Compounds of metabolites of 3-(4-biphenylylcarbonyl)propionic acid useful as anti-inflammatory and anti-platelet aggregation agents.

BACKGROUND OF THE INVENTION 
This invention is concerned with the preparation and use of antithrombotic 
and anti-inflammatory drugs. Some prior examples of related compounds will 
now be considered. U.S. Pat. No. 3,966,978 to Ellenbogen et al. discloses 
the use of 4-biphenylacetic acid in the amelioration of blood platelet 
aggregation. U.S. Pat. No. 3,784,701 to Tomcufcik et al. discloses a group 
of anti-inflammatory drugs, substituted benzoylpropionic acids, including 
a biphenyl which is a starting material of some of the compounds of the 
invention. 
U.S. Pat. No. 3,966,960 to Ellenbogen et al. is to the use of 
3-(4-biphenylcarbonyl)propionic acid as an inhibitor of platelet 
aggregation. U.S. Pat. No. 3,784,704 to Cohen et al. describes the use and 
preparation of 4-biphenylacetic acid in the amelioration of pain. 
U.S. Pat. No. 3,969,402 to Adams et al. discloses the preparation and use 
of 2-(hydroxy substituted-4-biphenylyl)propionic acids as 
anti-inflammatory agents. 
British Pat. No. 1,390,091 describes the preparation and use of 
5-(4-biphenylyl)-2-hydro-2-(3H)-furanone. The activity of this compound is 
indicated as "antiphlogistic" and inhibitory toward "the aggregation of 
thromocytes". 
Finally, an article by H. Yoshizawa, Y. Tada, T. Naruke and M. Mizumura; 
Basic Pharmacology and Therapeutics, 2, No. 11, Dec. 5, 1974, pp. 31-40, 
describes 3-(4-biphenylylcarbonyl)propionic acid and derivatives. 
SUMMARY OF THE INVENTION 
This invention is concerned with compounds of the formula: 
##STR1## 
wherein R, R.sub.1 and R.sub.2 are each selected from the group consisting 
of hydrogen and hydroxy, said formula being intended to include both the 
erythro and threo configurations. The compound wherein R, R.sub.1 and 
R.sub.2 are all hydrogen is disclosed in Mizumura (above). 
Specifically, the new compounds of this invention are: 
.gamma.,4'-Dihydroxy-4-biphenylbutyric acid 
.beta.,.gamma.-Dihydroxy-threo-4-biphenylbutyric acid* 
.beta.,.gamma.-Dihydroxy-erythro-4-biphenylbutyric acid* 
.alpha.,.gamma.-Dihydroxy-erythro-4-biphenylbutyric acid 
.alpha.,.gamma.-Dihydroxy-threo-4-biphenylbutyric acid 
FNT *Disclosed in the racemic form only in Mizumura. 
This invention is further directed to methods of treating inflammation and 
platelet aggregation, by the administration of compounds of the formula: 
##STR2## 
wherein R, R.sub.1 and R.sub.2 are each selected from the group comprising 
hydrogen and hydroxy. 
Specifically, these compounds include the aforementioned plus 
.gamma.-hydroxy-4-biphenylbutyric acid, which has been described in 
Chemical Abstracts 78, 3966r (1973). 
DESCRIPTION OF THE INVENTION 
.gamma.-hydroxy-4-biphenylbutyric acid may be prepared by treating 
3-(4-biphenylylcarbonyl)propionic acid with sodium borohydride in an 
alkaline medium and then acidifying the reaction mixture. 
.gamma.,4-dihydroxy-4-biphenylbutyric acid may be prepared by treating 
p-methoxybiphenyl with succinic anhydride in nitrobenzene. The mixture is 
treated with aluminum chloride at reduced temperature. The solid is 
dissolved in aqueous base with heat, chilled to reprecipitate and 
acidified giving 4'-methoxy-.gamma.-oxo-4-biphenylbutyric acid. This 
intermediate is refluxed with hydrogen bromide for several hours, diluted 
with water and cooled. The precipitate is 
4'-hydroxy-.gamma.-oxo-biphenylbutyric acid. This intermediate is treated 
with sodium borohydride in an alkaline medium and then acidified giving 
the desired product. 
