4-(3-Chloro-4-methoxybenzyl)aminophthalazines: Synthesis and inhibitory activity toward phosphodiesterase 5

Article abstract of DOI:10.1021/jm9905054

We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1-and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) and their vasorelaxant activity in isolated porcine coronary arteries preco

Full text of DOI:10.1021/jm9905054

J . Med. Chem. 2000, 43, 2523-2529
2523
4-(3-Ch lor o-4-m eth oxyben zyl)a m in op h th a la zin es: Syn th esis a n d In h ibitor y
Activity tow a r d P h osp h od iester a se 5
Nobuhisa Watanabe,* Hideyuki Adachi, Yasutaka Takase, Hirofumi Ozaki, Masayuki Matsukura,
Kazuki Miyazaki, Keiji Ishibashi, Hiroki Ishihara, Kohtarou Kodama, Mayu Nishino, Motoharu Kakiki, and
Yasuhiro Kabasawa
Tsukuba Research Laboratories, Eisai Company, Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, J apan
Received February 1, 2000
We synthesized various 4-(3-chloro-4-methoxybenzyl)aminophthalazines substituted at the 1-
and 6-positions and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5)
and their vasorelaxant activity in isolated porcine coronary arteries precontracted with
prostaglandin F2R (10-5 M). The preferred substituents at the 1-position of the phthalazine
were 4-hydroxypiperidino, 4-hydroxymethylpiperidino, 4-(2-hydroxyethyl)piperidino, and 4-oxo-
piperidino. Among these compounds, [4-(3-chloro-4-methoxybenzyl)amino-1-(4-hydroxy)piperi-
dino]-6-phthalazinecarbonitrile monohydrochloride (13) exhibited potent PDE5 inhibitory
activity (IC50 ) 0.56 nM) with >1700-fold high selectivity over other PDE isozymes (PDE1-4).
Compound 13 exhibited the most potent vasorelaxant action (EC50 ) 13 nM) in this series of
compounds. Compound 13 reduced mean pulmonary arterial pressure by 29.9 ( 3.1% when
administered intravenously at 30 µg/kg to the chronically hypoxic rats and had an apparent
oral bioavailability of about 19.5% in rats and was selected for further biological evaluation.
In tr od u ction
Cyclic nucleotide phosphodiesterases (PDEs) have
been classified into at least seven distinct isozyme
families,1 of which one, cGMP-PDE (PDE5), specifically
hydrolyzes cGMP. Zaprinast2 and MY-54453 are clas-
sical PDE inhibitors possessing a moderate selectivity
for PDE5. Recently, selective and potent PDE5 inhibi-
tors such as UK-92480,4 1,3-dimethyl-6-(2-propoxy-5-
methanesulfonylamidophenyl)-1,5-dihydropyrazolo[3,4-
d]pyrimidin-4-one (DMPPO),5 1-arylnaphthalene lig-
nans,6 and quinazoline derivatives7-9 (Figure 1) have
been reported to show vasorelaxing, antihypertensive,
and penile-erection-promoting activities.
In the previous paper we reported that some 4-ben-
zylamino-1-chloro-6-substituted-phthalazines (2-4) po-
tently inhibited PDE5 and proposed a pharmacophore
for PDE5 inhibition.10 In the present study, to find more
potent PDE5 inhibitors we synthesized a variety of 4-(3-
chloro-4-methoxybenzyl)aminophthalazines substituted
at the 1- and 6-positions, via the chlorides (2-6). We
evaluated their inhibitory activities toward PDE5. Some
of these compounds were also examined for relaxant
activity in isolated porcine coronary arteries precon-
tracted with prostaglandin F2R (PGF2R), in comparison
with E4021.
Ch em istr y
The general synthetic procedure was as follows
(Scheme 1). 1,4,6-Trisubstituted phthalazines were
synthesized by the reaction of 4-benzylamino-1-chloro-
6-substituted-phthalazine (2-5) and amine in the pres-
ence of diisopropylethylamine.
The syntheses of 2-5 were reported in the previous
paper.10 In the case of 6,7-dichlorophthalazine (6),
* To whom correspondence should be addressed. Tel: 81-298-47-
5873. Fax: 81-298-47-4012. E-mail: n5-watanabe@hhc.eisai.co.jp.
F igu r e 1.
