Organic nitrates and method for their preparation

New organic nitrate compounds, formed by condensing a nitrato alkanoic acid with a sulfur-containing amino acid or peptide, which prevent nitrate tolerance or overcome existing tolerance and which are useful for the treatment of cardiac diseases including circulatory diseases, high blood pressure, cardiac insufficiency and for dilating the peripheral vessels.

This invention is concerned with new organic nitrates and with a method for 
their preparation. 
BACKGROUND OF THE INVENTION 
Organic nitrates (nitric acid esters) have been proven useful in the 
therapy of cardiac diseases. 
They act both by alleviating the before and after effects of a load on the 
heart, as well as through improvement of the oxygen supply to the heart by 
dilation of the coronary vessels. 
In any case, it has been found in recent years that the organic nitrates 
which have been used so far in therapy, such as glycerol trinitrate (GTN), 
isosorbid-5-mononitrate or isosorbid dinitrate, because of nitrate 
tolerance, exhibit a clear drop in efficacy in a relatively short time 
when continuous high dosages are administered to the patient. Numerous 
experiments indicate that the presence of sulfhydryl (--SH) groups 
prevents the development of nitrate tolerance and that an existing 
tolerance can be reduced. 
The development of tolerance is presently understood to be as follows: 
According to the present state of knowledge, the pharmacological action of 
organic nitrate compounds depends on the presence of cysteine. The organic 
nitrate forms a common precursor with cysteine and, when it decomposes, 
--NO radicals among others, are liberated and activate soluble guanylate 
cyclase, the target enzyme, of the smooth muscle cells. Subsequent 
reactions triggered by the formation of cGMP lead to relaxation or 
dilation of the vessels. 
The reactive and short-lived, and so far, only hypothetical intermediate 
product would have to be a thioester of nitric acid or a thionitrate. 
Through intramolecular rearrangement and other subsequent reactions which 
have not yet been established, the final formation of a nitroso thiol is 
postulated, from which nitrogen monoxide or nitrite ions are liberated. On 
the other hand, the enzyme-dependent degradation with the aid of GSH 
reductase would not be of significance for the pharmacological action, 
because it leads exclusively to the formation of nitrite ions. As already 
stated, the nonenzymatic degradation needs cysteine and can thus be 
exhausted in a dose-dependent manner (exhaustion of the SH group pool), so 
that over a long term sufficient NO, which is the actual activator of 
guanyl cyclase, can no longer be formed so that the clinical effectiveness 
will be reduced. 
SUMMARY OF THE INVENTION 
The compounds provided by the invention for the therapy of cardiac diseases 
have a specific structure which includes a group from a nitrato fatty acid 
(nitratoalkanoic acid) and a group from a sulfur-containing amino acid or 
peptide. 
Therefore, an object of the present invention is to provide new organic 
nitrates which, based on the general structural principle that they 
include a sulfhydryl group, are characterized by the fact that they 
prevent or resist development of nitrate tolerance and/or reverse existing 
nitrate tolerance when used for the therapy of cardiac diseases. 
DETAILED DESCRIPTION OF THE INVENTION 
This object is achieved by the fact that compounds are provided having the 
general formula: 
##STR1## 
wherein the symbols have the following meaning: R represents hydroxy, a 
lower alkoxy, a lower alkenoxy, a di-lower alkylamino-lower alkoxy, 
acylamino-lower alkoxy, acyloxy-lower alkoxy, aryloxy, aryl-lower alkoxy, 
substituted aryloxy or substituted aryl lower alkoxy groups, where the 
substituent is methyl, methoxy or a halogen such as chloro, bromo or 
fluoro; amino, lower alkylamino, di-lower alkylamino, aryl-lower 
alkylamino, hydroxy-lower alkylamino or amino acid residues as present in 
peptide bonds, 
R.sup.1 represents hydrogen, an alkyl having 1 to 6 carbon atoms, a 
substituted lower alkyl in which the substituent is a halogen, groups 
defined by R containing hydroxy, lower alkoxy, aryloxy, amino, lower 
alkylamino, furthermore acylamino, acyloxy, arylamino, mercapto, lower 
alkylthio or arylthio, 
R.sup.2 represents hydrogen, lower alkyl and the additional groups 
represented by R.sup.1, 
R.sup.3 represents hydrogen or lower alkyl, 
R.sup.4 represents hydrogen, lower alkyl, phenyl, methoxy phenyl, 
phenyl-lower alkyl, methoxyphenyl-lower alkyl, hydroxyphenyl-lower alkyl, 
hydroxy-lower alkyl, alkoxy-lower alkyl, amino-lower alkyl, 
acylamino-lower alkyl, mercapto-lower alkyl or lower alkylthio-lower 
alkyl, 
R.sup.5 represents lower alkyl thiol, --SH, S--acyl and particularly 
--S-acetyl, --S-propionyl, --S-butyryl, --S-isobutyryl, --S-capryl, 
--S-pivaloyl and --S-benzoyl; 
##STR2## 
and lower alkylthio-lower alkanoic acid and esters and amides thereof, 
and lower alkylthio-lower alkyl, wherein R.sup.6 represents hydrogen and 
lower alkyl 
groups in which R and R.sup.5 are bonded together and form part of a 
thiolactone group, 
groups in which R and R.sup.4 are bonded together in the form of an ester 
or amide, 
groups in which R.sup.3 and R.sup.4 are bonded together in the form of an 
alkylene bridge with 2 to 4 carbon atoms, an alkylene bridge with 2 to 3 
carbon atoms and a sulfur atom, an alkylene bridge with 3 to 4 carbon 
atoms, which contains a double bond or an alkylene bridge as above, which 
can be substituted by one or more hydroxy, lower alkoxy, lower alkyl or 
di-lower alkyl groups, 
m, n and q are whole numbers or integers from 0 to 10, desirably 0 to 3, p 
is 0 or 1, and 
pharmaceutically acceptable salts thereof. 
According to a further feature of the invention, the nitrato alkanoic acid 
components can have a chain length of C.sub.2 -C.sub.6, they may be 
straight-chain or branched chain, and they may be racemic or optical 
isomers. 
Preferably, the amino acids cysteine, methionine or homocysteine are used 
in making the organic nitrates so that groups from such amino acids are 
present therein. 
Advantageously, the amino acids are of the stereo-chemical L-form. 
Cysteine and/or methionine are desirably present in the form of their 
methyl, ethyl or propyl esters. 
The --SH group of cysteine can be esterified with a lower alkanoic acid 
having 2 to 8 carbon atoms. 
