Synthesis and phosphodiesterase 5 inhibitory activity of new 5-phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one derivatives containing an N-acylamido group on a phenyl ring

Article abstract of DOI:10.1016/S0968-0896(01)00095-5

New sildenafil analogues with an N-acylamido group at the 5′-position of the phenyl ring, 6a-e, were prepared from the readily available starting compound 2 in four straightforward steps. Enzyme assays demonstrated that all the target compounds 6a-e showed higher PDE5 inhibitory activities than sildenafil. It was observed that the PDE5 inhibitory activity was enhanced as the chain length of R group increased, but introduction of the branched alkyl groups such as isopropyl (6d) and cyclohexyl (6e) resulted in the drop of potency compared with 6c. In particular the N-butyrylamido derivative 6c exhibited the highest PDE5 inhibitory activity, and was about 6-fold more potent than sildenafil. However, all the compounds exhibited somewhat weak selectivity (1-3-fold) over PDE6, indicating that the compounds 6a-e have intrinsically lower selectivity than sildenafil. Copyright

Full text of DOI:10.1016/S0968-0896(01)00095-5

Bioorganic & Medicinal Chemistry 9 (2001) 1895–1899
Synthesis and Phosphodiesterase 5 Inhibitory Activity of New
5-Phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
Derivatives Containing an N-Acylamido Group on a Phenyl Ring
Dae-Kee Kim,* Do Hyun Ryu, Namkyu Lee, Ju Young Lee, Jae-Sun Kim, Sukho Lee,
Jin-Young Choi, Je-Ho Ryu, Nam-Ho Kim, Guang-Jin Im, Won-Son Choi
and Tae-Kon Kim
Life Science Research Center, SK Chemicals, 600 Jungja-Dong, Changan-Ku, Suwon-Si, Kyungki-Do 440-745, Republic of Korea
Received 9January 2001; accepted 26 March 2001
Abstract—New sildenafil analogues with an N-acylamido group at the 50-position of the phenyl ring, 6a–e, were prepared from the
readily available starting compound 2 in four straightforward steps. Enzyme assays demonstrated that all the target compounds 6a–
e showed higher PDE5 inhibitory activities than sildenafil. It was observed that the PDE5 inhibitory activity was enhanced as the
chain length of R group increased, but introduction of the branched alkyl groups such as isopropyl (6d) and cyclohexyl (6e) resulted
in the drop of potency compared with 6c. In particular the N-butyrylamido derivative 6c exhibited the highest PDE5 inhibitory
activity, and was about 6-fold more potent than sildenafil. However, all the compounds exhibited somewhat weak selectivity (1–3-
fold) over PDE6, indicating that the compounds 6a–e have intrinsically lower selectivity than sildenafil. # 2001 Elsevier Science
Ltd. All rights reserved.
Introduction
hydrolyzed by PDE5. Inhibition of PDE5 elevates levels
of the cyclic nucleotide, leading to enhanced relaxation
of smooth muscle, increased arterial inflow, venous
congestion, and ultimately resulting in improved penile
erection in men with erectile dysfunction. Despite the
efficacy of sildenafil as a treatment for MED, there are
some notable drawbacks associated with its use. Clini-
cally significant adverse effects such as headache (16%),
facial flushing (10%), dyspepsia (7%) and visual dis-
turbances (3%) have been reported, and their incidence
is dose-dependent.3 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 It has been reported that over half of men
at 40–70 years of age suffered from erectile dysfunction
according to
a random community-based sample
study.2 Recent development of sildenafil citrate3 (Via-
gra; Chart 1) as an effective and orally active agent for
the treatment of MED has spurred significant interest in
the discovery of additional phosphodiesterase type 5
(PDE5) inhibitors.4 PDE5 inhibition is a particularly
attractive target because PDE5 is the predominant
cGMP-hydrolyzing enzyme present in the corpus caver-
nosum, the smooth muscle in the penis which helps
control vascular tone. Under normal physiological con-
ditions, nitric oxide is released from the cavernosal
nerve upon sexual stimulation. This activates soluble
guanylyl cyclase in the corpus cavernosum, causing an
increase in intracellular cGMP, which is normally