10.1021/jm9905054 CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/06/2000

2524 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Watanabe et al.
Sch em e 1a
a
Reagents: (a) 3-chloro-4-methoxybenzylamine hydrochloride, Et3N, 2-propanol; (b) amine, iPr2EtN, NMP (then HCl).
Sch em e 2a
respectively (Scheme 2). Compounds 40 and 41 were
synthesized from a cyano compound (13). Compound 42
was synthesized by the coupling of 40 and dimethyl-
amine (Scheme 3).
P h a r m a cologica l Resu lts a n d Discu ssion
The biological methods used are described in the
Experimental Section. E4021 or sildenafil citrate were
used as the active control in each PDE5-inhibitory
activity assay.
The pharmacological results obtained for the 4-[(3-
chloro-4-methoxybenzyl)amino]-6-phthalazinecarboni-
trile derivatives are listed in Table 1. 1-Piperidine
derivatives (9-20) and thiomorpholino derivatives (21-
23) generally exhibited more potent PDE5-inhibitory
activity than 2. The carboxylic acid (11) and sulfonamide
(20), which have an acidic proton, were weaker inhibi-
tors than the unsubstituted piperidine (9). The effects
of neutral and relatively small substituents at the
4-position of piperidine and 1-position of thiomorpholine,
i.e., unsubstituted (9), hydroxyl (13), keto (19), sulfide
(21), S-oxide (22), and S-dioxide (23), were almost
equivalent as regards PDE5-inhibitory activity. Some
of the hydroxy compounds (13-15) exhibited more
potent vasorelaxant effects than 2, and the 4-hydroxyl
compound (13) exhibited the most potent vasorelaxant
effect (EC50 ) 13 nM) among them. The 4-ketone (19)
was equipotent to the 4-hydroxyl compound (13) in
vasorelaxant effect.
a
Reagents: (a) HCOOH then HCl; (b) TFA then HCl.
a known 1,4,6,7-tetrachlorophthalazine (1)11 and 3-chloro-
4-methoxybenzylamine hydrochloride were refluxed in
2-propanol in the presence of triethylamine.
Compounds 11 and 19 were synthesized via the
protected tert-butyl ester (7) and ethyleneketal (8),
Sch em e 3a
a
Reagents: (a) aq 1 N NaOH, MeOH, reflux; (b) aq 1 N NaOH, EtOH, THF, rt; (c) Me2NH, DCC, HOBt, Et3N, CH3CN, H2O then HCl.

4-(3-Chloro-4-methoxybenzyl)aminophthalazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2525
Ta ble 1. Pharmacological Results of 1-Substituted-4-[(3-chloro-4-methoxybenzyl)amino]-6-phthalazinecarbonitriles
a
b
IC50 values were determined from the logarithmic concentration-inhibition curve (at least four points). EC50 values were determined
from the logarithmic concentration-inhibition curve. The value is given as the mean of two experiments. c nt means not tested.

2526 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Watanabe et al.
Ta ble 2. Pharmacological Results of 6-Substituted-4-[(3-chloro-
4-methoxybenzyl)amino]-1-(4-hydroxypiperidino)phthalazines
PDE5
vasorelaxant effect
EC50, nMb
compd
R6
IC50, nMa
13
36
37
38
39
40
41
42
CN
NO2
CF3
Cl
6,7-Cl2
COOH
CONH2
CONMe2
0.56
0.30
0.73
4.4
0.86
600
2.0
13
30
120
780
ntc
nt
nt
nt
58
a-c See footnotes in Table 1.
Among piperazine derivatives (24-28), 24-26 exhib-
ited potent inhibitory activity. The substituent effects
were similar to those of piperidines (24 vs 9, 26 vs
15), though the piperazine derivatives were slightly
weaker PDE5 inhibitors than the piperidine derivatives.
4-(2-Pyridyl)piperazine (27) and 4-(2-pyrimidyl)pipera-
zine (28) lacked potent inhibitory activity, so a bulky
substituent at the 4-position of piperazine seems to
decrease PDE5-inhibitory ability. Compounds (30-35)
substituted with a primary amino group were less
potent than 2.