According to an especially advantageous further development the invention 
provides compounds having the following chemical names: 
N-(2-nitratoacetyl)-cysteine ethyl ester 
N-(2-nitratoacetyl)-S-acetyl-cysteine ethyl ester 
N-(2-nitratoacetyl)-S-propionyl-cysteine ethyl ester 
N-(2-nitratoacetyl)-S-pivaloyl-cysteine ethyl ester 
N-(2-nitratoacetyl)-methionine methyl ester 
N-(2-nitratopropionyl)-cysteine 
N-(2-nitratopropionyl)-cysteine ethyl ester 
N-(2-nitratopropionyl)-methionine ethyl ester 
N-(2-nitratobutyryl)-cysteine 
N-(2-nitratobutyryl)-cysteine ethyl ester 
N-(2-nitratobutyryl)-S-acetyl-cysteine ethyl ester 
N-(2-nitratobutyryl)-S-butyryl-cysteine ethyl ester 
N-(2-nitratobutyryl)-methionine ethyl ester 
N-(2-nitratoisobutyryl)-cysteine 
N-(2-nitratoisobutyryl)-cysteine ethyl ester 
N-(2-nitratoisobutyryl)-S-benzoyl-cysteine ethyl ester 
N-(2-nitratoisobutyryl)-S-acetyl-cysteine ethyl ester 
N-(2-nitratoisobutyryl)-S-pivaloyl-cysteine ethyl ester 
N-(2-nitratoisobutyryl)-methionine ethyl ester 
N-(3-nitratobutyryl)-cysteine 
N-(3-nitratobutyryl)-cysteine ethyl ester 
N-(3-nitratobutyryl)-S-acetyl-cysteine ethyl ester 
N-(3-nitratobutyryl)-S-propionyl-cysteine ethyl ester 
N-(3-nitratobutyryl)-methionine ethyl ester 
N-(3-nitratobutyryl)-homocysteine thiolactone 
N-(3-nitratopivaloyl)-cysteine 
N-(3-nitratopivaloyl)-cysteine ethyl ester 
N-(3-nitratopivaloyl)-cysteine ethyl ester-S-ethyl carbonate 
N-(3-nitratopivaloyl)-S-acetyl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-propionyl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-butyryl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-isobutyryl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-pivaloyl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-benzoyl-cysteine ethyl ester 
N-(3-nitratopivaloyl)-methionine ethyl ester 
N-(3-nitratopivaloyl)-methionine 
N-(3-nitratopivaloyl)-homocysteine thiolactone 
N-(2-nitratohexanoyl)-cysteine ethyl ester 
N-(2-nitratohexanoyl)-S-propionyl-cysteine ethyl ester 
N-(3-nitratohexanoyl)-cysteine ethyl ester 
N-(3-nitratohexanoyl)-methionine methyl ester 
N-(12-nitratolauroyl)-cysteine 
N-(12-nitratolauroyl)-cysteine ethyl ester 
N-(12-nitratolauroyl)-S-acetyl-cysteine 
N-(12-nitratolauroyl)-S-pivaloyl-cysteine 
and other esters thereof, particularly lower alkyl esters such as the 
methyl, propyl, isopropyl, butyl and pentyl esters. 
According to another aspect of the invention, drugs or medicines containing 
one or more of the compounds are provided. 
The compounds provided by this invention can be administered to a patient 
as pure esterified or non-esterified compounds, or in the form of nontoxic 
acid addition salts, orally or intravenously. The patient may be a human 
or a lower animal. To obtain a practical size to dosage relationship one 
or more of the compounds may be combined with a suitable pharmaceutical 
carrier and made into unit-dosage forms for administration to a patient. 
Pharmaceutical carriers which are liquid or solid can be used. The 
preferred liquid carriers are water, ethanol, propyleneglycol, 
polyethyleneglycol and mixtures thereof. Flavoring materials can be 
included in the solutions as desired. A solution for intravenous injection 
may contain about 1 to 40 mg of active compound per liter. 
Solid pharmaceutical carriers such as starch, sugar and talc can be used to 
form powders. The powders can be used as such or be tableted, or be used 
to fill gelatin capsules. Suitable lubricants like magnesium stearate, 
binders such as gelatin and disintergrating agents like sodium carbonate 
in combination with citric acid can be used to form the tablets. 
Unit dosage forms such as tablets and capsules can contain any suitable 
predetermined amount of one or more of the compounds and can be 
administered one or more at a time at regular intervals. Such forms, 
however, may contain a concentration of about 0.1 to 10% by weight of a 
compound of this invention. 
A typical tablet can have the composition: 
______________________________________ 
Mg. 
______________________________________ 
1. N-(3-nitrato-S-pivaloyl)-cysteine ethyl ester 
25 
2. Starch, U.S.P. 57 
3. Lactose, U.S.P. 73 
4. Talc, U.S.P. 9 
5. Stearic acid 6 
______________________________________ 
Powders 1, 2 and 3 are slugged, then granulated, mixed with 4 and 5, and 
tableted. 
Daily administration of about 1 to 500 mg, and preferably about 10 to 200 
mg, of N-(3-nitrato-S-pivaloyl)-cysteine ethyl ester and other compounds 
of this invention, including those disclosed and named above, is usually 
satisfactory. However, since some variation between compounds is to be 
expected, the precise dosages of each is to be evaluated prior to 
administration. Furthermore, the differences in patients normally will 
require prescription of various amounts of the active drugs from case to 
case. In general, treatment may be achieved with the administration of 
unit dosage forms containing about 1 to 50 mg. of the active compound 
administered during a day. The usual unit dosage may be about 10 to 30 mg. 
administered one to three times a day. 
These organic nitrates, desirably in the form of a pharmaceutical 
composition, can be used for the treatment of cardiac disease as 
manifested by circulatory diseases, for example, as coronary dilators, as 
agents for the treatment of high blood pressure, cardiac insufficiency and 
for dilating the peripheral vessels, including the vessels of the brain 
and kidneys. 
Finally, the compounds can be prepared by condensing the corresponding 
nitrato fatty acids or their reactive derivatives with the amino group of 
an amino acid or a peptide. If desired, the compounds obtained can be 
subjected to side-chain alkylation or side-chain acylation in a subsequent 
reaction step. 
The process thus comprises reacting a compound of the formula: 
##STR3## 
in the form of a member of the group consisting of the free acid, a 
reactive halide, azide, ester and anhydride, with a compound of the 
formula 
##STR4## 
to produce a compound of the formula 
##STR5## 
wherein R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, m, n, o and p have 
the meaning previously set forth herein. 
Reactive derivatives of the nitrato fatty acids which can be used as 
reactants according to the invention are, for example, acid halides, acid 
anhydrides, activated amides and activated esters. Preferably, acid 
chlorides, acid azides, symmetrical acid anhydrides, activated esters and 
mixed anhydrides with organic or inorganic acids can be used. 
