This invention is related to novel vasomolol, compounds of this invention have the following formula wherein R.sub.1 represents C.sub.1-4 alkyl group. ##STR1## vasomolol is an ultra short-acting and vasodilatory selective .beta..sub.1 -adrenoceptor antagonist, and is devoid of ISA. Vasomolol is an utra short acting and vasodilatory selective .beta..sub.1 -adrenoceptor antagonist, and is devoid of IA. The ultra short-acting and vsodilatory .beta..sub.1 -adrenoceptor blocking activities of vasomolol-a guaiacoxypropanolamine derivative of vanillic acid ethyl ester--were studied. Vasomolol (0.5, 1.0, 3.0 mg/kg, I.v.) produced a dose-dependent bradycardia rsponse, and particularly a hypotensive action with an ultra short-acting property in pentobarbital-anesthetized normotensive rats. Vasomolol's steady-state of .beta.-blockade was attained within 10 min after initiation of an infuson and a rapid recovery from blockade took place after termination of the infusion. In isolated rat aorta, vasomolol (10.sup.-6 M-10.sup.-5 M) inhibit both henylephrine (10.sup.-6 M) and High K.sup.+ (75 mM)--induced smooth muscle contractions, concentration-dependently. This inhibitory effect of vasomolol was more sensitive on K.sup.+ than on phenylephrine-induced contractions.

BACKGROUND OF THE INVENTION 
.beta.-Adrenoceptor blockers are widely used in the treatment of 
cardiovascular disease, including hypertension, angina pectoris, 
supraventricular arrhythmias, hypertrophic cardiomyopathy, and myocardial 
infarction. All these .beta.-adrenoceptor blockers share the common 
feature of being competitive antagonists of .beta.-adrenoceptor. They 
differ, however, in additional pharmacological properties, such as in the 
.beta..sub.1 -/.beta..sub.2 -selectivity, in the presence or absence of 
intrinsic sympathomimetic activity(ISA), in the membrane stabilizing 
activity, in the lipid versus water solubility is reported in Mount Sinai 
Journal of Medicine 52(7), 553, 1985(by Squire, A. et al.) and in the 
pharmacokinetic properties is reported in Arzneim-Forch/Drug Res. 36(1), 
200, 1986 (by Harting, J. et al.). .beta.-adrenoceptor blockers with 
vasodilatory effects are also important for their calcium channel blocking 
properties as reported in Gen. Pharmacol. 24. 1425, 1993 (by inventor 
group). 
The ultra short-acting .beta.-blocker(USABB) was described to be much safer 
than longer-acting antagonists in critically ill patients who require a 
.beta.-adrenoceptor antagonist is reported in Med. Chem. 25, 1402, 1982 
(by Erhardt, P. W. et al.). This benefit is attributable to the fact that 
continuous infusion of USABB may achieve its steady-state concentrations 
quickly, and that the therapeutic actions terminate rapidly when the 
infusion is discontinued is reported in J. Pharm. Exp. Ther. 237, 912, 
1986 (by Quon, C. Y. et al.). Esmolol (formula I), a .beta..sub.1 
-selective adrenoceptor blocker rapidly metabolized by blood and liver 
esterase, was thus suggested for use as a USABB in controlling 
supraventricular arrhythmias, preoperative hypertension, and myocardial 
ischemia in acutely ill patients is reported in Drugs 33, 392, 1987 (by 
Benfield, P. et al.). USABB with vasodilatory properties was also of 
interest and seems to be of much greater significance than can be 
accounted for simply by .beta.-blockade alone is reported in Clin. 
Pharmacol. Ther. 38, 579, 1985 (by Reilly, C. S. et al.). Esterification 
of inactive metoprolol metabolite has shown various reduced and shorter 
duration systemic and .beta.-adrenergic blocking activities than 
metoprolol is reported by Bodor et al. 1988). Other esmolol-type, 
morpholino-type, and urea-type .beta.-blockers with USABB activities have 
also been reported recently in Chem. Pharm. Bull. 40, 1462, 1992 (by 
lguchi, S. H. et al.). 
