NO149064B - ANALOGUE PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE 4-ACETAMIDOPHENYL-1`- (PARA-CHLOROBENZOYL) -2`-METHYL-5`-METHOXYINDOL-3`-YL-ACETATE - Google Patents

Description

The present invention relates to an analog method

for the preparation of therapeutically active 4-acetamidophenyl-11-(para-chlorobenzoyl)-2'-methyl-5<1->methoxyindol-3'-yl-acetate with the formula:

and acid addition salts thereof.

Analgesics are widely used today, and many compounds

of various types have been proposed and marketed for use as such. Among the most effective and fast-acting of the currently used analgesics is 4-acetamidophenol of structural formula:

Despite its effectiveness as an analgesic pre-

4-acetamidophenol is not usually prescribed for the treatment of chronic conditions such as e.g. rheumatism and arthritis, except occasionally to relieve pain, due to the fact that it does not exhibit any anti-inflammatory effect in addition to the analgesic activity.

To alleviate chronic arthritis, rheumatism and similar conditions, it is currently very common to use one of the known anti-inflammatory substituted acetic acid analgesics.

A class of existing anti-inflammatory analgesics

which are widely used at present are the well-known substituted α-(2-methyl-ind[en/ol]-3-yl)-acetic acids of the general formula:

where A denotes a

group,

R denotes a fluorine atom or a methoxy group, n is 0 or 1 and where either X denotes a nitrogen atom and Y denotes a -C0 bond and the dashed line has no meaning, or where X denotes a carbon atom, Y denotes a =CH group and the dashed line indicates another C-C bond, as well as their pharmacologically acceptable salts.

The following compounds are examples of these known ones

anti-inflammatory analgesics of general formula II: 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid,

1-(para-methylsulfinyl-benzylidene)-2-methyl-5-fluoro-inden-3-yl-acetic acid,

1-(3-phenyl-acryloyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid, and

1-isonicotinyl-2-methyl-5-methoxy-indol-3-yl-acetic acid.

Unfortunately, the desirable anti-inflammatory and analgesic properties of these substituted acetic acids of general formula II suffer from the disadvantage that they give rise to disturbances in the gastrointestinal tract. It has been hypothesized that this may be due to the acidic nature of their carboxyl functions, but this explanation is by no means reliable, as there is little or no attenuation of this tendency when these anti-inflammatory analgesics are administered in the form of salts.

It is therefore particularly surprising that it has been found that it is not only possible to form an ester bond with the carboxyl group of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and the hydroxyl group present in the 4-acetamido-phenol, but also that the resulting ester is predominantly or completely free of unwanted side effects on the gut, but nevertheless is assimilated from the gut predominantly or completely unchanged into the body while being an effective and excellent anti-inflammatory, analgesic agent.

From DE official publication 2 234 651 a compound is known which can be regarded as an ester of indomethacin with hydroxyacetic acid (known as glycolic acid).

A comparison of the 1, D^Q and ED^Q values given in this publication with the corresponding values for paracetamol indomethacinate (IV) prepared according to the invention shows that the latter compound is superior in activity.

Furthermore, it appears from the Merck Index, page 583, that hydroxyacetic acid acts as a mild irritant on the skin and mucous membranes, so that even the slightest cleavage of the known ester could lead to serious irritation of the gastrointestinal tract, possibly more than what indomethacin alone could cause to.

British patent document 1 428 803 relates to esters of paracetamol, but with arylalkane carboxylic acids, and which are stated to be less toxic than the parent acids.

However, these known esters are completely different from the present ester, and there is nothing indicated in this publication which would lead the person skilled in the art to believe that the particular problems associated with indomethacin could be solved by using the present ester.

The acid addition salts according to the invention are all usable for preparative purposes, but for pharmaceutical applications the salts must be formed with pharmacologically acceptable acids. There are a large number of pharmacologically acceptable acids with which the acid addition salts can be formed. These can e.g. include hydrochloric acid, hydrobromic acid, acetic acid, methanesulfonic acid, tartaric acid, succinic acid, phosphoric acid and sulfuric acid.

