Novel 4-(substituted thiazolyl)-3-hydroxy-3-pyrroline-2,5-diones are disclosed which inhibit glycolic acid oxidase and thus are useful in the treatment and prevention of calcium oxalate renal lithiasis.

BACKGROUND OF THE INVENTION 
Close to 70% of kidney stones in man are composed partially or 
predominantly of calcium oxalate. There is no satisfactory drug specific 
for the treatment of calcium oxalate renal lithiasis, nor for prophylactic 
use by patients prone to recurrent attacks of this disease. 
The most common treatment for renal lithiasis due to calcium oxalate 
consists of surgical removal of stones, control of the diet to restrict 
calcium or oxalate, and ingestion of large quantities of water to dilute 
the urine. Attempts at chemotherapy have included the administration of 
magnesium oxide, calcium carbimide, orthophosphate, cellulose phosphate, 
isocarboxazide, thiazide diuretics, allopurinol and succinimide. Limited 
success has been realized by these drug approaches. No drug which 
specifically inhibits the biosynthetic formation of oxalic acid has 
previously been developed for the treatment of calcium oxalate renal 
lithiasis. 
The immediate metabolic precursor of the majority of the oxalate in the 
urine of a typical patient is glyoxylic acid. In turn its most important 
precursor is glycolic acid. The enzyme glycolate oxidase is able to carry 
out the oxidation of glycolic acid, through glyoxylic acid, to oxalic 
acid. Inhibition of this enzyme will, therefore, reduce the concentration 
of oxalic acid in the kidney and bladder, reducing the probability that 
calcium oxalate crystallization will occur. Thus inhibitors of glycolate 
oxidase provide a specific approach to the prevention and treatment of 
calcium oxalate renal lithiasis. 
Liao, et al, Arch. Biochem. Biophys., 154, 68-75 (1973) have shown that 
phenyllactic acid and n-heptanoic acid, which are inhibitors of glycolate 
oxidase, inhibit oxalate biosynthesis in isolated perfused rat liver. 
These compounds are not sufficiently potent to be useful as drugs. 
The preparation of 3-hydroxy-4-phenyl-3-pyrroline-2,5-dione 
##STR1## 
has been described by Harlay, J. Pharm. Chim., 24, 537-48 (1936). 
3-Hydroxy-4-aryl-3-pyrroline-2,5-diones are described in U.S. Pat. No. 
3,349,263 as intermediates in the preparation of antiphlogistic 
substances. A number of 
3-hydroxy-4-substitutedphenyl-3-pyrroline-2,5-diones are reported by G. S. 
Skinner et al., J. Am. Chem. Soc., 73, 2230 (1951). (In this paper these 
compounds are referred to as pyrrolidine-2,3,5-trione derivatives). 
3-Hydroxy-4-(4-bromo-1-naphthyl)-3-pyrroline-2,5-dione is described by G. 
S. Skinner et al., J. Am. Chem. Soc., 70, 4011 (1948). 
SUMMARY OF THE INVENTION 
It has now been found that novel compounds of the formula: 
##STR2## 
wherein n is 0 to 2; 
R is; 
##STR3## 
wherein 
R.sub.1 and R.sub.2 on the pyridine ring are independently hydrogen, 
loweralkyl containing 1 to 4 carbons or halogen and pharmaceutically 
acceptable salts thereof, are potent inhibitors of glycolate oxidase. They 
are, therefore, useful in the treatment and prevention of calcium oxalate 
kidney and bladder stone formation. 
Preferred compounds are those wherein n is 0 having the structure: 
##STR4## 
wherein 
R.sub.1 and R.sub.2 are independently hydrogen, halogen or loweralkyl 
containing 1 to 4 carbons. 
Still further preferred compounds are those wherein R.sub.1 and R.sub.2 are 
hydrogen having the structure: 
##STR5## 
Still further preferred compounds are those having the structure: 
##STR6## 
DETAILED DESCRIPTION 
About 70% of all renal calculi contain oxalate as the main component of the 
matrix. In the majority of patients the condition is associated with a 
higher than average level of metabolically produced oxalate. The major 
pathway for biosynthesis of oxalate can be represented as follows: 
##STR7## 
Glyoxylate is the major immediate forerunner of oxalate. An inhibitor of 
glycolate oxidase (G.O.) will inhibit both the conversion of glyoxylate to 
oxalate as well as the production of glyoxylate from glycolate. By 
reducing oxalic acid levels in the urine with the compounds of this 
invention, the formation of oxalate calculi will be reduced or prevented. 
