Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles and their evaluation as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.06.055

A series of hybrid aza heterocycles containing pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazole scaffold were synthesized from 1,3-dipolar cycloaddition reaction of pyrazolopyrimidinone based alkyne with azides using Cu(II) catalyst in presence of sodium ascorbate and evaluated for their anticancer efficacy in vitro against C6 rat and U87 human glioma cell lines. These compounds induced a concentration dependent inhibition of C6 rat and U87 human glioma cell proliferation. Compound 5f arrested the cells at S-phase of the cell cycle and induced apoptosis in U87 GBM cell lines. Further, apoptosis was evidenced by the cleavage of Caspase-3, PARP and up regulation of p53. In silico docking studies reveal that the compounds 5a, 5f and 5l were more effective in binding with TGFBR2 than other compounds.

Full text of DOI:10.1016/j.ejmech.2018.06.055

Accepted Manuscript
Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles and their
evaluation as potential anticancer agents
Muralidhar Allam, A.K.D. Bhavani, Anwita Mudiraj, Nikhil Ranjan, Mallikarjuna
Thippana, Phanithi Prakash Babu
PII:
S0223-5234(18)30546-4
10.1016/j.ejmech.2018.06.055
EJMECH 10525
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2018
Revised Date: 23 June 2018
Accepted Date: 23 June 2018
Please cite this article as: M. Allam, A.K.D. Bhavani, A. Mudiraj, N. Ranjan, M. Thippana, P.P. Babu,
Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles and their evaluation
as potential anticancer agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.06.055.
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ACCEPTED MANUSCRIPT
Graphical abstract
Specimen_001_control.fcs
DMSO
Specimen_001_50uM.fcs
50 µM
4
92
4
3
37 M1
0.51
%
Apoptotic cells/ Debris= 0.51% Cells in
S-phase= 8.95%
Apoptotic cells/ Debris= 26.64%
Cells in S-phase= 37.4%
M1
6.64
28
19
09
0
369
246
123
0
2
%
2
1
0
65.5
131.1
196.6
262.1
0
65.5
131.1
196.6
262.1
PE-H (x 1000)
PE-H (x 1000)
DMSO treated cells
5f treated cells
compound 5f:
•
•
•
Induces cell death in glioma cells.
Cells are arrested in S- phase of cell cycle.
triggers p53 mediated caspase dependent apoptotic pathway.

ACCEPTED MANUSCRIPT
Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles
and their evaluation as potential anticancer agents
a
a
b
b
Muralidhar Allam , Bhavani A. K. D. *, Anwita Mudiraj , Nikhil Ranjan ,
b
b*
Mallikarjuna Thippana Phanithi Prakash Babu
a
Department of Chemistry, Osmania University, Hyderabad, India-500007.
b
Department of Biotechnology, School of Life Sciences, University of Hyderabad,
Hyderabad, India-500046.
*
Corresponding Author: Mobile: 919177389748; E-mail: akdbhavani4ou@gmail.com
*
Mobile 9441583525 E-mail: prakash@uohyd.ac.in
Abstract
A series of hybrid aza heterocycles containing pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered
to 1,2,3-triazole scaffold were synthesized from 1,3-dipolar cycloaddition reaction of
pyrazolopyrimidinone based alkyne with azides using Cu(II) catalyst in presence of sodium
ascorbate and evaluated for their anticancer efficacy in vitro against C6 rat and U87 human
glioma cell lines. These compounds induced a concentration dependent inhibition of C6 rat
and U87 human glioma cell proliferation. Compound 5f arrested the cells at S-phase of the
cell cycle and induced apoptosis in U87 GBM cell lines. Further, apoptosis was evidenced by
the cleavage of Caspase-3, PARP and up regulation of p53. In silico docking studies reveal
that the compounds 5a, 5f and 5l were more effective in binding with TGFBR2 than other
compounds.
Keywords: Pyrazolo[3,4-d]pyrimidin-4(5H)-one, 1,2,3-Triazole, Glioma, Anti-cancer
activity, Apoptosis.
1
. Introduction
Pyrazolo[3,4-d]pyrimidin-4(5H)-one is a bioisostere of natural purine nucleoside heterocyclic
base, considered as privileged core skeleton in biologically active compounds. A broad
spectrum of pharmacological properties imparts in diversely substituted pyrazolo[3,4-
d]pyrimidin-4(5H)-ones, which are exemplified as anti-inflammatory [1], anti-bacterial [2],
anti-fungal [3], anti-viral [4], anti-microbial [5], CNS-modulating [6], anti-oxidant [7] and
cardiovascular agents [8]. Some derivatives of pyrazoloprymidinones are also used to treat
insomnia [9] and diabetes type-II [10]. Additionally, the pyrazolo[3,4-d]pyrimidin-4(5H)-
ones are also best regarded as potential pharmacophores of several compounds which exhibit
1

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the anti-cancer activity against human colon (HCT-116) [11], human leukemia (HL-60) [12],
human estrogenic sarcoma [13], non-small cell lung cancer (NCL-H 460), CNS cancer (SF-
2
3
68) [14], human laryngeal epidermal carcinoma (HeP2) [15] and skin cancer cell lines (G-
61) [16].
To the best of our knowledge, very few types of hybrid molecules containing both
pyrazolo[3,4-d]pyrimidin-4(5H)-one and 1,2,3-triazole scaffolds are known in literature.
Pyrazolo[3,4-d]pyrimidin-4(5H)-ones with 1,2,3-triazole moiety at 3-position I, II which
were prepared by using step wise or bis click reaction, are used as key intermediates in
constructing the cross-linked deoxyoligonucliotide duplex. The stability of the resulted cross-
linked duplexes are evaluated [17]. 3,6-Dimethyl-1-phenyl-5-((1-aryl-1H-1,2,3-triazol-4-yl)-
methyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones III were reported for moderate anticancer
activity against HCT-116, MCF-6 cell lines at 100 µM concentration and for 5-lipoxygenase
inhibition activities [18] (Fig. 1).
<<Fig. 1>>
Gliomas are the most common primary brain tumors of the Central Nervous System (CNS).
Glioblastoma multiforme is the malignant form of glioma characterized by extensive
proliferation and metastasis. They show a highly aggressive course, poor treatment response
with patients having short-term survival expectancies. The current standard of care is
maximal surgical resection followed by radiotherapy and chemotherapy. Activation of
apoptosis is one of the mechanisms of action of anticancer drugs since escaping apoptotic cell
death leading to unregulated cell proliferation and eventual tumor development is considered
as one of the hallmarks of cancer. Thus, the identification of new drugs to curb uncontrolled
cell growth remains an ongoing effort.
Very few pyrazolo[3,4-d]pyrimidines were explored for anti-cancer activity against
glioblastoma. A pyrazolopyrimidine derivative, MK-1775 is the most promising inhibitor of
Wee-1 kinase which is over expressed in primary diffuse intrinsic pontine glioma (DIPG)
tissue [19]. WAY-001, WAY-600, WYE-687 and WYE-354 and PP242 are pyrazolo-
pyrimidine derivatives, reported as mTOR inhibitors and exhibiting anti proliferative effect
against glioblastoma cells [20]. To the best of our knowledge, none of the pyrazolo[3,4-
d]pyrimidin-4(5H)-ones are explored against glioblastoma cell lines. In view of all these
findings, we became interested in developing the bioactive hybrid molecules wherein, the
pyrazolo[3,4-d]pyrimidin-4(5H)-one is flanked by 1,2,3-triazole through methylene linkage
and evaluating these compounds as potential anti-cancer agents on the glioma cell lines and
2

