Synthesis and evaluation of polycyclic pyrazolo[3,4-d]pyrimidines as PDE1 and PDE5 cGMP phosphodiesterase inhibitors

Article abstract of DOI:10.1021/jm970495b

Polycyclic pyrazolo[3,4-d]pyrimidines (represented by 3 and 4) were synthesized as analogues of the recently reported polycyclic guanine phosphodiesterase (PDE) inhibitors. From the structure-activity relationship (SAR) development of a series of compounds, it was discovered that C-3 benzyl and N-2 methyl disubstitution on the pyrazole ring gave the best combination of potency and selectivity for PDE1 and PDE5 cGMP PDEs as represented by compound 4c: PDE1, IC₅₀ = 60 nM; PDE3, IC₅₀ = 55 000 nM; PDE5, IC₅₀ = 75 nM. These compounds were also evaluated in vivo and found to be good orally active antihypertensives in laboratory animal models. Finally, comparisons were made of the in vitro and in vivo profiles of the pyrazolo- [3,4-d]pyrimidine compound 4c with those of two representative guanine compounds.

Full text of DOI:10.1021/jm970495b

4372
J . Med. Chem. 1997, 40, 4372-4377
Syn th esis a n d Eva lu a tion of P olycyclic P yr a zolo[3,4-d ]p yr im id in es a s P DE1 a n d
P DE5 cGMP P h osp h od iester a se In h ibitor s
Yan Xia,* Samuel Chackalamannil, Michael Czarniecki, Hsingan Tsai, Henry Vaccaro, Renee Cleven,
J ohn Cook, Ahmad Fawzi, Robert Watkins, and Hongtao Zhang
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New J ersey 07033
Received J uly 29, 1997X
Polycyclic pyrazolo[3,4-d]pyrimidines (represented by 3 and 4) were synthesized as analogues
of the recently reported polycyclic guanine phosphodiesterase (PDE) inhibitors. From the
structure-activity relationship (SAR) development of a series of compounds, it was discovered
that C-3 benzyl and N-2 methyl disubstitution on the pyrazole ring gave the best combination
of potency and selectivity for PDE1 and PDE5 cGMP PDEs as represented by compound 4c:
PDE1, IC50 ) 60 nM; PDE3, IC50 ) 55 000 nM; PDE5, IC50 ) 75 nM. These compounds were
also evaluated in vivo and found to be good orally active antihypertensives in laboratory animal
models. Finally, comparisons were made of the in vitro and in vivo profiles of the pyrazolo-
[3,4-d]pyrimidine compound 4c with those of two representative guanine compounds.
Ch a r t 1. Polycyclic Guanine PDE Inhibitors
In tr od u ction
The emerging field of cyclic nucleotide level regulation
by the isozyme family of phosphodiesterases (PDE) has
brought renewed interest in PDE inhibition as a target
for therapeutic intervention.1 Recently the role of cGMP
levels and the potential beneficial effects of inhibition
of the cGMP PDE for the treatment of cardiovascular
diseases have been reviewed.2 The considerable interest
in this area has been the focus of a large number of
programs directed at the identification of new chemical
classes of inhibitors of cGMP PDE.3 We have recently
disclosed our discovery of a new class of polycyclic
guanine PDE inhibitors which are illustrated in Chart
1.4 These compounds are potent inhibitors of PDE1 and
PDE5 cGMP PDEs in vitro and potent oral antihyper-
tensives in vivo. The desirable properties of the poly-
cyclic guanine compounds prompted us to investigate
analogues of these guanines to discover other related
classes of PDE inhibitors. In particular, we were
interested in pyrazolo[3,4-d]pyrimidine analogues (such
as 3 and 4 in Chart 2) where an isomeric pyrazole ring
replaces the imidazole ring in the polycyclic guanine
structure.5 Prior to our work, there was a brief patent
description of 5-hexyl-7-methylpyrazolo[3,4-d]pyrimi-
dine-4,6-dione as a PDE inhibitor.6 Since the initiation
of our work, there have been several recent reports of
pyrazolo[3,4-d]pyrimidines and pyrazolo[4,3-d]pyrim-
idines as potent and selective PDE5 inhibitors.7
Ch a r t 2. Pyrazolo[3,4-d]pyrimidine Analogues
Sch em e 1a
Here we describe the synthesis of polycyclic pyrazolo-
[3,4-d]pyrimidines and their in vitro and in vivo evalu-
ations as PDE1 and PDE5 cGMP PDE inhibitors. From
the structure-activity relationship (SAR) development
of this series of compounds, a new class of potent PDE1
and PDE5 cGMP PDE inhibitors with oral antihyper-
tensive activity was discovered.
a
Reagents: (i) CH3C(OEt)3, Ac2O, reflux, 23%; (ii) BnCOCl,
NaH, THF; (iii) Ag2CO3, EtI, 41% (from ethyl cyanoacetate); (iv)
POCl3, n-Bu3N, 40%.
gave 5b.8 Condensation of ethyl cyanoacetate with
phenylacetyl chloride gave an enol intermediate (5c)
which was O-alkylated to give 5d or converted to the
chloro intermediate 5e by treatment with POCl3 and
tributylamine.9
Syn th esis
The syntheses of the polycyclic pyrazolo[3,4-d]pyri-
midine compounds are outlined in Schemes 1-3. Scheme
1 shows the preparation of starting materials 5b-e.