Next, we examined the effect of substituents at the
6-position of phthalazine with the 1-substituent fixed
as 4-hydroxypiperidine. The results are listed in Table
2. The nitro (36) and trifluoromethyl (37) compounds
were almost equipotent as PDE5 inhibitors to the nitrile
(13). The chloro compound (38) was less potent. These
results were parallel to our previous findings.10 The 6,7-
dichloro compound (39) was unexpectedly potent. Car-
boxylic acid (40), carbamoyl (41), and dimethylamide
(42) derivatives of 13 showed loss of PDE5-inhibitory
activity. These results were parallel to our findings on
quinazoline derivatives.8b
F igu r e 2. Time courses of effects of compound 13 on mean
pulmonary (MPAP) and systemic (MSAP) arterial pressures
in chronically hypoxic pulmonary hypertensive rats. Com-
pound 13 at a dose of 30 µg/kg (closed circles, n ) 6) or vehicle
(open circles, n ) 6) was intravenously injected to conscious
rats. Values are mean ( SEM. *Significant difference from
corresponding vehicle values, P < 0.05 (by repeated-measures
ANOVA).
over 1700 times versus PDE1-4. This result confirms
that the pharmacophore we proposed in the previous
paper10 is appropriate for selective PDE5-inhibitory
activity. Compound 13 exhibited slight inhibitory activ-
ity toward PDE2 (IC50 ) 0.984 µM) and PDE4 (IC50
)
1.17 µM) but was ineffective toward PDE1 (IC50 ) 26.2
µM) and PDE2 (IC50 ) 17.5 µM).
Next, we examined the effects of 13 on mean pulmo-
nary (MPAP) and systemic (MSAP) arterial pressures
in chronically hypoxic pulmonary hypertensive rats. The
time courses of the hemodynamic effects of an intrave-
nous injection of 13 at 30 µg/kg are shown in Figure 2.
In chronically hypoxic rats before an intravenous injec-
We selected 13 as the optimum compound for PDE5-
inhibitory activity and vasorelaxant effect. It is com-
pared with other PDE inhibitors in Table 3. Compound
13 exhibited highly selective PDE5-inhibitory activity
Ta ble 3. Inhibitory Activities of 13 on Five PDE Isozymesa
IC50, µMb
(95% confidence interval)
compd
PDE5
PDE1
PDE2
PDE3
PDE4
13
0.000560
(0.000442-0.000710)
1.11
26.2
(20.0-34.4)
31.2
0.984
(0.841-1.15)
90.2
17.5
(11.4-26.9)
>100
1.17
(1.01-1.36)
>100
zaprinast
milrinone
dipyridamole
rolipram
(0.733-1.68)
(26.5-36.7)
(68.1-119)
>100
>100
>100
0.843
(0.581-1.23)
44.0
13.0
(10.4-16.3)
5.27
0.574
(0.244-1.35)
>100
74.5
(51.1-108)
>100
3.24
(2.54-4.14)
>100
(31.8-60.7)
(3.58-7.78)
>100
3.63
(2.56-5.13)
a
The five PDE isozymes were isolated from porcine tissues as follows: PDE1, aorta; PDE2, adrenal; PDE3, platelets; PDE4, liver;
b
PDE5, platelets. IC50 values were determined from the logarithmic concentration-inhibition curves (at least four points) and are mean
values of three experiments.

4-(3-Chloro-4-methoxybenzyl)aminophthalazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2527
Ta ble 4. Pharmacokinetic Parameters of 13 after 0.3 mg/kg
Administration in Rats
chromatography to give the free base (620 mg, 55%). The free
base (620 mg, 1.5 mmol) was recrystallized from a mixture of
1 N HCl (1.8 mL), H2O (6.4 mL), and EtOH (8 mL) to give 10
Cmax
,
AUC,
1
(420 mg, 62%) as a yellow powder: mp 240-243 °C (dec); H
route
n
tmax, h
t1/2, h
ng/mL
ng h/mL
BA, %
NMR (CDCl3) δ 2.38-2.46 (2H, m), 3.35-3.38 (2H, m), 3.80-
3.84 (2H, m), 3.85 (3H, s), 4.74 (2H, d, J ) 5.6 Hz), 5.85 (1H,
d, J ) 10 Hz), 5.93 (1H, d, J ) 10 Hz), 7.16 (1H, d, J ) 8.4
Hz), 7.48 (1H, dd, J ) 8.4, 2.0 Hz), 7.63 (1H, d, J ) 2.0 Hz),
8.23 (1H, d, J ) 8.4 Hz), 8.47 (1H, d, J ) 8.4 Hz), 9.49 (1H, s),
10.46 (1H, br s); MS m/e (FAB) 406 (MH+). Anal. (C22H20ClN5O‚
HCl) C, H, N.
iv
po
4
4
-
2.20
-
-
166.0
32.4
-
0.25
25.54
19.5
tion of 13 at a dose of 30 µg/kg, the mean pulmonary
and systemic arterial pressures were 27.7 ( 1.5 and 104
( 3 mmHg, respectively. Compound 13 caused signifi-
cant decreases in MPAP at doses of more than 3 µg/kg
and MSAP only at a dose of 30 µg/kg.
Maximum hypotensive responses of MPAP and MSAP
to 13 were 29.9 ( 3.1% and 9.2 ( 1.4% and were noted
5 and 3 min after the intravenous injection, respectively.
The lowering effect of 13 on MPAP lasted over 45 min,
whereas its effect on MSAP was transient. No changes
in heart rate were observed.
1-[4-[(3-Ch lor o-4-m e t h oxyb e n zyl)a m in o]-6-cya n o-1-
p h th a la zin yl]-4-p ip er id in eca r boxylic Acid Hyd r och lo-
r id e (11). A mixture of 7 (1.20 g, 2.4 mmol) and formic acid
(20 mL) was stirred at room temperature for 20 h. The reaction
mixture was concentrated at reduced pressure. The residue
was purified by silica gel column chromatography to give the
free base (1.05 g, 98%). The free base (1.0 g, 2.2 mmol) was
recrystallized from a mixture of 1 N HCl (2.3 mL), H2O (2.7
mL), and EtOH (5 mL) to give 11 (890 mg, 82%) as a yellow
1
powder: mp >270 °C; H NMR (DMSO-d6) δ 1.88-1.93 (2H,
m), 1.96-2.03 (2H, m), 2.50-2.59 (1H, m), 2.92-3.01 (2H, m),
3.50-3.58 (2H, m), 3.85 (3H, s), 4.74 (2H, d, J ) 5.2 Hz), 7.16
(1H, d, J ) 8.4 Hz), 7.48 (1H, dd, J ) 8.4, 2.4 Hz), 7.63 (1H,
d, J ) 2.4 Hz), 8.26 (1H, d, J ) 8.4 Hz), 8.46 (1H, dd, J ) 8.4,
1.2 Hz), 9.49 (1H, d, J ) 1.2 Hz); MS m/e (FAB) 452 (MH+).
Anal. (C23H22ClN5O3‚HCl‚0.5H2O) C, H, N.
We evaluated the pharmacokinetic parameters of 13.
As shown in Table 4, 13 had an apparent oral bioavail-
ability of about 19.5%. The plasma protein binding for
30 ng/mL of 13 was 95.7 ( 1.3%.