.beta.,.gamma.-hydroxy-threo-4-biphenylbutyric acid may be prepared by 
treating 3-(4-biphenylylcarbonyl)propionic acid with bromine in methylene 
chloride giving .beta.-bromo-.gamma.-4-biphenylbutyric acid. This 
intermediate is treated with sodium borohydride in alkaline medium and 
acidified giving cis-5-(4-biphenylyl)dihydro-4-hydroxy-2(3H)-furanone. 
This intermediate is treated with base in ethanol and then acidified 
giving the desired product. 
.beta.,.gamma.-hydroxy-erythro-4-biphenylbutyric acid is produced by the 
same process as the threo derivative except that the 
trans-5-(4-biphenylyl)dihydro-4-hydroxy-2(3H)-furanone is isolated by 
conventional chromatographic techniques. 
For therapeutic administration, the compounds of this invention may be 
incorporated with excipients and used, for example, in the form of 
tablets, dragees, capsules, liquids, elixirs, emulsions, suspensions, 
syrups, chocolate, candy, wafers, chewing gum and the like. Such 
compositions and preparations should contain at least 0.1% of active 
ingredient. The percentage in the compositions and preparations may, of 
course, be varied, and may conveniently be between about 2% and 60% or 
more of the weight of the unit. The amount of active component in such 
therapeutically useful compositions or preparations is such that a 
suitable dosage will be obtained. This dosage can also be obtained by the 
use of sustained release preparations. Preferred compositions or 
preparations according to the present invention are prepared so that a 
dosage unit form contains between about 1 and about 250 mg. of active 
component. 
Tablets, pills, dragees and the like may contain the following: a binder 
such as gum tragacanth, acadia, corn starch or gelatin; a disintegrating 
agent such as corn starch, potato starch, alginic acid or the like; a 
lubricant such as stearic acid, magnesium stearate, talc or the like; a 
sweetening agent such as sucaryl or saccharin may be added, as well as a 
flavoring such as peppermint, oil of wintergreen or cherry flavoring. 
The compounds of the present invention are active in vivo as inhibitors of 
platelet aggregation. 
The compounds were administered orally to male rats in various 
concentrations. After one to 2 hours the rats were bled and platelet rich 
plasma obtained. Collagen was added at a concentration of 500 mcg./ml. to 
induce platelet aggregation and comparisons were made between control and 
treated samples. The percent inhibition of aggregation produced by the 
test compound was recorded. The results of this test on a typical compound 
of this invention appear in Table I. 
TABLE I 
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Compound Dose mg./kg. 
% Inhibition 
______________________________________ 
.gamma.-Hydroxy-4-biphenyl- 
50 19 
butyric acid 25 25 
10 25 
______________________________________ 
In a second test the in vivo activity is determined in mice. Active 
compounds inhibit the respiratory depression associated with platelet 
aggregation and thrombosis induced by arachidonic acid. Mice were treated 
orally by gastric lavage with the test compounds in a starch suspension at 
various dosage levels. Two hours later a challenge dose of arachidonic 
acid was given to the mice intravenously at a concentration of 50 mg./kg. 
The period of respiratory distress for each animal was recorded in seconds 
by observation. The results of a typical compound of this invention appear 
in Table II in terms of percent inhibition in comparison with controls. 
TABLE II 
______________________________________ 
Compound Dose mg./kg. 
% Inhibition 
______________________________________ 
.gamma.-Hydroxy-4-biphenyl- 
1 46 
butyric acid 10 51 
25 66 
50 63 
______________________________________ 
The compounds of the present invention are active in vivo as 
anti-inflammatory agents. 
In determining the acute anti-inflammatory activity, Royal Hart, Wistar 
strain rats ranging in weight from 80 to 90 grams were used. The rats were 
fasted overnight prior to dosing but had free access to water. The 
compounds were administered in aqueous suspension, by gavage, in a column 
of 1.7 ml. per 50 g. of rat [corresponds to the hydration volume used by 
Winter, et al., Proc. Soc. Exp. Biol. and Med. 111, 544-547 (1962)]. 
The phlogistic agent used was carrageenin prepared as a sterile 1% 
suspension in 0.9% aqueous sodium chloride for routine testing. A volume 
of 0.05 ml. was injected through a 26 gauge needle into the plantar tissue 
of the right hind paw. Measurements were made 5 hours after drug 
administration (4 hours after carrageenin challenge). 
