Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
)t38949~
1 This invention relates to a novel pyridine derivative,
process for preparing the same and a pharmaceutical composition.
More particularly, this invention relates to a pyridine deriva-
tive represented ~y the formula
~ CONHC~2CI - R2 ~I)
wherein Rl is hydrogen, lower alkyl or -ON02 and R2 is -ON02 or
-CH20N02, or its salt; a process for preparing the same and a
pharmaceutical composition containing the derivative or its salt.
Some nicotinic acid derivatives or nicotinamide der-
ivatives have ~een reported in some references, for example,
' Japanese Patent Disclosure 1624/1976, U.S. Patent 3,092,634,
U.S. Patent 3,168,438 and "Mie Medical Journal" Vol. 16(3), pp.
- 207-211, (1967).
The Japanese Patent Disclosure discloses that nicotinic
ester derivatives are prepared ~y reacting l-nicotinyl-glycerine
or l-nicotinyl-2,3~isopropylidene glycerine with fuming nitric
acid, to o~tain 1 nicotinyl glycerine-2,3-dinitrate and then con-
verting the compound to its dioxane addition compound which has-
a coronary vasodilating action.
- U.S. Patent 3,092,634 and U.S. Patent 3,168,438 dis-
close that bis-nitric ester of N,N-bis-(~-hydroxyethyl)nicotin-
~ amide which has coronary vasodilating action is prepared by re-
-. acting nitric es~er of diethanolamine with nicotinic acid chlor-
:. : ....
idec
However, the compounds which have been reported have
short term ac~ion or adverse action against blood pressure or
function of heart and, thus, do not suffice
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1 as a drug to stimulate the circulatory system, for example, in
the case of ischemic heart disease~ Under the circumstances,
the development of an ideal druy has been desired.
In "Mie Medical Journal", Vol. 16(3), pp. 207 - 211 -~
(1967), 2-nicotinamido ethanol is mentioned. However, the
compound is merely shown as a test compound for antitumor
action and the Journal reported no remarkable pharmacological
action.
The inventors of this invention have researched and
tested for a long time to find a compound useful for a
circulatory disease and finally completed the present invention.
To this end, in one of its aspects, the invention
relates to a compound represented by the formula (I)
~ CO~HCH2lH 2
wherein Rl and R2 are as defined above and its salts, a process
for preparing the same and a pharmaceutical composition contain-
2~ ing the compound above or its salt.
In another of its aspects, the invention provides aprocess for preparing a nitric ester of N-(2-hydroxyethyl)nico-
tinamide of the formula
2CH2 - ON02
NJ
or a pharmaceutically acceptable salt thereof, which comprises
a process selectecl from the group of processes consisting of:
~a) reacting a compound of the formula
,, .,. 1 '
1~8~9~
-' ..
COOH
N
or its functional derivative at the carboxyl group, with a :~
compound of the forrnula
NH2 ~ CH2CH2 ON02
or its functional derivative at the amino group; ~nd
(b) reacting a compound of the formula
CONHCH2CH2 - OH
N . :
with a nitrating agent.
In yet another of its aspects, the invention provides
.
a novel compound of a nitric ester of N- (2-hydroxyethyl)nicotin- :--
amide of the formula
NHCH2CH2 - ONO~
' . '
The compound of the present invention or its salt is
novel and has improved actions for treating circulatory disease,
such as coronary vasodilating action, antihypertensive action,
antiarrhythmic action, anticoagulative action and peripheral
vasodilating action, and, thus, it is use~ul for treating ischemic
heart disease, as an antihypertensive drug, anticoagulative drug, ~ .
antiarrhythmic drug, and as a peripheral vasodilator including
cerebral vasodilator and renal vasodilator. .
According to the present invention the compound
1~)889~9~
1 of the formula (I~ may ~e prepared ~y one of the following
methods:
(1) A compound represented ~y the formula
1~ .
l N ~ COOH tII)
or its functional derivative at carboxyl group is reacted with
a compound represented ~y the formula
,~
NH2 - CH21H R2 (III)
Rl '
wherein Rl and R2 are as defined above or its functional deriv-
ative at amino group; and
(2) ~ compound represented ~y the formula
~ ~ONHCH2C~ ~ H4 (IV)
wherein H3 is hydrogen, lower alkyl or hydroxy and R4 i~ hydroxy
or -CH2OH is reacted with a nitrating agent.
The functional derivatives of the compound (II~ at
car~oxyl group include, for example, acid halide, acid anhydride,
active amide, active ester and the like. The derivatives which
may ~e commonly used are acid chlorides; acid azides; acidanhyd-
rides such as anhydrides derived from two moles of the compound
CII~ and anhydrides of the compound wîth another acid for example
dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric
acid, benzylphosphoric acid, halogenated phosphoric acid, di-
alkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric
--4--
.. . ~ '
10~8g~4
1 acid, alkylcarbonic acid, fatty acid such as pi~alic acid, pen-
tanoic acid! isopentanoic acid, 2-ethylbutanoic acid or trichloro-
acetic acid, or aromatic carboxylic acid such as benzoic acid;
amides for example, amides with imidazole, 4-substituted imid-
azole, dimethylpyrazole, triazole or tetrazole; and esters, for
example, Gyanomethyl ester, 4-Nitrophenyl ester, 2,4-dinitro-
phenyl ester, trichlorophenyl ester, pentachlorophenyl ester,
methanesulfonylphenyl ester, phenylazophenyl ester, phenylthio
ester, 4-nitrophenylthio ester, p-cresylthio ester, carboxymethyl-
thio ester, pyranyl ester, pyridyl ester, 8-quinolylthio ester
and esters with N,N-dimethylhydroxyamine, l-hydroxyl-2-~lH)-pyri-
done, N-hydroxysu~cinimide or N-hydroxyph*halimide.
