2-amino-3-aroyl-gamma-oxobenzenebutanoic acids and esters

2-Amino-3-aroyl-.gamma.-oxobenzenebutanoic acids and derivatives having the formula: ##STR1## wherein X is hydrogen, halogen, or loweralkyl; Y is hydrogen, halogen, loweralkyl, loweralkoxy, nitro, or trifluoromethyl; n is 1 or 2; and R is hydrogen, loweralkyl, or a pharmaceutically acceptable cation, are disclosed having anti-inflammatory activity.

BACKGROUND OF THE INVENTION 
1. Field of Invention 
The present invention relates to novel 
2-amino-3-aroyl-.gamma.-oxobenzenebutanoic acids, novel methods of 
preparation thereof, novel intermediates therefor, and pharmaceutical 
methods and compositions for treating living animals for pain and 
inflammation therewith. 
2. Description of the Prior Art 
2-Amino-3-aroyl-benzeneacetic acids, esters, and metal salts thereof have 
been disclosed as having anti-inflammatory activity in U.S. Pat. No. 
4,045,576 and as having anti-inflammatory and analgesic activity by 
Sancilio, L. F. et al. in AGENTS AND ACTIONS, Vol. 7/1 (1977) Birkauser 
Verlag Basel Schweiz. These compounds do not have a keto group on the 
alkanoic acid chain. 
3-Aroyl-.gamma.-oxobenzenebutanoic acids have been disclosed in U.S. Pat. 
No. 3,784,701 as having anti-inflammatory and analgesic activity. These 
compounds do not have a 2-amino group on the primary benzene ring as do 
the compounds of the present invention. 
SUMMARY OF THE INVENTION 
The novel 2-amino-3-aroyl-.gamma.-oxobenzenebutanoic acids of this 
invention have the formula: 
##STR2## 
wherein X is selected from hydrogen, halogen, or loweralkyl; Y is selected 
from hydrogen, halogen, loweralkyl, loweralkoxy, nitro, or 
trifluoromethyl; n is 1 or 2; R is H, loweralkyl, or a pharmaceutically 
acceptable cation. 
In the further definition of symbols and in the formulas hereof and where 
they appear elsewhere throughout this specification and in the claims, the 
terms have the following significance. 
The term "loweralkyl" as used herein, unless otherwise specified, includes 
straight and branched chain radicals of up to eight carbons inclusive and 
is exemplified by such groups as methyl, ethyl, propyl, isopropyl, butyl, 
sec. butyl, tert. butyl, amyl, isoamyl, hexyl, heptyl, and octyl radicals, 
and the like. The term "loweralkoxy" has the formula -O-loweralkyl. 
The terms "halo" or "halogen" as used herein includes fluorine, chlorine, 
bromine, and iodine. Preferably, the halogen is chlorine or bromine. 
The term "pharmaceutically acceptable cation" includes cations selected 
from such as sodium, potassium, calcium, magnesium, zinc, aluminum, 
copper, and the hydrates of the salts formed therewith when they occur. 
The compounds of the present invention are useful in controlling 
inflammation and pain and in inhibiting blood platelet aggregation. 
The anti-inflammatory utility of the novel compounds of this invention was 
determined using a modification of the Evans Blue-Carrageenan Pleural 
Effusion Assay of Sancilio, L. F., H. PHARMACOL. EXP. THER. 168, 199-204 
(1969), and a modification of the Adjuvant-Induced Arthritis Method of 
Walz, D. T. et al., J. PHARMAC. EXP. THER. 178, 223-231 (1971). See 
Pharmacology hereinbelow for description of tests. 
The analgesic utility of the compounds of Formula I was determined by a 
modification of the method of Collier et al., J. BR. PHARMAC. CHEMOTHER. 
32, 295-310 (1968). (See Pharmacology hereinbelow for description of 
test). 
Novel intermediates used in the preparation of compounds of Formula I are 
the compounds of Formulas II, III and IV below 
##STR3## 
wherein X and Y are as defined under Formula I; R.sup.1 and R.sup.2 are 
selected from loweralkyl or, when taken together with the adjacent 
nitrogen atom, may form a heterocyclic amine selected from 1-pyrrolidinyl, 
1-piperidinyl or 4-morpholinyl; and R.sup.4 is selected from hydrogen, 
loweralkyl or a metal cation selected from sodium, potassium, barium or 
calcium. 
