Compounds of the general formula: ##STR1## wherein, R.sup.1 and R.sup.2 are, the same or different, hydrogen, a halogen, a lower alkyl, a cycloalkyl, a lower alkoxy, trifluoromethyl or a phenylalkyloxy whose phenyl ring may be substituted by one to three substituents selected from the group consisting of a halogen, a lower alkyl, a lower alkoxy, methylenedioxy and trifluoromethyl; R.sup.3 is hydrogen or methyl; R.sup.4 is carboxyl or an esterified carboxyl; and X is oxygen or sulfur, or a salt thereof. These compounds possess aldose reductase inhibitory and platelet aggregation inhibitory activities, and are of use as drugs for prevention and treatment of diabetic complications such as diabetic cataract, retinophathy, nephropathy, and neuropathy.

This invention relates to a novel condensed thiolactam-N-acetic acid 
derivative having excellent aldose reductase inhibitory and platelet 
aggregation inhibitory activities. The compound of this invention is 
useful for prevention and treatment of diabetic complications such as 
diabetic cataract, retinopathy, nephropathy, and neuropathy. 
Japanese patent application Laid-Open No. 40264/86 describes that various 
3-thioxo-1,4-benzoxazine-4-acetic acid derivatives and 
3-thioxo-1,4-benzothiazine-4-acetic acid derivatives exert aldose 
reductase inhibition and are useful for treatment of peripheral 
disturbance due to diabetes (cataract, diabetic neuropathy). However all 
of the compounds concretely described in this publication are only weakly 
inhibitory to sorbitol accumulation when administered orally to diabetic 
animals, and therefore are not satisfactory for practical application as 
medicines. 
The inventors found, as a result of their research on the compounds 
described above and related compounds that special compounds which are not 
described in Japanese patent application Laid-Open No. 40264/86 are 
strongly inhibitory in vivo to sorbitol accumulation and also have 
platelet aggregation inhibitory property. 
This invention relates to 
1. A compound having the general formula 
##STR2## 
wherein, R.sup.1 and R.sup.2 are, the same or different, hydrogen, a 
halogen, a lower alkyl, a cycloalkyl, a lower alkoxy, trifluoromethyl or a 
phenylalkyloxy whose phenyl ring may be substituted by one to three 
substituents selected from the group consisting of a halogen, a lower 
alkyl, a lower alkoxy, methylenedioxy and trifluoromethyl; R.sup.3 is a 
hydrogen or methyl; R.sup.4 is carboxyl or an esterified carboxyl; and X 
is oxygen or sulfur, or a salt thereof, 
2. A therapeutic agent for diabetic complications, containing a compound 
having the general formula (I), or a pharmaceutically acceptable salt 
thereof, 
3. A method for producing a compound having the general formula: 
##STR3## 
wherein each symbol has the meaning given above, or a salt thereof, which 
comprises hydrolyzing a compound having the general formula: 
##STR4## 
wherein, R.sup.1, R.sup.2, R.sup.3 and X are the same as described above, 
and R.sup.5 is an esterified carboxyl, and 
4. A method for producing a compound having the general formula (Ib), which 
comprises reacting a compound having the general formula: 
##STR5## 
wherein each symbol has the meaning given above, with a compound having 
the general formula: 
EQU Y.sup.1 CH.sub.2 R.sup.5 (III) 
wherein Y.sup.1 is a halogen and R.sup.5 has the meaning given above, to 
give a compound having the general formula: 
##STR6## 
wherein each symbol has the meaning given above, and then reacting the 
resulting compound with a thionating agent. 