Condensation of ethyl cyanoacetate with orthoacetate
The pyrazole compounds of type 3 were prepared as
outlined in Scheme 2. Intermediates 7b-e were pre-
pared based on a literature synthesis of pyrazolo[3,4-
d]pyrimidine 7a from 5a via the pyrazole intermediate
6a .10 Condensation of 5a ,b,d with methylhydrazine or
benzylhydrazine gave pyrazoles 6b-e. Treatment with
X Abstract published in Advance ACS Abstracts, December 1, 1997.
S0022-2623(97)00495-0 CCC: $14.00 © 1997 American Chemical Society

PDE1 and PDE5 cGMP Phosphodiesterase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4373
Sch em e 2a
a
Reagents: (i) CH3NHNH2 or PhCH2NHNH2, MeOH, reflux, 84%; (ii) MeNCO, NEt3, PhH, 100 °C; NaOMe, MeOH, reflux, 37%; (iii)
POCl3, heat, 67%; (iv) trans-2-aminocyclopentanol, DMF, heat, 94%; (v) SOCl2, CH2Cl2, 49%; (vi) Pd(OH)2/C, H2, MeOH, 30%.
Sch em e 3a
a
Reagents: (i) benzaldehyde methylhydrazone, PhH; HCl, EtOH, reflux, 73%; (ii) MeNCO, NEt3, PhH, 100 °C; NaOMe, MeOH, reflux;
(iii) same as steps iii-v in Scheme 2.
methyl isocyanate followed by base-catalyzed cyclization
formed 7b-e. Intermediates 7b-e were then elabo-
rated to pyrazole compounds 3a -e according to the
procedures developed for the polycyclic guanine com-
pounds.4
of the pyrazole ring N-methyl proton resonance (δ 3.88)
of 3e gave an enhancement of the methine proton signal
(δ 4.83) at the C,D-ring junction but not of the benzylic
protons (δ 4.15). For compound 4a , irradiation of the
pyrazole ring N-methyl proton resonance (δ 3.61) gave
an enhancement of the benzylic proton signal (δ 4.28)
but not of the methine proton (δ 4.65-4.72) at the C,D-
ring junction.
The pyrazole compounds of type 4 were prepared as
shown in Scheme 3. It has been reported that conden-
sation of 5a with benzaldehyde methylhydrazone fol-
lowed by acid hydrolysis gave 10a .10 Our attempt to
apply the same reactions using the benzyl-substituted
intermediate 5d did not give the desired pyrazole
intermediate 10 in good yield. However, a more reactive
chloro intermediate (5e) gave 10 in 73% yield under
these conditions. Intermediate 10 was converted to the
pyrazole compound 4a according to the aforementioned
synthetic sequence (Scheme 2: 6 f 3). Use of other
amino alcohols to replace trans-2-aminocyclopentanol
in the sequence gave other polycyclic pyrazole com-
pounds (4c-e) (Table 1). Pyrazole compound 4f was
prepared similarly from the known intermediate 11a .11
Use of (-)-trans-2-aminocyclopentanol12 in Schemes 2
and 3 gave the optically active pyrazole compounds 3b,c,
and 4b,f.
Resu lts a n d Discu ssion
The SAR development of our polycyclic pyrazolo[3,4-
d]pyrimidine compounds was directed mostly toward
substitutions on the pyrazole ring. The choice of
substitutions was influenced by the SAR developed for
the polycyclic guanine compounds.4 In the evaluation
of selective cGMP PDE inhibitors, we were looking for
potent inhibition against the cGMP PDEs (PDE1 and
PDE5) and selectivity over the undesired cAMP PDE
(PDE3). Table 1 lists the in vitro PDE inhibition
results. Similar to the polycyclic guanine compounds,
all of the pyrazole compounds are either inactive or
weak inhibitors against PDE3, displaying IC50 values
from 29 µM to greater than 100 µM. Mono N-1 benzyl-
substituted (3a ) and mono C-3 methyl-substituted (3c)
compounds gave weak inhibition of PDE1 and PDE5.
However, N-1 benzyl- and C-3 methyl-disubstituted
The substitution pattern on the pyrazole ring of
representative pyrazole compounds 3e and 4a was
confirmed by NOE experiments (Chart 3).13 Irradiation

4374 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Xia et al.