The compounds of Formula I are pharmacologically active 
anti-inflammatory/analgesic prodrugs of 2-amino-3-benzoyl-benzeneacetic 
acids described in U.S. Pat. No. 4,045,576 and as such are capable of 
providing effectiveness in controlling inflammation and pain in living 
animals at a later time interval than the benzeneacetic acids. The 
effectiveness as an anti-inflammatory and analgesic of 
3-benzoyl-benzeneacetic acid (AHR-5850) is described by Sancilio, L. F. et 
al. in AGENTS AND ACTIONS (See reference above). 
It is therefore an object of the present invention to provide novel 
compounds via novel intermediates and compositions useful in the control 
of inflammation, pain, and blood-platelet aggregation. 
A further object is to provide prodrugs which break down to the 
2-amino-3-benzoyl-benzeneacetic acids after administration to living 
animals which are primarily responsible for the pharmacological activity, 
thus providing a more gradual and longer lasting effect than would be 
obtained from administration of the 2-amino-3-benzoyl-benzeneacetic acids. 
Additional objects will be apparent to one skilled in the art and still 
other objects will be apparent hereinafter. 
DETAILED DESCRIPTION OF THE INVENTION 
The compounds of Formula I are prepared by reactions represented by 
equations given in Chart I. 
##STR4## 
The 2-amino-3-aroyl-.gamma.-oxobenzenebutanoic acids and esters are 
prepared by a novel method comprising the following sequence of steps: 
Step 1, reacting an indole having the formula: 
##STR5## 
wherein X, Y and n are as defined under Formula I with formaldehyde and an 
amine having the formula: 
##STR6## 
wherein R.sup.1 and R.sup.2 are selected from loweralkyl or when taken 
together with the nitrogen atom, may form a heterocyclic amine such as 
1-pyrrolidinyl, 1-piperidinyl or 4-morpholinyl to give a compound having 
the formula: 
##STR7## 
wherein X, Y, n R.sup.1 and R.sup.2 have the starting values, 
Step 2, reacting the compound prepared in Step 1 with an alkali-metal salt 
of a diloweralkyl ester of malonic acid having the formula: 
EQU MCH(COOR.sup.3).sub.2 
wherein M is an alkali-metal and R.sup.3 is loweralkyl to give a compound 
having the formula 
##STR8## 
wherein X, Y, n and R.sup.3 have the values assigned above, 
Step 3, de-esterifying a compound prepared in Step 2 by heating it in 
aqueous basic solution and thereafter adding acid to give a compound of 
the formula: 
##STR9## 
wherein X, Y, and n have the values assigned above, 
Step 4, decarboxylating a compound prepared in Step 3 by heating to 
liberate carbon dioxide to give a compound having the formula: 
##STR10## 
wherein X, Y and n have the values assigned above, 
Step 5, oxidizing a compound prepared in Step 4 with ozone to give a 
compound having the formula: 
##STR11## 
wherein R is hydrogen or loweralkyl and X, Y, and n have the values 
assigned above and thereafter decomposing excess ozone, 
Step 6, esterifying a compound prepared in Step 5 with a loweralkanol 
wherein R is H to give a compound wherein R is loweralkyl 
Step 7, de-esterifying a compound prepared in Step 5 wherein R is 
loweralkyl by hydrolysis in aqueous base and neutralizing with a strong 
acid. 
In reference to the process steps summarized above as they apply to the 
preparation of compounds of Formulas I, II, III, and IV, the following 
further description is applicable: 
In Step 1, illustrated by the preparation of Intermediate 1 and 5, an 
aqueous solution of the HNR.sup.1 R.sup.2 amine and formaldehyde solution 
are reacted in the cold ca. 0.degree.-10.degree. C. and added to a mixture 
of the 7-benzoyl-1H-indole in acetic acid. Ethanol is added to the mixture 
which is then warmed. The preferred amine is dimethylamine as any excess 
amine is easily evaporated off. 
The product is separated by partitioning into the organic phase of a 
mixture of aqueous base and a solvent such as methylene chloride and 
evaporating the solvent and recrystallizing. 
In Step 2, illustrated by the preparation of Intermediates 2 and 6, the 
3-aminoalkyl-indolo-phenylmethanone is reacted with diloweralkyl 
alkali-metal malonate formed, for example, by sodium hydride and 
diloweralkyl malonate ester by refluxing in an aprotic solvent such as 
xylene until reaction has occurred. The product is isolated by 
conventional means such as evaporation of solvent and recrystallizing or 
by high pressure liquid chromatography. 