In the above general formulas the lower alkyl represented by R.sup.1 and 
R.sup.2 is favorably a straight chain or branched one having 1 to 6 carbon 
atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 
sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, 
and isohexyl; the lower alkoxy group is one having 1 to 6 carbon atoms and 
includes those formed by binding an oxygen atom to the alkyl group 
described above. The cycloalkyl represented by R.sup.1 and R.sup.2 is 
favorably the one having 3-7 carbon atoms, such as cyclopropyl, 
cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The halogen 
represented by R.sup.1 and R.sup.2 includes fluorine, chlorine, bromine 
and iodine, among which fluorine and chlorine are preferable. The 
phenylalkyloxy represented by R.sup.1 and R.sup.2 is favorably one having 
7-9 carbon atoms, such as benzyloxy, 1-phenylethoxy, 2-phenylethoxy, 
1-phenylpropyloxy, 2-phenylpropyloxy, and 3-phenylpropyloxy. These groups 
may have a substituent or substituents on their benzene ring, and such 
substituent includes a halogen (e.g. fluorine, chlorine, bromine, iodine), 
a lower alkyl (e.g. methyl, ethyl, propyl, isopropyl), a lower alkoxy 
(e.g. methoxy, ethoxy, propoxy, isopropoxy), methylenedioxy, and 
trifluoromethyl. One to three of these substituents may be present on the 
benzene ring, and two or more substituents on the ring may be the same or 
different. R.sup.1 and R.sup.2 may be present at any position in the 
benzene ring, but favorably at 6-, 7- or 8-position. In particular, when 
either R.sup.1 or R.sup.2 is alkyl or cycloalkyl, the 8-position is the 
most favorable position for alkyl or cycloalkyl. Among compounds (I), 
1,4-benzoxazine where X is oxygen is preferable to 1,4-benzothiazine where 
X is sulfur. 
The esterified carboxyl represented by R.sup.4 and R.sup.5 includes an 
alkoxycarbonyl having 2 to 6 carbon atoms (e.g. methoxycarbonyl, 
ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, 
isobutoxycarbonyl), an aryloxycarbonyl having 7 to 9 carbon atoms (e.g. 
phenoxycarbonyl, p-methylphenyloxy carbonyl) and aralkyloxycarbonyl having 
8 to 10 carbon atoms (e.g. benzyloxycaronyl). 
As the halogen represented by Y.sup.1 in the formula (III), chlorine, 
bromine and iodine are mentioned. 
The salt of the compound having the general formula (Ia), particularly a 
pharmaceutically acceptable salt, includes an alkali metal salt (e.g. 
sodium salt, potassium salt), an alkaline earth metal salt (e.g. calcium 
salt), and aluminum salt. 
The compound having the general formula (I) or the salt thereof has an 
asymmetric carbon atom, and hence forms optical isomers or diastereomers. 
The isomers may be obtained in their pure form if necessary. Resolution of 
a pair of diastereomers may be accomplished by a usual method such as 
fractional crystallization and chromatography on, for example, silica gel. 
Optical resolution of a racemate into its mirror image components may also 
be accomplished by a usual method such as salt formation of (Ia) with an 
optically active base (e.g. quinine, quinidine, cinchonine, cinchonidine, 
1-phenylethylamine) followed by selective crystallization or fractional 
crystallization and then neutralization with an appropriate acid (e.g. 
hydrochloric acid, hydrobromic acid, sulfuric acid) to give a free acid. 
Optical resolution of (Ia) may also be carried out by (1) converting (Ia) 
to its ester with an optically active alcohol (e.g. methyl lactate, ethyl 
lactate, 1-phenylethyl alcohol, menthol), (2) separating the diastereo 
esters by recrystallization or by chromatography, and then (3) hydrolyzing 
each diastereo ester to give each mirror image isomer of (Ia). 
Condensation of (Ia) with an optically active alcohol is, for example, 
carried out in a solvent (e.g. dichloromethane, chloroform, ethyl acetate, 
dimethylformamide), using a condensing agent (e.g. 
dicyclohexylcarbodiimide) in the presence of 4-dimethylaminopyridine at 
about 0.degree. -60.degree. C. Hydrolysis of the diastereo ester of (Ia) 
to the optically active (Ia) is conducted in the same manner as the 
hydrolysis of (Ib) which will be mentioned later. The (S)-isomer is more 
preferable to (R)-isomer in inhibiting aldose reductase. 
