Synthesis and phosphodiesterase inhibitory activity of new sildenafil analogues containing a carboxylic acid group in the 5′-sulfonamide moiety of a phenyl ring

Article abstract of DOI:10.1016/S0968-0896(01)00200-0

New sildenafil analogues possessing a carboxylic acid group in the 5′-sulfonamide of the phenyl ring, 9a-l, were prepared from the readily available starting compounds 6a-b and cyclic amines 3-5 in a three-step sequence. In the enzyme assays, it has been shown that all the target compounds 9a-l proved to be more potent in inhibiting phosphodiesterase type 5 (PDE5) than sildenafil by 4-38-fold. The effect on the IC₅₀ values were investigated by varying the alkoxy group (R) of the phenyl ring, the sulfonamide type (X), and the length of the methylene chain linking the carboxylic acid, and the results were discussed in detail. From this study, we have clearly demonstrated that introduction of a carboxylic acid group to the 5′-sulfonamide moiety of the phenyl ring greatly enhanced PDE5 inhibitory activity, probably by mimicking the phosphate group of cGMP. The piperidinyl propionic acid derivative 9i, which showed the highest PDE5 inhibitory activity and comparable to better selectivity over PDE isozymes in comparison with sildenafil, has been selected for more detailed biological investigations.

Full text of DOI:10.1016/S0968-0896(01)00200-0

Bioorganic & Medicinal Chemistry 9 (2001) 3013–3021
Synthesis and Phosphodiesterase Inhibitory Activity of New
Sildenafil Analogues Containing a Carboxylic Acid Group in the
50-Sulfonamide Moiety of a Phenyl Ring
Dae-Kee Kim,* Ju Young Lee, Namkyu Lee, Do Hyun Ryu, 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, South Korea
Received 19 February 2001; accepted 13 June 2001
Abstract—New sildenafil analogues possessing a carboxylic acid group in the 50-sulfonamide of the phenyl ring, 9a–l, were prepared
from the readily available starting compounds 6a–b and cyclic amines 3–5 in a three-step sequence. In the enzyme assays, it has been
shown that all the target compounds 9a–l proved to be more potent in inhibiting phosphodiesterase type 5 (PDE5) than sildenafil
by 4–38-fold. The effects on the IC50 values were investigated by varying the alkoxy group (R) of the phenyl ring, the sulfonamide
type (X), and the length of the methylene chain linking the carboxylic acid, and the results were discussed in detail. From this study,
we have clearly demonstrated that introduction of a carboxylic acid group to the 50-sulfonamide moiety of the phenyl ring greatly
enhanced PDE5 inhibitory activity, probably by mimicking the phosphate group of cGMP. The piperidinyl propionic acid deriva-
tive 9i, which showed the highest PDE5 inhibitory activity and comparable to better selectivity over PDE isozymes in comparison
with sildenafil, has been selected for more detailed biological investigations. # 2001Elsevier Science Ltd. All rights reserved.
Introduction
physiological conditions, nitric oxide (NO) 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
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
headache (16%), facial flushing (10%), dyspepsia (7%)
and visual disturbances (3%) have been reported, and
their incidence is dose-dependent.3 Certain of these side
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 Ten percent of the
respondents claimed complete dysfunction, while 25%
and 17% were diagnosed as moderate and minimal
dysfunction, respectively. Recent development of silde-
nafil citrate3 (Viagra1; Chart 1) as an orally effective
agent for the treatment of MED has spurred significant
interest in the discovery of additional phosphodiesterase
type 5 (PDE5) inhibitors.4 Sildenafil citrate is a potent,
reversible and selective PDE5 inhibitor that blocks
cGMP hydrolysis effectively (Kiꢀ3 nM). 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
effects are thought to be due to nonspecific inhibition of
5,6
other PDEs, specifically PDE1and PDE6.
Therefore,
the search for potent and more selective PDE5 inhibitors
is of primary interest.
It has been our research goal to develop new sildenafil
analogues that could alleviate the drawbacks of silde-
nafil, especially those with a modified phenyl ring, and a
couple of our recent results on the sildenafil derivatives
*Corresponding author. Tel.: +82-31-240-8400; fax: +82-31-252-
1379; e-mail: dkkim@skchemicals.com
0968-0896/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00200-0

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D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
have been submitted for publication.7 In the earlier
work of the sildenafil series, it was suggested that
appropriate substituents on the 50-position of the phenyl
ring could, depending on the actual conformation in the
enzyme active site, reproduce the role of the cyclic
phosphate of cGMP in binding. In the same report, it
was also revealed that two 50-sulfonamide derivatives
(Chart 1) with a hydroxyl (1: IC50=1.9 nM) or a car-
boxamide (2: IC50=2.1nM) group exhibited higher
PDE5 inhibitory activity compared with sildenafil
(IC50=3.6 nM).3c From these data, we deduced that
hydrogen bonding or polar interactions might be pre-
sent inside the space of the enzyme occupied by the
phosphate of cGMP, which could be important for
increasing the potency and selectivity. On the basis of
these findings, it was reasoned that carboxylic acid
functionality in the 50-sulfonamide moiety could
enhance the ability to mimic the role of the phosphate
of cGMP, probably due to its polar nature and capa-
bility for hydrogen bonding interaction. Therefore, we
decided to examine if introducing a carboxylic acid
group to the 50-sulfonamide of the phenyl ring could
enhance the PDE inhibitory activity and selectivity of
new analogues, when compared with sildenafil. In this
paper, we disclose the synthesis of new sildenafil analo-
gues possessing a carboxylic acid group in the 50-sulfo-
namide of the phenyl ring, 9a–l, and evaluation of their
in vitro PDE inhibitory activity.
readily prepared according to the literature procedures.8
With the starting cyclic amines 3–5 in hand, the target
compounds 9a–l were synthesized in a straightforward
three-step sequence from the known pyrazole-5-carbox-
amides 6a–b9 as shown in Scheme 1. Chlorosulfonyla-
tion reactions of compounds 6a–b in neat chlorosulfonic
acid at 0 ꢁC proceeded smoothly and selectively at the
50-position of the phenyl ring to give the desired pro-
ducts 7a–b in 82–83% yields. Two chlorosulfonyl deri-
vatives 7a–b were readily coupled with selected cyclic
amines 3–5 in EtOH at room temperature to produce
the sulfonamides 8a–l in the yields of 61–96%. Cycliza-
tion and the concomitant hydrolysis of the ester group,
when present in the sulfonamide moiety of the starting
mono-cyclic compounds, were efficiently affected under
basic conditions at 90 ꢁC using a mixture of aqueous 1N
NaOH solution and EtOH (2:1, v/v) to afford the cor-
responding pyrazolopyrimidinones 9a–l in 66–93%
yields.
Phosphodiesterase inhibitory activity was assessed in
vitro against two different forms of PDEs, PDE5 and
PDE6, which were isolated from rabbit platelet and
bovine retina, respectively, and the IC50 values for the
Results and Discussion
All the requisite piperidine and piperazine derivatives 3–
5 (Fig. 1) are either commercially available or were
Figure 1. Structures of starting cyclic amines.
Chart 1.