The condensation reaction of a nitrato fatty acid with an amino group of an 
amino acid can also be carried out in an inert solvent and in the presence 
of a condensing agent which promotes the formation of an acid amide bond, 
a carbodiimide such as N,N'-dicyclohexyl carbodiimide or a similar 
carbodiimide, an imine compound such as diphenylketene-N-cyclohexylimine 
or pentamethylene-ketene-N-cyclohexylimine, or a phosphate or phosphite 
such as triethyl phosphite, ethyl polyphosphate or isopropyl 
polyphosphate, over a period of 1-48 hours at temperatures from 
-10.degree. C. to the refluxing temperature of the solvent used.

The following examples illustrate without limiting the invention. 
Example 1 
Preparation of N-(3-nitratobutyryl)cysteine ethyl ester 
First Step 
Saponification of 3-hydroxybutyric acid ethyl ester 
3-Hydroxybutyric acid ethyl ester (Aldrich), 13.2 g (0.1 mole), was reacted 
with 4.0 g (0.1 mole) of NaOH dissolved in 100 ml of water. The reaction 
was complete when the solution became homogeneous. 
The reaction mixture was acidified with 10 ml of concentrated HCl and then 
extracted twice using 100 ml of ethyl acetate each time. Then the solution 
was evaporated on a rotary evaporator; a thin flowing oil remained. 
The yield was 8.81 g (theory: 10.4 g) of 3-hydroxybutyric acid. 
Second Step 
Nitration of the 3-hydroxybutyric acid 
3-Hydroxybutyric acid, 8.81 g (0.08 mole), and 50 mg of urea were dissolved 
in 50 ml of acetic acid at 5.degree. C. First 6.27 ml (0.15 mole) of 
HNO.sub.3 was added dropwise and then 14.17 ml (0.15 mole) of Ac.sub.2 O 
was added under cooling. The reaction mixture was stirred overnight. 
The mixture was worked up by adding 200 ml of ice water to the solution 
obtained and then extracting it with ethyl acetate. The organic phase was 
then extracted with NaHCO.sub.3. The NaHCO.sub.3 phase was acidified with 
concentrated HCl and extracted with ethyl acetate. Finally, the solution 
was evaporated on a rotary evaporator, whereupon a thin flowing oil 
remained. 
The yield was 9.4 g (theory 11.9 g) of 3-nitratobutyric acid. 
Third Step 
Preparation of N-(3-nitratobutyryl)-cysteine ethyl ester 
3-Nitratobutyric acid, 16.6 g (0.11 mole), was dissolved in 100 ml of 
dichloromethane. While passing N.sub.2 through the mixture, 17.9 g (0.12) 
of cysteine ethyl ester was added slowly at 15.degree. C. Then 24.7 g 
(0.12 mole) of dicyclohexyl carbodiimide (DCC) dissolved in 80 ml of 
dichloromethane was added dropwise slowly at 15.degree. C. while N.sub.2 
was passed through. The dicyclohexylurea formed was filtered off at the 
end of the reaction under suction and the solution was washed with 150 ml 
of 0.1N HCl. Then the solution was evaporated on a rotary evaporator. 
A purified sample of the substance was prepared by column chromatography 
and recrystallization from ethanol/n-hexane. 
Yield: 6.88 g (theory: 30.83 g) M.p. 77.8.degree. C. 
Example 2 
Preparation of N-(3-nitratobutyryl)-methionine ethyl ester 
3-Nitratobutyric acid, 6.35 g (0.043 mole), 7.47 g (0.043 mole) of 
methionine ethyl ester and an amount of dimethylaminopyridine (DMAP), that 
the tip of a spatula holds, were dissolved in 100 ml of dichloromethane 
with stirring and cooling to 10.degree. C. Then 10.31 g (0.05 mole) of DCC 
was dissolved in 80 ml of CH.sub.2 Cl.sub.2 and added dropwise slowly 
under simultaneous introduction of nitrogen. After the reaction was 
completed, the solution was filtered off under suction, washed with 
NaHCO.sub.3 and finally with HCl. The solution was evaporated in a rotary 
evaporator, whereupon an oil remained. 
Purification of a sample was carried out by column chromatography or by 
crystallization in the cold. 
The yield was 1.95 g (theory 12.05 g) of N-(3-nitratobutyryl)-methionine 
ethyl ester as a colorless oil. 
Example 3 
Preparation of N-(3-nitratopivaloyl)-cysteine ethyl ester 
First Step 
Preparation of nitratopivalic acid methyl ester 
Hydroxypivalic acid methyl ester, 25.0 g (0.19 mole) and 0.12 g of urea 
were dissolved at room temperature in 250 ml of CH.sub.2 Cl.sub.2 and 
cooled to 5.degree. C. under stirring. Then 23.8 g (0.38 mole) of 
HNO.sub.3 (100%) was added dropwise with stirring, so that the temperature 
did not exceed 10.degree. C. The mixture was cooled to 5.degree. C. and 
38.6 g (0.38 mole) of acetic anhydride was added dropwise with stirring in 
such a way that the temperature did not exceed 10.degree. C., the mixture 
was then stirred into an ice bath with cooling for a 15 minute period and 
then slowly warmed to room temperature and further stirred overnight at 
room temperature. The batch was introduced slowly into 500 ml of ice water 
under stirring. The CH.sub.2 Cl.sub.2 phase was separated and was washed 
once with each of 100 ml of distilled H.sub.2 O, 100 ml of saturated 
aqueous NaHCO.sub.3 solution and again with 100 ml of distilled H.sub.2 O. 
Then the CH.sub.2 Cl.sub.2 extract was evaporated to dryness on a 
Rotavapor at a maximum bath temperature of 40.degree. C. in the vacuum of 
a water jet pump. The light-yellow oily residue distilled at the vacuum of 
an oil pump at a bath temperature of 60.degree. C. as a clear thin flowing 
oil. 
Yield: 31.5 g, corresponding to 94.0% of theory 
Second Step 
Preparation of nitratopivalic acid 
NaOH, 14.0 g (0.350 mole), was dissolved in H.sub.2 O and cooled to about 
10.degree. C. Then a solution of 31.0 g (0.175 mole) of nitratopivalic 
acid methyl ester in 250 ml of methanol was added with stirring whereupon 
the reaction mixture developed a yellow color and the temperature 
increased to about 25.degree. C. After 90 minutes of stirring, the batch 
was neutralized with 29.5 ml (0.35 mole) of 37% HCl and the methanol was 
completely distilled off in a Rotavapor. The aqueous phase was extracted 
twice using 200 ml of methylene chloride each time. The combined methylene 
chloride extracts were washed once with 50 ml of distilled H.sub.2 O and 
the methylene chloride phase was evaporated to dryness on the Rotavapor. 