We have been interested in new sildenafil analogues that
could alleviate the drawbacks of sildenafil, especially
those with a modified phenyl ring, and have just sub-
mitted for publication one of our recent results on the
sildenafil derivatives containing an ether ring fused into
the phenyl group.7 Earlier, Dumaıtre and Dodic have
published a rather interesting report on new PDE5
inhibitors, 6-phenylpyrazolo[3,4-d]pyrimidinones, which
feature the isomeric structures of sildenafil.8 From the
*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)00095-5

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D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1895–1899
structure–activity relationship study of 6-phenylpyr-
azolo[3,4-d]pyrimidinone series, it was claimed that a
n-propoxy group at the 20-position of the phenyl ring is
necessary for inhibitory activity and the 50-position of
the phenyl ring can accommodate a great range of
unrelated groups without loss of potency. Among the
compounds reported above, the acetamido derivative 1
(Chart 1) caught our attention since it showed very high
in vitro PDE5 inhibitory activity as well as the highest
in vivo antihypertensive activity, in spite of its some-
what low metabolic stability. Based on these findings, it
was our peculiar interest to investigate if the PDE
inhibitory activity and selectivity, when compared with
sildenafil, could be manipulated by introducing the
N-acylamido substituent at the 50-position of the phenyl
ring. In this report, preparation of new sildenafil
analogues containing an N-acylamido moiety on the
phenyl ring, 6a–e, and evaluation of their in vitro PDE
inhibitory activity are disclosed.
Chemistry
Since it has been reported by Dumaıtre and Dodic that
a n-propoxy group at the 20-position of the phenyl ring
is essential for PDE5 inhibitory activity,8 we decided to
focus only on the n-propoxy derivatives. Target
compounds 6a–e were synthesized in four steps
from 1-methyl-4-(2-n-propoxybenzamido)-3-n-propyl-
pyrazole-5-carboxamide (2) which was readily prepared
by using known procedures in the literature.9 As shown
in Scheme 1, the synthesis began with nitration of com-
pound 2, and the reaction proceeded smoothly to pro-
duce the desired mono-nitro product 3 in 84% yield
when trifluoroacetic acid and concentrated nitric acid
were used. On the other hand, it was observed that di-
nitration of the phenyl ring took place predominantly
under the most common reaction conditions using con-
centrated sulfuric acid and nitric acid even at 0 ꢀC.
Cyclization of the nitro compound 3 was carried out
under typical basic conditions using NaOH (2.0 equiv)
in a mixture of H2O–EtOH (2:1, v/v) at 60 ꢀC to afford
the corresponding pyrazolopyrimidinone 4 in a rather
low yield of 41%. Under this basic condition, hydrolysis
of the secondary amide bond in compound 3 seemed to
be competing with cyclization because a strongly elec-
tron-withdrawing nitro group on the phenyl ring would
activate the amide moiety toward nucleophilic addi-
tions. Thus, this facile hydrolysis of compound 3 might
account for the low yield of cyclization reaction. Sub-
sequent reduction of a nitro group of compound 4
occurred readily at room temperature under hydro-
genation conditions (10% Pd/C, 1 atm of H2) to afford
the amino derivative 5 in an excellent yield of 97%.
Final acylations of the amino compound 5 were per-
formed with an appropriate acylating agent such as an
anhydride (acetic anhydride, propionic anhydride,
Chart 1.
Scheme 1. (a) Concd HNO3, CF3CO2H, 0 ꢀC to rt, 3 h; (b) 1 N NaOH in H2O, EtOH, 60 ꢀC, 18 h; (c) H2 (1 atm), 10% Pd/C, THF–EtOH (1:1),
rt, 3 h; (e) (RCO)2O (for 6a–d), or RCOCl (for 6e), Et3N, CH2Cl2, rt, 1 h.

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1895–1899
1897
butyric anhydride and isobutyric anhydride) or an acyl
Experimental
chloride (cyclohexanecarbonyl chloride). All the acyla-
tion reactions using excess amounts of an appropriate
acylating agent (1.9equiv) and Et 3N (2.5 equiv) in
anhydrous CH2Cl2 at room temperature were almost
quantitative, producing the target compounds 6a–e in
98–99% yields.
