Studies of cardiotonic agents. 8. Synthesis and biological activities of optically active 6-(4-(benzylamino)-7-quinazolinyl)-4,5-dihydro-5-methyl- 3(2H)-pyridazinone (KF15232)

Article abstract of DOI:10.1021/jm950197j

We previously reported that (±)-6-(4-(benzylamino)-7-quinazolinyl)-4,5- dihydro-5-methyl-3(2H)-pyridazinone ((±)-1, KF15232) showed potent cardiotonic activity with a strong myofibrillar Ca²⁺-sensitizing effect. As an extension of our work, we

Full text of DOI:10.1021/jm950197j

J . Med. Chem. 1996, 39, 297-303
297
Stu d ies of Ca r d ioton ic Agen ts. 8. Syn th esis a n d Biologica l Activities of
Op tica lly Active 6-(4-(Ben zyla m in o)-7-qu in a zolin yl)-4,5-d ih yd r o-5-m eth yl-
3(2H)-p yr id a zin on e (KF 15232)
Yuji Nomoto,* Haruki Takai, Tetsuji Ohno, Ken Nagashima, Kozo Yao, Koji Yamada, Kazuhiro Kubo,
Michio Ichimura, Akira Mihara, and Hiroshi Kase
Pharmaceutical Research Laboratories, Fuji, Kyowa Hakka Co., Ltd., 1188 Shimotogari, Nagaizumi-cho,
Sunto-gun, Shizuoka 411, J apan
Received March 17, 1995X
We previously reported that (()-6-(4-(benzylamino)-7-quinazolinyl)-4,5-dihydro-5-methyl-3(2H)-
pyridazinone ((()-1, KF15232) showed potent cardiotonic activity with a strong myofibrillar
Ca2+-sensitizing effect. As an extension of our work, we attempted to synthesize optically active
1. (()-4-(4-(Benzylamino)-7-quinazolinyl)-3-methyl-4-oxobutyric acid (-)-menthyl ester (6) was
separated into both diastereoisomers, and each was converted to optically pure 1 (>99% ee) in
an enantioselective manner. In order to determine the absolute configuration of the isomers,
an alternative synthesis of optically active 1 was employed. The precursor of (-)-1 ((+)-9)
was obtained by enantioselective synthesis from (R)-D-alanine. Consequently, we concluded
that the absolute configuration of (-)-1 at the 5-position of the pyridazinone ring was R. The
cardiotonic effects and inhibitory activities to PDE III and V of racemic 1 and (-)-1 were more
potent than those of (+)-1. These compounds also demonstrated greater vasorelaxant effects
in guinea pig aorta. In contrast, (+)-1 showed only weak cardiotonic and vasodilating effects,
although the compound displayed potent Ca2+-sensitizing activity. Racemic and (-)-1 attracted
our interest for the treatment of congestive heart failure.
In tr od u ction
failure using dual PDE III and V inhibitors, because
such compounds may be expected to have combined
inotropic-vasodilatory and diuretic action.
The extensive search to find a non-glycoside, non-
catecholamine replacement for digitalis led to the
discovery of several new cardiotonic drugs.1 We have
been investigating novel pyridazinones as cardiotonic
agents, and we previously reported the synthesis and
structure-activity relationships for the cardiotonic and
myofibrillar Ca2+-sensitizing activities of (()-6-(4-(ben-
zylamino)-7-quinazolinyl)-4,5-dihydro-5-methyl-3(2H)-
pyridazinone ((()-1, KF15232) and related compounds.2,3
In these studies, we found that (()-1 showed the most
potent cardiotonic and myofibrillar Ca2+-sensitizing
activities in the series, but structural requirements for
cardiotonic activity were different from those for myo-
fibrillar Ca2+-sensitizing activities on the basis of the
structure-activity relationship study of the related
compounds.3
Previously, the myofibrillar Ca2+-sensitizing effects
of some 4,5-dihydro-3(2H)-pyridazinone cardiotonics,
such as pimobendan, and MCI-154 were reported.4,5 It
can be speculated that the Ca2+-sensitizing effect is
responsible, at least in part, for the mechanism of the
inotropic action, and the risk of arrhythmogenic action
of compounds which show a Ca2+-sensitizing effect in
addition to a cardiac PDE III inhibitory activity that
might be lower than that of pure PDE III inhibitors.6-8
In a previous paper, we described (()-1 as having
inhibitory properties on bovine aortic PDE III and PDE
V.11 The present paper describes the synthesis and
biological activities of the racemic and optically active
1.
Ch em istr y
Resolu tion of th e Men th yl Ester 6 a n d Cycliza -
tion of Its Dia ster eom er s to Op tica lly Active 1.
Optically active 1 were synthesized by the route shown
in Scheme 1. (()-4-(4-Bromo-3-nitrophenyl)-3-methyl-
4-oxobutyric acid (2)12 was converted to the diastereo-
meric mixture of (-)-menthyl ester 3. Without sepa-
ration of the mixture, 3 was treated with CuCN to give
cyanide 4, which was reduced with TiCl3 to anthra-
nilonitrile 5. Treatment of 5 with triethyl orthoformate
in the presence of pyridinium hydrochloride followed by
heating with benzylamine afforded quinazoline 6. The
diastereomixture (6) was separated by flash column
chromatography to give 6a and 6b, which were purified
by recrystallization to >99% de. Compound 6a , which
was formerly eluated with hexane-AcOEt, was cyclized
by treatment with a mixture of hydrazine hydrate and
acetic acid (1:2 v/v) in EtOH at 10 °C to afford optically
pure (+)-1 (>99% ee). The other diastereomer (6b) was
converted to (-)-1 (>99% ee) under similar conditions.