Ta ble 1. In Vitro PDE Inhibition and in Vivo Antihypertensive Activities of Polycyclic Pyrazolo[3,4-d]pyrimidinesa
IC50 (nM)
compd
R
R′
PDE1
PDE3
PDE5
∆ mmHg @ mg/kg
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
H
Me
Me
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
H
H
Me
29000
2000
18000
1100
850
160
55
60
18
20
540
55000
50000
>100000
>100000
29000
3100
1000
2900
190
400
140
100
75
-14 @25
-3 @10
-7 @10
-16 @25
not tested
-18 @10
-26 @10
-33 @10
not tested
not tested
1 @ 10
Me
Me
85000
80000
55000
42000
90000
55000
140
100
2100
phenyl
benzyl
a
Compounds 3a ,d ,e and 4a are racemic mixtures, compounds 3b,c and 4b,f are 6aR,9aS-enantiomers, and compound 4e is the 7R-
enantiomer.
Ta ble 2. PDE1, PDE2, PDE3, PDE4, and PDE5 Inhibition of
Compound 4c and Its Comparison with Polycyclic Guanine
PDE Inhibitorsa
Ch a r t 3. Confirmation of Structures by NOE
Experiments
IC50 (nM)
∆ mmHg
PDE5 @ 10 mg/kg
compd PDE1
PDE2
PDE3
PDE4
1b
2b
4c
205 not tested not tested not tested 225
-28
-47
-33
100 1500
60 11000
80000
55000
8100
230
80
75
a
The polycyclic guanine compounds 1 and 2 are 6aR,9aS-
enantiomers.4 From ref 4.
b
compound 3b showed greater PDE1 inhibition. Chang-
ing the C-3 substituent from methyl to benzyl produced
greater inhibition of PDE1 and PDE5 as exemplified by
compound 3d . An additional methyl substitution at the
N-2 position (4a ,b) gave the best dual inhibition against
PDE1 and PDE5 among the different pyrazole substitu-
tions examined (e.g., 4a vs 3e). A comparison of the
racemic compound 4a and the 6aR,9aS-isomer 4b
reveals the absolute stereochemical preference for the
6aR,9aS-enantiomer. This absolute stereochemical pref-
erence is consistent with our earlier findings in the
polycyclic guanine series that the 6aR,9aS-enantiomer
is inherently more potent against PDE1 and PDE5.4 The
C-3 phenyl- and N-2 benzyl-disubstituted compound 4f
is a mediocre PDE1 and PDE5 inhibitor. Compounds
4c-e represent different substitution patterns of the
dihydroimidazole ring of these polycyclic pyrazole com-
pounds. Among these, no substantial effects on in vitro
potency and selectivity were observed. The in vivo
efficacy of the polycyclic pyrazolo[3,4-d]pyrimidine com-
pounds was evaluated in spontaneously hypertensive
rats (SHR) by oral administration. As shown in Table
1, compounds 4a -c exhibited good oral antihyperten-
sive activity, roughly consistent with their in vitro
potency for PDE1 and PDE5. The less potent com-
pounds 3a -d and 4f, however, showed weak antihy-
pertensive activity.
Chart 1). By comparison, 4c shows a similar profile to
the guanine compound 1 in terms of both in vitro
potency for PDE1 and PDE5 and in vivo antihyperten-
sive activity. When comparing 4c to guanine compound
2, even though the in vitro profile is similar (except that
4c appears to be more potent for PDE4 and is thus
somewhat less selective), the in vivo antihypertensive
activity of 4c is much weaker than that of 2. It is also
worth noting that the pyrazolo[3,4-d]pyrimidine com-
pound 3d , which is isomeric with guanine compound 2,
also displays much weaker antihypertensive activity
than 2.
In summary, new polycyclic pyrazolo[3,4-d]pyrimidine
compounds were synthesized as analogues of the poly-
cyclic guanine PDE inhibitors. By varying substitutions
on the pyrazole ring, these compounds can achieve
similar potency and selectivity for the cGMP PDEs
(PDE1 and PDE5). In vivo evaluations using SHR
indicated that they are bioavailable. The polycyclic
pyrazolo[3,4-d]pyrimidine compounds described here
represent potential therapeutic agents through cGMP
potentiations.
