Substituted phenyl-2-cyano-2-thioalkenoic acid esters which are useful as anti-inflammatory agents and as inhibitors of the progressive joint deterioration characteristic of arthritic disease.

DESCRIPTION OF THE INVENTION 
This invention is concerned with compounds of the formula: 
##STR1## 
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each selected 
from the group comprising hydrogen, halogen, lower alkyl (C.sub.1 
-C.sub.4), lower alkoxy (C.sub.1 -C.sub.4), trifluoromethyl and 
trichloromethyl, R.sub.6 is lower alkyl (C.sub.1 -C.sub.4); M is hydrogen 
or a pharmaceutically acceptable cation; and n is an integer one, two or 
three. 
The useful pharmaceutically acceptable salts of the compounds of the above 
structural formula wherein M is hydrogen are those with pharmacologically 
acceptable metal cations, ammonium, amine cations or quaternary ammonium 
cations. Preferred metal cations are those derived from the alkali metals, 
e.g. lithium, sodium and potassium, and from the alkaline earth metals, 
e.g. magnesium and calcium, although cationic forms of other metals, e.g. 
aluminum, iron, zinc and in particular copper are within the scope of this 
invention. 
Pharmacologically acceptable amine cations and those derived from primary, 
secondary or tertiary amines such as mono-, di- or trimethylamine, 
ethylamine, dibutylamine, triisopropylamine, N-methylhexylamine, 
decylamine, dodecylamine, allylamine, crotylamine, cyclopentylamine, 
dicyclohexylamine, mono- or dibenzylamine, .alpha.- or 
.beta.-phenylethylamine, ethylenediamine, diethylenetriamine, and 
aryliphatic amines containing up to and including 18 carbon atmos, as well 
as heterocyclic amines, e.g. piperidine, morpholine, pyrrolidine, 
piperazine and lower alkyl derivatives thereof, e.g. 1-methylpiperidine, 
4-ethylmorpholine, 1-isopropylpyrrolidine, 2-methylpyrrolidine, 
1,4-dimethylpiperazine, 2-methylpiperidine, and the like, as well as 
amines containing water-solubilizng or hydrophilic groups, e.g. mono-, di- 
or triethanolamine, ethyldiethanolamine, N-butylethanolamine, 
2-amino-1-butanol, 2-amino-2-ethyl-1, 3-propanediol, 
2-amino-2-methyl-1-propanol, tris (hydroxymethyl)aminomethane, 
N-phenylethanolamine, N-(p-tert-amylphenyl)diethanolamine, galactamine, 
N-methylglucamine, N-methylglucosamine, ephedrine, phenylephrine, 
epinephrine, procaine, and the like. 
Examples of suitable pharmacologically acceptable quaternary ammonium 
cations are tetramethylammonium, tetraethylammonium, 
benzyltrimethylammonium, phenyltriethylammonium and the like. 
The compounds of the present invention may be prepared according to the 
following Flowchart A. 
##STR2## 
In accordance with Flowchart A thionyl chloride and sodium carbonate in 
chloroform are used to convert a 5-alkylisoxazole 4-carboxylic acid (1) to 
the corresponding 5-alkylisoxazole carbonyl chloride (2) by heating for 
several hours. The compound (2) is then reacted with a substituted 
thiophenol (3) and one molar equivalent of triethylamine in ether at 
reduced temperature for several hours. The resulting alkylthio 
isoxazolecarboxylic acid-S-phenyl ester (4) is extracted in 
methylenechloride and crystallized with hexane at reduced temperature. The 
isoxazole (4) is treated with triethylamine in ether for several hours 
giving the 2-cyano-3-hydroxythio alkenoic acid-5-phenyl ester 
triethylamine salt (5) which is then acidified in aqueous solution with 
hydrochloric acid to provide (6) where R.sub.1, R.sub.2, R.sub.3, R.sub.4, 
R.sub.5, R.sub.6 and M are as described above. 
DETAILED DESCRIPTION OF THE INVENTION 
The compounds of the present invention have been found to be highly useful 
for meliorating inflammation and inhibiting joint deterioration in mammals 
when administered in amounts ranging from about one milligram to about 250 
mg. per kilogram of body weight per day. A preferred dosage regimen for 
optimum results would be from about 5 mg. to about 100 mg. per kilogram of 
body weight per day, and such dosage units are employed that a total of 
from about 0.35 gram to about 7.0 grams of the active ingredient for a 
subject of about 70 kg. of body weight are administered in a 24 hour 
period. This dosage regimen may be adjusted to provide the optimum 
therapeutic response. For example, several divided doses may be 
administered daily or the dose may be proportionally reduced as indicated 
by the exigencies of the therapeutic situation. A decided practical 
advantage of this invention is that the active ingredient may be 
administered in any convenient manner such as by the oral, intravenous, 
intramuscular, topical or subcutaneous routes. 