Zaprinast, a PDE V (cGMP-PDE) inhibitor, was
reported to have depressor and natriuretic effects9 and
was also known as an enhancer of the natriuretic
activity of atriopeptin by inhibition of cGMP degrada-
tion.10 Tradiational therapy for congestive heart failure
includes a combination of diuretics and digitalis. We
are interested in drug therapy for congestive heart
Deter m in a tion of Absolu te Con figu r a tion of
Op tica lly Active 1. We attempted to synthesize opti-
cally active 9 from the chiral amino acid in order to
determine the absolute configuration of 1.
Friedel-Crafts acylation of bromobenzene with (R)-
(-)-2-chloropropionyl chloride (7)13 derived from D-
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0022-2623/96/1839-0297$12.00/0 © 1996 American Chemical Society

298 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Nomoto et al.
Sch em e 1
Sch em e 2
Sch em e 3
alanine afforded (R)-(-)-4-bromo-R-chloropropiophenone
(8) (>99% ee). Compound (R)-(-)-8 was converted to
the 4-oxobutyric acid methyl ester ((R)-(+)-9) (32% ee)
using the method described by Owings et al.,14 although
a significant loss of enantiomeric purity occurred (Scheme
2).
Biologica l Resu lts
Ca r d ioton ic Activities. The in vitro properties of
racemic and optically active 1 were evaluated in isolated
guinea pig papillary muscles (Figure 1). All the test
compounds produced concentration-dependent increases
in developed tension. Racemic and (-)-1 produced
about 30% increases at a concentration of 30 µM, which
were significantly more potent than (+)-1.
We next evaluated the compounds for cardiotonic
activities in anesthetized guinea pigs. The effects of the
compounds were determined by measuring the percent
increase in the maximum dP/dt left ventricular pressure
(LV dP/dtmax, ∆%) after iv administration. The results
are expressed as absolute percentage increases in dP/
dtmax as shown in Figure 2. The administration of
0.001-0.1 mg/kg of racemic 1 and (-)-1 and 0.01-1.0
mg/kg of (+)-1 showed a dose-dependent increase in dP/
dtmax. The positive inotropic effects of the compounds
were probably not due to reflex but direct effects,
because there were minor increases in heart rate (less
than 16% even at maximum dose, data not shown).
Racemic 1 and (-)-1 also demonstrated potent and
On the other hand, the 4-oxobutyric acid ((+)-10
(>99% ee)) was obtained by recrystallization of the
diastereomeric salt formed by treatment of racemic 1022
with (+)-benzylmethylamine followed by hydrolysis.
After esterification of (+)-10 with TMSCH2N2, the chiral
HPLC behavior of the product was identical with that
of the methyl ester ((R)-(+)-9), and not identical with
(-)-9. Therefore, the absolute configuration of (+)-10
was R. The nitration of (R)-(+)-10 afforded (R)-(+)-2
(>99% ee). This compound was converted to 6b ((R)-6,
>99% de), which is the precursor of (-)-1, in several
steps using a similar method as described for (RS)-6
without racemization (Scheme 3).
On the basis of these findings, we concluded that the
absolute configuration of (-)-1 at the 5-position of the
pyridazinone ring was R.

Studies of Cardiotonic Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 299
Ta ble 1. Inhibitory Effects of 1 on Various PDEs
inhibn activities (IC50, nM)
canine
bovine
bovine
bovine
compd
heart III
arterial III
arterial IV
arterial V
rolipram
cGMP
milrinone
(()-1
100
>10000
2400
3000
47
4.9
>1000
100
1000
50
5
300
10000
>10000
>10000
>10000
5000
46
10
(-)-1
(+)-1
1000
F igu r e 1. Positive inotropic effects of 1 in electrically driven
papillary muscles from guinea pigs. Each point represents the
mean ( SEM of seven preparations. *p < 0.05, **p < 0.01 vs
(S)-(+)-1.
F igu r e 4. Effects of 1 on calcium-activated force in skinned
papillary muscle fibers from guinea pigs. Data are expressed
as percentage of the force development induced by 10 µM
trifluoperazine. Each point represents the mean ( SEM of
six preparations. *p < 0.05 vs (S)-(+)-1.
Myofibr illa r Ca 2+ Sen sitizin g Activity. We ex-
amined the myofibrillar Ca2+-sensitizing effect in skinned
muscle fibers from guinea pig papillary muscles on the
tension development induced by pCa (-log[Ca2+] M)
5.8.3,15 The potency of Ca2+-sensitizing activity of the
test compounds was compared with trifluoperazine (5
× 10-5 M). Relative potency was calculated as the
change in tension of each compound to that of trifluo-
perazine (TFP)16 (TFP ) 100%). This protocol compen-
sates for any variation in fiber and facilitates compari-
son of activity among the test compounds. The relative
tension developement was significantly increased by all
the test compounds (1 × 10-6 to 1 × 10-4 M) in a
concentration-related manner (Figure 4). In contrast
to the cardiotonic activity, (+)-1 showed the most potent
Ca2+-sensitizing effect of these compounds.
F igu r e 2. Effects of intravenously injected 1 on LV dP/dtmax
in anesthetized guinea pigs. Each point represents the mean
( SEM of four experiments. *p < 0.05, **p < 0.01 vs (S)-(+)-
1.
Va sor ela xa n t Activity. Compounds (-)-1 and (+)-1
relaxed the aortic rings, which were contracted by 10
µM of l-phenylephrine (PHE), in a concentration-de-
pendent manner (Figure 5). The vasorelaxant effect of
(-)-1 was more potent than that of (+)-1 and was
significant at 1 µM.