The colorless, oily residue was dissolved in 100 ml of ethyl acetate and 
again evaporated to dryness on the Rotavapor, whereupon a white solid 
residue remained from which the solvent residues were removed at the 
vacuum of an oil pump (0.4 torr) at a bath temperature of about 40.degree. 
C. over a period of 15 minutes in a Rotavapor. The solid, white residue of 
25.44 g (89.1% of theory) was dissolved in 100 ml of boiling n-hexane and 
2 ml of diisopropyl ether was added. After cooling to room temperature 
and addition of nucleating crystals, the product crystallized out. The 
product was allowed to stand at 0.degree. C. for 72 hours, the crystals 
were filtered off under suction and, after washing twice using 10 ml of 
n-hexane each time, it was dried in a vacuum drying oven to a constant 
weight at room temperature at about 2 torr. 
M.p. 54.2.degree. C. Yield: 23.66 g, corresponding to 82.9% of the theory 
Third Step 
Preparation of N-(3-nitratopivaloyl)-cysteine ethyl ester 
L-cysteine ethyl ester base, 10.7 g (71.7 mmoles), was dissolved in 200 ml 
of methylene chloride at room temperature under stirring in an N.sub.2 
atmosphere. Then 11.4 g (70.0 mmoles) of crystalline nitratopivalic acid 
were added and dissolved under stirring at room temperature. Then a 
solution of 14.8 g (71.7 mmoles) of N,N-dicyclohexylurea (DCC) in 50 ml of 
methylene chloride was added to this mixture with stirring in a nitrogen 
atmosphere at room temperature. The addition was carried out dropwise over 
a period of about 15 minutes whereupon the temperature increased to 
35.degree. C. After further stirring, white dicyclohexylurea precipitated. 
The batch was cooled to room temperature and stirred overnight under a 
nitrogen atmosphere. The dicyclohexylurea was then filtered off through a 
sintered glass filter and was washed once with 50 ml of CH.sub.2 Cl.sub.2. 
The combined methylene chloride solutions were washed once with 100 ml of 
1N HCl and twice using 100 ml of distilled H.sub.2 O each time (under an 
N.sub.2 atmosphere) and then evaporated in a Rotavapor at a bath 
temperature of about 40.degree. C. in the vacuum of a water jet pump at an 
initial pressure of 550 mbar to approximately 20 mbar. A yellow-brown oil 
was obtained. 
Yield: 21.2 g, corresponding to 102.9% of theory. 
The substance was purified by recrystallization from ethanol/hexane in the 
cold. 
Yield: 13.42 g of N-(3-nitratopivaloyl)-cysteine ethyl ester as a 
light-pink oil, corresponding to 65.1% of theory. 
Fourth Step 
Preparation of N-(3-nitratopivaloyl)-S-acetyl-cysteine ethyl ester 
To 10.3 g (35.0 mmoles) of N-(3-nitratopivaloyl)cysteine ethyl ester 
dissolved in 70 ml of dichloromethane was added a solution of 4.3 g (42.0 
mmoles) of acetic anhydride in 10 ml of dichloromethane in the cold 
dropwise with stirring. Then a solution of 5.0 g (49.0 mmoles) of 
triethylamine in 20 ml of dichloromethane was added dropwise in the cold 
with stirring. After the end the reaction, the batch was washed with 1N 
HCl, 10% aqueous sodium bicarbonate solution and water. The 
dichloromethane extract was evaporated to dryness on the Rotavapor. 11.6 
grams of a light-yellow, oily product thus was obtained from which 7.8 g 
of crystalline product (66.3% of theory) was obtained by recrystallization 
from ethanol/water in the cold and with the addition of nucleating 
crystals. 
M.p. &lt;5.degree. C. 
Fourth Step/Variation 1 
Preparation of N-(3-nitratopivaloyl)-S-butyryl-cysteine ethyl ester 
If one uses 6.7 g (42.0 mmoles) of butyric acid anhydride instead of acetic 
anhydride as described in Step 4, and if the reaction and the work-up is 
conducted in the same way, 13.0 g of a light-yellow, oily product is 
obtained from which 9.7 g of crystalline product (corresponding to 76.2% 
of theory) was obtained by recrystallization in the cold, as described in 
Step 4. 
M.p.:&lt;5.degree. C. 
Fourth Step, Variation 2 
Preparation of N-(3-nitratopivaloyl)-S-pivaloyl-cysteine ethyl ester 
If one uses 7.8 g (42.0 mmoles) of pivalic acid anhydride instead of the 
acetic anhydride described in Step 4, and if one conducts the reaction and 
the work-up in the same way, 14.1 g of a light-yellow, oily product is 
obtained from which one obtains 10.5 g of crystalline product 
(corresponding to 79.5% of theory) by recrystallization as described in 
Step 4. 
M.p. 45.degree. C. 
Fourth Step/Variation, 3 
Preparation of N-(3-nitratopivaloyl)-cysteine ethyl ester-S-ethyl carbonate 
If one uses 4.3 g (42.0 mmoles) of ethyl chloroformate instead of the 
acetic anhydride described in Step 4, and if one conducts the reaction and 
the work-up in the same way, 11.5 g of a light-yellow, oily product is 
obtained from which one obtains 9.5 g of crystalline product 
(corresponding to 74.1% of theory) by recrystallization as described in 
Step 4. 
M.p. 36.degree. C. 
Example 4 
Preparation of N-(3-nitratopivaloyl)-methionine ethyl ester 
L-methionine ethyl ester base, 12.4 g (70.0 mmoles), was dissolved in 250 
ml of methylene chloride at room temperature in an N.sub.2 atmosphere. 
Then 11.4 g (70.0 mmoles) of crystalline nitratopivalic acid was added and 
dissolved with stirring at room temperature. To the mixture was added 
dropwise a solution of 14.8 g (71.7 mmoles) of N,N-dicyclohexylurea (DCC) 
in 50 ml of methylene chloride in about 15 minutes at room temperature 
under stirring and in a nitrogen atmosphere, whereupon the temperature 
increased to 35.degree. C. After further stirring, white dicyclohexylurea 
precipitated. The batch was cooled to room temperature and was stirred 
overnight in a nitrogen atmosphere. The DCC urea was then filtered off 
through a sintered glass filter and was washed once with 50 ml of CH.sub.2 
Cl.sub.2. The combined methylene chloride solutions were washed once with 
100 ml of 1N HCl and twice with 100 ml of distilled H.sub.2 O each time 
(under an N.sub.2 atmosphere) and then evaporated in the Rotavapor at a 
bath temperature of about 40.degree. C. and in the vacuum of a water jet 
pump at an initial pressure of 550 mbar, which went to about 20 mbar. A 
light-yellow oil was obtained. 