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the possible mechanisms involved. Further, we present the molecular docking studies of the
target molecules with Transforming growth factor-beta receptor type 2 protein (TGFBR2)
which is overexpressed in glioblastoma cells.
2
. Results and discussion
2
.1 Chemistry
The required key precursor for the target pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to
,2,3-triazole was synthesized in three steps starting from 5-amino-1-aryl-1H-pyrazole-4-
1
carbonitrile (1a, 1b). The carbonitriles 1a and 1b were prepared according to the procedure
available in the literature [21]. 1-Aryl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (2a, 2b)
were obtained by cyclization of the corresponding carbonitrile 1a and 1b with formic acid
under reflux for 4 h [22]. Then, amide NH in 2a, 2b was propargylated with propargyl
bromide in the presence of 3 equivalents of potassium carbonate as mild base in dry acetone
°
at 0 C for 4 h to give the corresponding compounds 3a, 3b respectively in 85% and 92%
yields (Scheme 1). The structures of compounds 3a and 3b were confirmed based on their
-1
spectral data. IR spectrum of 3a showed acetylene band at 2228 cm and carbonyl band at
-1
1
1
663 cm . The H NMR spectrum exhibited characteristic signals at δ 4.85 as doublet due to
N-CH and at δ 2.53 as triplet due to acetylene proton. The presence of N-CH and acetylene
2
2
1
3
carbons also indicated in C NMR spectrum by showing signals at δ 34.8, 75.6 and 76.3
ppm.
<<Scheme 1>>
Recently, Rahmouni et. al., reported the propargylation at N-3 position of 3,6-
°
dimethylpyrazolo[3,4-d]pyrimidin-4(5H)-one using NaH as strong base in DMF at 20 C and
subsequent 1,3-dipolar cycloaddition of the propargylated compound with azide in toluene in
the presence of a catalytic amount of triethylamine under reflux for 8 h leading to a binary
mixture of 3,6-dimethyl-1-phenylpyrazolo[3,4-d]pyrimidin-4(5H)-one based triazoles (4- and
5
-regioisomers) [18]. Herein, we report pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1-
aryl-1,2,3-triazol-4-yl)methylene moiety as sole products using click reaction between
pyrazolopyrimidinone based alkyne and aromatic azides. The azide-alkyne reaction of 3a
with an azide 4a was carried out by using various mol% of CuSO in presence of sodium
4
ascorbate (20 mol %) in DMF-H O solvent mixture at room temperature for 6 h (Scheme 2).
2
Employing 1, 5, 10 mol % of CuSO .5H O resulted in the formation of triazole derivative 5a
4
2
3

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with inferior yields. Finally, good yield of the desired product was obtained when 15 mol %
of Cu(II) catalyst is used (Table 1).
<
<Scheme 2>>
<Table 1>>
The formation of triazole moiety in the compound 5a is indicated by the absence of peaks due
<
1
13
1
to acetylene group in IR, H NMR and C NMR spectra. H NMR spectrum of 5a exhibited
one singlet signal in aliphatic region at δ 5.39 due to N-CH protons and the remaining
2
signals are observed in aromatic region. Similarly, one signal in aliphatic region (δ 41.2) and
the remaining carbon signals in aromatic region (δ 107.2-151.2ppm) are observed along with
1
3
carbonyl carbon signal at δ 157.0 in C NMR spectrum. The ESI-HRMS also confirmed the
+
structure for 5a by showing [M+H] peak at m/z 370.1417. The structure for 5a was further
unambiguously confirmed by a single crystal X-ray analysis with 50% ellipsoid contour
percent probability (Fig. 2).
<<Fig. 2>>
Employing optimized conditions, 1,3-dipolar cycloaddition reaction of 3a-b with various
aromatic azides 4b-g in the presence of 15 mol % of the CuSO .5H O, 20 mol % of sodium
4
2
ascorbate afforded the corresponding target compounds 5b-5l in 78 to 92 % yields.
2
2
.2 Anticancer activity
.2.1 MTT assay
The target hybrid molecules 5a-l were screened for anti-proliferative activity in two glioma
cell lines (rat-C6 and human-U87) by MTT assay [23]. This assay provides a quantitative
measure of the number of cells with metabolically active mitochondria and is based on the
reduction of a tetrazolium bromide salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide]. The results of the inhibitory effect of all compounds against the growth
of both C6 and U87 glioma cell proliferation are presented in Table 2. The results are
compared at a concentration of 10 µM. Most of the derivatives are active and activity is more
prominent in U87 cell lines. The compounds 5a, 5f and 5l displayed high inhibition of cell
proliferation for both cell line cultures (Table 2). At 10 µM concentration in C6 glioma cell
lines, % inhibition of 5a, 5f and 5l were 34.10±2.5, 17.87±1.7 and 35.91±1.5 respectively.
These compounds also displayed more pronounced inhibition in U87 cell lines (5a, %
4

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inhibition = 52.34±4.9) (5f, % inhibition = 47.69±2.05) (5l, % inhibition =54.42±4.6). This
shows that the U87 cell lines are more sensitive towards these compounds. The compounds
5
e, 5g, 5h, 5j exhibited good anti-proliferative effect against U 87 cell lines with percentage
inhibition values of 46.35±1.25, 40.60±5.5, 40.98±2.1 and 21.31±4.9 respectively but did not
exert any inhibition of C6 glioma cell lines up to 100 µM concentration. Compound 5a, 5f
and 5l showed cytotoxicity in both C6 and U87 cells but compounds 5a and 5l were not
freely soluble in DMSO hence we have chosen 5f for further evaluation. IC50 value of
compound 5f in C6 cell line is 21.3 µM whereas in U87 cell line is 12.5 µM. Because of its
greater effectiveness on U87 cells viability we investigated the mechanistic aspects of 5f
mediated inhibition of U87 cell lines.
<<Table 2>>
2
.2.2 Flow Cytometry
The effect of 5f on cell cycle arrest of glioma U87 cells was determined by flow cytometry.
Glioma cells were treated with different concentrations of 5f (10 µM, 25 µM, 50 µM) or
DMSO (vehicle control). In the post treatment, the cells were stained with propidium iodide.
Analysis showed that compound 5f is inducing arrest in S phase of cell cycle, this implies
that the cell is unable to duplicate its DNA, as a result a decrease in M-phase is also observed
and the number of apoptotic cells are progressively increasing with increasing concentration
of 5f (Fig. 3).
<<Fig. 3>>
2
.2.3. Activated caspase-3 as execution factor for apoptosis
Further we investigated the activation of caspase-3 in compound 5f treated cells. Caspases are
a family of conserved cysteine-aspartic acid proteases which plays an important role in
apoptosis. Caspase-3 is called as executioner caspase [24]. They are synthesized as pro-
enzymes and are activated upon cleavage. Hence caspase-3 activation indicates apoptotic
mode of cell death. The activity of caspase-3 was evaluated by western blotting. Fig. 3
indicates caspase-3 expression levels increased with increase in concentration of compound
5
f treated cells. The functional expression of caspase-3 was studied for its substrate Poly
(ADP-ribose) polymerase (PARP). PARP acts as the main cleavage site of caspase-3. PARP
helps the cells to maintain their viability hence its cleavage is indicative of cells undergoing
apoptosis which is evident in western blotting. The results show increase in the cleaved
PARP levels in a concentration dependent manner. This is in consistence with the earlier
5

