Design, synthesis and biological evaluation of pyrazolopyrimidinone based potent and selective PDE5 inhibitors for treatment of erectile dysfunction

Article abstract of DOI:10.1016/j.bioorg.2019.103022

Our previous discovery of series of pyrazolopyrimidinone based PDE5 inhibitors led to find potent leads but with low aqueous solubility and poor bioavailability, and low selectivity. Now, a new series of same pyrazolopyrimidinone scaffold is designed, synthesized and evaluated for its PDE5 inhibitory potential. In this study, some of the molecules are found more potent and selective PDE5 inhibitors in vitro than sildenafil. The studies revealed that compound 5 is 20 fold selective to PDE5 against PDE6. As PDE6 enzyme is involved in the phototransduction pathway in the retina and creates distortion problem, the selectivity for PDE5 specifically against PDE6 enzyme is preferred for any development candidate and in present study, compound 5 has been found to be devoid of this liability of selectivity issue. Moreover, compound 5 has shown excellent in vivo efficacy in conscious rabbit model, it's almost comparable to sildenafil. The preclinical pharmacology including pharmacokinetic and physicochemical parameter studies were also performed for compound 5, it was found to have good PK properties and other physicochemical parameters. The development of these selective PDE5 inhibitors can further lead to draw strategies for the novel preclinical and/or clinical candidates based on pyrazolopyrimidinone scaffold.

Full text of DOI:10.1016/j.bioorg.2019.103022

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Pyrazolopyrimidinone Based
Potent and Selective PDE5 Inhibitors for Treatment of Erectile Dysfunction
G. Lakshma Reddy, Mohd. Ishaq Dar, Abhinandan D. Hudwekar, Priya
Mahajan, Amit Nargotra, Adil Manzoor Baba, Utpal Nandi, Priya Wazir,
Gurdarshan Singh, Ram A Vishwakarma, Sajad Hussain Syed, Sanghapal D.
Sawant
PII:
S0045-2068(19)30222-6
https://doi.org/10.1016/j.bioorg.2019.103022
103022
DOI:
Article Number:
Reference:
YBIOO 103022
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
11 February 2019
14 March 2019
29 May 2019
Please cite this article as: G. Lakshma Reddy, Mohd. Ishaq Dar, A.D. Hudwekar, P. Mahajan, A. Nargotra, A.
Manzoor Baba, U. Nandi, P. Wazir, G. Singh, R.A. Vishwakarma, S. Hussain Syed, S.D. Sawant, Design, Synthesis
and Biological Evaluation of Pyrazolopyrimidinone Based Potent and Selective PDE5 Inhibitors for Treatment of
Erectile Dysfunction, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103022
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, Synthesis and Biological Evaluation of Pyrazolopyrimidinone Based Potent and
Selective PDE5 Inhibitors for Treatment of Erectile Dysfunction
a,e†
b,e†
a,e
c,e
G. Lakshma Reddy,
Mohd. Ishaq Dar,
Abhinandan D. Hudwekar,
Priya Mahajan,
c,e
b,f
d,e
d
d,e
Amit Nargotra, Adil Manzoor Baba, Utpal Nandi, Priya Wazir, Gurdarshan Singh, Ram
A Vishwakarma,a,e Sajad Hussain Syed,b,e,f* Sanghapal D. Sawanta,e*
aMedicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180 001, India
bCancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180 001, India
cDiscovery Informatics, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180
0
01, India
dPK-PD-Tox Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu-180
01, India
eAcademy of Scientific and Innovative Research, Anusandhan Bhawan, 2 Rafi Marg, New Delhi-
10 001, India
fPharmacology Division, CSIR-Indian Institute of Integrative Medicine, Sanat Nagar, Srinagar-
90 005, India
0
1
1
E-mail: SDS- sdsawant@iiim.ac.in ; sdsawant@iiim.res.in ; SSH- sshussain@iiim.ac.in ;
sshussain@iiim.res.in
Tel: +91-191-2584999, 2585222 ; Fax: +91-1912586333
†Equal contribution as first author
IIIM communication No.: IIIM/2229/2018

Abstract
Our previous discovery of series of pyrazolopyrimidinone based PDE5 inhibitors led to find
potent leads but with low aqueous solubility and poor bioavailability, and low selectivity. Now, a
new series of same pyrazolopyrimidinone scaffold is designed, synthesized and evaluated for its
PDE5 inhibitory potential. In this study, some of the molecules are found more potent and
selective PDE5 inhibitors in vitro than sildenafil. The studies revealed that compound 5 is 20
fold selective to PDE5 against PDE6. As PDE6 enzyme is involved in the phototransduction
pathway in the retina and creates distortion problem, the selectivity for PDE5 specifically against
PDE6 enzyme is preferred for any development candidate and in present study, compound 5 has
been found to be devoid of this liability of selectivity issue. Moreover, compound 5 has shown
excellent in vivo efficacy in conscious rabbit model, it's almost comparable to sildenafil. The
preclinical pharmacology including pharmacokinetic and physicochemical parameter studies
were also performed for compound 5, it was found to have good PK properties and other
physicochemical parameters. The development of these selective PDE5 inhibitors can further
lead to draw strategies for the novel preclinical and/or clinical candidates based on
pyrazolopyrimidinone scaffold.
Keywords: Pyrazolopyrimidinone derivatives, PDE5 inhibitors, PDE6 enzyme, Sildenafil,
Erectile dysfunction

Introduction
PDE5 is the major cGMP-hydrolyzing enzyme in human corpus carvernosal tissue. Elevation of
cGMP is directly correlated with vascular smooth muscle relaxation. This enzyme is distributed
in lung, kidney, spleen, endothelial cells, and smooth muscle cells, etc. and plays a key role in
the regulation of the cellular level of cGMP.1
As reported in literature pyrazolopyrimidinones are very well exploited for their PDE inhibitory
activities.2-9 The first successful drug ‘Sildenafil’ was structurally based on
1
0
pyrazolopyrimidinone scaffold. Effectiveness of sildenafil in treating erectile dysfunction (ED)
was discovered serendipitously in phase II angina trials and this discovery has led to put several
molecules from pyrazolopyrimidinone class in different preclinical and clinical developmental
1
1
stages. Still, this molecule is being used as effective therapy for ED besides its several notable
side effects such as headache, nausea, cutaneous flushing, and the major one is visual
disturbance caused due to this molecule, and these side effects are because of its non selectivity
and off-target activity. This limited selectivity of sildenafil mostly against PDE1 and PDE6
1
2
needs improvement. Now, the drugs like vardenafil or tadalafil have come up with reduced side
effects because of their higher selectivity towards PDE5 isozyme.13 Looking into this aspect,
there is a need to develop more selective new candidates as PDE5 inhibitors. In connection with
our continuous efforts in search of newer molecules as PDE5 inhibitors, we have discovered a
series of molecules based on pyrazolopyrimidinone scaffold as potent and selective PDE5
inhibitors.
Previously, we reported the discovery of a series of pyrazolopyrimidinones as potent inhibitors
1
4
of PDE5, which was shown to be as efficacious as sildenafil. In an attempt to improve the
potency and selectivity of present series, modification or replacement towords the piperazine
ring of the structure were undertaken with the inputs of informatics in designing. A series of
designer molecules derived from pyrazolopyrimidinone scaffold that are further augmented with
better PDE5 activity and selectivity conducted in wet lab experiments and supported with
significant in vivo efficacy than sildenafil is presented here. Our studies commenced with the
design of molecules in silico by studying their interactions with PDE5 protein followed by their
chemical synthesis and biological studies, the details are described below.