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
3015
compounds 9a–l were determined from concentration–
response curves. The effects on the IC50 values as a
result of varying the alkoxy group (R) of the phenyl
ring, the sulfonamide type (X), and the length of the
methylene chain linking the carboxylic acid (n), are
shown in Table 1. Gratifyingly, all the target com-
pounds 9a–l proved to be more potent (4–38-fold) in
inhibiting PDE5 enzyme than sildenafil (IC50:
1.90Æ0.17 nM), and their IC50 values ranged from 0.05
to 0.51nM. In the view of the structure–activity rela-
tionship study, there were three important aspects
worth commenting. First, there was about 2–3-fold
increase in the activity when the ethoxy group of the
phenyl ring was changed into n-propyl group (9a–f vs
9g–l). Second, the compounds with the piper-
idinylsulfonamide group showed higher activity than
the corresponding piperazinyl derivatives (e.g., 9h–i vs
9k–l). Thirdly, it was clearly observed in the piper-
idinylsulfonamide series, 9a–d and 9g–j, that the PDE5
inhibitory activity was enhanced gradually as the
methylene chain length increased from n=0 to n=2, but
the activity decreased with longer chain (n=3). In con-
trast, the effects of the methylene chain length seemed to
be negligible in the piperazinylsulfonamide derivatives,
9e–f and 9k–l. Among the target compounds, the pro-
pionic acid derivative 9i exhibited the highest PDE5
Scheme 1. (a) ClSO3H, 0 ꢁC to rt, 2 h; (b) 3 (for 8a and 8g), 4 or 5 (for the rest), Et3N, EtOH, rt, 2 h; (c) 1N NaOH in H 2O–EtOH (2:1), 90 ꢁC, 17 h.
Table 1. In vitro PDEa inhibitory activities of compounds 9a–l
Compd
R
X
n
IC50 (nM)
PDE5
IC50 ratio
PDE1PDE3
PDE6
PDE6/PDE5
NDb
ND
0.63Æ0.03
0.72Æ0.13
ND
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
Sildenafil
Et
Et
Et
Et
Et
Et
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
—
CH
CH
CH
CH
N
0
1ND
2
3
1ND
2
ND
ND
ND
ND
0.51Æ0.10
ND
ND
4
0.33
0.41
Æ0.04
0.15Æ0.04
0.22Æ0.03
Æ0.05
ND
ND
ND
3
ND
ND
ND
11
6
10
ND
ND
5
N
ND
93Æ34
ND
1800Æ330
0.41Æ0.03
0.15Æ0.03
Æ0.03
ND
CH
CH
CH
CH
N
0
1.65Æ0.22
0.64Æ0.19
0.49Æ0.06
ND
1ND
2
3
1ND
2
—
ND
1
0.1
85Æ36
ND
1000Æ310
0.05Æ0.02
0.50Æ0.12
Æ0.05
ND
ND
0.23
ND
14600Æ2020
ND
0.96Æ0.07
N
—
ND
739Æ163
0.21Æ0.04
1.90Æ0.17
20.5Æ2.33
11
aPDEs were obtained from bovine heart (PDE1), rabbit platelet (PDE3 and PDE5), and bovine retina (PDE6), respectively, and assayed using [3H]-
cGMP or [3H]-cAMP SPA kit. IC50 values of sildenafil and test compounds were determined from the logarithmic concentration–inhibition curve.
The value is the meanÆSEM from three independent experiments.
bNot determined.

3016
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
inhibitory activity, and was about 38-fold more potent
than sildenafil. The inhibitory activities toward PDE6
have been also determined for some of selected com-
pounds with relatively high PDE5 inhibitory activity,
and their PDE6/PDE5 selectivity was compared with
that of sildenafil. Whereas two compounds, 9g and 9i,
showed comparable selectivity (10–11) to sildenafil (11),
the other four derivatives exhibited somewhat lower
selectivity (3–6-fold) over PDE6. Furthermore, selectiv-
ity over other isozymes (PDE1and PDE3) was obtained
for two analogues, 9g and 9i, which showed high PDE5
inhibitory activity and comparable selectivity over
PDE6. As shown in Table 1, there were small increases
in their selectivity over PDE1(1.6–4.3-fold) and PDE3
(1.5–2.6-fold) compared with those of sildenafil, and
compound 9i exhibited better selectivity than 9g and
sildenafil. In conclusion, we have clearly demonstrated
that the presence of a carboxylic acid group in the 50-
sulfonamide moiety of the phenyl ring indeed enhanced
PDE5 inhibitory activity to the great extent, presumably
by mimicking the phosphate group of cGMP. The
piperidinyl propionic acid derivative 9i, which showed
the highest PDE5 inhibitory activity and comparable to
better selectivity over PDE isozymes in comparison with
sildenafil, has been selected for more detailed biological
investigations.
solidified by dissolving in CH2Cl2 (20 mL) and pouring
CH2Cl2 solution to a large quantity of hexanes (800
mL) to afford the titled compound 7a–b as a white solid,
which was crystallized from a suitable solvent.
4-(5-Chlorosulfonyl-2-ethoxybenzamido)-1-methyl-3-n-
propylpyrazole-5-carboxamide (7a). Yield 82%; mp
158–159 ꢁC (CHCl3/hexanes); IR (neat) 3350, 3190
1
(NH), 1665, 1641 (C¼O), 1176 (SO2) cmꢂ1; H NMR
(CDCl3) d 0.95 (t, J=7.4 Hz, 3H, CH2CH2CH3), 1.60–
1.72 (m, 2H, CH2CH2CH3), 1.62 (t, J=6.9 Hz, 3H,
OCH2CH3), 2.54 (dd, J=7.8 Hz, 7.2 Hz, 2H,
CH2CH2CH3), 4.06 (s, 3H, NCH3), 4.46 (q, J=6.9 Hz,
2H, OCH2CH3), 5.71(br s, H1 , CONH 2), 7.26 (d,
J=9.0 Hz, 1H, H-30), 7.61(br s, 1H, CONH 2), 8.19
(dd, J=9.0 Hz, 2.7 Hz, 1H, H-40), 8.95 (d, J=2.7 Hz,
1H, H-60), 9.19 (br s, 1H, NH); MS (FAB) m/z 429
(MH+).
4-(5-Chlorosulfonyl-2-n-propoxybenzamido)-1-methyl-3-
n-propylpyrazole-5-carboxamide (7b). Yield 83%; mp
140–141 ꢁC (CH2Cl2/hexanes); IR (neat) 3469, 3286
1
(NH), 1683, 1651 (C¼O), 1177 (SO2) cmꢂ1; H NMR
(CDCl3) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.11
(t, J=7.5 Hz, 3H, OCH2CH2CH3), 1.59–1.72 (m, 2H,
CH2CH2CH3), 1.94–2.06 (m, 2H, OCH2CH2CH3), 2.52
(t, J=7.5 Hz, 2H, CH2CH2CH3), 4.06 (s, 3H, NCH3),
4.34 (t, J=6.6 Hz, 2H, OCH2CH2CH3), 5.68 (br s, 1H,
CONH2), 7.27 (d, J=9.0 Hz, 1H, H-30), 7.56 (br s, 1H,
CONH2), 8.19 (dd, J=9.0 Hz, 2.7 Hz, 1H, H-40), 8.96
(d, J=2.7 Hz, 1H, H-60), 9.19 (br s, 1H, NH); MS
(FAB) m/z 443 (MH+).
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
spectrophotometer.1H NMR spectra were recorded on a
Varian Unity 300 spectrometer. The chemical shifts are
reported in parts per million (ppm) relative to internal
tetramethylsilane in CDCl3 or DMSO-d6. Fast-atom
bombardment mass spectra (FAB-MS) 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 chro-
matography (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
sulfonamides, 8a–l: coupling reactions
A mixture of chlorosulfonyl derivative 7a–b (1.51
mmol), an appropriate cyclic amine 3–5 (1.81 mmol),
and triethylamine (0.63 mL, 4.53 mmol) in anhydrous
EtOH (25 mL) was stirred at room temperature under
nitrogen atmosphere for 2 h. The reaction mixture was
evaporated to dryness under reduced pressure, and the
resulting residue was purified by MPLC on silica gel
using MeOH in CHCl3 to afford the titled compound
8a–l, which was crystallized from a suitable solvent.