The compound having the general formula (I) and the salt thereof are novel 
compounds which are not found in the literature, and are strongly 
inhibitory to aldose reductase of mammals (e.g. mouse, rat, rabbit, dog, 
cat, bovine, human). Aldose reductase is known to reduce, for example, 
glucose into sorbitol and therefore to accelerate accumulation of sorbitol 
in the tissues such as blood vessels, nerve and lens, causing various 
complications in diabetic patients. The compound (I) of this invention and 
the salt thereof have been proved not only to inhibit strongly the said 
enzyme in vitro, but also to be chemically stable, be absorbed well 
through the digestive tract, and be well distributed into the tissues. 
Therefore the compound (I) and salt thereof are very effective in 
inhibiting sorbitol accumulation in the tissues of streptozocininduced 
diabetic rat when given orally. The compound (I) and salt thereof also 
have platelet aggregation inhibitory activity. It is assumed that 
increased platelet aggregation often seen in diabetic patients may be one 
of the causes of diabetic microangiopathy such as retinopathy and 
nephropathy. Therefore compound (I) and salt thereof are expected to be 
useful in treating the increased platelet aggregation to prevent such 
complications. 
The compound having the general formula (I) and the pharmaceutically 
acceptable salt thereof are only slightly toxic whether given acutely or 
chronically, and therefore very useful as medicines for prevention and 
treatment of diabetic complications in man (cataract, retinopathy, 
nephropathy, neuropathy, etc.). 
The compound having the general formula (I) and the pharmaceutically 
acceptable salt thereof may, when used as the medicines described above, 
be orally or parenterally given in the form of powder, granule, tablet, 
capsule or injection, prepared by mixing with pharmaceutically acceptable 
carriers, excipients (e.g. lactose, starch, sugar, magnesium stearate), or 
diluents (e.g. water), and also in the form of eye drop or eye ointment 
when used as therapeutic agents for cataract. The dose varies according to 
the nature of the compound, route of administration, symptoms, age and 
body weight of the patient, etc.; for example, for oral administration to 
an adult diabetic patient, the daily dose is about 50 to about 1500 mg, 
preferably about, 100 mg to about 1000 mg, which is favorably divided into 
1 to 3 doses. In the case of an eye drop composition, it is desirable that 
a formulation or a suspension containing about 0.001-1% of the active 
component is given 3-5 times a day at the dose of one to several drops at 
a time. In the case of eye ointment, a formulation prepared by mixing 
about 0.001-1% of the active component in a usual eye ointment base is 
given about 1-4 times a day according to the symptoms. 
The compound having the general formula (Ia) and the salt thereof can be 
produced by hydrolysis of the compound having the general formula (Ib). 
The said hydrolysis is conducted favorably in a solvent in the presence of 
an alkali such as sodium hydroxide and potassium hydroxide. Such solvents 
include alcohols such as methanol, ethanol, propanol, 2-propanol, and 
methoxyethanol, ethers such as dioxane, tetrahydrofuran, and 
dimethoxyethane, and mixtures of these solvents with water. The hydrolysis 
can be conducted usually at about 0.degree.-100.degree. C., preferably 
about 10.degree.-60.degree. C., and the amount of the alkali used is about 
1-5 moles, preferably about 1.1-3 moles, per 1 mole of the compound (Ib) 
used. In the case of 3,4-dihydro-3-thioxo-2H-1,4- benzoxazine-4-acetic 
acid esters having no substituent or a straight chain alkyl group at the 
2-position, the 2-oxo derivative is also produced abundantly as by-product 
owing to simultaneous hydrolysis of the 2-thioxo group while the esters 
are hydrolyzed. Therefore the yield is low and purification is difficult 
with such compounds. On the other hand, the compound (Ib) having an oxygen 
atom as X is resistant to such side reaction because of the 
.alpha.-branched structure of the substituent at the 2-position, and 
therefore has an advantage in production that not only the desired product 
(Ia) is obtained in good yield but also purification of (Ia) is easy. For 
example, crystalline product is obtained either by concentration of the 
reaction mixture followed by dilution with water and then acidification 
with hydrochloric acid, or by extraction with an appropriate solvent (e.g. 
ethyl acetate) after acidification, followed by concentration. The 
crystalline product can be purified further by recrystallization or by 
chromatography if necessary. The compound (Ia) having a sulfur atom as X 
is also chemically stable, and can be isolated and purified in a similar 
manner. The compound (Ia) can be converted into a salt because it has a 
carboxylic acid residue. When an optically active starting compound (Ib) 
is used, an optically active product (Ia) or a salt thereof can be 
obtained. 