Melting points were determined on a Thomas-Hoover
or Mettler melting point apparatus and are uncorrected.
Infra-red spectra were recorded on a Magna 750 FTIR
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. Fast-atom bombard-
ment mass spectra (FAB-MS) were obtained on a VG
Quattro mass spectrometer. Analytical thin-layer chro-
matography (TLC) was performed on Merck silica gel
60F-254 glass plates. Medium-pressure chromatography
(MPLC) was performed using Merck silica gel 60 (230–
400 mesh) with a VSP-2200 ceramic pump (Eyela). Ele-
mental analyses were performed on a Carlo Erba 1106
elemental analyzer.
Results and Discussion
The in vitro inhibitory activity was assessed against two
different forms of PDEs, PDE5 and PDE6, which were
isolated from rabbit platelet and bovine retina, respec-
tively, and the IC50 values for the compounds 6a–e were
determined from concentration-response curves using
concentrations ranging from 0.03 to 100 nM. It was
demonstrated by Terrett et al. that PDE5 from human
corpus cavernosal tissue was essentially identical to the
rabbit platelet enzyme and sildenafil showed similar
affinities for both enzymes (IC50=3.0 nM for corpus
cavernosum and 3.6 nM for rabbit).3c As summarized in
Table 1, all the target compounds 6a–e showed higher
PDE5 inhibitory activities than sildenafil (IC50: 1.76
nM), and their IC50 values ranged from 0.27 to 0.81
nM. It was observed that the PDE5 inhibitory activity
was enhanced as the chain length of R group increased.
In other words, there was about a 3-fold increase in the
activity when the methyl group (6a) was displaced with
n-propyl chain (6c). Especially, the N-butyrylamido
derivative 6c exhibited the highest PDE5 inhibitory
activity, and was about 6-fold more potent than silde-
nafil. In contrast, introduction of the branched alkyl
groups such as isopropyl (6d) and cyclohexyl (6e) resul-
ted in a slight decrease in the potency for PDE5 com-
pared with 6c. The inhibitory activities of these
compounds toward PDE6 have been also determined
and compared with sildenafil. Unfortunately, all the
compounds exhibited somewhat weak selectivity (1–3-
fold) over PDE6, indicating that compounds 6a–e
have intrinsically lower selectivity than sildenafil.
Based on these data, it was concluded that the new
sildenafil analogues with an N-acylamido moiety at
the 50-position of the phenyl ring, 6a–e, although
they showed higher PDE5 inhibitory activity com-
pared with sildenafil, would not be pursued any fur-
ther due to their disappointingly low selectivity over
PDE6.
1-Methyl-4-(5-nitro-2-n-propoxybenzamido)-3-n-propyl-
pyrazole-5-carboxamide (3). To a stirred solution of
1-methyl-4-(2-n-propoxybenzamido)-3-n-propylpyrazole-5-
carboxamide 2 (100 mg, 0.31 mmol) in trifluoroacetic
o
acid (1.0 mL) at 0 C was added slowly concentrated
HNO3 (0.36 mL), and the mixture was stirred at room
temperature for 3 h. The reaction mixture was poured
carefully into ice (10 g) and was extracted with CHCl3
(3Â20 mL). Combined organic layer was dried (MgSO4)
and concentrated in vacuo to afford a yellow oil.