Compositions according to the present invention having the desired clarity, 
stability and adaptability for parenteral use are obtained by dissolving 
from 0.10% to 10.0% by weight of active compound in a vehicle consisting 
of a polyhydric aliphatic alcohol or mixtures thereof. Especially 
satisfactory are glycerin, propylene glycol, and polyethylene glycols. The 
polyethylene glycols consist of a mixture of non-volatile, normally 
liquid, polyethylene glycols which are soluble in both water and organic 
liquids and which have molecular weights of from about 200 to 1500. 
Although the amount of active compound dissolved in the above vehicle may 
vary from 0.10 to 10.0% by weight, it is preferred that the amount of 
active compound employed be from about 3.0 to about 9.0% by weight. 
Although various mixtures of the aforementioned non-volatile polyethylene 
glycols may be employed, it is preferred to use a mixture having an 
average molecular weight of from about 200 to about 400. 
In addition to the active compound, the parenteral solutions may also 
contain various preservatives which may be used to prevent bacterial and 
fungal contamination. The preservatives which may be used for these 
purposes are, for example, myristyl-gamma-picolinium chloride, phenyl 
mercuric nitrate, benzalkonium chloride, phenethyl alcohol, 
p-chlorophenyl-.alpha.-glycerol ether, methyl and propyl parabens, and 
thimerosal. As a practical matter it is also convenient to employ 
antioxidants. Suitable antioxidants include, for example, sodium 
bisulfite, sodium metabisulfite, and sodium formaldehyde sulfoxylate. 
Generally, from about 0.05 to about 0.2% concentrations of antioxidant are 
employed. 
For intramuscular injection, the preferred concentration of active compound 
is 0.25 to 0.50 mg./ml. of the finished compositions. The compounds of 
this invention are equally adapted to intravenous administration when 
diluted with water or diluents employed in intravenous therapy such as 
isotonic glucose in appropriate quantities. For intravenous use, initial 
concentrations down to about 0.05 to 0.25 mg./ml. of active compound are 
satisfactory. 
The active compounds of the present invention may be orally administered, 
for example, with an inert diluent or with an assimilable edible carrier, 
or they may be enclosed in hard or soft shell gelatin capsules, or they 
may be compressed into tablets, or they may be incorporated directly with 
the food of the diet. For oral therapeutic administration, the active 
compounds may be incorporated with excipients and used in the form of 
tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the 
like. Such compositions and preparations should contain at least 0.1% of 
active compound. The percentage of the compositions and preparations may, 
of course, be varied and may conveniently be between about 2% to about 60% 
of the weight of the unit. The amount of active ingredient in such 
therapeutically useful compositions is such that a suitable dosage will be 
obtained. Preferred compositions or preparations according to the present 
invention are prepared so that an oral dosage unit form contains between 
about 50 and 250 milligrams of active compound. 
The tablets, troches, pills, capsules and the like may also contain the 
following: a binder such as gum tragacanth, acacia, corn starch or 
gelatin; excipients such as dicalcium phosphate; a disintegrating agent 
such as corn starch, potato starch, alginic acid and the like; a lubricant 
such as magnesium stearate; and a sweetening agent such as sucrose, 
lactose or saccharin may be added or a flavoring agent such as peppermint, 
oil of wintergreen, or cherry flavoring. When the dosage unit form is a 
capsule, it may contain, in addition to materials of the above type, a 
liquid carrier such as a fatty oil. Various other materials may be present 
as coatings or to otherwise modify the physical form of the dosage unit. 
For instance, tablets, pills, or capsules may be coated with shellac, 
sugar or both. A syrup or elixir may contain the active compound, sucrose 
as a sweetening agent, methyl and propylparabens as preservatives, a dye 
and flavoring such as cherry or orange flavor. Of course, any material 
used in preparing any dosage unit form should be pharmaceutically pure and 
substantially non-toxic in the amounts employed. 
Those compounds of the invention which have anti-inflammatory activity have 
had their activity confirmed in the synovium-cartilage test which is a 
modification of the method described by Dumonde, D. C. and Glynn, L. E., 
The Production of Arthritis in Rabbits by an Immunological Reaction to 
Fibrin, Brit. J. Exp. Pathol., 43, 373 (1962). 