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reports that activation of caspase-3 cleaves PARP into 89 kDa fragment leading to apoptosis
[
25]. In addition, we studied the involvement of p53 in inducing apoptosis in compound 5f
treated cells. Western blot analysis represents that p53 is up regulated with increase in the
concentration of 5f, confirming its involvement in the apoptosis (Fig. 4). All these results
confirm that compound 5f shows significant anti-proliferative and pro apoptotic effect on
glioma cells by triggering p53 mediated and caspase dependent apoptosis pathway.
<<Fig. 4>>
2
.3 Molecular Docking Studies
Molecular docking is a method to predict the interaction of a ligand to the binding site of
receptor. The protein Transforming growth factor-beta receptor type 2 (TGFBR2) was chosen
for the study. TGFBR2 is localized on the membrane and is one of the important receptors
that activates downstream apoptosis signaling cascade [26,27,28,29]. This can be targeted to
activate apoptotic cascade leading to caspase cleavage, DNA fragmentation and ultimately
apoptotic cell death. Molecular docking studies of 5a-l with TGFBR2 were carried out using
MVD software. The compounds 5a, 5f and 5l adopted U-shape conformation at the active site
of the target protein and the results which were obtained in the molecular docking studies are
presented in Table 3.
<<Table 3>>
Based on the docking studies, compounds 5a, 5f and 5l interaction with the receptor were
analyzed.
The protein-ligand complex analysis shows that ligand binds in the hydrophobic pocket of the
protein kinase domain [30] and binding mode is stabilized by the formation of hydrogen
o
bonds between ligand and active site residues of the protein. H-bond distance is below 4 A
for all the three compounds. Three compounds 5a, 5f and 5l are interacted with same amino
acid residues by showing similar H-bonding interactions at the active site of the target
protein, which are shown as follows: a Hydrogen bonding interaction between i) Lys 277 and
N-2 of pyrazole moiety ii) Thr 325 and carbonyl oxygen of pyrimidine ring iii) two Hydrogen
bonding interactions between His 328 and N-2 as well as N-3 of triazole ring (Fig. 5, 6 and
7
). The compound 5l also showed additional Hydrogen bonding interaction between Lys 277
and N-1 of pyrazole moiety. Besides the three interactions, the halogen of 5f showed
hydrophobic interaction with the protein (Fig. 8).
<<Fig. 5>>
6

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<<Fig. 6>>
<<Fig. 7>>
<<Fig. 8>>
3
. Conclusion
A series of new pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to triazoles were synthesized
from 1,3-dipolar cycloaddition reaction of 3 with azides 4 using Cu(II) catalyst in the
presence of sodium ascorbate. All compounds showed moderate to good cytotoxic activity
against U87 glioma cell lines at 10 µM concentration. But in C6 glioma cell lines 5e, 5g, 5h
and 5j did not show any activity up to 100 µm concentration. The compounds 5a, 5f and 5l
displayed significant anticancer activity against C6 and U87 glioma cell lines. The compound
5
f induced concentration dependent apoptosis in U87 cell lines predominantly through
activation of caspase-3 along with PARP cleavage. The molecular docking studies also
demonstrated the potent activity of 5a, 5f and 5l against glioma cell lines by providing the
strong interaction of these compounds with TGFBR2 protein and it gives the potent insight in
the development of good candidate for gliomas.
4
4
4
. Experimental section
.1 Chemistry
.1.1 General Chemistry
Melting points were determined with an electro thermal capillary melting point apparatus and
are uncorrected. TLC was performed on E-Merck pre-coated 60 F254 plates and spots were
rendered visible by exposing to UV light and iodine vapor. All the products were purified by
column chromatography using Silica gel (60-120 Mesh). The proton magnetic resonance
spectra were recorded on a Bruker Avance II (400 MHz) instrument using TMS as an internal
1
3
standard and CDCl as solvent. C NMR spectra were recorded on Bruker Avance II (100
3
MHz) instrument. Chemical shifts are given in parts per million (δ ppm). High Resolution
Mass Spectra were recorded on Agilent 6500 Q-TOF and 6200 Series TOF LC-MS solution,
and IR spectra were recorded on Shimadzu FTIR 8400S spectrophotometer.
4
.1.2 General procedure for the preparation of 1-aryl-5-(prop-2-yn-1-yl)-1H-pyrazolo[3,4-
d]pyrimidine-4(5H)-one (3a, 3b)
To the solution of 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 2a/b (9.43mmol) in dry
acetone, potassium carbonate (3.9g, 28.29mmol) and 3-bromoprop-1-yne (1.19g, 10.3mmol)
7

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o
was added at 0-5 C. The reaction mixture was stirred at room temperature. After 3-4 h, the
excess solvent in the reaction mixture was evaporated under reduced pressure, the resulting
residue was diluted with water and extracted by using EtOAc (3x50ml). The combined
organic layers were dried over anhydrous Na SO . After evaporation of the solvent, the crude
2
4
product was purified by silica gel column chromatography to give the corresponding
compound 3a/3b.
4
.1.2.1 1-Phenyl-5-(prop-2-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-one (3a)
-1
1
Yield 85 %; mp 297-299 °C; IR (cm ): 2228, 1663; H NMR (400 MHz, CDCl3): δ 8.33 (s,
1
7
1
7
4
H, Ar-H), 8.28 (s, 1H, Ar-H), 8.06 – 8.00 (m, 2H, Ar-H), 7.57 – 7.49 (m, 2H, Ar-H), 7.42 –
.35 (m, 1H, Ar-H), 4.85 (d, J = 2.6 Hz, 2H, N-CH2), 2.53 (t, J = 2.6 Hz, 1H, acetylene-H);
3C NMR (100 MHz, CDCl ) δ 156.1, 151.0, 148.0, 138.2, 136.2, 129.1, 127.4, 122.1, 107.0,
3
+
6.3, 75.6, 34.7; ESI MS: m/z = 251 [M+H] .
.1.2.2 1-(4-Chlorophenyl)-5-prop-2-yn-1-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-one (3b)
-1
1
Yield: 92 %; mp >300 °C; IR (cm ): 2210, 1694; H NMR (400 MHz, CDCl ): δ 8.33 (s, 1H,
3
Ar-H), 8.26 (s, 1H, Ar-H), 8.05 – 8.02 (dd, J = 2.13, 3.05 Hz, 2H, Ar-H), 7.46 – 7.49 (dd, J =
2
.13, 3.05 Hz, 2H, Ar-H), 4.84 (d, J = 2.6 Hz, 2H, N-CH ), 2.53 (t, J = 2.6 Hz, 1H, acetylene-
2
1
3
H); C NMR (100 MHz, CDCl ) δ 156.3, 151.0, 148.0, 138.2, 136.2, 129.2, 127.4, 122.2,
3
1
07.0, 76.3, 75.5, 34.8.
4
4
.1.3 General procedure for the synthesis of 1,2,3-triazol-4-yl pyrazolo[3,4-d]pyrimidin-
(5H)-ones 5a-l
To a stirred solution of compound (3a / 3b) (0.4mmol) in DMF: H O (4:1, v/v) (5 mL),
2
sodium ascorbate (20mol %), CuSO .5H O (15mol %) and appropriate azide 4a-g (1.2eq,
4
2
0
.48mmol) were added in succession and stirred at room temperature for 6-8 h. The progress
of the reaction was monitored using TLC. After completion of the reaction, ice cold water
100 mL) was added to the reaction mixture and extracted with EtOAc (3x50 mL). Combined
organic layers were dried over anhydrous Na SO and concentrated under reduced pressure.
(
2
4
The crude product obtained was purified by silica gel chromatography using pet.ether: EtOAc
7:3) to afford the corresponding products 5a-l.
.1.3.1 1-Phenyl-5-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl-1H-pyrazolo[3,4-d]
(
4
pyrimidin4(5H)-one (5a)
°
-1
1
Yield: 85%; mp: 194-196 C; IR (cm ): 1694, 1575, 1502; H NMR (400 MHz, CDCl ): δ 8.41
3
(s, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.21 (s, 1H, Ar-H), 8.04 – 7.98 (m, 2H, Ar-H), 7.74 – 7.68
(m, 2H, Ar-H), 7.56 – 7.48 (m, 4H, Ar-H), 7.48 – 7.41 (m, 1H, Ar-H), 7.37 (t, J = 7.4 Hz, 1H,
8