Compounds of formula (I) are potent inhibitors of glycolate oxidase and 
thus are useful in restricting oxalate levels in the blood and urine. 
Further, they are useful in the treatment and prevention of renal disease 
due to calcium oxalate stone formation in the kidney and bladder. They may 
be useful in the treatment of the genetically inherited diseases termed 
Hyperoxaluria types I and II in which very high levels of metabolic oxalic 
acid are present. 
Compounds of formula (I) have been unexpectedly found to block the 
contractions of guinea pig ileum induced by Slow Reacting Substance of 
Anaphylaxis (SRS-A). They are ineffective against contractions caused by 
histamine, which demonstrates specificity against SRS-A. SRS-A is 
considered a major mediator in human allergic asthma. Thus the compounds 
of formula (I) are useful in the treatment of allergy, especially allergic 
asthma. 
Compounds of formula (I) can be prepared according to the following scheme: 
##STR8## 
wherein 
R.sub.1 and R.sub.2 are as defined above. 
The compounds (I) wherein the heterocyclic substituent is at the 2-position 
of the thiazole ring are prepared generally by the method of Fairfull, 
Lowe and Peak, J. Chem. Soc., 742 (1952). The nitrile (II), prepared by 
known methods, is reacted with excess hydrogen sulfide gas in the presence 
of excess triethylamine in a basic organic solvent such as pyridine. When 
the reaction is complete, the reaction mixture is poured into ice-water 
and the thiobenzamide (III) collected by filtration. 
When n=0 the ethyl 4-thiazolylacetates (IV) are prepared by the classic 
Hantzsch procedure using the thiobenzamide (III) and ethyl 
4-chloroacetoacetate. When n=1 or 2 the homologous chloro- or bromomethyl 
ketones are utilized. 
Stirring the esters (IV) in concentrated ammonium hydroxide and dioxane or 
ammonia in methanol or ethanol for varying lengths of time, and 
temperature yield the corresponding amides (V). 
Preparation of the pyrrolinediones from the amides (V) is accomplished by 
two different routes. 
1. When n=0 the amides react with diethyl oxalate in a solvent such as DMF 
in the presence of strong base (generally alkali metal alkoxide) under an 
inert atmosphere. Acidification provides the desired hydroxypyrrolinedione 
derivative. 
2. When n=0, 1 or 2, the intermediate amide is converted to the nitrile by 
dehydration using prior art procedures (e.g. thionyl chloride in DMF or 
pyridine, or p-toluenesulfonyl chloride in pyridine or DMF). The nitrile 
is reacted with diethyl oxalate and strong base (alkali metal alkoxide) in 
a solvent such as DMF or toluene. The resulting 3-cyano-2-ketoacid ethyl 
ester is converted to the desired 3-hydroxy-3-pyrroline-2,5-dione 
derivative by dissolving in sulfuric or methanesulfonic acid, allowing the 
mixture to stand at room temperature overnight, and then pouring into 
ethanol containing 5-10% water. 
Alternatively, the 3-cyano-2-ketoacid ethyl ester intermediate may be 
converted to the iminoether using cold ethanolic hydrogen chloride for 
20-48 hours. The iminoether hydrochloride on heating in refluxing 
chloroform is converted to the hydroxypyrrolinedione derivative. 
For compounds of this invention wherein the hydroxypyrrolinedione moiety is 
attached at the 5-position of the thiazole ring, the above procedures are 
followed with the exception that for the thiazole ring-forming step there 
is utilized instead of the .chi.-haloketone, the isomeric .chi.-halo 
aldehydic ester intermediate. For example, in place of 4-chloroacetoacetic 
ethyl ester, 4-oxo-3-bromobutyric acid ethyl ester is employed. 
The following examples, given by way of illustration and not to be 
construed as limiting, further clarify the invention.

EXAMPLE 1 
General Method for the Preparation of 
3-Hydroxy-4-substituted-3-pyrroline-2,5-diones from Thiazoleacetamide 
Intermediates 
A mixture of the substituted acetamide (10 mmole), diethyl oxalate (1.533 
g, 10.5 mmole) and dry dimethylformamide (20 ml) is stirred under nitrogen 
or argon and cooled in an ice-bath. Potassium t-butoxide (2.464 g, 22 
mmole) is added in two equal portions 15 minutes apart and the reaction 
mixture is stirred for about 30 minutes in the ice-bath and then at room 
temperature overnight. The reaction mixture is poured into ice-water (100 
ml). If the potassium salt of the product dissolves, the aqueous mixture 
is extracted with ethyl acetate (2.times.35 ml) and then acidified with 6 
N hydrochloric acid in order to precipitate the product. The product is 
either collected by filtration or by extraction with ethyl acetate. 