F igu r e 3. Effects of orally administered 1 (0.1 mg/kg) on LV
dp/dtmax in conscious dogs.
Va scu la r Cyclic Nu cleotid e Levels. The aortic
rings treated with either (-)-1 or milrinone showed
higher cAMP and cGMP levels than those treated with
DMSO (control). Cyclic AMP levels increased with 1
and 10 µM of (-)-1 and were almost equal to those with
10 and 100 µM milrinone, respectively. In contrast,
(-)-1 increased cGMP levels more potently than mil-
rinone (Figure 6).
long-lasting cardiotonic activity in conscious dogs fol-
lowing oral administration at a dose of 0.1 mg/kg, while
(+)-1 showed no activity at the same dose (Figure 3).
In h ibitor y Activities for P DEs. We investigated
the ability of the compounds to inhibit canine heart PDE
III17 and bovine arterial PDE III, IV, and V18 (Table 1).
Racemic 1 and (-)-1 potently inhibited bovine arterial
PDE III and V and canine heart PDE III but not PDE
IV. The IC50 values of (-)-1 for these enzymes were 5,
10, and 4.9 nM and >10 µM, respectively, and were
lower than those of (+)- and (()-1. The inhibitory effect
of (+)-1 on PDE III was not detected even at 1000 nM.
Discu ssion
The inhibition of cardiac PDE III represents the
mechanism of action of cardiotonic activity of mil-
rinone19 and pyridazinones such as imazodan20 and

300 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Nomoto et al.
rinone increased cAMP levels in guinea pig aorta.
Increase in guinea pig aortic cAMP levels of (-)-1 and
milrinone showed only 10-fold difference, whereas IC50
values of them for inhibition of bovine aortic PDE III
showed 200-fold difference. We cannot clearly explain
this discrepancy. However, milrinone inhibited PDE IV,
which also modulates vascular cAMP level, with IC50
value of 10 µM. Not only PDE III but also PDE IV
dependent mechanism may be involved in the increasing
effects of milrinone on guinea pig aortic cAMP.
Zaprinast, a PDE V specific inhibitor, also decreases
vascular tone by elevation of the cGMP level.9 In both
bovine aortic PDE III and PDE V the inhibitory activi-
ties of (-)-1 were more potent than those of (+)-1. The
PDE inhibitory activities probably account for the
difference in vasorelaxant activities between (-)-1 and
(+)-1. The cAMP-dependent vasodilating effects of PDE
III inhibitors are beneficial for treatment of congestive
heart failure by reducing preload and/or afterload. A
guanylate cyclase activator isosorbide dinitrate also
improves congestive heart failure.23 Furthermore, the
depressor and natriuretic actions of zaprinast appear
to be mediated by an increase in cGMP. We, therefore,
consider that a dual inhibitor of PDE III and PDE V
may be a more effective agent for congestive heart
failure than currently available pure PDE III inhibitors.
Few compounds are known which have potent inhibitory
activities on both PDE III and V. Racemic 1 and (-)-1
demonstrated potent inhibitory activity on vascular
PDE V in addition to both cardiac PDE III and vascular
PDE III. Compound (-)-1 actually increased cGMP
levels in guinea pig aorta more potently than milrinone.
Consequently, the inhibition of PDE V by racemic 1 and
(-)-1 might play an additional beneficial role in the
treatment of congestive heart failure. Further biological
and pharmacological studies of these compounds will
be reported elsewhere.
F igu r e 5. Vasorelaxant effects of 1 on the phenylephrine-
induced contraction in guinea pig aorta. Each point represents
the mean ( SEM of five or six preparations. *p < 0.01 vs (S)-
(+)-1.
F igu r e 6. Increases in cyclic nucleotide levels (cAMP and
cGMP) by (-)-1 and milrinone in guinea pig aortic rings. Each
column is the mean of three or four experiments and vertical
bar indicates SEM.
indolidan21 and is believed to be the principal component
of the inotropic-vasodilatory action. The in vitro and
in vivo positive inotropic effects of (-)-1 in both dogs
and guinea pigs were more potent than those of (+)-1.
In addition, the order of the positive inotropic activity
of the compounds was consistent with that of the
inhibitory activity of heart PDE III. These results
indicate that the PDE III inhibitory activity of (-)-1
plays an important role in the cardiotonic activity of
racemic 1. It had been earlier demonstrated at Smith-
Kline Beecham that (R)-(-)-4,5-dihydro-6-[4-(1,4-dihy-
dro-4-oxo-1-pyridyl)phenyl]-5-methyl-3(2H)-pyridazino-
ne (SK&F 95654) is about 80-100 times more active
toward inhibition of PDE III than the corresponding (S)-
isomer.14 The (R)-configuration may, therefore, be
essential for the inhibition of PDE III in the pyridazi-
none series.
In summary, we synthesized optically active 1 by the
resolution of precursor 6 followed by enantioselective
cyclization and determined the absolute configuration
of (-)-1. Compound (-)-1 was more potent in its
cardiotonic and inhibitory activities on PDE III and V
than (+)-1. Compound (+)-1 showed only weak cardio-
tonic activities although the compound demonstrated
potent Ca2+ sensitizing activity. Racemic 1 and (-)-1
attracted our interest for the treatment of congestive
heart failure.
Exp er im en ta l Section
All melting points were determined on a Bu¨chi 510 micro
melting point apparatus and are uncorrected. Infrared (IR)
spectra were measured on a Shimadzu IR-27G spectropho-
tometer. 1H-NMR spectra were measured on a Varian EM390,
a Hitachi R-90H, and a J EOL J NM-GX-270 spectrometer using
tetramethylsilane (TMS) as an internal standard. Mass
spectra (MS) were run on a J EOL-J MS-01SG-2 and a J MS-
SX102 spectrometer. Optical purity was determined by HPLC
(CHIRALCEL OD, 0.46 cm i.d. × 25 cm; eluent, EtOH-
hexane) (Daicel Chemistry Co., Ltd.). Carboxylic acids were
analyzed after esterification by treatment with TMSCH2N2.
