Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs and their biological evaluation as anticancer agents: mTOR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.051

A microwave assisted strategy for synthesis of series of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-ones has been developed and their biological evaluation as anticancer agents is described. The synthetic protocol involves simple procedure by oxidative coupling of 4-amino-1-methyl-3-propyl-1H-pyrazole- 5-carboxamide with different aldehydes in presence of K₂S ₂O₈ offering 5-substituted-1H-pyrazolo[4,3-d]pyrimidin- 7(6H)-one compounds in excellent yields. The in vitro anticancer activity screening against human cancer cell lines HeLa, CAKI-I, PC-3, MiaPaca-2, A549 gave good results. The in detailed mechanistic correlation studies of compound 3m revealed that the compound shows anticancer activity through apoptosis mechanism and also inhibits mTOR with nonomolar potency. The design was based on docking with mTOR protein. The concentration dependent cell cycle analysis, western blotting experiment and nuclear cell morphology studies have been described.

Full text of DOI:10.1016/j.ejmech.2014.04.051

Accepted Manuscript
Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs and their
biological evaluation as anticancer agents: mTOR inhibitors
G. Lakshma Reddy, Santosh Kumar Guru, M. Srinivas, Anup Singh Pathania, Priya
Mahajan, Amit Nargotra, Shashi Bhushan, Ram A. Vishwakarma, Sanghapal D.
Sawant
PII:
S0223-5234(14)00372-9
10.1016/j.ejmech.2014.04.051
EJMECH 6922
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 October 2013
Revised Date: 14 April 2014
Accepted Date: 15 April 2014
Please cite this article as: G.L. Reddy, S.K. Guru, M. Srinivas, A.S. Pathania, P. Mahajan,
A. Nargotra, S. Bhushan, R.A. Vishwakarma, S.D. Sawant, Synthesis of 5-substituted-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs and their biological evaluation as anticancer agents: mTOR
inhibitors, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.04.051.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs and their
biological evaluation as anticancer agents: mTOR inhibitors
G. Lakshma Santosh Kumar Guru, Reddy, M. Srinivas, Anup Singh Pathania, Priya Mahajan,
Amit Nargotra, Shashi Bhushan,* Ram A. Vishwakarma,* Sanghapal D. Sawant*
Series of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-ones are synthesized using microwave assisted
strategy. Screened against HeLa, CAKI-I, PC-3, MiaPaca-2, A549 for anticancer activity and 3m
was found to be an mTOR inhibitor.

ACCEPTED MANUSCRIPT
Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs
and their biological evaluation as anticancer agents: mTOR inhibitors
a,d
b
a,d
b,d
G. Lakshma Reddy, Santosh Kumar Guru, M. Srinivas, Anup Singh Pathania, Priya
c,d
c,d
b,d
a,d,
Mahajan, Amit Nargotra, Shashi Bhushan, * Ram A. Vishwakarma, * Sanghapal D.
a,d,
Sawant, *
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road-
Jammu-180 001, India
b
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road-
Jammu-180 001, India
c
Discovery Informatics, CSIR-Indian Institute of Integrative Medicine, Canal Road-Jammu-180
0
01, India
d
Academy of Scientific and Innovative Research, India
Tel.: +91-191-2569111, Fax: +91-191-2569333; IIIM Communication No. IIIM/1610/2013
E-mail: sdsawant@iiim.ac.in , sawant.rrl@gmail.com
1

ACCEPTED MANUSCRIPT
Abstract:
A microwave assisted strategy for synthesis of series of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-
ones has been developed and their biological evaluation as anticancer agents is described. The
synthetic protocol involves simple procedure by oxidative coupling of 4-amino-1-methyl-3-
propyl-1H-pyrazole-5-carboxamide with different aldehydes in presence of K S O offering 5-
2
2
8
substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one compounds in excellent yields. The in vitro
anticancer activity screening against human cancer cell lines HeLa, CAKI-I, PC-3, MiaPaca-2,
A549 gave good results. The in detailed mechanistic correlation studies of compound 3m
revealed that the compound shows anticancer activity through apoptosis mechanism and also
inhibits mTOR with nonomolar potency. The design was based on docking with mTOR protein.
The concentration dependent cell cycle analysis, western blotting experiment and nuclear cell
morphology studies have been described.
2 2 8
Keywords: Pyrazolo[4,3-d]pyrimidin-7(6H)-one, K S O catalyst, Microwave irradiation,
Cytotoxicity, mTOR inhibitor
2

ACCEPTED MANUSCRIPT
1
.0. Introduction
The pyrazolo[4,3-d]pyrimidin-7(6H)-ones and their bioisosteres are heterocyclic compounds
1
with important biological functions including antitumor activity and many other activities. 6-
2
cycloalkyl-pyrazolopyrimidinones are reported for CNS disorders, GHS-R1a antagonists and
3
inverse agonists for the treatment of obesity is also repoted. Recently, imine-
4
pyrazolopyrimidinones are presented as anticancer derivatives. 1-aryl-4,5-dihydro-1H-
pyrazolo[3,4-d]pyrimidin-4-ones were identified as inhibitors of cyclin-dependent kinase (CDK)
5
with IC50 in the low micromolar range and several other reports are available for various
activities for this scaffold. On the basis of biological data reported in literature, pyrazolo[4,3-
d]pyrimidin-7(6H)-one class of compounds are very important in the treatment of impotence, as
6
can be evidenced by the top selling PDE5 inhibitory drug in market i.e., sildenafil. Few more to
include in pyrazolopyrimidinone class other than sildenafil as PDE5 inhibitors are
acetildenafil (hongdenafil), aildenafil (methisosildenafil), sulfoaildenafil (thioaildenafil), etc.
7
A pyrazolopyrimidine scaffold based molecule i.e. PP242, PP30 and some others (Fig. 1), are
reported as highly potent, selective and ATP-competitive mTORC1/mTORC2 inhibitor (IC = 8
5
0
nM for PP242 and 80 nM for PP30). PP242 has >10 folds selectivity over the other PI3K family
kinases (IC 0.102 µM, 0.408 µM, 1.27 µM, 1.96 µM and 2.2 ꢀM for p110γ, DNA-PK, p110δ,
5
0
p110α and p110β, respectively). PP242 is also reported to exhibits excellent selectivity over 215
other protein kinases. PP242 differentially inhibits insulin-stimulated phosphorylations of
cellular proteins both in vitro and in vivo in a manner distinctly different from that seen in
-/-
mTORC2-functional knockout SIN1 cells or in cultures treated with Rapamycin, which targets
only mTORC1, but not mTORC2. Moreover, it is reported that PP242 can significantly enhance
iPSC generation, which is experimentally confirmed by J.A Menendez et. al. that this mTOR
inhibitor (PP242) is the most powerful longevity-promoting molecule that enhances iPSC
8
generation, and robustly decelerates the cellular senescence imposed by a DDR equivalent to
senescence that is caused by pluripotency associated transcription factor expression. However,
support for this hypothesis was evidenced by recent findings that well-characterized mTOR
inhibitors and autophagy activators (e.g., PP242, rapamycin and resveratrol) notably improve the
8
speed and efficiency of iPSC generation.
3