Yield: 24.9 g of crude N-(3-nitratopivaloyl-L-methionine methyl ester, 
corresponding to 110.3% of theory. 
The crude product was purified by sample chromatography on a column. 
Yield: 17.6 g of N-(3-nitratopivaloyl)-methionine ethyl ester as a 
colorless oil, corresponding to 78.0% of theory. 
Example 5 
N-(12-nitratolauroyl)-S-acetyl-cysteine 
First Step 
Preparation of 12-nitratolauric acid 
12-Hydroxylauric acid, 54.1 g (0.250 mole), and 0.3 g of urea were 
dissolved in 1.3 liter of CHl.sub.3 with slight warming and then cooled to 
20.degree. C. with stirring. Then, with stirring, 23.6 g (0.375 mole) of 
HNO.sub.3 (100%) was added slowly, dropwise, whereupon the temperature 
rose to 27.degree. C. The mixture was cooled to 20.degree. C. and 38.3 g 
(0.375 mole) of acetic acid anhydride was added dropwise under stirring 
and cooling, whereby a temperature limit of 25.degree. C. was observed. 
The mixture was stirred overnight at room temperature. Finally, it was 
washed five times using 0.5 liter of distilled H.sub.2 O each time. The 
CHCl.sub.3 phase, which had been dried over Na2SO.sub.4 and clarified with 
pulverized activated carbon, was evaporated to dryness in a Rotavapor at a 
bath temperature of 50.degree. C. at the vacuum of a water jet pump. The 
oily residue of 60.8 g was dissolved in 500 ml of boiling n-hexane and, 
after cooling to room temperature, it was allowed to stand overnight in a 
refrigerator at 0.degree. C. The crystalline product was precipitated and 
was washed twice using 50 ml of n-hexane each time. Finally, the product 
was evaporated to constant weight in a vacuum drying oven at room 
temperature and at about 2 torr. 
M.p. 29.degree. C. 
Yield: 39.4 g, corresponding to 60.3% of theory. 
Second Step 
Preparation of 12-nitratolauric acid chloride 
Nitratolauric acid, 2.61 g (10 mmoles), was dissolved in 50 ml of methylene 
chloride and 4.44 g (35 mmoles) of oxyalyl chloride in 50 ml of methylene 
chloride was added dropwise at room temperature with stirring. The mixture 
was stirred overnight. Finally, the product was evaporated to dryness in a 
rotary evaporator. 
Yield: 3 g, corresponding to 93.2% of theory. 
Third Step 
Preparation of N-(12-nitratolaurol)-cysteine 
Under a nitrogen atmosphere, 6.06 g (50 mmoles) of L-cysteine was 
introduced into 300 ml of DMF with stirring. Then 5.60 g (20 mmoles) of 
12-nitratolauric acid chloride in 50 ml of dichloromethane was added 
dropwise. Since a clear solution was not obtained, the mixture was heated 
to 60.degree. C. Finally, 100 ml of distilled H.sub.2 O was added and the 
mixture was stirred overnight at room temperature. Finally, the mixture 
was diluted with 300 ml of H.sub.2 O and was extracted four times using 
200 ml of ethyl acetate each time. The organic phase was dried over 
Na.sub.2 SO.sub.4 and then evaporated. The residue was taken up in 100 ml 
of ether and was allowed to stand in a refrigerator overnight at 0.degree. 
C. White crystals were obtained. 
M.p. 74.degree.-75.degree. C. 
Yield: 4.1 g of N-(12-nitratolauroyl)-cysteine. 
Fourth Step 
Preparation of N-(12-nitratolauroyl )-S-acetyl-cysteine 
Under a nitrogen atmosphere, 1.82 g (5 mmoles) of 
N-(12-nitratolauroyl)-cysteine was placed in 20 ml of ethyl acetate. The 
mixture was cooled to 0.degree. C. and 2.5 ml of acetic anhydride was 
added dropwise. Then at -5.degree. C., 1.52 g (15 mmoles) of triethylamine 
dissolved in 5 ml of ethyl acetate was added dropwise. The reaction 
solution was washed with water and evaporated to dryness. 
M.p.: oil at room temperature. 
Yield: 2 g, corresponding to 98.4% of theory. 
Example 6 
Preparation of N-(12-nitratolauroyl)-cysteine ethyl ester 
Cysteine ethyl ester base, 4 g (26.8 mmoles), was dissolved in 50 ml of 
methylene chloride and 2.8 g (10 mmoles) of 12-nitratolauric acid chloride 
dissolved in 50 ml of methylene chloride was added dropwise with stirring 
and the mixture then was stirred overnight. The precipitated cysteine 
ethyl ester HCl salt was filtered off under suction and the solvent was 
removed in a Rotavapor. The oily residue (6 g) was dissolved in 100 ml of 
ether and was allowed to stand in a refrigerator overnight at 0.degree. C. 
The precipitated product was filtered off. 
M.p. 59.degree.-60.degree. C. 
Yield: 1.6 g, corresponding to 40.0% of theory. 
Example 7 
Preparation of N-(2-nitratopropionyl)-cysteine ethyl ester 
First Step 
Preparation of nitratolactic acid ethyl ester 
Lactic acid ethyl ester, 33 g (0.28 mole), was dissolved in 300 ml of 
dichloromethane. After addition of 100 mg of urea, 22.5 ml (0.56 mole) of 
100% nitric acid was added dropwise at a temperature of 
5.degree.-10.degree. C. The solution was cooled to 0.degree. C. Then 52.8 
ml (0.56 mole) of acetic anhydride was added dropwise in such a way that 
the temperature did not rise above 5.degree. C. The solution was allowed 
to stand overnight at room temperature and then was washed with 250 ml of 
water. The organic phase was separated and dried over sodium sulfate. 
After filtration, the dichloromethane was distilled off. The oily residue 
obtained was processed by distillation. 
Yield: 30.34 g, corresponding to 66.4% of theory. 
B.p.: 34.degree. C. (0.25 torr). 
Second Step 
Preparation of nitratolactic acid 
Nitratolactic acid ethyl ester, 30 g (0.18 mole), was dissolved in 80 ml of 
dioxane. Then 30 ml of water and 2 g (0.02 mole) of sulfuric acid were 
added to the solution and the mixture was refluxed for 19 hours. The 
solution was evaporated to a volume of about 50 ml and then diluted with 
300 ml of water. The pH value was adjusted to 7-8 by the addition of 
sodium bicarbonate. The unreacted ester was removed by extraction with 
dichloromethane. 
