N-Acylcarnosine aluminum salt, its preparation, and a digestive ulcer remedy containing such salt

An N-acylcarnosine aluminum salt of the formula, ##STR1## wherein R represents a lower alkyl group having 1 to 6 carbon atoms, a phenyl group or a lower alkoxy group-substituted phenyl group, and n is an integer of 1 to 3 is effective for use as a digestive ulcer-remedying or anti-ulcer agent.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to novel N-acylcarnosine compounds, and more 
particularly, to an N-acylcarnosine aluminum salt, its preparation, and a 
digestive ulcer remedy containing such salt. 
2. Description of the Prior Art 
In recent years, there has been a trend toward the increase of patients 
suffering from a digestive or peptic ulcer, and various attempts have been 
made to develop remedies for the ulcer. 
The present inventors have synthesized a number of compounds and have 
investigated their pharmaceutical effects. As a result of this 
investigation, it has been found that N-acylcarnosine compounds of a 
specific type, which will appear hereinafter, exhibit a significantly 
excellent digestive ulcer-remedying or anti-ulcer effect and are low in 
toxicity and hence are satisfactory for actual use. Based upon this 
finding, the present invention has been accomplished. 
SUMMARY OF THE INVENTION 
Therefore, one object of the present invention is to provide a novel 
N-acylcarnosine aluminum salt. 
Another object of the invention is to provide a novel process for preparing 
an N-acylcarnosine aluminum salt. 
A further object of the invention is to provide a novel digestive ulcer 
remedy which comprises an effective amount of an N-acylcarnosine aluminum 
salt. 
Briefly, these objects and other objects and advantages of this invention 
can be attained by an N-acylcarnosine aluminum salt of the formula (I), 
##STR2## 
wherein R represents a lower alkyl group containing 1 to 6 carbon atoms, a 
phenyl group or a lower alkoxy group-substituted phenyl group, and n is an 
integer of 1 to 3. 
DETAILED DESCRIPTION OF THE INVENTION 
The N-acylcarnosine aluminum salt of the formula (I) according to the 
present invention can be prepared, for example, by interacting an 
N-acylcarnosine of the formula (II), 
##STR3## 
wherein R has the same meaning as defined above, which an aluminum 
alkoxide or an inorganic aluminum salt by any of the methods described 
below. 
Method 1 
An N-acylcarnosine is reacted with an aluminum alkoxide to obtain an 
N-acylcarnosine aluminum salt of the formula (I). 
The acylcarnosine useful as one of the starting materials is prepared, for 
example, by converting the corresponding carboxylic acid to an acid 
halogenide in any usual manner and then reacting the halogenide with 
carnosine. 
Typical examples of the aluminum alkoxide include aluminum methoxide, 
aluminum ethoxide, aluminum isopropoxide, aluminum t-butoxide, aluminum 
cyclohexyloxide and the like. When the aluminum alkoxide contains any 
impurity such as aluminum hydroxide or a polymer thereof, it is preferable 
to remove the impurity by distillation, solvent extraction or the like. 
The reaction is favorably conducted in a suitable solvent at a temperature 
ranging from room temperature to 80.degree. C. Suitable solvents include 
water, an organic solvent such as methanol, ethanol, isopropanol or 
butanol, and a mixture thereof. After completion of the reaction, the 
solvent and secondarily produced alcohols are removed from the reaction 
solution to obtain the desired N-acylcarnosine aluminum salt of the 
formula (I). 
Method 2 
An N-acylcarnosine is reacted with an inorganic aluminum salt, and the 
resulting aqueous reaction solution is passed through a column packed with 
an anion exchange resin to obtain the desired N-acylcarnosine aluminum 
salt of the formula (I). 
Typical examples of the inorganic aluminum salt useful in method 2 include 
mineral acid salts of aluminum such as aluminum sulfate, aluminum nitrate 
and aluminum chloride. 
The anion exchange resin useful in method 2 may be either a weakly basic 
resin or a strongly basic resin, and preferably, a weakly basic anion 
exchange resin such as Amberlite IR-45 is used. 
The amount of the anion exchange resin depends on the amount of the ions of 
a mineral acid and on the type of the ions in an aqueous solution of the 
N-acylcarnosine and the mineral acid. Too small amounts of such resin 
adversely cause the mineral acid ions to introduce into an effluent, while 
excessive amounts induce the adsorption of the N-acylcarnosine to an 
objectionable degree. Accordingly, it is desirable that the anion exchange 
resin be used twice or three times equivalent of the N-acylcarnosine. 