Volumes of both the normal and carrageenin inflamed feet were determined. 
The difference between the two measurements is considered to be the 
increased edema due to the carrageenin administration. Results are 
expressed as a C/T efficacy ratio (edema of control animals/edema of 
treated animals) and a C/T ratio of greater than 1.41 is considered 
active. 
The results of this test on a typical compound of this invention is 
recorded in Table III. 
TABLE III 
______________________________________ 
Compound Dose mg./kg. 
C/T Edema Ratio 
______________________________________ 
.gamma.-Hydroxy-4-bi- 
250 2.0 
phenylbutyric acid 
______________________________________ 
In a second test the activity against chronic inflammation in adjuvant 
induced arthritis was determined. Groups of 3 Royal Hart, Wistar strain 
rats, weighing 200.+-.10 g. each were injected intradermally in the right 
hind paw with Freund's adjuvant (dried human tubercle bacilli in a mineral 
oil vehicle) at a dose of 2 mg./kg. of body weight. Test compounds were 
administered orally in a 1.5% starch vehicle at 50 mg./kg. of body weight 
once daily on days 0 through 13 post challenge. Control rats were treated 
in a similar manner, but given only starch vehicle. On the 14th day post 
challenge the diameter of the injected paw (primary lesion) was measured 
by micrometer caliper. The volume of inflamed paws was estimated from 
these measurements and the results are expressed as percent inhibition of 
swelling as compared to controls. At the same time, the other inflamed 
sites, such as ears, paws and tail (secondary lesions) were observed and 
each rat was graded as to the degree of inflammation and swelling present. 
The grading is based on a scale of 0 to 24, where 0 represents a complete 
absence of induced arthritic nodules and 24 represents the maximum degree 
of inflammation. The mean grade for each treated group is calculated and 
the effects of each compound are expressed as percent inhibition of the 
control grade. Table IV records the results of this test on a typical 
compound of this invention. 
TABLE IV 
______________________________________ 
% Inhibition of 
% Inhibition of 
Swelling Control grade 
Compound (primary lesion) 
(secondary lesion) 
______________________________________ 
.gamma.-Hydroxy-4- 
51 30 
biphenylbu- 
tyric acid 
______________________________________ 
Another method of determining a drug effect on conditions which result in 
inflammation is by measuring the effect on ultraviolet induced erythema in 
guinea pigs. Albino guinea pigs were depilitated on their flanks, the 
evening before testing, with a standard mixture of barium sulfide and gum 
acacia. On the morning of the test, groups of 4 guinea pigs were dosed by 
gavage one hour prior (-1 hour) to ultraviolet exposure. At 0 hour they 
were restrained in a plastic container which allows exposure of 3 circular 
spots. They were then exposed to ultraviolet irradiation from a "Hanovia" 
Kromayer lamp, model 10, for 60 seconds. At one and four hours, the degree 
of erythema for each of the three sites was assessed according to the 
following scoring system: 0=no erythema, 0.5=incomplete circle or faint 
erythema and 1.0=incomplete circle of distinct erythema. Thus, the maximum 
score for each animal was 3.0. The results of this test on a typical 
compound of the present invention appears in Table V. 
TABLE V 
______________________________________ 
Dose No. of Score (avg.) 
Compound mg./kg. animals 1 hour 4 hour 
______________________________________ 
.gamma.-Hydroxy-4-bi- 
250 8 0 2.0 
phenylbutyric 
125 4 0.4 2.9 
acid 62.5 4 0 2.1 
______________________________________

DETAILED DESCRIPTION OF THE INVENTION 
Particular embodiments of this invention are detailed in the following 
examples. 
EXAMPLE 1 
.gamma.-Hydroxy-4-biphenylbutyric acid 
A 25.4 g. portion of 3-(4-biphenylylcarbonyl)propionic acid is dissolved in 
2 liters of water made alkaline with sodium hydroxide. A 37.8 g. portion 
of sodium borohydride is added and the mixture is stirred for 2 hours. The 
suspension is then slowly poured into 100 ml. of glacial acetic acid. The 
mixture is cooled and the resulting solid is filtered, washed with water 
and dried giving the desired product, m.p. 139.degree.-140.degree. C. 