The compound having the general formula (Ib) can be produced by the 
following procedure. (Process 1) 
The compound (IV) can be produced by the reaction of the compound (II) with 
a halogenoacetic acid ester (III). This reaction is conducted in an 
appropriate solvent in the presence of a base. Such solvents include 
tetrahydrofuran, dimethoxyethane, dioxane, N,N-dimethylformamide, and 
dimethylsulfoxide, and such bases include sodium methoxide, sodium 
ethoxide, potassium t-butoxide, sodium hydride, potassium hydride, sodium 
amide, etc. It is desirable that in this reaction the compound (II) is 
allowed to react with a base to form an anion of compound (II) which is 
then allowed to react with the compound (III). The reaction temperature is 
about -10.degree.-100.degree. C., preferably about 0.degree.-60.degree. C. 
The amounts of the base and the compound (III) used are about 1-1.5 moles 
per 1 mole of the compound (II). 
(Process 2) 
The compound (IV) obtained in the (Process 1) is allowed to react with a 
thionating agent to give the compound (Ib). This reaction can be conducted 
in a solvent in the presence of a thionating agent. As the solvent, 
aromatic hydrocarbons such as benzene, toluene, xylene, and chlorobenzene, 
ethers such as tetrahydrofuran and methoxyethane, halogenated hydrocarbons 
such as chloroform, dichloromethane, and dichloroethane, and pyridine are 
mentioned. Thionating agent used includes phosphorus pentasulfide, 
Lawesson reagent, and Davy reagent. The reaction is conducted usually at 
about 10.degree.-150.degree. C., preferably at about 
20.degree.-130.degree. C. The amount of the thionating agent used is about 
0.5-3 moles, preferably about 1-2 moles, per 1 mole of the compound (IV). 
The compound (II) in which X is an oxygen atom can be produced, for 
example, by the method described in Journal of Medicinal and 
Pharmaceutical Chemistry, 5, 1378 (1962) or by a method in accordance with 
said method, via the route (Procedure A) described in the following. 
##STR7## 
wherein Z is a chlorine or a bromine atom and the other symbols are the 
same as described above. 
The compound (II) in which X is a sulfur atom can be produced, for example, 
by the following methods (Procedure B or Procedure C). 
##STR8## 
wherein R.sup.6 is a hydrogen or a lower alkyl having 1 to 4 carbon atoms 
and the other symbols are the same as described above. 
##STR9## 
wherein Y.sup.2 is a halogen atom and the other symbols are the same as 
described above. 
In the reaction formulas described above, the lower alkyl group represented 
by R.sup.6 includes methyl, ethyl, propyl, isopropyl, and butyl, and the 
halogen atom represented by Y.sup.2 includes fluorine, chlorine and 
bromine. 
In the Procedure B, the reaction between (VIII) and (IX) is conducted in an 
appropriate solvent (e.g. methanol, ethanol, propanol, dioxane, 
tetrahydrofuran, dimethoxyethane, dimethylsulfoxide, 
N,N-dimethylformamide), in the presence of a base (e.g. sodium carbonate, 
potassium carbonate, triethylamine) if necessary, at about 0.degree. 
-about 100.degree. C. In this reaction a compound (XIII) represented by 
the formula: 
##STR10## 
wherein the symbols are the same as described above is first formed as an 
intermediate, followed by ring closure reaction to give (II-2). Therefore, 
when this ring closure reaction proceeds only slowly, the ring closure 
reaction may be accelerated by appropriate heating or by adding an acid 
(e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic 
acid, p-toluenesulfonic acid, acetic acid), either without or after 
isolation of (XIII). 