The aqueous phase was adjusted to pH 1 with concentrated hydrochloric acid 
and extracted three times using 150 ml of ethyl acetate each time. The 
extracts were combined and dried over sodium sulfate. After filtration the 
ethyl acetate was removed completely in a rotary evaporator. 
Yield: 14.6 g of colorless oil, corresponding to 59.2% of theory. 
Third Step 
Preparation of N-(2-nitratopropionyl)-cysteine ethyl ester 
In a nitrogen atmosphere, 17 g (0.13 mole) of nitratolactic acid and 18.9 g 
(0.13 mole) of cysteine ethyl ester were dissolved in 200 ml of 
dichloromethane at 10.degree.-15.degree. C. At 15.degree.-20.degree. C. a 
solution of 28.6 g (0.14 mole) of N,N-dicyclohexyl carbodiimide and 75 ml 
of dichloromethane was added dropwise. After 1 hour, the precipitated 
N,N-dicyclohexylurea was filtered off and was washed with 75 ml of 
dichloromethane. The filtrate was extracted twice using 50 ml of 0.1N 
hydrochloric acid each time. The organic phase was evaporated completely 
on a rotary evaporator. The crude crystalline product (22.4 g) was 
recrystallized from 100 ml of ethanol/n-hexane (1:1). 
Yield: 7.6 g, corresponding to 22.6% of theory. 
M.p.: 92.8.degree. C. 
EXAMPLE 8 
Preparation of N-(3 -Nitratohexanoyl)-L-cysteine ethyl ester 
5.1 g of 3-nitratohexanoic acid, 5.94 g of cysteine ethyl ester 
hydrochloride, and a catalytic amount of dimethylaminopyridine were 
suspended in 50 ml of dioxan. At room temperature a solution of 6.58 g of 
N,N-dicyclohexylcarbodiimide in 20 ml of dioxan was added dropwise. The 
batch was stirred for two days at room temperature and then filtered. The 
dioxan was evaporated, the residue was dissolved in 100 ml of ethylacetate 
and extracted twice with 100 ml aliquots of 0.1N HCl. Then the solution 
was evaporated on a rotary evaporator. Yield: 4.6 g (51.44% of theory) of 
the product as a light brown oily product. 
EXAMPLE 9 
Preparation of N-(3-Nitratopivaloyl)-homocysteine thiolactone 
5 g of 3-nitratopivalic acid and 4.7 g of homocysteine thiolactone HCl were 
suspended in 50 ml of tetrahydrofuran. After the addition of 2.4 g of 
pyridine, at a temperature of 5.degree.-10.degree. C., a solution of 6.2 g 
of N,N-dicyclohexylcarbodiimide in 20 ml of tetrahydrofuran was added 
dropwise. The mixture was stirred overnight at room temperature and then 
filtered. The filtrate was evaporated on a rotary evaporator. 150 ml of 1N 
NaOH were added and extracted twice with 100 ml aliquots of CH.sub.2 
Cl.sub.2. The organic phase was washed with 100 ml of 1N HCl and 
evaporated on a rotary evaporator. Yield: 2.6 g of oil. The product was 
crystallized from 20 ml of a mixture of n-hexane and 10 ml of ethanol. 
Yield: 510 mg (6.5% of theory). M.p.: 88.6.degree. 
EXAMPLE 10 
Preparation of N-(2-Nitratoacetyl)-L-cysteine ethyl ester 
12.6 g of cysteine ethyl ester was dissolved in a solution of 11.35 g of 
2-nitratoacetic acid in 200 ml of ethyl acetate. At a temperature of 
20.degree.-25.degree. C., a solution of 19.4 g of 
N,N-dicyclohexylcarbodiimide in 20 ml of CH.sub.2 Cl.sub.2 was added 
dropwise. The mixture was stirred overnight at room temperature and 
filtered. The filtrate was evaporated on a rotary evaporator. Yield: 17.9 
g. The product was recrystallized twice from isopropanol. Yield: 2.5 g 
(11.66% of theory). M.p.: 76.1.degree. C. 
EXAMPLE 11 
Preparation of N-(3-Nitratopivaloyl)-S-propionyl-L-cysteine ethyl ester 
N-(3-nitratopivaloyl)-cysteine ethyl ester (10.3 g) and 5.5 g of propionic 
anhydride are combined in 60 ml of dichloromethane at room temperature in 
a nitrogen atmosphere with stirring. Then 5 g of triethylamine in 
dichloromethane is added dropwise under stirring. After the end of the 
reaction, the solution is washed with 1N HCl, 10% aqueous sodium 
bicarbonate solution and water. The dichloromethane extract is evaporated 
to dryness on a rotavapor. 12.46 grams (101.6% of theory) of a light brown 
oily product is obtained. The identity of the product is verified by mass 
spectroscopy. 
EXAMPLE 12 
Preparation of N-(2-Nitratohexanoyl)-cysteine ethyl ester 
2.74 g of 2-nitratohexanoic acid, 2.78 g of cysteine ethyl ester HCl and a 
catalytic amount of dimethylaminopyridine were dissolved in 50 ml of 
dioxan under stirring and heating. Then 3.09 g of 
N,N-dicyclohexylcarbodiimide dissolved in 20 ml of dioxan are added 
dropwise in a nitrogen atmosphere. After cooling the crystals were 
filtered off and the filtrate was evaporated on a rotary evaporator. The 
residue was dissolved in 100 ml of ethyl acetate under a nitrogen 
atmosphere and washed with 100 ml of water. The solution was then 
evaporated on a rotary evaporator to yield 4.63 g of product (theory 5.58 
g). The product was crystallized from 50 ml of ethanol and 60 mg of pure 
product obtained. M.p.: 51.4.degree. C. 
EXAMPLE 13 
Preparation of N-(2-Nitratobutyrl)-cysteine ethyl ester 
2-nitratobutyric acid (5 g), 8.59 g of cysteine ethyl ester HCl and a 
catalytic amount of dimethylaminopyridine were dissolved in 100 ml of 
dioxan under heating and stirring in a nitrogen atmosphere. Then 9.49 g of 
N,N-dicyclohexylcarbodiimide in 80 ml of dioxan are added dropwise. The 
reaction mixture is stirred overnight and filtered. The filtrate was 
evaporated on a rotary evaporator. The residue was dissolved in ethyl 
acetate and washed with water. The solution was then evaporated on a 
rotary evaporator to yield 8.03 g of an oily product (9.24 g theory). 