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Synthesis of pyrazolo[4,3-d]pyrimidin-7(6H)-one is well exploited and there are various methods
already reported for the synthesis of pyrazolo[4,3-d]pyrimidin-7(6H)-one class of compounds
9
using traditional as well as microwave mediated approaches. The aim of this study was to
synthesize and evaluate the biological potential of pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs
for their anticancer potential. In this direction, we initiated our efforts towards its synthesis and
biological activity. The detailed chemistry and biological evaluation of these compounds as
anticancer agents is explained in the present study.
2
2
2
.0. Result and discussion
.1. Chemistry:
.1.1. Synthesis of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs:
We intended to synthesize compounds based on 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
scaffold by using microwave assisted protocol (Scheme 1). In this direction we started the
studies for optimization of synthesis of 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-
d]pyrimidin-7(6H)-one 3a.
The optimization studies were initiated by screening of different oxidizing agents as depicted in
table 1 using DMSO:Water in 1:1 proportion to see the conversion in desired product. Amongst
all oxidants, the best result was observed with K S O , in equivalence studies for catalyst, 3 eq.
2
2
8
of catalyst has given maximum yields (Table 1, see Supplementary data). Therefore, all reactions
were conducted using this condition after optimization of catalyst. However, oxone has also
given the product 3a with minor yields.
After screening of the catalyst we started study of selectivity for solvent that could affect the
formation of 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one 3a.
The solvent screening was carried out to find out the best conversion, the mixture of DMSO:H O
2
in 1:1 proportion has given the best results with excellent yields (Table 2, see Supplementary
data).
The microwave protocols were optimized for this reaction as mentioned in the table 3; the
reactions carried under different microwave Watt powers have given varied results. Wherein,
entry 3(b) (Table 3, see Supplementary data) was found to be the best condition for maximum
conversion.
4

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A series of compounds based on 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one scaffold was
synthesized using these optimized conditions, wherein, all kind of substrates with diversity
around aryl ring were chosen for conversion and in all cases products obtained in good to
excellent yields (Table 4).
2
.2. Biology:
2
.2.1. In vitro anticancer activity
These compounds were taken up for in vitro cell based cytotoxicity screening against various
human cancer cell lines and the results for this screening are mentioned in the Table 5.
2
.2.2. Compound 3m inhibits significant cell growth inhibition in different panel of human
cancer cell lines:
Cytotoxicity assay was performed by using tetrazolium based calorimetric method (MTT assay)
against human lung cancer cell lines A549, kidney cancer cell line Caki-1, pancreateic cancer
cell line MiaPaCa-2, prostate cancer cell line PC-3 and cervical cancer cell line HeLa.
Compound 3m caused concentration dependent inhibition of cell proliferation in these cell lines
in 48 h (Fig.2). Compound 3m has IC value of approximately 14 µM in A549 cell line, 17 µM
5
0
in Caki-1 cells, 24 µM in MiaPaCa-2 cells, 38 µM in PC-3 cells, in 19µM HeLa cells after 48 h.
.2.3. Compound 3m altered whole cell and nuclear morphology
2
Treatment of human leukaemia HL-60 cells with compound 3m at 10, 30, 50, and 70µM
concentrations caused cell wall deformation, shrinkage of cell size, nuclear condensation and
formation of scattered apoptotic bodies as shown by arrows in the Fig. 3A and B, while the
nuclei of untreated cells are healthy and round in shape. The number of apoptotic bodies
increased with increased concentration of compound 3m in A549 cells. This revealed that
compound 3m induce cell death through induction of apoptosis in A549 cells.
2
.2.4. Compound 3m increases sub-G0 DNA fraction of cell cycle phase in concentration
dependent manner
A549 cells treated with compound 3m exhibited concentration dependent increase in hypo
diploid sub-G0 DNA fraction (<2nDNA) indicative of apoptotic population as analyzed by
modfit software (Fig. 4). Control cells showed 1% sub-G0 DNA fraction while A549 cells
treated with compound 3m for 24 h exhibited continuous increase in sub-G0 fraction which may
comprise both apoptotic and debris fraction implying together the extent of cell death.
5