In silico studies:
It is reported that the hydrophobic favored regions around the alkoxy group of the phenyl ring
and methyl piperazine group of sildenafil are favorable for substitution.15 The ‘Q’ pocket
accommodates the pyrazolopyrimidinone group of sildenafil, strongly suggests that the
chemically similar guanidine group of cGMP also binds at this region. The amide moiety of the
pyrazolopyrimidinone group forms a bidentate hydrogen bond with the γ-amide group of
Gln817. The ethoxyphenyl group of sildenafil fits into the hydrophobic H-pocket formed by
Phe786, Ala783, Leu804 and Val782. The ‘L’ region composed of residues Tyr664, Met816,
Ala823 and Gly819 surrounds the methylpiperazine group of sildenafil.
1
6
Using the PDB code 2H42, the binding site analysis of PDE5 was carried out with respect to
the standard molecule sildenafil. It was observed that in lid region there is a scope of
modification at N-methyl piperazine ring position by the substitution of hydrophobic moiety
followed by the hydrogen bond donor or accepter (Figure 1A). Studies have shown that
piperazine moiety is not crucial for PDE inhibitory activity and compounds with replacement of
piperazine substituents charged or larger than a methyl group is of benefit for better inhibitory
1
7
activity. Opening the piperazine ring represents a crucial alteration that gives more flexibility to
1
7
the molecules allowing different conformations of the supported functional groups. Based on
the in silico findings and above available literature reports, sildenafil derivatives were designed
and synthesized. The sildenafil and its two most active derivatives i.e. compound 5 and 6
(Figures 1B, 1C and 1D) showed similar π-π and H-bonding interaction with Tyr612, and the
bidentate H-bond formation with the Gln817. In case of compound 5, Phe786 forms π-π
interaction (Figure 1C) whereas in compound 6, Met816 forms H-bonds, and stabilize the PDE5-
sildenafil derivatives complex. Moreover, the active compound 5 and 6 showed comparable
docking scores of -10.298 and -10.757 w.r.t. sildenafil (-10.844). The best docked conformations
of sildenafil (23) and its most active derivatives are represented in figure 1E. It was observed that
the selected derivatives retain the same orientation as that of sildenafil in the active pocket of
PDE5, therefore most of the interacting residues are similar in all the three studied systems.

A
B
C
D
E
Fig. 1: Sildenafil and its derivatives (A) Scope of modification around the sildenafil (standard
compound), Interaction of PDE5 in complex with (B) Sildenafil 23, (C) compound 5, (D)
compound 6 (with the enzyme residues shown within 4Å from the centroid of the active site).
(E) Docked conformation of Sildenafil (grey), compound 5 (green), and 6 (orange) within the
active site of PDE5.

A
B
C
D
Fig. 2. 2D Interaction diagram of sildenafil and its derivatives in complex with PDE6. (A)
compound 6, (B) Sildenafil 23, (C) Compound 5 (D) 3D docked conformation of compound 5
(green), and 6 (orange) and sildenafil 23 (grey) within the active site of PDE6.
To address the selectivity of sildenafil and its derivatives among PDE5 and PDE6, further
molecular interaction studies were carried out with PDE6 using PDB ID 5ML3 (Figure 2). It was
observed that sildenafil derivatives did not show any interaction with Arg61, Gln78, Tyr149
(
Figure 2A and 2B), which are reported to be pivotal residues for PDE6 inhibition.18 The
standard, sildenafil showed H-bonding with Arg61 and Gln78, and core moiety i.e. pyrazolo-
pyrimidione form π-π interaction with Trp90 (Figure 2C). The sildenafil derivative compound 6
formed H-bond with the Glu88 (Figure 2B). The docking score of these derivatives with PDE6
(Sildenafil (23): -8.806, compound 5: -8.211, and compound 6: -8.564) is also less in comparison
to PDE5. The orientation difference of sildenafil derivatives w.r.t. sildenafil and absence of any

interactions with the important residues Arg61, Gln78, Tyr149 (for PDE6) may be the reason for
their selectivity towards PDE5.
Chemistry and Biological activity:
The synthesis of inhibitors of PDE5 based on pyrazolopyrimidinone is described here. The
synthesis was started from reaction of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide
o
with 2-ethoxybenzaldehyde in the presence of CuCl in EtOH solvent at 70 C for 3h gave
2
cyclized compound 3 i.e. 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-
o
7
7
(6H)-one. Chlorosulfonation of 3, in the presence of chlorosulfonic acid at 0 C for 5 h afforded
5% of compound 4 i.e. 4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-
d]pyrimidin-5-yl)benzene-1-sulfonyl chloride. Compound 4 was then treated with different
secondary amines in the presence of DIPEA as base in dry DCM, at room temperature for 4-6 h,
which furnished final compounds (5-22), Scheme 1. All modifications were carried out towards
piperazine ring of the structure and the final step was modified accordingly.
O
O
O
N
N
HN
X
R
HN
O
S
H2NOC
H2N
2-ethoxybenz
aldehyde(2),
N
O
S
O
HN
N
HN
N
O
N
ClSO3H
N
N
N
0 oC, 5 h.
Cl
N
DIPEA, DCM
rt, 4-6 h
N
CuCl2, EtOH
nPr 70 oC, 3 h.
N
n-Pr
nPr
nPr
OEt
OEt
X
OEt
4
1
3
5-22
R
X = C or N
Scheme 1: Synthesis of sildenafil based analogs
The newly synthesized all sildenafil analogs (compounds 5-22) were screened for PDE5 and
PDE6 (for selectivity) inhibition by in vitro PDE5 and PDE6 enzyme based assay and the IC50
values were calculated. Interestingly, all the analogs were found to be very potent (in nanomolar
range) inhibitors of the PDE5 and some of the molecules found better than sildenafil (Table 1).
All compounds were screened for PDE5 selectivity against PDE6 enzyme. Some compounds 5,
1
7, 21 and 22 have shown good PDE5 fold selectivity like 20, 33, 18.5 and 18 respectively
against PDE6, which is better than sildenafil (4.5 fold selectivity for PDE5 against PDE6).
The effect of substitutions at R position in structure A (Table 1) were found to be crucial. Some
of the cyclic piperidine ring based modified analogs have shown excellent IC value and good
5
0
PDE5 selectivity compared to sildenafil (23).

Table 1: PDE5 potency (IC ), PDE6 selctivity (Fold) and solubility of synthesized compounds
5
0
O
N
O
HN
O
N
S
R
N
nPr
OEt
A
Sr No.
Compound
R
IC50(nM) Fold selectivity
Solubility (µM)
PDE5A1
against PDE6
Water PBS SGF SIF
1
2
3
4
5
6
7
8
9
1
1
1
1
1
.
5
N
O
0
.8
20
2.6
10
20
5
90
20
5
91
10
1
169
5
.
6
N
OH
O
4
.1
.
7
N
4
.27
9.78
6.23
1.5
1
.
8
OH
N
N
1
.3
.5
5
5
5
1
.
9
OH
N
7
5
5
1
1
.
10
11
12
13
14
15
16
17
18
1
0
7.5
ND
5
ND
5
ND
1
ND
1
Ph
.
N
1
8.5
10.6
7.89
7.3
.
N
NHBoc
NH2
1
.9
ND
ND
40
1
ND
ND
20
1
ND
ND
800
1
ND
ND
1
.
N
2
6
0.
1.
2.
3.
4.
N
N
1
07
2.0
N
N
F
1
.1
.1
.0
6.09
6.19
33
1
F
F
2
1
1
1
5
N
CF3
6
1
1
1
1
O
1
6
1.6
10
1
1
1
N
N
NH
1
1
1
1
1
5.
6.
7.
8.
9.
19
N
O
6.4
.7
9.3
7.9
4.9
1
200
10
10
40
1
120
1
1
5
20
1
20
N
N
N
N
5
200
200
1
21
22
N
N
N
N
18.5
18.0
4.2
N
5
5
.8
.6
10
10
1
N
23 (Sildenafil)
N
N
Me
800
20
ND: not determined