In Step 3, illustrated by the preparation of Intermediates 3 and 7, the 
propanedioic acid ethyl ester derivative is heated in intermediate 
strength base such as 3N sodium hydroxide. Other bases (MOH) which are 
suitable are those such as potassium, calcium, and barium hydroxides. At 
this point the liberated propanedioic acid is in the form of its metal 
salt corresponding to the base used. A strong acid such as hydrochloric 
acid is added to give the free propanedioic acid derivative and the 
product is separated by conventional precipitation and filtration. 
In Step 4, illustrated by the preparation of Intermediates 4 and 8, the 
propanedioic acid derivative is heated at about 180.degree.-210.degree. C. 
under reduced pressure until liberation of carbon dioxide ceases. The 
product is obtained by cooling the resulting melt. 
In Step 5, illustrated by Examples 1 and 2, the propanoic acid derivative 
is oxidized with ozone in a mixture of ethanol and ethyl acetate. Aqueous 
potassium iodide is added. Liberated iodine is removed by washing with 
sodium thiosulfate solution and the organic layer is concentrated. The 
residue is dissolved in a suitable solvent; e.g., ethanol, and heated with 
6N hydrochloric acid under reflux. The solution is again concentrated and 
the residue is partitioned between dilute aqueous base and methylene 
chloride and the product is isolated by conventional means. In this step, 
the more concentrated the initial solution is, the more likely an ester 
will result. 
Steps 6 and 7 are conventional esterification and de-esterification steps 
which are employed depending on whether an acid is obtained in Step 5 and 
an ester is desired or whether an ester is obtained in Step 5 and an acid 
is desired. 
Metal salts of acids obtained in Steps 5 or 7 may be obtained by 
conventional means. 
The procedure for the synthesis of the starting indole derivatives is as 
disclosed in U.S. Pat. No. 4,221,716. The routine is 
indolines.fwdarw.7-benzoylindolines.fwdarw.7-benzoylindoles. 
The following Intermediates 1-8 illustrate the synthesis of compounds of 
Formulas II, III, and IV, and the following Examples 1-3 illustrate the 
synthesis of compounds of Formula I and should in no way be regarded as 
limiting, the limiting factors being only the definitions given under 
Formulas I, II, III, and IV. Variations in X and Y are brought about by 
starting with the appropriate corresponding indole as would be 
recognizable by one skilled in the art. 
INTERMEDIATE 1 
[3-[(Dimethylamino)methyl]-1H-indol-7-yl]phenylmethanone 
An 18.0 g (0.16 mole) portion of 40% aqueous dimethylamine was cooled to 
5.degree. C. and 24 g (0.4 mole) of glacial acetic acid was added. To this 
mixture, held at 5.degree. C., was added 12.2 g (0.15 mole) of 37% 
formalin. This aqueous mixture was added to a mixture of 33.1 g (0.15 
mole) of 7-benzoyl-1H-indole and 20 ml of acetic acid. After 100 ml of 
absolute ethanol was added to the mixture, it was warmed on a steam bath 
for 1/2 hr. Isolation of the product was accomplished by concentrating the 
mixture under reduced pressure, partitioning between dilute sodium 
hydroxide and methylene chloride and concentrating the organic solution. 
The residue was recrystallized from isopropyl alcohol to give 29 g (70%) 
of light yellow crystals, m.p. 111.0.degree.-113.5.degree. C. 
Analysis: Calculated for C.sub.18 H.sub.18 N.sub.2 O: C, 77.67; H, 6.52; N, 
10.06 Found: C, 78.07; H, 6.49; N, 10.07 
INTERMEDIATE 2 
2-[(7-Benzoyl-1H-indol-3-yl)methyl]propanedioic acid, diethyl ester 
A mixture of 19.4 g (0.07 mole) of 
[3-[(dimethylamino)methyl]-1H-indol-7-yl]phenylmethanone and 32.5 g (0.2 
mole) of diethyl sodiomalonate prepared from 0.85 g (0.2 mole) of 57% 
sodium hydride in oil and 32 g (0.2 mole) of diethyl malonate in 50 ml of 
xylene was heated at reflux for 17 hr. The mixture was cooled, diluted 
with diethyl ether and washed with water. The solvent and excess reagents 
were removed by distillation at high vacuum. The residue was crystallized 
first from isopropyl alcohol and then from isopropyl ether to give 12.6 g 
(46%) of light yellow powder, m.p. 86.0.degree.-88.0.degree. C. 
