Synthesis and phosphodiesterase 5 inhibitory activity of novel phenyl ring modified sildenafil analogues

Article abstract of DOI:10.1016/S0968-0896(01)00055-4

New sildenafil analogues containing an ether ring fused into the phenyl moiety, 6a-d and 7a-d, were efficiently synthesized from the readily available starting materials, 1a-d and 2, in five steps. Ab initio calculations indicated that introduction of a cyclic ether to the phenyl group might enhance the co-planarity of the molecule. The torsional angles were calculated to be 2-3° for the 5-membered cyclic ether derivatives, 6a, 6c, 7a, and 7c, and 12-16° for the 6-membered ones, 6b, 6d, 7b, and 7d. On the other hand, sildenafil showed the least co-planarity with the torsional angle of 23° compared with the target compounds, 6a-d and 7a-d. in the enzyme assay, however, the in vitro PDE 5 inhibitory activity was found out to be inversely related to the degree of co-planarity. In other words, the least planar sildenafil showed the highest actiivty, and the most planar 5-membered cyclic ether derivatives were least active by 100-200-fold compared with sildenafil. Our study clearly demonstrated that the open chain 2'-alkoxy group of the phenyl ring, although less effective for inducing the co-planarity, seemed to act as a much better lipophilic requirement than the cyclic alkoxy moiety. Copyright

Full text of DOI:10.1016/S0968-0896(01)00055-4

Bioorganic & Medicinal Chemistry 9 (2001) 1609–1616
Synthesis and Phosphodiesterase 5 Inhibitory Activity of Novel
Phenyl Ring Modified Sildenafil Analogues
Dae-Kee Kim,* Namkyu Lee, Ju Young Lee, Do Hyun Ryu, Jae-Sun Kim, Suk-Ho Lee,
Jin-Young Choi, Kieyoung Chang, Young-Woo Kim, Guang-Jin Im, Won-Son Choi,
Tae-Kon Kim, Je-Ho Ryu, Nam-Ho Kim and Kyoungrim Lee
Life Science Research Center, SK Chemicals, 600 Jungja-Dong, Changan-Ku, Suwon-Si, Kyungki-Do 440-745, South Korea
Received 28 December 2000; accepted 16 February 2001
Abstract—New sildenafil analogues containing an ether ring fused into the phenyl moiety, 6a–d and 7a–d, were efficiently synthe-
sized from the readily available starting materials, 1a–d and 2, in five steps. Ab initio calculations indicated that introduction of a
cyclic ether to the phenyl group might enhance the co-planarity of the molecule. The torsional angles were calculated to be 2ꢀ3ꢁ for
the 5-membered cyclic ether derivatives, 6a, 6c, 7a, and 7c, and 12ꢀ16ꢁ for the 6-membered ones, 6b, 6d, 7b, and 7d. On the other
hand, sildenafil showed the least co-planarity with the torsional angle of 23ꢁ compared with the target compounds, 6a–d and 7a–d.
In the enzyme assay, however, the in vitro PDE 5 inhibitory activity was found out to be inversely related to the degree of co-
planarity. In other words, the least planar sildenafil showed the highest activity, and the most planar 5-membered cyclic ether
derivatives were least active by 100–200-fold compared with sildenafil. Our study clearly demonstrated that the open chain 20-
alkoxy group of the phenyl ring, although less effective for inducing the co-planarity, seemed to act as a much better lipophilic
requirement than the cyclic alkoxy moiety. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
PDE5 elevates levels of the cyclic nucleotide, leading to
enhanced relaxation of smooth muscle, increased arter-
ial inflow, venous congestion, and ultimately resulting in
improved penile erection in men with erectile dysfunc-
tion. Despite the efficacy of sildenafil as a treatment for
MED, there are some notable drawbacks associated
with its use. Clinically significant adverse effects such as
nausea, headache, cutaneous flushing and visual dis-
turbances have been reported, and their incidence is
dose-dependent. Certain of these side-effects are
thought to be due to nonspecific inhibition of other
PDEs, specifically PDE1 and PDE6.5,6 Therefore, the
search for potent and more selective PDE5 inhibitors is
of primary interest.
Male erectile dysfunction (MED), the persistent inabil-
ity to achieve or maintain an erection for satisfactory
sexual performance, is a common and important medi-
cal problem.1 According to a random community-based
sample study, over half of men at 40 to 70 years of age
suffered from erectile dysfunction.2 Recent development
of sildenafil citrate3 (Viagra; Chart 1) as an effective and
orally active agent for the treatment of MED has spur-
red significant interest in the discovery of additional
phosphodiesterase type 5 (PDE5) inhibitors.4 PDE5 is
the predominant cGMP-hydrolyzing enzyme present in
the corpus cavernosum, the smooth muscle in the penis
which helps control vascular tone. Under normal
physiological conditions, nitric oxide is released from
the cavernosal nerve upon sexual stimulation. This
activates soluble guanylyl cyclase in the corpus caver-
nosum, causing an increase in intracellular cGMP,
which is normally hydrolyzed by PDE5. Inhibition of
Intramolecular hydrogen bonding has been shown to
play an important role for high biological activity by
maintaining co-planarity between the phenyl and purine
ring in the sildenafil analogues. It has been demon-
strated that the 20-substituent containing a group, which
is capable of forming a hydrogen bonding to the pyr-
imidinone NH, enhances activity of the drugs in the
earlier work of antiallergenic 8-azapurines (Chart 1).7 In
the structure–activity relationship study of sildenafil
series, it was also suggested that the 20-alkoxy moiety of
*Corresponding author. Tel.: +82-31-240-8400; fax: +82-31-252-
1379; e-mail: dkkim@skchemicals.com
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00055-4

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D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
the phenyl ring not only provided a hydrogen bonding
between the pyrimidinone NH and oxygen lone pair of
the alkoxy group maintaining co-planarity between the
phenyl and purine ring, but also served as a requirement
for a small lipophilic substituent.3c Based on these find-
ings, it was our particular interest to investigate if fur-
ther modification of the 20-alkoxy group of the phenyl
ring to a fused-ring system could enhance the PDE
inhibitory activity and selectivity compared with silde-
nafil. In this paper, the preparation of new sildenafil
analogues possessing an oxygen-containing ring on the
phenyl moiety, 6a–d and 7a–d, and evaluation of their in
vitro PDE inhibitory activity are disclosed.