Based on these results, the compound with lower PDE5 IC and good selectivity against PDE6,
5
0
compound 5 was taken for further biological investigational studies (i.e. isozymes selectivity,
physicochemical properties, in vivo efficacy and pharmacokinetic profiling).
The substitutions on piperidine or piperazine ring affected the activity of compounds. Piperidone,
the C4-carbonyl piperidine based analog (compound 5) was found to be highly active among all
synthesized compounds and its reduced analog 6 (C4-OH) was comparatively found little less
active and less selective but improved water solubility was noted for this compound, could be
because of hydrogen bonding. However, there was not much difference found in activity when
this reduced analog was protected with methyl group (compound 7), but it increased the
selectivity for PDE5. The terminal hydroxylated analogs (compound 8 and 9) with varying alkyl
chain lengths on C4 of piperidine ring were active against PDE5 but were having less selectivity.
The benzyl (compound 10) and methyl (compound 11) substituted C4 piperidine analog was less
active and selective. C4 amino substituted Boc protected analog (compound 12) was active but
free amino group bearing (compound 13) and hetero cyclic ring (compound 14) were found
comparatively less active on C4 position of piperidine was less active, both compounds were
found with equal selectivity. The fluorinated compounds 15 to 17, did not show much alteration
in activity, but one of the -CF bearing analog (compound 17) has shown better selectivity (33
3
fold, the best in series) towards PDE5 against PDE6 but this has not given good results when
tested for in vivo efficacy, and this could be because of less solubility. The 2-piperazone analog
(compound 18) was little less active and lacks selectivity as well. The extended bulkier group
substitutions like aryl and heteroaryl (compounds 19 to 22) on C4 of piperazine were less active.
The increased selectivity was observed for compounds 21 and 22, which suggest that pyrimidyl
kind of substitutions towards N-piperazine ring could be a good choice to work further for
medicinal chemistry to generate better PDE5 selective candidates. Eventhough, the overall
observation on pattern of substitutions could not be concluded with a proper structure activity
relationship on the substitutions towards piperidine or piperazine ring of the sildenafil core, but
all designed and synthesized analogs were found to have good activity. Based on these inputs,
from in silico and in vitro activity profile, compound 5 was chosen for further detailed studies.
Isozyme selectivity profiling of compound 5:
From the in vitro PDE5 inhibition screening results compound 5 (low IC 0.8 nM and 20 fold
5
0
selectivity against PDE6) was taken for further studies. We tested compound 5 for isozymes

selectivity against all PDEs. The screening results are shown in table 2. Even though, the
selectivity of compound 5 towards PDE5 against PDE6 was better, however, it has shown poor
selectivity towards PDE1 isozyme, this issue could be further addressed as a future activity with
the help of in silico approach.
Table 2: Isozymes selectivity profile of compound 5 and sildenafil against all 11 PDEs
Sildenafil (23)[a]
Compound 5
Enzyme
Mean
IC50, nM
PDE / PDE5
selectivity, fold
difference
Mean
IC50, nM
PDE/PDE5
selectivity, fold
difference
PDE1A1
PDE2A1
PDE3A
600
63,000
26,000
5,000
5.6
107
11250
4642
892
41
51
9000
7200
>10,000
>10,000
0.8
>12500
>12500
1
PDE4A1
PDE5A1
PDE6C
1
24
4.2
16
20
PDE7A1
PDE8A1
PDE9A2
PDE10A1
PDE11A4
22,000
>100,000
3,600
5,400
7,800
3928
>17,857
642
>10,000
>10,000
4,300
8,000
2200
>12500
>12500
5375
964
10000
2750
1392
[
a
]
5
0
1
9
I
C
v
a
l
u
e
s
o
f
s
i
l
d
e
n
a
f
i
l
a
g
a
i
n
s
t
a
l
l
o
t
h
e
r
P
D
E
s
,
e
x
c
e
p
t
P
D
E
5
a
n
d
P
D
E
6
h
a
v
e
b
e
e
n
t
a
k
e
n
f
r
o
m
p
u
b
l
i
s
h
e
d
d
a
t
a
Physicochemical properties of compound 5:
Significant results of in vitro study prompted us to carry out detailed preclinical characterization
of compound 5. In physicochemical properties we checked the plasma protein binding affinity of
compound 5. Human serum protein binding affinity, affects the drug distribution in body and this
also have impact on the drug-drug interaction. In plasma; drug exist in bound  unbound form.
Compound 5 was found to have fraction unbound = 0.039 and % bound = 96.13. In the context
of compounds partitioning and dissociation, pKa (Dissociation constant) and Log P (Partition
coefficient) are useful parameters to anticipate behavior of drug molecules in body. At different
pH, a molecule exist in different ionic species which impact on the ADME (pharmacokinetic).
Thus partition coefficient (Log P) and dissociation constant (pKa) are useful parameter to predict
the distribution of a drug compound in a biological system. Compound 5 has optimum pKa = 8.7

and LogP =2.5 respectively. Caco-2 permeability is also an important parameter to predict
human intestinal permeability and bioavailability of orally administered drugs. An efflux ratio of
1
is indicative of passive diffusion, value less than 0.5 and greater than 2 regarded as active
-6
influx and active efflux, respectively. For compound 5, Caco-2 permeability (10 cm/sec)
showed; A to B as 0.211; B to A as 0.30, and efflux ratio of compound 5 in Caco-2 monolayer
permeability model was (B/A/A/B=1.4; Table 3). Solubility is one of the important parameters to
achieve desired concentration of drug in systemic circulation for desired pharmacological
response and it is also an important parameter to be considered during dosing, formulation and
administration of drug. Compound 5 was having moderate solubility in water = 10, PBS = 90,
SGF = 91 and SIF = 169 µM respectively. Compound 5 has little low solubility in water but has
good solubility in PBS, SGF and SIF. However, sildenafil has low solubility in water and PBS,
moderate to good solubility in SIF and has high solubility in SGF. Further to know the
metabolism and involvement of CYPs, we carried CYP inhibition of compound 5 and 23.
Cytochrome P450 is a drug metabolizing enzyme, its induction or inhibition affects the blood
level of a drug, and hence the toxicity and efficacy also. In further study, compound 5 showed no
significant liability against CYP3A4 (66% inhibition) and CYP2C9 (21% inhibition) at 10 μM
concentration.
Table 3: Study of physicochemical properties and pre-formulation data for compound 5
Plasma protein
binding
Caco-2 permeability
-6
Compound
Code
Solubility (µM)
Water PB
(10 cm/sec)
%
Fraction
%
Efflux Metabolized
ratio
SGF SIF
A to B B to A
S
unbound (fu) Bound
in MLM
LogP pKa
5
10 90 91 169
0.039
96.13 0.211
0.30
1.4
>95
2.5
8.7
2
3
(Sildenafil) 40 10 800 20
0.066
96.0 37.9
45.5
1.2
90
2.69 4.86
Pharmacokinetic studies of compound 5:
Pharmacokinetics study was carried out following an oral administration of compound 5 and 23
sildenafil) individually at a single dose of 30 mg/kg in BALB/c mice. Mean plasma
(
concentration versus time profiles and pharmacokinetic parameters of 5 and 23 are represented in
Fig. 3 and Table 4, respectively. Both 5 and 23 was absorbed rapidly as maximum plasma
concentration (1024 and 1452 ng/mL) was achieved within 0.5 h of dose administration that can
satisfy the proposed therapeutic application. Areas under the curve (AUC & AUC ) were 803
0
-t
0-∞
and 1462 ng.h/mL, respectively for 5 and 1111 & 1650 ng.h/mL, respectively for 23. Difference

in oral exposure of 5 with 23 is possibly related to the lower solubility in SGF and higher value
of pKa. However, compound 5 is expected to be better in vivo efficacy based on the
comprehensive results of oral exposure and in vitro efficacy. Half life of 5 (1.3 h) is lower in
comparison to 23 (1.5 h). Moreover, half life of both the compounds is correlated to its
corresponding clearance rate from the body. The clearance of this molecule with an effective
time frame of around 2 hr will be extremely suitable for the patients because too high lead to
therapeutic failure and too low clearance may be unnecessary for patients compliance or increase
in liability if associated to it. Overall, compound 5 has satisfactory pharmacokinetic profile that
insinuates further development.
Table 4: Pharmacokinetic parameters of compound 5 and 23 (sildenafil) after oral administration
at 30 mg/kg in BALB/c mice
Pharmacokinetic parameter 5
23
Cmax (ng/mL)
Tmax (h)
1024 1452
0.50 0.25
T (h)
1.3
1.5
1
/2
AUC (ng.h/mL)
AUC0-∞ (ng.h/mL)
803 1462
1111 1650
49.2 39.6
27.0 18.2
1.61 1.64
0
-t
V /F (l/kg)
d
Cl/F (l/h/kg)
MRT (h)
Fig. 3: Comparative plasma concentration vs. time profiles after oral administration of 5 and 23
Sildenafil) individually at 30 mg/kg in BALB/c mice
(
In vivo efficacy study of compound 5 in conscious rabbit model:
The animal studies for compound 5 and sildenafil were carried out in rabbits after approval from
the IIIM-Institutional biosafety committee. Nitric oxide is the main neurotransmitter which