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The damage was more apparent with higher concentration of compound 3m over the period of
study. The sub-G0 fraction increased from ~1% of control to ~28% after 24 h of treatment of
compound 3m. There was hardly any significant effect after 24h of treatment on G1, S and
G2/M, which indicated that decrease in DNA fluorescence is not cell cycle selective.
2
.2.5. Compound 3m inhibits mTOR-p70S6Kinase signaling and induces apoptosis in A549
cells
Compound 3m at indicated concentrations inhibits phosphorylated (Serine 2448) and non
phosphorylated form of mammalian target of rapamycin (mTOR), its two other subunits rictor
and raptor and their two main substrate p70S6K and 4EBP1in A549 cells after 24h (Fig.5).
Compound 3m inhibits active phosphorylated form of eIF4E (Serine 209) and p-p70S6Kinase
(T389) at all concentrations (Fig.5).
Compound 3m also inhibits mTOR kinase in a cell free enzyme assay (K-LISA™ mTOR kit)
and IC value was found to be 203 nM (Fig. 6). The mammalian target of rapamycin (mTOR) is
5
0
a serine/threonine kinase present downstream of phosphatidylinositol 3-kinase/Akt signalling
pathway and it involves in regulating basic cellular functions including cellular growth and
1
0
proliferation (Wullschleger etal, 2006). Aberrant activation of the PI3K/Akt/mTOR pathway is
1
1
found in many types of cancer including small cell lung cancer (Marinov etal. 2009). AKT can
activate mTOR by phosphorylating at serine 2448. Activated mTOR have two well characterized
downstream targets, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic translation initiation
factor 4E binding protein 1 (4EBP1), both of which are involve in the regulation of protein
1
2
synthesis (Hay and Sonenberg, 2004). Simultaneously, compound 3m also alter the expression
of key apoptotic proteins like caspas-3 activation and PARP-1 (Poly-ADP-ribose-polymerase)
cleavage (Fig. 5).
2
.3. Docking studies
Based on the docking studies, the molecule 3m shows one H-bond interaction with Val2240 at a
0
distance of 2.203 A (Fig. 7). Further, this ligand also shows more stability within the binding
pocket due to the hydrophobic cleft formed by Leu 2185, Trp 2239, Met 2345 and Ile 2356
around the ligand. The best dock score and the calculated binding energy of this complex were -
6
.8 and -80.95 respectively. Based on this evidence and the preliminary biological activity, the in
detailed biological activity for 3m was conducted.
6

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3
.0. Conclusion
In present study, 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one scaffold have been constructed using
microwave assisted protocol and the biological evaluation results as anticancer agents are
promising. The synthetic protocol can be applied for preparation of analogs of active compound
i.e. 3m which involves a simple procedure to obtain 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-
7
(6H)-one compounds in excellent yields. Moreover, 3m also acts as mTOR inhibitor and the
concentration dependent cell cycle analysis, western blotting experiment and nuclear cell
morphology studies suggests that the mechanism through which 3m acts as anticancer agent is
apoptosis. 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one is an excellent scaffold and can
be exploited for further study in the field of cancer.
4
4
.0. Experimental protocols
.1.1. Chemistry
All reactions were performed in a sealed tube under mentioned microwave irradiation conditions.
Analytical thin layer chromatography was performed using TLC pre-coated silica gel 60 F254 (20
x 20 cm). TLC plates were visualized by exposing UV light or by iodine vapors or immersion in
anisaldehyde charring reagent or in 2,4-dinitrophenyl hydrazine or ninhydrin followed by heating
on hot plate. Organic solvent were concentrated by rotary evaporation and dried using high
vacuum suction pump.
C
ompounds were purified by column chromatography using normal phase
1
silica-gel (100-200 mesh size). H NMR spectra were recorded with 400 and 500 MHz NMR
instruments. Chemical data for protons are reported in parts per million (ppm, scale) downfield
3
from tetramethylsilane and are referenced to the residual proton in the NMR solvent (CDCl : δ
7
.26, DMSO-d δ 2.50 or other solvents as mentioned).
6
4
.1.1.1.
Synthesis of 1-methyl-3-propyl-5-(2,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-
d]pyrimidin-7(6H)-one
In a typical procedure, a solution of aromatic aldehyde i.e. 2,4,5-trimethoxybenzaldehyde (0.196
g, 1 eq.) and 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide (0.191 g, 1.05 eq) in
2 2 2 8
DMSO:H O (1:1) add K S O (0.810 g, 3 eq.) was taken in a sealed reaction tube and the
reaction mixture was irradiated under microwave conditions for 3 min with a power of 350
o
Watts at 100 C. After completion, the reaction mass was diluted with EtOAc (20 mL) and added
water (30 mL). Separated the organic layer and extracted with EtOAc (2x10ml). The combined
7

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organic layer was then washed with brine solution, concentrated under vacuum and purified on
silica-gel (100-200 mesh) column chromatography, affording white solid 1-methyl-3-propyl-5-
(2,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one i.e., compound 3j in (0.351 g)
9
8% yield.
4
.1.1.2.
Representative analytical data for compound 3j i.e., 1-methyl-3-propyl-5-(2,4,5-
1
trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one. H NMR (400 MHz, CDCl
3
) δ
1
0.99 (s, 1H), 8.03 (s, 1H), 6.59 (s, 1H), 4.27 (s, 3H), 4.05 (s, 3H), 3.97 (s, 6H), 2.93 (t, J = 7.5
1
3
Hz, 2H), 1.93 – 1.83 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). C NMR (126 MHz, DMSO) δ 153.61,
1
5
3
4
52.09, 151.83, 149.06, 144.66, 142.60, 137.96, 123.88, 113.50, 112.81, 97.89, 56.57,
+
6.15, 55.89, 37.77, 27.07, 21.59, 13.81. HRMS (ESI) calcd for C H N O [M-H ]
1
8
23
4
4
59.17193, found 359.17139.
.1.2. Molecular modeling studies
The computational studies on mTOR were carried out using the Schrodinger suite 2012
molecular modeling software. Crystal structure 4JT5 was taken for docking studies. The
coordinates of mTOR protein in complex with the co-crystalized ligand (2-[4-amino-1-(propan-
2
-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-1H-indol-5-ol) that was obtained from protein data
1
3
bank. The protein was prepared for docking using the protein preparation wizard. Hydrogens
were added to the protein and water residues were removed beyond 5A°from the heteroatom.
Further, only those water residues, having interactions with the protein and heteroatom were
kept, and the rest were deleted. Then the ligand was extracted and protein was refined by
assigning H-bonds and minimization at OPLS 2005 force field. A grid was generated at active
site, identified on the bases of already co-crystalised ligand to the receptor using receptor grid
generation module. The docking protocol was standardized using the co-crytalized ligand
conformation.
4
.1.3. Biological Studies
a) Reagents/chemicals: RPMI-1640, streptomycin, kanamycin, penicillin, L-glutamine,
phenylmethanesulfonyl fluoride (PMSF), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), Propidium iodide (PI), Fetal bovine serum, pyruvic acid were purchased from
Sigma-Aldrich (Bangalore, India). Anti-human antibodies to PARP-1, caspase-3 and β-actin
were purchased from SantaCruz Biotechnology (SantaCruz, CA). Anti-human antibodies to
8