In conclusion, we have synthesized various 4-(3-
chloro-4-methoxybenzyl)aminophthalazines substituted
at the 1- and 6-positions via the 1-chlorides. In this
series of compounds, 13 exhibited potent PDE5-inhibi-
tory activity (IC50 ) 0.56 nM) and high selectivity for
PDE5 over other PDE isozymes (PDE1-4), amounting
to >1700-fold. Compound 13 also exhibited the most
potent vasorelaxant action (EC50 ) 13 nM) in this series
of compounds. Compound 13 reduced mean pulmonary
arterial pressure by 29.9 ( 3.1% when administered
intravenously at 30 µg/kg to the chronically hypoxic rats
and had an apparent oral bioavailability of about 19.5%
in rats and was selected for further biological evalua-
tion.12,13
4-[(3-Ch lor o-4-m eth oxyben zyl)a m in o]-1-(4-oxop ip er i-
d in o)-6-p h th a la zin eca r bon itr ile Hyd r och lor id e (19). A
mixture of 8 (10.0 g, 21.5 mmol) and TFA (40 mL) was stirred
at room temperature for 12 h. The reaction mixture was
concentrated at reduced pressure. The residue was taken up
in water and extracted with EtOAc. The organic layer was
washed with aqueous NaHCO3, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography and recrystallized from EtOH-H2O to give
the free base (7.29 g, 80%). The free base (7.20 g, 17.1 mmol)
was recrystallized from a mixture of 1 N HCl (20.5 mL) and
EtOH (50 mL) to give 19 (4.45 g, 57%) as a yellow powder:
mp 206 °C (dec); 1H NMR (DMSO-d6) δ 2.62-2.68 (4H, m),
3.55-3.61 (4H, m), 3.85 (3H, s), 4.77 (2H, d, J ) 5.6 Hz), 7.15
(1H, d, J ) 8.6 Hz), 7.49 (1H, dd, J ) 8.6, 2.0 Hz), 7.64 (1H,
d, J ) 2.0 Hz), 8.40 (1H, d, J ) 8.4 Hz), 8.50 (1H, dd, J ) 8.4,
1.2 Hz), 9.55 (1H, d, J ) 1.2 Hz); MS m/e (FAB) 422 (MH+).
Anal. (C22H20ClN5O2‚HCl‚0.25H2O) C, H, N.
Exp er im en ta l Section
Melting points (mp) were determined on an electrothermal
capillary melting point apparatus and on a hot-stage ap-
paratus, without correction. All 1H NMR spectra were mea-
sured on a Varian (400 MHz) spectrometer with tetrameth-
ylsilane (TMS) as an internal standard. Mass spectra (MS)
and elemental analyses were performed at the Analytical
Chemistry Section of Eisai Tsukuba Research Laboratories.
N -(3-Ch lor o-4-m e t h oxyb e n zyl)-N -(4,6,7-t r ich lor o-1-
p h th a la zin yl)a m in e (6). A mixture of 1 (2.2 g, 8.2 mmol),
3-chloro-4-methoxybenzylamine hydrochloride (1.8 g, 8.7 mmol),
Et3N (2.6 mL, 18.7 mmol), and 2-propanol (100 mL) was heated
at reflux for 6 h. The mixture was concentrated under reduced
pressure, and the residue was dissolved in EtOAc and THF.
The solution was washed with H2O and brine, dried over
anhydrous MgSO4, and concentrated under reduced pressure.
The residue was purified by recrystallization (EtOH-THF) to
give 6 (2.16 g, 66%) as a pale yellow powder: 1H NMR (CDCl3)
δ 3.90 (3H, s), 4.77 (2H, d, J ) 4.8 Hz), 5.29 (1H, t, J ) 4.8
Hz), 6.91 (1H, d, J ) 8.0 Hz), 7.32 (1H, dd, J ) 8.0, 2.4 Hz),
7.45 (1H, d, J ) 2.4 Hz), 7.89 (1H, s), 8.28 (1H, s); MS m/e
(FAB) 404 (MH+).