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1
3
Ar-H), 5.39 (s, 2H, N-CH ); C NMR (100 MHz, CDCl ) δ 157.0, 151.2, 149.0, 142.7,
2
3
1
38.2, 136.7, 135.9, 129.7, 129.1, 129.0, 127.4, 122.1, 120.5, 107.2, 41.2; HRMS(ESI) m/z:
+
calculated for C H N O [M+H] : 370.1416; Found: 370.1417
.
2
0
16
7
Crystal data for 5a: Empirical formula, C H N O; Formula weight, 369.39; Crystal color,
2
0
15
7
3
Colorless; habit, needle; Crystal dimensions, 0.36 x 0.24 x0.12 mm ; Crystal system,
Monoclinic; lattice type, primitive; lattice parameters, a = 18.000(4) Å, b = 13.519(3) Å, c =
7
.2202(15) Å, α = 90°, β = 98.373(15)°, γ = 90°; V = 1738.2(7) Å3; Space group, P 21/c; Z
4; D (cald) = 1.411 Mg/m3; F(000) = 768; λ(Mo-Kα) = 0.71073 Å; R[I>2σ(I)] = 0.0984 (
324), wR2 = 0.2139 (3313). Detailed X-ray crystallographic data is available from the
=
1
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK (for
compound 5a CCDC #1571323).
4
.1.3.2 1-Phenyl-5-((1-(o-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-
4
(5H)-one (5b)
-1
1
Yield: 89%; mp: 190-192°C; IR ( cm ): 1654, 1555, 1575; H NMR (400 MHz, CDCl ): δ
3
8
.40 (s, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.17 (s, 1H, Ar-H), 8.02-8.01 (m, 2H, Ar-H), 7.58-
.57 (m, 2H, Ar-H), 7.53 – 7.50 (m, 2H, Ar-H), 7.38-7.36 (m, 1H, Ar-H), 7.31-7.21 (m, 2H,
7
1
3
Ar-H), 5.38 (s, 2H, N-CH ), 2.41 (s, 3H, CH ); C NMR (100 MHz, CDCl ): δ 156.5, 151.4,
2
3
3
1
45.4, 142.3, 140.7, 138.4, 136.5, 136.3, 131.6, 125.3, 126.2, 125.7, 123.8, 123.1, 108.3,
+
1
06.5, 41.2, 20.3, 14.3. HRMS (ESI) m/z: calculated for C H N O [M+H] : 384.1573;
2
1
18
7
Found: 384.1565.
4
4
.1.3.3 1-Phenyl-5-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrazolo[3,4-d] pyrimidin-
(5H)-one (5c)
-1
1
Yield: 85%; mp: 194-196°C; IR (cm ): 1685, 1582, 1527; H NMR (400 MHz, CDCl ): δ 8.40
3
(s, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.16 (s, 1H, Ar-H), 8.03-8.01 (m, 2H, Ar-H), 7.57-7.56 (m,
2
5
1
4
H, Ar-H), 7.53 – 7.46 (m, 2H, Ar-H), 7.36-7.34 (m, 1H, Ar-H), 7.25-7.27 (m, 2H, Ar-H),
1
3
.38 (s, 2H, N-CH ), 2.41 (s, 3H, CH ); C NMR (100 MHz, CDCl ): δ 157.0, 151.2, 145.1,
2
3
3
42.6, 135.2, 138.2, 136.0, 134.4, 130.2, 125.1, 127.3, 122.1, 122.1, 120.5, 107.3, 41.2, 21.0.
.1.3.4 5-((1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (5d)
°
-1
1
Yield: 80%; mp: 182-184 C; IR (cm ): 1653, 1580, 1505; H NMR (400 MHz, CDCl ): δ 8.44
3
(s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.25 (s, 1H, Ar-H), 8.06 – 7.58 (m, 2H, Ar-H), 7.72 (dd, J
=
8.3, 1.6 Hz, 1H, Ar-H), 7.52 (dd, J = 10.8, 5.1 Hz, 2H, Ar-H), 7.46 – 7.33 (m, 2H, Ar-H),
13
7
.05 (dd, J = 11.6, 4.5 Hz, 2H, Ar-H), 5.40 (s, 2H, N-CH ), 3.85 (s, 3H, O-CH ); C NMR
2 3
9

ACCEPTED MANUSCRIPT
(
100 MHz, CDCl ) δ 156.5, 151.2, 151.0, 145.2, 141.8, 141.4, 138.2, 136.0, 130.3, 125.1,
3
1
27.3, 125.5, 125.4, 122.1, 121.2, 112.1, 107.3, 55.5, 41.0; HRMS (ESI) m/z: calculated for
+
C H N O [M+H] : 400.1522; Found: 400.1515.
21
18
7
2
4
.1.3.5 5-((1-(2-Florophenyl-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-1H-pyrazolo[3,4-d]-
pyrimidin-4(5H)-one (5e)
°
-1
1
Yield: 81%; mp: 186-188 C; IR (cm ): 1706, 1575, 1506; H NMR(CDCl 400MHz): δ 8.42(s,
3,
1
1
7
1
1
3
4
H, Ar-H), 8.25-8.28(m, 1H, Ar-H), 8.24(s, 1H, Ar-H), 8.02-7.55(m, 2H, Ar-H),7.50-7.86(m,
H, Ar-H), 7.53-7.45(m, 2H, Ar-H), 7.47-7.42(m, 1H, Ar-H), 7.38-7.35(m, 1H, Ar-H),7.33-
1
3
.28(m, 2H, Ar-H), 5.40(s, 2H, N-CH ); C NMR (100 MHz, CDCl ): δ156.5, 154.3, 152.3,
2
3
51.2, 145.0, 142.4, 138.2, 136.0, 130.5, 130.4, 125.1, 127.3, 125.1, 124.8, 122.1, 117.1,
+
16.5, 107.2, 41.0; HRMS (ESI) m/z: calculated for C H FN O [M+H] : 388.1322; Found:
2
0
15
7
88.1319.
.1.3.6 5-((1-(3-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-1H-pyrazolo[3,4-d]-
pyrimidin-4(5H)-one (5f)
°
-1
1
Yield: 79 %; mp: 180-182 C; IR (cm ): 1688, 1578, 1526; H NMR (400 MHz, CDCl ): δ 8.39
3
(s, 1H,Ar-H), 8.25 (s, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 8.04 – 7.58 (m, 2H, Ar-H), 7.77 (t, J =
1
.5 Hz, 1H, Ar-H), 7.64 – 7.55 (m, 1H, Ar-H), 7.55 – 7.34 (m, 5H, Ar-H), 5.38 (s, 2H, N-
13
CH ); C NMR (100 MHz, CDCl ): δ 157.0, 151.2, 148.5, 143.0, 138.2, 137.5, 136.0, 135.6,
2
3
1
30.8, 125.1, 125.1, 127.4, 122.1, 120.8, 118.5, 107.2, 41.3; HRMS(ESI) m/z: for
+
C H ClN O [M+H] : 404.1027; Found: 404.1022
.
20
15
7
4
.1.3.7 5-((1-(2,3-Dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5g)
°
-1
1
Yield: 88% ; mp: 191-193 C; IR (cm ): 1658, 1574, 1505; H NMR (400MHz, CDCl ): δ 8.44
3
(s, 1H, Ar-H), 8.25(s, 1H, Ar-H), 8.04-8.01(m, 2H, Ar-H), 7.52(s, 1H, Ar-H), 7.54-7.45(m,
2
3
1
4
3
4
H, Ar-H), 7.35-7.25(m, 2H, Ar-H), 7.23-7.10(m, 2H, Ar-H), 5.40(s, 2H, N-CH ), 2.35 (s,
2
13
H, ortho-CH ), 2.01(s, 3H, meta-CH ); C NMR (100 MHz, CDCl ): δ 156.8, 151.3, 145.3,
3
3
3
41.7, 138.8, 136.5, 136.2, 132.8, 132.5, 131.5, 125.2, 126.1, 125.7, 123.8, 123.0, 107.5,
+
1.1, 20.3, 14.3; HRMS (ESI) m/z: calculated for C H N O [M+H] : 358.1725; Found:
2
2
20
7
58.1717.
.1.3.8 1-(4-Chlorophenyl-5-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (5h)
°
-1
1
Yield: 81%; mp: 190-192 C; IR (cm ): 1658, 1575, 1506; H NMR (400 MHz, CDCl ): δ 8.42
3
(s, 1H, Ar-H), 8.22 (d, J = 7.1 Hz, 2H, Ar-H), 8.02 (s, 2H, Ar-H), 7.70 (s, 2H, Ar-H), 7.47 (s,
1
3
5
H, Ar-H), 5.35 (s, 2H, N-CH ); C NMR (100 MHz, CDCl ): δ156.8, 151.3, 145.2, 142.6,
2 3
1
0