4-(2-Ethoxy-5-((4-hydroxycarbonyl)piperidinylsulfonyl)-
benzamido)-1-methyl-3-n-propylpyrazole-5-carboxamide
(8a). Yield 97%; mp 247 ꢁC dec (MeOH/CHCl3/hex-
anes); IR (neat) 3345, 3160 (NH, CO2H), 1706, 1656,
General procedures for the preparation of the
chlorosulfonyl derivatives 7a–b: chlorosulfonylation
reactions
1
To a stirred and cooled chlorosulfonic acid (30 mL) in
an ice bath under nitrogen atmosphere was added por-
tionwise carboxamide compound 6a–b (45.40 mmol),
and the reaction mixture was warmed to room tem-
perature gradually for 2 h after the addition. The
resulting mixture was added, carefully and dropwise, to
the cooled mixture of CHCl3 (500 mL) and ice water
(100 g) in an ice bath, and the organic layer was sepa-
rated. The aqueous layer was extracted further with
CHCl3 (2Â100 mL), and the combined extracts were
dried (Na2SO4), filtered, and evaporated to dryness
under reduced pressure to give the desired sulfonyl
chloride as an off-white solid. The crude product was
1640 (C¼O), 1156 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.90 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.42 (t, J=6.9
Hz, 3H, OCH2CH3), 1.51–1.65 (m, 4H, 2CHax and
CH2CH2CH3), 1.84–1.94 (m, 2H, 2 CHeq), 2.17–2.27
(m, 1H, CHCO2), 2.36–2.52 (m, 4H, 2NCHax and
CH2CH2CH3), 3.40–3.52 (m, 2H, 2NCHeq), 3.92 (s, 3H,
NCH3), 4.30 (q, J=6.9 Hz, 2H, OCH2CH3), 7.34 (br s,
1H, CONH2), 7.41(d, J=8.7 Hz, 1H, H-30), 7.81(br s,
1H, CONH2), 7.84 (dd, J=8.7 Hz, 2.4 Hz, 1H, H-40),
7.88 (d, J=2.4 Hz, 1H, H-60), 9.61(br s, 1H, NH); MS
(FAB) m/z 522 (MH+). Anal. calcd for C23H31N5O7S:
C, 52.96; H, 5.99; N, 13.43. Found: C, 53.12; H, 6.08; N,
13.27.

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
3017
4-(2-Ethoxy-5-(4-(ethoxycarbonylmethyl)piperidinylsulfo-
nyl)benzamido)-1-methyl-3-n-propylpyrazole-5-carboxa-
mide (8b). Yield 93%; mp 177–178 ꢁC (EtOAc/
hexanes); IR (neat) 3357, 3180, 3075 (NH), 1733, 1670,
4-(2-Ethoxy-5-(4-(ethoxycarbonylmethyl)piperazinylsul-
fonyl)benzamido)-1-methyl-3-n-propylpyrazole-5-carbox-
amide (8e). Yield 68%; mp 178–178.5 ꢁC (CHCl3/
EtOAc/hexanes); IR (neat) 3359, 3182 (NH), 1747,
1674, 1640 (C¼O), 1169 (SO2) cmꢂ1; 1H NMR (DMSO-
d6) d 0.90 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.16 (t,
J=7.2 Hz, 3H, CO2CH2CH3), 1.42 (t, J=6.9 Hz, 3H,
OCH2CH3), 1.53–1.65 (m, 2H, CH2CH2CH3), 2.46 (t,
J=7.5 Hz, 2H, CH2CH2CH3), 2.60 (br s, 4H, 2 NCH2),
2.88 (br s, 4H, 2 NCH2), 3.24 (s, 2H, NCH2CO2), 3.92
(s, 3H, NCH3), 4.05 (q, J=7.2 Hz, 2H, CO2CH2CH3),
4.31(q, J=6.9 Hz, 2H, OCH2CH3), 7.31(br s, H1 ,
CONH2), 7.43 (d, J=8.7 Hz, 1H, H-30), 7.78–7.90 (m,
3H, H-40, H-60 and CONH2), 9.64 (br s, 1H, NH); MS
(FAB) m/z 565 (MH+). Anal. calcd for C25H36N6O7S:
C, 53.18; H, 6.43; N, 14.88. Found: C, 53.01; H, 6.32; N,
15.03.
1
1640 (C¼O), 1168 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.89 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.15 (t, J=7.2
Hz, 3H, CO2CH2CH3), 1.16–1.29 (m, 2H, 2CHax), 1.42
(t, J=6.9 Hz, 3H, OCH2CH3), 1.53–1.65 (m, 3H, CH
and CH2CH2CH3), 1.71 (br d, J=12.6 Hz, 2H, 2 CHeq),
2.18–2.26 (m, 2H, 2NCHax), 2.21(d, J=6.6 Hz, 2H,
CH2CO2), 2.46 (dd, J=7.8, 7.5 Hz, 2H, CH2CH2CH3),
3.61(br d, J=11.4 Hz, 2H, 2NCHeq), 3.92 (s, 3H,
NCH3), 4.02 (q, J=7.2 Hz, 2H, CO2CH2CH3), 4.30 (q,
J=6.9 Hz, 2H, OCH2CH3), 7.33 (br s, 1H, CONH2),
7.41(d, J=8.7 Hz, 1H, H-30), 7.83 (dd, J=8.7, 2.4
Hz, 1H, H-40), 7.87 (br s, 1H, CONH2), 7.88 (d,
J=2.4 Hz, 1H, H-60), 9.60 (br s, 1H, NH); MS (FAB)
m/z 564 (MH+). Anal. calcd for C26H37N5O7S: C,
55.40; H, 6.62; N, 12.42. Found: C, 55.22; H, 6.69; N,
12.58.
4-(2-Ethoxy-5-(4-(2-ethoxycarbonylethyl)piperazinylsul-
fonyl)benzamido)-1-methyl-3-n-propylpyrazole-5-carbox-
amide (8f). Yield 95%; mp 160.5–161 ꢁC (EtOAc/
hexanes); IR (neat) 3365, 3191 (NH), 1726, 1673, 1643
4-(2-Ethoxy-5-(4-(2-ethoxycarbonylethyl)piperidinylsulfo-
nyl)benzamido)-1-methyl-3-n-propylpyrazole-5-carboxa-
mide (8c). Yield 90%; mp 174–175 ꢁC (EtOAc/
hexanes); IR (neat) 3358, 3202, 3180 (NH), 1730, 1668,
1
(C¼O), 1169 (SO2) cmꢂ1; H NMR (CDCl3) d 0.96 (t,
J=7.2 Hz, 3H, CH2CH2CH3), 1.22 (t, J=7.2 Hz, 3H,
CO2CH2CH3), 1.60 (t, J=7.2 Hz, 3H, OCH2CH3),
1.60–1.72 (m, 2H, CH2CH2CH3), 2.42 (t, J=7.2 Hz,
2H, CH2CH2CH3), 2.52–2.57 (m, 6H, 3 NCH2), 2.69 (t,
J=7.2 Hz, 2H, CH2CO2), 3.05 (br s, 4H, 2 NCH2), 4.07
(s, 3H, NCH3), 4.10 (q, J=7.2 Hz, 2H, CO2CH2CH3),
4.40 (q, J=7.2 Hz, 2H, OCH2CH3), 5.62 (br s, 1H,
CONH2), 7.17 (d, J=9.0 Hz, 1H, H-30), 7.69 (br s, 1H,
CONH2), 7.91(dd, J=9.0, 2.4 Hz, 1H, H-40), 8.63 (d,
J=7.2 Hz, 1H, H-60), 9.28 (br s, 1H, NH); MS (FAB)
m/z 579 (MH+). Anal. calcd for C26H38N6O7S: C,
53.96; H, 6.62; N, 14.52. Found: C, 54.03; H, 6.52; N,
14.39.