4-[(3-Ch lor o-4-m eth oxyben zyl)am in o]-1-(1,2,3,6-tetr ah y-
d r o-1-p yr id in yl)-6-p h th a la zin eca r bon itr ile Hyd r och lo-
r id e (10). Gen er a l P r oced u r e. A mixture of 2 (1.0 g, 2.8
mmol), 1,2,3,6-tetrahydro-1-pyridine (1.2 g, 14.5 mmol),
iPr2EtN (2.5 mL, 14.4 mmol), and NMP (10 mL) was heated
at 170 °C for 4.5 h and then poured into water and extracted
with EtOAc. The organic layer was washed with brine, dried
over anhydrous MgSO4, and concentrated under reduced
pressure. The residue was purified by silica gel column
4-[(3-Ch lor o-4-m eth oxyben zyl)a m in o]-1-(4-h yd r oxyp i-
p er id in o)-6-p h th a la zin eca r boxylic Acid (40). A mixture
of 13 (1.0 g, 2.4 mmol), aqueous 1 N-NaOH (40 mL), and
MeOH (100 mL) was heated at reflux for 6 h. To this mix-
ture was added H2O (100 mL) and aqueous 1 N HCl (40
mL) to give a precipitate. The resultant precipitate was
collected by filtration and washed with H2O to give 40 (860
mg, 82%) as a yellow solid: mp 205-207 °C; 1H NMR (DMSO-
d6) δ 1.62-1.73 (2H, m), 1.90-1.96 (2H, m), 2.90-2.97 (2H,
m), 3.35-3.45 (2H, m), 3.66-3.74 (1H, m), 3.82 (3H, s),
4.64 (2H, s), 4.77 (1H, bs), 7.10 (1H, d, J ) 8.4 Hz), 7.36 (1H,
dd, J ) 8.4, 2.0 Hz), 7.48 (1H, d, J ) 2.0 Hz), 8.08 (1H, d, J )
8.6 Hz), 8.38 (1H, dd, J ) 8.6, 1.4 Hz), 8.54 (1H, bs), 9.03 (1H,
s); MS m/e (FAB) 443 (MH+). Anal. (C22H23ClN4O4‚0.7H2O) C,
H, N.
4-[(3-Ch lor o-4-m eth oxyben zyl)a m in o]-1-(4-h yd r oxyp i-
p er id in o)-6-p h th a la zin eca r boxa m id e (41). To a solution
of 13 (500 mg, 1.2 mmol) in EtOH (6 mL) and THF (6 mL)
was added aqueous 1 N NaOH (2.4 mL). The mixture was
stirred for 15 h at room temperature. It was then diluted with
EtOAc, washed with brine, dried over anhydrous MgSO4, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography and recrystallized from
CH3CN to give 41 (230 mg, 44%) as a yellow powder: mp 167-
1
169 °C; H NMR (DMSO-d6) δ 1.62-1.72 (2H, m), 1.88-1.96
(2H, m), 2.85-2.95 (2H, m), 3.30-3.43 (2H, m), 3.68 (1H, m),
3.82 (3H, s), 4.63 (2H, d, J ) 5.7 Hz), 4.73 (1H, d, J ) 4.0 Hz),
7.09 (1H, d, J ) 8.4 Hz), 7.36 (1H, dd, J ) 8.4, 2.0 Hz), 7.46
(1H, d, J ) 2.0 Hz), 7.72 (1H, br s), 7.74 (1H, t, J ) 5.7 Hz),

2528 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Watanabe et al.
7.98 (1H, d, J ) 8.6 Hz), 8.11 (1H, br s), 8.25 (1H, dd, J ) 8.4,
1.3 Hz), 8.78 (1H, d, J ) 1.3 Hz); MS m/e (FAB) 442 (MH+).
Anal. (C22H24ClN5O3‚0.8H2O) C, H, N.
All data are expressed as mean ( SEM. The value for each
hemodynamic parameter after the injection of 13 was com-
pared with the value after the injection of vehicle by using
repeated-measures ANOVA followed by Dunn-type multiple
comparison. P values of <0.05 were considered significant.
P h a r m a cok in etic Stu d ies. The pharmacokinetics of 13
was studied in male Sprague-Dawley rats. In the iv and po
studies, 13 was dissolved in 10% lactose-phosphoric acid (pH
2). Rats were sacrificed by exsanguination under ether anaes-
thetized at 5 (iv only), 15, and 30 min and 1, 1.5 (po only), 2,
4, 6, 8, and 24 h after dosing, and blood samples (1 mL) were
collected. Plasma was obtained by centrifugation of blood at
3000g for 15 min and stored at -20 °C until analysis. The
concentration of 13 was determined by HPLC following liquid-
liquid extraction from plasma.
N6,N6-Dim eth yl-4-[(3-ch lor o-4-m eth oxyben zyl)a m in o]-
1-(4-h yd r oxyp ip er id in o)-6-p h th a la zin eca r boxa m id e Hy-
d r och lor id e (42). To a suspension of 40 (300 mg, 0.68 mmol),
dimethylamine hydrochloride (110 mg, 1.35 mmol), DCC (210
mg, 1.0 mmol), and 1-hydroxybenzotriazole (140 mg) in tri-
ethylamine (0.24 mL) and acetonitrile (10 mL) was added H2O
(1 mL), affording a clear solution. The mixture was heated at
60 °C for 8 h, during which time a precipitate was formed.