The concentration of the aqueous solution which is passed through the 
column is not critical but is preferably held at such a level that the 
concentration of the mineral acid ions is in the range of about 0.5 to 1 
equivalent per liter of the solution. The space velocity is suitably in 
the range of about 0.5 to 2. 
The aqueous solution which has come out of the column is concentrated under 
reduced pressure and evaporated to dryness to obtain the desired compound 
of the formula (I). 
In either method 1 or method 2 above, when the molar ratio of an 
N-acylcarnosine to an aluminum alkoxide or an inorganic aluminum salt is 
varied, an N-acylcarnosine aluminum salt can be prepared which corresponds 
to such molar ratio. 
The N-acylcarnosine aluminum salts typical of and practical for the present 
invention which were listed as samples A or I were tested to determine 
their digestive ulcer-curing effects and degrees of toxicity with the 
results tabulated in the following experimental examples. 
Samples: 
__________________________________________________________________________ 
A: 
##STR4## (DL-form) 
B: 
##STR5## (L-form) 
C: 
##STR6## (L-form) 
D: 
##STR7## (L-form) 
E: 
##STR8## (L-form) 
F: 
##STR9## (L-form) 
G: 
##STR10## (L-form) 
H: 
##STR11## (L-form) 
I: 
##STR12## (L-form) 
__________________________________________________________________________ 
It will be noted that as control compounds for the purposes of comparison, 
use was made of L-carnosine, L-glutamine, N-acetyl-L-glutamine aluminum 
and aluminum sucrose sulfate which are known to have an anti-ulcer effect. 
The anti-ulcer effects and degrees of toxicity of the N-acylcarnosine 
aluminum compounds according to the invention were determined using rats 
in which instances various gastric ulcer models of rats were utilized to 
estimate the anti-ulcer effects. 
The experimental methods of ulcers and toxicity will be apparent from the 
following description. 
EXPERIMENT 1 
Shay's ulcer: Groups of ten male Donryu strain rats each weighing 210 to 
230 g were deprived of food for 48 hours. The pylorus of each rat was 
ligated according to the method of Shay et al [Gastroenterology, 5, 43-61 
(1945)]. Each of the animals was allowed to stand abstained from food and 
water for further 14 hours and then sacrificed to remove its stomach. 
After collection of the gastric juice, the ulcerated area (mm.sup.2) in 
the forestomach of each rat was measured under a dissecting microscope 
(10x). The total area (mm.sup.2) of all the lesions of each rat was 
indicated as an ulcer index. Test medicine samples were given orally 
immediately after the pylorus ligation. 
The experimental results are shown in Table 1. 
TABLE 1 
______________________________________ 
Inhibitory Effects on Shay's Ulcer on Rats 
Dose 
Samples (mg/kg, P.O.) 
Ulcer index 
% Inhibition 
______________________________________ 
Control -- 2.4 .+-. 0.7 
-- 
A 1,000 0.5 .+-. 0.5 
79.2 
L-Carnosine 1,000 2.1 .+-. 0.7 
12.5 
L-Glutamine 1,000 2.2 .+-. 0.8 
8.3 
Aluminum sucrose 
sulfate 1,000 0.7 .+-. 0.8 
71.7 
Control -- 2.5 .+-. 0.7 
-- 
B 1,000 0.6 .+-. 0.4 
76.0 
L-Carnosine 1,000 2.2 .+-. 0.8 
12.0 
L-Glutamine 1,000 2.3 .+-. 0.7 
8.0 
N-Acetyl-L- 
glutamine 
aluminum 2,000 0.7 .+-. 0.6 
72.0 
Aluminum sucrose 
sulfate 1,000 0.8 .+-. 0.9 
68.0 
Control -- 2.9 .+-. 0.4 
-- 
C 1,000 1.0 .+-. 0.7 
65.5 
D 1,000 0.9 .+-. 0.9 
69.0 
N-Acetyl-L- 
glutamine 
aluminum 2,000 0.9 .+-. 0.7 
69.0 
Aluminum sucrose 
sulfate 1,000 0.8 .+-. 0.6 
72.4 
Control -- 3.1 .+-. 1.1 
-- 
E 1,000 0.7 .+-. 0.5 
77.4 
F 1,000 0.9 .+-. 0.7 
71.0 
G 1,000 0.9 .+-. 1.0 
71.0 
H 1,000 1.0 .+-. 0.9 
67.7 
I 1,000 1.1 .+-. 0.8 
64.5 
N-Acetyl-L 
glutamine 
aluminum 2,000 1.0 .+-. 0.6 
67.7 
Aluminum sucrose 
sulfate 1,000 0.9 .+-. 0.5 
71.0 
______________________________________ 
EXPERIMENT 2 
Stress ulcer: Groups of ten male Donryu strain rats each weighing 240 to 
260 g were placed in a stress cage which served to immobilize the animals 
therein and then immersed in a water bath of 23.degree. C. on a level with 
the xiphoid process according to the method of K. Takagi et al [Jap. J. 