Diastereomeric purity was determined by normal phase HPLC
(YMC Pack SIL, 1.0 cm i.d. × 30 cm; eluent, AcOEt-hexane).
(()-4-(4-B r o m o -3-n it r o p h e n y l)-3-m e t h y l-4-o x o b u -
tyr ic Acid (-)-Men th yl Ester (3). A mixture of (()-4-(4-
bromo-3-nitrophenyl)-3-methyl-4-oxobutyric acid ((()-2) (75.1
g, 238 mmol), (-)-menthol (225 g, 1.4 mol), and p-TsOH‚H2O
(15 g) was heated to 110 °C for 6 h. AcOEt and H2O were
added, and the mixture was partitioned. The organic layer
In contrast to PDE III inhibitory and cardiotonic
activities, (+)-1 showed the most potent Ca2+-sensitizing
activity of these compounds. This result corresponds
well with our finding3 that the structural requirements
for cardiotonic activity were different from those for
myofibrillar Ca2+-sensitizing activity. In this study, we
found that the Ca2+-sensitizing activity of the optical
isomers was not consistent with the cardiotonic activi-
ties of them, although it was previously reported that
both the positive inotropic and Ca2+-sensitizing effects
of (-)-pimobendan were more potent than those of its
(+)-isomer.7 This finding indicates that the Ca2+ sen-
sitizing effect is not a major mechanism of cardiotonic
action of (-)-1 and racemic 1.
PDE III inhibitors generally dilate blood vessels by
increasing cAMP levels. In this study, (-)-1 and mil-

Studies of Cardiotonic Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 301
was dried over MgSO4 and concentrated, and then (-)-menthol
was removed by distillation at 170 °C under reduced pressure
to afford residual crude 3 as an oil. The crude product was
used in the next reaction without further purification. An
analytical sample was prepared by silica gel column chroma-
tography purification (hexane-AcOEt ) 3:1). HRMS: calcd
for C21H29BrNO5 454.1230, found 454.1223.
ee; mp 66 °C; [R]25 ) -40.4° (c ) 1.0, MeOH). Anal. (C9H8-
D
BrClO) C, H, N.
(R)-(+)-Meth yl 4-(4-Br om op h en yl)-3-m eth yl-4-oxobu -
tyr a te ((R)-(+)-9). Potassium tert-butoxide (0.4 g) was added
to a solution of diethyl malonate (0.46 mL) in DMF (10 mL),
and the mixture was stirred for 30 min at room temperature.
After addition of (R)-(-)-8 (>99% ee, 0.25 g, 1.0 mmol), the
reaction mixture was stirred for 15 min at same temperature,
and then the reaction was quenched with phosphate buffer
(pH 7). After stirring for 15 min, the mixture was partitioned
between AcOEt and H2O. The water layer was adjusted to
pH 4 with 1 N HCl and extracted with CHCl3. The extract
was dried over MgSO4 and concentrated to dryness. The
residue was purified by silica gel column chromatography to
(()-4-(4-Cya n o-3-n it r op h en yl)-3-m et h yl-4-oxob u t yr ic
Acid (-)-Men th yl Ester (4). A mixture of 3 (11.8 g, 26 mmol)
and CuCN (3.0 g, 31 mmol) in DMF (50 mL) was stirred at
130 °C for 30 min. AcOEt (50 mL) and hexane (200 mL) were
added to the mixture. After removal of the precipitated copper
salt, the mixture was washed with H2O, dried over MgSO4,
and concentrated under reduced pressure to give crude 4 (10.0
g) as an oil. The crude 4 was used in the next reaction without
further purification. An analytical sample was obtained by
silica gel column chromatography purification (hexane-AcOEt
) 2:1). HRMS: calcd for C22H29N2O5 401.2077, found 401.2083.
(()-4-(3-Am in o-4-c ya n op h e n yl)-3-m e t h yl-4-oxob u -
tyr ic Acid (-)-Men th yl Ester (5). TiCl3 (20% aqueous
solution) (85 mL) was added to a solution of 4 (10.0 g) in
acetone (350 mL) portionwise. The mixture was concentrated
to half volume and partitioned between AcOEt and H2O. The
organic layer was washed with saturated NaHCO3 and H2O,
dried over MgSO4, and concentrated to dryness to afford crude
5 (11.0 g, 84%). The crude product was used in the next
reaction without further purification. An analytical sample
was obtained by silica gel column chromatography purification
(hexane-AcOEt ) 2:1). HRMS: calcd for C22H30N2O3 370.2256,
found 370.2262.
give 0.05 g (18%) of oily (R)-(+)-9: 32% ee; [R]25 ) 3.3° (c )
D
1.0, MeOH); HRMS calcd for C12H13 79BrO3 284.0048, found
284.0033.
The chiral HPLC behavior of this compound was identical
with that of the methyl ester ((+)-9) derived from (+)-10 which
was obtained by optical resolution (see below).
Resolu tion of (()-4-(4-Br om op h en yl)-3-m eth yl-4-ox-
obu tyr ic Acid ((()-10). A mixture of (()-10 (271 mg, 1.0
mmol) and (()-R-methylbenzylamine (0.13 mL, 1.0 mmol) was
suspended in EtOH (3 mL) and stirred for 2 h with ice cooling.