mediates smooth muscle relaxation, necessary for penile erection, by activating the guanylyl
cyclase and raising the cGMP levels. In penile tissue, the cGMP is predominantly metabolized
by PDE5, hence an inhibitor of PDE5 increases cGMP levels, enhances relaxation of smooth
2
0
muscle in the corpus cavernosum, and induces penile erection. In order to check and compare
the activity of the compound 5 with sildenafil in animal model of erectile dysfunction, we
followed a well accepted model for penile erection called conscious rabbit model.21 Briefly,
compound 5 and sildenafil were injected into the ear vein of a group of five healthy adult male
rabbits each. Another injection of sodium nitroprusside (SNP), which acts as a donor of nitric
oxide, was given five minutes later. The SNP injection induced the penile erection in all the
animals and length of the uncovered penile mucosa was measured at different time points, by
using vernier calliper. The effect of SNP in all the experiments was removed from the readings
obtained from the control experiments performed with the SNP alone injections. A graph was
plotted between the length of uncovered penile mucosa verses the time and the area under the
curve was calculated for comparison. The results (Fig. 4) show that compound 5 is indeed able to
sustain the penile erection in the rabbit model, with almost the same, rather slightly higher
efficiency as that of sildenafil. The area under curve of compound 5 (640.2) is almost same to
that of sildenafil (601.9).
Time (min)
Fig. 4: Compound 5 is able to sustain SNP induced penile erection in male rabbits with comparable AUC
to that of sildenafil. A single dose (3mg/kg) of sildenafil and compound 5 were given intravenously to a
group of 5 male rabbits each, which was followed, 5 minutes later, by an injection of sodium
nitroprusside (0.2mg/kg) in all animals. Another group of 5 male rabbits received only SNP and served as
a control group. Length of uncovered penile mucosa in all the rabbit groups was measured at different
time points. A graph of the length verses time was plotted to calculate the area under curve (AUC) for all
experiments.

The overall observation on the studies conducted on compound 5 have produced relevant data as
per design of the analogs based on pyrazolopyrimidinone scaffold. To summarize this work with
achieved end results, we have identified a series of potent and selective PDE5 inhibitors based on
a pyrazolopyrimidinone scaffold. Compound 5, has shown comparatively excellent in vitro
activity with greater selectivity for PDE5 against PDE6 along with comparable in vivo efficacy
to sildenafil. Compound 5 has also shown good PK profile and found good or rather better, in
other physico-chemical parameter studies also, like solubility, CYP liabilities, permeability
metabolic stability (MLM) and plasma protein binding (PPB). This series of molecules are useful
for extending further efforts in medicinal chemistry for identifying better lead candidate for ED.

1
1
.1.
Experimental Section
.1.1. Chemistry:
General Procedure for synthesis of compound-5: Synthesis of 5-(2-ethoxyphenyl)-1-methyl-
-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (3):- 4-amino-1-methyl-3-propyl-1H-
3
pyrazole-5-carboxamide (5.0 g, 2.74 mmol) and 2-ethoxybenzaldehyde (4.32 g, 2.88 mmol) were
o
suspended in ethanol and the mixture was heated at 70 C for 1.5 hours after confirmation of
forming an imine by TLC. Added CuCl (10.87 g, 8.2 mmol) and the reaction mixture again
2
o
heated at 70 C under O for 1.5 hours. After completion of reaction, ethanol was removed under
2
vacuum and to this crude residue was added ethyl acetate (50 mL) and water (50 mL). The
organic layer was separated and water layer was re-extracted with (2x50 mL) ethyl acetate. The
combined organic layers were washed with brine solution, concentrated under vacuum and
purified by using Column chromatography afforded the title compound as a white solid. Yield
8
5%.
Synthesis of 4-ethoxy-3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d]
pyrimidin-5-yl) benzene-1-sulfonyl chloride (4):- To the chlorosulphonic acid (3.0 mL, 36.58
mmol) was added 5-(2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-
o
one (1 g, 2.4 mmol) while maintaining the temperature 0 C, then reaction was allowed to
o
proceed at 0-5 C until TLC analysis indicated the absence of starting material. After completion,
to the reaction mixture, cold CHCl was added and to it ice was added in portions. Organic layer
3
was separated and re-extracted the water layer with 2×100 mL cold CHCl and the combined
3
organic layer are washed with brine solution, concentrated under vacuum; Yield 75%.
Synthesis of 5-(2-ethoxy-5-((4-oxopiperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one) (5):- Piperidin-4-one (18.0 mg, 0.18 mmol) was
dissolved in dry DCM and added DIPEA (95.0µL, 0.54 mmol) stirred the reaction mixture 10
o
minutes at 15 C added 4-ethoxy-3-(1- methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-
d]pyrimidin-5-yl)benzene-1-sulfonyl chloride (75 mg, 0.18 mmol) and stirred the reaction for 6
o
hours at 25 C. After completion of reaction, added 30 mL DCM, 30 mL water and organic layer
was separated. Water layer re-extracted with 20 mL DCM and the combined organic layer
washed with brine solution, concentrated under vacuum and purified by using Column
chromatography afforded the title compound as a white solid. Yield 90%.

5
-(2-Ethoxy-5-((4-hydroxypiperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d]pyrimidin-7(6H)-one (6): White solid; yield 85%; TLC (MeOH:CHCl ; 1:9): R = 0.3;
3
f
o
1
m.p. 180 - 181 C; H NMR (500 MHz CDCl ): δ 10.84 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 7.84
3
(dd, J = 8.8, 2.4 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 4.34 (q, J = 6.8 Hz, 2H), 4.27 (s, 3H), 3.85 –
3
5
1
.76 (m, 1H), 3.61 (t, J = 7.6 Hz, 2H), 2.97 – 2.90 (m, 4H), 1.96 – 1.87 (m, 4H), 1.69 – 1.63 (m,
1
3
H) 1.02 (t, J = 7.2 Hz, 3H); C NMR (126 MHz, CDCl ) δ 159.2, 153.6, 146.9, 146.5, 138.3,
3
31.5, 130.9, 129.7, 124.4, 121.0, 113.0, 66.0, 65.6, 43.1, 38.2, 33.2, 27.7, 22.2, 14.5, 14.0;
+
HRMS (ESI) m/z calcd. for C H N O S [M+H] : 476.1968, found 476.1967.
2
2
30
5
5
5
-(2-Ethoxy-5-((4-methoxypiperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d]pyrimidin-7(6H)-one (7): White solid; yield 80%; TLC (MeOH:CHCl ; 1:9): R = 0.5;
3
f
o
1
m.p. 172 - 173 C; H NMR (400 MHz, CDCl ) δ 10.84 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 7.84
3
(dd, J = 8.7, 2.4 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 4.28 (s, 3H), 3.34 –
3
1
.28 (m, 1H), 3.26 (s, 3H), 3.22 – 3.16 (m, 2H), 3.12 – 3.04 (m, 2H), 2.93 (t, J = 7.5 Hz, 2H),
1
3
.95 – 1.71 (m, 6H), 1.65 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.4 Hz, 3H); C NMR (126 MHz,
CDCl ) δ 157.5, 152.0, 145.3, 144.9, 136.8, 129.9, 129.3, 128.3, 122.9, 119.4, 111.5, 72.2, 64.4,
3
+
5
4
5
4.2, 41.3, 36.6, 28.1, 26.1, 20.7, 13.0, 12.4; HRMS (ESI) m/z calcd. for C H N O S [M+H] :
2
3
32
5
5
90.2124, found 490.2128.
-(2-Ethoxy-5-((4-(hydroxymethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (8): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9): R
3
f
o
1
=
0.3; m.p. 205 - 206 C; H NMR (400 MHz, CDCl ) δ 10.82 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H),
3
7
3
–
.85 (dd, J = 8.7, 2.3 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.38 (q, J = 6.9 Hz, 2H), 4.27 (s, 3H),
.89 (d, J = 11.6 Hz, 2H), 3.51 – 3.45 (m, 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.40 – 2.32 (m, 2H), 1.91
1
3
1.80 (m, 2H), 1.67 (t, J = 6.9 Hz, 3H), 1.51 – 1.12 (m, 5H), 1.03 (t, J = 7.4 Hz, 3H); C NMR
(
101 MHz, CDCl ) δ 159.1, 153.6, 146.9, 146.5, 138.3, 131.5, 130.8, 129.9, 124.4, 121.0, 113.0,
3
6
7.0, 66.0, 46.1, 38.2, 37.8, 28.0, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for
+
C H N O S [M+H] : 490.2124, found 490.2124.
2
3
32
5
5
5
-(2-Ethoxy-5-((4-(2-hydroxyethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (9): White solid; yield 65%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.25; m.p. 195 - 197 C; H NMR (400 MHz, CDCl ) δ 10.83 (s, 1H), 8.82 (d, J = 2.3 Hz,
f
3
1
3
H), 7.84 (dd, J = 8.7, 2.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 4.27 (s,
H), 3.85-3.82 (m, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.35 (t, J = 11.5 Hz,