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mTOR and its phosphorylated form (S2448), p-eIF4E (S209), Raptor, Rictor and p-p70S6Kinase
(
T389) were purchased from Cell signaling technology (Danvers, MA). K-LISA™ mTOR kit
#CBA055) was purchased from Calbiochem, USA. Electrophoresis reagents, Protein estimation
(
kit and protein marker were from Bio-Rad Laboratories (Hercules, CA).
b) Cell culture and treatment: Human lung cancer cell lines A549, kidney cancer cell line
Caki-1, pancreatic cancer cell line MIA-PaCa-2, prostate cancer cell line PC3 and cervical
cancer cell line HeLa were purchased from ECACC. Cells were grown in RPMI/DMEM/MEM
growth medium containing 10% FCS, 100U penicillin and 100 mg/ml streptomycin. Cells were
o
grown in CO
2
incubator (Thermocon Electron Corporation, Houston, TX) at 37 C temperature,
9
5% humidity and 5% CO gas environment. Cells treated with compounds were dissolved in
2
DMSO while the untreated control cultures received only the vehicle (DMSO<0.2%).
.1.3.1. Cell proliferation assay
4
Cells were seeded in 96 well flat bottom plates. Next day when they attained 60-70%
confluency, they were treated with compounds at different concentrations for 24h and 48h. MTT
dye (2.5 mg/ml in PBS) was added 4 hours priors to experiment termination. The supernatant
was discarded and the MTT formazan crystals were dissolved in 150 µL of DMSO. The OD
1
4
measured at 570 nm with reference wavelength of 620 nm (Bhushan et al 2006). The most
active compound 3m was taken up for in detailed biological evaluation.
4
.1.3.2.
Hoechst Staining
Human lung cancer A549 cells were treated with compound 3m for 24 h at 10, 30, 50 and 70 µM
concentrations. After treatment cells were collected, washed twice with PBS and fixed in 400 µl
of fixing solution composed of cold acetic acid: methanol (1+3, v/v) overnight at 4 °C. Cells
were washed with fixing solution and dispensed in 50 µl of fixing solution. Spread cells on a
clean cold slide and dried overnight at room temperature. Cells were stained with Hoechst 33258
(5 µg/ml in 0.01 M citric acid and 0.45 M disodium phosphate containing 0.05% Tween 20) for
2
0-30 min at room temperature. After that slides were washed with distilled water followed by in
PBS. While wet, pour 40 µl of mounting fluid (PBS: glycerol, 1/1) over the slide and covered
with glass cover slip and sealed with nail polished. Cells were observed under microscope for
1
5
any nuclear morphological changes occur in apoptosis (Saxena et al. 2010).
.1.3.3. Cell cycle analysis
4
9

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2
Human lung cancer cell line A549 was seeded in 60mm dishes and after attaining 60-70%
confluency, treated with compound 3m for 24 hours at 10, 30, 50 and 70 µM concentrations.
Cells were washed with PBS twice after 24h and fixed overnight in 70% alcohol. Next day cells
were washed twice with PBS and thereafter they are subjected to RNase digestion (200 µg/mL)
0
at 37 C for 1.30 h and then incubated with PI (10 µg/mL) for 30 min. Cells were analyzed
immediately on flow cytometer (BD Biosciences, San Jose, CA). The data were collected in list
mode on 10,000 events and illustrated in a histogram, where the number of cells (counts) is
plotted against the relative fluorescence intensity of PI (FL-2; λem: 585 nm; red fluorescence).
Resulting DNA distributions were analyzed by Modfit (Verity Software House Inc., Topsham,
ME) for the proportions of cells in G -G , S- phase, and G -M phases of the cell cycle (Chanda et
0
1
2
1
6
al 2012).
.1.3.4.
4
Cell lysates preparation for western blots analysis
Human lung cancer cell line A549 was treated with compound 3m for 24h at 10, 30, 50 and 70
0
µM concentrations. Cells were collected by centrifugation at 400×g at 4 C, washed with PBS
twice and processed for preparation of whole cell lysates. Cells were lysed with cold Cell lysis
buffer (RIPA, Sigma with 50 mM NaF, 0.5 mM NaVO4, 2 mM PMSF and 1% protease inhibitor
0
cocktail) for 40 min. Cells were centrifuged at 12000× g for 15 min at 4 C and the supernatant
1
7
was collected as whole cell lysates for western blot analysis (Bhushan et al 2007).
.1.3.5. Western blot analysis
4
Protein was measured using Bio-Rad protein assay kit and protein lysates (30-70 µg) were
0
subjected to SDS-PAGE analysis. They are transferred to PVDF membrane for 2 hours at 4 C at
1
00V using Bio-Rad electrode assembly. The membrane was blocked by incubation with 3%
BSA or 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at
room temperature to prevent any non-specific binding. After an hour the blots were incubated
with respective primary antibodies for 3-4 h at room temperature. They are washed three times
with TBST and were incubated with horseradish peroxidase conjugated secondary antibodies for
another 1 h. At the end, membranes were washed three times with TBST buffer with 15 min
interval and signals detected using ECL plus chemiluminescence’s kit on X-ray film (Bhushan et
1
7
al 2007).
.1.3.6.
4
mTOR kinase essay
1
0