1
1640 (C¼O), 1170 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.89 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.09–1.21 (m,
3H, CH and 2CHax), 1.15 (t, J=6.9 Hz, 3H,
CO2CH2CH3), 1.39–1.47 (m, 2H, CHCH2CH2), 1.42 (t,
J=6.9 Hz, 3H, OCH2CH3), 1.52–1.65 (m, 2H,
CH2CH2CH3), 1.70 (br d, J=9.3 Hz, 2H, 2 CHeq), 2.16
(br t, J=10.5 Hz, 2H, 2NCHax), 2.25 (t, J=7.5 Hz, 2H,
CH2CO2), 2.46 (dd, J=8.1, 7.2 Hz, 2H, CH2CH2CH3),
3.61(br d, J=11.1 Hz, 2H, 2NCHeq), 3.92 (s, 3H,
NCH3), 4.01(q, J=6.9 Hz, 2H, CO2CH2CH3), 4.30 (q,
J=6.9 Hz, 2H, OCH2CH3), 7.33 (br s, 1H, CONH2),
7.41(d, J=8.7 Hz, 1H, H-30), 7.84 (dd, J=8.7, 2.4 Hz,
1H, H-40), 7.87 (br s, 1H, CONH2), 7.88 (d, J=2.4
Hz, 1H, H-60), 9.60 (br s, 1H, NH); MS (FAB) m/z
578 (MH+). Anal. calcd for C27H39N5O7S: C, 56.14;
H, 6.80; N, 12.12. Found: C, 56.29; H, 6.91; N,
11.90.
4-(5-(4-(Hydroxycarbonyl)piperidinylsulfonyl)-2-n-pro-
poxybenzamido)-1-methyl-3-n-propylpyrazole-5-carboxa-
mide (8g). Yield 99%; mp 171–172 ꢁC (MeOH/CHCl3);
IR (neat) 3346, 3183, 3075 (NH, CO2H), 1673, 1641
1
(C¼O), 1167 (SO2) cmꢂ1; H NMR (DMSO-d6) d 0.89
(t, J=7.5 Hz, 3H, CH2CH2CH3), 0.99 (t, J=7.5 Hz,
3H, OCH2CH2CH3), 1.51–1.65 (m, 4H, 2CHax and
4-(2-Ethoxy-5-(4-(3-ethoxycarbonylpropyl)piperidinylsul-
fonyl)benzamido)-1-methyl-3-n-propylpyrazole-5-carbox-
amide (8d). Yield 76%; mp 168–168.5 ꢁC (MeOH/
Et2O); IR (neat) 3365, 3179, 3074 (NH), 1733, 1670,
CH2CH2CH3), 1.76–1.85 (m, 4H,
2
CHeq and
OCH2CH2CH3), 2.00–2.02 (m, 1H, CHCO2), 2.41–2.52
(m, 4H, 2NCHax and CH2CH2CH3), 3.38–3.43 (m, 2H,
1
1639 (C¼O), 1167 (SO2) cmꢂ1; H NMR (DMSO-d6) d
2NCHeq), 3.91(s, 3H, NCH ), 4.19 (t, J=6.6 Hz, 2H,
3
0.89 (t, J=7.2 Hz, 3H, CH2CH2CH3), 1.15 (t, J=7.2
Hz, 3H, CO2CH2CH3), 1.06–1.18 (m, 5H, CHCH2CH2
and 2CHax), 1.40–1.51 (m, 2H, CHCH2CH2), 1.42 (t,
J=6.9 Hz, 3H, OCH2CH3), 1.52–1.62 (m, 2H,
CH2CH2CH3), 1.70 (br d, J=9.9 Hz, 2H, 2 CHeq), 2.18
(br t, J=10.8 Hz, 2H, 2NCHax), 2.23 (t, J=7.2 Hz, 2H,
CH2CO2), 2.46 (dd, J=8.1, 7.2 Hz, 2H, CH2CH2CH3),
3.61(br d, J=11.4 Hz, 2H, 2NCHeq), 3.92 (s, 3H,
NCH3), 4.02 (q, J=7.2 Hz, 2H, CO2CH2CH3), 4.30 (q,
J=6.9 Hz, 2H, OCH2CH3), 7.33 (br s, 1H, CONH2),
7.41(d, J=8.7 Hz, 1H, H-30), 7.82 (br s, 1H, CONH2),
7.84 (dd, J=8.7, 2.4 Hz, 1H, H-40), 7.88 (d, J=2.4 Hz,
1H, H-60), 9.60 (br s, 1H, NH); MS (FAB) m/z 592
(MH+). Anal. calcd for C28H41N5O7S: C, 56.83; H,
6.98; N, 11.84. Found: C, 56.99; H, 7.07; N, 11.71.
OCH2CH2CH3), 7.40 (d, J=8.7 Hz, 1H, H-30), 7.44 (br
s, 1H, CONH2), 7.83 (dd, J=8.7, 2.4 Hz, 1H, H-40),
7.81–7.87 (m, 2H, CONH2 and H-60), 9.67 (br s, 1H,
NH); MS (FAB) m/z 536 (MH+). Anal. calcd for
C24H33N5O7S: C, 53.82; H, 6.21; N, 13.08. Found: C,
54.01; H, 6.16; N, 12.96.
4-(5-(4-(Ethoxycarbonylmethyl)piperidinylsulfonyl)-2-n-
propoxybenzamido)-1-methyl-3-n-propylpyrazole-5-car-
boxamide (8h). Yield 95%; mp 171–172 ꢁC (EtOAc/
hexanes); IR (neat) 3348, 3182, 3074 (NH), 1741, 1670,
1
1641 (C¼O), 1168 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.89 (t, J=7.5 Hz, 3H, CH2CH2CH3), 0.99 (t, J=7.5
Hz, 3H, OCH2CH2CH3), 1.15 (t, J=7.2 Hz, 3H,
CO2CH2CH3), 1.17–1.29 (m, 2H, 2CHax), 1.52–1.65 (m,

3018
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
3H, CH and CH2CH2CH3), 1.71 (br d, J=12.3 Hz, 2H,
2 CHeq), 1.78–1.88 (m, 2H, OCH2CH2CH3), 2.17–2.27
(m, 2H, 2NCHax), 2.22 (d, J=6.6 Hz, 2H, CH2CO2),
2.46 (t, J=7.5 Hz, 2H, CH2CH2CH3), 3.61(br d,
J=11.4 Hz, 2H, 2NCHeq), 3.92 (s, 3H, NCH3), 4.02 (q,
J=7.2 Hz, 2H, CO2CH2CH3), 4.20 (t, J=6.6 Hz, 2H,
OCH2CH2CH3), 7.33 (br s, 1H, CONH2), 7.41(d,
J=8.7 Hz, 1H, H-30), 7.83 (dd, J=8.7, 2.4 Hz, 1H, H-
40), 7.86 (br s, 1H, CONH2), 7.87 (d, J=2.4 Hz, 1H, H-
60), 9.57 (br s, 1H, NH); MS (FAB) m/z 578 (MH+).