If the potassium salt is not soluble when the reaction mixture is quenched 
in ice-water, then it is necessary to acidify the resulting suspension and 
collect the product by filtration. The crude product is generally less 
pure when obtained in this way. 
Compounds may be solvated after recrystallization (with either DMF, 
dioxane, isopropanol or acetonitrile) and require drying at 110.degree. 
C./0.05 Torr in order to remove the solvate. 
EXAMPLE 2 
Preparation of 
3-Hydroxy-4-[2-(4-pyridyl)thiazol-5-yl]-3-pyrroline-2,5-dione 
Pyridine-4-thioamide (1.38 g, 0.01 mole) and 3-bromo-4-oxobutyric acid 
ethyl ester (3.1 g., 0.015 mole) in ethanol (50 ml) are heated at reflux 
for six hours. After cooling the solvent is evaporated and the residue is 
neutralized with sodium bicarbonate solution and then extracted with 
chloroform (300 ml.). The chloroform solution is washed with water, dried 
with (MgSO.sub.4) and evaporated to yield crude 
2-(4-pyridyl)-thiazole-5-acetic acid ethyl ester. Purification is carried 
out by dissolving in acetone and adding petroleum ether to induce 
crystallization. When this ester intermediate is treated with methanol 
solution saturated with ammonia gas (25 ml./g. of ester) for 3 days at 
room temperature 2-(4-pyridyl)thiazol-5-ylacetamide is obtained after 
partial evaporation of the methanol. Purification is carried out by 
recrystallization from ethanol. When the amide is treated according to the 
procedure in Example 1, there is obtained 
3-hydroxy-4-[2-(4-pyridyl)thiazol-5-yl]-3-pyrroline-2,5-dione. 
When the above procedure is carried out starting with 3-chloro or 
3-bromopyridine-4-thioamide, but substituting 4-chloro-3-oxobutyric acid 
ethyl ester for the 3-bromo-4-oxobutyric acid ethyl ester, there are 
obtained 
3-hydroxy-4-[2-(3-chloro-4-pyridyl)thiazol-4-yl]-3-pyrroline-2,5-dione and 
3-hydroxy-4-[3-bromo-4-pyridyl)thiazol-4-yl]-3-pyrroline-2,5-dione 
respectively. 
EXAMPLE 3 
3-Hydroxy-4-[2-(4-pyridyl)thiazol-4-yl]-3-pyrroline-2,5-dione 
When pyridine-4-thioamide is reacted with 6-chloro-5-oxohexanenitrile, 
according to the procedure of Example 2 (in place of 3-bromo-4-oxobutyric 
acid ethyl ester) there is obtained 
4-[2-(4-pyridiyl)thiazol-4-yl]-butanenitrile. To a solution of this 
nitrile (2.29 g., 0.01 mole) in dimethylformamide (30 ml.) is added 
diethyloxalate (1.74 g., 0.012 mole), and potassium t-butoxide (2.48 g., 
0.022 mole). The mixture is stirred overnight. Following evaporation under 
vacuum to one-half volume, chloroform (500 ml.) is added plus water (200 
ml.), and the mixture acidified with conc. HCl to pH 2-3. The chloroform 
is separated, washed well with water, and evaporated to yield 
2-oxo-3-cyano-5-[2-(4-pyridyl)thiazol-5-yl]pentanoic acid ethyl ester. The 
ester (3.24 g., 0.01 mole) is dissolved in methanesulfuric acid (30 ml.) 
and stirred for 24 hours. To the acidic mixture is added 80% 
ethanol-water. After standing for 2 hours, the ethanol is removed under 
vacuum. The residual aqueous mixture is neutralized with pyridine to pH 
2-3, and the title product obtained on filtration. 
EXAMPLE 4 
2-[2-(4-Pyridyl)thiazol-4-yl]acetonitrile 
To 2-[2-(4-pyridyl)thiazol-4-yl]acetamide (2.19 g., 0.01 mole) in pyridine 
(30 ml.) is added gradually p-toluenesulfuryl chloride (1.91 g., 0.01 
mole). After stirring for one hour the mixture is poured into excess 
ice-water to give the title compound. 
The physical constants of certain intermediates and end-product 
hydroxypyrrolinediones of this invention are tabulated below: 
__________________________________________________________________________ 
##STR9## 
##STR10## 
##STR11## 
RCN Calc. 