.beta.-Adrenoceptor blockers derived from vanilloid type compounds have 
been partly described in previous reports (by inventor group, 1993). 
Vanilloid type .beta.-blockers are a family of guaiacoxypropanolamines, 
chemically with 3-methoxy, 4-hydroxy benzyl nucleus that are found in 
pungent principles. Those vanilloid type compounds may cause the 
activation of sensory and autonomic cardiovascular system in vivo, but 
alkylation of its 4-hydroxy benzyl nucleus are devoid of pungent activity 
and maintain vasodilatory effect, there reported in Eur. J. Med. Chem. 27, 
187, 1992 and 37, 938, 1994 (by inventor group). To date, very few 
.beta.-blockers with vanilloid base was described for its USABB activity 
(Iguchi, S. H. et al, 1992). The present study was further aimed at 
investigating the pharmacological properties of vasomolol, a derivative of 
vanillic acid or its ethyl ester with a vanilloid base, for its possible 
ability to bind to .beta.-adrenoceptors, its relative selectivity for 
.beta.-adrenoceptors, intrinsic sympathomimetic activity, and particularly 
its USABB and vasodilatory activities 
DETAILED DESCRIPTION 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide new vasomolol derivatives 
of formula II, their pharmaceutically acceptable salt and compositions 
comprising the same which are ultra short acting, vasodilatory selective 
.beta..sub.1 -drenoceptor antagonist, and is devoid of ISA in wherein 
R.sub.1 represents hydrogen, C.sub.1-4 alkyl group. 
Another object of the present invention is to provide vasodilatory 
selective .beta..sub.1 -adrenoceptor antagonist compositions and a method 
of treatment of patients in need of treatment. 
Another object of the present invention is to provide process for the 
preparation of the novel compounds and to pharmaceutically compositions 
comprising the same. 
I. Preparation Methods 
The preparation methods of formula II are shown in FIG. 2. The synthetic 
reactions are preferably carried out in the alkaline solution of starting 
materials, like vanillin, vanilliic acid and its ester compounds. 
According to the reaction scheme herein below (FIG. 2) vanilliic acid was 
synthesized from vanillin, then treated with epichlorohydrine gave 
compound 3. Amination compound 3 with butylamine in alcoholic solution 
produced the compound 4; esterized compound 4 with compound of formula III 
give compound of formula II. 
According to the reaction other scheme herein below (FIG. 2) compound of 
formula IV was synthesized from vanillin, or vanillic acid with compound 
of formula III separately, then treated with epichlorohydrine gave 
compound of formula V. Amination compound of formula V with butylamine in 
alcoholic solution produced the compound of formula II. on responds the 
process, the epichlorohydrine may be replaceable with epibromohydrine give 
the result for same alike produced. 
In the compound of formula II, III, IV and V in wherein R.sub.1 represents 
hydrogen, C.sub.1-4 alkyl group. These structures of all compounds 
described above were assigned according to the IR, .sup.1 H--NMR, .sup.13 
C--NMR, MS, elemental analytical data. 
The vasomolol derivatives of formula II according to the present invention 
and also their salts display useful pharmacological properties, Suitable 
salts of compounds of the formula II are the sodium salts, calcium salts, 
potassium salts, or magnesium salts etc. pharmaceutically acceptable 
salts. 
The formula II derivatives and pharmaceutically acceptable salt are ultra 
short acting, vasodilatory selective .beta..sub.1 -adrenoceptor 
antagonist, and is devoid of ISA. Those compounds are useful as a medicine 
for the prevention arrhythmia. The following tests are given for the 
pharmacological activity. 
II. Phamacological Activity 
Measurement of blood pressure and heart rate 
The method reported in Gen. Pharmacol. 24, 1425, 1993 (by inventor group), 
drug 
vasomolol (0.5, 1.0, 3.0 mg/kg) 
esmolol (300 .mu.g/kg/min) 
phenylephrine (10 .mu.g/klg, i.v.) 