Anal. calcd for C27H39N5O7S: C, 56.14; H, 6.80; N,
12.12. Found: C, 56.27; H, 6.78; N, 11.99.
3H, OCH2CH2CH3), 1.16 (t, J=7.2 Hz, 3H,
CO2CH2CH3), 1.53–1.65 (m, 2H, CH2CH2CH3), 1.76–
1.88 (m, 2H, OCH2CH2CH3), 2.46 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 2.59 (br s, 4H, 2 NCH2), 2.89 (br s, 4H,
2 NCH2), 3.23 (s, 2H, NCH2CO2), 3.92 (s, 3H, NCH3),
4.05 (q, J=7.2 Hz, 2H, CO2CH2CH3), 4.22 (t, J=6.3
Hz, 2H, OCH2CH2CH3), 7.32 (br s, 1H, CONH2), 7.43
(d, J=9.6 Hz, 1H, H-30), 7.78–7.84 (m, 3H, H-40, H-60
and CONH2), 9.62 (br s, 1H, NH); MS (FAB) m/z 579
(MH+). Anal. calcd for C26H38N6O7S: C, 53.96; H,
6.62; N, 14.52. Found: C, 54.11; H, 6.60; N, 14.39.
4-(5-(4-(2-Ethoxycarbonylethyl)piperazinylsulfonyl)-2-n-
propoxybenzamido)-1-methyl-3-n-propylpyrazole-5-car-
boxamide (8l). Yield 90%; mp 177–177.5 ꢁC (EtOAc/
hexanes); IR (neat) 3422, 3190 (NH), 1723, 1670, 1643
4-(5-(4-(2-Ethoxycarbonylethyl)piperidinylsulfonyl)-2-n-
propoxybenzamido)-1-methyl-3-n-propylpyrazole-5-car-
boxamide (8i). Yield 91%; mp 170–171 ꢁC (EtOAc/hex-
anes); IR (neat) 3349, 3208, 3077 (NH), 1731, 1669,
1
(C¼O), 1170 (SO2) cmꢂ1; H NMR (CDCl3) d 0.95 (t,
1
1640 (C¼O), 1166 (SO2) cmꢂ1; H NMR (DMSO-d6) d
J=7.2 Hz, 3H, CH2CH2CH3), 1.10 (t, J=7.2 Hz, 3H,
OCH2CH2CH3), 1.22 (t, J=7.2 Hz, 3H, CO2CH2CH3),
1.60–1.72 (m, 2H, CH2CH2CH3), 1.91–2.03 (m, 2H,
OCH2CH2CH3), 2.42 (t, J=7.2 Hz, 2H, CH2CH2CH3),
2.52–2.57 (m, 6H, 3 NCH2), 2.69 (t, J=7.2 Hz, 2H,
CH2CO2), 3.05 (br s, 4H, 2 NCH2), 4.07 (s, 3H, NCH3),
4.10 (q, J=7.2 Hz, 2H, CO2CH2CH3), 4.28 (t, J=6.6
Hz, 2H, OCH2CH2CH3), 5.60 (br s, 1H, CONH2), 7.18
(d, J=9.0 Hz, 1H, H-30), 7.64 (br s, 1H, CONH2), 7.91
(dd, J=9.0, 2.4 Hz, 1H, H-40), 8.63 (d, J=7.2 Hz, 1H,
H-60), 9.27 (br s, 1H, NH); MS (FAB) m/z 593 (MH+).
Anal. calcd for C27H40N6O7S: C, 54.71; H, 6.80; N,
14.18. Found: C, 54.55; H, 6.88; N, 14.31.
0.89 (t, J=7.5 Hz, 3H, CH2CH2CH3), 0.97 (t, J=7.5
Hz, 3H, OCH2CH2CH3), 1.15 (t, J=7.2 Hz, 3H,
CO2CH2CH3), 1.10–1.20 (m, 3H, CH and 2CHax), 1.39–
1.47 (m, 2H, CHCH2CH2), 1.52–1.62 (m, 2H,
CH2CH2CH3), 1.65–1.72 (m, 2H, 2 CHeq), 1.75–1.86
(m, 2H, OCH2CH2CH3), 2.17 (br t, J=10.5 Hz, 2H,
2NCHax), 2.26 (t, J=7.5 Hz, 2H, CH2CO2), 2.45 (t,
J=7.5 Hz, 2H, CH2CH2CH3), 3.61(br d, J=11.1 Hz,
2H, 2NCHeq), 3.92 (s, 3H, NCH3), 4.02 (q, J=7.2 Hz,
2H, CO2CH2CH3), 4.19 (t, J=6.3 Hz, 2H,
OCH2CH2CH3), 7.33 (br s, 1H, CONH2), 7.41(d,
J=8.7 Hz, 1H, H-30), 7.81–7.86 (m, 3H, CONH2, H-40
and H-60), 9.57 (br s, 1H, NH); MS (FAB) m/z 592
(MH+). Anal. calcd for C28H41N5O7S: C, 56.83; H,
6.98; N, 11.84. Found: C, 56.66; H, 6.91; N, 11.97.
General procedures for the preparation of the
pyrazolopyrimidinones 9a–l: cyclization reactions
4-(5-(4-(3-Ethoxycarbonylpropyl)piperidinylsulfonyl)-2-n-
propoxybenzamido)-1-methyl-3-n-propylpyrazole-5-car-
boxamide (8j). Yield 75%; mp 153–154.5 ꢁC (MeOH/
Et2O); IR (neat) 3350, 3184, 3074 (NH), 1735, 1668,
A suspension of carboxamide 8a–l (4.79 mmol) in 1N
NaOH aqueous solution (34 mL, 34.00 mmol) and
EtOH (17 mL) was heated at 90 ꢁC under nitrogen
atmosphere for 17 h. The reaction mixture was cooled,
evaporated in vacuo and acidified to about pH 2–3 with
2 N aqueous HCl solution. The resulting solution was
extracted with 10% MeOH in CHCl3 (3Â20 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 an off-white
solid. The crude product was purified by MPLC on
silica gel using MeOH in CHCl3 to afford the titled
compound as a white solid, which was crystallized from
a suitable solvent.
1
1640 (C¼O), 1167 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.89 (t, J=7.2 Hz, 3H, CH2CH2CH3), 0.99 (t, J=7.2
Hz, 3H, OCH2CH2CH3), 1.12–1.21 (m, 5H,
CHCH2CH2 and 2CHax), 1.15 (t, J=7.2 Hz, 3H,
CO2CH2CH3), 1.43–1.52 (m, 2H, CHCH2CH2), 1.52–
1.65 (m, 2H, CH2CH2CH3), 1.70 (br d, J=10.2 Hz, 2H,
2 CHeq), 1.76–1.88 (m, 2H, OCH2CH2CH3), 2.19 (br t,
J=11.1 Hz, 2H, 2NCHax), 2.23 (t, J=7.2 Hz, 2H,
CH2CO2), 2.45 (dd, J=7.8, 7.2 Hz, 2H, CH2CH2CH3),
3.61(br d, J=11.4 Hz, 2H, 2NCHeq), 3.92 (s, 3H,
NCH3), 4.02 (q, J=7.2 Hz, 2H, CO2CH2CH3), 4.20 (t,
J=6.6 Hz, 2H, OCH2CH2CH3), 7.33 (br s, 1H,
CONH2), 7.42 (d, J=8.7 Hz, 1H, H-30), 7.80 (br s, 1H,
CONH2), 7.83 (dd, J=8.7, 2.7 Hz, 1H, H-40), 7.87 (d,
J=2.7 Hz, 1H, H-60), 9.57 (br s, 1H, NH); MS (FAB)
m/z 606 (MH+). Anal. calcd for C29H43N5O7S: C,
57.50; H, 7.16; N, 11.56. Found: C, 57.63; H, 7.10; N,
11.45.