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mTOR inhibition of compound 3m was found out by using K-LISA™ mTOR kit from
Calbiochem (#CBA055). It is an ELISA-based assay that utilizes a p70S6K-GST fusion protein
as a specific mTOR substrate. The assay was carried out according to the manufacturer’s
protocol. Briefly, 100 ꢀl of recombinant p70S6K-GST fusion protein was pre-incubated at room
temperature in the glutathione coated 96-well plate for 1h after that a mixture of 49µl of ice-
chilled mTOR kinase and 1µl of test compounds or DMSO was added. The reaction was initiated
by the addition of 50 ꢀl of mTOR kinase assay buffer containing 100 ꢀM ATP and 1µM DTT.
The plate was treated first with 100 ꢀl of anti-p70S6K-T389 for 1h and then with 100 ꢀl of HRP-
conjugated antibody for 1h to detect the T389-phosphorylated p70S6K. Absorbance was
measured at 450 nm and 595 nm using microplate spectrophotometer. The IC50 values were
calculated by analysis non linear regression with variable slope by using GraphPad Prism-5
software.
Acknowledgment
GLR thanks CSIR-New Delhi for the award of fellowship. The support of funding for carrying
th
present research work from CSIR 12 FYP project BSC0108 is gratefully acknowledged.
Appendix. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
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Figure captions
Figure 1: Pyrazolo[4,3-d]pyrimidin-7(6H)-one scaffold based potential candidates and drugs
Figure 2: Compound 3m inhibits cell proliferation in different panel of human cancer cell lines. Cells
were grown in 96-well culture plate and when 60-70% confluent was treated with 1, 10, 30, 50, 70 and
1
00 µM concentration for 48h. Cells were incubated with MTT and OD measured as described in
Materials and Methods. Data are Mean ± SD (n= 8 wells), and representative of three similar experiments
Figure 3: Effect of compound 3m on the cell wall and nuclear morphology of A549 cells. A) A549 cells
were treated with 10, 30, 50 and 70µM concentrations of compound 3m for 24h and visualized under
phase contrast inverted microscope (Olympus 1X 70, 30X). B) Subsequently cells were stained with
Hoechst 33258 and visualized for nuclear morphology and apoptotic bodies’ formation. Compound 3m
induced the formation of apoptotic bodies as indicated by arrows in concentration dependent manner.
Data are representative of one of three similar experiments
Figure 4: Cell cycle analyses of lung cancer A549 cells through PI staining. Cells were seeded in six
well plate and after 60-70% confluence, treated with compounds 3m for 24h time period at indicated
concentrations. After treatment cells were stained with PI (10µg/ml) to determine DNA Fluorescence and
cell cycle phase distribution as described in Materials and methods. Data were analyzed by Modfit
software (Verity Software House Inc., Topsham, ME) for the proportions of different cell cycle phases.
Fraction of cells from apoptotic, G1, S and G2 phases analyzed from FL2- A vs. cell counts is shown in
%. Data are representative of one of three similar experiments.
Figure 5: Inhibition of mTOR signalling cascade in lung cancer A549 cells by compounds 3m. A549
cells treated with compound 3m at 10, 30, 50 and 70 µM concentrations for 24 h. Total cell lysates were
prepared and protein samples (40-80 µg) were loaded on SDS-PAGE gel for western blot analysis, β-actin
was used as internal control to represent the same amount of proteins applied for SDS-PAGE. Specific
antibodies were used for detection of mTOR, its activated phosphorylated form at serine 2448, Raptor,
Rictor along with downstream substrates of mTOR, eIF4E and p70S6kinase. Compound 3m also induces
apoptotic caspase-3 level and PARP cleavage. Immunoblots were representative of one of three similar
experiments.
Figure 6: Compound 3m inhibits mTOR kinase in a cell free enzyme essay (K-LISA™ mTOR kit)
showing IC value as 203 nM.
50
Figure 7: A cartoon showing the interactions of compound 3m with active site of mTOR using PDB4JT5
0
(
The carbonyl group of molecule showing H-bonding with Val2240 (2.203 A ))
1
4

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Table 4: Synthesis of compounds based on 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one scaffold
a
All yields are isolated yields after column purification
Table 5: In vitro cell based screening of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one compounds
Cell lines
HeLa
(Cervix cancer)
CAKI-I
(Renal cancer)
PC-3
(Prostate Cancer)
MiaPaca-2
(Pancreatic
Cancer)
A549
(Lung cancer)
Code
Conc,
%
IC50
Conc,
%
IC50
Conc,
%
IC50
Conc,
%
IC50
Conc,
%
IC50
(
µM) GI (µM) (µM) GI (µM) (µM) GI (µM) (µM) GI (µM) (µM) GI (µM)
1
0
36
10 12
30 18
100 19
10
30 10
100 14
10
30
100 14
10 33
30 48
100 98
10 14
30 16
7
10
7
10
30
100
10
30
100
10
30
100
10
21
3a
3b
3c
3d
30 37
00 40
1
30 34
00 42
1
30 43
00 59
1
>100
>100
92
>100
>100
>100
57
>100
>100
32
30 25
100 38
10 18
30 20
100 32
10 11
30 20
100 44
10 11
30 15
>100
>100
>100
>100
30 >100
37
0
15
36
30
45
80
0
1
1
1
0
31
10
8
5
6
>100
38
30 25
100 52
10 26
30 33
100 46
10 24
30 38
0
22
0
0
90
30 21
>100
>100
30
17
1
1
00 36
100 74
10 31
30 50
100 73
100 19
10 22
30 27
100 47
100 22
10 14
30 17
100 45
100
10
30
58
1
0
21
12 >100
25
40
3
e
30 22
00 39
>100
30
>100
>100
100
1
5