Results 
Intravenous injection of vasomolol(0.5, 1.0, 3.0 mg/kg) produced a 
hypotensive action and a decrease in heart rate in 
pentobarbital-anesthetized Wistar rats (Table 1). Administrating esmolol 
in the same doses also showed similar to those of vasomolol. The 
bradycardia duration of both vasomolol and esmolol were a little less than 
10 min. In the contrast, propranolol produced a longer bradicardia effect 
which persisted for more than 60 min. Intravenous infusion of vasomolol 
and esmolol (300 .mu.g/kg/min) could not significantly inhibit 
phenylephrine 10 .mu.g /klg, i.v.)--induced pressor response in 
reserpinized rats (data not shown). 
Effects of vasomolol on .beta.-blockade and duration of action method 
Vasomolol (100 .mu.g/kg/min) was infused consecutively into a femoral vein 
in pentobarbital-anesthetized rats. Each perfusion was maintained for 60 
min. Before and during the infusion of vasomolol and esmolol, at 10-min 
intervals, bolus i.v. injections of (-) isoproterenol (0.5. .mu.g/kg) were 
administered to assess the level of .beta.-blockade. (-) lsoproterenol 
injections were also continued for 60 min. after termination of the 
highest dose of vasomolol and esmolol to assess the duration of 
.beta.-blockade reported in J. Pharm. Exp. Ther. 237, 912, 1986 (by Quon, 
C. Y. et al.). The percentage of inhibition of the 
(-)isoproterenol-induced tachycardia parameter of .beta.-blockade--was 
calculated during and after infusions of vasomolol. 
drug 
isoproterenol (0.5 .mu.g/kg) 
vasomolol (0.5, 1.0, 3.0 mg/kg) 
esmolol (300 .mu.g/kg/min) 
Results 
Repeated isoproterenol administrations in rats at 10 min intervals produced 
increases in heart rate that were reproducible in magnitude for a 30 min. 
period. The time courses of .beta.-adrenergic receptor blockade during 
infusions of vasomolol and esmolol are shown in FIG. 2. The onset of 
.beta.-blockade was rapid and steady-state level of blockade occurred 
within 10 min. after initiating each infusion rate and remained constant 
through each infusion period. .beta.-blockade caused by vasomolol and 
esmolol disappeared rapidly after the termination of infusion. The time 
required for 50% recovery averaged 9.5.+-.1.0 min.(mean.+-.S.E.M.). 
Effects of vasomolol on pheylephrine and high K.sup.+ induced contractions 
of isolated rat aorta 
Measurement of vasodilatation effect method 
Male rats weighing 200-250 g were sacrificed by a blow on the head and the 
thoracic aorta was removed and cleaned of all loosely adhering tissue. The 
thoracic aorta was cut from an area close to the aortic arch, immediately 
placed in a physiological solution, and trimmed to 2-4 mm in length as 
described in Gen. Pharmacol. 24, 1425, 1993 (by inventor group) The rings 
were then mounted with a tension of 1.0 g between parallel hooks in a 10 
ml organ bath containing a physiological solution(mM):NaCl 112; KCl5.0; 
NaHCO.sub.3 25; KH.sub.2 PO.sub.4 1.0; CaCl.sub.2 1.25; MgSO.sub.4 1.2; 
and glucose 11.5!. The bath was maintained at 37.degree. C. and aerated 
with a mixture of 95% O.sub.2 and 5% CO.sub.2. The tension of the aortic 
rings were recorded isometrically by means of a force displacement 
transducer (UGO, Model 7004, Italy) connected to an amplifier (COULBOURN, 
Model S72-25, U.S.A.). Before the start of the experiments, all 
preparations were allowed to equilibrate for 1.0 hr in the physiologic 
solution. 
Measurement of atrial rate and tension 
Guinea-pigs (Hartley) of either sex weighing between 350 and 500 g were 
sacrificed by a blow on the head. Their hearts and trachea were quickly 
excised and excess tissue was removed. Spontaneously-beating right atria 
were removed from the hearts and mounted in a 10 ml organ bath with one 
end fixed and the other end connected to a force displacement transducer 
(Grass, Model 13-6615-50, U.S.A). The frequency of contraction was 
measured on a separate channel by a tachometer (GOULD WindoGraf, Model 
40-8474-20, U.S.A). The experiments were carried out at 32.5.degree. C. in 
a Krebs solution of the following composition (mM) : NaCl 113, KCl 4.8, 
CaCl.sub.2 2.2, KH.sub.2 PO.sub.4 1.2, MgCl.sub.2 1.2, NaHCO.sub.3 25, 
Dextrose 11.0; bubbled with a 95% O.sub.2 -5% CO.sub.2 mixture. The atrial 
strip was prestretched to a baseline tension of 0.2 g. The atria were 
equilibrated for 90 min in an aerated (95% O.sub.2 -5% CO.sub.2) Krebs 
solution before the experimental protocols were initiated, For the 
assessment of .beta.-adrenergic blocking activity, a control cumulative 
concentration-response curve to the chronotropic effect of (-) 
isoproterenol was established. The atria were then allowed a 30-60 min 
washout period to estabilize, after which time various concentrations of 
the test compound were incubated with the atrium 30 min before the 
cumulative concentrations of the (-) isoproterenol (3.times.10.sup.10 
-10.sup.5 M) was added. All responses to (-) isoproterenol were calculated 
as a percentage of the maximum control response to (-) isoproterenol. 
In the isolated left atria, quiescent left atria were dissected free of 
connective tissue and mounted in organ chambers under a resting tension of 
0.5 g. Atria were bathed in an aerated Kreb's solution(32.5.degree. C.) 
and were driven at 2-s intervals via two platinum electrodes placed at 
either side of the atrium. .beta.-Adrenoceptor antagonist activity was 
determined as follows. Cumulative concentration-response curves to the 
positive inotropic effects of (-) isoproterenol were obtained in the 
absence and presence of various concentrations of a test compound. An 
incubation time of 30 min was allowed for the test compound. Data were 
calculated as a percentage of the increase in force induced by (-) 
isoproterenol. 
Results 
In the isolated rat thoracic aorta, contractions induced by phenylephrine 
(10.sup.5 M) and high K.sup.+ (75 mM) were significantly inhibited by the 
adding with vasomolol (10.sup.6 -10.sup.5 M) in the isolated rat aorta. 
The relaxation profit of vasomolol was more sensitive in high K.sup.+ 
than in phenylephrine-contracted aorta preparations (FIG. 3). 
Effects of vasomolol on .beta.-adrenoceptor activity in guinea-pig atria 
and trachea 
Contractility of isolated tracheal strips method 
Guinea-pig trachea were cleaned of extraneous connective tissue and cut 
into spiral strips as described in Gen. Pharmacol. 24, 1425, 1993 (by 
inventor group). This spiral strip was cut into two equal segments and 
both were suspended in organ baths filled with 20 ml of Kreb's solution. 
Temperature was maintained at 32.5.degree. C. and the solution was gassed 
with 95% O.sub.2 -5% CO.sub.2. An initial basal tension of 2 g was applied 
to each tracheal strip and the tissue was allowed to gain tone 
spontaneously until a steady level was reached (60 min). The tracheal 
preparations were pretreated with phenoxybenzamine (50 .mu.M for 30 min 
followed by thorough wash-out as described in Br. J. Pharmacol. 57, 369, 
1976 (by O'Donnell, S. R. et al.) to prevent extraneuronal uptake and to 
block .alpha.-drenoceptors. For the determination of .beta.-adrenoceptor 
antagonist activity, cumulative concentration-response curves to the 
relaxant effects of isoproterenol were obtained in the absence and 
presence of a test compound (60 min incubation time). Data were calculated 
as a percentage of the maximum relaxation induced by (-) isoproterenol. 
drug 
Vasomolol (5.times.10.sup.-8 -10.sup.-6 M) 
isoproterenol 
propranolol 
Results 
Vasomolol antagonized isoproterenol-induced positive chronotropic actions 
in isolated guinea-pig right atrial strips. Vasomolol (5.times.10.sup.-8 
-10.sup.-6 M) caused a dose-dependent parallel shift to the right of the 
isoproterenol concentration-response curves. The results of a typical 
experiment with right atria is illustrated in FIG. 4A. In electrically 
driven guinea-pig left atrial strips, vasomolol also antagonized 
isoproterenol-induced positive inotropic responses and produced 
dose-dependent rightward shifts of the cumulative concentration-response 
curves to isoproterenol. Potential time-dependent changes in agonist 
potency were monitored by control experiments in which both the first and 
second isoproterenol concentration-response curves were carried out 
without antagonist. There was a decrease in the potency of isoproterenol 
in the second concentration-response curve that was statistically 
significant (data not shown). The CR for antagonists was corrected for 
this change in sensitivity. Vasomolol was more potent than atenolol, 
slightly more potent than metoprolol, and was less potent than propranolol 
in .beta.-adrenoceptor blocking activity. The pA.sub.2 values and slopes 
of regression lines are indicated in Table 2. Vasomolol (5.times.10.sup.8 
-10.sup.6 M) also competitively antagonized (-) isoproterenol-induced 
relaxation from the spontaneous tone of reserpinized guinea-pig tracheal 
strips and thus produced parallel shifts to the right of the agonist 
concentration-response curves (FIG. 4B). The CR for antagonists was 
adjusted for the same reason that it was for left atrial strips. Vasomolol 
was less potent than propranolol in .beta..sub.2 -adrenoceptor blocking 
action. The pA.sub.2 values and slopes of regression lines are indicated 
in Table 2. 
.beta..sub.1 :.beta..sub.2 -selectivity of vasomolol. 
Propranolol Intrinsic sympathomimetic activity method 
Animals were pretreated with reserpine (10 mg/kg, i.p.) 24 hr prior to the 
experiment described in Naunyn-Schmiedeberg's Arch. Pharmacol. 311, 237, 
1980 (by Kaumann, A. J. et al.). All preparations including isolated right 
atria and left atria strips were studied. The concentration-response curve 
was obtained by cumulative addition of (-) isoproterenol, propranolol, 
atenolol or vasomolol. 
Results 
The .beta..sub.1 /.beta..sub.2 -selectivity ratio was obtained from the 
antilogarithm of the difference between the mean pA.sub.2 values obtained 
from the right atria and trachea as described in J. Pharm. Pharmacol. 57, 
369, 1972 (by Baird, J. R. C. et al.). Vasomolol was 39.8 times more 
potent on right atria than on trachea, I.e. was selective for .beta..sub.1 
-adrenoceptors. Propranolol was only 1.3 times more potent on right atria 
than on trachea and was, therefore, considered to be non-selective (Table 
2). 
Lack of intrinsic sympathomimetic activity of vasomolol drug 
esmolol 
vasomolol, 
atenolol, 
propranolol 
isoproterenol 
Results 
The frequency of contraction of right atria and tension developed by left 
atrial strips from reserpinized guinea-pigs were measured against 
cumulatively increasing concentrations of vasomolol, atenolol, propranolol 
or isoproterenol. As shown in FIG. 5, isoproterenol produced 
concentration-dependent increases in heart rate and contractility with a 
maximum increase at 10.sup.6 M. Vasomolol did not induce an increase in 
the heart rate or contractility, but caused negative inotropic and 
chronotropic effects in concentrations at 10.sup.-5 M or above. 
Propranolol also produced negative inotropic and chronotropic effects, and 
such depressant effects usually increased steeply with concentration, 
leading in most cases to arrest or unexcitability of the preparation at 
concentrations between 10.sup.-4 and 10.sup.-3 M. 
Effects of vasomolol on radioligand binding studies. 
Receptor binding experiments. 
method 
Pig's heart were obtained from a local slaughterhouse immediately after the 
animals had been killed and transported to our laboratory in ice-cold 
buffer (250 mM sucrose/50 mM Tris hydroxymethyl! aminomethane HCl/1 mM 
magnesium chloride, pH 7.4). Various heart tissues were removed and 
prepared as detailed as described in Cardiovasc. Res. 25, 764, 1991 (by 
Bjornerheim, R. et al.). In the test for receptor binding, the ventricles 
were placed in ice-cold buffer and all subsequent procedures were carried 
out at 4.degree. C. The tissue was homogenised in 20 volumes of TE buffer 
(10 mmole.litre.sup.-1 Tris-HCl, 1 mmole.litre.sup.-1 EDTA, 0.1 
mmole.litre.sup.-1 ascorbic acid, pH 7.4) with three 12-s pulses using a 
Polytron homogenizer (Kinematica, Model PT 3000, Switzerland). The 
homogenate was filtered with pressure through muslin and the filtrate 
centrifuged for 10 min at 1000 g to remove connective tissue, unbroken 
cells and cell debris. The supernatant was centrifuged again at 10,000 g 
for 12 min. This second supernatant was then centrifuged for 15 min at 
30,000 g and the final pellet was suspended in a assay buffer (75 mM Tris 
HCl/25 mM MgCl.sub.2, pH 7.4). 
Protein was determined by the method in Anal. Biochem. 72, 248, 1976 (by 
Bradford, M. M. et al.). This membrane preparation has been described in 
Biochem. Biophys. Acta. 541, 334, 1978 (by Ciaraldi, T. et al.). The 
binding assay of .sup.3 H!-DHA was carried out as described in Biochem. 
Biophys. Res. Commun. 60, 703, 1974 ( by Lefkowitz, R. J. et al.) with 
slight modifications. .sup.3 !-DHA and ventricular membranes (200-300 
.mu.g) were incubated for 60 min at 25.degree. C. with and without the 
addition of 1 .mu.M alprenolol, in 75 mM Tris-HCI buffer comprising 
MgCl.sub.2 25 mM, to make a final volume of 250 .mu.l. In competitive 
binding experiments, the competing agent was added directly to the 
incubation mixture. The incubation was terminated by addition of 1 ml of 
ice-cold assay buffer followed by immediate filtration through Whatman 
GF/C glass fiber filters supported on a 12-port filter manifold 
(Millipore). The filters were immediately washed three times with 5 ml of 
ice-cold assay buffer and dried in an oven at 60.degree. C. for 2 hr prior 
to adding 5 ml of Triton-toluene based scintillation fluid. 
Membrane-bound 
.sup.3 H!-DHA trapped in the filters was counted in a Wallac LKB 1211 
rackbeta liquid scintillation counter with an efficiency of 41%. In each 
experiment, non-specifically bound .sup.3 H!-DHA was determined by 
incubating membrane protein and .sup.3 H!-DHA with 1 .mu.M alprenolol. 
Specific binding was thus obtained by deducing this value from the total 
binding of .sup.3 H!-DHA for each sample. 
Result 
.sup.3 H!-DHA was bound to guinea-pig ventricular membranes in a saturable 
manner as illustrated in FIG. 6. The concentration dependence of .sup.3 
H!-DHA binding was studied with labeled compound concentrations ranging 
from 0.1 to 30 nM. Scatchard analysis (Ann. N. Y. Acad. Sci. 51, 660, 
1949) to determine the affinity and number of binding sites is shown in 
the inset. The equilibrium dissociation constant (Kd) was 8.1.+-.0.9 nM 
(mean.+-.S.E.M.), and the maximum binding capacity (B.sub.max) was 
120.3.+-.6.0 fmol/mg protein (mean.+-.S.E.M.) at 25.degree. C. The binding 
of .sup.3 H!-DHA reached equilibrium in approximately 20 min and 
maintained it for up to 90 min (data not shown). FIG. 7 demonstrates the 
competition curves of .beta.-adrenoceptor antagonists for .sup.3 H!-DHA 
binding sites in the ventricular membranes. The lC.sub.50 value 
(mean.+-.S.E.M.) of (-) propranolol, a nonselective .beta.-antagonist, was 
33.1.+-.4.5 nM. The lC.sub.50 values of vasomolol and esmolol were 
4.7.+-.1.5 and 5.0.+-.1.3 (uM), respectively. The order of potency of 
.beta.-adrenoceptor inhibiting .sup.3 H!-DHA binding was (-) 
propranolol&gt;&gt;vasomolol=esmolol.