2
2
1
3
5
5
H), 1.90 – 1.81 (m, 2H), 1.77 (d, J = 11.5 Hz, 2H), 1.65 (t, J = 7.0 Hz, 3H), 1.53 – 1.40 (m,
1
3
H), 1.14 – 1.25 (m, 3H), 1.03 (t, J = 7.4 Hz, 3H); C NMR (126 MHz, CDCl ) δ 159.1, 153.6,
3
46.9, 146.5, 138.4, 131.6, 130.9, 129.6, 124.4, 120.9, 112.9, 66.0, 60.0, 46.4, 38.6, 38.2, 31.7,
+
1.4, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for C H N O S [M+H] : 504.2281, found
2
4
34
5
5
04.2287.
-(5-((4-Benzylpiperidin-1-yl)sulfonyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo
4,3-d]pyrimidin-7(6H)-one (10): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9): R = 0.6;
[
3
f
o
1
m.p. 186 - 188 C; H NMR (400 MHz, CDCl ) δ 10.84 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 7.84
3
(
dd, J = 8.7, 2.3 Hz, 1H), 7.28 – 7.24 (m, 2H) 7.20 – 7.15 (m, 2H), 7.09 (d, J = 7.1 Hz, 2H), 4.39
q, J = 6.9 Hz, 2H), 4.29 (s, 3H), 3.86 (t, J = 11.5 Hz, 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.54 (d, J =
(
6
2
1
3
5
5
.6 Hz, 2H), 2.29 (t, J = 11.5 Hz, 2H), 1.91 – 1.80 (m, 2H), 1.76 – 1.61 (m, 6H), 1.46 – 1.36 (m,
1
3
H), 1.02 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, CDCl ) δ 159.0, 153.6, 146.9, 146.5, 139.7,
3
38.4, 131.5, 130.9, 129.8, 128.9, 128.2, 126.0, 124.4, 120.9, 112.9, 66.0, 46.5, 42.6, 38.2, 37.3,
+
1.2, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for C H N O S [M+H] : 550.2488, found
2
9
36
5
4
50.2495.
-(2-Ethoxy-5-((4-methylpiperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo
4,3-d]pyrimidin-7(6H)-one (11): White solid; yield 80%; TLC (MeOH:CHCl ; 1:9): R = 0.5;
[
3
f
o
1
m.p. 206 - 208 C; H NMR (400 MHz, CDCl ) δ 10.83 (s, 1H), 8.82 (d, J = 2.4 Hz, 1H), 7.85
3
(dd, J = 8.8, 2.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 4.28 (s, 3H), 3.80 (d,
J = 11.0 Hz, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.34 (t, J = 11.0 Hz, 2H), 1.91 – 1.80 (m, 2H), 1.71 –
1
3
1
.60 (m, 6H), 1.36 – 1.28 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H), 0.92 (d, J = 5.0 Hz, 3H); C NMR
(
101 MHz, CDCl ) δ 159.1, 153.6, 147.0, 146.6, 138.4, 131.6, 130.9, 130.2, 124.5, 121.0, 113.0,
3
6
6.0, 46.4, 38.2, 33.4, 30.1, 27.7, 22.2, 21.4, 14.5, 14.0; HRMS (ESI) m/z calcd. for
+
C H N O S [M+H] : 474.2175, found 474.2173.
2
3
32
5
4
tert-Butyl(1-((4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimi
din-5-yl)phenyl)sulfonyl)piperidin-4-yl)carbamate (12): White solid; yield 75%; TLC
o
1
(
MeOH:CHCl ; 1:9): R = 0.4; m.p. 227 - 228 C; HNMR (400 MHz, CDCl ) δ 10.85 (s, 1H),
3
f
3
8
.82 (d, J = 1.9 Hz, 1H), 7.84 (dd, J = 8.7, 1.8 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 4.39 (q, J = 6.9
Hz, 2H), 4.28 (s, 3H), 3.79 – 3.70 (m, 2H), 3.49 – 3.38 (m, 1H), 2.94 (t, J = 7.5 Hz, 2H), 2.57 –
2
2
.46 (m, 2H), 2.02 – 1.96 (m, 2H), 1.91 – 1.80 (m, 2H), 1.66 (t, J = 6.9 Hz, 3H), 1.56 – 1.46 (m,
1
3
H), 1.41 (s, 9H), 1.03 (t, J = 7.3 Hz, 3H); C NMR (126 MHz, CDCl ) δ 156.8, 152.5, 151.2,
3

1
2
5
5
44.5, 144.0, 135.9, 129.0, 128.5, 127.2, 122.0, 118.7, 110.7, 77.2, 63.6, 44.5, 42.8, 35.8, 29.3,
+
5.9, 25.3, 19.8, 12.1, 11.6; HRMS (ESI) m/z calcd. for C H N O S [M+H] : 575.2652, found
2
7
39
6
6
75.2629.
-(5-((4-Aminopiperidin-1-yl)sulfonyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d]pyrimidin-7(6H)-one (13): To a solution of 12 (1 mmol) in CH Cl was added 30% TFA
2 2
in CH Cl solution and stirred for 2 h at rt. The reaction mixture poured onto crushed ice and
2
2
adjust the pH = 12 with NaOH solution and extracted with CH Cl . The combined organic layer
2
2
was dried on Na SO and concentrated in vacuo, affording white solid with 80% yield. TLC
2
4
o
1
(
MeOH:CHCl ; 1:9): R = 0.2; m.p. 206 - 207 C; H NMR (400 MHz, CDCl ) δ 8.81 (d, J = 2.3
3
f
3
Hz, 1H), 7.85 (dd, J = 8.7, 2.4 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 4.38 (q, J = 6.9 Hz, 2H), 4.27
(s, 3H), 3.75 –3.72 (m, , 2H), 2.93 (t, J = 7.5 Hz, 2H), 2.69 (m, 1H), 2.56 – 2.50 (m, 2H), 1.92 –
1
3
1
.81 (m, 4H), 1.64 (t, J = 7.0 Hz, 3H), 1.50 – 1.46 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H); C NMR
(
126 MHz, CDCl ) δ 159.2, 153.6, 146.9, 146.5, 138.3, 131.5, 130.8, 129.8, 124.4, 121.0, 113.0,
3
6
6.0, 47.6, 44.9, 38.2, 34.7, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for C H N O S
22 31 6 4
+
[
M+H] : 475.2127, found 475.2127.
5
-(5-([1,4'-Bipiperidin]-1'-ylsulfonyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo [4,3-
d] pyrimidin-7(6H)-one (14): White solid; yield 65%; TLC (MeOH:CHCl ; 1:9): R = 0.3; m.p.
3
f
o
1
2
8
17 - 218 C; H NMR (400 MHz, CDCl ) δ 10.83 (s, 1H), 8.84 (d, J = 2.2 Hz, 1H), 7.87 (dd, J =
3
.8, 2.2 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.30 (s, 3H), 3.94 (d, J = 11.5
Hz, 2H), 2.95 (t, J = 7.5 Hz, 2H), 2.51 – 2.49 (m, 4H), 2.35 (s, 2H), 1.93 – 1.82 (m, 4H), 1.75 –
1
3
1
.27 (m, 12H), 1.05 (t, J = 7.3 Hz, 3H); C NMR (126 MHz, CDCl ) δ 156.9, 151.4, 144.7,
3
1
44.2, 136.1, 129.4, 128.7, 127.3, 122.2, 118.8, 110.8, 63.8, 59.5, 47.7, 44.0, 36.0, 25.5, 24.8,
+
2
2
3.8, 22.3, 20.0, 12.3, 11.8; HRMS (ESI) m/z calcd for C H N O S [M+3H] : 272.6455,
27 41 6 4
found 272.6431.
5
-(2-Ethoxy-5-((4-fluoropiperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d]pyrimidin-7(6H)-one (15): White solid; yield 75%; TLC (MeOH:CHCl ; 1:9): R = 0.55;
3
f
o
1
m.p. 199 - 200 C; H NMR (400 MHz, CDCl ) δ 10.83 (s, 1H), 8.84 (d, J = 2.1 Hz, 1H), 7.85
3
(dd, J = 8.8, 2.1 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 4.85 – 4.71 (m, 1H), 4.39 (q, J = 6.9 Hz, 2H),
4
1
1
.28 (s, 3H), 3.50 – 3.40 (m, 2H), 3.0 – 2.90 (m, 4H), 2.04 – 1.91 (m, 4H), 1.92 – 1.80 (m, 2H),
1
3
.65 (t, J = 6.9 Hz, 3H), 1.02 (t, J = 7.3 Hz, 3H); C NMR (126 MHz, CDCl ) δ 158.4, 152.8,
3
46.1, 145.6, 137.5, 130.6, 130.1, 128.7, 123.6, 120.2, 112.3, 85.2 (d, J = 177.66 Hz), 65.2, 40.9