Chemistry
Requisite starting materials, 7-carboxy-2,3-dihydro-
benzofuran (1a),8 8-chromancarboxylic acid (1b),9 2,3-
methylenedioxybenzoic acid (1c),10 2,3-ethylenediox-
ybenzoic acid (1d)11 and 4-amino-1-methyl-3-n-pro-
pylpyrazole-5-carboxamide (2),3c,12 were readily
available by using known procedures in the literature.
As shown in Scheme 1, the synthesis commenced with
the conversion of each carboxylic acid 1a–d to the cor-
responding acyl chloride in refluxing thionyl chloride
(SOCl2), and the resulting acyl chloride was used for the
next step after complete removal of excess thionyl chlo-
ride under reduced pressure. Selective acylations of the
amine moiety in 2 with cyclic benzoyl chlorides were
carried out in CH2Cl2 in the presence of a catalytic
amount of DMAP (0.1 equiv) at 0 ꢁC to produce the
amides 3a–d in high yields of 82–99%. Cyclization
reactions of the amides 3a–d were efficiently affected
under basic conditions at reflux temperature using
NaOH (2.0 equiv) in a mixture of H2O–EtOH (3:1, v/v)
to afford the corresponding pyrazolopyrimidinones 4a–
d in 70–77% yields. It was observed that under basic
cyclization conditions, the primary amide group of 3a–d
was also hydrolyzed, and the corresponding crude car-
boxylic acid as a major by-product was isolated in
Chart 1.
Scheme 1. (a) SOCl2, reflux, 3 h; (b) DMAP, Et3N, CH2Cl, 0 ꢁC, 1 h; (c) NaOH, H2O/EtOH (3:1), 110 ꢁC, 7–12 h; (d) C1SO3H, 0 ꢁC, 2 h; (e) 1-
methylpiperazine (for 6a–d), or 1-(2-hydroxyethyl)piperazine (for 7a–d), EtOH, room temperature, overnight.

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
1611
about 20–29%. Chlorosulfonylations of the pyr-
Results and Discussion
azolopyrimidinones 4a–d in chlorosulfonic acid at 0 ꢁC
proceeded smoothly and selectively at the 50-position of
the phenyl ring to give the desired compounds 5a–d in
50–95% yields. In these reactions, the yields were found
to be highly variable, seemingly depending on the
structure of the substrate and the reaction scale. These
chlorosulfonyl derivatives 5a–d were readily coupled
with two cyclic amines to produce the sulfonamides as
the target compounds. 1-Methylpiperazine and 1-(2-
hydroxyethyl)piperazine were selected as starting mate-
rials since they have been reported to provide most sui-
table PDE5 inhibitory activities and physical properties
in the earlier related work of Terrett and his co-work-
ers.3c The coupling reactions with chlorosulfonyl deri-
vatives 5a–d were performed using excess amounts of an
appropriate cyclic amine (3–4 equiv) in EtOH at room
temperature to afford the target products, 6a–d and 7a–d,
in 70–97% yields.
Prior to actual synthesis, ab initio calculations using
Gaussian program13 were carried out for sildenafil and
the target compounds, 6a–d and 7a–d, to examine the
co-planarity between the phenyl and the heterocyclic
ring at the HF/3-21G and HF/6-31G(d) levels. In the
processes, the geometries of the total nine compounds
were first optimized using 3-21G basis set, and then the
resulting structures were taken for geometry optimiza-
tion calculation at HF/6-31G(d) level. The fully opti-
mized structures and their key parameters are shown in
Figure 1 and Table 1, respectively, and the torsional
angles depicted in Figure 1 can be used to evaluate the
co-planarity. The torsional angles from HF/3-21G cal-
culation featured the perfect planar structures for all
nine compounds since the angle values were between
ꢀ1.1 and 1.3ꢁ. On the other hand, when the higher level of
theory, HF/6-31G(d), was implemented into calculation,
Figure 1. Optimized structures of sildenafil and 7d using HF/6-31G(d) method as visualized by CS Chem3D Pro.
Table 2. In vitro PDEa inhibitory activities of 6a–d and 7a–d
Table 1. Estimated co-planarity of sildenafil and target compounds,
6a–d and 7a–d, by ab initio calculationsa
Compd
IC50 (nM)
PDE6
IC50 ratio
Method
PDE5
PDE6/PDE5
HF/3-21G
Torsion (ꢁ)
HF/6-31G(d)
Torsion (ꢁ)a
6a
6b
6c
6d
7a
7b
7c
7d
238
12.8
423
7.90
204
12.7
330
6.25
1.90
NDb
53.9
ND
10.6
ND
41.1
ND
ND
4
ND
1
ND
3
ND
1
Compd
Sildenafil
14.3
0.4
23.0
3.2
16.5
2.4
12.5
3.2
16.0
2.3
6a
6b
6c
6d
7a
7b
7c
7d
ꢀ0.9
0.5
ꢀ1.3
0.4
7.34
20.5
Sildenafil
11
ꢀ0.9
0.5
ꢀ1.1
aPDE5 and PDE6 were prepared from rabbit platelet and bovine
retina, respectively, and assayed using [3H]-cGMP SPA kit. IC50
values were determined from the logarithmic concentration-inhibition
curve. The value is the mean from three experiments.
12.4
aTorsional angles are shown in Figure 1.

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D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
the torsional difference of the object compounds became
clear. The computed results showed that introduction of
a cyclic ether to the phenyl group indeed enhanced the
co-planarity between the phenyl and the heterocyclic
ring. Particularly, the co-planarity was greater in the
compounds, 6a, 6c, 7a, and 7c, which contain the 5-
membered cyclic ether, than in the 6-membered ring
compounds, 6b, 6d, 7b, and 7d. The torsional angles
were 2–3ꢁ for the 5-membered cyclic ether derivatives,
6a, 6c, 7a, and 7c, and 12–16ꢁ for the 6-membered ones,
6b, 6d, 7b, and 7d. On the other hand, sildenafil showed
the least co-planarity with the torsional angle of 23ꢁ
compared with the target compounds, 6a–d and 7a–d.