Precipitated crystals were collected by filtration and were
recrystallized from EtOH to give 76 mg (20%) of ((+)-R-
methylbenzyl)ammonium salt of crude (R)-(+)-10 (85% ee). The
product was recrystallized two times from EtOH to afford pure
(R)-(+)-10 (>99% ee): mp 134-136 °C; [R]
25D ) 8.7° (c ) 1.0,
DMF). This compound (0.70 g, 1.8 mmol) was dissolved in
MeOH (5 mL) and saturated NaHCO3 (50 mL) and partitioned
with CHCl3. The water layer was adjusted to pH 4 with 1 N
HCl and extracted with CHCl3. The organic layer was dried
over MgSO4 and concentrated. The residue was crystallized
(1R,2S,5R)-Men th yl (S)-4-(4-(Ben zylam in o)-7-qu in azoli-
n yl)-3-m eth yl-4-oxobu tyr a te (6a ) a n d (1R,2S,5R)-Men -
th yl (R)-4-(4-(Ben zyla m in o)-7-qu in a zolin yl)-3-m eth yl-4-
oxobu tyr a te (6b). A mixture of 5 (41.0 g, 111 mmol), triethyl
orthoformate (50 mL), and pyridinium hydrochloride (1.5 g)
in DMF (250 mL) was stirred for 30 min at room temperature.
Benzylamine (90 mL) was added to the mixture, and the
resultant mixture was stirred at 110 °C for 2 h and then
concentrated to dryness under reduced pressure. The residue
was separated by silica gel flash column chromatography (2.5
kg, hexane-AcOEt ) 3:1-1:1) to give 14.4 g (90% de) of 6a ,
15 g of 6, and 18.7 g (95% de) of 6b. Crude 6a (90% de) was
recrystallized four times from Et2O-hexane to afford 3.8 g
(7.0%) of pure 6a (>99% de), and crude 6b (95% de) was
recrystallized from Et2O-hexane to give 12.5 g (23%) of pure
6b (>99% de).
from hexane to give 0.33 g of (R)-(+)-10 (68%): >99% ee; [R]25
) 13.5° (c ) 1.0, MeOH).
D
E n a n t ioselect ive Con ver sion of (R)-(+)-10 t o (R)-6.
Nitr a tion of (R)-(+)-10. Compound (R)-(+)-10 (>99% ee)
(0.20 g, 0.74 mmol) was added portionwise to fuming HNO3
(2 mL), and the reaction mixture was stirred at room temper-
ature for 15 min. H2O was added to the mixture and then
extracted with AcOEt. The organic layer was dried over
MgSO4 and evaporated to dryness. The residue was crystal-
lized from Et2O-hexane to give 0.16 g of (R)-(+)-2 (68%):
>99% ee; [R]25 ) 17.1° (c ) 1.0, MeOH).
D
Con ver sion of (R)-(+)-2 t o (R)-6. Compound (R)-(+)-2
(>99% ee) was converted to (R)-6 (>99% de) in several steps
using the method described for (RS)-6.
6a : mp 69-72 °C; [R]25 ) -28.1° (c ) 1.0, DMF). Anal.
D
(C30H37N3O3) C, H, N. 6b: mp 112-120 °C. [R]25 ) -29.7°
D
(c ) 1.0, DMF). Anal. (C30H37N3O3) C, H, N.
P h a r m a cologica l Meth od s. Ma ter ia l. The optical puri-
ties of the test compounds used in pharmacological tests were
99% ee for (R)-(-)-1 and 96% ee for (S)-(+)-1.
Sta tistics. Intergroup differences were statistically ana-
lyzed using Mann-Whitney’s U-test (two groups) or Tukey’s
test (three groups). Differences were considered significant
at p < 0.05.
(R )-(-)-4,5-Dih yd r o-5-m e t h yl-6-(4-(b e n zyla m in o)-7-
qu in a zolin yl)-3(2H)-p yr id a zin on e ((R)-(-)-1). A mixture
of hydrazine monohydrate-acetic acid (1:2 v/v) (60 mL) was
added to a solution of 6b (10.0 g, 21 mmol) in EtOH (300 mL)
at 10 °C and stirred for 60 h. AcOEt (1 L) was added, and the
mixture was washed with H2O, saturated NaHCO3, and H2O.
The organic layer was dried over MgSO4 and concentrated to
dryness under reduced pressure. The residue was crystallized
from MeOH-H2O to give 3.63 g (51%) of (-)-1 (>99% ee). An
analytical sample was obtained by recrystallization from
In Vitr o Stu d ies. P ositive In otr op ic Activity. Male
guinea pigs, weighing 250-400 g, were anesthetized with
sodium pentobarbital (50 mg/kg iv) and exsanguinated. The
hearts were rapidly excised. Right ventricular papillary
muscles were dissected and suspended isometrically in organ
baths containing Krebs-Henseleit solution (composition in
mM: NaCl, 133.5; KCl, 4.96; CaCl2, 2.5; NaH2PO4, 1.35;
NaHCO3, 16.3; MgSO4, 0.61; glucose, 7.77 (pH 7.4)) maintained
at 35 °C, gassed with 95% O2-5% CO2. Isometric contractile
force was measured with a force transducer (Nihon Kohden,
TB-611T, Tokyo, J apan) under a resting tension of 0.5 g and
recorded on a chart recorder (Yokogawa, Type 3066). The
papillary muscles were placed between two parallel platinum
plates and stimulated to contract by square-wave pulses of 2
ms at 50% above threshold voltage (Nihon Kohden, SEN-2201).