ACCEPTED MANUSCRIPT
1
36.5, 136.7, 136.2, 132.5, 125.7, 125.2, 125.0, 123.0, 122.1, 120.6, 107.4, 41.2; HRMS
+
(
ESI) m/z: calculated for C H ClN O [M+ H] : 404.1027; Found: 404.1016
.
20
15
7
4
.1.3.9 1-(4-Chlorophenyl)-5-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5i)
°
-1
1
Yield: 78% ; mp: 202-204 C; IR (cm ): 1656, 1577, 1531; H NMR (CDCl 400MHz): δ 8.45
3
,
(
s, 1H, Ar-H,), 8.30 (s, 1H, Ar-H), 8.23 (s, 1H, Ar-H), 8.04 (d, J = 8.8 Hz, 2H, Ar-H), 7.72
d, J = 7.2 Hz, 1H, Ar-H), 7.45 (dd, J = 22.3, 8.3 Hz, 3H, Ar-H), 7.05 (t, J = 7.5 Hz, 2H, Ar-
(
1
3
H), 5.40 (s, 2H, N-CH ), 3.85 (s, 3H, O-CH ); C NMR (100 MHz, CDCl ): δ 156.8, 151.3,
2
3
3
1
1
4
4
51.0, 145.4, 141.3, 136.5, 136.2, 132.8, 130.3, 125.2, 125.5, 125.4, 123.0, 121.1, 112.1,
+
07.5, 55.5, 41.0; HRMS (ESI) m/z: calculated for C H ClN O [M+H] : 434.1132; Found:
2
1
17
7
2
34.1125.
.1.3.10 1-(4-Chlorophenyl)-5-((1-(2-florophenyl-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5j)
°
-1
1
Yield: 85%; mp: 203-205 C; IR (cm ): 1656, 1580, 1530; H NMR (400 MHz, CDCl ): δ 8.43
3
(s, 1H, Ar-H), 8.26 (d, J = 7.9 Hz, 2H, Ar-H), 8.03 (d, J = 8.5 Hz, 2H, Ar-H), 7.88 (t, J = 7.2
Hz, 1H, Ar-H), 7.47 (d, J = 8.6 Hz, 3H, Ar-H), 7.31 (d, J = 7.2 Hz, 2H, Ar-H), 5.40 (s, 2H);
1
3
C NMR (100 MHz, CDCl ): δ 160.3, 156.7, 151.3, 145.2, 142.3, 136.8, 136.2, 132.8, 130.6,
3
1
25.2, 126.3, 125.2, 125.0, 124.5. 124.8, 123.0, 117.1, 116.5, 107.4, 41.0; HRMS (ESI) m/z:
+
calculated for C H ClFN O [M+H] : 422.0932; Found: 422.0924
.
2
0
14
7
4
.1.3.11 1-(4-Chlorophenyl)-5-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (5k)
°
-1
1
Yield: 88%; mp: 204-206 C; IR (cm ): 1655, 1585, 1525; H NMR (400 MHz, CDCl ): δ 8.41
3
(s, 1H, Ar-H), 8.23 (d, J = 5.5 Hz, 2H, Ar-H), 8.03 (d, J = 8.7 Hz, 2H, Ar-H), 7.77 (s, 1H, Ar-
1
3
H), 7.61 (d, J = 7.4 Hz, 1H, Ar-H), 7.50 – 7.41 (m, 4H, Ar-H), 5.38 (s, 2H); C NMR (100
MHz, CDCl ): δ 156.5, 151.3, 145.1, 142.5, 137.5, 136.8, 136.2, 135.7, 132.5, 130.8, 125.2,
3
1
25.1, 123.0, 122.1, 121.6, 120.5, 118.5, 107.4, 41.3; HRMS (ESI) m/z: calculated for
+
C H Cl N O [M+H] : 438.0637; Found: 438.0630.
20
14
2
7
4
.1.3.12 1-(4-Chlorophenyl)-5-((1-(2,3-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-1H-
pyrazolo[3,4-d] pyrimidin-4(5H)-one (5l)
°
-1
1
Yield: 80%; mp : 190-192 C; IR (cm ): 1652, 1576, 1525; H NMR (400 MHz, CDCl ): δ 8.46
3
(s, 1H, Ar-H), 8.24 (s, 1H, Ar-H), 8.05 – 8.01 (m, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.52 – 7.43
(m, 2H, Ar-H), 7.30 (t, J = 8.6 Hz, 1H, Ar-H), 7.21 (t, J = 7.7 Hz, 1H, Ar-H), 7.12 (d, J = 7.8
Hz, 1H, Ar-H), 5.35 (d, J = 8.6 Hz, 2H, N-CH ), 2.35 (s, 3H, ortho-CH ), 2.01 (s, 3H, meta-
2
3
13
CH ); C NMR (100 MHz, CDCl ) δ 156.8, 151.3, 145.3, 141.7, 138.8, 136.5, 136.2, 132.8,
3
3
1
1

ACCEPTED MANUSCRIPT
1
32.5, 131.5, 125.2, 126.1, 125.7, 123.8, 123.0, 107.5, 41.1, 20.3, 14.3. HRMS (ESI) m/z:
+
calculated for C H ClN O [M+H] : 432.133; Found: 432.1340.
2
2
19
7
4
.2 X-ray crystallography
X-ray reflections for compound 5a were collected at 298 K on Bruker D8 QUEST Eco. The
structure was solved and refined using APEX 3 software. All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms on heteroatoms were located from difference
electron density maps and all C−H hydrogens were fixed geometrically.
4
4
4
.3 Biological protocols
.3.1 Materials and Methods
.3.1.1 Cells and cell culture conditions
Rat glioma cell lines (C6) and human glioma cell lines (U87) were cultured in Dulbecco’s
Modified Eagles Medium (DMEM) media, supplemented with 10% heat inactivated fetal
bovine serum (Gibco, NY, USA), 100 IU/mL penicillin, and 100 mg/mL streptomycin
o
maintained in a humidified atmosphere with 5% CO at 37 C.
2
4
.3.1.2 Cell viability assay
The cell viability assessment was done by reduction of yellow MTT to formazan. Glioma
4
cells (C6 and U87) were plated at a density of 1×10 cells/ well in 96 well plate and allowed
to adhere. After overnight incubation, cells were treated with respective concentration of the
compounds and vehicle (0.2% DMSO) alone for 48 h. Then the medium was removed and 50
o
µl of MTT (1mg/ml in PBS) was added in each well and further incubated for 4 h at 37 C.
Formazan crystals were solubilized in 100 µl of isopropanol, and absorbance was measured at
5
70 nm. The experiment was performed in triplicate and results were expressed as mean ±
SD.
4
.3.1.3 Flow cytometry assay
6
Approximately 1×10 U87 cells were plated in 60 mm plate and allowed to adhere for 24 h.
After overnight incubation cells were treated with respective concentration of compound 5f
and vehicle (0.2% DMSO) for 48 hours. Cells were then washed with 1X chilled PBS and
collected in 1ml DMEM media after trypsinization, centrifuged at 2000 rpm for 5 min, fixed
in 70% ethanol, stained with propidium iodide (PI) mixture (50 mg/mL PI, 1% triton × 100,
5
0 mg/mL RNase A) and incubated for 30 min in the dark at room temperature. Then, PI
1
2