Analysis: Calculated for C.sub.23 H.sub.23 NO.sub.5 : C, 70.22; H, 5.89; N, 
3.56 Found: C, 70.50; H 5.93; N, 3.61 
INTERMEDIATE 3 
2-[(7-Benzoyl-1H-indol-3-yl)methyl]propanedioic acid 
A mixture of 10.0 g (0.025 mole) of 
2-[(7-benzoyl-1H-indol-3-yl)methyl]propanedioic acid diethyl ester in 150 
ml of 3N sodium hydroxide was heated at reflux for 18 hr, then treated 
with charcoal, cooled and filtered. The dark yellow filtrate was acidified 
by the dropwise addition of 50 ml of concentrated hydrochloric acid. The 
addition of 20 ml of methylene chloride caused the formation of a ppt., 
which was collected and recrystallized from chloroform-methanol to give 
6.0 g (70%) of off-white crystals, m.p. 188.degree.-189.degree. C. 
Analysis: Calculated for C.sub.19 H.sub.15 NO.sub.5 : C, 67.65; N, 4.48; N, 
4.15 Found: C, 67.91; H, 4.50; N, 4.20 
INTERMEDIATE 4 
7-Benzoyl-1H-indole-3-propanoic acid 
A 2.4 g (0.07 mole) sample of 
2-[(7-benzoyl-1H-indol-3-yl)methyl]propanedioic acid was heated at 
190.degree. C. under vacuum until carbon dioxide evolution ceased (1/2 
hr). The syrup was cooled to give 2.1 g (100%) of a yellow solid, m.p. 
166.5.degree.-168-5.degree. C. 
Analysis: Calculated for C.sub.18 H.sub.15 NO.sub.3 : C, 73.71; H, 5.16; N, 
4.78 Found: C, 73.76; H, 5.11; N, 4.84 
INTERMEDIATE 5 
(4-Chlorophenyl)-[3-[(dimethylamino)methyl]-1H-indol-7-yl]methanone 
This compound was prepared by the procedure used to synthesize the compound 
of Intermediate 1. A combination of 13.5 g (0.12 mole) of 40% aqueous 
dimethylamine, 16.5 g (0.275 mole) of acetic acid, 9.3 g (0.115 mole) of 
37% formalin, and 28.1 g (0.11 mole) of 
(4-chlorophenyl)(1H-indol-7-yl)methanone gave 35.3 g (99%) of crude title 
compound. Two recrystallizations of a small sample from 2-propanol gave 
white crystals, m.p. 95.degree.-99.degree. C. 
Analysis: Calculated for C.sub.18 H.sub.17 ClN.sub.2 O: C, 69.12; H, 5.48; 
N,. 8.96 Found: C, 69.27; H, 5.51; N, 8.82 
INTERMEDIATE 6 
2-[[7-(4-Chlorobenzoyl)-1H-indol-3-yl]methyl]propanedioic acid, diethyl 
ester 
This compound was prepared by the procedure used to synthesize the compound 
of Intermediate 2, substituting dimethylsulfoxide to replace xylene as 
solvent. A combination of 4.2 g (0.1 mole) of 57% sodium hydride, 30 ml of 
dimethyl sulfoxide, 80 g (0.5 mole) of diethylmalonate and 31.2 g (0.1 
mole) of 
(4-chlorophenyl)-[3-[(dimethylamino)methyl]-1H-indol-7-yl]methanone, gave 
45 g of crude title compound. A small sample (3.1 g) was purified by HPLC 
to give, after a recrystallization from 90% aqueous ethanol, 2.0 g (67%) 
of yellow crystals, m.p. 102.0.degree.-102.5.degree. C. 
Analysis: Calculated for C.sub.23 H.sub.22 ClNO.sub.5 : C, 64.56; H, 5.18; 
N, 3.27 Found: C, 64.71; H, 5.21; N, 3.38 
INTERMEDIATE 7 
2-[[7-(4-Chlorobenzoyl)-1H-indol-3-yl]methyl]propanedioic acid 
This compound was prepared by the procedure used to synthesize the compound 
of Intermediate 3. A batch of 28.8 g (0.067 mole) of 
2-[[7-(4-chlorobenzoyl)-1H-indol-3-yl]methyl]propanedioic acid diethyl 
ester and 600 ml of 3N sodium hydroxide gave, after a recrystallization 
from chloroform-methanol, 19.5 g (79%) of pale yellow crystals, m.p. 