As expected, the structural variation of the 50-sub-
stituent of the phenyl ring in the compounds 6 and 7 did
not affect the planarity of the molecule. We found these
results from ab initio calculations to be really encoura-
ging since it has been known that greater co-planarity of
the molecule could improve the biological activity.
reach into the right lipophilic pocket of the enzyme. On
the other hand, the ethylenedioxy unit of compound 7d
is tied onto the phenyl ring and therefore might not
provide proper spatial orientation for the appropriate
lipophilic interactions with the enzyme, which could
lead to the diminished activity.
In conclusion, our extensive studies on new sildenafil
analogues containing an ether ring fused into the phenyl
moiety, 6a–d and 7a–d, clearly demonstrated that the
open chain 20-alkoxy group of the phenyl ring, although
less effective for inducing the co-planarity, seemed to
fulfill an important role as a lipophilic requirement
much more efficiently than the cyclic ones.
Experimental
Melting points were determined on a Thomas–Hoover
or Mettler melting point apparatus and are uncorrected.
Infrared spectra were recorded on a Magna 750 FTIR
Target compounds, 6a–d and 7a–d, were evaluated for
in vitro inhibitory activities against two different forms
of PDEs, PDE5 and PDE6, and their IC50 values were
determined from concentration–response curves (Table 2).
Disappointingly, all the compounds with the modified
alkoxy group of the phenyl ring showed lower PDE5
inhibitory activities than sildenafil. Replacement of the
alkoxy side chain with 5-membered ether ring (6a and
7a) resulted in about 100-fold drop of PDE5 inhibitory
activities, and the activities decreased even further
(about 200-fold) with the addition of one more oxygen
into the ether ring (6c and 7c). In contrast, compounds
with a 6-membered ring (6b and 7b) showed about 6-
fold decrease in PDE5 inhibitory activities compared
with sildenafil, and introduction of additional oxygen
(6d and 7d) slightly improved their potency. Sub-
stituents on the piperazine did not seem to produce any
significant change in their PDE5 inhibitory activities,
although slight increases in potency were observed with
the 2-hydroxyethyl moiety. In addition, the inhibitory
activity toward PDE6 has been determined for the
selected compounds, but they exhibited no (6d and 7d)
or rather low (6b and 7b) selectivity over PDE6, indi-
cating that these series of compounds have intrinsically
lower selectivity than sildenafil.
1
spectrophotometer. H NMR spectra were recorded on
a Varian Unity 300 spectrometer. The chemical shifts
are reported in parts per million (ppm) relative to inter-
nal tetramethylsilane in CDCl3 or DMSO-d6. Fast-atom
bombardment mass spectra (FABMS) were obtained on
a VG Quattro mass spectrometer. Analytical thin-layer
chromatography (TLC) was performed on Merck silica
gel 60F-254 glass plates. Medium-pressure chromato-
graphy (MPLC) was performed using Merck silica gel
60 (230–400 mesh) with a VSP-2200 ceramic pump
(Eyela). Elemental analyses were performed on a Carlo
Erba 1106 elemental analyzer.
General procedures for the preparation of the amides 3a–d:
acylation reactions
A solution of a cyclic benzoic acid 1a–d (46.0 mmol) in
SOCl2 (45 mL) was refluxed at 90 ꢁC for 3 h under N2
atmosphere. Excess SOCl2 was distilled out from the
reaction mixture, and the residual SOCl2 was removed
completely under vacuum for 1 h. The crude acyl chlo-
ride was dissolved in anhydrous CH2Cl2 (110 mL), and
was slowly added to a cooled mixture of 2 (7.28 g,
40.0 mmol), Et3N (11.2 mL, 80.0 mmol) and a catalytic
amount of DMAP (0.49 g, 4.0 mmol) in anhydrous
CH2Cl2 (110 mL) at 0 ꢁC under N2 atmosphere over 30
min. After additional stirring for 1 h at 0 ꢁC, the mixture
was washed with 1 N HCl aqueous solution (150 mL),
and the aqueous layer was further extracted with 3%
MeOH in CHCl3 (2Â150 mL). The combined organic
layer was dried (MgSO4) and evaporated to dryness
under reduced pressure to afford the desired amide as a
yellow to brown solid. The crude product was purified
by MPLC over SiO2 using a mixture of MeOH/CHCl3
as eluent to give the titled compound, which was crys-
tallized from a suitable solvent.
It should be noted that while the modification of the
alkoxy chain to a ring system increased the degree of co-
planarity according to the quantum mechanical calcu-
lations, it significantly reduced the enzyme inhibitory
activity. In other words, compounds with the 5-mem-
bered ether ring (6a, 6c, 7a and 7c) were expected to
have the highest co-planarity but they exhibited the least
activity, which is contrary to our initial anticipation that
the higher co-planarity would produce higher inhibitory
activity through more favorable hydrogen bonding
interaction. Although highly speculative, this dis-
crepancy might be explained in terms of the spatial
rearrangement of the lipophilic 20-substituent on the
phenyl ring. By scrutinizing the structures of sildenafil
and the representative compound 7d in Figure 1, the
ethyl group of sildenafil seems to be freely rotating and
spans away from the phenyl ring, which could help
4-((20,30-Dihydrobenzofuran-70-carbonyl)amino)-1-methyl-
3-n-propylpyrazole-5-carboxamide (3a). Yield 99%; mp
169.4–170.2 ꢁC (EtOAc); IR (neat) 3399, 3335, 3306
1
(NH), 1677, 1647 (C¼O) cmꢀ1; H NMR (CDCl3) d
0.93 (t, J=7.2 Hz, 3H, CH2CH2CH3), 1.57–1.70 (m, 2H,

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
1613
CH2CH2CH3), 2.54 (dd, J=7.8 Hz, 7.2Hz, 2H,
CH2CH2CH3), 3.36 (t, J=8.7 Hz, 2H, OCH2CH2), 4.06
(s, 3H, NCH3), 4.81 (t, J=8.7 Hz, 2H, OCH2CH2), 5.55
(br s, 1H, CONH2), 7.03 (dd, J=7.8 Hz, 7.2 Hz, 1H, H-
50), 7.43 (dd, J=7.2 Hz, 1.5 Hz, 1H, H-40), 7.89 (br s,
1H, CONH2), 7.95 (dd, J=7.8 Hz, 1.5 Hz, 1H, H-60),
8.96 (br s, 1H, NH); MS (FAB) m/z 329 (MH+). Anal.
calcd for C17H20N4O3: C, 62.18; H, 6.14; N, 17.06.