The analogy method according to the invention is characterized in that

a) 4-acetamidophenol is reacted with 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetyl chloride, or b) 4-acetamidophenol is reacted with the mixed anhydride of 1-(para-chlorobenzoyl) -2-methyl-5-methoxy-indol-3-yl-acetic acid and para-toluenesulfonyl chloride, c) para-aminophenyl-11-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3'- yl acetate is reacted with acetic anhydride,

or

d) 4-acetamidophenol is reacted with the symmetrical anhydride of 1-(para-chlorobenzoyl)-2-methyl-5-methoxyindol-3-yl-acetic acid.

The reaction between 4-acetamidophenol and 1-(para-chloro-benzoyl)-2-methyl-5-methoxy-indol-3-yl-acetyl chloride (method alternative a)) is preferably carried out in an anhydrous inert solvent. The inert solvent will normally be an organic solvent, and is preferably a substituted aromatic hydrocarbon such as toluene, one of the many halogenated aromatic solvents/ an aliphatic hydrocarbon, a halogenated aliphatic hydrocarbon such as chloroform, or an ether such as diethyl ether.

As the reaction is relatively vigorous, it can be carried out at ambient temperature without heating, or when the reaction is very vigorous, which it sometimes is, it can possibly be carried out under cooling.

The reaction is preferably carried out in the presence of a base,

and suitably in a nitrogen-containing aromatic heterocyclic compound. The base that is frequently preferred above all others is pyridine.

The procedure can also be advantageous

involve a preliminary step, namely preparation of the substituted acetic acid chloride starting material, by reacting a solution and/or suspension in an anhydrous inert solvent of the acetic acid or a salt thereof with thionyl chloride.

When a salt of the acetic acid is used, this is preferably the pyridine salt.

The reaction with thionyl chloride can be carried out at a

any suitable temperature between ambient temperature and the solvent reflux temperature, and will usually be carried out at a temperature within the range of 40 to 65°C.

The solvent used in the reaction is preferably the same as that used in the above-described subsequent step in the preparation. Thus, it will normally be an organic solvent and preferably a substituted aromatic hydrocarbon such as toluene, one of the many halogenated aromatic solvents, an aliphatic hydrocarbon, a halogenated aliphatic hydrocarbon such as chloroform or dichloromethane, or an ether such as diethyl ether.

Preferably, the solvent used is anhydrous dichloromethane, and the reaction is then suitably carried out under reflux at a temperature of approx. 41° C. The quantity of the reactants will preferably be approx. 1 mol of thionyl chloride per moles of the substituted acetic acid, 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid.

It has been found advantageous to carry out the reaction between the acetic acid and the thionyl chloride in the presence of a catalytic amount of N,N-dimethylformamide.

According to method alternative b), the mixed anhydride of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and para-toluenesulfonyl chloride is reacted with 4-acetamidophenol.

The reaction between the mixed anhydride and 5-acetamidophenol can and preferably should be carried out in an anhydrous inert solvent, and this is preferably an organic solvent as described in connection with the first method described.

Furthermore, the reaction can also be carried out in the presence of a base, and preferably a tertiary amine such as triethylamine or pyridine.

The reaction between the acetic acid and para-toluenesulfonyl chloride is conveniently carried out in an anhydrous solvent, and this is preferably an organic solvent as previously defined in connection with the subsequent step in the process.

The reaction must be carried out in the presence of a base, which is preferably a tertiary amine and preferably a triethylamine or pyridine.

It is not necessary to isolate the mixed anhydride from the reaction mixture in which it was prepared. Instead, the solution of the mixed anhydride obtained from the first step of the method can advantageously be subjected to the second step of the method in the same reaction mixture without removal of the base, and

r

in the same reactions.

According to process alternative c) para-aminophenyl-1'-((para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3'-yl-acetate is reacted with acetic anhydride.

The para-aminophenyl compound can conveniently be prepared by reduction of para-nitrophenyl-1'-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3<1->yl-acetate using nascent hydrogen, developed f .ex. with iron powder'and an acid. The acid is suitably warm acetic acid.

The para-nitrophenyl compound can in turn be prepared by reacting 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid or a salt thereof with bis-(4-nitrophenyl)-sulphite.