Exp er im en ta l Section
Melting points were taken on a Thomas-Hoover or Mel-
Temp II melting point apparatus and are uncorrected. Chro-
matography was performed over Universal Scientific or Selecto
Scientific flash silica gel (32-63 µm). Analytical HPLC
analyses were done on Dynamax columns (silica gel for normal
phase and C8 for reverse phase) eluting with appropriate
solvents as indicated. 1H NMR spectra were determined with
a Varian VXR 200, Gemini 300, or Gemini 400 MHz instru-
ment using either Me4Si or residual solvent signal as internal
Compound 4c was further evaluated against two
other PDE isozymes (PDE2 and PDE4) to get a more
comprehensive PDE inhibition profile. The result is
listed in Table 2 along with those of the polycyclic
guanine compounds 1 and 2 (structures are shown in

PDE1 and PDE5 cGMP Phosphodiesterase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4375
standards. Rotations were determined on a Rudolph Autopol
III or Perkin-Elmer 243B polarimeter. Mass spectra were
obtained on a VG-ZAB-SE, Extrel-401, HP-MS Engine, J EOL
HX-110, or Sciex API 100 mass spectrometer. Elemental
analyses were determined by the Physical-Analytical Depart-
ment of Schering-Plough Research Institute using either CEC
240-HA, CEC CE-440, or Fisons EA 1108 CHNS elemental
analyzers and are within 0.4% of the theoretical value unless
otherwise noted. Fractional solvent impurities reported in the
molecular composition were identified by the characteristic
mL) at reflux until no more water separated in the Dean-
Stark trap. The intermediate 5e (20 g, 0.080 mol) was added,
and the resulting solution was stirred at room temperature
for 2 h. The solution was concentrated in vacuo. The residue
was heated with concentrated HCl (14.5 mL) in refluxing
EtOH (150 mL) for 45 min. The solution was concentrated in
vacuo. The residue was partitioned between EtOAc and
NaHCO3 (saturated). The organic layer was washed with
brine, dried (MgSO4), and concentrated in vacuo. Flash
chromatography on a silica gel column with EtOAc-hexane
(4-6) as eluent gave 10 (9.0 g, 73%): 1H NMR (300 MHz,
CDCl3) δ 1.18 (t, 3H, J ) 7 Hz), 3.45 (s, 3H), 4.19 (q, 2H, J )
7 Hz), 4.22 (s, 2H), 4.48 (br s, 2H), 5.18 (s, 2H), 7.01-7.25 (m,
5H).
5-Meth yl-1-(p h en ylm eth yl)-1H-p yr a zolo[3,4-d ]p yr im i-
d in e-4,6(5H,7H)-d ion e (7b). The pyrazole intermediate 6b
(2.28 g, 9.3 mmol) was heated with methyl isocyanate (2.2 mL,
37 mmol) and Et3N (0.26 mL, 1.9 mmol) in benzene (20 mL)
at 100 °C for 10 h. The solution was concentrated in vacuo.
The residue was heated with NaOMe (1.5 g, 28 mmol) in
refluxing MeOH (50 mL) for 1 h. The solution was concen-
trated in vacuo. The residue was dissolved in water, washed
with CH2Cl2, and adjusted to pH 6 with 10% HCl. The
precipitates were filtered, washed with water and ether, and
dried in vacuo to give 7b (0.87 g, 37%) as white solids: 1H
NMR (300 MHz, CDCl3) δ 3.38 (s, 3H), 5.43 (s, 2H), 7.35 (s,
5H), 7.98 (s, 1H), 12.07 (br s, 1H); MS (CI) m/z 257 (MH+, 100).
Anal. (C13H12N4O2) C, H, N.
1
peaks in the H NMR and were quantitated by integration of
the 1H resonances. Ethyl (ethoxymethylene)cyanoacetate (5a )
was purchased from Aldrich Chemical Co. and used as such.
Eth yl 2-Cya n o-3-eth oxy-2-bu ten oa te (5b). A solution of
ethyl cyanoacetate (51 mL, 0.48 mol) and triethyl orthoacetate
(88 mL, 0.48 mol) in Ac2O (150 mL) was refluxed for 3 h. The
solution was concentrated in vacuo. The residue was kept in
a refrigerator for 3 days to precipitate the product 5b (20 g,
23%) as white crystals: 1H NMR (200 MHz, CDCl3) δ 1.31 (t,
3H, J ) 7 Hz), 1.43 (t, 3H, J ) 7 Hz), 2.61 (s, 3H), 4.23 (q, 2H,
J ) 7 Hz), 4.27 (q, 2H, J ) 7 Hz).
Eth yl 2-Cya n o-3-h yd r oxy-4-p h en yl-2-bu ten oa te (5c).
NaH (4.0 g, 0.10 mol, 60% dispersion in mineral oil) was added
to a solution of ethyl cyanoacetate (10.64 mL, 0.10 mol) in dry
THF (100 mL) at room temperature and stirred for 10 min. A
solution of phenylacetyl chloride (13.6 g, 0.088 mol, generated
from phenylacetic acid and SOCl2 by standard procedures) in
dry THF (10 mL) was added dropwise. The mixture was
stirred for 1 h at room temperature. The mixture was
concentrated in vacuo. The residue was partitioned between
CH2Cl2 and water. The organic layer was dried (MgSO4) and
concentrated in vacuo to give crude 5c (22 g). Distillation at
140 °C/0.5 mmHg gave pure 5c as an oil: 1H NMR (300 MHz,
CDCl3) δ 1.28 (t, 3H, J ) 7 Hz), 3.81 (s, 2H), 4.25 (q, 2H, J )
7 Hz), 7.10-7.35 (m, 5H), 13.60 (br s, 1H); MS (FAB) m/z 232
(MH+, 100).
Eth yl 2-Cyan o-3-eth oxy-4-ph en yl-2-bu ten oate (5d). The
crude 5c (11 g) was stirred with EtI (19 mL, 0.238 mol) and
Ag2CO3 (26.27 g, 0.0952 mol) in CH2Cl2 (500 mL) at room
temperature for 16 h. The mixture was filtered. The filtrate
was concentrated in vacuo. Flash chromatography of the
residue on a silica gel column with EtOAc-hexane (10-90
then 15-85) as eluent gave 5d (5.36 g, 41% from ethyl
cyanoacetate) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ
1.30 (t, 3H, J ) 7 Hz), 1.33 (t, 3H, J ) 7 Hz), 4.22 (q, 2H, J )
7 Hz), 4.26 (q, 2H, J ) 7 Hz), 4.50 (s, 2H), 7.19 (d, 2H, J ) 7
Hz), 7.25-7.38 (m, 3H); MS (EI) m/z 259 (M+, 100).
Eth yl 3-Ch lor o-2-cya n o-4-p h en yl-2-bu ten oa te (5e). Tri-
n-butylamine (5.2 mL, 22 mmol) was added dropwise to a
solution of pure 5c (4.62 g, 20 mmol) in POCl3 (10 mL) at 0
°C. The resulting red solution was heated at 88 °C for 1 h.
The solution was extracted with ether (200 mL). The extracts
were washed with 10% HCl, H2O, and saturated NaHCO3
solution. The organic layer was dried (MgSO4) and concen-
trated in vacuo. Flash chromatography of the residue on a
silica gel column with EtOAc-hexane (5-95) as eluent gave
5e (2.00 g, 40%) as a yellow oil: 1H NMR (300 MHz, CDCl3) δ
1.31 (t, 3H, J ) 7 Hz), 4.29 (q, 2H, J ) 7 Hz), 4.41 (s, 2H),
7.18-7.33 (m, 5H).
E t h yl 5-Am in o-1-(p h en ylm et h yl)-1H -p yr a zole-4-ca r -
boxyla te (6b). A mixture of ethyl (ethoxymethylene)cyano-
acetate (5a ; 1.69 g, 10.0 mmol), benzylhydrazine dihydrochlo-
ride (2.14 g, 11.0 mmol), and Et3N (7.0 mL, 50 mmol) in MeOH
(25 mL) was refluxed for 15 h. The mixture was concentrated
in vacuo. The residue was partitioned between H2O and CH2-
Cl2. The organic layer was dried (MgSO4) and concentrated
in vacuo. Flash chromatography on a silica gel column with
EtOAc-hexane (2-8 then 3-7) as eluent gave 6b (2.05 g, 84%)
as white solids. Recrystallization from benzene gave white
crystal flakes: mp 108-110 °C; 1H NMR (300 MHz, CDCl3) δ
1.35 (t, 3H, J ) 7 Hz), 4.28 (q, 2H, J ) 7 Hz), 4.89 (br s, 2H),
5.18 (s, 2H), 7.18-7.40 (m, 5H), 7.71 (s, 1H); MS (CI) m/z 246
(MH+, 100). Anal. (C13H15N3O5) C, H, N.
2,5-Dim eth yl-3-(p h en ylm eth yl)-2H-p yr a zolo[3,4-d ]p y-
r im id in e-4,6(5H,7H)-d ion e (11): similarly prepared from 10;
1H NMR (200 MHz, DMSO-d6) δ 3.12 (s, 3H), 3.65 (s, 3H), 4.32
(s, 2H), 7.15-7.32 (m, 5H), 11.57 (s, 1H).
6-Ch lor o-1,5-d ih yd r o-5-m et h yl-1-(p h en ylm et h yl)-4H -
p yr a zolo[3,4-d ]p yr im id in -4-on e (8a ). The intermediate 7b
(7.00 g, 27.3 mmol) was heated with POCl3 (100 mL) at reflux
for 48 h. The solution was cooled and diluted with hexane
(1000 mL). The liquid layer was decanted. The residue was
dissolved in CH2Cl2, washed (carefully!) with saturated NaH-
CO3 solution, dried (MgSO4), and concentrated in vacuo.
Recrystallization from benzene gave 8a (5.05 g, 67%) as white
solids: 1H NMR (300 MHz, CDCl3) δ 3.71 (s, 3H), 5.45 (s, 2H),
7.26-7.34 (m, 5H), 8.06 (s, 1H); MS (CI) m/z 275 (MH+). Anal.
(C13H11ClN4O) C, H, N.
(()-1,5-Dih yd r o-6-[(2-h yd r oxycyclop en t yl)a m in o]-5-
m eth yl-1-(p h en ylm eth yl)-4H-p yr a zolo[3,4-d ]p yr im id in -
4-on e (9a ). The chloro intermediate 8a (4.60 g, 16.7 mmol)
was heated with (()-trans-2-aminocyclopentanol (3.38 g, 33.4
mmol) in DMF (100 mL) at 100 °C under N2 for 15 h. The
solution was concentrated in vacuo. The residue was dissolved
in CH2Cl2, washed with saturated NaHCO3 solution, dried
(MgSO4), and concentrated in vacuo. Flash chromatography
of the residue on a silica gel column with CHCl3-MeOH (95-
5) as eluent gave 9a (5.32 g, 94%) as white solids: 1H NMR
(300 MHz, CDCl3) δ 1.51-2.36 (m, 6H), 3.43 (s, 3H), 3.98-
4.11 (m, 1H), 4.88 (br s, 1H), 5.31 (d, 1H, J ) 18 Hz), 5.36 (d,
1H, J ) 18 Hz), 7.23-7.36 (m, 5H), 7.96 (s, 1H); MS (CI) m/z
340 (MH+). Anal. (C18H21N5O2) C, H, N.
r a c-(6a R*,9a S*)-5,6a ,7,8,9,9a -H exa h yd r o-5-m et h yl-1-
(ph en ylm eth yl)cyclopen t[4,5]im idazo[1,2-a ]pyr azolo[4,3-
e]p yr im id in -4(1H)-on e (3a ). Thionyl chloride (3.2 mL, 44
mmol) was added dropwise to a solution of intermediate 9a
(5.00 g, 14.7 mmol) in CH2Cl2 (200 mL) and stirred at room
temperature for 11 h. The solution was diluted with CH2Cl2,
washed with saturated NaHCO3 solution, dried (MgSO4), and
concentrated in vacuo. Flash chromatography of the residue
on a silica gel column with CHCl3-MeOH (99-1) as eluent
gave 3a (2.31 g, 49%) as white solids: 1H NMR (300 MHz,
CDCl3) δ 1.55-2.07 (m, 6H), 3.34 (s, 3H), 4.55 (t, 1H, J ) 7
Hz), 4.66 (t, 1H, J ) 7 Hz), 5.31 (d, 1H, J ) 17 Hz), 5.58 (d,
1H, J ) 18 Hz), 7.06 (d, 2H, J ) 6 Hz), 7.32-7.40 (m, 3H),
7.91 (s, 1H); MS (FAB) m/z 322 (MH+). Anal. (C18H19N5O) C,
H, N.
Similarly prepared were the following compounds.
Eth yl 3-Am in o-1-m eth yl-5-(ph en ylm eth yl)-1H-pyr azole-
4-ca r boxyla te (10). Methylhydrazine (7.36 g, 0.16 mol) was
heated with benzaldehyde (18.8 g, 0.18 mol) in benzene (250
(6a R ,9a S )-3,5-Dim e t h yl-5,6a ,7,8,9,9a -h e xa h yd r o-1-
(p h en ylm et h yl)cyclop en t [4,5]im id a zo[1,2-a ]p yr a zolo-
[4,3-e]p yr im id in -4(1H)-on e (3b): [R]22 +200.7° (c 0.27,
D

4376 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Xia et al.
EtOH); 1H NMR (200 MHz, CDCl3) δ 1.48-2.05 (m, 6H), 2.47
(s, 3H), 3.32 (s, 3H), 4.51 (t, 1H, J ) 6 Hz), 4.64 (t, 1H, J ) 6
Hz), 5.24 (d, 1H, J ) 17 Hz), 5.48 (d, 1H, J ) 17 Hz), 7.04 (d,
2H, J ) 8 Hz), 7.28-7.40 (m, 3H); MS (FAB) m/z 336 (MH+,
100%); Anal. (C19H21N5O) C, H, N.
DMSO-d6) δ 1.20-2.05 (m, 6H), 2.32 (s, 3H), 3.09 (s, 3H), 4.48
(t, 1H, J ) 6 Hz), 4.55 (t, 1H, J ) 7 Hz), 12.64 (br s, 1H); MS
(CI) m/z 246 (MH+, 100). Anal. (C12H15N5O‚0.08CH2Cl2) C,
H, N.
P DE In h ibition Assa ys. PDE assays were performed in
a reaction medium containing 50 mM Tris-HCl (pH 7.5), 2 mM
MgCl2, 0.1 mg/mL BSA, and 1 µM cGMP or cAMP, respec-
tively. Assays were carried out for 30 min at 30 °C using the
methods described by Thompson.14 Reaction mixture for assay
of PDE1 activity also contained 1 mM CaCl2 and 0.1 µM
calmodulin. The reaction mixture for assay of PDE2 contained
5 µM cGMP. [3H]cGMP was used as the substrate in the
assays for PDE1 and PDE5, while [3H]cAMP was used as the
substrate for PDE2, PDE3, and PDE4. The concentration of
compounds that produce 50% inhibition of enzyme activity
(IC50) was determined from the curve of percentage inhibition
of enzyme activity vs log molar concentration of the com-
pounds. All assays were carried out in duplicate, and reported
values represent the mean of the two determinations. Mea-
surements were reproducible on the average to (25%, except
for the very weakest inhibitors where solubility limits were
occasionally exceeded. PDE1, PDE2, PDE3, PDE4, and PDE5
utilized in the assays were purified from bovine aorta,15
recombinant bovine adrenal cortex,16 bovine heart,17 canine
kidney,18 and bovine lung,19 respectively. These preparations
were free of substantial contaminating phosphodiesterase
activities.
An tih yp er ten sive Activity. PDE inhibitors were evalu-
ated in the spontaneously hypertensive rat using the meth-
odology previously described by Smith et al.20 and more
recently by Vemulapalli et al.21 Reported values reflect peak
changes in mean arterial pressure in comparison to a control
group to which vehicle was administered. The compounds
were adminstered orally as aqueous solutions or suspensions
in 0.4% methylcellulose as the vehicle. In general differences
of g10 mmHg are considered statsitically signficant. The
drugs verapamil and nifedipine were routinely run as positive
controls in this assay. Two hours after a 30 mg/kg oral dose
of verapamil or nifedipine, reproducible falls in blood pressure
of 70 ( 4 and 58 ( 5 mmHg (mean ( SEM), respectively, were
produced.
r a c-(6a R*,9a S*)-5,6a ,7,8,9,9a -Hexa h yd r o-1,5-d im eth yl-
3-(p h en ylm eth yl)cyclop en t[4,5]im id a zo[1,2-a ]p yr a zolo-
1
[4,3-e]p yr im id in -4(1H)-on e (3e): H NMR (300 MHz, CD3-
OD) δ 1.55-2.10 (m, 6H), 3.32 (s, 3H), 3.88 (s, 3H), 4.15 (AB
q, 2H), 4.77 (t, 1H, J ) 7 Hz), 4.83 (t, 1H, J ) 7 Hz), 7.15-
7.47 (m, 5H); MS (CI) m/z 336 (MH+, 100). Anal. (C19H21N5O)
C, H; N: calcd, 20.88; found, 19.80.
r a c-(6a R*,9a S*)-1,3-Bis(p h en ylm et h yl)-5,6a ,7,8,9,9a -
h exah ydr o-5-m eth ylcyclopen t[4,5]im idazo[1,2-a ]pyr azolo-
1
[4,3-e]p yr im id in -4(1H)-on e (3f): H NMR (300 MHz, CD3-
OD) δ 1.50-2.10 (m, 6H), 3.34 (s, 3H), 4.17 (d, 1H, J ) 14
Hz), 4.28 (d, 1H, J ) 14 Hz), 4.52 (t, 1H, J ) 7 Hz), 4.64 (t,
1H, J ) 7 Hz), 5.27 (d, 1H, J ) 17 Hz), 5.55 (d, 1H, J ) 17
Hz), 7.00-7.50 (m, 10H); MS (EI) m/z 411 (M+, 15). Anal.
(C18H19N5O‚0.24CHCl3) C, H; N: calcd, 15.91; found, 15.42.
r a c-(6a R*,9a S*)-5,6a ,7,8,9,9a -Hexa h yd r o-2,5-d im eth yl-
3-(p h en ylm eth yl)cyclop en t[4,5]im id a zo[1,2-a ]p yr a zolo-
[4,3-e]p yr im id in -4(1H)-on e (4a ): 1H NMR (300 MHz, CDCl3)
δ 1.44-2.24 (m, 6H), 3.38 (s, 3H), 3.61 (s, 3H), 4.28 (s, 2H),
4.65-4.72 (m, 2H), 7.12-7.27 (m, 5H); MS (FAB) m/z 336
(MH+, 100). Anal. (C19H21N5O) C, H, N.
(6a R ,9a S )-5,6a ,7,8,9,9a -H e xa h yd r o-2,5-d im e t h yl-3-
(ph en ylm eth yl)cyclopen t[4,5]im idazo[1,2-a ]pyr azolo[4,3-
e]p yr im id in -4(1H)-on e (4b): [R]22 +187.9° (c 0.74, EtOH);
D
1H NMR (300 MHz, CDCl3) δ 1.44-2.24 (m, 6H), 3.38 (s, 3H),
3.61 (s, 3H), 4.28 (s, 2H), 4.65-4.72 (m, 2H), 7.12-7.27 (m,
5H); MS (EI) m/z 335 (M+, 39), 306 (100). Anal.
(C19H21N5O‚0.10H2O) C, H, N.