197.degree.-201.degree. C. with decomposition. 
Analysis: Calculated for C.sub.19 H.sub.14 ClNO.sub.5 : C, 61.38; H, 3.80; 
N, 3.77 Found: C, 61.58; H, 3.81; N, 3.83 
INTERMEDIATE 8 
7-(4-Chlorobenzoyl)-1H-indole-3-propanoic acid 
This compound was prepared by the procedure used to synthesize the compound 
of Intermediate 4. A batch of 18.9 g (0.51 mole) of 
2-[[7-(4-chlorobenzoyl)-1H-indol-3-yl]methyl]propanedioic acid heated to 
200.degree. C. gave 16.6 g (100%) of a dark yellow solid, m.p. 
190.degree.-202.degree. C. 
Analysis: Calculated for C.sub.18 H.sub.14 ClNO.sub.3 : C, 65.96; H, 4.31; 
N, 4.27 Found: C, 66.13; H, 4.25; N, 4.28

EXAMPLE 1 
2-Amino-3-benzoyl-.gamma.-oxobenzenebutanoic acid 
A solution of 8.7 g (0.03 mole) of 7-benzoyl-1H-indole-3-propanoic acid in 
300 ml of ethyl acetate and 100 ml of abs. ethanol was ozonized until 
ozone was present above the solution. The yellow solution was then treated 
with 16.6 g (0.1 mole) of potassium iodide in 30 ml of acetic acid and 30 
ml of water. After stirring 1 hr, the liberated iodine was removed by 
washing with a 15% sodium thiosulfate solution and the yellow organic 
layer was concentrated. The residue was dissolved in 100 ml of ethanol and 
20 ml of 6N hydrochloric acid and heated at reflux for 16 hr. The dark red 
solution was concentrated and the residue was partitioned between dil. 
sodium hydroxide and methylene chloride. The basic aqueous layer was made 
acidic and the pH was adjusted to 2-3 by the addition of dil. sodium 
hydroxide. The solid was collected and dried, then recrystallized from 
benzene isopropyl ether to give 5.9 g (67 %) of yellow powder, m.p. 
161.0.degree.-2.5.degree. C. 
Analysis: Calculated for C.sub.17 H.sub.15 NO.sub.4 : C, 68.68; H, 5.09; N, 
4.71 Found: C, 68.82; H, 5.11; N, 4.67 
EXAMPLE 2 
2-Amino-3-(4-chlorobenzoyl)-.gamma.-oxobenzenebutanoic acid, ethyl ester 
A solution of 13.1 g (0.04 mole) of 
7-(4-chlorobenzoyl)-1-H-indole-3-propanoic acid in 450 ml of ethyl acetate 
and 150 ml of absolute ethanol was treated with ozone until ozone was 
present above the solution. The solution was then stirred with an aqueous 
solution of potassium iodide, followed by a wash with aqueous sodium 
thiosulfate. The organic fraction was concentrated and the residue was 
dissolved in 250 ml of 190 proof ethanol. The solution was heated to 
reflux, 150 ml of 6N hydrochloric acid was added, and heating was 
continued for 18 hr. The mixture was diluted with 400 ml of water and a 
gummy solid separated. The gum was partitioned between dilute sodium 
hydroxide solution and methylene chloride. The basic aqueous fraction 
contained only a small amount of acidic material upon acidification, so it 
was discarded. The methylene chloride layer was dried over anhydrous 
sodium sulfate and passed through a column of silica gel. The 
yellow-colored eluant was concentrated and the crystalline residue was 
recrystallized from cyclohexane to give 3.5 g (26%) of bright yellow 
powder, m.p. 112.degree.-115.degree. C. 