(
d, J = 3.78 Hz), 37.4, 29.6 (d, J = 22.68 Hz), 26.9, 21.4, 13.7, 13.2; 19F NMR (376 MHz,
+
CDCl ) δ -185.8; HRMS (ESI) m/z calcd. for C H FN O S [M+H] : 478.1924, found
3
22 29
5
4
4
78.1928.
5
-(5-((4,4-Difluoropiperidin-1-yl)sulfonyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d]pyrimidin-7(6H)-one (16): White solid; yield 75%; TLC (MeOH:CHCl ; 1:9): R = 0.55;
3 f
o
1
m.p. 205 - 207 C; H NMR (400 MHz, CDCl ) δ 10.81 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 7.85
3
(dd, J = 8.8, 2.4 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 4.39 (q, J = 7.0 Hz, 2H), 4.28 (s, 3H), 3.32 –
3
.24 (m, 4H), 2.93 (t, J = 7.5 Hz, 2H), 2.15 – 2.05 (m, 4H), 1.92 – 1.80 (m, 2H), 1.66 (t, J = 7.0
1
3
Hz, 3H), 1.03 (t, J = 7.4 Hz, 3H); C NMR (126 MHz, CDCl ) δ 159.4, 153.6, 146.9, 146.3,
3
1
38.3, 131.3, 130.8, 129.6, 124.4, 121.2, 120.7 (t, J = 241.92 Hz), 113.2, 66.1, 43.3 (t, J = 5.04
1
9
Hz), 38.2, 33.5 (t, J = 23.94 Hz), 27.7, 22.2, 14.5, 14.0; F NMR (376 MHz, CDCl ) δ -99.2;
3
+
HRMS (ESI) m/z calcd. For C H F N O S [M+H] : 496.1830, found 496.1819.
2
2
28
2
5
4
5
-(2-Ethoxy-5-((4-(trifluoromethyl)piperidin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (17): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.55; m.p. 203 - 204 C; H NMR (400 MHz, CDCl ) δ 10.75 (s, 1H), 8.74 (d, J = 2.2 Hz,
f
3
1
3
3
H), 7.77 (dd, J = 8.8, 2.3 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 4.31 (q, J = 6.9 Hz, 2H), 4.20 (s,
H), 3.88 (d, J = 11.3 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 2.31 (t, J = 11.3 Hz, 2H), 1.97 – 1.83 (m,
H), 1.83 – 1.74 (m, 2H), 1.70 – 1.62 (m, 2H), 1.58 (t, J = 6.9 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H);
1
3
C NMR (126 MHz, CDCl ) δ 159.3, 153.6, 146.9, 146.4, 138.3, 131.5, 130.9, 129.5, 124.4,
3
1
21.1, 120.6, 113.1, 66.1, 45.0, 39.6, 38.2, 27.7, 24.1, 22.2, 14.5, 14.0; 19F NMR (376 MHz,
+
CDCl ) δ -73.7; HRMS (ESI) m/z calcd. for C H F N O S [M+H] : 528.1892, found 528.1894.
3
23 29
3
5
4
5
-(2-Ethoxy-5-((3-oxopiperazin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-pyrazolo
[
4,3-d] pyrimidin-7(6H)-one (18): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9): R = 0.3;
3
f
o
1
m.p. 262 - 263 C; H NMR (400 MHz, CDCl ) δ 10.88 (s, 1H), 8.82 (d, J = 2.4 Hz, 1H), 7.88
3
(dd, J = 8.8, 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.43 (s, 1H), 4.39 (q, J = 6.9 Hz, 2H), 4.28 (s,
3
H), 3.74 (s, 2H), 3.54 – 3.44 (m, 2H), 3.43 – 3.34 (m, 2H), 2.94 (t, J = 7.5 Hz, 2H), 1.91 – 1.81
1
3
(
m, 2H), 1.65 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, CDCl ) δ 165.8,
3
1
3
4
59.7, 153.6, 147.0, 146.2, 138.3, 131.7, 131.1, 128.6, 124.5, 121.6, 113.4, 66.2, 48.6, 42.6, 41.2,
+
8.2, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for C H N O S [M+H] : 475.1764 found
2
1
27
6
5
75.1767.