ACCEPTED MANUSCRIPT
1
0
34
10 30
30 68
100 78
10 27
30 32
100 41
10 22
30 25
100 35
10 34
30 56
100 66
10 16
30 16
100 17
10 25
30 31
100 35
10 32
30 62
100 74
10 10
30 23
100 39
10 19
30 24
100 32
10 22
30 31
100 47
10 34
30 45
100 98
10
30
100
10
30
100
10
30
100
10
30
100
10
20
57
98
0
28
>100
90
3
f
30 69
00 81
1
30 17
00 48
1
30 44
00 78
1
30 19
00 14
1
30 33
00 35
1
30 35
00 43
24
>100
34
18
>100
>100
28
27
>100
>100
>100
>100
>100
38
>100
>100
>100
>100
>100
1
1
1
1
1
1
0
12
10
6
3
3
g
h
30 16
100 32
10 23
30 29
100 43
10 11
30 22
100 43
10 12
30 21
100 32
8
43
11
28
59
19
39
58
0
0
29
0
12
87
3
3
i
j
>100
>100
>100
0
30
10
30
5
7
>100
>100
>100
30
7
10
30
7
21
100 10
0
32
10
30
0
6
10
3
11 >100
31
47
3
k
30 24
100 44
100 13
100
1
0
49
10 35
30 65
100 87
10 39
30 67
100 81
10 35
30 51
100 76
10 24
30 26
100 49
10 33
30 78
100 97
10 12
30 44
100 84
10 16
30 17
100 25
10 17
30 23
100 43
10 38
30 57
100 87
10 31
30 57
100 90
10 12
30 16
100 23
10 11
30 20
100 43
10
30
100
10
30
100
10
30
100
10
30
100
10
30
100
10
30
100
10
30
100
10
30
100
10
26
57
90
45
68
98
0
27
65
0
28
14
3
l
30 66
00 87
1
30 63
00 85
1
30 31
00 49
1
30 43
00 65
13
19
20
17
29
31
37
35
24
1
1
1
1
0
36
3
m
0
22
87
3
n
>100
>100
>100
>100
0
11
>100
86
3
o
p
35
>100
96
>100
95
8
23
22
37
68
0
23
46
9
14
48
0
10
29
10
37
78
3
10
0
00
1
30
1
18
33
0
>100
10
30
100
34
47
54
10
30
100
23
44
56
10
30
100
15 >100
21
44
3
1
0
10 23
30 35
100 44
10 21
30 28
100 36
10 21
30 22
100 31
10 46
30 55
100 61
10 21
30 25
100 29
10 11
30 14
100 27
10 17
30 22
>100
>100
>100
54
3
q
7
>100
>100
>100
88
>100
>100
>100
27
>100
>100
>100
98
>100
96
1
1
1
1
00 12
1
30 21
00 44
1
30 23
00 38
1
30 44
00 61
100 48
10 21
30 32
100 55
10 21
30 32
100 45
10 11
30 13
100 34
0
13
3
r
0
0
10
30
1
3
3
s
>100
>100
100 14
10 23
30 31
100 51
0
32
3
t
30
100
1
6

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Figure 1
Figure 2
1
7

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Figure 3
Figure 4
1
8

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Figure 5
Figure 6
1
9

ACCEPTED MANUSCRIPT
Figure 7
Scheme 1: Microwave assisted synthesis of 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one
2
0

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Highlights
Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs and their
biological evaluation as anticancer agents: mTOR inhibitors
G. Lakshma Reddy, Santosh Kumar Guru, M. Srinivas, Anup Singh Pathania, Priya Mahajan,
Amit Nargotra, Shashi Bhushan,* Ram A. Vishwakarma,* Sanghapal D. Sawant*
Highlights
•
•
•
•
Microwave assisted synthesis of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one scaffold
In vitro anticancer activity: HeLa, CAKI-I, PC-3, MiaPaca-2, A549 cancer cell lines
Compound 3m: cell cycle analysis, western blotting, nuclear cell morphology
mTOR inhibitory potential of compound 3m with nonomolar potency: IC50= 203 nM

ACCEPTED MANUSCRIPT
Appendix
Supplementary data
Synthesis of 5-substituted-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one analogs
and their biological evaluation as anticancer agents: mTOR inhibitors
a,d
b
a,d
b,d
G. Lakshma Reddy, Santosh Kumar Guru, M. Srinivas, Anup Singh Pathania, Priya
c,d
c,d
b,d
a,d,
Mahajan, Amit Nargotra, Shashi Bhushan, * Ram A. Vishwakarma, * Sanghapal D.
a,d,
Sawant, *
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road-
Jammu-180 001, India
b
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road-
Jammu-180 001, India
c
Discovery Informatics, CSIR-Indian Institute of Integrative Medicine, Canal Road-Jammu-180
0
01, India
d
Academy of Scientific and Innovative Research, India
Tel.: +91-191-2569111, Fax: +91-191-2569333; IIIM Communication No. IIIM/1610/2013
E-mail: sdsawant@iiim.ac.in , sawant.rrl@gmail.com
S1

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Table of Contents
S.No
Content
Table 1: Screening of catalyst for oxidative
cyclization and formation of 3a
Table 2: Solvent screening for the formation of 3a
Table 3: Optimization of microwave protocols for
the synthesis of 3a
Page Nos
1
.1.
S3
1
1
.2.
.3.
S3
S4
1
1
.4.
.5.
Analytical data
S4-S7
S8-S61
S62
Scanned spectral data of compounds
References
S2

ACCEPTED MANUSCRIPT
1
.1. Table 1: Screening of catalyst for oxidative cyclization and formation of 3a
S.No
Solvent
Oxidizing
agent
Yield(%)
3a
4
DMSO/H O(1:1)
H O
2 2
0
75
1
2
3
4
2
DMSO/H2O(1:1)
TBHP
0
0
70
75
15
DMSO/H O(1:1)
m-CPBA
Oxone
2
DMSO/H O(1:1)
15
2
0
5
DMSO/H O(1:1)
K S O
8
98
2
2
2
1
.2.
Table 2: Solvent screening for the formation of 3a
Yield(%)a
Sr. No Solvent
Oxidant
3a
4
1
EtOH
ACN
0
95
0
0
90
85
2
3
THF
PhCl
4
5
6
7
0
50
H O
2
20
45
75
EtOH/H O(1:1) K S O
2 2 8
20
2
ACN/H O(1:1)
85
65
10
2
8
9
THF/H2O(1:1)
30
10
5
DMF
85
10
DMSO
90
11
DMF/H O(1:1)
5
2
90
1
2
DMSO/H O(1:1)
98
2
0
a
All yields are isolated yields after column purification
S3