Pharmac., 18 (9), 9-19 (1968)], thereby imposing stress on each rat. Seven 
hours after the immersion, each of the animals was withdrawn from the 
water bath and immediately sacrificed by a blow on the head, followed by 
removal of the stomach of each rat. The stomach was slightly inflated by 
injecting a 1% formalin solution and then immediately immersed in a 1% 
formalin solution, followed by incising the stomach along its greater 
curvature to measure the length (mm) of each lesion in the glandular 
portion under a dissecting microscope (10x). The total length (mm) of all 
the lesions of each rat was indicated as an ulcer index. Test medicine 
samples were given orally ten minutes before the immersion in water. 
The results are shown in Table 2. 
TABLE 2 
______________________________________ 
Inhibitory Effects on Stress Ulcer in Rats 
Dose 
Samples (mg/kg, P.O.) 
Ulcer index 
% Inhibition 
______________________________________ 
Control -- 15.2 .+-. 3.2 
-- 
A 300 8.0 .+-. 2.8 
47.4 
1,000 4.4 .+-. 2.2 
71.1 
L-Carnosine 300 14.2 .+-. 3.7 
6.6 
1,000 10.7 .+-. 2.2 
29.6 
Aluminum sucrose 
sulfate 1,000 6.5 .+-. 6.0 
56.7 
Control -- 17.5 .+-. 3.5 
-- 
B 300 9.2 .+-. 3.0 
47.4 
1,000 4.8 .+-. 2.6 
72.6 
L-Carnosine 300 16.0 .+-. 4.0 
8.6 
1,000 12.1 .+-. 2.5 
30.9 
N-Acetyl-L- 
glutamine 
aluminum 2,000 5.5 .+-. 4.8 
68.6 
Aluminum sucrose 
sulfate 1,000 7.3 .+-. 6.8 
58.3 
______________________________________ 
A and B have the same meaning as defined above. 
EXPERIMENT 3 
Aspirin-induced ulcer: Groups of ten male Donryu strain rats each weighing 
220 to 230 g were deprived of food for 24 hours. The pylorus of each rat 
was ligated under ether anethesia according to the method of S. Okabe et 
al [Jap. J. Pharmac. 24, 357-361 (1974)]. After the pylorus ligation, 
aspirin (100 mg/kg) was administered orally to each rat. Seven hours after 
the administration, each of the animals was sacrificed under ether 
anesthesia to remove its stomach. After collection of the gastric juice 
and treatment with a 1% formalin solution, the length (mm) of each lesion 
formed in the glandular portion was measured. The total length (mm) of all 
the lesions of each rat was indicated as an ulcer index. Test medicine 
samples were given orally immediately after the pylorus ligation. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Inhibitory Effects on Aspirin-induced Ulcer in Rats 
Dose 
Samples (mg/kg, P.O.) 
Ulcer index 
% Inhibition 
______________________________________ 
Control -- 17.7 .+-. 2.3 
-- 
A 1,000 0.6 .+-. 0.6 
96.6 
L-Glutamine 1,000 6.0 .+-. 2.2 
66.1 
Aluminum sucrose 
sulfate 1,000 3.7 .+-. 2.1 
79.1 
Control -- 17.5 .+-. 3.5 
-- 
B 1,000 0.6 .+-. 0.6 
96.4 
L-Glutamine 1,000 4.5 .+-. 1.5 
73.2 
Aluminum sucrose 
sulfate 1,000 2.0 .+-. 1.1 
88.1 
______________________________________ 
A and B have the same meaning as defined above. 
EXPERIMENT 4 
Indomethacin-induced ulcer: Groups of ten male Donryu strain rats each 
weighing 200 to 215 g were deprived of food for 24 hours. Then, the 
pylorus was ligated under ether anethesia, after which indomethacin was 
administered subcutaneously in an amount of 25 mg/kg to each rat. Seven 
hours after the administration, each of the animals was scarificed under 
ether anethesia to remove its stomach, followed by immersion in a 1% 
formalin solution for ten minutes. The stomach which had been semi-fixed 
was incised along its greater curvature, and the length (mm) of each 
lesion formed in the mucous membrane was measured under a dissecting 
microscope (10.times.). The total length (mm) of all the lesions of each 
rat was indicated as an ulcer index. Test medicine samples were given 
orally ten minutes before the pylorus legation. 
The results are shown in Table 4. 