ACCEPTED MANUSCRIPT
stained cells were subjected to flow cytometry using a FACS Calibur (BD Biosciences). Cell
cycle parameters were analyzed using flowjo software.
4
.3.1.4 Western blotting
Glioma cells were pre-treated with different concentrations compound 5f. Cells were then
washed three times in ice-cold PBS and harvested in cold RIPA lysis buffer for 20 min.
followed by sonication. After sonication cells were centrifuged at 12000 rpm for 15 min. at
o
o
4
C and the clear supernatant was collected as whole cell lysate and stored at -80 C. Protein
concentrations were determined by Bradford assay reagent (Sigma, USA). Equal amount of
proteins were electrophoretically separated through 10% SDS-polyacrylamide gel and electro
transferred to nitrocellulose membrane. Membrane was blocked for 1 h at room temperature
o
in PBST containing 5% skimmed milk and were incubated overnight at 4 C with primary
antibodies against cleaved caspase-3, cleaved PARP, caspase-3, p53 and actin. After
washing, the membranes were incubated with HRP-conjugated secondary antibodies (1:2000)
and blots were developed using luminescence detection reagents (Thermo scientific, USA).
4
.3.1.5 Statistical analysis
Statistical analysis was performed using Graph Pad Prism 7.0 (Graph Pad Software Inc., CA,
and USA). All the data are presented as the mean ± standard deviation (S.D.). For in vitro
experiments, bars represent variations within the wells of the experiment. The representative
experimental data shown in here were repeated at least three times. The difference between
two groups was evaluated using Student's t test. Significant difference among three or more
groups was determined by one-way ANOVA with a post hoc analysis (Turkey test). The
values of p < 0.05, p < 0.01 and p < 0.001 were considered as statistically significant (*), very
significant (**), highly significant difference (***) respectively, unless otherwise mentioned.
4
.4 Molecular docking studies
To investigate the potent activity of the synthesized compounds for a protein target,
molecular docking was done using molecular docking algorithm MolDock with Molegro
Virtual Docker (MVD) software [31]. The docking scoring function of MolDock is based on
a piecewise linear potential (PLP). The re-ranking score is the highest ranked poses to further
increase docking accuracy.
4
.4.1 Ligand Preparation
1
3

ACCEPTED MANUSCRIPT
The 2D structure of the small molecule was built using MarvinSketch 5.2.6 software [32].
The structure was cleaned using fine build 3D cleaning method. This 3D structure was energy
minimized using SYBYL-X software [27].
4
.4.2. Protein Preparation
The crystal structure of TGFBR2 (PDB ID: 5E8Y) was downloaded from the RCSB Protein
Data Bank (PDB) [26], and its functional groups were protonated in Molegro Virtual Docker
(MVD) protein preparation wizard.
The protein receptor was treated as rigid and the grid centering was done at the binding site
with a box having the dimensions 18.36 Å, -0.86 Å and 6.91 Å for the detected cavity of the
protein. Further redocking was done for the most possible conformations of ligand with
protein. The grid box was centered at 11.52 Å, -1.30 Å and 10.13 Å in dimensions with
radius 10 Å.
Docking results were analyzed based on the docking scores of the most stable conformations
and its interactions with active site residues.
Acknowledgements
Authors are grateful to the Director, Central Facilities for Research and Development
(CFRD), Osmania University, Hyderabad for providing laboratory facilities. This work was
also financially supported by the project under UGC-UPE/FAR/OU/2017, No.63 program.
One of the authors, Muralidhar Allam thanks to UGC, New Delhi for award of SRF.
References
[
1] S. B. Yewale, S. B. Ganorkar, K. G. Baheti, R. U. Shelke, Novel 3-substituted-1-aryl-5-
phenyl-6-anilinopyrazolo[3,4-d]pyrimidin-4-ones: Docking, synthesis and
pharmacological evaluation as a potential anti-inflammatory agents. Bioorg. Med. Chem.
Lett. 22 (2012) 6616-6620.
[
2] M. Bakavoli, G. Bagherzadeh, M. Vaseghifar, A. Shiri, M. Pordel, M. Mashreghi, P.
Pordeli, M. Araghi, Molecular iodine promoted synthesis of new pyrazolo[3,4-
d]pyrimidine derivatives as potential antibacterial agents. Eur.J.Med.Chem. 45 (2010)
6
47-650.
1
4

ACCEPTED MANUSCRIPT
[
[
3] B. S. Holla, M. Mahalinga, M. S. Karthikeyan, P. M. Akberali, N. S. Shetty, Synthesis of
some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents.
Bioorg. Med. Chem. 14 (2006) 2040-2047.
4] J.-H. Chern, K.-S. Shia, T.-A. Hsu, C.-L. Tai, C.-C. Lee, Y.-C. Lee, C.-S. Chang, S.-N.
Tseng, S.-R. Shih, Design, synthesis, and structure–activity relationships of
pyrazolo[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors. Bioorg. Med.
Chem. Lett. 14 (2004) 2519-2525.
[
[
5] A. Bazgir, M. M. Khanaposhtani, A. A. Soorki, One-pot synthesis and antibacterial
activities of pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine-dione derivatives. Bioorg. Med.
Chem. Lett. 18 (2008) 5800-5803.
6] B. R. Neustadt, J. Hao, N. Lindo, W. J. Greenlee, A. W. Stamford, D. Tulshian, E.
Ongini, J. Hunter, A. Monopoli, R. Bertorelli, C. Foster, L. Arik, J. Lachowicz, K. Ng,
K.-I. Feng, Potent, selective, and orally active adenosine A2A receptor antagonists:
Arylpiperazine derivatives of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines. Bioorg.
Med. Chem. Lett. 17 (2007) 1376-1380.
[
7] A. El-Mekabaty, Synthesis and Antioxidant Activity of Some New Heterocycles
Incorporating the Pyrazolo[3,4-d]pyrimidin-4-one Moiety. Chem. Heterocycl. Compd.
5
0 (2015) 1698-1706.
[
[
8] P. Gong, Y.F. Zhao, D. Wang, Synthesis and vasodilatory activities of new pyrazolo[3,4-
d]pyrimidin-4-one derivatives, Chin. Chem. Lett. 13 (2002) 613-616.
9] K. W. Weitzel, J. M. Wickman, S. G. Augustin, J. G. Strom, Zaleplon: a pyrazolopyrimi-
dine sedative-hypnotic agent for the treatment of insomnia. Clin. Ther. 22 (2018) 1254-
1
267.
[
[
10] B. D. Manning, Challenges and Opportunities in Defining the Essential Cancer Kinome.
Sci. Signal. 2 (2009) pe15-pe15.
11] M. M. El-Enany, M. M. Kamel, O. M. Khalil, H. B. El-Nassan, Synthesis and antitumor
activity of novel 6-aryl and 6-alkylpyrazolo[3,4-d]pyrimidin-4-one derivatives.
Eur.J.Med.Chem. 45 (2010) 5286-5291.
[
[
12] X. J. Song, Y. Shao, X. G. Dong, Microwave-assisted synthesis of some novel
fluorinated pyrazolo[3,4-d]pyrimidine derivatives containing 1,3,4-thiadiazole as
potential antitumor agents. Chin. Chem. Lett. 22 (2011) 1036-1038
13] F. Manetti, A. Santucci, G. A. Locatelli, G. Maga, A. Spreafico, T. Serchi, M.
Orlandini, G. Bernardini, N. P. Caradonna, A. Spallarossa, C. Brullo, S. Schenone, O.
Bruno, A. Ranise, F. Bondavalli, O. Hoffmann, M. Bologna, A. Angelucci, M. Botta,
1
5