EXAMPLE 2 
.gamma.-4'-Dihydroxy-4-biphenylbutyric acid 
A 50 g. portion of p-methoxybiphenyl and 30 g. of succinic anhydride are 
mixed in 600 ml. of nitrobenzene and cooled to about 5.degree. C. A 73 g. 
portion of aluminum chloride is added in portions keeping the temperature 
at about 5.degree. C. The mixture is then stirred for 15 hours and allowed 
to stand at room temperature. The mixture is poured into ice and then 
steam distilled until no more nitrobenzene comes over. The residue 
solidifies and is dissolved in 1,250 ml. of water containing 63 g. sodium 
carbonate. The solution is boiled with charcoal, filtered and the filtrate 
is chilled. The precipitate is filtered at room temperature and the gummy 
mixture is acidified with HCl and cooled. The precipitate is triturated 
with 50% acetic acid and the insoluble portion is recovered by filtration 
giving 4'-methoxy-.gamma.-oxo-4-biphenyl-butyric acid. 
A 3.0 g. portion of the preceding product, 20 ml. of glacial acetic acid 
and 6 ml. of hydrogen bromide is refluxed for 16 hours. The resulting 
mixture is cooled, diluted with 25 ml. of water and the precipitate is 
collected by filtration. The material is recrystallized from ethyl alcohol 
after treatment with activated charcoal. The initial acidic filtrate is 
evaporated, combined with the recrystallized material and refluxed again 
with 20 ml. of acetic acid and 6 ml. of hydrogen bromide. The resulting 
mixture is diluted with a small amount of water, cooled and filtered. The 
precipitate is collected and recrystallized from ethyl alcohol to give 1.4 
g. of 4'-hydroxy-.gamma.-oxo-biphenylbutyric acid. 
A 0.54 g. portion of the above product is dissolved in 80 ml. of water and 
2.4 ml. of N sodium hydroxide with stirring. To the stirred solution is 
added 0.76 g. of sodium borohydride. Stirring is continued for 16 hours at 
room temperature. The resulting clear solution is cooled in ice and 3 ml. 
of glacial acetic acid is added to give a gelatinous precipitate. The 
mixture is filtered through diatomaceous earth, and the filtered material 
is rinsed successively with diethyl ether. The combined ethereal filtrates 
are dried over magnesium sulfate and evaporated to dryness. The residue is 
crystallized from ethyl acetate-benzene and the product of the Example 
(0.3 g.) is collected by filtration and air dried, mp. 
141.5.degree.-143.5.degree. C. 
EXAMPLE 3 
.beta.,.gamma.-Dihydroxy-threo-4-biphenylbutyric acid 
To a 10.0 g. portion of 3-(4-biphenylylcarbonyl) propionic acid stirred in 
one liter of methylene dichloride at room temperature is added 6.8 g. of 
bromine in 50 ml. of methylene dichloride. Stirring is continued at room 
temperature for 21/2 hours. The solvent is evaporated resulting in 13 g. 
of .beta.-bromo-.gamma.-oxo-4-biphenylbutyric acid. 
To a 12.5 g. portion of the above compound stirred in 100 ml. of water at 
room temperature is added 50 ml. of N sodium hydroxide, then 1.0 g. of 
sodium borohydride is added and stirring is continued for 16 hours. The 
resulting solution is filtered and the pH of the filtrate is adjusted to 
pH 6 with glacial acetic acid. The solution is extracted twice with 
chloroform and the combined chloroform extract is rinsed with water, dried 
over magnesium sulfate and evaporated to an oil. The oil is triturated 
with ether to yield 0.9 g. of curde 
cis-5-(4-biphenylyl)dihydro-4-hydroxy-2(3H)-furanone. 
A 1.6 g. portion of the crude product (prepared as described above) is 
recrystallized twice from ethyl alcohol to give 0.8 g. of purified 
product. 
A 0.5 g. amount of the purified material above in 50 ml. of ethyl alcohol 
and 125 ml. of a 10% aqueous solution of sodium carbonate is heated 
briefly on a steam bath, then is stirred at room temperature for 16 hours. 