ACCEPTED MANUSCRIPT
1
.3. Table 3: Optimization of microwave protocols for the synthesis of 3a
a
All yields are isolated yields after column purification
1
3
.4. Analytical data
1
1
a) 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
NMR (400 MHz, CDCl ) δ 11.14 (s, 1H), 8.48 (d, J = 8.0 Hz, 1H), 7.46 (m 1H), 7.15 (m 1H),
3
7
.04 (d, J = 8.4Hz, 1H), 4.35 – 4.23 (m, 5H), 2.95 (t, J = 7.6 Hz, 2H), 1.95 – 1.83 (m, 2H), 1.61
+
(
t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.2Hz, 3H). HRMS (ESI) calcd for C H N O ([M-H ]
1
7
21
4
2
3
13.16645, found 313.16580.
b) 5-(4-methoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one :
1
,3
3
1
H NMR (400 MHz, CDCl ) δ 10.71 (s, 1H), 8.06 (d, J = 8.8Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H),
3
4
.28 (s, 3H), 3.89 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H), 1.93 – 1.82 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H).
+
HRMS (ESI) calcd for C H N O [M-H ] 299.15080, found 299.15035
1
6
19
4
2 (
1
3
c) (E)-1-methyl-3-propyl-5-styryl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one: H NMR (400
MHz, CDCl ) δ 11.50 (s, 1H), 7.75 (d, J = 16.4Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.46 – 7.34 (m,
H), 6.97 (d, J = 16.4 Hz, 1H), 4.29 (s, 3H), 2.92 (t, J = 7.6 Hz, 2H), 1.92 – 1.80 (m, 2H), 1.04
3
3
1
3
(
t, J = 7.2 Hz, 3H). C NMR (126 MHz, DMSO) δ 154.15, 149.09, 144.71, 137.97, 136.22,
S4