The reaction between the acetic acid and bis-(4-nitrophenyl) sulphite is preferably carried out in solution in a tertiary amine, preferably pyridine, and at ambient or near ambient temperature.

According to process alternative d) 4-acetamidophenol is reacted with the symmetrical anhydride of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indor-3-yl-acetic acid in the presence of a strong aqueous base, so that the reaction mixture forms a two-phase system which is stirred while forming the desired product.

The reaction between 4-acetamidophenol and the acid anhydride forms the corresponding acetic acid as a by-product, and must therefore be carried out in the presence of a strong base, which can be an organic base such as triethylamine, but will preferably be aqueous sodium hydroxide. The anhydride is somewhat unstable in the presence of water and is therefore brought to the reaction in solution in a water-immiscible solvent and preferably an anhydrous inert organic solvent, suitably benzene, toluene, chloroform, dichloromethane or ethers such as diethyl ether. The reaction is carried out with stirring in a two-phase reaction system, so that any decomposition of the anhydride before it has taken part in the specified reaction is reduced to a minimum. This reaction can conveniently be carried out at ambient or near ambient temperatures, but not above 30°C.

The acid anhydride is produced by reacting 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and N,N'-dicyclohexylcarbodiimide in solution in an anhydrous, non-hydroxyl or on otherwise inert organic solvent at ambient or near ambient temperature, usually not above 30°C.

4-acetamidophenyl-1'-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3<1->yl-acetate of general formula IV exhibits excellent anti-inflammatory and analgesic activity when tested by generally accepted tests for anti-inflammatory drugs, and preliminary studies establish that when administered orally, it will be largely free of gastrointestinal side effects.

For use in human and veterinary medicine as an anti-inflammatory analgesic, 4-acetamidophenyl-1<1->

(para-chlorobenzoyl)-2'-methyl-5<1->methoxy-indol-3'-yl-acetate is first accurately formulated in suitable pharmaceutical preparations by connection with suitable pharmaceutical carriers.

The term "pharmaceutical" is used herein to exclude any possibility that the nature of the carrier (obviously considered in relation to the method of administration for which the composition is intended) may be harmful. The selection of a suitable carrier for any chosen method of administration is known to those skilled in the art.

Although the dose of the anti-inflammatory analgesic will depend to a certain extent on the method of administration and of course the condition of the person being treated, it can be stated as a general indication that the applicable dose varies from 30 to 750 mg (in divided doses) of the active analgesic per day for an adult patient, a unit dose usually containing from 10 mg to 250 mg and preferably approx. 50 mg.

The following examples and formulations illustrate the invention.

Example 1 Preparation of 4-acetamidophenyl-1'-(para-chloro-.benzoyl)-2'-methyl-5'-methoxy-indol-3'-yl-acetate of general formula IV

Step A:

A solution of 6.0 g of thionyl chloride in 10 ml of dichloromethane was slowly added to a stirred solution of 7.9 g of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and 4, 8 g of pyridine in 80 ml of dichloromethane. The resulting red solution was refluxed for 15 minutes and contained 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetyl chloride which, however, was not isolated. When a sample was isolated, this acid chloride was found to have a melting point of 107 - 109°C.

Step B:

A suspension of 7.55 g of 4-acetamidophenol in 5.0 g of pyridine and 10 mL of dichloromethane was carefully added to the refluxed solution from step A. This mixture was then refluxed for 10 minutes, cooled, washed twice with 2M hydrochloric acid and once with 20% sodium carbonate solution, and was then dried over magnesium sulfate.

Concentration of the dichloromethane solution under reduced pressure gave a yellow solid which was recrystallized from aqueous acetone as cream needles (yield 11.6 g, 48%), m.p. = 198 - 199° C.

Example 2 Preparation of 4-acetamidophenyl-1'-(para-klpr-benzoyl-2'-methyl-5'-methoxy-indol-3'-yl-acetate of formula IV via the para-nitrophenyl ester

Step A: Preparation of para-nitrophenyl ester

A solution of 10 g of bis-(4-nitrophenyl) sulphite in 60

ml of pyridine was added to a solution of 4.0 g of 1-(para-chloro-

benzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid in 50 ml of pyridine. The mixture was kept at room temperature overnight under a nitrogen atmosphere, and then concentrated under reduced pressure.

