‐1‐[4′‐ethyl‐(1,1′‐biphenyl)‐4‐yl]‐3‐methyl‐1H‐pyrazolo[3,4‐
d]pyrimidin‐4‐amine (PP‐33d)
White solid (74% yield); mp: 185–188°C; 1H NMR (400 MHz, DMSO‐
d6) δ 8.31 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.2 Hz,
2H), 7.62–7.13 (m, 9H), 4.05 (s, 2H), 2.71–2.60 (m, 5H), and 1.23 (t,
J = 7.6 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 167.23, 158.77, 155.69,
143.39, 138.74, 138.61, 138.38, 135.12, 130.94, 128.16, 128.03,
127.89, 127.30, 126.25, 123.85, 121.62, 99.19, 45.28, 35.36, 14.46,
and 12.93; GCMS: m/z 419.
|
4.2.4
Detection of apoptosis by annexin‐V/PI
staining
NCI‐H460 cells (0.1 × 106) were cultured in 12‐well plates and
treated with or without compound PP‐31d for 24 hr. After treatment,
cells were harvested, washed, and resuspended in the binding buffer
from BD annexin‐V kit. Incubation of cell suspension with FITC
annexin‐V (5 µl) and PI (5 µl) was carried out in the dark for 5 min at
37°C. Cells undergoing apoptosis were then analyzed using flow
cytometry (FACS Calibur; BD). The percentages of cells positive for
annexin‐V, PI alone and both annexin‐V and PI were calculated by
dot blot analysis using Cell Quest Pro software (BD Biosciences).
|
4.2
In vitro anticancer activity
|
4.2.1
Cell lines and culture conditions
Human large cell lung cancer cell lines (NCI‐H460) were purchased
from National Center for Cell Science, Pune, India and cultured RPMI‐
1640 containing 10% fetal bovine serum, 100 µg/ml of penicillin, and
100 µg/ml of streptomycin at 37°C in a humidified atmosphere
containing 5% CO2.
|
4.2.5
Analysis of the mitochondrial membrane
potential
|
4.2.2
Assessment of cell viability and morphology
Changes in the mitochondrial membrane potential were determined
using lipophilic and cationic dye 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐
tetraethylbenzimidazolylcarbocyanine iodide (JC‐1). Compound
PP‐31d was incubated at different concentrations (5, 10, and 20 µM)
with NCI‐H460 cells (0.1 × 106) for 24 hr. Cells were then harvested
by gentle trypsinization, washed with PBS, and resuspended in
freshly prepared 1 ml JC‐1 (2.5 µM) solution at 37°C for 20 min. The
mitochondrial membrane potential of NCI‐H460 cells was analyzed
The viability of NCI‐H460 cells was analyzed after treatment with
compounds PP‐31d and PP‐33d using MTT assay. Briefly, cells were
seeded at a density of 5 × 104 cells/ml in 96‐well plates followed by
treatment with different concentrations (2.5, 5, 10, and 20 µM) of
compounds for 24 hr. DMSO‐treated cells were used as the vehicle
control. Further, to each well, 10 µl of MTT (5 mg/ml) solution was

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GAONKAR ET AL.
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by flow cytometer (FACS Calibur, BD) with an excitation of 488 nm
and an emission of 530 nm.
is thankful to UGC, New Delhi, for providing Senior Research Fel-
lowship (SRF).
CONFLICT OF INTERESTS
|
4.2.6
Detection of intracellular ROS generation
The authors declare that there are no conflicts of interests.
DCFDA‐H2, a cell‐permeable fluorescent probe, was used to de-
termine intracellular ROS production. Five micrometers of the com-
pound PP‐31d was incubated with NCI‐H460 cells for 24 hr, followed
by washing and staining with 15 µM DCFDA‐H2 for 10 min at 30°C.
The relative fluorescence intensity was analyzed using fluorescence
microscopy.
ORCID
Mohammed Azharuddin Savanur
http://orcid.org/0000-0002-
8903-2265
Imtiyaz Ahmed M. Khazi
http://orcid.org/0000-0001-8216-1913
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Additional supporting information may be found online in the
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How to cite this article: Gaonkar S, Savanur MA, Nadaf AA,
et al. Novel pyrazolo[3,4‐d]pyrimidine derivatives inhibit
human cancer cell proliferation and induce apoptosis by ROS
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