The solution is evaporated to a small volume and the reaction mixture is 
cooled and filtered. The resultant precipitate is stirred at room 
temperature for 3 hours in 100 ml. of 10% acetic acid and is filtered. The 
filtered acid is crystallized from ethanol-water yielding off-white 
crystals. This material is recrystallized from ethyl alcohol after 
treatment with activated charcoal to yield 180 mg. of the product of the 
Example as a white solid m.p. 178.degree.-180.degree. C. 
EXAMPLE 4 
.beta.,.gamma.-Dihydroxy-erythro-4-biphenylbutyric acid 
To a solution of 15 g. of -bromo- -oxo-4-biphenylbutyric acid in 800 ml. of 
water and 25 ml. of N sodium hydroxide, stirred for 10 minutes at room 
temperature, is added 7.6 g. of sodium borohydride. Stirring is continued 
for 16 hours and the solution is filtered. The filtrate is poured onto a 
mixture of 20 ml. of glacial acetic acid and ice with some diethyl ether 
added to minimize foaming. The aqueous layer is then extracted twice with 
ether. The combined ether solution is dried over magnesium sulfate and 
evaporated. The resultant solid is rinsed twice with ether. The rinsings 
are evaporated to dryness and the residue obtained is combined with the 
residue which is collected by filtering the previously ether extracted 
aqueous layer after standing overnight, to yield a total 2.2 g. of 
material. The preceding material is chromatographed. Fractions 9-17 are 
collected and evaporated to dryness to give 420 mg. of 
trans-5-(4-biphenylyl)dihydro-4-hydroxy-2(3H)-furanone. 
A 400 mg. portion of the preceding product in 80 ml. of absolute ethyl 
alcohol and 50 ml. of a 10% aqueous solution of sodium carbonate is 
stirred at room temperature for 16 hours. The solution is evaporated to 
dryness and the resulting solid is triturated with water and acetic acid, 
cooled and filtered. The material is air dried and is twice recrystallized 
from isopropyl alcohol to yield the product of the example as colorless 
crystals, m.p. 158.degree.-160.degree. C. 
EXAMPLE 5 
.alpha.,.gamma.-Dihydroxy-erythro-4-biphenylbutyric acid and 
.alpha.,.gamma.-Dihydroxy-threo-4-biphenylbutyric acid 
A total of 9 guinea pigs are each dosed with 100 ml./kg. of 
3-(4-biphenylylcarbonyl)-propionic acid. Their combined 24 hour urinary 
excretion (230 ml.) is acidified and extracted with chloroform:ether 
(8:3). The solvent is evaporated under nitrogen and the residue is 
partitioned on a celite column eluting with heptane:ethyl 
acetate:methanol:water:acetic acid (300:200:80:20:1.5), resulting in the 
separation and recovery of the desired threo and erythro compounds. 
EXAMPLE 6 
Preparation of Compressed Tablets 
______________________________________ 
Ingredient mg./Tablet 
______________________________________ 
Active compound 0.5-250 
Dibasic Calcium Phosphate NF 
qs 
Starch U.S.P. 20 
Modified Starch 5 
Magnesium stearate U.S.P. 
1-3 
______________________________________ 
EXAMPLE 7 
Preparation of Hard Shell Capsule 
______________________________________ 
Ingredient mg./Capsule 
______________________________________ 
Active Compound 0.5-250 
Lactose Spray dried qs 
Magnesium stearate 1-5 
______________________________________ 
EXAMPLE 8 
Preparation of Oral Suspension 
______________________________________ 
Ingredient % W/V 
______________________________________ 
Active compound 0.5-5 
Polysorbate 80 U.S.P. 0.1 
Flavoring agent qs 
Methylparaben U.S.P. 0.18 
Propylparaben U.S.P. 0.02 
Liquid sugar 75.0 
Purified water qs ad 100.0 
______________________________________ 
These novel compounds possess asymmetric centers and thus can be produced 
as racemic mixtures or as individual enantiomers. The racemic mixtures can 
be resolved if desired at appropriate stages by methods known to those 
skilled in the art, to obtain the respective individual enantiomers. It is 
to be understood that the racemic mixtures and the individual enantiomers 
are encompassed within the scope of the present invention. 
Other embodiments of this invention will be obvious to those skilled in the 
art without departing from the spirit of the invention. The foregoing 
examples are merely illustrative of the invention which is limited solely 
by the claims.