5-(2-Ethoxy-5-(4-(hydroxycarbonyl)piperidinylsulfonyl)-
phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (9a). Yield 68%; mp 203–204.5 ꢁC
(MeOH/CHCl3/hexanes); IR (neat) 3294, 3101 (NH,
1
CO2H), 1706, 1684 (C¼O), 1164 (SO2) cmꢂ1; H NMR
(DMSO-d6) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.33 (t, J=6.9 Hz, 3H, OCH2CH3), 1.53–1.63 (m, 2H,
2CHax), 1.64–1.80 (m, 2H, CH2CH2CH3), 1.81–1.89 (m,
2H, 2 CHeq), 2.16–2.23 (m, 1H, CHCO2), 2.47 (br t,
J=9.0 Hz, 2H, 2NCHax), 2.78 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 3.43 (br d, J=11.7 Hz, 2H, 2NCHeq),
4.16 (s, 3H, NCH3), 4.22 (q, J=6.9 Hz, 2H,
OCH2CH3), 7.36 (d, J=8.7 Hz, 1H, H-30), 7.83 (d0d,
J=8.7, 2.4 Hz, 1H, H-40), 7.87 (d, J=2.1Hz, 1H, H-6 ),
4-(5-(4-(Ethoxycarbonylmethyl)piperazinylsulfonyl)-2-n-
propoxybenzamido)-1-methyl-3-n-propylpyrazole-5-car-
boxamide (8k). Yield 61%; mp 130 ꢁC dec (EtOAc/hex-
anes); IR (neat) 3345, 3186 (NH), 1739, 1671, 1642
1
(C¼O), 1171 (SO2) cmꢂ1; H NMR (DMSO-d6) d 0.89
(t, J=7.5 Hz, 3H, CH2CH2CH3), 0.99 (t, J=7.5 Hz,

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
3019
12.20 (br s, 1H, NH); MS (FAB) m/z 504 (MH+). Anal.
calcd for C23H29N5O6S: C, 54.86; H, 5.80; N, 13.91.
Found: C, 54.98; H, 5.72; N, 13.77.
dec (CHCl3/Et2O); IR (neat) 3311 (NH, CO2H), 1735,
1
1701 (C¼O), 1169 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3), 1.42 (t, J=6.9
Hz, 3H, OCH2CH3), 1.69–1.81 (m, 2H, CH2CH2CH3),
2.78 (t, J=7.5 Hz, 2H, CH2CH2CH3), 3.20–3.60 (m,
10H, 5 NCH2), 4.17 (s, 3H, NCH3), 4.23 (q, J=6.9 Hz,
2H, OCH2CH3), 7.43 (d, J=9.0 Hz, 1H, H-30), 7.86–
7.92 (m, 2H, H-40 and H-60), 12.30 (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, 53.12; H, 5.90; N,
16.38.
5-(2-Ethoxy-5-(4-(hydroxycarbonylmethyl)piperidinylsul-
fony)phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (9b). Yield 84%; mp 219–
220 ꢁC (CHCl3/Et2O); IR (neat) 3306, 3123 (NH,
1
CO2H), 1729, 1674 (C¼O), 1163 (SO2) cmꢂ1; H NMR
(DMSO-d6) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.14–1.27 (m, 2H, 2CHax), 1.33 (t, J=6.9 Hz, 3H,
OCH2CH3), 1.51–1.65 (m, 1H, CH), 1.68–1.80 (m, 4H,
2 CHeq and CH2CH2CH3), 2.12 (d, J=6.9 Hz, 2H,
CH2CO2), 2.26 (br t, J=12.0 Hz, 2H, 2NCHax), 2.78 (t,
J=7.5 Hz, 2H, CH2CH2CH3), 3.61(br d, J=12.0 Hz,
2H, 2NCHeq), 4.16 (s, 3H, NCH3), 4.21(q, J=6.9 Hz,
2H, OCH2CH3), 7.36 (d, J=8.7 Hz, 1H, H-30), 7.82 (dd,
J=8.7, 2.4 Hz, 1H, H-40), 7.86 (d, J=2.4 Hz, 1H, H-60),
12.18 (br s, 1H, NH); MS (FAB) m/z 518 (MH+). Anal.
calcd for C24H31N5O6S: C, 55.69; H, 6.04; N, 13.53.
Found: C, 55.83; H, 5.96; N, 13.36.
5-(2-Ethoxy-5-(4-(2-hydroxycarbonylethyl)piperazinylsul-
fonyl)phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (9f). Yield: 74%; mp 236 ꢁC
dec (CHCl3/MeOH/hexanes); IR (neat) 3318, 3068
1
(NH, CO2H), 1730, 1693 (C¼O), 1161 (SO2) cmꢂ1; H
NMR (DMSO-d6)
d
0.95 (t, J=7.5 Hz, 3H,
CH2CH2CH3), 1.34 (t, J=6.9 Hz, 3H, OCH2CH3),
1.69–1.81 (m, 2H, CH2CH2CH3), 2.75–2.81(m, 4H,
CH2CH2CH3 and CH2CO2), 3.09–3.90 (m, 10H,
5 NCH2), 4.17 (s, 3H, NCH3), 4.22 (q, J=6.9 Hz, 2H,
OCH2CH3), 7.42 (d, J=8.7 Hz, 1H, H-30), 7.88–7.93
(m, 2H, H-40 and H-60), 12.28 (br s, 1H, NH); MS
(FAB) m/z 533 (MH+). Anal. calcd for C24H32N6O6S:
C, 54.12; H, 6.06; N, 15.78. Found: C, 54.31; H, 6.00; N,
15.55.
5-(2-Ethoxy-5-(4-(2-hydroxycarbonylethyl)piperidinylsul-
fonyl)phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (9c). Yield 93%; mp 208–
209 ꢁC (CHCl3/Et2O); IR (neat) 3294, 3103 (NH,
1
CO2H), 1706, 1689 (C¼O), 1164 (SO2) cmꢂ1; H NMR
(DMSO-d6) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),
1.05–1.25 (m, 3H, CH and 2CHax), 1.33 (t, J=6.9 Hz,
3H, OCH2CH3), 1.36–1.47 (m, 2H, CHCH2CH2), 1.62–
1.81 (m, 4H, 2 CHeq and CH2CH2CH3), 2.12–2.29 (m,
4H, 2NCHax and CH2CO2), 2.78 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 3.61(br d, J=11.1 Hz, 2H, 2NCHeq),
4.16 (s, 3H, NCH3), 4.20 (q, J=6.9 Hz, 2H,
OCH2CH3), 7.36 (d, J=8.7 Hz, 1H, H-30), 7.82 (dd,
J=8.7 Hz, 2.4 Hz, 1H, H-40), 7.85 (d, J=2.4 Hz, 1H, H-
60), 11.97 (br s, 1H, CO2H), 12.19 (br s, 1H, NH); MS
(FAB) m/z 532 (MH+). Anal. calcd for C25H33N5O6S:
C, 56.48; H, 6.26; N, 13.17. Found: C, 56.63; H, 6.24; N,
13.04.