After a 90-min equilibration period, increasing concentrations
of racemic 1, (R)-(-)-1, or (S)-(+)-1 dissolved in dimethyl
sulfoxide (DMSO) were added cumulatively to baths at 10-
min intervals to provide concentration-response curves. In-
creases in developed tension are expressed as percent of the
predrug-developed tension.
EtOH-CH2Cl2: [R]25 ) -371° (c ) 1.0, DMF).
D
(S )-(+)-4,5-Dih yd r o-5-m e t h yl-6-(4-(b e n zyla m in o)-7-
qu in a zolin yl)-3(2H)-p yr id a zin on e ((S)-(+)-1). Compound
(S-)-(+)-1 (>99% ee) (0.47 g, 66%) was obtained from 6a (1.0
g, 2.1 mmol) using same procedure described above: [R]25
375° (c ) 1.0, DMF).
)
D
(R)-(-)-4-Br om o-r-ch lor opr opioph en on e ((R)-(-)-8). (R)-
(-)-2-Chloropropionyl chloride (7), bromobenzene (7.6 mL, 72
mmol), finely powdered AlCl3 (13.8 g, 100 mmol), and CH2Cl2
were mixed at 10 °C, and then the mixture was warmed to
room temperature and stirred for 2 h. The reaction mixture
was poured onto a mixture of 3 N HCl (100 mL) and ice (500
mL) and then extracted with CH2Cl2. The organic layer was
dried over MgSO4 and concentrated to dryness to afford crude
(R)-(-)-8 (12.1 g, 49%) as a solid. The crude product was used
in the next reaction without further purification. An analytical
sample was obtained by recrystallization from hexane: >99%

302 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Nomoto et al.
Ca 2+-Sen sitizin g Activity. Experiments were performed
at 24 °C. Male guinea pigs (Hartley) weighing 300-400 g were
killed by a blow on the head, and the hearts were excised. The
papillary muscles were isolated from the right ventricle in the
relaxing solution (RS) of the following composition (in mM):
KSO3CH3, 129; Mg(SO3CH3)2, 5.1; PIPES, 20; Na2ATP, 4.2;
EGTA, 2 (adjusted to pH 7.00 with KOH at 24 °C). A small
bundle (about 0.1-0.2 mm in diameter and 2-3 mm in length)
of muscle was prepared from the papillary muscles. One end
of the bundle was connected to a strain gauge transducer
(Nihon Koden, TB-611T) for measurement of isometric tension,
and the other end was fixed in an organ bath. Developed
tension was recorded on a chart recorder (Yokogawa). To
obtain a skinned fiber, the small bundle was treated with RS
containing 250 µg/mL saponin for 30 min.
At the beginning of the experiment, the fiber stretched in
RS until resting tension was 1-2 mg. After RS was removed,
the saponin-treated fiber was contracted with the activating
solution (AS, pCa(-log[Ca2+], M) ) 6.00) of the following
composition (in mM): KSO3CH3, 90; Mg(SO3CH3)2, 5.2; PIPES,
20; Ca(SO3CH3)2, 7.2; Na2ATP, 4.2; EGTA, 10; (adjusted to pH
7.00 with KOH at 24 °C) and then relaxed with RS. For
subsequent experiments, we used the fibers, which were
immediately relaxed to the basal level by the RS treatment,
as the skinned fibers.
that caused 50% inhibition of substrate hydrolysis (IC50) were
calculated from the concentration-inhibition curves.
Va sor ela xa n t Activity. Thoracic aortas were isolated
from male Hartley guinea pigs (500-600 g) and cut into 3-4-
mm rings. The aortic rings were mounted on metal holders
and suspended in Krebs-Henseleit solution (KHS) maintained
at 35 °C, gassed with 95% O2-5% CO2. Isometric contraction
was measured under a resting tension of 1.5 g.
The rings were contracted with 10 µM PHE. The contrac-
tion reached a plateau, after which cumulatively increasing
concentrations of the drugs tested, dissolved in DMSO, were
added to the bath. The vasorelaxation induced by the drugs
was measured.
Mea su r em en t of Va scu la r cAMP a n d cGMP Con ten ts.
Aortic rings from guinea pigs were contracted with 10 µM
PHE. Subsequently, each concentration of (-)-1 or milrinone,
dissolved in DMSO, was added to the organ bath. After the
maximum vasorelaxant effects were measured, the tissues
were immediately frozen in liquid nitrogen. The frozen tissues
were homogenized in ice-cold 6% trichloroacetic acid (TCA).
After centrifugation, TCA in the supernatant was extracted
with water-saturated ether. The content of cyclic nucleotides
in the TCA-removed supernatant was assayed by a radioim-
munoassay method.
In Vivo Stu d ies. An esth etized Gu in ea P igs. Male
Hartley guinea pigs (460-570 g) were anesthetized with
halothane-nitrous oxide and were artificially ventilated (60
breaths/min with a tidal volume of 10 mL/kg) with a respirator
pump. The left jugular vein was cannulated with a polyeth-
ylene tube for the administration of the drugs. To measure
left ventricular pressure (LVP), a heparin-filled polyethylene
catheter was inserted into the left ventricle through the left
carotid artery and coupled to a pressure transducer (MPC-
500, Millar Instruments). LV dp/dtmax was derived with an
electric differentiator. Increasing doses of racemic and opti-
cally active 1 were administered intravenously at 5-min
intervals. The maximal increase in LV dp/dtmax for each
concentration was recorded.
The skinned fiber was contracted with AS. When the
contraction reached a stable plateau (predrug value), 10 µM
TFP‚2HCl (Boeringer Mannheim) was added to the bath. The
skinned fiber was washed with RS and contracted again with
RS. The contacted fiber was treated with racemic or optically
active 1 dissolved in DMSO. The calcium-sensitizing effects
induced by the drugs were expressed as a percentage against
of the TFP-induced contraction.