5
-(5-((4-(4-Acetylphenyl)piperazin-1-yl)sulfonyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (19): White solid; yield 65%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.4; m.p. 228 - 229 C; H NMR (400 MHz CDCl ): δ 10.80 (s, 1H), 8.87(d, J =2.4 Hz,
f
3
1
2
3
H), 7.88 – 7.81 (m, 3H), 7.17 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 4.38 (q, J =7.0 Hz,
H), 4.27 (s, 3H), 3.45 – 3.43 (m, 4H), 3.25 – 3.23 (m, 4H), 2.94 (t, J = 7.6 Hz, 2H), 2.50 (s,
1
3
H), 1.92 – 1.81 (m, 2H), 1.64 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H); C NMR (126 MHz,
CDCl ) δ 196.5, 159.5, 153.5, 153.3, 146.9, 146.3, 138.3, 131.6, 131.1, 130.3, 128.6, 124.4,
3
1
21.2, 114.2, 113.2, 66.1, 47.3, 45.7, 38.2, 27.7, 26.2, 22.3, 14.5, 14.1; HRMS (ESI) m/z calcd.
+
For C H N O S [M+H] : 579.2390, found 579.2397.
2
9
35
6
5
5
-(2-Ethoxy-5-((4-(pyridin-4-yl)piperazin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (20): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.3; m.p. 213 - 215 C; H NMR (400 MHz, CDCl ) δ 10.82 (s, 1H), 8.84 (s, 1H), 8.26 (d, J
f
3
=
4.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 1H), 7.17 (d, J = 8.8 Hz, 1H), 6.60 (d, J = 5.0 Hz, 2H), 4.38
(q, J = 6.9 Hz, 2H), 4.27 (s, 3H), 3.45 (t, J = 4.8 Hz, 4H), 3.20 (t, J = 4.8 Hz, 4H), 2.94 (t, J = 7.5
1
3
Hz, 2H), 1.91 – 1.81 (m, 2H), 1.64 (t, J = 6.9 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H); C NMR (101
MHz, CDCl ) δ 159.5, 154.2, 153.5, 150.3, 146.9, 146.3, 138.3, 131.5, 131.1, 128.7, 124.5,
3
1
21.4, 113.2, 108.7, 66.1, 45.7, 45.4, 38.2, 27.7, 22.2, 14.5, 14.0; HRMS (ESI) m/z calcd. for
+
C H N O S [M+H] : 538.2236, found 538.2239.
2
6
32
7
4
5
-(2-Ethoxy-5-((4-(pyridin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (21): White solid; yield 70%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.35; m.p. 186 - 187 C; H NMR (400 MHz, CDCl ) δ 10.80 (s, 1H), 8.85 (d, J = 2.3 Hz,
f
3
1
H), 8.14 (d, J = 3.6 Hz, 1H), 7.86 (dd, J = 8.7, 2.3 Hz, 1H), 7.48 – 7.40 (m, 1H), 7.15 (d, J = 8.7
Hz, 1H), 6.64 – 6.58 (m, 2H), 4.37 (q, J = 7.0 Hz, 2H), 4.27 (s, 3H), 3.67 (t, J = 4.8 Hz, 4H),
3
1
1
2
5
.18 (t, J = 4.8 Hz, 4H), 2.94 (t, J = 7.5 Hz, 2H), 1.91 – 1.81 (m, 2H), 1.64 (t, J = 7.0 Hz, 3H),
1
3
.04 (t, J = 7.4 Hz, 3H); C NMR (126 MHz, CDCl ) δ 159.4, 158.6, 153.6, 148.0, 147.0, 146.3,
3
38.3, 137.7, 131.6, 131.1, 128.7, 124.4, 121.1, 114.0, 113.1, 107.2, 66.1, 45.8, 44.7, 38.2, 27.7,
+
2.3, 14.5, 14.1; HRMS (ESI) m/z calcd. for C H N O S [M+H] : 538.2236, found 538.2244.
2
6
32
7
4
-(2-Ethoxy-5-((4-(pyrimidin-2-yl)piperazin-1-yl)sulfonyl)phenyl)-1-methyl-3-propyl-1H -
pyrazolo[4,3-d]pyrimidin-7(6H)-one (22): White solid; yield 65%; TLC (MeOH:CHCl ; 1:9):
3
o
1
R = 0.35; m.p. 209 - 210 C; H NMR (400 MHz, CDCl ) δ 10.81 (s, 1H), 8.86 (d, J = 2.3 Hz,
f
3
1
H), 8.28 (d, J = 4.7 Hz, 2H), 7.87 (dd, J = 8.7, 2.3 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 6.51 (t, J =

4
4
3
1
.7 Hz, 1H), 4.38 (q, J = 7.0 Hz, 2H), 4.29 (s, 3H), 3.98 (t, J = 4.8 Hz, 4H), 3.15 (t, J = 4.8 Hz,
H), 2.95 (t, J = 7.5 Hz, 2H), 1.95 – 1.82 (m, 2H), 1.65 (t, J = 7.0 Hz, 3H), 1.06 (t, J = 7.4 Hz,
1
3
H); C NMR (126 MHz, CDCl ) δ 161.1, 159.4, 157.7, 153.6, 147.0, 146.3, 138.3, 131.6,
3
31.0, 128.7, 124.4, 121.1, 113.1, 110.5, 66.0, 45.9, 42.9, 38.2, 27.7, 22.2, 14.5, 14.0; HRMS
+
(
ESI) m/z calcd. for C H N O S [M+H] : 539.2189, found 539.2196.
2
5
31
8
4
In silico22-23 Methodology
The computational studies against PDE5 and PDE6 were carried out using the Schrodinger suite
015-1 molecular modeling software. The coordinates of PDE5 in complex with sildenafil
inhibitor (PDB code: 2H42, having 2.3Å resolution) and PDE6 in complex with Deltasonamide1
PDB code: 5ML3, having 1.4Å resolution) were obtained from protein data bank.22-23 The
2
(
selected protein-inhibitor complexes were prepared for molecular docking studies using the
protein preparation wizard, where hydrogen were added to the protein with assigning bond order
and only that water residues were kept which were interacting with protein as well as
2
3
heteroatom. Then the heteroatom 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
2
4
bases of already co-crystallized ligand to the receptor using receptor grid generation module. In
order to standardize the docking protocol, the co-crystallized ligand was re-docked on to the
prepared protein structure and RMSD was calculated between its docked and bound
conformation. Herein at XP scoring function of Glide an RMSD of 1.02Å (for PDE5) and 0.973
(for PDE6) was observed which was better than SP and HTVS. Thus all the molecular docking
studies were performed using XP scoring function of Glide.
Pharmacological studies:
(
a) In vitro PDE screening: The PDE5 inhibitory activity (IC ) of different compounds was
50
checked by using commercially available purified human PDE5A active (Signalchem, Canada:
Cat No. P93-31G), expressed by baculovirus in sf9 insect cells and PDE Glo Phosphodiesterase
Assay kit from Promega (Cat No.V1361). The assays were performed by following the
manufacturer recommended protocol. In order to determine the IC of the active molecules a
5
0
nine point titration in duplicate against the PDE5A was performed. Data analysis was performed
using GraphPad Prism®, version 5.00, for Windows using a sigmoidal dose-response (variable

slope) equation. Each point represents an average of two replicates per concentration. IC values
5
0
of Compound 5 against all other PDE isoforms was carried out at BPS Biosciences, USA.
b) In vivo activity: The in vivo study for compound 5 was carried out at Reliance Life Sciences,
(
Mumbai. In order to check the in vivo activity of the most effective molecule, we followed a well
accepted model for penile erection called conscious rabbit model.19,20 Briefly, compound 5 as
well as sildenafil 23 were dissolved in Transcutol and diluted with 20% cremophor-EL in
distilled water at a ratio of 3:7. This solution was injected into the ear vein of a group of five
healthy male rabbit (3-3.5kg weight) in a volume of 0.5ml/kg. The event was followed 5 minutes
later by another equal volume saline injection containing sodium nitroprusside (0.2 mg/kg),
which acts as a donor of nitric oxide (NO). Length of uncovered penile mucosa was measured
using a sliding caliper at different time points for 70 minutes after administration of the test
compounds. The area under the curve (AUC) was calculated by Graph Pad Software. Controls
experiments were performed with the SNP alone injections.
Pharmacokinetic study:24 Compounds were administered by oral route to Swiss mice as
mentioned (5 mice in each group) at a dose of 1, 2.5 and 10 mg/kg respectively. For p.o.
compound 5 was triturated in 2% gum acacia (w/v) and 10ml distilled water added to form
1
0mg/10ml as suspension. Samples derived from plasma at different time points were then
analyzed by LC-MS/MS to generate the required pharmacokinetic parameters.
Acknowledgements
GLR, MID thanks CSIR/UGC for the award of senior research fellowship. The funding support
from HCP0008 is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://