EXAMPLE 14 
Preparation of N-(3-Nitratobutyryl)-S-acetyl-cysteine ethyl ester 
Seven grams of N-(3-nitratobutyryl)-cysteine ethyl ester are dissolved in 
150 ml of dichloromethane. Then 27.9 ml of acetic anhydride are slowly 
added at 5.degree.-10.degree. C. under a nitrogen atmosphere. A solution 
of 30.4 g of triethylamine in 100 ml of dichloromethane is added dropwise. 
The mixture is then stirred overnight and then washed with a sodium 
bicarbonate solution and subsequently with water. The solution is then 
carefully evaporated on a rotary evaporator. Yield: 9.8 g. The product is 
crystallized from 50 ml of ethanol. Yield: 3.13 g; 38.84% of theory. M.p.: 
81.9.degree. C. 
EXAMPLE 15 
Preparation of N-(3-Nitratopivaloyl)-S-benzoyl-cysteine ethyl ester 
10 g of N-(3-nitratopivaloyl)-cysteine ethyl ester are dissolved in 100 ml 
of dichloromethane at 5.degree.-10.degree. C. under a nitrogen atmosphere. 
Subsequently, 4.8 ml of benzoyl chloride are added dropwise. 4.7 ml of 
triethylamine dissolved in 50 ml of dichloromethane are added dropwise at 
5.degree.-10.degree. C. The reaction mixture is stirred for three days. 
Ice water is added, the organic phase is separated and evaporated. The 
product is purified by column chromatography using a mixture of 75 parts 
of methanol and 25 parts of water as the elutant. The eluted fraction is 
evaporated on a rotary evaporator. Yield: 10.7 g of purified oily product; 
79% of theory. 
EXAMPLE 16 
Preparation of N-(2-Nitratoisobutyryl)-L-cysteine ethyl ester 
First Step 
Nitration of 2-hydroxyisobutyric acid ethyl ester 
16.6 ml concentrated nitric acid at 10.degree.-15.degree. C. are added 
dropwise to a solution of 26.5 g of 2-hydroxyisobutyric acid ethyl ester 
in 250 ml of dichloromethane. The solution is cooled to 
0.degree.-5.degree. C. and at this temperature 37.5 ml of acetic anhydride 
were added dropwise. The solution is stirred overnight at room temperature 
and then 500 ml of ice water are added. The organic phase is separated and 
successively washed with 100 ml of water, 100 ml of an aqueous 
concentrated sodium bicarbonate solution and 100 ml of water. The 
dichloromethane is removed by use of a rotary evaporator. Yield: 34 g of 
the product as an oil; 96% of theory. 
Second Step 
Saponification of 2-Nitratoisobutyric acid ethyl ester 
30.4 g of 2-nitratoisobutyric acid ethyl ester are dissolved in 120 ml of 
ethanol. Then a solution of 13.7 g of sodium hydroxide in 100 ml of water 
is added. The resulting mixture is stirred for 4 hours at room temperature 
and then neutralized by the addition of 29 ml of 37% hydrochloric acid. 
After evaporation on a rotary evaporator the residue (21 g) is suspended 
in 75 ml of boiling n-hexane and filtered. The product is isolated by 
crystallization at room temperature. Yield: 18.99 g; 66.1% of theory. 
Third Step 
Preparation of N-(2-Nitratoisobutyryl)-cysteine ethyl ester 
A solution of 14.5 g of dicyclohexylcarbodiimide (DCC) in 50 ml of 
dichloromethane at 5.degree.-10.degree. C. is added dropwise to a solution 
of 8.7 g of 2-nitratoisobutyric acid and 9.6 g of cysteine ethyl ester in 
50 ml of dichloromethane. The mixture is stirred for four days at room 
temperature, the DCC-urea is filtered off and the filtrate is washed with 
100 ml of 1N hydrochloric acid and 100 ml of water. The dichloromethane is 
removed by use of a rotary evaporator, yielding an oily residue which was 
purified twice by column chromatography using 765 g of kieselgel Li 
Chroprep RP18 and 70 methanol/30 water as the eluting solvent. Yield: 1.48 
g of oil; 9.1% of theory. 
EXAMPLE 17 
Preparation of N-(2-Nitratoisobutyryl)-L-methione ethyl ester 
N-(2-nitratoisobutyryl)-L-methione ethyl ester was produced using the 
following materials: 
______________________________________ 
1. L-methione ethyl ester base 
10.6 g 
2. 2-nitratoisobutyric acid 
8.9 g 
3. Dicyclohexylcarbodiimide (98%) 
13 g 
4. Dimethylaminopyridine 20 mg 
5. Dichloromethane 185 ml 
______________________________________ 
The process of Example 16, Third Step, was followed using L-methione ethyl 
ester instead of cysteine ethyl ester. Yield: 20.53 g of a light yellow 
oil; 111% of theory. 
The resulting product contained about 10% of a --O--NO.sub.2 -containing 
impurity. A solution was prepared by dissolving 20.5 g of the oil in 51.5 
ml of methanol and 20 ml of distilled water for purification by column 
chromatography. (See Example 16, Third Step). 16.75 g of the product as a 
colorless oil were obtained. Yield: 90.5% of theory. 
EXAMPLE 18 
Preparation of N-(3-Nitratopivaloyl)-S-isobutyryl-L-cysteine ethyl ester 
N-(3-nitratopivaloyl)-S-isobutyryl-L-cysteine ethyl ester was prepared 
using the following materials: 
______________________________________ 
1. 3-Nitratopivaloyl-L-cysteine ethyl ester 
10.3 g 
2. Isobutyric anhydride 6.7 g 
3. Triethylamine (D = 0.73) 
5 g 
4. Dichloromethane 60 ml 
______________________________________ 
The process of Example 11 was followed. 
3-Nitratopivaloyl-L-cysteine ethyl ester (10.3 mg) and 6.7 g of isobutyric 
anhydride are combined in 60 ml of dichloromethane at room temperature in 
a nitrogen atmosphere with stirring. Then 5 g of triethylamine in 
dichloromethane is added dropwise under stirring. After the end of the 
reaction, the solution is washed with 1N HCl, 10% aqueous sodium 
bicarbonate solution and water. The dichloromethane extract is evaporated 
to dryness on a rotavapor. 10.4 grams (81.6% of theory) of an oily product 
is obtained. The identity of the product is verified by mass spectroscopy. 
PHARMACOLOGICAL ACTIVITY TESTING 
The pharmacological action of the compounds provided by the invention is 
illustrated further by the following experiments. 
Pharmacological Experiment Method 1 
Administration of new organic nitrates to detect the nitrate action on the 
circulatory parameters of a dog that is awake. 