The starting compounds (III) may be used as an activ-
- ated form at amino group which can be activated by the use of
phosphorous trichloride, ethyl chlorophosphite, methyl chloro~
phosphite or the like.
The process as defined in method ~1~ above may be
carried out ~y reacting, for example, a reactive derivati~e of
the compound (II2 at car~oxyl group with the compound (III) to
condense them at a temperature of ~rom -10 to 50C, preferably
~ - 10C for 0.5 - 4 hours. The solvents which may be used for
this reaction include water, benzene, toluene, tetrahydrofuran,
diethyl ether, dioxane, dimethyl~formamide, chloroform, methylene
chloride, acetonitrile~ acetone~ carbon tetrachloride ethyl
acetate and the like. An accelerator for the condensation re-
action may be useclr which includes inorganic basic substances,
for example~ hydroxide, carbonate or acetate of an alkali metal
or alkaline earth metal, such as sodium acetate, sodium carbonate,
potassium acetate, potassium car~onate, sodium hydroxide, calcium
acetate, calcium carbonate; or amine compounds such as pyridine,
--5--
.
., ' ' ' '. ' ' , ', ` '
~L0889~4
1 triethylamine, dimethylaniline, picoline or the like~
The condensation reaction of the compound (II) with
the compound (III) the amino group of which has been activated
with phosphorous trichloride, èthyl chlorophosphite, methyl
chlorophosph-ite or the like may ~e conveniently carried out at
from room temperature to the reflux temperature of a solvent used
for 0.5 - 3 hours. The solvent usually used for this invention
includes a neutral solvent, such as ~en~ene, toluene, xylene, di-
oxane or tetrahydrofuran; or a ~asic solvent such as pyridine,
triethylamine, dimethylamine, dimethylaniline or picoline. In
case the neutral solvent is used, it is preferable to add aminecompound such as pyridine, triethylamine dimethylaniline, picoline
or the like.
In another em~odiment of the invention, the compound
(III may ~e reacted with the compound (III) in an inert solvent
in the presence of an amide-formation accelerator, for example,
an imide compound such as N~N~-dicyclohexylcarbodimide~ N-cyclo-
~exyl-N'-morpholino-et~ylcar~odiimide, N~N'-diethylcar~odiimide
or the like; an imine compound such as diphenylketene-N-cyclo-
hexylimine~ pentameth~leneketene-N-cyclohexylimine or the like;
or a phosphate or phosphite such as triethyl phosphite,~ethyl
polyphosphate, isopropyl polyphosphate or the like at from room
temperature to the reflux temperature of the solvent used for
1 - 5 hours. The inert solvent which may be used in this react-
ion includes, for example~ benzene, toluene, tetrahydrofuran,
chloroform, dioxane, acetonitrile and dimethylformamide.
-6
.
" 1~88S~4~
,
The reaction as previously defined in Method (2)
may be carried out by reacting the compound (IV) with a
nitrating agent in an inert solvent such as chloro~orm,
dichloromethane or the like at a temperature of ~rom -5C to
room temperature for 1 - 3 hours. The nitrating agent which may
be generally used in this reaction is fuming nitric acid,
nitril chloride or the like.
The compound (I) prepared by Method (1) or Method
(2) may be converted to its organic or inorganic acid addition
' 10 salt such as hydrochloride, nitrate, oxalate, p-tolunensulfonate,
maleate or the like.'
The object compound, pyridine derivative (I) may
be formulated by a conventional way into a pharmaceutical
composition in the form of tablet, granule, powder, capsule,
suspension, parenteral injection, suppository or the like. For
the preparation of tablet, powder, granule or capsule filled
with powder or granule, the object compound may be mixed with
one or more p~armaceutical carriers such as lactose, starch, '
mannitol, kaolin, crystaline cellulose, talc, calcium caxbonate, '
magnesium stearate or the like. For'the preparation o~ soft
capsule filled with liquid preparation, the object compound may
be dissolved in an oil. The object compound may also be suspended
in an arabic gum or sucrose aqueous solution and the pH adjusted.
On the other hand the object compound may be blended with
mannitol to make it suitable for parenteral injection.
The pyridine derivative according to this invention
may be present in any form of the pharmace'utical composi-
tion in an amount sufficient to exhibit the actions for
treating or preventing circulatory disease but not to
_ 7 _
~.:
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1 exhibit any adverse action ~y the administration of the composi-
tion. A unit dosage form such as a tablet or capsule may contain
usually 5 - 20 mg of the active compound when the composition is
orally administered~ In case the composition is given parent-
erally, a unit dosage such as a vial may usually contain about
1 - 10 mg of the compound.
It will be understood that the actual dosage changes,
as a matter of course, depend upon.the conditions of individual
patient and, therefore, it should be specifically determined when
used~ However, it will ~e safe and convenient if the dosage in
terms of the active cr~mpound is usually 10 - 100 mg, preferably
- 60 mg per day for an adult when administered orally, and
usually 1 - 100 mg, prefera~ly 1 - 50 mg, per day for an adult
in the case of the parenteral injection.
Fig. 1 is a graph showing percent increase in coronary
~lood flow when the compound prepared according to Example 1 or
nitroglycerin was intravenously administered in accordance with
Experiment 1.
Fig. 2 is a graph showing the duration of increase in
coronary ~lood flow when the compound of Example 1 or nitrogly-
cerin was intravenously administered in accordance with Experi-
ment 1.
Fig. 3 is a graph showing the change in the left vent-
ricular tension when the compound of Example 1 or nitroglycerin
was intravenously administered in accordance with Experiment 1.