The residue was triturated with 200 ml of ethyl acetate and the mixture was filtered. The filtrate was washed with 100 ml of dilute hydrochloric acid and then with 50 ml of saturated aqueous sodium carbonate solution and finally with water, and was then dried over magnesium sulfate.

Concentration of the ethyl acetate solution gave a yellow solid which was triturated with 50 ml of ether and filtered. In this way, a yield of 5.0 g (93%) of the para-nitrophenyl ester was obtained, which melted at 153-154° C. and was pure enough to be used in the next step without further purification.

Step B: Preparation of 4-acetamidophenyl ester

A solution of 1.0 g of para-nitro-phenyl-1'-(para-chloro-benzoyl)-2'-methyl-5'-methoxy-indol-3'-yl acetate in 100 ml of ethyl acetate was shaken under hydrogen at 2 atmospheres of pressure with 500 mg -5% palladiumized bone charcoal catalyst for 5 hours.

The catalyst was removed by filtration through a pad of Celite, and the ethyl acetate solution was concentrated under reduced pressure. 2 ml of acetic anhydride was added to the remaining oil, and after the exothermic reaction had subsided, chloroform was added until all of the contents of the flask were in solution.

The chloroform was removed under reduced pressure and the residual oil was triturated with 50 ml of ether and quickly filtered. Addition of a small amount of petroleum ether (40-60°) produced crystallization of 4-acetamidophenyl-1'-(para-chlorobenzoyl-2'-methyl-5'-methoxy-indol-3<1->yl- acetate as cream-colored needles. Yield = 0.75 g. This was identical to the sample prepared via the acid chloride in Example 1.

E xample 3 Preparation of 4-acetamidophenyl-1'-(para-chloro-benzoyl-2'-methyl-5'-methoxy-indol-3'-yl-acetate of the formula

A mixture of 3.58 g of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and 1.9 g of para-toluenesulfonyl chloride in 20 ml of dichloromethane was stirred and slowly treated with 1, 01 g of triethylamine in 5 ml of dichloromethane and stirred for a further 10 minutes.

A solution of 1.51 g of 4-acetamidophenol and 1.0 g of pyridine in 30 ml of dichloromethane was added, and the mixture was refluxed for 10 minutes and then cooled to room temperature. The dichloromethane solution was washed first with 2M hydrochloric acid, then with 20% sodium carbonate solution and dried over magnesium sulfate.

Concentration of the dichloromethane solution under reduced pressure gave a yellow solid which was crystallized from aqueous acetone as cream needles. Yield = 3.51 g, 71%. This sample was identical to the sample that was prepared via the acid chloride in example 1.

Example 4 Preparation of 4-acetamidophenyl-1'-(para-chloro-benzoyl)-2'-methyl-5'-methoxy-indol-3-yl-acetate Step A: Preparation of the symmetrical anhydride of 1-( para-chlorobenzoyl) — 2-methyl-5-methoxy-indol-3-yl-acetic acid 16.5 g (0.08 mol) N,N<1->dicyclohexylcarbodiimide was added to a solution of 28.62 g (0.08 mol) of 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid in 150 ml of tetrahydrofuran. The resulting mixture was allowed to stand at room temperature overnight. It was then filtered, the filtrate was evaporated to dryness and the residue taken up in ethanol and recrystallized to give 6.5 g of the desired 2-acetoxy-benzoic anhydride.

Step B: Preparation of 4-acetamidophenyl-1'-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3-yl-acetate 1.51 g (0.01 mol) 4-acetamidophenol was dissolved in 20 ml of IM sodium hydroxide. A solution of 6.98 g (0.01 mol) of the anhydride obtained in step A in 20 ml of benzene was added to this solution. The biphasic mixture was shaken vigorously for 30 minutes and then filtered to give the desired product.

Evaluation of the stability of the compound

To assess the stability of 4-acetamidophenyl-1'-(para-chlorobenzoyl)-2<1->methyl-5<1->methoxy-indol-3<1->yl-acetate in the gastro-intestinal tract, simulated gastric and intestinal fluids prepared as follows:

Preparation of simulated gastric fluid

2 g of sodium chloride was mixed with 7.0 ml of concentrated hydrochloric acid and the mixture was then made up to a volume of 1000 ml of distilled water, forming a simulated gastric fluid with a pH of approx. 1.2.