ACCEPTED MANUSCRIPT
1
35.16, 129.31, 128.95, 127.31, 124.28, 120.99, 37.77, 27.09, 21.65, 13.85. HRMS (ESI) calcd
+
for C H N O [M-H ] 295.15589, found 295.15622.
1
7
19
4
3
7
8
1
3
3
d) 5-(3-bromo-4-methoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-
1
(6H)-one: H NMR (400 MHz, DMSO) δ 11.80 (s, 1H), 8.26 (d, J = 1.8 Hz, 1H), 8.05 (dd, J =
.8, 1.7 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 4.08 (s, 3H), 3.86 (s, 3H), 2.73 (t, J = 7.6 Hz, 2H),
+
.77 – 1.62 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C16
H18BrN
4
O
2
[M-H ]
77.06131, found 377.05963.
1
1
e) 1-methyl-5-(3-nitrophenyl)-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
NMR (400 MHz, DMSO) δ 8.90 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.38 (dd, J = 8.0, 1.4 Hz, 1H),
7
7
3
.81 (t, J = 8.0 Hz, 1H), 4.17 (s, 3H), 2.82 (t, J = 7.4 Hz, 2H), 1.85 – 1.71 (m, 2H), 0.96 (t, J =
+
.4 Hz, 3H). HRMS (ESI) calcd for C15
H
16
N
5
O
3
[M-H ] 314.12531, found 314.12430.
f) 5-(3-bromo-4-fluorophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-
1
one: H NMR (400 MHz, DMSO) δ 12.51 (s, 1H), 8.41 (d, J = 5.2 Hz, 1H), 8.13 (s, 1H), 7.54 (m
1
H), 4.16 (s, 3H), 2.81 (t, J = 7.5 Hz, 2H), 1.84 – 1.69 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). HRMS
+
(
ESI) calcd for C H BrFN O [M-H ] 365.04133, found 365.04088.
1
5
15
4
1
3
g) 1-methyl-5-(5-nitrofuran-2-yl)-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one: H
NMR (400 MHz, DMSO) δ 8.32 (s, 1H), 7.88 (s, 1H), 4.16 (s, 3H), 2.82 (t, J = 7.4 Hz, 2H), 1.84
1
3
–
1
1.68 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H). C NMR (101 MHz, DMSO) δ 153.75, 152.07, 147.43,
45.67, 139.96, 136.88, 124.72, 115.02, 114.06, 37.92, 27.02, 21.52, 13.79. HRMS (ESI)
+
calcd for C H N O [M-H ] 304.10458, found 304.10409.
1
3
14
5
4
1
1
3
h) 5-(4-hydroxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
NMR (400 MHz, DMSO) δ 12.16 (s, 1H), 10.04 (s, 1H), 7.95 (d, J = 8.7 Hz, 2H), 6.87 (d, J =
8
.7 Hz, 2H), 4.14 (s, 3H), 2.79 (t, J = 7.5 Hz, 2H), 1.88 – 1.62 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H).
+
HRMS (ESI) calcd for C15
H
17
N
4
O
2
[M-H ] 285.13515, found 285.13382.
2
, 3
1
3
i) 5-(4-chlorophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
3
NMR (400 MHz, CDCl ) δ 10.94 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.30
(s, 3H), 2.93 ((t, J = 7.6 Hz, 3H), 1.92 – 1.81 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). HRMS (ESI)
+
calcd for C H ClN O [M-H ] 303.10126, found 303.0991.
1
5
16
4
3
j) 1-methyl-3-propyl-5-(2,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one:
1
H NMR (400 MHz, CDCl ) δ 10.99 (s, 1H), 8.03 (s, 1H), 6.59 (s, 1H), 4.27 (s, 3H), 4.05 (s,
3
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ACCEPTED MANUSCRIPT
13
3
H), 3.97 (s, 6H), 2.93 (t, J = 7.5 Hz, 2H), 1.93 – 1.83 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). C
NMR (126 MHz, DMSO) δ 153.61, 152.09, 151.83, 149.06, 144.66, 142.60, 137.96, 123.88,
1
13.50, 112.81, 97.89, 56.57, 56.15, 55.89, 37.77, 27.07, 21.59, 13.81. HRMS (ESI) calcd
+
for C18
H
23
N
4
O
4
[M-H ] 359.17193, found 359.17139.
3
k) 5-(4-methoxy-3-nitrophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-
1
one: H NMR (400 MHz, DMSO) δ 12.54 (s, 1H), 8.62 (d, J = 2.3 Hz, 1H), 8.38 (dd, J = 8.9, 2.3
Hz, 1H), 7.52 (d, J = 9.0 Hz, 1H), 4.16 (s, 3H), 4.01 (s, 3H), 2.81 (t, J = 7.5 Hz, 2H), 1.83 – 1.71
+
(
m, 2H), 0.96 (t, J = 7.4 Hz, 3H). HRMS (ESI) calcd for C H N O [M-H ] 344.13588, found
1
6
18
5
4
3
3
44.13441.
l) 5-(3-fluoro-4-methoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-
1
one: H NMR (400 MHz, CDCl
3
) δ 11.36 (s, 1H), 8.07 (dd, J = 10.8 ,1.8Hz, 1H), 7.92 (d, J = 8.4
Hz, 1H), 7.07 (t, J = 8.4 Hz, 1H), 4.31 (s, 3H), 3.98 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H), 1.96 – 1.76
1
3
(
m, 2H), 1.04 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, DMSO) δ 154.55, 152.26, 149.25,
1
1
48.61, 144.85, 137.76, 125.44, 124.26, 114.90, 114.70, 113.57, 56.20, 37.80, 27.09, 21.61,
+
3.83; HRMS (ESI) calcd for C H FN O [M-H ] 317.14138, found 317.14025.
1
6
18
4
2
3
m) 5-(3,5-dimethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one:
1
H NMR (400 MHz, CDCl ) δ 10.92 (s, 1H), 7.25 (d, J = 2.0 Hz, 2H), 6.61 (t, J = 2.0 Hz, 1H),
3
1
3
4
.30 (s, 3H), 3.89 (s, 6H), 2.98 – 2.89 (m, 2H), 1.93 – 1.80 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). C
(
126 MHz, CDCl
3
) δ 161.27, 155.58, 149.29, 146.87, 139.14, 134.88, 124.55, 105.53, 102.65,
+
5
3
3
5.60, 38.18, 27.73, 22.35, 14.05 HRMS (ESI) calcd for C H N O [M-H ] 329.16137, found
17
21
4
3
29.16031.
n) 5-(3-chlorophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
3
1
NMR (400 MHz, CDCl ) δ 11.76 (s, 1H), 8.23 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.54 – 7.43 (m,
3
2
H), 4.33 (s, 3H), 2.94 (t, J = 7.6 Hz, 2H), 1.93 – 1.82 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). HRMS
+
(
ESI) calcd for C H ClN O [M-H ] 303.10126, found 303.09995.
1
5
16
4
1
3
o) 5-(2-fluorophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one: H
NMR (400 MHz, CDCl ) δ 9.73 (d, J = 10.4 Hz, 1H), 8.34-8.28 (m, 1H), 7.51 (dd, J = 13.7, 7.1
3
Hz, 1H), 7.37-7.31 (m,1H), 7.25-7.18 (m, 1H), 4.27 (s, 3H), 2.92 (d, J = 7.6 Hz, 2H), 1.92 – 1.81
1
3
(
m, 2H), 1.03 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, DMSO) δ 160.70, 158.22, 154.01,
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1
1
3
47.04, 144.99, 137.70, 132.39, 131.01, 124.52, 122.27, 116.13, 37.83, 27.09, 21.66,
+
3.76. HRMS (ESI) calcd for C H FN O [M-H ] 287.13081, found 287.12944.
1
5
16
4
4
1
p) 5-(3-fluorophenyl)-1-methyl-3-propyl-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H
NMR (400 MHz, CDCl
3
) δ 11.71 (s, 1H), 8.05 -7.87(m, 2H), 7.54-7.46 (m, 1H), 7.26-7.20 (m,
1
H), 4.31 (s, 3H), 2.94 (t, J = 7.6 Hz, 2H), 1.93 – 1.82 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). HRMS
+
(ESI) calcd for C15
H
4
16FN O [M-H ] 287.13081, found 287.1304.
3
q)
5-(4-hydroxy-3-nitrophenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-
1
one: H NMR (400 MHz, DMSO) δ 12.49 (s, 1H), 8.64 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.24 (d,
J = 8.0 Hz, 1H), 4.14 (s, 3H), 2.79 (t, J = 7.5 Hz, 2H), 1.86 – 1.65 (m, 2H), 0.95 (t, J = 7.4 Hz,
1
3
3
1
H). C NMR (101 MHz, DMSO) δ 154.47, 153.82, 148.10, 144.86, 137.62, 136.67, 133.76,
24.47, 123.80, 119.21, 79.11, 37.78, 27.08, 21.57, 13.82. HRMS (ESI) calcd for
+
C H N O [M-H ] 330.12023, found 330.11878.
15
16
5
4
2
3
r) 5-(3, 4-dimethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one :
H NMR (400 MHz, CDCl ) δ 11.03 (s, 1H), 7.69 – 7.62 (m, 2H), 6.95 (d, J = 8.4 Hz, 1H), 4.26
1
3
(s, 3H), 4.01 (s, 3H), 3.95 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H), 1.93 – 1.80 (m, 2H), 1.03 (t, J = 7.4
+
Hz, 3H). HRMS (ESI) calcd for C17
H
21
N
4
O
3
[M-H ] 329.16137, found 329.16032.
1
1
3
s) 1-methyl-3-propyl-5-(pyridin-3-yl)-1H-pyrazolo [4, 3-d] pyrimidin-7(6H)-one : H NMR
(
400 MHz, CDCl ) δ 12.13 (s, 1H), 9.43 (d, J = 1.8 Hz, 1H), 8.78 (dd, J = 4.7, 1.1 Hz, 1H),
3
8
.58–8.43 (m, 1H), 7.47 (dd, J = 7.9, 4.8 Hz, 1H), 4.32 (s, 3H), 2.95 (t, J = 7.6 Hz, 2H), 1.95 –
+
1
.82 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). HRMS (ESI) calcd for C H N O [M-H ] 270.13549,
1
4
16
5
found 270.13503.
3
t) 5-(benzo[d][1,3]dioxol-5-yl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one:
1
H NMR (400 MHz, DMSO) δ 12.25 (s, 1H), 7.67 (dd, J = 8.2, 1.8 Hz, 1H), 7.62 (d, J = 1.7 Hz,
1
H), 7.05 (d, J = 8.2 Hz, 1H), 6.12 (s, 2H), 4.14 (s, 3H), 2.79 (t, J = 7.5 Hz, 2H), 1.83 – 1.70 (m,
+
2
3
H), 0.95 (t, J = 7.4 Hz, 3H). HRMS (ESI) calcd for C H N O [M-H ] 313.13007, found
1
6
17
4
3
13.12878.
S7