Analysis: Calculated for C.sub.19 H.sub.18 ClNO.sub.4 : C, 63.43; H, 5.04; 
N, 3.89 Found: C, 63.52; H, 5.04; N, 3.92 
EXAMPLE 3 
2-Amino-3-(4-chlorobenzoyl)-.gamma.-oxobenzenebutanoic acid 
A solution of 3.3 g (0.0092 mole) of 
2-amino-3-(4-chlorobenzoyl)-.gamma.-oxobenzenebutanoic acid ethyl ester in 
70 ml of hot 190 proof ethanol was treated with 40 ml of 4N aqueous sodium 
hydroxide solution and the mixture was heated at reflux for 18 hr. The hot 
mixture was filtered and the insoluble material was discarded. The 
filtrate was cooled and the precipitate collected by filtration. This 
precipitate was partitioned between dilute hydrochloric acid and methylene 
chloride. The organic layer was separated, dried over magnesium sulfate 
and concentrated to give 2.7 g (89%) of bright yellow crystals, m.p. 
172.degree.-177.degree. C. 
Analysis: Calculated for C.sub.17 H.sub.14 ClNO.sub.4 : C, 61.55; H, 4.25; 
N, 4.22 Found: C, 61.43; H, 4.24; N, 4.24 
PHARMACOLOGY 
Acute Anti-inflammatory Test-Evans Blue-Carrageenen Pleural Effusion Assay 
The method is that of Sancilio and Fishman in TOXICOL. APPL. PHARMAC. 26, 
575-584 (1973). Fasted Sprague-Dawley male rats, weighing between 250-500 
g were randomly divided into control and experimental groups of six 
animals one hour after oral administration of the compounds, e.g., Formula 
I compounds or indomethacin, the rats were etherized and 5 ml of a mild 
irritant solution (0.075% Evans blue and 0.5% carrageenan type 7) was 
administered intrapleurally. Five hours later, the animals were sacrificed 
with carbon dioxide, pleural fluids were collected in calibrated 
centrifuge tubes and measured. Results were expressed as the average 
percent decrease in volume of pleural fluid from that of the control 
group. The carrier was 0.5% Tween 80 in distilled water and was also the 
control article. Potency as compared to indomethacin was determined by 
regressional analysis by the method of Bliss, C. (1951) VITAMIN METHODS 
VOL. 2, pp 445-610, Ed. by Gyorgy, N.Y. Academic Press. Using this 
procedure, it was determined that the compound of Example 1 was 1.73 
(0.78-3.62) times the potency of indomethacin or considering the overlap 
of confidence limits, it is about as potent as indomethacin in the 
foregoing pleural effusion anti-inflammatory assay over the range of 0.16 
to 4.0 mg/kg body weight. 
Chronic Anti-inflammatory Test-Adjuvant-Induced Arthritis Assay 
A modification of the method of Walz et al, J. PHARMAC. EXP. THER. 178, pp 
223-31 (1971), was used. This consisted of a therapeutic rather than a 
prophylactic dosing regimen. 
Female Lewis Wistar rats, weighing between 150 and 235 g, were used. On day 
0 a tattoo was made on each leg at the point where the Achilles tendon 
enters the gastrocnemius muscle. This served as a reference point for 
measuring the limb volume, plethysmographically. Several hours later, 0.05 
ml of a suspension of 1.5% Mycobacterium butyricum in mineral oil was 
injected into the subplantar surface of the right hind foot. On day 18 the 
hind limb volumes of both feet were determined. Animals with significant 
swelling of the uninjected feet were randomized by block design into 
groups of seven or eight. They were dosed orally five days/week, starting 
on day 18, with the last dose being given on day 28. Twenty-four hours 
after the last dose, the edema of the injected and uninjected feet was 
determined by difference. Results were expressed as milliliter of edema of 
the injected and uninjected feet. 
In this test, the compound of Example 3 was found to be 0.86 (0.47-1.7) 
times as potent as indomethacin. 
Analgesia Test-Acetylcholine-induced Abdominal Constriction in Mice 
The method is a modification of that of Collier, H. O. J. et al., J. BR. 
PHARMAC. CHEMOTHER. 32, 295-310 (1968). Fed female mice are randomized 
into groups of 10. Group 1 received the control article (carrier) which 
was 0.5% Tween 80 in distilled water (10 ml/kg). Test agent was suspended 
in 10 ml/kg of the carrier and administered by gavage to the mice and 180 
min later acetylcholine bromide in 0.06% saline was administered 
intraperitoneally. Immediately thereafter, each mouse was placed under an 
inverted 1-liter beaker and observed for 3 min for the presence of 
abdominal constriction. The compound of Example 3 prevented abdominal 
constriction in 70% of the mice when administered at 4.0 mg/kg in 10 ml/kg 
of the carrier. This compared to 60% blocked by 1.0 mg/kg indomethacin 
under the same conditions. 