Fig. 4 is a graph.showing the change in heart rate
- when the compound of Example 1 or nitroglycerin was intravenously
administered in accordance with Experiment 1.
Figs. 5, 6 and 7 are graphs showing the change in elec-
trocardiograms when the compound of Example 1 was intravenously
administered.
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88944
1Fig, 8 is a graph showing the change in systemic blood
pressure when ~he compound of Examp:Le 1 or nitroglycerin was
intravenously administered in accordance with Experiment 1.
Figs. g and 10 are graphs showing the change in systemic
blood pressure when the compound of Example 1 was intravenously
administered according to Experimenl 1.
Figs. 11 and 12 are graphs showing the change in aortic
~lood flo~ when the compound of Exa~ple 1 was intravenously
administered.
lOFig. 13 is a test chart showing the effect of the com-
pound of Example 1 to the platelet coagulation caused by adeno-
sine diphosphate in accordance with Experiment 7.
Fig~ 14 is a test chart showing the effect of the com-
pound to the platelet coagulation caused by collagen in accordance
with Experiment 7,
Experiment 1
.. , . . , :
The acute tQXiCity ~LD5Qi of the compound of Example
2 was determined ~y the use of SD strain male and female rats
~4 weeks oldl which were orally or intravenously administered
the compound. LD50 against both male and female rats ranged
from 1,20Q to 1,3Q0 mg/kg for oral administration and from 800
to l,OOQ mg/kg for intravenous administration,
Ex~eriment 2
Adult mongrel dogs which had ~een anestetized by the
intravenous administration of 30 - 40 mgjkg of pento~arbital
sodium were subjected to thoracotomy under oxygenic ventilation
by use of Bird's respirator and thereafter various physical
phenomena were measured in the following manner.
12 Coronary ~lood flow ~CBF~
An electromagnetic flowmeter probe ~as attached to a
_g_
' ' ' ' ' '
.. . . . .
~{118l~5~44
1 circumflex branch or an origin of an anterior descending branch
of the left coronary artery.
2) Coronary perfusion pressure ~CBP)
The fine catheter connected to a pressure transducer
was inserted into the distal side of the probe in the circumflex
branch of the left coronary artery,
3) Aortic blood flow (AoBF)
An electromagnetic flowmeter probe was attached to the
origin of aorta.
1~ 4~ Left ventricular tension (LVT)
A strain gauge was sewn on the anterior wall of the
left ventricle.
51 Systemic blood pressure (SBP)
A catheter connected to a pressure transducer was in-
serted into the right femoral artery,
6) Heart rate (HR)
Heart rate was measured by a cardiotachometer using
pulse pressure as trigger pulse.
7) Electrocardiogram ~ECG~
An electrocardiogram was recorded according to an uni-
polar lead in the cardiac surface using different electrode set
on a portion of anterior wall of left ventricle.
In addition to test Items 1 - 7 above, some of the test
animals were subjected to the measurement of renal blood flow
(RB~) and femoral blood flow ~BF) by attaching an electromagnetic
flowmeter probe to the left renal artery and the left femoral
artery.
The test compound of Example 1 was dissolved in the
physiological saline solution ox in the distilled water and ad-
ministered intravenously, orally or sublingually, Nitroglycerin
,
10~ 4
1 was administered in the same manner as an acti~e control drug to
compare with the test compound.
Results
~I) Intravenous Administration
a~ Change in coronary ~lood f:Low
Diastolic coronary flow began to increase 10 - 20 se-
- conds after the intravenous administration of the test compound
with a dose of 10 ~g/kg or more. Systolic coronary flow was in-
creased with a dose of 250 ~g/kg or more, accompanied by tran-
sient decrease just after the administxation. Mean coronary flowshowed a persistent increase after the administration of the test
cQmpound.
The increasing degree of coronary flow is shown in
Figs. 1 and 2 in terms of maximum percent change and duration
against the values ~efore administration.
The administration of the compound of this invention
even in a dose level of 10 ~g/kg i.v. produced a significant in-
crease in coronary flow and in a dose level of 500 ~g/kg i v.
caused a increase so remarkable as to appear to be reactive
hyperemia. In the latter case, the duration of increase in cor-
onar~ flow ~ecame to near 3 hours. The effect of the compound of
this invention was superior to that of nitroglycerin, particularly
in the duration of action.
~2 Chan~e in left ventricular tension
As shown in Fig. 3, the left ventricular tension caused
a slight decrease after the intravenous administration of the
compound of Example 1 in a dose level of 50 ~g/kg or more. The
decrease was almost equivalent to t~at o~ nitroglycerin in the
tested dose range. However, the maximum percent decrease was
33~ or less even in a dose level of 500 ~g/kg i,v,
.
.: . . .. . .
.
. . .