Isola tion of cAMP P h osp h od iester a ses. Canine cardiac
cAMP PDE (PDE III and IV) were prepared by the procedure
of Weishaar and colleagues.17 Bovine arterial PDE III, IV, and
V were prepared by the method of Torphy and Cieslinski.18a
Assa y of Ca n in e Ca r d ia c P DE Activity. The reaction
buffer contained 50 mM BES-NaOH (pH 7.2) and 20 mM
MgCl2. The peak III fraction (40 µL) diluted with 60 µL of
diluent (50 mM BES-NaOH, 0.1 mg/mL soybean tryprin
inhibitor, pH 7.2) was added to the buffer in a final volume of
300 µL. The PDE activity of PDE III and PDE IV was
measured with 1 µM substrate ([3H]cAMP) in the presence of
25 µM cGMP and 10 µM rolipram, respectively.
After incubation at 30 °C for 10 min, the reaction was
stopped by the addition of 100 µL of HCl solution (0.25 M)
and then neutralized in an ice bath. The reaction mixture was
incubated at 30 °C for additional 10 min with 100 µL of 100
µg/mL 5′-nucleotidase and subsequently applied to a 1 mL
DEAE-Sephadex A-25 column. The radioactivity of the eluate
containing [3H]adenosine was determined by a liquid scintil-
lation counter.
Con sciou s Dogs. Adult mongrel dogs of either sex weigh-
ing 10-15 kg were anesthetized with pentobarbital sodium
(35 mg/kg iv). A Millar Micro-Tip pressure transducer (MPC-
500, Millar Instruments) was tunneled subcutaneously from
the back to the neck and implanted into the left ventricle
through the right carotid artery. After 2 days for recovery,
the concious dogs were placed consciously in a small cage. The
pressure transducer was coupled to a connector, and LVP and
LV dp/dtmax were measured. Racemic and optically active 1,
diluted 100 times with lactose, were administered orally in
gelatin capsules. The parameters were recorded for 8 h after
administration of the drugs.
Refer en ces
Assa y of Bovin e Aor tic P DE Activity. The reaction
buffer contained 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1
mM dithiothreitol, and 20 mM NaCl. The PDE V fraction or
the PDE III and IV mixture fraction (40 µL) was added to the
buffer in a final volume of 300 µL. The PDE V activity was
measured with 1 µM [3H]cGMP in the presence of 3 mM EGTA.
The PDE activity of PDE III and PDE IV was assayed with 1
µM [3H]cAMP in the presence of 20 µM cGMP and 20 µM
rolipram, respectively. After incubation at 30 °C for 30 min,
the reaction was stopped by the addition of 100 µL of HCl
solution (0.25 M) and then neutralized in an ice bath. The
reaction mixture was incubated at 30 °C for additional 30 min
with 100 µL of 100 µg/mL 5′-nucleotidase and subsequently
applied to a 1 mL DEAE-Sephadex A-25 column. The radio-
activity of the eluate containing [3H]guanosine or [3H]adenos-
ine was determined by a liquid scintillation counter.
(1) Taylor, M. D.; Sircar, I.; Steffen, R. P. Agents for the treatment
of congestive heart failure. Annu. Rep. Med. Chem. 1987, 22,
85-94.
(2) Nomoto, Y.; Takai, H.; Ohno, T.; Kubo, K. Studies on cardiotonic
agents. VI. Synthesis of novel 4,5-dihydro-3(2H)-pyridazinone
derivatives carrying some benzoheterocycles at the 6-position.
Chem. Pharm. Bull. 1991, 39, 352-357.
(3) Nomoto, Y.; Takai, H.; Ohno, T.; Kubo, K. Studies on cardiotonic
agents. VII. Potent cardiotonic agent KF15232 with myofibril-
lar Ca2+ sensitizing effect. Chem. Pharm. Bull. 1991, 39, 900-
910.
(4) Austel, V.; Heider, J .; Eberlein, W.; Diederen, W.; Haarman, W.
U.S. Patent 4 361 563; Chem. Abstr. 1983, 98, 72123p.
(5) Narimatsu, A.; Kitada, Y.; Satoh, N.; Suzuki, R.; Okushima, H.
Cardiovascular pharmacology of 6-[4-(4′-pyridyl)aminophenyl]-
4,5-dihydro-3(2H)-pyridazinone hydrochloride, a novel and po-
tent cardiotonic agent with vasodilator properties. Arzneim.-
Forsch. 1987, 37, 398-406.
(6) Kitada, Y.; Narimatsu, A.; Suzuki, R.; Endoh, M.; Taira, N. Does
the positive inotropic action of a novel cardiotonic agent, MCI-
154, involve mechanisms other than cyclic AMP? J . Pharmacol.
Exp. Ther. 1987, 243, 639-645.
(7) Fujino, K.; Sperelakis, N.; Solaro, R. J . Differential effects of d-
and l-pimobendan on cardiac myofilament calcium sensitivity.
J . Pharmacol. Exp. Ther. 1988, 247, 519-523.
Mea su r em en t of P DE In h ibitor y Activity. The PDE
inhibitors tested were dissolved in dimethyl sulfoxide (DMSO).
The final concentration of DMSO was 1% in the reaction
mixture and inhibited PDE activity by less than 10%. In the
presence of the drugs, PDE activities of the four PDE fractions
were measured in duplicate. The concentrations of the drugs

Studies of Cardiotonic Agents
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 303
(8) Mu¨ller-Beckmann, B.; Freund, P.; Honerja¨ger, P.; Kling, L.;
Ru¨egg, J . C. In vitro investigation on a new positive inotropic
and vasodilating agent (BM 14.478) that increases myocardial
cyclic AMP content and myofibrillar calcium sensitivity. J .