References
[1] J.D. Corbin, S.H. Francis, Cyclic GMP Phosphodiesterase-5: Target of Sildenafil, J. Biol.
Chem. 274 (1999) 13729-13732.
[
2] H. Zheng, L. Li, B. Sun, Y. Gao, W. Song, X. Zhao, Y. Gao, Z. Xie, N. Zhang, J. Ji, H.
Yuan, H. Lou, Design and synthesis of furyl/thineyl pyrroloquinolones based on natural
alkaloid perlolyrine, lead to the discovery of potent and selective PDE5 inhibitors, Eur. J.
Med. Chem. 150 (2018) 30-38.
[
3] O. Rabal, J.A. Sanchez-Arias, M. Cuadrado-Tejedor, I. Miguel, M. Perez-Gonzalez, C.
García-Barroso, A. Ugarte, A.E. de Mendoza, E. Saez, M. Espelosin, S. Ursua, T.
Haizhong, W. Wei, X. Musheng, A. Garcia-Osta, J.Oyarzabal, Design, synthesis,
biological evaluation and in vivo testing of dual phosphodiesterase 5 (PDE5) and histone
deacetylase 6 (HDAC6)- selective inhibitors for the treatment of Alzheimer's disease,
Eur. J. Med. Chem. 150 (2018) 506-524.
[
4] J. Liu, A. Maisonial-Besset, B. Wenzel, D. Canitrot, A. Baufond, J.M. Chezal, P. Brust,
E. Moreau, Synthesis and in vitro evaluation of new fluorinated quinoline derivatives
with high affinity for PDE5: Towards the development of new PET neuroimaging probes,
Eur. J. Med. Chem. 136 (2017) 548-560.
[
[
[
[
5] H.A. Ghofrani, I.H. Osterloh, F. Grimminger, Sildenafil: from angina to erectile
dysfunction to pulmonary hypertension and beyond, Nat. Rev. Drug Discov. 5 (2006)
6
89-702.
6] A. Garcia-Osta, M. Cuadrado-Tejedor, C. Garcia-Barroso, J. Oyarzabal, R. Franco,
Phosphodiesterases as therapeutic targets for Alzheimer's disease, ACS Chem. Neurosci.
3
(2012) 832-844.
7] F. Wunder, A. Tersteegen, A. Rebmann, C. Erb, T. Fahrig, M. Hendrix, Characterization
of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line, Mol.
Pharmacol. 68N (2005) 1775-1781.
8] P.R. Verhoest, C. Proulx-Lafrance, M. Corman, L. Chenard, C.J. Helal, X. Hou, R.
Kleiman, S. Liu, E. Marr, F.S. Menniti, C.J. Schmidt, M. Vanase-Frawley, A.W.
Schmidt, R.D. Williams, F.R. Nelson, K.R. Fonseca, S. Liras, Identification of a brain
penetrant PDE9A inhibitor utilizing prospective design and chemical enablement as a
rapid lead optimization strategy, J. Med. Chem. 52 (2009) 7946-7949.

[
9] P.R. Verhoest, K.R. Fonseca, X. Hou, C. Proulx-Lafrance, M. Corman, C.J. Helal, M.M.
Claffey, J.B. Tuttle, K.J. Coffman, S. Liu, F. Nelson, R.J. Kleiman, F.S. Menniti, C.J.
Schmidt, M. Vanase-Frawley, S Liras, Design and discovery of 6-[(3S,4S)-4-methyl-1-
(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1, 5 dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943), a selective brain penetrant PDE9A
inhibitor for the treatment of cognitive disorders, J. Med. Chem. 55 (2012) 9045-9054.
10] N.K. Terrett, A.S. Bell, D. Brown, P. Ellis, Sildenafil (VIAGRA™), a potent and
selective inhibitor of type 5 cGMP phosphodiesterase with utility for the treatment of
male erectile dysfunction, Bioorg. Med. Chem. Lett. 6 (1996) 1819-1824.
[
[
[
[
[
11] S.G. Kang, J.J. Kim, Udenafil: efficacy and tolerability in the management of erectile
dysfunction, Ther. Adv. Urol. 5 (2013) 101-110.
12] P.J . Wright, Comparison of phosphodiesterase type 5 (PDE5) inhibitors, Int. J. Clin.
Pract. 60 (8) (2006) 967–975
13] S. Doggrell, Do vardenafil and tadalafil have advantages over sildenafil in the treatment
of erectile dysfunction? Int. J. Impot. Res. 19 (2006) 281-295.
14] (a) S.D. Sawant, G.L. Reddy, I.M. Dar, M. Srinivas, G. Gupta, P.K. Sahu, P. Mahajan, S.
Singh, S.C. Sharma, M. Tikoo, G.D. Singh, A. Nargotra, R.A. Vishwakarma, S.H. Syed,
Discovery of Novel Pyrazolopyrimidinone Analogs as Potent Inhibitors of
Phosphodiesterase Type-5, Bioorg. Med. Chem. 23 (9) (2015) 2121-2128; (b) S.D.
Sawant, G.L. Reddy, M. Srinivas, S.S. Hussain, M.I. Dar, A. Nargotra, P. Mahajan, R.A.
Vishwakarma, Pyrazolopyrimidinones for the treatment of impotence and process for the
preparation thereof. (WO 2015/114647 A1, 08/2015)
[
[
[
15] J. Yoo, K.M. Thai, D.K. Kim, J.Y. Lee, H.J. Park, 3D-QSAR studies on sildenafil
analogues, selective phosphodiesterase 5 inhibitors, Bioorg. Med. Chem. Lett. 17 (15)
(2007) 4271-4274.
16] H. Wang, Y. Liu, Q. Huai, J. Cai, R. Zoraghi, S.H. Francis, J.D. Corbin, H. Robinson, Z.
Xin, G. Lin, H. Ke, Multiple conformations of phosphodiesterase-5: implications for
enzyme function and drug development, J. Biol Chem. 281 (30) (2006) 21469-21479.
17] H.A. Flores Toque, F.B. Priviero, C.E. Teixeira, E. Perissutti, F. Fiorino, B. Severino, F.
Frecentese, R. Lorenzetti, J.S. Baracat, V. Santagada, G. Caliendo, E. Antunes, G. De

Nucci, Synthesis and pharmacological evaluations of sildenafil analogues for treatment of
erectile dysfunction, J. Med. Chem. 51 (9) (2008) 2807-2815.
[
18] P. Martin-Gago, E.K. Fansa, C.H. Klein, S. Murarka, P. Janning, M. Schürmann, M.
Metz, S. Ismail, C. Schultz-Fademrecht, M. Baumann, P.I. Bastiaens, A. Wittinghofer, H.
Waldmann. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and
Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2. Angew. Chem. Int.
Ed. Engl. 56 (9) (2017) 2423-2428.
[
19] J. Kotera, H. Mochida, H. Inoue, T. Noto, K. Fujishige, T. Sasaki, T. Kobayashi, K.
Kojima, S. Yee, Y. Yamada, K. Kikkawa, K. Omori, Avanafil, a potent and highly
selective phosphodiesterase-5 inhibitor for erectile dysfunction, J. Urol. 188 (2012) 668-
6
74.
[20] R.B. Moreland, I. Goldstein, A. Traish, Sildenafil, a novel inhibitor of phosphodiesterase
type 5 in human corpus cavernosum smooth muscle cells, Life Sci. 62 (1998) 309-318.
[21] E. Bischoff, K. Schneider, A conscious-rabbit model to study vardenafil hydrochloride
and other agents that influence penile erection, Int. J. Impot. Res. 13 (2001) 230-235.
[22] G.M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju, W. Sherman, Protein and ligand
preparation: Parameters, protocols, and influence on virtual screening enrichments, J.
Comput. Aid. Mol. Des. 27 (3) (2013) 221-234
[
23] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz, M.P.
Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis, P.S. Shenkin, Glide:
a new approach for rapid, accurate docking and scoring. 1. Method and assessment of
docking accuracy, J Med Chem. 47 (7) (2004) 1739-49.
[
24] A. Sharma, A. Magotra, A. Dogra, S. K. Rath, S. Rayees, P. Wazir , S. Sharma , P. L.
Sangwan, S. Singh, G. Singh, U. Nandi, Pharmacokinetics, pharmacodynamics and safety
profiling of IS01957, a preclinical candidate possessing dual activity against
inflammation and nociception, Regul. Toxicol. Pharmacol. 91 (2017) 216–225.

Graphical Abstract
Design, Synthesis and Biological Evaluation of Pyrazolopyrimidinone Based Potent and
Selective PDE5 Inhibitors for Treatment of Erectile Dysfunction

Design, Synthesis and Biological Evaluation of Pyrazolopyrimidinone Based Potent and Selective PDE5
Inhibitors for Treatment of Erectile Dysfunction
Highlights
Pyrazolopyrimidinone scaffold based molecules as potent PDE5 inhibitors
Twenty fold more selective towards PDE5 against PDE6 isozyme
Excellent in vivo efficacy profile in conscious rabbit model
Good preclinical pharmacology profile