~lL01~89~4
c) Change in heart rate
As shown in Fig. 4, the heart rate showed almost no
change ~y the administration of the compound of this invention
in a dose level up to 20 ~g/kg i.v., while, in a dose level of
50 yg/kg i.v. or more, presented a s,light dose-dependent decrease
to reach 17% in a dose level of 500 ~g/kg i.v. In contrast, the
administration of nitroglycerin in a dose level of more than 10
~g/kg i.v. increased the heart rate.
d~ Change in electrocardiogram
As shown in Figs. 5 and 7, PP and QTc intervals were
prolonged dose-dependently by the-intravenous administration of
the compound of Example 1 with a dose more than 50 ~g/kg, while
the PQ interval had almost no change ~y the administration of
dose up to 1,000 ~g/kg i.v,
e~ Chan~e in systemic blood pressure
~ As shown in Figs. 8, q and 10, the systemic blood pres-
sure presented a significant, dose-dependent fall by the intra-
venous a*ministration of the test compound in a dose level of
more than 50 ~g/kg. The effect of the compound on blood pressure
was not stronger thah that of nitroglycerin, but the duration
was longer than that produced with nitroglycerin.
f) Change in aortic blood flow
As shown in Figs. 11 and 12, the aortic blood flow pre-
sented a slight, dose-dependent increase by the intravenous ad-
ministration of the compound of Example 1 in a dose level of 10
~g/ky or more. The percent increase in a dose level of 500 ~g/kg
i~Y, reached maxi~um 40%, ~ut the duration was not so long in
comparison with the degree of increase in coronary flow.
g) Ch~nge in renal or femoral flow
The renal and femoral flows were increased in spite of
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.
' '. : . . .~ : ' ',
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.. . . .
1()8~944
1 a fall in systemic blood pressure hy the intravenous administra-
tion of the compound of Example 1 in a dose level of 100 to 250
yg/kg. However, the increase in the renal and femoral flows
were less than that in coronary flow concerning the degree and
the duration.
From the test results stated a~ove, it was confirmed
that the compound of this invention had an increasing effect on
coronary flow far superior to that of nitroglycerin and, in
addition, it produced a fall in blood pressure, a decrease in ''
heart rate and a reduction in cardiac tension, which were not
excessive e~en in a high dose level.
Moreover, the fact that the compound of this ln~ention
did not disturb the conduction of excitation in the heart as
shown in no prolonged PQ interval of elctrocardiogram, indicates
that it may be very useful as a drug for ischemic heart disease,
The compound could ~e used as an antihypertensive drug
or a peripheral vasodilator ~ecause it has a longlasting and
mild a~tih~pertensive action and a dilating action ~or femoral
and renal vessels, as well as a desira~le effect on ischemic
2~ heart.
~I~ Oral and Su~lingual Administration
The compound prepared according to Example 2 was sub-
lingually administered to dogs as a tablet containing 1 - 10 mg
of the compound. About 2 minutes after the administration~ cor- ''
onary flow was apparently increased~ The compound in a dose
le~el of 10 mg showed almost no change in systemic blood pressure,
cardiac tension and aortic flow. In contrast, the sublingual
administration of a tablet containing the hydrochloride of the
acti~e compound did not show any substantial increase in coronary
flow~ When the compound prepared according to Example 1 or 2 in
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. ,' ',
,. , '.
1081~ 4
1 either its free form or hydrochloride was intraduodenally admin-
istered in a dose level of 50 ~g/kg or more, an apparent, long-
lasting increase in coronary flow was observed,
From the test results, the compound of this invention
may be used as various preparations, such as for parenteral in-
jection; or in the form of capsule, tablet, granule or powder
for oral administration or in sublingually administrative form.
In contrast, nitroglycerin is not absorbed through the intestinal
wall and, therefore, only sublingual administration is available.
Experiment 3
Adultmongrel dogs which had been anesthetized by the
intravenous administration of 30 - 40 mg/kg of pentobarbital sod-
ium were subjected to thoracotomy under oxygenic ventilation by
the use of Bird's respitator. A catheter was inserted towards a
proximal direction into a branch of an origin of the left coron-
ary artery and a contrast media such as Conlaxin H ~ or Angio-
conrei ~ was injected through the catheter. The morphological
change in the left coronary artery was filmed using a 35 mm cine-
film before and after the injection of the compound. From the
analysis, it was found that the coronary artery was remarkably
dilated by the intravenous adminstration of the compound of this
invention in a dose level of 100 ~g/kg or more.
Experiment 4
The same preparation as in Experiment 2 was set up using
adult mongrel dogs anesthestized by the intravenous administration
; of pentobar~ital sodium. The left anterior descending branch or
ci-rcumflex branch of the coronary artery was mechanically stric-
tured or occluded in the distal side of the portion attaching the
electromagnetic flowmeter probe to experimentally induce cardiac
ischemic phenomenon, and then the efEect of the compound of
Example 1 on the phenomenon was observed.
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.. ; ' . ' , ': .
~ 8944
1 Under incomplete stricture, the coronary flow was
slightly increased when the compound of Example 1 was intraven-
~usly a~ministered in a dose level of 100 ~g/kg or less, while
slightly decreased when administrated in a dose level of more
than 250 ~g/kg. After the administration of the compound of
this invention, ST elevation of the electrocardiogram in the car-
diac ischemic portion was apparently improved and, at the same
time, the recovery of ventricular tension in an ischemic part
was also observed. Under complete occlusion, the compound did
not improve ST elevation or ventricular tension in the midpoint
of the ischemic pGrtion, but the improvement in the surrounding
point of the ischemic portion was recognized.
Similarly, when the tablet containing 10 mg bf the
free form of the compound prepared in Example 2 was sublingually
administered, the coronary flow was increased and ST elevation
in the electrocardiogram and a reduction inventricular tension
. ,, i
`were improved.
F~periment 5
~ A papillary muscle isolated from a guinea pig was set
in the organ hath filled with the oxygeni~ed Tyrode's solution
~Ca; 1 8 mM, K: 2,7 mMl at 30 C. One end of the muscle was
fixed to the organ bath and the other end was connected to a
tension meter by a thread. The effect of the compound of Example
1 on the muscle contraction force was observed by giving electric
stimulation (2~V, 5 msec, lHz~ to the muscle with an Ag-AgCl
electrode. The antagonistic action of the compound against
calcium ion or isoproterenol was also investigated.