ACCEPTED MANUSCRIPT
Identification of a Novel Pyrazolo[3,4-d]pyrimidine Able To Inhibit Cell Proliferation of
a Human Osteogenic Sarcoma in Vitro and in a Xenograft Model in Mice. J. Med.
Chem. 50 (2007) 5579-5588.
[
14] S. Gupta, L. M. Rodrigues, A. P. Esteves, A. M. F. Oliveira-Campos, M. S. J.
Nascimento, N. Nazareth, H. Cidade, M. P. Neves, E. Fernandes, M. Pinto, N. M. F. S.
A. Cerqueira, N. Brás, Synthesis of N-aryl-5-amino-4-cyanopyrazole derivatives as
potent xanthine oxidase inhibitors. Eur.J.Med.Chem. 43 (2008) 771-780.
[
[
[
15] A. H. Shamroukh, A. E. Rashad, R. E. Abdel-Megeid, H. S. Ali, M. M. Ali, Some
Pyrazole and Pyrazolo[3,4-d]pyrimidine Derivatives: Synthesis and Anticancer
Evaluation. Arch. Pharm. 347 (2014) 559-565.
16] M. Devarakonda, R. Doonaboina, S. Vanga, J. Vemu, S. Boni, R. P. Mailavaram,
Synthesis of novel 2-alkyl-4-substituted-amino-pyrazolo[3,4-d]pyrimidines as new leads
for anti-bacterial and anti-cancer activity. Med. Chem. Res. 22 (2013) 1090-1101.
17] H. Xiong, F. Seela, Stepwise “Click” Chemistry for the Template Independent
Construction of a Broad Variety of Cross-Linked Oligonucleotides: Influence of Linker
Length, Position, and Linking Number on DNA Duplex Stability. J. Org. Chem. 76
(2011) 5584-5597.
[
[
18] A. Rahmouni, S. Souiei, M. A. Belkacem, A. Romdhane, J. Bouajila, H. Ben Jannet,
Synthesis and biological evaluation of novel pyrazolopyrimidines derivatives as
anticancer and anti-5-lipoxygenase agents. Bioorg. Chem. 66 (2016) 160-168.
19] J. M. Kreahling, P. Foroutan, D. Reed, G. Martinez, T. Razabdouski, M. M. Bui, M.
Raghavan, D. Letson, R. J. Gillies, S. Altiok, Wee1 Inhibition by MK-1775 Leads to
Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas. PLOS
One, 8 (2013) e57523.
[
[
20] Q. Liu, C. Thoreen, J. Wang, D. Sabatini, N. S. Gray, mTOR mediated anti-cancer drug
discovery. Drug Discov Today Ther Strateg. 6 (2009) 47-55.
21] F.A. Harden, R.J. Quinn, P.J. Scammells, Synthesis and adenosine receptor affinity of a
series of pyrazolo[3,4-d]pyrimidine analogs of 1-methylisoguanosine, J. Med. Chem. 34
(1991) 2892-2898.
[
22] M. Kandeel, L. Mohamed, M. Abd El Hamid, A. Negmeldin, design, synthesis, and
antitumor evaluation of novel pyrazolo[3,4-d]pyrimidine derivatives. Sci. Pharm. 80
(2012) 531-546.
1
6

ACCEPTED MANUSCRIPT
[
23] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of a
tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity
testing, Cancer Res. 47 (1987) 936-942.
[
[
24] D.J. McConkey, S. Orrenius, M. Jondal, Cellular signalling in programmed cell death
(apoptosis), Immunol today, 11 (1990) 120-121.
25] G.V. Chaitanya, P.P. Babu, Differential PARP cleavage: an indication of heterogeneous
forms of cell death and involvement of multiple proteases in the infarct of focal cerebral
ischemia in rat, Cell Mol Neurobiol. 29 (2009) 563-573.
[
[
26] N. Schuster, K. Krieglstein, Mechanisms of TGF-β-mediated apoptosis, Cell Tissue
Res. 307 (2002) 1-14.
27] D.R. Principe, B. DeCant , E. Mascarinas , E. Wayne ,A. M. Diaz, N. Akagi , R.F.
Hwang , B. Pasche , D. W. Dawson , D. Fang , D.J. Bentrem , H.G. Munshi , B. Jung,
P.J. Grippo, TGFβ signaling in the pancreatic tumor microenvironment promotes
fibrosis and immune evasion to facilitate tumorigenesis. Cancer Res. 76(2016),
2
525–2539.
[
[
[
28] M. Schiffer, M. Bitzer, I. S.D. Roberts, J. B. Kopp, P. Dijke, P. Mundel, E. P. Böttinger,
Apoptosis in podocytes induced by TGF-b and Smad7. J Clin Invest. 108 (2001), 807-
8
16.
29] Y. Li, Y. Liu, Y. Fu, T. Wei, L. Le Guyader, G. Gao, RS Liu, YZ Chang, C. Chen, The
triggering of apoptosis in macrophages by pristine graphene through the MAPK and
TGF-beta signaling pathways. Biomaterials, 33 (2012), 402-411.
30] A. Marchler-Bauer, S. Lu, J. B. Anderson, F. Chitsaz, M. K. Derbyshire, C. DeWeese-
Scott, J. H. Fong, L. Y. Geer, R. C. Geer, N. R. Gonzales, M. Gwadz, D. I. Hurwitz, J.
D. Jackson, Z. Ke, C. J. Lanczycki, F. Lu, G. H. Marchler, M. Mullokandov, M. V.
Omelchenko, C. L. Robertson, J. S. Song, N. Thanki, R. A. Yamashita, D. Zhang, N.
Zhang, C. Zheng, S. H. Bryant, CDD: a Conserved Domain Database for the functional
annotation of proteins. Nucleic Acids Res. 39 (2011), D225-D229.
[
[
31] R. Thomsen, M. H. Christensen, MolDock:ꢀ A New Technique for High-Accuracy
Molecular Docking. J. Med. Chem. 49 (2006), 3315-3321.
32] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N.
Shindyalov, P. E. Bourne, The Protein Data Bank. Nucleic Acids Res. 28 (2000) 235-
2
42.
Supplementary data
1
7