The purpose of the test is to determine how the new organic nitrates act on 
various circulatory parameters of a dog that is awake after intravenous or 
oral administration. All experiments were carried out on trained beagle 
dogs; the circulatory parameters were measured with an arterial catheter 
tip manometer and a balloon catheter introduced into the V. jugularis. In 
order to describe the action on the arterial system, the systolic, mean 
and diastolic blood pressure (BP) and heart rate (HR) were measured. From 
these, the peripheral resistance (TPR) and the extensibility of the 
arterial air chamber (COMPL) were calculated. The low pressure system was 
measured through the central venous pressure (CVP) and the pulmonary 
arterial pressure (PAP). The control or reference product used was 
isosorbid-5-mononitrate (ISM-5). 
DESCRIPTION OF THE DRAWINGS 
The drawing figures illustrate graphically the activity spectrum of 
specific organic nitrates. 
The abbreviations used in the drawings have the following meanings: 
CO means cardiac output 
SV means stroke volume 
BP means diastolic blood pressure 
HR means heart rate 
CVP means central venous pressure 
PAP means pulmonary arterial pressure 
COMPL means extensibility of the arterial air chamber 
TPR means peripheral resistance 
In "Mean.+-.sem.N=5" for example, mean is mean value, sem. is standard 
error of the mean, and N is the number of errors of the mean value. 
FIGS. 1 to 8 show the action of orally and intravenously administered 
ISM-5. After administration by each route, the ISM-5 caused a slight 
decrease in systolic blood pressure, the mean pressure was hardly 
effected, the extensibility of the air chamber increased significantly and 
the low pressure system pressure decreased. 
FIGS. 9 to 16 show the corresponding effects of 
N-(3-nitratopivaloyl)-methionine ethyl ester (Nitrato-Piv-Meth-Et) for the 
corresponding circulatory areas. Here, again, the comparison between 
intravenous and oral administration indicates good bioavailability. 
The novel substance N-(3-nitratopivaloyl)-cysteine ethyl ester was also 
tested and it shows good bioavailability and a course of action typical of 
a nitrate. 
The results for both of the tested novel compounds show they have an action 
which is comparable to that of ISM-5 and have good bioavailability. 
Pharmacological Experiment Method 2 
Determining the lack of tolerance to the action of new organic nitrates on 
increasing coronary flow in isolated perfused heart. 
The purpose of the present investigation was to study the action and the 
development of tolerance to new organic nitrate compounds on isolated 
perfused rat heart. For this purpose, a rat heart was isolated and 
prepared as a "working heart". 
In this experimental arrangement, the heart performs defined circulatory 
work from which a defined oxygen consumption and coronary flow result. The 
action of nitrate-like compounds can be measured in this model by the 
drug-induced coronary flow increase. 
The resistance of the coronary vessels on an isolated working rat heart was 
chosen as the parameter for the detection of the action of the nitrate. A 
rat heart with a weight of about one gram is perfused through the left 
auricle with a plasma-like solution which contains nutrients and is 
saturated with oxygen. The left ventricle pumps the solution into the 
aorta against a defined pressure. Corresponding to the physiological 
conditions, a part of this solution flows through the coronary vessels for 
supplying the heart itself. At a defined work of the heart, this fraction, 
from which the coronary resistance can be calculated, is constant. The 
addition of a nitrate or other coronary dilating drug causes a drop in 
this coronary resistance. Therefore, if a constant concentration of an 
organic nitrate is introduced to the heart, then, after an initial 
decrease of the resistance, a partial loss of activity occurs within 20 
minutes. On this model, the substances also show a coronary dilating 
effect, but this is not followed by a loss in activity. The maximum drop 
of the coronary resistance is still completely present after 60 minutes. 
The tested compounds were compared in an equimolar dosage with 10.sup.-4 M 
nitroglycerin trinitrate. Continuous infusion of nitroglycerin causes a 
rapid coronary flow increase by 7.6.+-.1.88 ml/min g.sub.ww (x.+-.SD). 
Within 20 minutes, the flow decreases by 55.9%. Upon further perfusion, 
the action of nitroglycerin is unchanged. In this experimental model, the 
new nitrates also show a coronary flow increase, but this is followed by 
only a very slight drop in the activity. This result indicates that the 
described new compounds do not show the tolerance behavior as do 
conventional nitrates. 
TABLE 1 
______________________________________ 
Action of new organic nitrates in comparison to nitro- 
glycerin on the coronary flow in isolated perfused 
rat hearts. - x .+-. SEM, n = 7 
Maximum 
Coronary Activity 
Increase Drop in 
(ml/min g.sub.ww) 
flow (%) 
______________________________________ 
100 .mu.M nitroglycerine 
7.6 .+-. 0.71 56.0 
100 .mu.M N-(3-nitratopivaloyl)- 
6.6 .+-. 0.88 5.2 
cysteine ethyl ester 
100 .mu.M N-(3-nitratopivaloyl)- 
8.3 .+-. 0.92 7.0 
methionine ethyl ester 
______________________________________ 
Finally, the action of N-(3-nitratopivaloyl)-cysteine ethyl ester 
(Nitrato-Piv-Cy-Et) was characterized on guinea pig heart. 
Nitrato-Piv-Cy-Et leads to a concentration-dependent increase of the 
coronary flow in a working heart model (guinea pig heart) even in a very 
low dose range. A flow increase of 25% is achieved even with 380 .mu.g of 
nitrato-Piv-Cy-Et per liter of perfusion medium, corresponding to 1.3 
.mu.mole per liter. The corresponding concentration for glycerol 
trinitrate (GTN) with 5 mg/liter is at least a factor of 12 times higher. 
Thus, in the case of Nitrato-Piv-Cy-Et we are dealing with an especially 
active compound with regard to the vessels. No weakening of the coronary 
dilating effect as a manifestation of tolerance development could be 
observed at any dose during a one hour drenching perfusion. It can be 
concluded from this that Nitrato-Piv-Cy-ET does not produce any vessel 
tolerance, in contrast to GTN. 
On isolated guanyl cyclase, Nitrato-Piv-Cy-Et leads to a 
concentration-dependent activation of the enzyme, corresponding to 
enhanced formation of cGMP per unit time. The special characteristic of 
this compound in comparison to the classical organic nitro compounds is 
that the activation occurs in vitro even in the absence of cysteine. This 
explains at the same time the observation that Nitrato-Piv-Cy-ET and the 
other chemical compounds, for example, those in the "Working Heart Model", 
do not cause any tolerance, a finding which is of especially great 
practical importance for clinical long-term administration. The 
concentration necessary for one-half of the maximum activity (ED.sub.50) 
of guanyl cyclase is around 200 .mu.mole/liter. The corresponding value 
for GTN (in the presence of 5 mmoles/liter of cysteine) is approximately 
80 .mu.moles/liter.