From the test results, it was found that the contraction
of the papillary muscle induced by the electric stimulation was
inhi~ited with the application of the compound of this invention
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.,
: ~ .
10~8944
1 in a concentration level of more than 1 ~g/ml and that calcium
ion had antagonistic effect against such inhibitory action. On
the other hand, when isoproterenol was applied in a concentration
level of 0.08 ~g/ml, the papillary muscle exhi~ited excessive
excitement to produce two or more irregular contractions per
single electric stimulation. Under this state, the addition of
the compound of this invention to the solution in a concentration
level of more than 2 ~g/ml inhi~ited the excessive excitement.
~II) A spiral strip isolated from the dog coronary artery
was set in the organ ~ath filled with a Ca -free Lock's solution.
One end of the strip was fixed to the organ bath and the other
end was connected to a tension meter, The effect of the compound
of Example l on the contraction of the strip induced by the polar-
ization of the solution with the addition of 43 mM of K and 1 mM
of Ca~+ was investigated.
The test results showed that the contraction induced
by K depolarization was inhibited by the compound of this
invention and such inhibitary action was diminished by the add-
ition of Ca++.
(III~ The effect of Ca on the increase in coronary flow wasinvestigated using the dog anesthetized with pento~arbital sodium
according to the same procedure as in Experiment 2-(Ij. The in-
crease in coronary 10w induced by the administration of the com-
pound of this invention was inhi~ited by a larger dose o Ca +.
(IV~ Taenia coli of a guinea pig was suspended in the organ
bath filled with Tyrode's solution and the effect or the compound
of Example 1 on the spontaneous contraction and the contraction
induced ~y K depolarization was investigated.
The test results showed that the spontaneous contraction
and the K depolarization induced contraction were inhi~ited ~y
-16-
.. . . . . .
.
1~8~?9~
1 application of the co~pound in a concentration level of 2 ~g/ml
and such inhibitary action was diminished ~ the addition of Ca
Analysis of the test results indicates that the com-
pound of the present invention has an antagonistic action to
Ca r 50 that the inhibitory action to cardiac contraction the
dilating action to smooth muscle of dog coronary artery and the
inhibitory action to contraction of taenia coli of guinea pig
induced by the application of the c~mpound were antagonized by
the addition of Ca~. Further, it was also found that the com-
pound of this invention has an antiarrhythmic action because thecompound inhibited the excessi~e excitement of papillary muscle
induced ~y the administration of isoproterenol,
Experiment 6
The ~lood flow was made insufficient by loading exper-
imentally the coronary artery of a dog, for example, ~y strict-
uring the coronary artery for a certain period of time, and the
^ ,
artery was allowed to sustain in this condition. According to
this treatment~ its peripheral ~lood pressure and peripheral
blood flow repeated periodical and spontaneous fluctuations with-
in minutes order period accompanied with ST elevation in electro-
cardiogram. This phenomenon was closely similar to the fit of
variant angina pectoris in a clinical case, The periodical
fluctuation has been proved to ~e caused by the periodical spas-
mus in the strictured coronary artery. The effect of the com-
pound on such a Yariant angina pectoris model was investigated.
The test results show that the periodical fluctuations
-in the coronary ~lood pressure and coronary flow was inhibited
~y the intravenous administration of the compound of Example 1
in a dose level of 25Q ~g/kg or more. That is, the periodical
fluctuation in blood flow apparently reduced, and only fine
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10~3944
1 fluctuations remained. The effective duration o~ the inhi~itory
action ranged from 25 to 40 minutes.
As stated ak,ove, the compound of this invention is
~elievea to ~e effective on the treatment of variant angina pect-
oris.
Experiment 7
According to the procedure explained below, the compound
of E~ample 1 was subjected to investigate as to whether or not
it has an anticoagulative action to platelets, namely antiembolic
1 0 action.
1. Preparation of the test solution
1-1 Adjustment of platelet-rich plasma ~RP solution)
A rabbit blood containing 38% sodium citrate (blood:
sodium citrate aqueous solution = 9:1~ was subjected to the cent-
rifugation at 1,~00 r.p.m. for 10 minutes, and the supernatant
fluid was used as PRP solution.
1-2 Adjustment of an adenosine diphosphate solution
(ADP solution)
Adenosine diphosphate CSigma Chemical Co., Ltd.) was
dissolved in a physiological saline solution at a concentration
of 10~ ~g/ml.
1-3 Adjustment o~ collagen suspension
- Fifty mg of collagen o~,tained from ~ovine tendon (Sigma
Chemical Co., Ltd.,3 was suspended in 5 ml of a physiological
saline solution and homogenized by a glassey homogenizer for 5
minutes. Next, it was subjected to centrifugation at 500 r.p.m.
for S minutes, and the supernatant fluid was used as a collagen
suspension,
1-4 The compound of Example 1 was dissolved in the
physiological saline solution to obtain the test solution,
-18-
2. Method of measurement
The measurement was performed by the use of Aggrego-
meter ~vans Electroselenium htd., Model 169).
PRP solution ~0.5 ml) and the test solution ~0.025 ml)
or a physiological saline solution as the control (0.025 ml)
were charged into two cuvetts, respectively, and then subjected
to incu~ation at 37 C for 2 minutes Further, after 0.025 ml of
ADP solution or 0.025 ml of the collagen suspension was added to
each cuvette, the e~fect of the compound on platelet aggregation
induced by ADP or collagen was measured.
As shown in Figs. 13 and 14, the initial rate of ADP-
induced aggregation in the test compound was the same as that in
the control/but the test compound accelerated the dissociation of
the aggregated platelets. On the other hand, the test c~mpound
delayed the onset of the collagen-induced aggregation and made a
maximum extent of the aggregation lower than the control. From
these results~ the inhibitory effect of the compound on the plate-
let aggregation was confirmed, so that the compound of this
invention was also found to be useful as an anticoagulant.