After cooling, the precipitate was filtered off, and the filtrate
was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to give the free
base (260 mg, 82%) as a yellow solid. The free base (260 mg,
0.55 mmol) was recrystallized from 1 N HCl, EtOH, EtOAc,
and IPE to give 42 (260 mg, 93%) as a yellow powder: mp
Ack n ow led gm en t. We thank Drs. S. Souda, K.
Miyake, S. Abe, T. Nagakura, and S. Suzuki for helpful
discussions and assistance during the course of this
work and the staff of Eisai Analytical Chemistry Section
for obtaining mass spectra and for elemental analysis.
1
179-184 °C (dec); H NMR (DMSO-d6) δ 1.65-1.75 (2H, m),
1.92-2.00 (2H, m), 2.94 (3H, s), 3.06 (3H, s), 3.00-3.10 (2H,
m), 3.45-3.53 (2H, m), 3.73-3.80 (1H, m), 3.85(3H, s), 4.71
(2H, d, J ) 5.6 Hz), 7.16 (1H, d, J ) 8.6 Hz), 7.44 (1H, d, J )
8.6 Hz), 7.59 (1H, bs), 8.12 (1H, dd, J ) 8.6, 1.0 Hz), 8.17 (1H,
d, J ) 8.6 Hz), 8.86 (1H, bs), 10.10 (1H, bs), 13.85 (1H,bs); MS
m/e (FAB) 470 (MH+). Anal. (C24H28ClN5O3‚HCl‚H2O) C, H,
N.
Refer en ces
(1) Beavo, J . A. Cyclic Nucleotide Phosphodiesterases: Functional
Implications of Multiple Isoforms. Physiol. Rev. 1995, 75, 725-
748.
En zym e Sou r ce a n d P h osp h od iester a se Activity As-
sa y. PDE5 was separated from the supernatant of a homoge-
nate of porcine platelets or porcine lung by DEAE-Toyopearl
650S chromatography.9
PDE activity was determined by a modification of a previ-
ously described two-step radioisotopic procedure.14 [3H]cGMP
at a concentration of 1 µM was used as a substrate. The tested
compounds were dissolved in DMSO and diluted with the
assay buffer to give concentrations ranging from 10-10 to 10-6
M. The final concentration of DMSO was 1% (v/v).
Va sor ela xa n t Effect in Isola ted P or cin e Cor on a r y
Ar ter ies P r econ tr a cted w ith P GF 2r. Porcine coronary
arteries were removed, freed from adjacent tissues, and cut
into rings with great care to avoid damage to the endothelium.
The rings were longitudinally opened and mounted in organ
baths containing 10 mL of Krebs-Henseleit solution (37 °C,
pH 7.4, bubbled with 95% O2-5% CO2). The coronary arterial
strips were allowed to equilibrate under a resting tension of 1
g. The presence of intact endothelial cells was confirmed by
bradykinin-induced (final concentration, 10-8 M) relaxation in
preparations precontracted with KCl (final concentration, 50
mM). The strips were contracted with PGF2R (final concentra-
tion, 10-5 M), and after the attainment of a plateau contrac-
tion, cumulative concentration-relaxation curves for a test
compound were constructed. Relaxation was calculated as a
percentage of the contractile response to PGF2R.
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pressure (380 mmHg) in a hypobaric chamber for 3 weeks to
induce hypoxic pulmonary hypertension as described previ-
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anesthetized with intramuscular ketamine (90 mg/kg) and
pentobarbital sodium (15 mg/kg) for catheterization of the
pulmonary and right carotid arteries and right jugular vein.
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Kohden, Tokyo, J apan).
The hemodynamic measurements were performed under
normoxic conditions. Either 1, 3, 10, or 30 µg/kg of 13 or its
vehicle (5% glucose solution containing 5 mM phosphoric acid)
was intravenously injected into conscious chronic hypoxic rats.
Pulmonary and systemic arterial pressures were monitored
for 90 min. At the end of the study, the rat was killed by an
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