5-(5-(4-(Hydroxycarbonyl)piperidinylsulfonyl)-2-n-propoxy-
pheny)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-
d]pyrimidin-7-one (9g). Yield 91%; mp 222–223 ꢁC
(EtOAc/hexanes); IR (neat) 3312 (CO2H), 1707 (C¼O),
1162 (SO2) cmꢂ1; 1H NMR (DMSO-d6) d 0.94 (t, J=7.5
Hz, 3H, CH2CH2CH3), 0.95 (t, J=7.5 Hz, 3H,
OCH2CH2CH3), 1.53–1.67 (m, 2H, 2CHax), 1.69–1.80
(m, 4H, 2 CH2CH2CH3), 1.85–1.95 (m, 2H, 2 CHeq),
2.19–2.29 (m, 1H, CHCO2), 2.24–2.45 (m, 2H,
2NCHax), 2.78 (t, J=7.5 Hz, 2H, CH2CH2CH3), 3.46
(br d, J=11.7 Hz, 2H, 2NCHeq), 4.11 (t, J=6.3 Hz, 2H,
OCH2CH2CH3), 4.16 (s, 3H, NCH3), 7.37 (d, J=9.0
Hz, 1H, H-30), 7.83 (dd, J=9.0, 2.1Hz, 1H, H-4 0), 7.87
(d, J=2.1Hz, 1H, H-6 0), 12.18 (br s, 1H, NH); MS
(FAB) m/z 518 (MH+). Anal. calcd for C24H31N5O6S:
C, 55.69; H, 6.04; N, 13.53. Found: C, 55.88; H, 5.98; N,
13.33.
5-(2-Ethoxy-5-(4-(3-hydroxycarbonylpropyl)piperidinyl-
sulfonyl)phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-py-
razolo[4,3-d]pyrimidin-7-one (9d). Yield 91%; mp
215.5–216.5 ꢁC (MeOH/CHCl3/Et2O); IR (neat) 3293
1
(NH, CO2H), 1705 (C¼O), 1164 (SO2) cmꢂ1; H NMR
(DMSO-d6) d 0.94 (t, J=7.2 Hz, 3H, CH2CH2CH3),
1.06–1.20 (m, 5H, CHCH2CH2 and 2CHax), 1.33 (t,
J=7.2 Hz, 3H, OCH2CH3), 1.40–1.50 (m, 2H,
5-(5-(4-(Hydroxycarbonylmethyl)piperidinylsulfonyl)-2-n-
propoxypheny)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (9h). Yield 66%; mp 179–
180 ꢁC (EtOAc/hexanes); IR (neat) 3286, 3079 (NH,
CHCH2CH2), 1.68–1.80 (m, 4H,
2
CHeq and
1
CH2CH2CH3), 2.14 (t, J=7.2 Hz, 2H, CH2CO2), 2.22
(br t, J=11.1 Hz, 2H, 2NCHax), 2.78 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 3.62 (br d, J=11.1 Hz, 2H, 2NCHeq),
CO2H), 1729, 1705 (C¼O), 1167 (SO2) cmꢂ1; H NMR
(DMSO-d6) d 0.93 (t, J=7.2 Hz, 3H, CH2CH2CH3),
0.94 (t, J=7.2 Hz, 3H, OCH2CH2CH3), 1.15–1.27 (m,
2H, 2CHax), 1.53–1.65 (m, 1H, CH), 1.66–1.80 (m, 6H,
2 CHeq and 2 CH2CH2CH3), 2.11 (d, J=6.9 Hz, 2H,
CH2CO2), 2.27–2.31(m, 2H, 2NCH ax), 2.77 (t, J=7.5
Hz, 2H, CH2CH2CH3), 3.61(br d, J=11.4 Hz, 2H,
2NCHeq), 4.11 (t, J=6.3 Hz, 2H, OCH2CH2CH3), 4.16
(s, 3H, NCH3), 7.37 (d, J=8.7 Hz, 1H, H-30), 7.82 (dd,
J=8.7, 2.4 Hz, 1H, H-40), 7.86 (d, J=2.4 Hz, 1H, H-60),
12.18 (br s, 1H, NH); MS (FAB) m/z 532 (MH+). Anal.
calcd for C25H33N5O6S: C, 56.48; H, 6.26; N, 13.17.
Found: C, 56.62; H, 6.00; N, 13.06.
4.16 (s, 3H, NCH3), 4.21(q,
J=7.2 Hz, 2H,
OCH2CH3), 7.36 (d, J=8.7 Hz, 1H, H-30), 7.82 (dd,
J=8.7, 2.4 Hz, 1H, H-40), 7.86 (d, J=2.4 Hz, 1H, H-60),
12.12 (br s, 1H, NH); MS (FAB) m/z 546 (MH+). Anal.
calcd for C26H35N5O6S: C, 57.23; H, 6.47; N, 12.83.
Found: C, 57.11; H, 6.50; N, 12.97.
5-(2-Ethoxy-5-(4-(hydroxycarbonylmethyl)piperazinylsul-
fonyl)phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyra-
zolo[4,3-d]pyrimidin-7-one (9e). Yield 83%; mp 212 ꢁC

3020
D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
5-(5-(4-(2-Hydroxycarbonylethyl)piperidinylsulfonyl)-2-n-
propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyr-
azolo[4,3-d]pyrimidin-7-one (9i). Yield 87%; mp 181–
182 ꢁC (EtOAc/hexanes); IR (neat) 3314, 3052 (NH,
(s, 3H, NCH3), 7.42 (d, J=8.7 Hz, 1H, H-30), 7.87–7.93
(m, 2H, H-40 and H-60), 12.26 (br s, 1H, NH); MS
(FAB) m/z 547 (MH+). Anal. calcd for C25H34N6O6S:
C, 54.93; H, 6.27; N, 15.37. Found: C, 55.02; H, 6.42; N,
15.21.
CO2H), 1702 (C¼O), 1163 (SO2) cmꢂ1
;
1H NMR
(DMSO-d6) d 0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3),
0.95 (t, J=7.5 Hz, 3H, OCH2CH2CH3), 1.10–1.25 (m,
3H, CH and 2CHax), 1.36–1.45 (m, 2H, CHCH2CH2),
1.68–1.78 (m, 6H, 2 CHeq and 2 CH2CH2CH3), 2.12–
2.25 (m, 4H, 2NCHax and CH2CO2), 2.77 (t, J=7.5 Hz,
Determination of PDE1, PDE3, PDE5 and PDE6
inhibitory activity
Bovine heart PDE1was purchased from Sigma (St.
Louis, MO, USA). PDE3 and PDE5 were prepared
from the rabbit platelet using the method described by
Hidaka et al.10 with minor modifications. Fresh rabbit
whole blood was centrifuged at 360g to obtain the pla-
telet-rich plasma (PRP). Platelets were isolated from
PRP by centrifugation at 1200g, sonicated (20 s per mL)
in 50 mM Tris–HCl buffer (pH 7.4) containing 1mM
MgCl2, and then centrifuged at 40,000g for 2 h at 4 ꢁC.
The supernatant was loaded on the DEAE-cellulose
column with a bed volume of 35 mL (Sigma Co.) 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, PDE3 and PDE5 were eluted using a con-
tinuous 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, 1mM EDTA, 1mM phenylmethyl sul-
fonylfluoride (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 40,000g for 60 min at 4 ꢁC. The super-
natant 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, Swe-
den) pre-equilibrated with 20 mM HEPES buffer (pH
7.2) containing 1mM 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 to 600 mM NaCl in equilibration buffer with a total
volume of 60 mL.