Found: C, 62.35; H, 6.27; N, 16.87.
extraction with CH2Cl2 (2Â350 mL) was performed.
The combined extracts were dried (MgSO4) and evapo-
rated to dryness under reduced pressure to afford the
desired pyrazolopyrimidinone as a white solid. The
crude product was purified by MPLC on SiO2 using a
mixture of MeOH/CHCl3 as eluent to give the titled
compound, which was crystallized from a suitable
solvent.
5-(20,30-Dihydrobenzofuran-70-yl)-1-methyl-3-n-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (4a). Yield
77%; mp 168.0–168.9 ꢁC (EtOAc/hexane); IR (neat)
3306 (NH), 1703 (C¼O) cmꢀ1; 1H NMR (CDCl3) d 1.03
(t, J=7.5 Hz, 3H, CH2CH2CH3), 1.81–1.94 (m, 2H,
CH2CH2CH3), 2.93 (dd, J=7.8 Hz, 7.5 Hz, 2H,
CH2CH2CH3), 3.32 (t, J=8.7 Hz, 2H, OCH2CH2), 4.27
(s, 3H, NCH3), 4.82 (t, J=8.7 Hz, 2H, OCH2CH2), 7.03
(dd, J=7.8 Hz, 7.5 Hz, 1H, H-50), 7.33 (dd, J=7.2 Hz,
1.2 Hz, 1H, H-40), 8.26 (dd, J=7.8 Hz, 1.2 Hz, 1H, H-
60), 10.81 (br s, 1H, NH); MS (FAB) m/z 311 (MH+).
Anal. calcd for C17H18N4O2: C, 65.79; H, 5.85; N,
18.05. Found: C, 65.92; H, 5.99; N, 18.01.
4-((80-Chromancarbonyl)amino)-1-methyl-3-n-propylpyr-
azole-5-carboxamide (3b). Yield 82%; mp 167.1–
168.1 ꢁC (EtOAc/hexane); IR (neat) 3427, 3231 (NH),
1
1670, 1649 (C=O) cmꢀ1; H NMR (CDCl3) d 0.94 (t,
J=7.5 Hz, 3H, CH2CH2CH3), 1.58–1.70 (m, 2H,
CH2CH2CH3), 2.09–2.17 (m, 2H, OCH2CH2CH2), 2.54
(t, J=7.5 Hz, 2H, CH2CH2CH3), 2.92 (t, J=6.3 Hz, 2H,
OCH2CH2CH2), 4.06 (s, 3H, NCH3), 4.41 (dd,
J=5.4 Hz, 5.1 Hz, 2H, OCH2CH2CH2), 5.58 (br s, 1H,
CONH2), 7.01 (t, J=7.8 Hz, 1H, H-60), 7.27 (dd,
J=7.8 Hz, 1.8 Hz, 1H, H-50), 7.98 (br s, 1H, CONH2),
8.06 (dd, J=7.8 Hz, 1.8 Hz, 1H, H-70), 9.37 (br s, 1H,
NH); MS (FAB) m/z 343 (MH+). Anal. calcd for
C18H22N4O3: C, 63.14; H, 6.48; N, 16.36. Found: C,
63.33; H, 6.71; N, 16.31.
5-(Chroman-80-yl)-1-methyl-3-n-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (4b). Yield 73%; mp
159.9–160.6 ꢁC (EtOAc/hexane); IR (neat) 3263 (NH),
;
1705 (C=O) cmꢀ1 1H NMR (CDCl3) d 1.03 (t,
J=7.5 Hz, 3H, CH2CH2CH3), 1.81–1.93 (m, 2H,
CH2CH2CH3), 2.05–2.15 (m, 2H, OCH2CH2CH2), 2.89
(t, J=6.3 Hz, 2H, OCH2CH2CH2), 2.93 (t, J=7.5 Hz,
2H, CH2CH2CH3), 4.27 (s, 3H, NCH3), 4.30 (dd,
J=5.4 Hz, 5.1Hz, 2H, OCH2CH2CH2), 7.01 (t,
J=7.8 Hz, 1H, H-60), 7.18 (dd, J=7.8 Hz, 1.8 Hz, 1H,
H-50), 8.28 (dd, J=7.8 Hz, 1.8Hz, 1H, H-70), 10.98 (br s,
1H, NH); MS (FAB) m/z 325 (MH+). Anal. calcd for
C18H20N4O2: C, 66.65; H, 6.21; N, 17.27. Found: C,
66.58; H, 6.31; N, 17.11.
4-((20,30-Methylenedioxybenzoyl)amino)-1-methyl-3-n-pro-
pylpyrazole-5-carboxamide (3c). Yield 91%; mp 158–
159 ꢁC (EtOAc); IR (neat) 3303, 3177 (NH), 1671, 1639
1
(C=O) cmꢀ1; H NMR (CDCl3) d 0.93 (t, J=7.5 Hz,
3H, CH2CH2CH3), 1.60–1.70 (m, 2H, CH2CH2CH3),
2.53 (t, J=7.8 Hz, 2H, CH2CH2CH3), 4.06 (s, 3H,
NCH3), 5.59 (br s, 1H, CONH2), 6.18 (s, 2H, OCH2O),
7.01 (t, J=7.8 Hz, 1H, H-50), 7.08 (dd, J=7.8 Hz,
1.5 Hz, 1H, H-40), 7.61 (dd, J=7.8 Hz, 1.5 Hz, 1H, H-
60), 7.67 (br s, 1H, CONH2), 8.27 (br s, 1H, NH); MS
(FAB) m/z 331 (MH+). Anal. calcd for C16H18N4O4: C,
58.17; H, 5.49; N, 16.96. Found: C, 58.01; H, 5.23; N,
17.18.