E~periment 8
By the same method as disclosed in Experiment 2, item
5, change in systemic blood pressure when test compounds were
intraYenously administered was measured. The results obtained
are shown in the follo~ing Ta~le.
TABLE
.. _ _
Test Compound Dose Percent Change Duration
C~s/kgl of S.B.P. ~1 ~in.)
. .
Compound of E~ample 50 16 22
Compound of Example 50 20 17
Nitroglycerin 50 45 8
. . . ....
,
~. ' ' ", ' '.
8894~
Example 1
To a mixture of 5 g of sodium hydrogen car~onate, 15 ml
of water, 1.69 g of the nitric ester of monoethanolamine nitrate
and 20 ml of chloroform was added slowly 2.5 g of nicotinyl
chloride hydrochloride o~er 10 to 30 minutes under stirring at
0 to 5C, The stirring was continued for an additional 30 min- ~-
utes and then the chloroform layer was separated. The remained
aqueous layer was extracted with chloroform and the extract was
com~ined with t~e separated chloroform layer. The organic layer
was washed with a potassium carbonate aqueous solution, dried
oYer anhydrous sodium sulfate-and evaporated under reduced pres- ''
sure to dryness. The residue was dissolved in ether-isopropanol
~ and then hydrogen chloride was ~u~led into the solution ' ~,
under cooling to give 2.35 ~ of the nitric ester of N-~2-hydroxy-
ethyl~ nicotinamide hydrochloride. Recrystallization from ethan- '
ol afforded colorless needle"crystals having a melting point of
132C.
Anal~sis:
Calcd. for C8HloN304Cl: C,38.80; H, 4~07; N, 16.96 (%~
Found C,38~89,-; H, 4.02; N, 16.72 (~)
IR~cm ~
NH, 3255; CoO~ 166~; ON02, 1640
Ex:ample 2
To a solution of 1.69 g of the nitric ester of mono-
ethanolamine in 20 ml of pyridine ~as added slowly 2.5 g of ni-
cotinyl chloride hydrochloride over ln to 30 minutes under
stirring at 5C. After stirring for an additional 30 minutes,
the xeaction mixture was evaporated to dryness. The residue was "
dissolved in chloroform and the solution was washed with a sod-
iumi hydrogen car~onate aqueous solution. The organic layer was
, :
:. ~, '
1()~894~4
1 separated, dried over anhydrous sodium sulfate and evaporated
under reduced pressure to dryness. The residue was chromato-
graphed on silica gel ~Takogel C-200; Wako Pure Chemical Indust-
ries, Ltd., Japan) and eluted with ~enzene-ethanol (5:1). The
eluent was evaporated to a semi-solid mass which was crystall-
ized from diethyl ether to gi~e 1.97 g of the nitric ester of
N-~2-h~dxox~rethyllnicotinamide~
Recrystallization of the crystals from diethyl ether-
ethanol afforded colorless needles having a melting point of 9210 to 23C.
E~ample 3
A solution of 10 g of the nitric ester of N-~2-hydroxy-
ethyl~ nicotinamide hydrochloride in water was neutralized with
a sodium hydrogen carhonate aqueous solution. The solution was
e~tracted with chloroform and the extract was dried over anhydrous
sodium sulfate and evaporated under reduced pressure to dryness.
The residue was crystallized from diethyl ether to give 7 g of
the nitric ester of N-C2-hydrQxyethyl) nicotinamide. Recrystall-
ization fro~ isopropanol-diethyl ether gave colorless needles
ha~in~ a melting point of ~3C,
IR ~m~l~
NH~ 3250, ONO2, 1630
Example 4
To a mixture of 1.6~ g of the nitric ester of mono-
ethanolamine nitrate in 5 ml of water~ 3.8 ml of 35~ potassium
carbonate solution and 17 ml of tetrahydrofuran were added alter-
nately 3 g of isonicotinyl chloride hydrochloride and 11~2 ml of
35% potassium car~onate aqueous solution over 30 minutes under
stirring at Q to 5C The reaction mixture was stirred for an
- 30 additional 15 minutes, and the organic layer was separated. The
-21-
lV~39~4
1 aqueous layer was extracted with tetrahydrofuran and the extrac~
was combined with the previously separated organic layer. The
combined organic layers were dried over anhydrous sodium sul-
fate and filtered. Evaporation of the solvent in vacuo and
crystallization of the residue aforded the nitric ester of N-
~2-hydroxyethyl) isonicotinamide.
The product was dissolved in diethyl ether and the
dropwise addition of diethyl ether-fuming nitric acid under
cooling gave 1.5 g of the nitric ester of N-~2-hydroxy-ethyl)
isonicotinamide nitrate. Recrystallization from isopropanol
afforded the crystals having a melting point of 105C ~decomposi-
tion~.
Analysis:
8 10N407: C, 35.04; H, 3i68; N 20 43 (%)
Found: C, 35.00; H, 3.59; N, 20.38 (~
IR~m~
- NH~ 328~; C=0, 1671; ON02, 1625
Example 5
To a mixture of 1.83 g of the nitric ester of isopropan-
20 olamine nitrate in 5 ml of water, 3.8 ml of 35% potassium carbon-
ate aqueous solution and 17 ml of tetrahydrofuran were added
alternately 3 g of nicotinyl chloride hydrochloride and 11.2 ml
of 35% potassium carbonate solution over 30 minutes under stirring
at Q to 5C. The reaction mixture was stirred for an additional
15 minutes and the organic layer was separated. The aqueous
layer was extracted with tetrahydrofuran and the extract was com- ;
~ined with the previously separated organic layer. The combined
extracts were dried over anhydrous sodium sulfate and filtered.