Resulting yellow residue dissolved in CHCl3 (1 mL) was
added to a solution of hexanes (40 mL) to obtain the
titled compound (96 mg, 84%) as a white solid. Analy-
tically pure compound was obtained by crystallization
from EtOAc/hexanes: mp 176–176.5 ꢀC; IR (neat) 3469,
1
3281 (NH), 1679, 1658 (C¼O), 1345 (NO2) cmꢁ1; H
NMR (CDCl3/TMS) d 0.94 (t, J=7.5 Hz, 3 H,
CH2CH2CH3), 1.11 (t, J=7.5 Hz, 3 H, OCH2CH2CH3),
1.57–1.71 (m, 2 H, CH2CH2CH3), 1.93–2.05 (m, 2 H,
OCH2CH2CH3), 2.53 (dd, J=7.8 Hz, 7.5 Hz, 2 H,
CH2CH2CH3), 4.06 (s, 3 H, NCH3), 4.33 (t, J=6.6 Hz,
2 H, OCH2CH2CH3), 5.67 (br s, 1 H, CONH2), 7.19(d,
J=9.0 Hz, 1 H, H-30), 7.60 (br s, 1 H, CONH2), 8.41
(dd, J=9.0 Hz, 3.0 Hz, 1 H, H-40), 9.15 (d, J=3.0 Hz, 1
H, H-60), 9.20 (s, 1 H, NH); MS (FAB) m/z 390 (MH+).
Anal. calcd for C18H23N5O5: C, 55.52; H, 5.95; N,
17.98. Found: C, 55.69; H, 6.02; N, 17.83.
1-Methyl-5-(5-nitro-2-n-propoxyphenyl)-3-n-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (4). A sus-
pension of 1-methyl-4-(5-nitro-2-n-propoxybenzamido)-
3-n-propylpyrazole-5-carboxamide
3 (76 mg, 0.20
Table 1. In vitro PDEa inhibitory activities of compounds 6a–e
mmol) in 1 N NaOH aqueous solution (0.41 mL, 0.41
mmol) and EtOH (0.5 mL) was heated at 60 ꢀC under
nitrogen atmosphere for 18 h. The reaction mixture was
cooled and acidified to about pH 2–3 with 1.0 N aqu-
eous HCl solution. Resulting mixture was extracted
with CHCl3 (3Â5 mL), and the combined extracts were
washed once with brine (20 mL). The organic layer was
dried (MgSO4), filtered, and evaporated to dryness in
vacuo to afford a yellow solid. The crude product was
purified by MPLC on silica gel (gradient elution: 1/3
EtOAc in CHCl3 followed by 1/2 EtOAc in CHCl3) to
afford the titled compound (30 mg, 41%) as a white
solid. Analytically pure compound was obtained by
Compound
IC50 (nM)
IC50 ratio
PDE6/5
PDE5
PDE6
6a
6b
6c
6d
6e
Sildenafil
0.81ꢂ0.05
0.38ꢂ0.06
0.27ꢂ0.03
0.35ꢂ0.07
0.42ꢂ0.03
1.76ꢂ0.12
2.09ꢂ0.35
0.69ꢂ0.21
0.43ꢂ0.13
0.49ꢂ0.07
0.79ꢂ0.13
24.6ꢂ6.2914
3
2
2
1
2
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 meanꢂSEM from three experiments.

1898
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1895–1899
o
crystallization from EtOAc/hexanes: mp 199–199.5 C;
CH2CH2CH3), 4.16 (t, J=6.5 Hz, 2 H, OCH2CH2CH3),
4.27 (s, 3 H, NCH3), 7.02 (d, J=9.0 Hz, 1 H, H-30), 7.35
(br s, 1 H, CONH), 8.01 (dd, J=9.0 Hz, 3.0 Hz, 1 H, H-
40), 8.20 (d, J=3.0 Hz, 1 H, H-60), 11.20 (br s, 1 H, 6-
NH); MS (FAB) m/z 384 (MH+). Anal. calcd for
C20H25N5O3: C, 62.65; H, 6.57; N, 18.26. Found: C,
62.77; H, 6.71; N, 18.11.
IR (neat) 3319(NH), 169(C ¼O), 1343 (NO2) cmꢁ1
;
1H NMR (CDCl3/TMS) d 1.05 (t, J=7.5 Hz, 3 H,
CH2CH2CH3), 1.20 (t, J=7.5 Hz, 3 H, OCH2CH2CH3),
1.82–1.94 (m, 2 H, CH2CH2CH3), 2.01–2.13 (m, 2 H,
OCH2CH2CH3), 2.96 (dd, J=7.8 Hz, 7.2 Hz, 2 H,
CH2CH2CH3), 4.28 (s, 3 H, NCH3), 4.31 (t, J=7.5 Hz,
2 H, OCH2CH2CH3), 7.16 (d, J=9.3 Hz, 1 H, H-30),
8.33 (dd, J=9.3 Hz, 3.0 Hz, 1 H, H-40), 9.34 (d, J=3.0
Hz, 1 H, H-60), 10.80 (br s, 1 H, NH); MS (FAB) m/z
372 (MH+). Anal. calcd for C18H21N5O4: C, 58.21; H,
5.70; N, 18.86. Found: C, 58.40; H, 5.79; N, 18.69.
1-Methyl-5-(5-propionylamino-2-n-propoxyphenyl)-1-
methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-
7-one (6b). Yield 99%; mp 212–213 ꢀC: IR (neat) 3314,
3288 (NH), 1705, 1659(C ¼O) cmꢁ1; 1H NMR (CDCl3/
TMS) d 1.03 (t, J=7.5 Hz, 3 H, CH2CH2CH3), 1.16 (t,
J=7.5 Hz, 3 H, OCH2CH2CH3), 1.28 (t, J=7.5 Hz, 3
H, CH3CH2CO), 1.80–1.92 (m, 2 H, CH2CH2CH3),
1.93–2.05 (m, 2 H, OCH2CH2CH3), 2.43 (q, J=7.5 Hz,
2 H, CH3CH2CO), 2.92 (dd, J=7.8 Hz, 7.5 Hz, 2 H,
CH2CH2CH3), 4.16 (t, J=6.6 Hz, 2 H, OCH2CH2CH3),
4.27 (s, 3 H, NCH3), 7.02 (d, J=9.0 Hz, 1 H, H-30), 7.34
(br s, 1 H, CONH), 8.07 (dd, J=9.0 Hz, 2.7 Hz, 1 H, H-
40), 8.18 (d, J=2.7 Hz, 1 H, H-60), 11.20 (br s, 1 H, 6-
NH); MS (FAB) m/z 398 (MH+). Anal. calcd for
C21H27N5O3: C, 63.46; H, 6.85; N, 17.62. Found: C,
63.63; H, 6.70; N, 17.49.
5-(5-Amino-2-n-propoxyphenyl)-1-methyl-3-n-propyl-1,6-
dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (5). A mix-
ture of 1-methyl-5-(5-nitro-2-n-propoxyphenyl)-3-n-
propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one 4
(2.29g, 6.17 mmol) and 10% Pd/C (0.20 g) in THF (70
mL) and EtOH (70 mL) was purged with hydrogen gas
three times, and stirred vigorously under hydrogen
atmosphere (a balloon) at room temperature for 3 h.
The mixture was filtered through a Celite pad, and the
filtrate was evaporated to dryness under reduced pres-
sure. Resulting yellow residue was purified by MPLC on
silica gel (gradient elution: 1/4 EtOAc in hexanes, 1/2
EtOAc in hexanes, followed by 1/1 EtOAc in CHCl3) to
afford the titled compound (2.08 g, 99%) as a pale yel-
low solid. Analytically pure compound was obtained by
crystallization from EtOAc/hexanes: mp 110–110.5 ꢀC;
IR (neat) 3422, 3349, 3279 (NH), 1694 (C¼O) cmꢁ1; 1H
NMR (CDCl3/TMS) d 1.04 (t, J=7.5 Hz, 3 H,
CH2CH2CH3), 1.14 (t, J=7.5 Hz, 3 H, OCH2CH2CH3),
1.81–1.92 (m, 2 H, CH2CH2CH3), 1.90–2.01 (m, 2 H,
OCH2CH2CH3), 2.93 (dd, J=7.8 Hz, 7.5 Hz, 2 H,
CH2CH2CH3), 4.08 (t, J=6.6 Hz, 2 H, OCH2CH2CH3),
4.27 (s, 3 H, NCH3), 6.79(dd, J=8.7 Hz, 3.0 Hz, 1 H,
H-40), 6.89(d, J=8.7 Hz, 1 H, H-30), 7.83 (d, J=3.0 Hz,
1 H, H-60), 11.30 (br s, 1 H, NH); MS (FAB) m/z 342
(MH+). Anal. calcd for C18H23N5O2: C, 63.32; H, 6.79;
N, 20.51. Found: C, 63.15; H, 6.88; N, 20.66.
5-(5-Butyrylamino-2-n-propoxyphenyl)-1-methyl-3-n-pro-
pyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (6c).
Yield: 98%; mp 207–207.5ꢀC; IR (neat) 3317, 3291 (NH),
1704, 1656 (C¼O) cmꢁ1; 1H NMR (CDCl3/TMS) d 1.03
(t, J=7.5 Hz, 6 H, CH2CH2CH3 and CH3CH2CH2CO),
1.16 (t, J=7.5 Hz, 3 H, OCH2CH2CH3), 1.73–1.93 (m,
4 H, CH2CH2CH3 and CH3CH2CH2CO),1.93–2.05
(m, 2 H, OCH2CH2CH3), 2.37 (t, J=7.5 Hz, 2 H,
CH3CH2CH2CO), 2.92 (t, J=7.5 Hz, 2 H, CH2CH2CH3),
4.16 (t, J=6.6 Hz, 2 H, OCH2CH2CH3), 4.27 (s, 3 H,
NCH3), 7.02 (d, J=9.0 Hz, 1 H, H-30), 7.29(br s, 1 H,
CONH), 8.07 (dd, J=9.0 Hz, 3.0 Hz, 1 H, H-40), 8.18
(d, J=3.0 Hz, 1 H, H-60), 11.20 (br s, 1 H, 6-NH); MS
(FAB) m/z 412 (MH+). Anal. calcd for C22H29N5O3: C,
64.21; H, 7.10; N, 17.02. Found: C, 63.99; H, 7.22; N,
17.17.
General procedures for the preparation of the pyrazolo-
pyrimidinones 6a–e. To a stirred solution of 5-(5-amino-
2-n-propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one 5 (0.92 mmol) and triethy-
lamine (0.33 mL, 2.34 mmol) in CH2Cl2 (4 mL) was
added an appropriate anhydride (1.76 mmol), and the
mixture was stirred at room temperature for 1 h. The
reaction mixture was evaporated to dryness under
reduced pressure, and the resulting yellow residue was
purified by MPLC on silica gel (gradient elution: 2%
MeOH in CHCl3 followed by 5% MeOH in CHCl3) to
afford the titled compound as a white solid, which was
crystallized from EtOAc/hexanes.
5-(5-Isobutyrylamino-2-n-propoxyphenyl)-1-methyl-3-n-pro-
pyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one
(6d).
Yield 98%; mp 223–223.5 ꢀC; IR (neat) 3314 (NH),
1703, 1661 (C¼O) cmꢁ1; 1H NMR (CDCl3/TMS) d 1.04
(t, J=7.5 Hz, 3 H, CH2CH2CH3), 1.16 (t, J=7.5 Hz, 3 H,
OCH2CH2CH3), 1.29(d, J=6.9Hz, 6 H, CH(C H3)2),
1.81–1.91 (m, 2 H, CH2CH2CH3), 1.91–2.05 (m, 2 H,
OCH2CH2CH3), 2.55 (septet, J=6.9Hz, 1 H, C H(CH3)2),
2.93 (dd, J=7.8 Hz, 7.5 Hz, 2 H, CH2CH2CH3), 4.17 (t,
J=6.6 Hz, 2 H, OCH2CH2CH3), 4.27 (s, 3 H, NCH3),
7.03 (d, J=9.0 Hz, 1 H, H-30), 7.28 (br s, 1 H, CONH),
8.11 (dd, J=9.0 Hz, 2.7 Hz, 1 H, H-40), 8.16 (d,
J=2.7 Hz, 1 H, H-60), 11.20 (br s, 1 H, 6-NH); MS
(FAB) m/z 412 (MH+). Anal. calcd for C22H29N5O3: C,
64.21; H, 7.10; N, 17.02. Found: C, 64.40; H, 7.19; N,
16.88.
5-(5-Acetylamino-2-n-propoxyphenyl)-1-methyl-3-n-pro-
pyl-1,6-dihydro-7H-pyrazolo[4ꢀ ,3-d]pyrimidin-7-one (6a).