Preparation of simulated intestinal fluid

6.8 g of monobasic potassium phosphate was dissolved in 250 ml of water, and to this was added a mixture of 190 ml of 0.2 M sodium hydroxide solution and 400 ml of water. The pH of the resulting solution was adjusted to 7.5 with 0.2M sodium hydroxide, and the solution was then diluted to 1000 ml with distilled water, forming a simulated intestinal fluid.

Assessment method

100 mg of 4-acetamidophenyl-1'-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3'-yl acetate was suspended in 5 ml of each of the simulated liquids and shaken for 16 hours at 37°C. The mixture was then allowed to stand and the supernatant was placed on silica TLC plates and developed in 90:10 chloroform/methanol. The mixture

was then extracted with 5 ml of chloroform, and the chloroform-containing mixture was filtered through phase-separating paper,

which only allowed the chloroform solution to pass through. The chloroform solution was also placed on silica TLC plates and developed in 90:10 chloroform/methanol.

The results of this procedure, performed for both simulated stomach and simulated intestinal fluid, showed that negligible degradation occurred in both of these fluids. This indicates that 4-acetamidophenyl-11-(para-chlorobenzoyl)-21-methyl-51-methoxy-indol-3'-yl-acetate will be predominantly, or completely free of stomach upset effect when administered orally.

Assessment of gastric tolerance in rats

Stomach tolerance is the main problem encountered with oral administration of known compounds exhibiting analgesic and anti-inflammatory properties, and the therapeutic index (or safety factor) of such compounds is best demonstrated via the gastric tolerance test in rats.

Accordingly, 4-acetamidophenyl-11-(para-chlorobenzoyl)-2'-methyl-5<1->methoxy-indol-3'-yl-acetate was tested for its acute (4 h) gastric tolerance in rats, following oral administration at two dose concentrations, as it was simultaneously compared with a smaller (but equimolar) amount of the most stomach-irritating and of the two anti-inflammatory drugs which are the two parts that make up the analgesic ester being tested.

In order for the experiments to be carried out under conditions that allowed the differences between the new compound and the known compounds used as a comparison standard to be reproducible and unequivocal, the effect of the reference drug in relation to the dose was also investigated in a series of preliminary experiments, which enabled that suitable doses could be determined before the testing of the compounds began.

Test procedure

Groups of 10 rats (Charles River, 180 - 250 g body weight) were fasted overnight and until sacrifice (approximately 21 hours). The drugs were administered orally as a suspension in 1% gum tragacanth. 4 hours after administration, animals were euthanized by bleeding from the neck, then their abdomens were removed, opened, gently washed with saline, and stretched flat for examination.

On examination, superficial mucosal bleeding was classified according to increasing severity according to their size and number as: dots, spots or large zones. Bleeding fusions penetrating the mucosa (submucosal lesions) were considered ulcers, and also graded in severity according to their size and number, spots (1-2 mm in size) and channels (with a longitudinal dimension). Perforations were not usually observed.

The animals were then ranked according to the most serious symptoms each one showed, and where the wounds (submucosal lesions) were as a rule considered more serious than mucosal bleeding (superficial). The control animals uniformly showed a certain degree of "hyperaemia" which corresponds to a congestive state of the blood vessels at the point of sacrifice and not a persistent functional hyperaemia. It was therefore not considered a sign of stomach irritation.

Statistical comparisons between the specified groups were performed by the Mann and Whitney U test, based on the graded severity of the observations. The results, listed as the percentage of rats exhibiting either superficial mucosal hemorrhage alone, or ulceration (submucosal lesion) generally associated with mucosal hemorrhage, are given in simplified form in the following Table I:

The results listed in Table I show that compared to 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-ind61-3-yl-acetic acid, Compound IV, i.e. 4-acetamidophenyl-1<1->

(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3'-yl-acetate,

a clearly improved tolerance of the stomach. A comparison of the stomach effect of 5 mg/kg 1-(para-chlorobenzoyl-2-methyl-5-methoxy-indol-3-yl-acetic acid) shows that the stomach tolerance on a molar basis is more than doubled.