Fd. Calc. 
Fd. Calc. 
Fd. 
__________________________________________________________________________ 
##STR12## 
N C H Cl 
8.96 53.75 5.48 11.33 
8.89 53.71 5.30 11.26 
N C H 
16.99 58.28 5.30 
16.86 58.49 5.28 
N C H 
13.95 55.80 3.68 
14.22 55.60 3.64 
MP 94-96.degree. C. 
MP 208-210.degree. C. 
MP 288.degree. C. 
##STR13## 
N C H 
11.28 58.04 4.87 
11.43 58.29 4.91 
N C H 
19.17 54.78 4.14 
18.87 54.72 4.26 
N C H 
15.38 52.74 2.58 
15.66 52.57 2.71 
MP 197-199.degree. C. 
MP 290.degree. C. (dec.) 
##STR14## 
N C H 
9.84 50.61 4.60 
10.04 50.43 4.62 
N C H 
19.17 54.78 4.14 
19.03 54.78 4.20 
N C H 
15.38 52.74 2.58 
15.62 52.97 2.44 
MP 123-125.degree. C. 
MP 175-177.degree. C. 
MP 269-270.degree. C. 
##STR15## Oil N C H 
25.44 49.08 3.66 
25,38 49.07 3.61 
N C H 
20.43 48.17 2.21 
20.44 48.05 2.24 
MP 169-171.degree. C. 
MP 289-291.degree. C. (dec.) 
##STR16## 
N C H 
11.02 47.22 3.96 
11.12 47.46 3.95 
N C H 
18.65 42.65 3.13 
18.53 42.71 3.08 
N C H 
15.05 43.00 1.80 
15.21 42.81 1.92 
MP 88-90.degree. C. 
MP 175-177.degree. C. 
MP 279-280.degree. C. 
__________________________________________________________________________ 
(dec.) 
NOTE: 
The compounds of this invention may also be termed 3(substituted 
thiazolyl)4-hydroxy-3-pyrroline-2,5-dione derivatives. 
Included within the scope of the invention are the pharmaceutically 
acceptable salts of formula (I) compounds. The compounds of formula (I) 
are strong organic acids with a pKa in the range 2-4. Thus salts are 
readily formed with the usual inorganic cations such as sodium, potassium 
and ammonium. Salts with organic amines such as trimethylamine, 
triethylamine, n-butylamine and the like are also very stable. The 
neutralization can be carried out by a variety of procedures known to the 
art to be generally useful for the preparation of such salts. The choice 
of the most suitable procedure will depend on a variety of factors 
including convenience of operation, economic considerations, and 
particularly the solubility characteristics of the particular free base, 
the acid, and the acid addition salt. 
The compounds of formula (I) are utilized for the stated utilities by 
formulating them in a composition such as tablet, capsule or elixir for 
oral administration. Sterile solutions or suspensions can be used for 
parenteral administration. About 10 to 200 mg of a compound of formula I 
or a physiologically acceptable salt is compounded with a physiologically 
acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, 
flavor, etc., in a unit dosage form as called for by accepted 
pharmaceutical practice. The amount of active substance in the composition 
is such that dosage in the range indicated is obtained. The total daily 
dose will be in the 30 to 2000 mg range, and preferably in the range of 50 
mg to 1000 mg. 
Illustrative of the adjuvants which may be incorporated in tablets, 
capsules and the like are the following: a binder such as gum tragacanth, 
acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; 
a disintegrating agent such as corn starch, potato starch, alginic acid 
and the like; a lubricant such as magnesium stearate; a sweetening agent 
such as sucrose or lactose; a flavoring agent such as peppermint, oil of 
wintergreen or cherry. When the dosage unit form is a capsule, it may 
contain in addition to materials of the above type a liquid carried such 
as a fatty oil. Various other materials may be present as coating or to 
otherwise enhance the pharmaceutical elegance of the preparation. For 
instance, tablets may be coated with shellac, sugar or the like. A syrup 
or elixir may contain the active compound, sucrose as a sweetening agent, 
methyl and propyl parabens as preservatives, a dye and a flavoring such as 
cherry or orange flavor. 
Sterile compositions for injection can be formulated according to 
conventional pharmaceutical practice by dissolving or suspending the 
active substance in a conventional vehicle such as water for injection, a 
naturally occurring vegetable oil like sesame oil, coconut oil, peanut 
oil, cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate 
or the like. Buffers, preservatives, antioxidants and the like can be 
incorporated as required.