Yield 99%; mp 233–233.5 C; IR (neat) 3310, 3285
(NH), 1703, 1661 (C¼O) cmꢁ1; 1H NMR (CDCl3/TMS)
d 1.03 (t, J=7.5 Hz, 3 H, CH2CH2CH3), 1.16 (t, J=7.5
5-(5-Cyclohexanecarbonylamino-2-n-propoxyphenyl)-1-
methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimi-
din-7-one (6e). Yield 99%; mp 213–214 ꢀC; IR (neat)
Hz,
3 H, OCH2CH2CH3), 1.80–1.92 (m, 2 H,
CH2CH2CH3), 1.93–2.05 (m, 2 H, OCH2CH2CH3), 2.21
(s, 3 H, CH3CO), 2.92 (dd, J=8.1 Hz, 7.5 Hz, 2 H,
1
3314, 3290 (NH), 1703, 1657 (C¼O) cmꢁ1; H NMR

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 1895–1899
1899
(CDCl3/TMS) d 1.03 (t, J=7.5 Hz, 3 H, CH2CH2CH3),
1.16 (t, J=7.5 Hz, 3 H, OCH2CH2CH3), 1.24–1.40 (m, 3
H, c-Hex), 1.50–1.59(m, 2 H, c-Hex), 1.70–1.76 (m, 1
H, c-Hex), 1.81–1.93 (m, 4 H, CH2CH2CH3 and c-Hex),
1.93–2.05 (m, 4 H, OCH2CH2CH3 and c-Hex), 2.44 (tt,
J=15.0 Hz, 3.3 Hz, 1 H, CHCO), 2.93 (dd, J=8.1 Hz,
7.2 Hz, 2 H, CH2CH2CH3), 4.16 (t, J=6.5 Hz, 2 H,
OCH2CH2CH3), 4.27 (s, 3 H, NCH3), 7.02 (d, J=9.0
Hz, 1 H, H-30), 7.27 (br s, 1 H, CONH), 8.11 (dd, J=9.0
Hz, 3.0 Hz, 1 H, H-40), 8.16 (d, J=3.0 Hz, 1 H, H-60),
11.20 (br s, 1 H, 6-NH); MS (FAB) m/z 452 (MH+).
Anal. calcd for C25H33N5O3: C, 66.50; H, 7.37; N,
15.51. Found: C, 66.71; H, 7.53; N, 15.34.
enzyme assay. Enzymatic activity was determined using
a PDE scintillation proximity assay (SPA) kit (Amer-
sham Pharmacia Biotech, Buckinghamshire, UK)
according to the protocol 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 reac-
tion buffer, the mixtures 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, silde-
nafil and test compounds were dissolved in DMSO and
diluted with distilled 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 concentra-
tion of compounds to produce a 50% inhibition of
enzyme activity and calculated by quantal probit analy-
sis in Pharmacological Calculation System.12
Determination of PDE 5 and PDE6 inhibitory activity
PDE5 was prepared from the rabbit platelet using the
method described by Hidaka et al.10 with minor mod-
ifications. Fresh rabbit whole blood was centrifuged at
360 g to obtain the platelet-rich plasma (PRP). Platelets
were isolated from PRP by centrifugation at 1200 g,
sonicated (20 s per mL) in 50 mM Tris–HCl buffer (pH
7.4) containing 1 mM MgCl2, and then centrifuged at
40,000 g 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–acetate contain-
ing 3.75 mM 2-mercaptoethanol, pH 6.0). After the
column was washed with 60 mL of equilibration buffer,
PDE5 was eluted using a continuous gradient of 0 to
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.11
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 homogenizer (Kinematica
AG, Switzerland) at 5000 rpm with two bursts for 10 s.
The homogenate was then centrifuged at 40,000 g 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) containing 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 collected at a flow rate of 60 mL/h and
characterized for cGMP and cAMP hydrolytic PDE
activities as described below. Fractions comprising the
main peaks of cGMP hydrolytic PDE activity were
pooled and stored at ꢁ20 ꢀC in 50% glycerol until the
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