2H, CH2CH2CH3), 3.61(br d,
J=11.1 Hz, 2H,
2NCHeq), 4.11 (t, J=6.3 Hz, 2H, OCH2CH2CH3), 4.16
(s, 3H, NCH3), 7.36 (d, J=8.7 Hz, 1H, H-30), 7.82 (dd,
J=8.7, 2.4 Hz, 1H, H-40), 7.86 (d, J=2.4 Hz, 1H, H-60),
12.10 (br s, 1H, NH); MS (FAB) m/z 546 (MH+). Anal.
calcd for C26H35N5O6S: C, 57.23; H, 6.47; N, 12.83.
Found: C, 57.03; H, 6.55; N, 13.01.
5-(5-(4-(3-Hydroxycarbonylpropyl)piperidinylsulfonyl)-2-
n-propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-
pyrazolo[4,3-d]pyrimidin-7-one (9j). Yield 71%; mp
183.5–184.5 ꢁC (MeOH/CHCl3/Et2O); IR (neat) 3290
(NH, CO2H), 1732, 1706 (C¼O), 1165 (SO2) cmꢂ1; H
1
NMR (DMSO-d6)
d
0.94 (t, J=7.2 Hz, 3H,
CH2CH2CH3), 0.95 (t, J=7.2 Hz, 3H, OCH2CH2CH3),
1.07–1.20 (m, 5H, CHCH2CH2 and 2CHax), 1.41–1.52
(m, 2H, CHCH2CH2), 1.68–1.78 (m, 6H, 2 CHeq and 2
CH2CH2CH3), 2.15 (t, J=7.5 Hz, 2H, CH2CO2), 2.22
(br t, J=10.5 Hz, 2H, 2NCHax), 2.77 (t, J=7.5 Hz, 2H,
CH2CH2CH3), 3.62 (br d, J=11.1 Hz, 2H, 2NCHeq),
4.11 (t, J=6.3 Hz, 2H, OCH2CH2CH3), 4.16 (s, 3H,
NCH3), 7.37 (d, J=9.0 Hz, 1H, H-30), 7.82 (dd, J=9.0,
2.4 Hz, 1H, H-40), 7.86 (d, J=2.4 Hz, 1H, H-60), 11.92
(br s, 1H, CO2H), 12.16 (br s, 1H, NH); MS (FAB) m/z
560 (MH+). Anal. calcd for C27H37N5O6S: C, 57.94; H,
6.66; N, 12.51. Found: C, 58.11; H, 6.58; N, 12.34.
5-(5-(4-(Hydroxycarbonylmethyl)piperazinylsulfonyl)-2-n-
propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyr-
azolo[4,3-d]pyrimidin-7-one (9k). Yield 87%; mp 189 ꢁC
dec (CHCl3/Et2O); IR (neat) 3317 (NH, CO2H), 1733,
1
1701 (C¼O), 1169 (SO2) cmꢂ1; H NMR (DMSO-d6) d
0.94 (t, J=7.5 Hz, 3H, CH2CH2CH3), 0.95 (t, J=7.5
Hz, 3H, OCH2CH2CH3), 1.65–1.82 (m, 4H,
2
CH2CH2CH3), 2.78 (t, J=7.5 Hz, 2H, CH2CH2CH3),
3.18–3.75 (m, 10H, 5NCH2), 4.13 (t, J=6.6 Hz, 2H,
OCH2CH2CH3), 4.17 (s, 3H, NCH3), 7.44 (d, J=8.7
Hz, 1H, H-30), 7.88 (dd, J=8.7 Hz, 2.4 Hz, 1H, H-40),
7.91(d, J=2.4 Hz, 1H, H-60), 12.26 (br s, 1H, NH); MS
(FAB) m/z 533 (MH+). Anal. calcd for C24H32N6O6S:
C, 54.12; H, 6.06; N, 15.78. Found: C, 54.30; H, 5.85; N,
15.61.
Fractions (1.0 mL each) collected at a flow rate of 60
mL/h were characterized for cGMP (PDE5 and PDE6)
or cAMP (PDE3) hydrolytic PDE activities as described
below. Fractions comprising the main peaks of cGMP
hydrolytic PDE activity were pooled and stored at
ꢂ20 ꢁC in 50% glycerol until the enzyme assay. Enzy-
matic activity was determined using a PDE scintillation
proximity assay (SPA) kit (Amersham Pharmacia Bio-
tech, Buckinghamshire, UK) according to the protocol
supplied by the manufacturer. The reaction buffer con-
tained [3H]-cGMP (5 mCi/mL) or [3H]-cAMP (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 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
5-(5-(4-(2-Hydroxycarbonylethyl)piperazinylsulfonyl)-2-n-
propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyr-
azolo[4,3-d]pyrimidin-7-one (9l). Yield 79%; mp 220 ꢁC
dec (CHCl3/MeOH/hexanes); IR (neat) 3315, 3061
1
(NH, CO2H), 1728, 1693 (C¼O), 1161 (SO2) cmꢂ1; H
NMR (DMSO-d6)
d
0.94 (t, J=7.5 Hz, 3H,
CH2CH2CH3), 0.95 (t, J=7.2 Hz, 3H, OCH2CH2CH3),
1.66–1.80 (m, 4H, 2 CH2CH2CH3), 2.70–2.82 (m, 4H,
CH2CH2CH3 and CH2CO2), 3.10–3.90 (m, 10H,
5 NCH2), 4.13 (t, J=6.3 Hz, 2H, OCH2CH2CH3), 4.17

D.-K. Kim et al. / Bioorg. Med. Chem. 9 (2001) 3013–3021
3021
sample was settled for 20 min. For the inhibitor studies,
sildenafil 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 or cAMP 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% inhibi-
tion of enzyme activity and calculated by quantal probit
analysis in Pharmacological Calculation System.12
1997, 11, 140. (c) Truss, M. C.; Stief, C. G. Drugs Today 1998,
34, 805.
5. Beavo, J. A. Physiol. Rev. 1995, 75, 725.
6. Scrip 1997, October 14, 24–25.
7. (a) Kim, D.-K.; Lee, N.; Lee, J. Y.; Ryu, D. H.; Kim, J.-S.;
Lee, S. H.; Choi, J.-Y.; Chang, K.; Kim, Y.-W.; Im, G.-J.;
Choi, W.-S.; Kim, T.-K.; Ryu, J.-H.; Kim, N.-H.; Lee, K.
Bioorg. Med. Chem. 2001, 9, 1609. (b) Kim, D.-K.; Ryu, D.
H.; Lee, N.; Lee, J. Y.; Kim, J.-S.; Lee, S. H.; Choi, J.-Y.;
Chang, K.; Kim, Y.-W.; Im, G.-J.; Choi, W.-S.; Kim, T.-K.;
Ryu, J.-H.; Kim, N.-H.; Lee, K. Bioorg. Med. Chem. 2001, 9,
1895.
8. (a) Brundish, D.; Bull, A.; Donovan, V.; Fullerton, J. D.;
Garman, S. M.; Hayler, J. F.; Janus, D.; Kane, P. D.;
McDonnell, M.; Smith, G. P.; Wakeford, R.; Walker, C. V.;
Howarth, G.; Hoyle, W.; Allen, M. C.; Ambler, J.; Butler, K.;
Talbot, M. D. J. Med. Chem. 1999, 42, 4584. (b) Fukumi, H.;
Sakamoto, T.; Sugiyama, M.; Yamaguchi, T.; Lisuka, Y. EP
470686, 1991. (c) Pieper, H.; Austel, V.; Himmelsbach, F.;
Linz, G.; Guth, B; Weisenberger, J. WO 9620173, 1996.
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