Formulation and Administration 
The present invention also contemplates novel therapeutic compositions 
containing the compounds of the invention as active ingredients. Effective 
quantities of any of the foregoing pharmacologically active compounds may 
be administered to a living animal body in any one of various ways; for 
example, orally as in capsules or tablets, parenterally in the form of 
sterile solutions or suspensions, and in some cases intravenously in the 
form of sterile solutions. In forming the novel compositions of this 
invention, the active ingredient is incorporated in a suitable carrier, 
illustratively, a pharmaceutical carrier. Suitable pharmaceutical carriers 
which are useful in formulating the compositions of this invention include 
starch, gelatin, glucose, magnesium carbonate, lactose, malt and the like. 
Liquid compositions are also within the purview of this invention and 
suitable liquid pharmaceutical carriers include ethyl alcohol, propylene 
glycol, glycerine, glucose syrup and the like. 
The pharmacologically active compounds may be advantageously employed in a 
unit dosage of from 0.1 to 250 milligrams or more depending on the size of 
the animal. For example, a large animal such as a horse may require 
tablets of 500-1000 milligrams active ingredient. The unit dosage may be 
given a suitable number of times daily so that the daily dosage may vary 
from 0.3 to 450 milligrams. Five to 25 milligrams appears optimum per unit 
dose. 
It is only necessary that the active ingredient constitute an effective 
amount, i.e., such that a suitable effective dosage will be obtained 
consistent with the dosage form employed. The exact individual dosages as 
well as daily dosages will, of course, be determined according to standard 
medical principles under the direction of a physician or veterinarian. 
The active agents of the invention may be combined with other 
pharmacologically active agents, or with buffers, antacids or the like, 
for administration and the proportion of the active agent in the 
compositions may be varied widely. 
The following are examples of compositions formed in accordance with this 
invention. 
1. Capsules 
Capsules of 5 mg., 25 mg., and 50 mg. of active ingredient per capsule are 
prepared. With the higher amounts of active ingredient, adjustment may be 
made in the amount of lactose. 
______________________________________ 
Typical blend for 
Per capsule, 
encapsulation mg. 
______________________________________ 
Active ingredient 
5.0 
Lactose 296.7 
Starch 129.0 
Magnesium stearate 
4.3 
Total 435.0 mg. 
______________________________________ 
Additional capsule formulations preferably contain a higher dosage of 
active ingredient and are as follows. 
______________________________________ 
Per capsule, 
Ingredients mg. 
______________________________________ 
Active ingredient 
25.0 
Lactose 306.5 
Starch 99.2 
Magnesium stearate 
4.3 
Total 435.0 mg. 
______________________________________ 
In each case, uniformly blend the selected active ingredient with lactose, 
starch, and magnesium stearate and encapsulate the blend. 
2. Tablets 
A typical formulation for a tablet containing 5.0 mg. of active ingredient 
per tablet follows. The formulation may be used for other strengths of 
active ingredient by adjustment of weight of dicalcium phosphate. 
______________________________________ 
Per tablet, mg. 
______________________________________ 
(1) Active ingredient 
5.0 
(2) Corn starch 13.6 
(3) Corn starch (paste) 
3.4 
(4) Lactose 79.2 
(5) Dicalcium phosphate 
68.0 
(6) Calcium stearate 
0.9 
170.1 mg. 
______________________________________ 
Uniformly blend 1, 2, 4, and 5. Prepare 3 as a 10 percent paste in water. 
Granulate the blend with starch paste and pass the wet mass through an 
eight mesh screen. The wet granulation is dried and sized through a twelve 
mesh screen. The dried granules are blended with the calcium stearate and 
pressed. 
3. Injectable-2% sterile solutions. 
______________________________________ 
Per cc. 
______________________________________ 
Active ingredient 
20 mg. 
Preservative, e.g., 
0.5% weight/volume 
chlorobutanol 
Water for injection 
q.s. 
______________________________________ 
Prepare solution, clarify by filtration, fill into vials, seal and 
autoclave. 
Various modifications and equivalents will be apparent to one skilled in 
the art and may be made in the compounds, compositions, and methods of the 
present invention without departing from the spirit or scope thereof, and 
it is therefore understood that the invention is to be limited only by the 
scope of the appended claims.