ACCEPTED MANUSCRIPT
1
13
Copies of IR, H NMR, C NMR and HRMS spectra for compounds.
MTT graphs of all compounds
Table/Figure/Scheme Captions:
Table 1: Optimized condition for azide-alkyne cyclization of 3a and 4a
Table 2: Inhibition of cell proliferation of 5a-l in C6 and U87 glioma cell lines at 10µM
concentration.
Table 3: Re-rank scores, MolDock scores, Hydrogen bond energies & interacting amino acid residues
of TGFBR2 protein with compounds were obtained through Molecular docking studies
a
using Molegro Virtual Docker (MVD)
Fig. 1: Biologically active pyrazolopyrimidinone - triazole hybrid molecules
Fig. 2: ORTEP diagram of 5a (CCDC No.: 1571323)
Fig. 3: a) IC 50 values of three active compounds.
b) 5f induced S phase arrest in U87 cells. Cells were treated with 5f at the indicated
concentrations for 48 h. Graphs represent cell arrest in S phase and percentage of apoptotic
cells.
Fig. 4: 5f treatment induced the pro-apoptotic genes in U87 glioma cells. 5f induced cleavage
of caspase-3 and PARP and increased p53 expression. Densitometric analysis represents the
expression of particular genes. The blots are normalized with actin and are representative of
three independent experiments.
Fig. 5: Docking conformation of compound 5a with active site residues of TGFBR2. Protein shown
in cartoon representation (left) and lines representation (right). Compound 5a shown in ball
and sticks representation. H-Bonds shown as green dotted lines.
Fig. 6: Docking conformation of compound 5f with active site residues of TGFBR2. Protein shown in
cartoon representation (left) and lines representation (right). Compound 5f shown in ball and
sticks representation. H-Bonds shown as green dotted lines.
Fig. 7: Docking conformation of compound 5l with active site residues of TGFBR2. Protein shown in
cartoon representation (left) and lines representation (right). Compound 5l shown in ball and
sticks representation. H-Bonds shown as green dotted lines.
Fig. 8: The halogen of 5f showed hydrophobic interaction with the protein.
Scheme 1: Synthetic route for the preparation of 3a, 3b. Reagents & conditions: i) HCOOH, 4 h,
o
reflux, ii) K CO , Acetone, 0 C, 3-4 h, reflux.
2
3
Scheme 2: Synthesis of pyrazolopyrimidinone - triazole hybrid molecules (5a-l). Reagents &
conditions: i) CuSO .5H O (15 mol %), Sodium ascorbate (20 mol %), DMF: H O (4:1,
4
2
2
5ml), room temperature, 6-8 h.
1
8

ACCEPTED MANUSCRIPT
a
Table 1: Optimized condition for azide-alkyne cyclization of 3a and 4a
b
S. No
Catalyst (mol %)
CuSO .5H O (1)
Time (h)
(%)Yield 5a
1
2
3
4
5
6
6
6
6
6
20
45
60
85
85
4
2
CuSO .5H O (5)
4
2
CuSO .5H O (10)
4
2
CuSO .5H O (15)
4
2
CuSO .5H O (20)
4
2
a
2 2
All the reactions were carried out using 0.4 mmol of 3a and 0.48 mmol of 4a in DMF: H O, 4:1,v/v(5ml),CuSO4.5H O
Sodium ascorbate (20 mol %), room temperature.
Isolated yield
b
Table 2: Inhibition of cell proliferation of 5a-l in C6 and U87 glioma cell lines at 10µM
concentration.
S. No. Compound
Substituents
C6
U87
%
(
inhibition
10 µM)
% inhibition
(10 µM)
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
R = R = R = R = H
34.10±2.5
13.64±15.45
16.87±7.45
4.91±9.6
NA
52.34±4.9
1
2
3
4
R = H , R = Me, R = R = H
21.32±4.9
41.62±1
2
3
4
R = R = R = H, R = Me
1
2
3
4
R = H, R = OMe, R =R = H
15.7±2.9
1
2
3
4
R = H, R = F, R = R = H
46.35±1.25
47.69±2.05
40.60±5.5
40.98±2.1
32.98±1.05
21.31±4.9
47.29±2.7
54.42±4.6
1
2
3
4
R = R = H, R = Cl , R = H
17.87±1.7
NA
1
2
3
4
5g
5h
5i
R = H, R = R = Me, R = H
1 2 3 4
R = Cl, R = R = R = H
NA
1
2
3
4
R = Cl, R = OMe, R = R = H
16.37±3.0
NA
1
2
3
4
1
0
1
2
5j
R = Cl, R =F, R =H, R = H
1 2 3 4
1
1
5k
5l
R = Cl, R =H, R = Cl, R = H
9.55±9.5
35.91±1.5
1
2
3
4
R = Cl, R = R = Me, R = H
1
2
3
4
Table 3: Re-rank scores, MolDock scores, Hydrogen bond energies & interacting amino acid residues
of TGFBR2 protein with compounds were obtained through Molecular docking studies
a
using Molegro Virtual Docker (MVD)
Compound
MolDock
Rerank
H-bond
Interacting amino acids
1
9

ACCEPTED MANUSCRIPT
Score
Score
energies
5
a
-136.846
-108.934
-7.30695
Lys-277, Thr-325, His-328
Thr-325, His-328
5b
-140.707
-130.808
-133.201
-136.373
-141.029
-142.788
-128.82
-115.369
-97.1456
-104.734
-107.926
-110.574
-114.412
-102.283
-105.703
-101.77
-0.757799
-0.98457
-1.32127
-0.803235
-7.28431
-1.89671
-2.454
5
c
Lys-277, Asn-332
Val-250, Lys-277
Thr-325, His-328
5d
5
e
5f
Lys-277, Thr-325, His-328
Thr-325, His-328, Cys-396
Val-250, Lys-277
Val-250, Lys-277
-
5
g
5h
5
i
-133.177
-129.938
-129.365
-153.056
-1.8525
-
5
j
5k
-100.752
-118.546
-2.21967
-7.6148
Val-250, Lys-277
Lys-277, Thr-325, His-328
5
l
a
The scores of the compounds were the best of five possible conformations obtained from independent
docking rounds.
Fig. 1: Biologically active pyrazolopyrimidinone - triazole hybrid molecules
2
0

ACCEPTED MANUSCRIPT
Fig. 2: ORTEP diagram of 5a (CCDC No.: 1571323)
2
1

ACCEPTED MANUSCRIPT
Fig. 3: a) IC 50 values of three active compounds.
b) 5f induced S phase arrest in U87 cells. Cells were treated with 5f at the indicated
concentrations for 48 h. Graphs represent cell arrest in S phase and percentage of apoptotic
cells.
Fig. 4: 5f induced the p53 mediated apoptotis in U87 glioma cells: cleavage of caspase-3, PARP and
increased p53 expression. Densitometric analysis is representing the expression of particular
genes. All the blots were normalized with actin.
2
2

ACCEPTED MANUSCRIPT
Fig. 5: Docking conformation of compound 5a with active site residues of TGFBR2 Protein shown in
cartoon representation (left) and lines representation (right). Compound 5a shown in ball and
sticks representation. H-Bonds shown as green dotted lines.
Fig. 6: Docking conformation of compound 5f with active site residues of TGFBR2 Protein shown in
cartoon representation (left) and lines representation (right). Compound 5f shown in ball and
sticks representation. H-Bonds shown as green dotted lines.
Fig. 7: Docking conformation of compound 5l with active site residues of TGFBR2 Protein shown in
cartoon representation (left) and lines representation (right). Compound 5l shown in ball and
sticks representation. H-Bonds shown as green dotted lines.
Fig. 8: The halogen of 5f showed hydrophobic interaction with the protein.
2
3

ACCEPTED MANUSCRIPT
Scheme 1: Synthetic route for the preparation of 3a, 3b. Reagents & conditions: i) HCOOH, 4 h,
o
reflux, ii) K CO , Acetone, 0 C, 3-4 h, reflux.
2
3
Scheme 2: Synthesis of pyrazolopyrimidinone - triazole hybrid molecules. Reagents & conditions: i)
CuSO .5H O (15 mol %), Sodium ascorbate (20 mol %), DMF: H O (4:1, 5ml), room
4
2
2
temperature, 6-8 h.
2
4

ACCEPTED MANUSCRIPT
•
•
Hybrid azaheterocycles - pyrazolopyrimidinones tethered to triazole ring were synthesized.
Good anticancer activity against C6 and U87 cell lines. 5a, 5f and 5l showed better inhibition
potential against U87 cell lines.
•
•
5f induces S phase arrest in U87 cells and triggers p53 mediated caspase dependent apoptotic
pathway.
Docking studies confirm the potent activity of 5a, 5f and 5l by providing the strong interaction of
these compounds with TGFBR2 protein as evidenced by downstream signaling.