5-(20,30-Methylenedioxyphenyl)-1-methyl-3-n-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (4c). Yield
70%; mp 186–187 ꢁC (CH2Cl2/hexane); IR (neat) 3177
4-((20,30-Ethylenedioxybenzoyl)amino)-1-methyl-3-n-pro-
pylpyrazole-5-carboxamide (3d). Yield 94%; mp 135–
137 ꢁC (EtOAc/hexane); IR (neat) 3579, 3351, 3277
1
(NH), 1686 (C=O) cmꢀ1; H NMR (CDCl3) d 1.03 (t,
J=7.2 Hz, 3H, CH2CH2CH3), 1.80–1.93 (m, 2 H,
CH2CH2CH3), 2.93 (dd, J=7.8 Hz, 7.5 Hz, 2 H,
CH2CH2CH3), 4.27 (s, 3H, NCH3), 6.17 (s, 2 H,
OCH2O), 6.96 (dd, J=7.8 Hz, 1.8 Hz, 1H, H-40), 7.02 (t,
J=7.8 Hz, 1H, H-50), 7.90 (dd, J=7.8 Hz, 1.8 Hz, 1H,
H-60), 10.07 (br s, 1H, NH); MS (FAB) m/z 313 (MH+).
Anal. calcd for C16H16N4O3: C, 61.53; H, 5.16; N,
17.94. Found: C, 61.71; H, 5.39; N, 17.82.
1
(NH), 1676, 1632 (C=O) cmꢀ1; H NMR (CDCl3) d
0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.55–1.75 (m, 2H,
CH2CH2CH3), 2.54 (dd, J=7.8 Hz, 7.2 Hz, 2H,
CH2CH2CH3), 4.06 (s, 3H, NCH3), 4.37–4.39 (m, 2H,
OCH2), 4.49–4.51 (m, 2H, OCH2), 5.59 (br s, 1H,
CONH2), 7.01 (dd, J=8.1 Hz, 7.8 Hz, 1H, H-50), 7.11
(dd, J=8.1 Hz, 1.8 Hz, 1H, H-40), 7.79 (dd, J=7.8 Hz,
1.8 Hz, 1H, H-60), 7.84 (br s, 1H, CONH2), 9.01 (br s,
1H, NH); MS (FAB) m/z 345 (MH+). Anal. calcd for
C17H20N4O4: C, 59.29; H, 5.85; N, 16.27. Found: C,
59.55; H, 5.97; N, 16.01.
5-(20,30-Ethylenedioxyphenyl)-1-methyl-3-n-propyl-1,6-di-
hydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (4d). Yield
74%; mp 154.5–157.0 ꢁC (EtOAc/hexane); IR (neat)
1
3279 (NH), 1700 (C=O) cmꢀ1; H NMR (CDCl3) d
General procedures for the preparation of the pyrazolo-
pyrimidinones 4a–d: cyclization reactions. A suspension
of the amide 3a–d (28.26 mmol) and NaOH (2.26 g,
56.51 mmol) in a mixture of H2O (96 mL) and EtOH
(32 mL) was refluxed at 110 ꢁC for 7–12 h, cooled to
room temperature, and followed by EtOH evaporation
in vacuo. The pH of the reaction mixture was adjusted
to 1L with 2 N HCl aqueous solution (18 mL), and
1.03 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.73–1.93 (m, 2H,
CH2CH2CH3), 2.92 (t, J=7.5 Hz, 2H, CH2CH2CH3),
4.27 (s, 3H, NCH3), 4.34–4.37 (m, 2H, OCH2), 4.49–
4.52 (m, 2H, OCH2), 6.97–7.04 (m, 2H, H-40 and H-50),
7.99 (dd, J=6.6 Hz, 3.0 Hz, 1H, H-60), 10.67 (br s, 1H,
NH); MS (FAB) m/z 327 (MH+). Anal. calcd for
C17H18N4O3: C, 62.57; H, 5.56; N, 17.17. Found: C,
62.36; H, 5.67; N, 17.41.

1614
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
General procedures for the preparation of the chloro-
sulfonyl derivatives 5a–d: chlorosulfonylation reactions.
To a well-stirred and cooled chlorosulfonic acid (3 mL)
in an ice bath under N2 atmosphere was added por-
tionwise the pyrazolopyrimidinone 4a–d (3.16 mmol)
over 30 min in order to prevent clumping, and the
resulting brownish solution was stirred in an ice bath
for an additional 2 h. The reaction mixture was carefully
poured into the well-stirred ice–water (200 g), and was
extracted with 10% MeOH in CH2Cl2 (2Â200 mL). The
combined organic phase was washed with brine, dried
(MgSO4) and evaporated to dryness under reduced
pressure to afford the desired chlorosulfonyl compound
as a yellowish solid. The crude product was triturated
with Et2O to give the titled compound, which was
crystallized from a suitable solvent.
1.67–1.79 (m, 2H, CH2CH2CH3), 2.79 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 4.16 (s, 3H, NCH3), 4.28–4.37 (m, 4H, 2
OCH2), 7.16 (d, J=1.8 Hz, 1H, H-40), 7.39 (d,
J=1.8 Hz, 1H, H-60), 7.57 (br s, 1H, NH); MS (FAB) m/
z 407 [(MꢀH2O)H+]. Anal. calcd for C17H17ClN4O5S:
C, 48.06; H, 4.03; N, 13.19. Found: C, 48.33; H, 4.29; N,
13.01.
General procedures for the preparation of the sulfona-
mides, 6a–d and 7a–d: coupling reactions. A mixture of
the chlorosulfonyl derivative 5a–d (0.4 mmol) and an
appropriate cyclic amine (1.2 mmol) in absolute EtOH
(12 mL) was stirred overnight at room temperature, and
then the reaction mixture was concentrated in vacuo to
afford the crude sulfonamides, 6a–d and 7a–d, as a yel-
low solid. The residue was purified by MPLC on SiO2
using a mixture of MeOH/CHCl3 as eluent to give the
titled compound, which was crystallized from a suitable
solvent.