Assessment of desired pharmacological effects

Three tests are commonly used to determine the efficacy of analgesic and anti-inflammatory drugs, and all three tests were used to evaluate Compound IV and 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indole-3- yl-acetic acid (Compound B). In all these tests which are set out below, the groups of rats used consisted of 10 rats each.

1) Provoked arthritis test (oblique view) on rats

Provoked disease was induced within several groups of rats.

When provoked arthritis was induced in rats (normally with a mycobacterial provoking agent), the provoking agent induces inflamed lesions in the area of the body remote from the injection site, after a period of 10-15 days, although the inflammation begins to subside by the 30th day.

One group remained untreated as a control group, other groups were treated by oral administration, S once per day for 18 days with different amounts of the parent compound, namely Compound B, used as a comparison standard, and of the compound to be tested, Compound IV. Connect-. then in each case it was administered to the rat in solution in 1 part polyoxyethylene oleic acid glyceride, filled up to 5 parts with 1% aqueous gum tragacanth, to which 1% ethanol was also added when this was necessary to effect dissolution. The amount of this solvent carrier administered was generally 5 ml per kg of the rat. The same vehicle (but without active ingredient) was also administered to the control group of rats.

The treatment was continued for 18 days, during which time the rats were tested at intervals using the usual oblique vision test. Briefly stated, this assesses the grasping function of the rat by placing it on a steel wire sieve which is turned up over an angle of 90° from the horizontal to the vertical, and further if necessary. The angle at which the rat loses its grip and falls from view is recorded. The greater the angle of inclination at which the rat falls off the sight, the more powerful its grip, and the less its grasping function is affected by the arthritis-like provoked disease, or in other words, the better relief of pain exhibited by the tested compound.

A group of rats serving as a control group received only the vehicle in the appropriate amount per day from day 0-18 (17 doses). Another group of rats received 1 mg/kg per day of Compound B for 18 days. A third group received 2 mg/kg per day of Compound B. A fourth group of rats received 2.7 mg/kg of Compound IV. The molar equivalent of a dose concentration of 2.7 mg/kg of Compound IV is a dose concentration of approximately 2 mg/kg of Compound B, and to the extent that the former proves to be superior to the latter, the application of this thereby established.

The results obtained are shown graphically in fig. 1 the following drawings, where the line of gradual decline of grasping function expressed in the angle of inclination of the sight against time is shown for each of the different compounds tested and for the controls. It should be noted that the observations were continued after the 18th day.

From fig. 1 it can be concluded that Compound IV, when tested at 2.7 mg/kg, is approximately as active as half the molar amount of Compound B in the grip function test. No deaths occurred in any of the treated rats.

2) Provoked arthritis test (body weight) in rats

The experimental determination of unknown side effects is much more difficult than the determination of stomach irritation, but their presence can be determined by a reduction in body weight. To ensure that the results of the slanted sight test were not unreliable due to other side effects of the compound being tested, the body weight of the rats suffering from the induced disease was also determined each day, and the mean of each group of rats is indicated in fig. 2 in the accompanying drawings.

From fig. 2 it can be concluded that the systemic effect on body weight of Compound IV is essentially equal to half of the molar amount (or negligible less) of Compound B.

3) Antipyretic test against yeast-induced hyperthermia

in rats

Using the conventional procedure, groups of rats were injected with yeast. Immediately after the injection, different doses of Compound IV and Compound B were administered orally to different groups of rats, except for the control group which received only the vehicle.

The body temperature of all rats was carefully monitored for a period of 8 hours after oral administration of the test compound. The results are shown in fig. 3 in the accompanying drawings. Compound IV was tested at 5.5 mg/kg.

Compound B was tested at its molar amount of 4 mg/kg and half molar amount of 2 mg/kg. As can be seen, Connection IV

as active as the quarter molar amount of Compound B.

4) Randall-Selitto test in rats

Using conventional methods, the various compounds to be tested, as identified hereinafter, were administered orally to the rats in each group (except a control group which received only the vehicle) and 30 minutes later each of the rats was injected into a bacpot of yeast.