In Procedure C, (X) and (XI) are allowed to react in the presence of a base 
to give (XII) which is then reduced to give (II-2). The reaction between 
(X) and (XI) is conducted in the same solvent as used in Procedure B in 
the presence of a base (e.g. sodium carbonate, potassium carbonate, sodium 
hydrogencarbonate, potassium hydrogencarbonate, triethylamine) at about 
0.degree.-100.degree. C. The reduction of (XII) is conducted 
advantageously by using a metal and an acid, such as iron-acetic acid, 
iron-hydrochloric acid, zinc-acetic acid and tin-hydrochloric acid. Also 
in this reaction the compound (XIII) is produced as an intermediate, and 
therefore the ring closure reaction is accelerated by a manner similar to 
that described in Procedure B. 
When optically active starting compounds (VI), (IX) and (XI) are used in 
the above-mentioned Procedures A-C, optically active products (II) are 
obtained. Inversion of the absolute configuration occurs in the 
ring-closure reaction of (VII) to form (II-1) in Procedure A and in the 
reaction of (VIII) with (IX) to form (II-2) in Procedure B. The absolute 
configuration of (XI) is retained in Procedure C. 
In the following, the results of pharmacological studies to prove the 
effectiveness of the compounds (I) of this invention in comparison with 
control compounds are described. 
1. Aldose reductase inhibition (in vitro study) 
Partially purified aldose reductase from human placenta was used for the 
test of aldose reductase inhibitory effect according to the method of S. 
Hayman et al. described in Journal of Biological Chemistry, 240, 877 
(1965) or the method of J. H. Kinoshita et al. described in Metabolism, 
28, Supplement 1, 462 (1979). The inhibitory activities of each compound 
at 10.sup.-6 M and at 10.sup.-7 M, expressed in inhibition percentage, are 
listed in Table 1. 
2. Inhibition of sorbitol accumulation in the tissue of experimentally 
induced diabetic rat 
Spraque-Dawley rats (male, 5-7-week-old, 5 animals per group) were fasted 
for 18 hours, and received injection of 70 mg/kg of streptozocin (Cal 
Biochem Co.) into the caudal vein under anesthesia with ether, so that 
diabetic rats were obtained. From immediately after the injection of 
streptozocin these rats were given orally the test compound of 50 mg/kg or 
30 mg/kg in the form of 5% gum arabic suspension twice a day (at 10 a.m. 
and 5 p.m.) for 2 days. During this period the rats were allowed to take 
CE-2 diet (Clea Japan, Inc.) and water ad libitum. In the morning on the 
3rd day (9 a.m.) the rats were killed (decapitation and venesection), and 
immediately the sciatic nerves were isolated, from which sorbitol was 
extracted according to the method of M. J. Peterson et al. described in 
Metabolism, 28, 456 (1979) and the sorbitol level was determined by the 
enzymatic method of P.S. Clements et al. in Science, 166, 1007 (1969). The 
results are expressed in percentage (%) taking the value of the untreated 
control group as 100, and shown in table 1. 
TABLE 1 
______________________________________ 
sorbitol 
Compound in vitro inhibition (%) 
accumulation (%) 
(Example No.) 
10.sup.-6 M 
10.sup.-7 M 
50 mg/kg 
30 mg/kg 
______________________________________ 
1 46.7 19.1 18**** 51*** 
2 43.9 8.7 12**** 33**** 
3 48.9 8.9 23**** -- 
4 45.6 3.8 14**** 45*** 
5 48.1 17.3 11**** 29**** 
6 48.2 9.4 16**** 33**** 
7 50.0 14.9 -- 27**** 
8 50.2 9.1 -- 30**** 
9 46.4 20.0 46**** 60*** 
11 47.1 15.2 38**** 67*** 
12 43.0 19.2 -- 58** 
13 46.8 14.9 -- 35*** 
17 45.7 2.6 -- 62* 
19 39.3 8.0 -- 15**** 
20 38.4 4.6 -- 25**** 
Control.sup.2 
A 50.8 25.0 60** 73 
B 43.1 15.7 78* -- 
C 48.7 19.6 71* -- 
______________________________________ 
(Note) 
(1) Student's ttest: 
*p &lt; 0.05 
**p &lt; 0.02 
***p &lt; 0.01 
****p &lt; 0.001 
(2) A: 6fluoro-3,4-dihydro-3-thioxo-2H--1,4benzoxazine-4-acetic acid 
B: 3,4dihydro-3-thioxo-2H--1,4benzothiazine-4-acetic acid 
C: 3,4dihydro-2-methyl-3-thioxo-2H--1,4benzothiazine-4-acetic acid 
(Results and Discussion) 
As shown in Table 1, although the compounds (I) of this invention were only 
as effective as or even slightly less effective than the control compounds 
in inhibition of aldose reductase in vitro, their inhibitory activities of 
sorbitol accumulation in vivo were superior to those of the control 
compounds. The difference is particularly evident when the comparison is 
made between the compound No. 2 with A, and between No. 9 and No. 11 with 
B and C, which are pairs of compounds having comparable substituents. 