2′,5′-Dim et h yl-3′-(p h en ylm et h yl)sp ir o[cyclop en t a n e-
1,7′(8′H)-[2H]im id a zo[1,2-a ]p yr a zolo[4,3-e]p yr im id in ]-4′-
1
(5′H)-on e (4c): H NMR (300 MHz, CDCl3) δ 1.52-1.95 (m,
8H), 3.34 (s, 3H), 3.56 (s, 3H), 3.70 (s, 2H), 4.28 (s, 2H), 7.10-
7.25 (m, 5H); MS (ESI) m/z 350 (MH+, 100). Anal. (C20H23N5O)
C, H, N.
7,8-Dih ydr o-3-(ph en ylm eth yl)-2,5,7,7-tetr am eth yl-[2H]-
im id a zo[1,2-a ]p yr a zolo[4,3-e]p yr im id in -4(5H)-on e (4d ):
1H NMR (400 MHz, CDCl3) δ 1.40 (s, 6H), 3.40 (s, 3H), 3.65
(s, 3H), 3.69 (s, 2H), 4.36 (s, 2H), 7.21-7.31 (m, 5H); MS (ESI)
m/z 324 (MH+, 100). Anal. (C18H21N5O) C, N; H: calcd, 6.67;
found, 6.25.
Ack n ow led gm en t . We thank Dr. M. Puar of our
Structural Chemistry Department for the NOE struc-
tural determinations.
(7R )-7,8-Dih yd r o-2,5-d im e t h yl-7-(1-m e t h yle t h yl)-3-
(p h en ylm et h yl)-[2H ]im id a zo[1,2-a ]p yr a zolo[4,3-e]p yr i-
1
m id in -4(5H)-on e (4e): [R]25 +89° (c 0.28, EtOH); H NMR
Su p p or tin g In for m a tion Ava ila ble: HPLC chromato-
grams (normal and reverse phases) of compounds 3c-e and
4b (8 pages). Ordering information is given on any current
masthead page.
D
(400 MHz, CDCl3) δ 0.92 (d, 3H, J ) 6.7 Hz), 1.02 (d, 3H, J )
6.7 Hz), 1.89 (m, 1H), 3.43 (s, 3H), 3.65 (s, 3H), 3.69 (m, 1H),
3.96 (t, 1H, J ) 9.5 Hz), 4.07 (m, 1H), 4.36 (s, 2H), 7.21-7.32
(m, 5H); MS (ESI) m/z 338 (MH+, 100). Anal. (C19H23N5O) C,
H, N.
Refer en ces
(6a R,9a S)-5,6a ,7,8,9,9a -Hexa h yd r o-5-m eth yl-3-p h en yl-
2-(p h en ylm eth yl)cyclop en t[4,5]im id a zo[1,2-a ]p yr a zolo-
[4,3-e]p yr im id in -4(1H)-on e (4f): [R]22D +147° (c 0.46, EtOH);
1H NMR (300 MHz, CDCl3) δ 1.45-2.30 (m, 6H), 3.35 (s, 3H),
4.72 (t, 1H, J ) 7 Hz), 4.79 (t, 1H, J ) 7 Hz), 5.15 (s, 2H),
6.95-7.45 (m, 10H); MS (EI) m/z 397 (M+, 51), 368 (100). Anal.
(C24H23N5O) C, H, N.
r a c-(6a R*,9a S*)-5,6a ,7,8,9,9a -H exa h yd r o-5-m et h yl-3-
(ph en ylm eth yl)cyclopen t[4,5]im idazo[1,2-a ]pyr azolo[4,3-
e]p yr im id in -4(1H)-on e (3d ). A mixture of 3f (425 mg, 1.03
mmol) and 20% Pd(OH)2/C (0.41 g) was hydrogenated at 60
psi for 24 h. The solids were filtered and washed with CH2-
Cl2-MeOH (5-1). The filtrate and washings were combined
and concentrated in vacuo. Flash chromatography of the
residue on a silica gel column with CH2Cl2-MeOH (95-5 then
90-10) as eluent gave 3d (100 mg, 30%) as white solids: 1H
NMR (300 MHz, CD3OD) δ 1.50-2.30 (m, 6H), 3.31 (s, 3H),
4.25 (s, 2H), 4.70 (t, 1H, J ) 7 Hz), 4.83 (t, 1H, J ) 7 Hz),
7.17-7.38 (m, 5H); HRMS EI (C18H19N5O) calcd 321.1590 (M+),
found 321.1588. Anal. (C18H19N5O‚0.05CH2Cl2) C, H, N.
(6a R ,9a S )-3,5-Dim e t h yl-5,6a ,7,8,9,9a -h e xa h yd r ocy-
clopen t[4,5]im idazo[1,2-a ]pyr azolo[4,3-e]pyr im idin -4(1H)-
on e (3c): similarly prepared from 3b; 1H NMR (300 MHz,
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