Synovium-cartilage Test 
Male, New Zealand rabbits, weighing 1.6-3.0 kg. were used. The rabbits were 
housed in individual cages and given food and water ad libitum. The 
rabbits were sensitized to bovine serum albumin (BSA) by injection of 20 
mg./kg. BSA in complete Freund's adjuvant. The emulsion was injected 
subcutaneously into multiple sites on the back. Total injection volume 
equaled one mg./kg. of body weight. Between 18 and 21 days following 
immunization, the rabbits were challenged with an intraarticular injection 
of 5 mg. BSA into the right knee joint. Unanesthetized animals were 
restrained on their backs, and a 20-gauge, one-inch needle, with a 5 ml. 
syringe, was introduced into the joint and synovial fluid aspirated to 
determine that the synovium had been penetrated. The needle was then left 
in place for injection of the antigen. A 0.5 ml. portion of 10 mg./ml. BSA 
in sterile saline was injected into the synovial sac. The rabbits received 
an additional five intraarticular injections of 5 mg. BSA into the right 
knee at intervals of 12-16 days. Following the sixth injection the rabbits 
were sacrificed by an intracardiac injection of 5 ml. of a saturated 
solution of potassium chloride. The right knee was shaved. A longitudinal 
incision was made over the patellar ligament and the skin reflected under 
sterile conditions. The patellar ligament was cut tranversely and 
reflected. Facia and capsular tissue were trimmed from the lateral and 
medial aspects of the knee exposing the synovial membrane. The membrane 
was grasped with forceps, stretched laterally and then excised in a single 
piece on each side of the knee. The infrapatellar fat pad was removed and 
the joint space widened by cutting the anterior attachments of the menisci 
and severing the collateral cruciate ligaments. The femur and tibia were 
then separated and the menisci excised along with sufficient amounts of 
synovial tissue from the popliteal area adhereing to the menisci. The 
tissue were immediately placed in a sterile Petri dish containing tissue 
culture medium composed of MEM (Earle's salts) without sodium bicarbonate, 
with 25 mM Hepes buffer, pH adjusted to 7.34-7.37, and antibiotics 
(streptomycin nd neomycin, 100 units/ml.). Ten percent normal rabbit serum 
(NRS) was added. The tissues were rinsed three times in fresh medium plus 
10% NRS, then cut into pieces of 20-30 mg. 
Articular cartilage was obtained from the knees of normal, young rabbits, 
weighing 1.0 to 1.5 kg. The knees were shaved, the rabbits sacrificed and 
the joint exposed as described above. Synovial tissue and the 
infrapatellar fat pad were removed. Ligaments were severed and the menisci 
excised. Femur and tibia were then separated and articular cartilage was 
cut from supracondylar lines, patellar surface and femoral condyles. Due 
to curvature of the bone, these pieces were not more than 6-7 mg. each. No 
cartilage was taken from the tibia. The cartilage was placed in a sterile 
Petri dish containing tissue culture medium plus 10% NRS and rinsed three 
times with fresh medium +10% NRS. The cartilage was then cut into 1-2 mg. 
pieces and stored at -70.degree. C. 
A 10 mg. portion of the test compound was dissolved or suspended in 
absolute ethanol. Ten .mu.l. were then transferred to the complete tissue 
culture medium. The final concentration of test compound was then 10 
mcg./ml. and the vehicle 0.1%. 
Tissue culture medium was added to 12.times.75 mm clear plastic culture 
tubes containing one piece of normal articular cartilage. A piece of 
arthritic synovial tissue was then added to all tubes except those tubes 
in which cartilage was incubated alone. All tubes were then incubated at 
37.degree. C. for 48 hours with constant rotation in a roller drum at 0.2 
rpm. After 48 hours the cartilage was removed, hydrolyzed and assayed for 
hexoseamine and hydroxyproline. Hexoseamine to hydroxyproline ratios were 
calculated for the three groups: 
1. Cartilage alone 
2. Cartilage+synovium 
3. Cartilage+synovium+test compound. 
Cartilage hexoseamine decreases when it is cultured in the presence of 
synovium but remains constant when cultured alone. Hydroxyproline remains 
constant in all groups and the amount assayed is a measure of the size of 
the incubated cartilage. Therefore, the hexoseamine/hydroxyproline ratio 
decreases in the cartilage+synovium group relative to the cartilage alone 
group. The decrease is approximately 50%. 
If a compound prevents the decrease in the hexoseamine/hydroxyproline ratio 
by greater than 50% it is retested. A compound is considered active if it 
averages greater than 50% suppression of break down in three separate 
tests. 
The compound 2-cyano-3-hydroxythiocrotonic acid-S-phenyl ester is active 
when tested by the above procedure having a mean percent inhibition of 
105.77% based on three tests.