TABLE 4 
______________________________________ 
Inhibitory Effects on Indomethacin-induced Ulcer in Rats 
Dose 
Samples (mg/kg, P.O.) 
Ulcer index 
% Inhibition 
______________________________________ 
Control -- 16.3 .+-. 2.4 
-- 
A 300 6.5 .+-. 2.2 
60.1 
L-Carnosine 300 17.3 .+-. 3.0 
.about.0 
L-Glutamine 300 9.5 .+-. 4.1 
41.8 
Aluminum sucrose 
sulfate 300 7.8 .+-. 3.0 
52.1 
Control -- 16.8 .+-. 2.1 
-- 
B 300 6.4 .+-. 2.2 
61.9 
L-Carnosine 300 17.1 .+-. 3.1 
.about. 0 
L-Glutamine 300 9.7 .+-. 3.9 
42.3 
Aluminum sucrose 
sulfate 300 7.4 .+-. 2.8 
56.0 
______________________________________ 
A and B have the same meaning as defined above. 
EXPERIMENT 5 
Histamine-induced ulcer: Groups of ten male Donryu strain rats each 
weighing 210 to 230 g were deprived of food for 48 hours and then 
administered intraperitoneally with histamine phosphate (300 mg/kg). Four 
hours after the administration, each of the animals was sacrificed under 
ether anesthesia, followed by removal of the stomach and immersion in a 1% 
formalin solution for ten minutes. The stomach which had been semi-fixed 
was incised along the greater curvature, and the length (mm) of each 
lesion was measured under a dissecting microscope (10.times.). The total 
length (mm) of all the lesions of each rat was indicated as an ulcer 
index. Test medicine samples were given orally before the histamine 
administration. 
The results are shown in Table 5. 
TABLE 5 
______________________________________ 
Inhibitory Effects on Histamine-induced Ulcer in Rats 
Dose 
Samples (mg/kg, P.O.) 
Ulcer index 
% Inhibition 
______________________________________ 
Control -- 24.1 .+-. 4.7 
-- 
A 300 9.6 .+-. 1.8 
60.2 
L-Carnosine 300 17.3 .+-. 3.2 
28.2 
Aluminum sucrose 
sulfate 300 14.4 .+-. 1.5 
40.2 
Control -- 23.0 .+-. 3.0 
-- 
B 300 9.1 .+-. 1.9 
60.4 
L-Carnosine 300 18.0 .+-. 2.7 
21.7 
N-Acetyl-L- 
glutamine 
aluminum 2,000 8.5 .+-. 3.0 
63.0 
Aluminum sucrose 
sulfate 300 10.7 .+-. 2.1 
53.5 
______________________________________ 
A and B have the same meaning as defined above. 
EXPERIMENT 6 
Acute toxicity: Male and female Wister strain rats each weighing 150 to 200 
g were divided into two groups, respectively, each group consisting of ten 
rats, and were administered orally with some of the N-acylcarnosine 
aluminum compounds according to the invention. The thus treated rats were 
visually observed for seven days after the administration. 
The results are shown in Table 6. 
TABLE 6 
______________________________________ 
Values of LD50 in Rats 
LD50 (mg/kg P.O.) 
Samples Male Female 
______________________________________ 
A &gt;10,000 &gt;10,000 
B &gt;10,000 &gt;10,000 
______________________________________ 
As is clearly seen from the experimental results of Tables 1 to 6, the 
N-acylcarnosine aluminum compounds or salts according to the invention 
exhibit an excellent inhibiting effect on various ulcer models. That is, 
when administered orally in an amount of 300 or 1,000 mg/kg to rats in the 
tests of Shay's ulcer, stress ulcer, aspirin-induced ulcer, 
indomethanocin-induced ulcer and histamine-induced ulcer, such salts are 
significantly effective for inhibiting any of the ulcers and hence are 
more excellent than any existing anti-ulcer agents. 
In the acute toxicity test, even when the N-acylcarnosine aluminum salts 
were administered orally in an amount as large as 10 g/kg, no death of 
rats was recognized, and no or little change in general symptoms was 
observed. 
Accordingly, the N-acylcarnosine aluminum salts of the present invention 
can be used as a digestive ulcer remedy which is higher in safety and more 
excellent in effectiveness than L-glutamine, N-acetyl-L-glutamine aluminum 
and aluminum sucrose sulfate which have now been widely used as anti-ulcer 
agents. The N-acylcarnosine aluminum salts may be administered either 
orally or parenterally and may be used in the form of, for example, 
tablets, capsules, powders, granules and syrups for oral administration 
and also in the form of injection for parenteral administration. 
The amount of administration is generally in the range of 500 to 5,000 
mg/day for adults, which may be varied depending both on the age and on 
the symptom.