5-(50 -Chlorosulfonyl-20,30 -dihydrobenzofuran-70 -yl)-1-
methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimi-
din-7-one (5a). Yield 61%; mp 228 ꢁC dec (CH2Cl2/hex-
ane); IR (neat) 3337 (NH), 1695 (C=O), 1174 (SO2)
5-(50 -(4-Methylpiperazinyl)sulfonyl-20,30 -dihydrobenzo-
furan-70-yl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo
4,3-d]pyrimidin-7-one (6a). Yield 97%; mp 188.8–
189.6 ꢁC (MeOH/Et2O); IR (neat) 3327 (NH), 1706
cmꢀ1 1H NMR (CDCl3) d 1.05 (t, J=7.5 Hz, 3H,
;
CH2CH2CH3), 1.80–1.93 (m, 2H, CH2CH2CH3), 2.96
(dd, J=7.8 Hz, 7.2Hz, 2H, CH2CH2CH3), 3.47 (t,
J=8.9 Hz, 2H, OCH2CH2), 4.28 (s, 3H, NCH3), 5.04 (t,
J=8.9 Hz, 2H, OCH2CH2), 7.95 (d, J=2.1 Hz, 1H, H-
40), 9.00 (d, J=2.1 Hz, 1H, H-60), 10.52 (br s, 1H, NH);
MS (FAB) m/z 409 (MH+). Anal. calcd for
C17H17ClN4O4S: C, 49.94; H, 4.19; N, 13.70. Found: C,
49.63; H, 4.46; N, 13.99.
1
(C¼O), 1170 (SO2) cmꢀ1; H NMR (DMSO-d6) d 0.95
(t, J=7.5 Hz, 3H, CH2CH2CH3), 1.70–1.82 (m, 2H,
CH2CH2CH3), 2.18 (br s, 3H, NCH3), 2.42 (br s, 4H, 2
NCH2), 2.80 (t, J=7.5 Hz, 2H, CH2CH2CH3), 2.93 (br
s, 4H, 2 SO2NCH2), 3.40 (t, J=8.7 Hz, 2H, OCH2CH2),
4.16 (s, 3H, NCH3), 4.84 (t, J=8.7 Hz, 2H, OCH2CH2),
7.74 (d, J=1.8 Hz, 1H, H-40), 7.95 (d, J=1.8 Hz, 1H, H-
60), 11.78 (br s, 1H, NH); MS (FAB) m/z 473 (MH+).
Anal. calcd for C22H28N6O4S: C, 55.92; H, 5.97; N,
17.78. Found: C, 56.13; H, 5.86; N, 17.92.
5-(60-Chlorosulfonylchroman-80-yl)-1-methyl-3-n-propyl-
1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(5b).
Yield 86%; mp 190.2–190.9 ꢁC dec (EtOAc/hexane); IR
(neat) 3327 (NH), 1711 (C¼O), 1168 (SO2) cmꢀ1; H
1
NMR (CDCl3) d 1.04 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.83–1.93 (m, 2H, CH2CH2CH3), 2.17–2.24 (m, 2H,
OCH2CH2CH2), 2.95 (t, J=7.5Hz, 2H, CH2CH2CH3),
3.02 (t, J=6.3 Hz, 2H, OCH2CH2CH2), 4.28 (s, 3H,
NCH3), 4.58 (dd, J=5.4 Hz, 5.1 Hz, 2H, OCH2CH2CH2),
7.85 (d, J=2.4 Hz, 1H, H-50), 8.94 (d, J=2.4 Hz, 1H, H-
70), 10.64 (br s, 1H, NH); MS (FAB) m/z 369 [(M–H2O–
Cl)+]. Anal. calcd for C18H19ClN4O4S: C, 51.12; H,
4.53; N, 13.25. Found: C, 51.46; H, 4.81; N, 12.93.
5 - (60 - (4 - Methylpiperazinyl)sulfonylchroman - 80 - yl) - 1-
methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]-pyrimi-
din-7-one (6b). Yield 97%; mp 221–222 ꢁC dec (EtOAc/
hexane); IR (neat) 3313 (NH), 1680 (C=O), 1164 (SO2)
1
cmꢀ1; H NMR (DMSO-d6) d 0.93 (t, J=7.2 Hz, 3H,
CH2CH2CH3), 1.65–1.79 (m, 2H, CH2CH2CH3), 1.95–
2.05 (m, 2H, OCH2CH2CH2), 2.15 (s, 3H, NCH3), 2.37
(br s, 4H,
2 NCH2), 2.77 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 2.85–2.95 (m, 6H, 2 SO2NCH2 and
OCH2CH2CH2), 4.15 (s, 3H, NCH3), 4.31 (dd,
J=5.1 Hz, 4.8 Hz, 2H, OCH2CH2CH2), 7.59 (d,
J=2.1 Hz, 1H, H-50), 7.65 (d, J=2.1 Hz, 1H, H-70),
12.20 (br s, 1H, NH); MS (FAB) m/z 487 (MH+). Anal.
calcd for C23H30N6O4S: C, 56.77; H, 6.21; N, 17.27.
Found: C, 56.89; H, 6.39; N, 17.07.
5-(50-Chlorosulfonyl-20,30-methylenedioxyphenyl)-1-methyl-
3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(5c). Yield 50%; mp 203 ꢁC dec (CH2Cl2/hexane); IR
1
(neat) 3236 (NH), 1680 (C¼O), 1173 (SO2) cmꢀ1; H
NMR (CDCl3) d 1.05 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.80–1.93 (m, 2H, CH2CH2CH3), 2.95 (dd, J=7.8 Hz,
7.5Hz, 2H, CH2CH2CH3), 4.28 (s, 3H, NCH3), 6.39 (s,
2H, OCH2O), 7.53 (d, J=1.8 Hz, 1H, H-40), 8.67 (d,
J=1.8 Hz, 1H, H-60), 9.93 (br s, 1H, NH); MS (FAB) m/
z 393 [(MꢀH2O)H+]. Anal. calcd for C16H15ClN4O5S:
C, 46.78; H, 3.68; N, 13.64. Found: C, 46.45; H, 3.83; N,
13.86.