Treatment of diabetic patients usually requires a long period, and 
therefore the compounds of this invention which are expected to show 
excellent effect at a lower dose are very useful as therapeutic agents. 
3. Inhibitory action on platelet aggregation 
Effect of 3,4-dihydro-2,8-diisopropyl-3-thioxo-2H-1,4-benzoxazine-4-acetic 
acid on platelet aggregation was examined using washed rat platelets. 
(1) Methods 
Rat blood was collected into a plastic syringe containing one-tenth volume 
of 3.8% trisodium citrate from the abdominal aorta of 6-7 weeks old, male 
Spraque-Dawley rats under ether anesthesia. Platelet rich plasma (PRP) was 
prepared by centrifuging the blood at 150 xg for 15 min at room 
temperature. To prepare washed platelets, the PRP was centrifuged at 1500 
xg for 10 min and the precipitated platelets were resuspended in Tyrode's 
solution. The number of platelets used was 7.times.10.sup.5 
cells/mm.sup.3. Platelet aggregation was measured by the turbidmetric 
method using an aggregometer (Nikko Bioscience, Model -4M, Tokyo). The 
test compound dissolved in 10 .mu.l of water was added to 250 .mu.l of 
washed platelet suspension and incubated at 37.degree. C. for 3 min before 
10 .mu.l of aggregating agents (5-10 .mu.l/ml of collagen, Nikko 
Bioscience or 10 .mu.M of ADP, Shigma, St. Louis, Mo., at the final 
concentration) was added. The percent inhibition of aggregation by the 
test compound was calculated by dividing the percent aggregation by that 
observed in the control run, then multiplying by 100. 
(2) Results 
The test compound inhibited aggregation of washed platelets induced by 
either collagen or ADP in a dose related manner (Table 2). Its inhibitory 
action was more potent than that of indomethacin in ADP-induced 
aggregation. 
TABLE 2 
__________________________________________________________________________ 
Test compound (M) Indomethacin (M) 
__________________________________________________________________________ 
Inducer 
3 .times. 10.sup.-5 
6 .times. 10.sup.-5 
10.sup.-4 
3 .times. 10.sup.-4 
10.sup.-4 
3 .times. 10.sup.-4 
Collagen 
74.5 .+-. 5.5* 
46.5 .+-. 1.3** 
31.8 .+-. 0.9** 
24.1 .+-. 3.4** 
28.5 .+-. 1.9** 
-- 
ADP -- -- 90.0 .+-. 6.0 
42.0 .+-. 7.1 
101.5 .+-. 8.4 
97.2 .+-. 6.4 
__________________________________________________________________________ 
Mean .+-. SD (N = 3). 
**p &lt; 0.001 and 
*p &lt; 0.01 vs control (water). 
4. Acute toxicity 
Acute toxicity of 
3,4-dihydro-2,8-diisopropyl-3-thioxo-2H-1,4-benzoxazine-4-acetic acid was 
examined in mice and rats. 
(1) Methods 
The various doses of the test compound suspended in 5% gum arabic solution 
were orally administered to male ICR mice (4 weeks old) and male Wistar 
rats (5 weeks old), and then mortality was observed during 14 days. Each 
group consisted of 5 animals. 
(2) Results 
The LD.sub.50 of the test compound is shown in Table 3. 
TABLE 3 
______________________________________ 
Animal 
LD.sub.50 (mg/kg) 
______________________________________ 
Mouse 1880 
Rat 2830 
______________________________________ 
The following Examples and Reference Examples illustrate the present 
invention more concretely.