To assess the effect of the treatment on pain thresholds, pressure was applied to both the injected hindpaw and the other non-injected hindpaw. The pain threshold was observed as the pressure that the rat does not receive, and the reading for each rat was expressed as the percentage of the pain threshold pressure of the injected hindpaw to the pain threshold pressure of the non-injected hindpaw.

These pain threshold percentages were determined at intervals of 3 hours, 5 hours, 7 hours and 24 hours after the injection. The results of this and the other tests are listed in the following table II:

It appears from Table II that Compound IV was tested at 5.5 mg/kg per day and compared to Compound B at 4 mg/kg per day. From the results thus obtained, it appears that this has the same analgesic effect as Compound B at these molar equivalent doses in this test.

Assessment of therapeutic index

From the relative stomach tolerances shown in Table I and the relative anti-inflammatory activities shown in fig. 1, it is possible to work out this relative separation of anti-inflammatory activity from the stomach toxicity of Compound IV compared to Compound B as shown in the following table

III.

It appears that Compound IV is at least as good as Compound B in terms of its analgesic effect (compared on a molar basis), but only a quarter as active on a molar basis as an antipyretic and only half as active on a molar basis as an anti-inflammatory. However, the important point is its lack of stomach toxicity. Although its analgesic effect is as good as Compound B, Compound IV has a gastric tolerance greater than double the molar amount of Compound B.

Comparison between it. therapeutic activity of paracetamol-indomethacinate (compound IV) and indomethacin

Compound IV and indomethacin were tested in the acetylcholine-induced writhing test in mice. The test compounds were administered orally to female Charles River mice (body weight 23 to 29 g) at intervals of 1, 3, and 5 hours before an intraperitoneal challenge with 3.5 mg/kg acetylcholine chloride. The incidence of writhing was observed 25 to 120 seconds after challenge and the percent protection against writhing was determined compared to controls. ED5Q values (and 0.95 reference limits) were calculated by the Litchfield and Wilcoxon method and were expressed in micromoles/kg. Each EDj-Q value was derived from results using

50 to 60 animals (4 to 5 dose levels). The ED5Q values for inhibition of torsion are given in the following table:

From the results stated above, it appears that:

(1) Indomethacin exhibits its maximal analgesic activity at 1 hour, and its ED^Q value remains essentially unchanged for 5 hours after administration, (2) compound IV exhibits a slower onset of action and is maximally effective at 5 hours after administration, at which time - point the analgesic potency is equal to or slightly better than that of indomethacin.

Further tests on rats have confirmed the very low propensity of compound IV to cause gastric irritation in rats, being found to be completely harmless at doses up to 20.5 mg/kg. In contrast, indomethacin produced gastric lesions in 50 and 90% of the tested rats at dose levels of 5 and 10 mg/kg.

Based on the overall data for compound IV, it can be determined that it exhibits a significant anti-inflammatory, analgesic and antipyretic effect in the dose range of from 1.37 to 10.96 mg/kg (2.8 to 22.4 mol/kg) with a very low propensity to cause gastric irritation in rats. These data, expressed in relative potency compared to indomethacin based on equally effective molar dose levels, can be summarized as follows:

Claims (1)

Analogous process for the preparation of therapeutically active 4-acetamidophenyl-1-(para-chlorobenzoyl)-2'-methyl-5'-methoxy-indol-3<1->yl-acetate with the formula: and acid addition salts thereof, characterized in that a) 4-acetamidophenol is reacted with 1-(para-chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetyl chloride, or b) 4-acetamidophenol is reacted with the mixed anhydride of 1-(para -chlorobenzoyl)-2-methyl-5-methoxy-indol-3-yl-acetic acid and para-toluenesulfonyl chloride, c) para-aminophenyl-1'-(para-chlorobenzoyl)-2<1->methyl-5'-methoxy -indol-3'-yl acetate is reacted with acetic anhydride, or d) 4-acetamidophenol is reacted with the symmetrical anhydride of 1-(para-chlorobenzoyl)-2-methyl-5-methoxyindol-3-yl-acetic acid.