5-(50-(4-Methylpiperazinyl)sulfonyl-20,30-methylenedioxy-
phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]-pyrimidin-7-one (6c). Yield 90%; mp 187–188 ꢁC
(MeOH/hexane); IR (neat) 3335 (NH), 1701 (C=O),
1172 (SO2) cmꢀ1 1H NMR (DMSO-d6) d 0.94 (t,
;
J=7.5 Hz, 3H, CH2CH2CH3), 1.69–1.82 (m, 2H,
CH2CH2CH3), 2.15 (s, 3H, NCH3), 2.33–2.40 (m, 4H, 2
NCH2), 2.79 (t, J=7.5 Hz, 2H, CH2CH2CH3), 2.90–
2.98 (m, 4H, 2 SO2NCH2), 4.16 (s, 3H, NCH3), 6.32 (s,
2H, OCH2O), 7.36 (d, J=1.8 Hz, 1H, H-40), 7.64 (d,
J=1.8 Hz, 1H, H-60), 12.15 (br s, 1H, NH); MS (FAB)
m/z 475 (MH+). Anal. calcd for C21H26N6O5S: C,
5-(50-Chlorosulfonyl-20,30-ethylenedioxyphenyl)-1-methyl-
3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(5d). Yield 95%; mp 209 ꢁC dec (CH2Cl2/hexane); IR
1
(neat) 3361 (NH), 1704 (C=O), 1178 (SO2) cmꢀ1; H
NMR (CDCl3) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
1615
53.15; H, 5.52; N, 17.71. Found: C, 53.11; H, 5.60; N,
17.66.
3H, NCH3), 4.37 (t, J=5.4 Hz, 1H, OH), 6.32 (s, 2H,
OCH2O), 7.36 (d, J=1.8 Hz, 1H, H-40), 7.64 (d,
J=1.8 Hz, 1H, H-60), 12.15 (br s, 1H, NH); MS (FAB)
m/z 505 (MH+). Anal. calcd for C22H28N6O6S: C,
52.37; H, 5.59; N, 16.66. Found: C, 52.47; H, 5.68; N,
16.51.
5-(50 -(4-Methylpiperazinyl)sulfonyl-20,30 -ethylenedioxy-
phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]-pyrimidin-7-one (6d). Yield 98%; mp 218.4–221.4 ꢁC
(MeOH/CHCl3/hexane); IR (neat) 3337 (NH), 1679
(C¼O), 1164 (SO2) cmꢀ1; H NMR (DMSO-d6) d 0.94
5-(50-(4-(2-Hydroxyethyl)piperazinyl)sulfonyl-20,30-ethyl-
enedioxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-py-
razolo[4,3-d]pyrimidin-7-one (7d). Yield 96%; mp 207.4–
209.2 ꢁC (MeOH/CHCl3/hexane); IR (neat) 3439, 3335
1
(t, J=7.5 Hz, 3H, CH2CH2CH3), 1.67–1.80 (m, 2H,
CH2CH2CH3), 2.15 (s, 3H, NCH3), 2.36 (br s, 4H, 2
NCH2), 2.78 (t, J=7.5 Hz, 2H, CH2CH2CH3), 2.80 (br
s, 4H, 2 SO2NCH2), 4.16 (s, 3H, NCH3), 4.36–4.47 (m,
4H, 2 OCH2), 7.30 (d, J=2.1 Hz, 1H, H-40), 7.42 (d,
J=2.1 Hz, 1H, H-60), 12.13 (br s, 1H, NH); MS (FAB)
m/z 489 (MH+). Anal. calcd for C22H28N6O5S: C,
54.09; H, 5.78; N, 17.20. Found: C, 53.88; H, 5.95; N,
17.37.
(NH and OH), 1689 (C=O), 1169 (SO2) cmꢀ1
;
1H
0.94 (t, J=7.5 Hz, 3H,
NMR (DMSO-d6)
d
CH2CH2CH3), 1.67–1.80 (m, 2H, CH2CH2CH3), 2.36
(t, J=6.0 Hz, 2H, NCH2CH2OH), 2.48 (br s, 4H, 2
NCH2), 2.78 (t, J=7.5 Hz, 2H, CH2CH2CH3), 2.80 (br
s, 4H,
2
SO2NCH2), 3.41 (t, J=6.0 Hz, 2H,
NCH2CH2OH), 4.16 (s, 3H, NCH3), 4.35–4.44 (m, 5H,
2 OCH2 and OH), 7.30 (d, J=2.1 Hz, 1H, H-40), 7.42 (d,
J=2.1 Hz, 1H, H-60), 12.25 (br s, 1H, NH); MS (FAB)
m/z 519 (MH+). Anal. calcd for C23H30N6O6S: C,
53.27; H, 5.83; N, 16.21. Found: C, 53.47; H, 5.70; N,
16.48.
5-(50-(4-(2-Hydroxyethyl)piperazinyl)sulfonyl-20,30-dihydro-
benzofuran-70 -yl)-1-methyl-3-n-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (7a). Yield 91%; mp
213.6–214.8 ꢁC (MeOH/Et2O); IR (neat) 3391, 3327
(NH and OH), 1707 (C¼O), 1165 (SO2) cmꢀ1; 1H NMR
(DMSO-d6) d 0.95 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.70–1.82 (m, 2H, CH2CH2CH3), 2.37 (br s, 2H,
NCH2CH2OH), 2.51 (br s, 4H, 2 NCH2), 2.80 (t,
J=7.5 Hz, 2H, CH2CH2CH3), 2.90 (br s, 4H, 2
SO2NCH2), 3.30–3.42 (m, 4H, NCH2CH2OH and
OCH2CH2), 4.16 (s, 3H, NCH3), 4.36 (br s, 1H, OH),
4.84 (t, J=8.7 Hz, 2H, OCH2CH2), 7.73 (d, J=1.8 Hz,
1H, H-40), 7.94 (d, J=1.8 Hz, 1H, H-60), 11.78 (br s, 1H,
NH); MS (FAB) m/z 503 (MH+). Anal. calcd for
C23H30N6O5S: C, 54.96; H, 6.02; N, 16.72. Found: C,
54.91; H, 6.13; N, 16.85.
Determination of PDE5 and PDE6 inhibitory activity.