E~aporation of the solvent under reduced pressure and crystall-
ization of the residue gave the nitric ester of N-(2-hydroxypropyl)
nicotinamide.
-22~
. .
- . ' '. ' , ,,;: ', ' " .. '.
08~9~4
The product was dissolved in acetone and then hydrogen
chloride was bubbled into the solution to give 1.5 g of the nit-
ric ester o~ N- (2-hydroxypropyl~ nicotinamide hydrochloride as
colorless crystals. (mp: 161C (decomposition)).
Analysis:
Calcd- for C~H12N3O4Cl: C, 41,31; H, 4.62; N, 16.06 (~)
Found : C, 41.38; H, 4.60; N, 16.11 (~)
IR ~cm 11:
NH, 323Q; C=O, 1672; ONO2, 1620
Example 6
By the procedure similar to that of Example 4~ nico-
tinyl chloride hydrochloride was reacted with nitric ester of
propanol amine nitrate to o~tain nitric ester of N-(3-hydroxy- ~
propyl~ nicotinamide hydrochloride. ~Yield: 75%; mp: 127C -
(decQmposition~
Analysis:
, . . -
Calcd, for C~H12N3O4Cl: C, 41,31; H, 4.62; N, 16.06 (%)
Found : C, 41,40; H, 4.53; N~ 16~15 (~)
NH, 3235; C=O~ 1672; ONO2, 1617 ;
F~xa~le 7
By the procedure similar to that of Example 4, isonico-
tin~l chloride hydrochloride was reacted with nitric ester of
propanolamine nitrate to o~tain nitric ester o~ N~(3-hydroxy-
propyll iæonicotinamide hydrochloride. (Yield: 72~, mp: 125C
~decomposition~.
Analysis:
Calcd- for CqH12N304Cl C, 41,31; H, 4.62; N, 16.06 (%)
Found : C, 41.40; H, 4.58; N, 16.01 (%)
IR (cm 12;
NH, 3230; C=O, 1672, ONO2, 1615
-23-
, . . . .
~l088~44
1 Example 8
By the procedure similar to that of Example 4, pico-
linyl chloride hydrochloride was reacted with nitric ester of
monoethanolamine nitric acid salt to obtain nitric ester of N-
C2~hydrQxyethyl~ picolinamide~ (Yield: 78%, mp: 55 - 56C)
Analysis:
Calcd, for C~HgN304: C, 45.5Q; H, 4~29; N, 19.89 ~)
Found ; C~ 45.25; H, 4~10; N, 19.17 (~)
IR ~cm-l~
NH, 3362; C=0, 1665; ON02, 162Q
Example ~ -
By the procedure similar to that of Example 4, nico-
tin~l chloride hydrochloride was reacted with 2,3-hydroxypropyl-
amine to obtain dinitric ester of N- ~,3-di-hydroxypropyl) nico-
tinamide as pale-yello~ oil.
Example lQ
,
- N-C2-hydro~yethyl~ nicotinamide nitrate ~1.145 g) was
gradually added to 3 ~1 of fuming nitric acid which had been
~ooled to a temperature of from -lQ to -5C while stirring.
~0 After stirrin~ for additional one hour at 0 - 5C, diethyl ether
was added to the solution to precipitate 1.15 g of nitric ester
of N- ~-hydrQxyethyl~ nicotinamide nitric acid ester as colorless
crystals. The crystaIs were dissolved in a sodium carbonate
aqueous solution and ethyl acetate was added to the solution.
The ethyl acetate layer was separated, dried over sodium sulfate
and evaporated under reduced pressure~ The residue was crystall-
ized from diethyl ether to give nitric ester of N-12-hydroxyethyl)
nicotinamide~ Recrystallization from diethyl ether afforded
colorless crystals havin~ a melting point of 90 - 92C.
-24-
, . . . .
.,: .
:
nalysis:
Calcd. for C8HgN3O4 C, 45.50; H, 4.29; N, 19.89 (~)
Found : C, 45.37; H, 4.09; N, 19.71 (~)
Example 11
By the procedure similar to that of Example 10, N-~2-
hydroxyethyl)isonicotinamide nitrate was reacted with fuming
nitric acid to obtain nitric ester of N-(2-hydroxy-ethyl) iso-
nicotinamide nitrate. (Yield: 76%, mp: 105C (decomposition)).
Analysis:
Calcd, for C8HloN4O7: C, 34.04; H, 3.68; N, 20.43 (~)
Found : C, 35.11; H, 3.57; N, 20.34 (~)
Example 12 ~harmaceutical Preparation~
- a~ Su~lingual Tablet
Compound prepared according 5 mg
to Example 2
Lactose 19.7 mg
Mannitol 25 mg
Magnesium Stearate 0.3 mg
Total 50 mg/
tablet
The ingredients were uniformly blended in the propor-
tions described above and the mixture was formed inta,tablets,
each tablet ~eing 5 mm in diameter and 50 mg in weight.
bL Table for Internal ~se
Compound prepared according 10 mg
to E~ample 1
Lactose 44.5 mg
Corn Starch 20 mg
Crystalline Cellulose 25 mg
Magnesium Stearate 0.5 mg
Total 100 mg/
tablet
-25-
~1088S~4
The ingredients were uniforml~ ~lended in the propor-
tions described a~oYe and the mixture was formed into tablets,
each ka~let ~eing 7 mm in diameter and 100 mg in weight.