Cardiovasc. Pharm. 1988, 11, 8-16.
(9) Mcmahon, E. G.; Palomo, M. A.; Mehta, P.; Olins, G. M.
Depressor and natriuretic effects of M&B22948, a guanosine
cyclic 3′,5′-monophosphate selective phosphodiesterase inhibitor.
J . Pharmacol. Exp. Ther. 1989, 251, 1000-1005.
(10) Wilkins, M. R.; Settle, S. L.; Needleman, P. Augmentation of
the natriuretic activity of exogenous and endogenous atriopeptin
in rats by inhibition of guanosine 3′,5′-cyclic monophosphate
degradation. J . Clin. Invest. 1990, 85, 1274-1279.
(11) Ohno, T.; Nagashima, K.; Moriyama, T.; Ishikawa, T.; Kubo, K.;
Yamada, K. Vasorelaxant properties of KF15232, a new phos-
phodiesterase inhibitor. Can. J . Physiol. Pharmacol. 1994, 72
(Suppl. 1), 153.
(12) Hauel, N.; Heider, J .; Stein, H.; Austel, V.; Riffen, M.; Diederen,
W.; Haarmann, W. Ger. Patent 3006671; Chem. Abstr. 1981, 95,
203981e.
(13) Fu, S.-C. J .; Birnbaum, S. M.; Greenstein, J . P. Influence of
optically active acyl groups on the enzyme hydrolysis of N-
acylated-L-amino acids. J . Am. Chem. Soc. 1954, 76, 6054-
6058.
(14) Owings, F. F.; Fox, M.; Kowalski, C. J .; Baine, N. H. An
enantioselective synthesis of SK&F 93505, a key intermediate
for preparing cardiotonic agents. J . Org. Chem. 1991, 56, 1963-
1966.
(15) Kitada, Y.; Narimatsu, A.; Matsumura, N.; Endoh, M. Increase
in Ca++ sensitivity of the contractile system by MCI-154, a novel
cardiotonic agent, in chemically skinned fibers from the guinea
pig papillary muscles. J . Pharmacol. Exp. Ther. 1987, 243, 633-
638.
(18) (a) Torphy, T. J .; Cieslinski, L. B. Characterization and selective
inhibition of cyclic nucleotide phosphodiesterase isozymes in
canine tracheal smooth muscle. Mol. Pharmacol. 1990, 37, 206-
214. (b) Kincarid, R. L.; Manganiello, V. C. Assay of cyclic
nucleotide phosphodiesterase using radiolabeled and fluorescent
substrates. Methods Enzymol. 1988, 159, 457-470.
(19) (a) Alousi, A. A.; Canter, J . M.; Montenaro, M. J .; Fort, D. J .;
Ferrari, R. A. Cardiotonic activity of milrinone, a new and potent
cardiac bipyridine, on the normal and failing heart of experi-
mental animals. J . Cardiovasc. Pharm. 1983, 5, 792-803. (b)
Alousi, A. A.; Stankus, G. P.; Stuart, J . C.; Walton, L. H.
Characterization of the cardiotonic effects of milrinone, a new
and potent cardiac bipyridine, on isolated tissues from several
animal species. J . Cardiovasc. Pharm. 1983, 5, 804-11.
(20) Bristol, J . A.; Sircar, I.; Moos, W. H.; Evans, D. B.; Weishaar,
R. E. Cardiotonic agents. 1. 4,5-Dihydro-6-[4-(1H-imidazol-
1-yl)phenyl]-3(2H)-pyridazinones: Novel positive inotropic agents
for the treatment of congestive heart failure. J . Med. Chem.
1984, 27, 1099-1101.
(21) Robertson, D. W.; Krushinski, J . H.; Beedle, E. E.; Wyss, V.; Don
Pollock, G.; Wilson, H.; Kauffman, R. F.; Hayes, J . S. Dihydro-
pyridazinone cardiotonics: The discovery and inotropic activity
of 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-py-
ridazinyl)-2H-indol-2-one. J . Med. Chem. 1986, 29, 1832-1840.
(22) McEvoy, F. J .; Allen, G. R., J r. 6-(Substituted phenyl)-5-
substituted-4,5-dihydro-3(2H)-pyridazinones. Antihypertensive
agents. J . Med. Chem. 1974, 17, 281-286.
(23) (a) Franciosa, J . A.; Goldsmith, S. R.; Chor, J . N. Contrasting
immediate and long-term effects of isosorbide dinitrate on
exercise capacity in congestive heart failure. Am. J . Med. 1980,
69, 559-566. (b) Leier, C. V.; Huss, P.; Magorien, R. D.;
Unverferth, D. V. Improved exercise capacity and differing
arterial and venous tolerance during chronic isosorbide dinitrate
for congestive heart failure. Circulation 1983, 67, 817-822.
(16) Sellin, L. C.; Alajoutsija¨rvi, A.; To¨rnquist, K.; Fraser, M.; Pippuri,
A.; Ojala, I. Inhibition of cardiac phosphodiesterase III by the
novel cardiotonic agent 6-[4-(4′-pyridyl)aminophenyl]-4,5-dihy-
dro-3(2H)-pyridazinone hydrochloride. Arzneim.-Forsh. 1988, 38
(II), 1787-1789.
(17) Weishaar, R. E.; Kobylarz-Singer, D. C.; Kaplan, H. R. Subclass
of cyclic AMP phosphodiesterase in cardiac muscle. J . Mol. Cell.
Cardiol. 1987, 19, 1025-1036.
J M950197J