PDE5 was prepared from the rabbit platelet using the
method described by Hidaka et al.14 with minor mod-
ifications. Fresh rabbit whole blood was centrifuged at
360Âg to obtain the platelet-rich plasma (PRP). Plate-
lets were isolated from PRP by centrifugation at
1200Âg, sonicated (20 s per mL) in 50 mM Tris–HCl
buffer (pH 7.4) containing 1 mM MgCl2, and then cen-
trifuged at 40,000Âg for 2 h at 4 ꢁC. The supernatant
was loaded on the DEAE-cellulose column with a bed
volume of 35 mL (Sigma Co., St. Louis, MO, USA) pre-
equilibrated with equilibration buffer (50 mM Tris-ace-
tate containing 3.75 mM 2-mercaptoethanol, pH 6.0).
After the column was washed with 60 mL of equilibra-
tion buffer, PDE5 was eluted using a continuous gra-
dient of 0–600 mM sodium acetate in equilibration
buffer with a total volume of 60 mL. The bovine retina
PDE6 was prepared using the method described by
Ballard et al.15 with minor modifications. Bovine retinas
were minced and homogenized in the homogenization
buffer (20 mM HEPES containing 0.25 M sucrose,
1 mM EDTA, 1 mM phenylmethyl sulfonylfluoride
[PMSF], pH 7.2) using a Polytron PT 10/35 homo-
genizer (Kinematica AG, Switzerland) at 5000 rpm with
2 bursts for 10 s. The homogenate was then centrifuged
at 40,000Âg for 60 min at 4 ꢁC. The supernatant was
recovered and filtered through 0.2 mm filter. The filtered
sample was loaded on the Hitrap Q column with a bed
volume of 5 mL (Pharmacia, Uppsala, Sweden) pre-
equilibrated with 20 mM HEPES buffer (pH 7.2) con-
taining 1 mM EDTA and 0.5 mM PMSF. The column
was then washed with 25 mL of equilibration buffer.
PDE6 was eluted using a continuous gradient of 0–
600 mM NaCl in equilibration buffer with a total
volume of 60 mL. Fractions (1.0 mL each) were col-
lected at a flow rate of 60 mL/h and characterized for
cGMP & cAMP hydrolytic PDE activities as described
below. Fractions comprising the main peaks of cGMP
hydrolytic PDE activity were pooled and stored at
5-(60-(4-(2-Hydroxyethyl)piperazinyl)sulfonylchroman-80-
yl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]-
pyrimidin-7-one (7b). Yield 85%; mp 211.7–212.3 ꢁC
dec (EtOAc/hexane); IR (neat) 3561, 3380 (NH and OH),
1
1675 (C¼O), 1174 (SO2) cmꢀ1; H NMR (DMSO-d6) d
0.93 (t, J=7.2 Hz, 3H, CH2CH2CH3), 1.67–1.80 (m, 2H,
CH2CH2CH3), 1.95–2.05 (m, 2H, OCH2CH2CH2), 2.36
(t, J=6.0 Hz, 2H, NCH2CH2OH), 2.40–2.55 (m, 4H, 2
NCH2), 2.77 (t, J=7.5 Hz, 2H, CH2CH2CH3), 2.83–
2.98 (m, 6H, 2 SO2NCH2 and OCH2CH2CH2), 3.39–
3.45 (m, 2H, NCH2CH2OH), 4.15 (s, 3H, NCH3), 4.31
(dd, J=5.1 Hz, 4.8 Hz, 2H, OCH2CH2CH2), 4.38 (t,
J=5.4 Hz, 1H, OH), 7.59 (d, J=2.1 Hz, 1H, H-50), 7.65
(d, J=2.1 Hz, 1H, H-70), 12.19 (br s, 1H, NH); MS
(FAB) m/z 517 (MH +). Anal. calcd for C24H32N6O5S:
C, 55.80; H, 6.24; N, 16.27. Found: C, 55.62; H, 6.45; N,
16.09.
5-(50-(4-(2-Hydroxyethyl)piperazinyl)sulfonyl-20,30-methyl-
enedioxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-py-
razolo[4,3-d]pyrimidin-7-one (7c). Yield 88%; mp 232–
234 ꢁC dec (MeOH/hexane); IR (neat) 3362, 3308 (NH
and OH), 1704 (C¼O), 1168 (SO2) cmꢀ1
;
1H NMR
(DMSO-d6) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.69–1.82 (m, 2H, CH2CH2CH3), 2.37 (t, J=6.3 Hz, 2H,
NCH2CH2OH), 2.45–2.52 (m, 4H, 2 NCH2), 2.79 (t,
J=7.5 Hz, 2H, CH2CH2CH3), 2.93 (br s, 4H, 2
SO2NCH2), 3.39–3.43 (m, 2H, NCH2CH2OH), 4.16 (s,

1616
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1609–1616
ꢀ20 ꢁC in 50% glycerol until the enzyme assay.
Enzymatic activity was determined using a PDE scintil-
lation proximity assay (SPA) kit (Amersham Pharmacia
Biotech, Buckinghamshire, UK) according to the pro-
tocol supplied by the manufacturer. The reaction buffer
contained [3H]-cGMP (5 mCi/mL), 1.7 mM EGTA, and
8.3 mM MgCl2 in 50 mM Tris–HCl buffer (pH 7.5).
After PDE was added to the reaction buffer, the mix-
tures were incubated at 30 ꢁC for 30 min. The reaction
was then stopped by the addition of 50 mL of SPA
beads, and the radioactivity was counted on the liquid
scintillation counter (Tri-Carb 1500, Packard Inc.,
Meriden, CT, USA) after each sample was settled for 20
min. For the inhibitor studies, sildenafil and test com-
pounds were dissolved in DMSO and diluted with dis-
tilled water. The final concentration of DMSO was less
than 0.2% (v/v). All the inhibition experiments were
conducted under the conditions where the level of cGMP
hydrolysis did not exceed 15%, and the product formation
increased linearly with time and amount of enzyme. IC50
was defined as the concentration of compounds to produce
a 50% inhibition of enzyme activity and calculated by
quantal probit analysis in Pharmacological Calculation
System.16
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