c) Hard Capsules
Compound prepared according20 mg
to Example 2
Lactose 176 mg
Magnesium Stearate 4 mg
Total 200 mg/capsule
- The ingredients were uniformly ~lended in the propor-
tions descri~ed a~ove and hard capsules each being identified
as No. 3 were filled with 200 mg each of mixture ~y a packing
machine to form capsules each weighing 250 mg.
d2 Granules
Compound prepared according10 mg
to Example 2
Lactose 710 mg
Paste of Cornstarch 280 mg -
Total 1,000 mg/wrapper
The ingredients were uniformly kneaded and then gran-
ulated to form granules, each granule having a diameter of about
1 ~n.
e~ Parenteral Injection
Compound prepared according5 mg
to Example 2
Mannitol 50 mg
Total 55 mg/vial
The ingredients were dissolved in 1 ml of distilled
water and the solution was sterilized and filtered. The solution
30 was filled in a vial andfreeze-dried and the vial was sealed to ;
form a parenteral injection. Thefreeze-dried mixture was dissolved
in 1 ml of distilled water when it is used as parenteral injection.
. :
-26-
- . . . .
... ' : . ,
:, . . .
88s~
SUPPLEMENTARY DISCLOSURE
The applicants have conducted comparative tests with
the nitric ester of N- (2-hydroxyethyl)nicotinamide (hereinafter,
the compound of this invention) and the nitrate of the acid
dinitrooxydiethyl amide nicotinate as disclosed in the prior
art reference, namely, United States Patent Number 3,036,074,
which issued on May 22, 1962 to Stieglitz et al (hereinafter, the
reference)O
Figure 15 is a graph showing the percent increase in
coronary blood flow when the compound of the present invention
used in the comparative tests was administered and when the
compound of the reference was administered.
Figure 16 is a graph showing the duration of increase
in coronary blood flow when the compound of the present invention
used in the comparative tests was administered and when the
compound of the reference was administered.
Figure 17 is a graph showing the degree of drop of the
systemic blood pressure induced by the administration of the
compound of the-present invention used in the comparative tests
and when the compound of the reference was used.
Figure 18 is a graph showing the change in heat rate
after the administration of the compound of the present invention
used in the comparative tests and when the compound of the
reference was used.
Figure 19 is a graph showing the change in coronary
blood flow under stricturing of the coronary artery after the
administration of the compound of the present invention used in
the comparative tests and when the compound of the reference
was used.
-27-
`" 1~i~894~
1 Comparative Test:
Nitric ester of N-(2-hydroxyethyl)nicotinamide was
compared in pharmacological activity with the nitrate of the
acid dinitrooxydiethyl amide nicotinate disclosed in the
prior art reference, United States Patent 3,036,074.
Experiment
In accordance with the methods illustrated in
Experiment 2, Items 1, 5 and 6, except that beagle dogs which
had been anesthetized by the intravenous administration of
30 - 40 mg/kg of pentobarbital sodium were used as *est animals,
the change of coronar~ blood flow (CBF), systemic blood pressure
(SBP) and heart rate were measured upon administrating the
compound of this invention or the compound of the reference.
Further, in accordance with the methods disclosed in Experiments
4 and 6, the effects of both compounds upon the change of
coronary blood flow under stricturing of the coronary artery and
upon the variant angina pectoris model were measured.
Results
a) Change in coronary blood flow (CBF~:
The increase in CBF and the duration of increase in
CBF by the administration of the compound of this invention or
the reference are shown in Fig. 15 and Fig. 16, respectively.
The intravenous administration of the compound of
this invention even in a dose of 20 ~g/kg showed a significant
increase in coronary flow. Further, the effect of the compound -
of this invention on the increase in coronary blood flow was
significantly superior to the effect of the compound of the
reference with respect to each different dose.
Similarly, the intravenous administration of the
compound of this invention in a dose of 100 pg/kg showed a
duration of action of over 20 minutes, while the duration of
-28-
~: , . . .
1~88S~ 4
1 action given by the intravenous administration of the compound
of the reference is remarkably short. Thus, the differences
in their action are significant.
b) Change in systemic blood pressure (SBP):
AS shown in Fig. 17, the degree of drop of the
systemic blood pressure induced by the administration of the
compound of this invention was lower than that of the compound
of the reference.
c) Change in heart rate (HR):
As shown in Fig. 18, the heart rate showed almost
no change by the administration of the compound of this
invention, while it was remarkably increased by the administra-
tion of the reference compound.
- d) Change in coronary blood flow under stricturing of coronary
artery:
As shown in Fig. 19, coronary flow showed an increase
by the intravenous administration of the compound of this
invention, but,~in contrast, it showed a drop by the admini-
stration of the reference compound.
e) Effects of the compound on variant angina pectoris:
The intravenous administration of the compound of
this invention in a dose of more than 150 ~g/kg had an inhibiting
effect on abnormal change in electrocardiogram (ST-elevation) and
on the periodical ~luctuation in the coronary blood flow. In
contrast, the intravenous administration of the reference com-
pound even in a dose of 300 ~g/kg showed no such inhibiting
effect.
Discussion:
In general, the pharmacological activities required
as an anti-anginal drug which is the subject of this invention
-29-
~lO~389~4
1 are to increase the coronary blood flow over a prolonged period
of time without significantly changing the heart rate and blood
pressure. From these viewpoints, the compound of this invention
is, as shown in the test results a), b) and c) described above,
far superior in the activity required for an antianginal drug
to the compound disclosed in the reference.
Further, in the tests using pathological models,
particularly, in the test using a variant angina pectoris model
by stricturing the coronary artery, the compound of this invention
showed excellent effects although no significant effects were
seen with use of the reference compound.
--~0--
:
