NO148780B - ANALOGY PROCEDURE FOR THE PREPARATION OF NEW PHARMACOLOGICAL ACTIVE 2-ACETOXY-BENZOIC ACID ESTERS OF 4-ALKYL-3,5-DIOXO-PYRAZOLIDE INGREDIATES - Google Patents

Description

The present invention relates to an analog method

in the preparation of new pharmacologically active 2-acetoxy-benzoic acid

esters of 4-alkyl-3,5-dioxo-pyrazolidine derivs.

Analgesics are widely used today, and many associated

Sees of various types have been proposed and marketed for use as such. For the relief of chronic conditions such as rheumatism and arthritis, it is normally desirable to

turn an agent that exhibits an anti-inflammatory effect in addition to the analgesic activity. For the relief of chronic arthritis, rheumatism and similar conditions, it is thus currently normal to use one of the known anti-inflammatory analgesics.

Some of the best-known, most effective and widely re-

of the existing anti-inflammatory analgesics can be considered as 4-alkyl-3,5-dioxo-pyrazolidine derivatives of gene-

rell formula:

but which can also be assumed to exist in the following alternative tautomeric forms:

in which general formulas I, Ia and Ib either X is phenyl and Y is phenyl or para-hydroxyphenyl, or where X and Y together with the

intermediate nitrogen atoms to which they are attached denote a 3-dimethylamino-7-methyl-[1,2,4]-benzotriazine ring system fused with the pyrazolidine ring, and Alk denotes either an n-butyl or an n-propyl group.

It should be noted that any reference to compounds of general formula I includes the tautomeric forms Ia and Ib.

The following compounds are typical of the known anti-inflammatory, analgesic pyrazolidine derivatives of general formula I: 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine,

1-phenyl-2-(p-hydroxyphenyl)-3,5-dioxo-4-n-butyl-pyrazolidine, and

5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazolo-[1,2-a][1,2,4]-benzotriazine-1,3(2H)-dione.

Even better known as an analgesic is the compound

of structural formula:

namely 2-acetoxy-benzoic acid.

Unfortunately, the desired anti-inflammatory and analgesic properties of the pyrazolidine derivatives of general formula I and the desired analgesic properties of 2-acetoxy-benzoic acid of structural formula II suffer from the disadvantage that they give rise to disturbances in the gastrointestinal tract. Furthermore, when both are administered simultaneously to the patient, they are found to be mutually antagonistic, each preventing assimilation of the other.

It is therefore particularly surprising that it has been found that it is not only possible to form esters between the carboxyl group present in 2-acetoxy-benzoic acid of structural formula II and the hydroxy group(s) present in the pyrazolidine derivatives of general formula I (or at least in the tautomeric forms la and Ib) but also that the resulting esters, even if these are formed between stomach-disturbing and mutually antagonistic groups, are nevertheless predominantly or practically completely free of unwanted side effects on the gastrointestinal tract, while at the same time being effective and excellent anti-inflammatory analgesics, which are satisfactorily assimilated by the patient.

The invention thus relates to an analogous process for the production of new, therapeutically active 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives with the general formula:

wherein either one of X or Y is phenyl and the other is phenyl, para-hydroxyphenyl, or a group of the formula: or wherein X and Y together with the nitrogen atoms to which they are attached represent a 3-dimethylamino-7-methyl-[ 1,2,4]-benzotriazine ring system, and "Alk" denotes an n-propyl or n-butyl group, as well as their acid addition salts with pharmacologically acceptable inorganic and organic acids. 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives produced according to the present invention are, for example: 1,2-diphenyl-3-oxo-4-n-butyl-5-(2 <1->acetoxy-benzoyloxy)-pyrazolid-4-ene of structural formula: 1-phenyl-2-(para-hydroxy-phenyl)-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy )-pyrazolid-4-ene of structural formula: or its 3-(2<1->acetoxy-benzoyloxy)-5-oxo isomer, namely 1-phenyl-2-(para-hydroxy-phenyl)-3-(2'-acetoxy-benzoyloxy)-4-n -butyl- 5-oxo-pyrazolid-3-ene which, however, can alternatively and more appropriately be called 1-(para-hydroxy-phenyl)-2-phenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy -benzoyloxy)-pyrazolid-4-ene of structural formula: 1-phenyl-2-(para-[2'-acetoxy-benzoyloxy]-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine of structural formula: or any of its tautomeric 5-hydroxy-4-en-3-one and 3-hydroxy-4-en-5-one forms, which may be considered to be encompassed by the name and structural formula C, 1-phenyl-2-(para-[2'-acetoxy-benzoyloxy]-phenyl)-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene of structural formula: or its 3-(2<1->acetoxy-benzoyloxy)-5-oxo isomer, namely 1-phenyl-2-(para-[2'-acetoxy-benzoyloxy]-phenyl)-3-(2 '-acetoxy-benzoyloxy)-4-n-butyl-5-oxo-pyrazolid-3-ene, which can alternatively and more appropriately be called 1-(para-[2'-acetoxy-benzoyloxy]-phenyl)-2- phenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene with structural formula: -1-(2'-acetoxy-benzoyloxy)-2-n -propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-en[1,2-a] [1,2,4]benzotriazine with structural formula: and acid addition salts thereof with pharmacologically acceptable acids or its l-oxo-3-hydroxy isomer, namely l-oxo-2-n-propyl-3-(2<1->acetoxy-benzoyloxy)-5-dimethylamino-9-methyl -pyrazolid-3-eno[1,2,4]benzotriazine of structural formula:

and acid addition salts thereof formed with pharmacologically acceptable acids.

It has further been found that these 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives produced according to the invention can be produced very easily and usually in relatively good yields by various preparative methods.

The analog method according to the invention is characterized by the fact that a compound of the general formula:

wherein either X is phenyl and Y is phenyl or para-hydroxyphenyl, or wherein X and Y together with the nitrogen atoms to which they are attached represent a 3-dimethylamino-7-methyl-[1,2,4]-benzotriazine ring system fused with the pyrazolidine ring, and "Alk" is as defined above, is reacted with either (a) 2-acetoxy-benzoic acid chloride in the presence of a base to form the desired compound of formula X or an acid addition salt thereof, or

(b) a mixed anhydride of general formula:

wherein "Ayl" denotes an alkyl group of 1-20 carbon atoms,

an aryl group of 6-20 carbon atoms, an alkaryl group of 7-20 carbon atoms or an aralkyl group of 7-20 carbon atoms; m is 0 or 1, and Q denotes a carbonate pm C or when m is 0, a sulfinyl group S=0, forming the desired compound of formula X or an acid addition salt thereof, whereupon the desired compound is isolated, if necessary, by washing both with diluted acid, preferably dilute hydrochloric acid, and with dilute alkali, preferably dilute aqueous sodium carbonate or dilute aqueous sodium hydroxide, or (c) a solution in a water-immiscible solvent of 2-acetoxy-benzoic anhydride in the presence of a strong base such that the reaction mixture forms a two-phase -reaction system which is stirred while forming the desired compound of formula X.

The reaction between the pyrazolidine derivative of general formula I and the 2-acetoxybenzoic acid chloride is preferably carried out in an anhydrous inert solvent.

Since the reaction is relatively vigorous, it can be carried out without heating at ambient temperature, or it may even be necessary or at least desirable when the reaction is very vigorous, which it sometimes is, to use cooling.

The inert solvent can advantageously be an organic solvent such as in particular (a) substituted aromatic hydrocarbons including halogenated hydrocarbons, e.g. toluene, (b) aliphatic hydrocarbons including halogenated hydrocarbons, e.g. chloroform, trichloromethane and dichloromethane, and (c) aliphatic ethers, e.g. diethyl ether. The

preferred solvents are chloroform and dichloromethane.

The reaction must be carried out in the presence of a base, which may conveniently be a tertiary amine. The base that is frequently preferred over all others is pyridine.

The first method according to the invention can also advantageously include a preliminary step, namely production of the 2-acetoxy-benzoic acid chloride starting material with structural formula III, by reacting 2-acetoxy-benzoic acid with structural formula II in solution and/or suspension in an anhydrous inert solution ning agent, with thionyl chloride.

The reaction with thionyl chloride can be carried out at any suitable temperature between ambient temperature and the solvent reflux temperature, and usually within the range from room temperature up to 65°C.

The solvent can advantageously be the same as that used in the above-described subsequent step in the preparation, as the obtained 2-acetoxy-benzoic acid chloride does not need to be isolated from the reaction product, and can thus, for example, be a relatively low-boiling organic solvent such as (a) a substituted aromatic hydrocarbon including halogenated hydrocarbons, e.g. toluene, (b) an aliphatic hydrocarbon, including combined aliphatic hydrocarbons, e.g. chloroform, trichloromethane or dichloromethane, or (c) an aliphatic ether, e.g. diethyl ether. The specifically preferred solvents are chloroform and dichloromethane.

When the solvent used is anhydrous dichloromethane, the reaction can conveniently be carried out under reflux at a temperature of 40° C. The amount of the reactants will preferably be approx. 1 mol of thionyl chloride per moles of 2-acetoxy-benzoic acid.

It has been found advantageous to carry out the reaction between 2-acetoxy-benzoic acid and the thionyl chloride in the presence of a catalytic amount of N,N-dimethylformamide.

The alternative reaction b) between the mixed anhydride of general formula IV and the 4-alkyl-3,5-dioxo-pyrazolidine derivative of general formula I can and preferably should be carried out in an anhydrous inert and usually organic solvent such as e.g. may be a substituted aromatic solvent, e.g. toluene, or any of the commonly used halogenated aromatic hydrocarbon solvents or halogenated aliphatic hydrocarbon solvents, for example chloroform (trichloromethane).

For reasons that will become apparent hereinafter, it should be noted that this reaction can also be carried out without harm in the presence of a base, such as a tertiary amine such as triethylamine or pyridine.

The mixed anhydrides of general formula IV can be advantageously prepared in a preliminary step in this second process, in which a reactive organic chloride of general formula:

where "Ayl", m and Q have the meanings given above, is reacted with 2-acetoxy-benzoic acid of structural formula II or a salt thereof in the presence of a base, forming the desired corresponding mixed anhydride.

The reactive organic chloride of general formula V can e.g. be: (a) an alkyl or aralkyl or aryl or alkaryl chloroformate of general formula: or (b) a sterically hindered alkyl or aralkyl, or aryl or alkaryl chloride of general formula: or (c) an alkyl or aralkyl or aryl or alkaryl sulfonyl chloride of general formula:

wherein "Ayl" denotes an alkyl, aralkyl, aryl or alkaryl group having the previously indicated carbon atoms therein.

The reaction (sometimes called "first step reaction") between 2-acetoxy-benzoic acid of structural formula II and the reactive organic chloride of general formula V (or specifically Va, Vb or Vc) is conveniently carried out in an anhydrous, usually inert organic solvent, like for example. may be a substituted aromatic solvent, e.g. toluene, or any suitably used halogenated aromatic hydrocarbon solvent or halogenated aliphatic hydrocarbon solvent, for example chloroform (trichloromethane).

This first step reaction must be carried out in the presence of a base, which will advantageously be a tertiary amine, and preferably either triethylamine or pyridine.

As previously indicated, the subsequent reaction between the mixed anhydride of formula IV and the 4-alkyl-3,5-dioxo-pyrazolidine derivative of formula I (sometimes hereinafter referred to as the "second step reaction") is carried out preferably in the same type of anhydrous, inert organic solvent which in the first stage reaction is carried out, and the second stage reaction is further unaffected by the presence of a base. It is therefore possible and also desirable to carry out both the first stage reaction and the second stage reaction in the same anhydrous inert organic solvent, without isolation of the first stage reaction product, and usually in the same reaction vessel.

Reaction c) between 4-alkyl-3,5-dioxo-pyrazolidine derivatives of general formula I and 2-acetoxy-benzoic anhydride gives 2-acetoxy-benzoic acid as a by-product and must

therefore performed in the presence of a strong base, such as e.g. can be an organic base such as triethylamine, but will preferably be aqueous sodium hydroxide. As the anhydride is somewhat unstable in the presence of water, it must be brought to the reaction in solution in an anhydrous inert water-immiscible organic solvent, suitably benzene, toluene, chloroform, dichloromethane or an aliphatic ether such as diethyl ether, so that the reaction can then be carried out at strong stirring in a two-phase reaction system and thus reduce any breakdown of the anhydride before it has taken part in the reaction. This reaction can conveniently be carried out at ambient or near-ambient temperatures that do not exceed 30° C.

2-acetoxy-benzoic anhydride has the structural formula:

This third process may further advantageously include a further preliminary step, namely the production of 2-acetoxy-benzoic anhydride of structural formula VI by reacting 2-acetoxy-benzoic acid of structural formula II with a carbodiimide in solution in an anhydrous, non-hydroxyl or otherwise inert organic solvent at ambient or near ambient temperature, usually not above 30°C.

In principle, any carbodiimide can be used, but in practice the carbodiimide used must of course be stable, and consequently it is recommended that the carbodiimide be used is an N,N'-di[alk/ar]yl-carbodiimide of the general formula:

wherein Z' and Z" each represent an alkyl or cycloalkyl or alkaryl or aryl or aralkyl group containing from 6 to 20 carbon atoms. Furthermore, it must be added that the only carbodiimide currently available at an economical price is N,N'-dicyclohexyl -carbodiimide, which is therefore preferred for use according to the present invention.

The solvent used in the method will advantageously be tetrahydrofuran.

The 2-acetoxy-benzoic acid esters of the 4-alkyl-3,5-dioxo-pyrazolidine derivatives prepared according to the invention by any of the methods described herein exhibit excellent anti-inflammatory and analgesic activity when tested by generally accepted tests for anti-inflammatory and analgesic drugs, and preliminary studies show that when the preferred compounds are administered orally, they will be essentially free of gastrointestinal side effects.

For use in human and veterinary medicine as anti-inflammatory analgesics, the 2-acetoxy-benzoic acid esters of the 4-alkyl-3,5-dioxo-pyrazolidine derivatives of general formula I, and specifically the compounds of formulas A, B and B<1>, C , D and D', as well as E and E<1>, however, are formulated in suitable pharmaceutical compositions in conjunction with suitable pharmaceutical carriers.

The term "pharmaceutical" is used herein to exclude any possibility that the nature of the carrier, considered of course in relation to the mode of administration contemplated, could be harmful rather than beneficial. The selection of a suitable carrier for any chosen mode of administration is believed to be known to those skilled in the art.

Although the preparations are primarily intended for administration via the digestive tract, including rectally,

they can also be administered parenterally. However, the preferred route of administration is oral, including perlingual. In terms of these different routes of administration, the "pharmaceutical carrier" is preferably:

(a) the ingestible excipient of a tablet, coated tablet, sublingual tablet or pill, the ingestible container of a capsule, the ingestible pulverulent solid carrier in a powder, or the ingestible liquid medium in a syrup, solution, suspension or elixir , (b) the solid or liquid medium of a paste, lotion,

ointment,

(c) a sterile injectable liquid solution or suspension medium, or (d) a base material with a low melting point capable of releasing the active ingredient to perform its pharmacological function, which base material forms a suppository when suitably shaped.

Although the administration methods stated above represent those that are likely to be used, they do not exclude other possibilities.

The anti-inflammatory analgesics produced according to the invention, and in particular the 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives as previously identified and with structural formula A, B and B', C, D and D<1> as well such as E and E', will most frequently be administered in the form of tablets, capsules and other solid dosage forms.

Although the doses of these anti-inflammatory analgesics may depend to a certain extent on the particular compound used, the method of administration and of course the condition of the person to be treated, it can be stated as a general indication that the applicable dose will usually be from 400 mg upwards to 5 g (in divided doses) of the active analgesic per day for an adult patient, as a unit dose usually contains from 150 mg to 400 mg of active ingredient.

The following examples illustrate preparation

of certain 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivs.

Group I Manufacture by "first" process via 2-acetoxy-benzoic acid chloride

Example 1 Preparation of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene (A)

Step A Preparation of 2-acetoxy-benzoic acid chloride (III)

100 g of 2-acetoxy-benzoic acid was suspended in 27 ml of chloroform containing 1 ml of N,N-dimethylformamide. 4 2 ml (or 68.9 grams) of thionyl chloride was added and the mixture was stirred at room temperature for 2 hours. Then a further 5 ml of thionyl chloride was added and the mixture was heated to 35°C for 20 minutes. At this point, the reaction mixture had become clear. The solvent was removed on a rotary evaporator and the residue was pumped out under high vacuum over 2 hours.

The acid chloride thus prepared, namely 2-acetoxy-benzoic acid chloride of formula III, was obtained in this way as an oil, in a yield of approx. 100 grams and which was used without further purification in the subsequent step B.

However, when the product was taken up in a mixture (20:1) of petroleum ether (40 - 60° C) and chloroform, the desired 2-acetoxy-benzoic acid chloride can be recovered in crystalline form. Yield 80 grams, m.p. = 48 - 50° C.

Step B Preparation of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene (A) 20 grams of 2-acetoxy-benzoic acid chloride obtained as described in step A, was dissolved in 300 ml of ether, and to this solution was then added 9.0 g of pyridine. Then 30.8 g of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine was added and the reaction mixture was vigorously stirred for 20 minutes and then boiled under reflux for 1 hour.

The mixture was filtered to remove the pyridine hydrochloride, after which the ether was removed and the residue was dissolved in 100 ml of dichloromethane. This solution was washed with dilute hydrochloric acid, then with 5% sodium carbonate solution. The dichloromethane solution was dried and evaporated to dryness under reduced pressure.

TLC (chloroform) indicated the presence of unreacted pyrazolidine derivative. This could be completely removed by dissolving the product in 75 ml of ether and vigorous shaking with 100 ml of 2.5 M sodium hydroxide solution for 20 minutes. An additional 100 mL of ether ole added to the mixture to extract the product. The ether layer was dried and evaporated to give a practically colorless oil which solidified rapidly on standing. TLC (chloroform) indicated that the product was pure.

A yield of 30 g of the desired 1,2-diphenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene was thus obtained. Temp. = 96 - 99° C.

Example 2 Preparation of 1-(2'-acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-eno-[1,2-a] [1, 2, 4]- benzotriazine ( E)

Step A Preparation of 2-acetoxybenzoic acid chloride

This was similar to step A in example 1.

Step B Preparation of 1-(2'-acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-eno-[1,2-a][1,2 , 4]- benzotriazine ( E)

A mixture of 9.0 g of 5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazolo-[1,2-a] [1,2,4]-benzotriazine-1,3(2H)-dione and 3.0 g of pyridine in 50 ml of dichloromethane was added to a stirred solution in 100 ml of dichloromethane of 60 g of 2-acetoxy-benzoic acid chloride, prepared as indicated in step A.

This reaction mixture was refluxed for 10 minutes, cooled and washed with 100 ml of 2M hydrochloric acid. The dichloromethane solution was then concentrated under reduced pressure, and the residual oil was dissolved in 200 ml of ether containing 5% ethyl acetate and washed twice with 100 ml of 2M aqueous sodium hydroxide solution, and then with 100 ml of water and dried over magnesium sulfate. Concentration of the ether solution under reduced pressure gave a yellow oil.

Addition of an ether/petroleum ether mixture (60 - 80°C) caused crystallization of the desired 1-(2<1->acetoxy-benzoyl-oxy)-2-n-propyl-3-oxo-5-dimethylamino-9-methyl -pyrazolid-2-eno-[1,2-a][1,2,4]-benzotriazine (E) in a yield of 6.4 g of cream-colored needles. Sm.p. 138 - 140° C.

Group II Manufacture by "other" process via the mixed

an anhydride of general formula IV

Example 3 Preparation of 1-phenyl-2-(para-[2'-acetoxy-benzoyl-oxy)-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine (C) Step A Preparation of the mixed anhydride of 2-acetoxy-benzoic acid and formic acid (IV where Ayl' = Et, m = 1, Q=C)

3.6 g (0.02 mol) of 2-acetoxy-benzoic acid and 2.18 g

(0.02 mol) of ethyl chloroformate was stirred together in 50 ml of dichloromethane at 5° C. A solution of 2.02 g (0.02 mol) of triethylamine in 25 ml of dichloromethane was added dropwise while maintaining the temperature at less than 10 °C, and the mixture was then stirred at room temperature for 30 minutes.

A solution of the desired mixed anhydride of 2-acetoxy-benzoic acid and formic acid of general formula IV was thus obtained where Ayl denotes an ethyl group, m is equal to 1 and Q denotes a carbon atom. This solution was used without isolation of the mixed anhydride in subsequent step B.

Step B Preparation of l-phenyl-2-(para-[2'-acetoxy-benzoyl-oxy]-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine (C)

To the solution obtained from step A, a solution of 3.24 g (0.01 mol) 1-phenyl-2-(para-hydroxyphenyl)-3,5-dioxo-4-n-butyl-pyrazolidine was slowly added and 2.02 g (0.02 mol) of triethylamine in 25 ml of dichloromethane. This mixture was then stirred for 30 minutes at 40°C, cooled and washed with 50 ml portions of first 2M hydrochloric acid and then 2M sodium hydroxide. The resulting solution was dried by passing it over anhydrous magnesium sulfate and the solvent was then evaporated to give the desired monoester, 1-phenyl-2-(para-[2<1->acetoxy-benzoyloxy]-phenyl)-3, 5-dioxo-4-n-butyl-pyrazolidine, as a white crystalline solid. Sm.p. = 156 - 157° C.

Group III Production by the "third" process via 2-acetoxy-benzoic anhydride

Example 4 Preparation of 1-phenyl-2-(para-[2'-acetoxy-benzoyl-oxy]-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine (C)

Step A Preparation of 2-acetoxy-benzoic anhydride (VI)

16.5 g (0.08 mol) of N,N<1->dicyclohexylcarbodiimide was added to a solution of 14.4 g (0.08 mol) of 2-acetoxybenzoic acid in 150 ml of tetrahydrofuran. The resulting mixture was allowed to stand at room temperature overnight. It was then filtered, the filtrate evaporated to dryness and the residue taken up in ethanol and recrystallized to give 6.5 g of the desired 2-acetoxy-benzoic anhydride. Sm.p. = 83 - 85° C.

Step B Preparation of 1-phenyl-2-(para-[2'-acetoxy-benzoyl-oxy]-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine (C)

3.24 g (0.01 mol) of 1-phenyl-2-(para-hydroxy-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine was dissolved in 20 ml of 1M sodium hydroxide. To this solution was then added a solution of 6.84 g (0.02 mol) of 2-acetoxybenzoic anhydride, obtained as described in step A, in 20 ml of benzene. The biphasic mixture was vigorously shaken for 30 minutes and then filtered to give the desired product. Sm.p. = 156 - 157° C.

It should be noted that regardless of the method used, the esterification of the 2-acetoxy-benzoic acid with l-phenyl-2-(para-hydroxy-phenyl)-3,5-dioxo-4-n-butyl-pyrazolidine results preferably in the monoester 1-phenyl-2-(para-[2'-acetoxy-benzoyloxy]-phenyl)-3,5-dioxo-4-n-butyl-pyrcizolidin (C) instead of the possible alternative isomeric monoesters (B) or ( B<1>) at least under the reaction conditions used in the previous examples 3 and 4. Small amounts of 1-phenyl-2-(para-hydroxy-phenyl)-3-oxo-4-n-butyl-5- (2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene (B) and of 1-(para-hydroxy-phenyl)-2-phenyl-3-oxo-4-n-butyl-5-(2 However, '-acetoxy-benzoyloxy)-pyrazolid-4-ene (B') is formed during these reactions,

as well as certain amounts of the diesters 1-phenyl-2-(para-[2<1->acetoxy-benzoyloxy]-phenyl)-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)- pyrazolid-4-ene (D) and 1-(para-[2'-acetoxy-benzoyloxy]-phenyl)-2-phenyl-3-(2<1->acetoxy-benzoyloxy)-4-n-butyl-5 -oxo-pyrazolid-4-ene

(D<1>), all of which can be recovered and isolated if the reactions are carried out on a suitable scale and under suitable operating conditions. However, the recovery and isolation of these various alternative compounds B, B', D and D' is not further described, as one cannot strive to prepare each of B, B', D and D<1> when C is readily available prepared and these have shown essentially the same pharmacological activity as its isomers.

It should also be noted that the same comments apply to the esterification of 2-acetoxy-benzoic acid with 5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazolo[1,2-a][1,2,4]-benzotriazine -1,3-(2H)-dione which, probably due to steric hindrance, preferentially results in 1-(2<1->acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9- methyl-pyrazolid-3-eno[1,2-a][1,2,4]-benzotriazine (E) instead of its isomer l-oxo-2-n-propyl-3-(2'-acetoxy-benzoyloxy )-5-dimethylamino-9-methyl-pyrazolid-3-eno-[1,2-a][1,2,4]-benzotriazine (E<1>), at least under the reaction conditions used in the previous example 2. However, small amounts of isomer E<1> are formed during the course of the reaction, and can be recovered and isolated if the reaction is carried out on a suitable scale and with suitable operating conditions, but this is not described as E can be easily prepared and appears to exhibit essentially the same activity.

In the following, some provisions are given which show that certain preferred 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives are free of unwanted side effects, and which show their excellent anti-inflammatory and/or analgesic properties.

Evaluation of stability

In order to assess the stability of the compounds prepared according to the invention in the gastro-intestinal tract, simulated gastric and intestinal fluids were prepared as follows:

Preparation of simulated gastric fluid

2 g of sodium chloride was mixed with 7.0 ml. concentrated hydrochloric acid and the mixture was then filled up to a volume of 1000 ml with distilled water, forming a simulated gastric fluid with a pH value 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 solution was added a mixture of 190 ml of 0.2M

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 the compounds to be tested were suspended in 5 ml of each of the simulated liquids, and shaken for 16 hours at 37° C. Then the mixture was allowed to stand, and the supernatant liquid 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 allowing only the chloroform solution to pass. 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 the simulated gastric and simulated intestinal fluids showed that negligible degradation occurred in both of these fluids. This indicates that all the tested compounds are predominantly or completely free of stomach-disturbing effects when administered per os.

Assessment of gastric tolerance in rats

Gastric tolerance is the main problem encountered in oral administration of the class of compounds that exhibit analgesic and anti-inflammatory activity, and the therapeutic index (or safety factor) of such compounds is best demonstrated via the gastric tolerance test in rats.

Accordingly, one of the preferred and typical compounds of the invention was tested for its acuteness

(4 hours) stomach tolerance in rats after oral administration at two or more dosage concentrations, as it was simultaneously compared with an equimolar mixture of the two anti-inflammatory drugs which are the two parts that make up the analgesic ester to be tested.

In order for the experiments to be carried out under conditions that allowed the differences between the new compounds 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 examined in a series of preliminary experiments, which enabled suitable doses to be determined before testing of the compounds according to the invention began.

Test procedure

Groups of 10 male 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, the animals were euthanized by bleeding from the neck, then their stomachs were removed, opened, gently washed with saline, and stretched flat for examination.

During the examination, superficial mucosal bleeding was classified according to increased 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), 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 the 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: Footnotes: (a) Compound Ph = 1, 2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine. (b) Compound A = 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-, acetoxy-benzoyloxy)-pyrazolid-4-ene.

(c) Aspirin = 2-acetoxy-benzoic acid.

The results listed in Table I show that compared to 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine alone, with a mixture of 1,2-diphenyl-3,5-dioxo-4- n-butyl-pyrazolidine and aspirin, or even with aspirin alone,

is compound A, i.e. 1,2-diphenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolidin-4-ene, remarkably non-irritating to the gastric mucosa, at doses up to 183 mg/kg, and is far better tolerated than aspirin administered at equal dosage concentrations on a weight basis.

Similar tests carried out with other compounds prepared according to the invention confirmed the gastric tolerance already indicated by the above-mentioned stability assessment.

Specifically, for example, acute stomach tolerance tests were carried out comparing the effects of 1-(2<1->acetoxy-benzoyloxy)-2-n-propy1-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2 -eno[1,2-a][1,2,4]-benzotriazine (E) with that of one of its parent compounds, namely 5-(dimethylamino)-9-methyl-2-n-propyl-1H-pyrazolo- [1,2-a] [1,2,4]-benzotriazine-1,3(2H)-dione (hereinafter referred to as "Compound Az"). After preliminary trials to determine the appropriate interval between dosing and killing (finally chosen as 6 hours), the compounds to be tested were ground and suspended in a 1% gum tragacanth vehicle, and a single dose was administered orally to groups of fasted rats in a concentration of 1 mmol/kg and in a volume of 10 ml/kg body weight. A control group received only the vehicle. The results are shown somewhat concentrated in the following table II:

It appears from the results indicated in Table II that the stomach irritation of compound E according to the invention is reduced compared to that of the well-known compound Az.

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 first evaluate Compound A, 1,2-diphenyl-3-oxo-4-n-butyl-5-( 2<1->acetoxy-benzoyloxy)-pyr-zolid-4-ene, in comparison with one of its parent compounds, namely 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine (Compound Ph) and also compound E, 1-(2<1->acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-eno[1,2-a]-[ 1,2,4]-benzotriazine, in comparison with one of its parent compounds, namely 5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazole-[1,2-a][1,2,4] -benzotriazine (Compound Az).

la) Provoked arthritis test (oblique view) in rats

(Compound A)

Provoked disease was induced in several groups of rats.

When provoked arthritis was induced in rats (normally with mycobacterial provocateur), the provocateur induces inflamed lesions in areas of the body distant from the injection site, after a period of 10 - 15 days, although

the inflammation starts to decrease by the 30th day.

One group was untreated as a control group, other groups were treated by oral administration, once per day for 18 days with different amounts of the parent compound, namely 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine used as a comparative standard, and of the compound to be tested, namely 1,2-diphenyl-3 -oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene. The compound was in each case administered to the rat in solution in 1 part polyoxyethylene-oleic acid glyceride filled up to 5 parts 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 by means of 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 the 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 40 mg/kg per day of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine for 18 days, and of this group 1/10 were dead by day 9. A third group received 80 mg/kg per day of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine, but none of these died, showing that the deaths in the second group were pure cases without significance. A fourth group of rats received 61 mg/kg of the compound to be tested, namely 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-

benzoyloxy)-pyrazolid-4-ene. The molar equivalent of a dose concentration of 61 mg/kg of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxybenzoyloxy)-pyrazolid-4-ene is a dose concentration of approximately 40 mg/ kg of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine, and to the extent that the former proves to be superior to the latter, its use is thereby established.

The results obtained are shown graphically in fig. 1 in

the accompanying drawings, in which the line of gradual impairment of grasping function expressed in the oblique angle of sight against time is shown for each of the different compounds tested and for the controls.

From fig. 1, it can be concluded that Compound A, 1,2-diphenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene, when tested by a concentration of 61 mg/kg,

is as active as 1.5 times its molar amount of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine in the grip function test. No deaths occurred among the rats treated with 1,2-diphenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene, but one rat died in that group which was treated with 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine.

lb Provoked arthritis test (body weight) in rats

(Compound A)

The experimental determination of unknown side effects is 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 oblique vision test were not unreliable due to other side effects of the tested compound, the body weight of the rats suffering from the induced disease was also determined each day, and the mean weight of each group of rats is given in fig. 2 in the accompanying drawings.

From fig. 2, it can be concluded that the systemic effect on body weight of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene is still greater than 2 times the molar amount of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine.

lc) Provoked arthritis (acute phase) in rats

(Compound E)

The anti-inflammatory activity was investigated by a subacute test involving 3-dose administration in rats using the following procedure: Groups of non-fasted female (Charles River) rats with a body weight of 170 g, each group consisting of 6 animals, were given a subplantar injection of 0.5 mg (per animal) of Mycobacterium butyr. in 0.1 ml of sterile paraffin oil in the right hind<p>paw (the opposite paw remained untreated) and the edema that subsequently developed was measured at the intervals indicated below by differential plethysmometry of injected and non-injected paws using an Ugo -Basile equipment. The following treatment schedule was used:

Day 0 - at 9.00: First dose of medicine

at 10.00: mycobacterial provocation injection (s.c.) at 15.00: edema volume determination

Day 1 - at 9.00: 2nd dose of medicine

at 14.00: edema volume

Day 2 - at 9.00: 3rd dose of medicine

at 14.00: edema volume

Day 3 - at 9.00: edema volume (no prior medication)

The drugs tested were:

in

Compound E = 1-(21-acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9-pyrazolid-2-eno[1,2-a] [1,2,4]-benzotriazine

Compound Az = 5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazolo[1,2-a][1,2,4]benzotriazine-1,3(2H)-dione These were administered orally to the animals. The administration concentration was 0.1 mmol/kg, 0.2 mmol/kg and 0.4 mmol/kg for Compound Az, and because the latter was found to exhibit a statistically significant effect at 0.2 mmol/kg kg, this concentration was also chosen for Compound E. The control group naturally received no drug.

The results thus obtained are listed in the following table III:

From the results listed in Table III, it appears that Compound Az exhibited sharply dose-dependent effects in the range 0.1 - 0.2 - 0.4 mmol/kg: the total effects (i.e. the sum of the % inhibition of edema observed at day 0 to 3) amounted to 53 - 87 - 160% for the respective dose concentrations. Compound E, with a total effect of 59%, was somewhat less active, being approximately equivalent in effect to 0.5 molar equivalent of Compound Az.

2a) Antipyretic test against yeast-induced hyperemia in rats (Compound A)

Using conventional procedures, groups of rats (each group consisted of 10 animals) were injected with yeast. Immediately after the injection, various doses of Compound A, 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyl-oxy)-pyrazolid-4-ene, as well as a mixture of aspirin with 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine, for comparison, administered orally to different groups of rats, except for the control group that 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 and 4 in the subsequent drawings.

It should be noted that 1,2-diphenyl-3-oxo-4-n-butyl-5-(2<1->acetoxy-benzoyloxy)-pyrazolid-4-ene was tested at 50.5,

61, 122 and 244 mg/kg and compared with aspirin at 61 mg/kg, with 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine at 20 and 40 mg/kg, and with a mixture of aspirin (12 mg/kg) and 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine (20 mg/kg). Compound A thus proved to have a dose-dependent effect in the range of 30 to 120 mg/kg, which is of relatively slow onset compared to the effects of aspirin or of a mixture of aspirin and 1,2-diphenyl-3,5- dioxo-4-n-butyl-pyrazolidine.

2b) Antipyretic test against yeast-induced hyperthermia

in rats (Compound E)

Applying substantially the same procedure as above to groups of male rats with a body weight of 215 - 235 g (each group consisted of 7 rats), Compound E was compared with Compound Az and the results obtained are shown in the following Table IV:

From the results listed in Table IV, it appears that at a dosage concentration of 0.5 mmol/kg of Compound E, this has an antipyretic activity which is comparable/ but insignificantly less than the activity of Compound Az.

3a) Randall-Selitto test in rats (Compound A)

Using conventional methods, the various compounds to be tested, as identified hereinafter, were administered orally to groups of rats (each group consisted of 10 rats), and 30 minutes later, each of the rats was injected into a hind paw with yeast. A control group of rats received only the vehicle.

To assess the effect of the treatment on the pain thresholds, pressure was applied to both the injected hindpaw and the other, non-injected, hindpaw. The pain thresholds were observed as the pressure that the rat did not receive, and the readings for each rat were 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 are listed in the following table V.

It appears from Table V that Compound A, 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene, was tested at 23 mg/kg, 46 mg/kg and 91 mg/kg per day, and was compared with 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine at 30 mg/kg and 60 mg/kg per day. From the results thus obtained, it appears that it has the same analgesic effect as 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine at the lower doses tested, e.g. 4 6 mg/kg of Compound A which is equimolar with 30 mg/kg of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine. At the highest doses administered, namely 91 mg/kg of Compound A, it is less active than the equimolar dose of 60 mg/kg of 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine at 3 -hour reading, suggesting a very low onset of action of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene.

3b) Randall-Selitto test in rats (Compound E)

Applying the same technique as described above to groups of female rats with a body weight of 135 - 155 g (each group consisted of 10 animals), the analgesic properties of Compound E were compared with those of the parent compound Az, and the results obtained are listed in Table VI :

From the results listed in Table VI, it appears that Compound E is insignificantly less active than Compound Az, equivalent to approx. 0.5 times at 0.2 mmol/kg dosage concentration.

Assessment of therapeutic index

From the relative stomach tolerances from Table I and the relative anti-inflammatory activities as shown in fig. 1 it is possible to work out the relative separation of the anti-inflammatory activity from the stomach toxicity for 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4- en (A) compared to 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine (Ph) as shown in the following Table VII:

It thus appears that 1,2-diphenyl-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene (Compound A) is significantly improved compared to 1,2-diphenyl-3 ,5-dioxo-4-n-butyl-pyrazolidine (Compound Ph), not only because it has a somewhat stronger anti-inflammatory and analgesic effect (compared on a molar basis), but also because it has much more reduced stomach toxicity, as these factors together give it an improved therapeutic index which is approx. 6 times better than for 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine.

The other compounds according to the invention have so far not been evaluated in the same detail, but preliminary pharmacological tests indicate that qualitatively similar results are obtained with these other compounds. In particular, it will be observed from the previously stated results that 1-(2<1->acetoxy-benzoyloxy)-2-n-t-propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-eno [1 , 2-a] -

[1,2,4]-benzotriazine (Compound E), although this is somewhat less active, also has a clearly better stomach tolerance compared to 5-dimethylamino-9-methyl-2-n-propyl-lH-pyrazolo[1,2 -a]-[1,2,4]-benzotriazine-1,4(2H)-dione, so that its therapeutic index is thus assumed to be at least as good or better.

Claims (3)

1. Analogous process for the preparation of new, therapeutically active 2-acetoxy-benzoic acid esters of 4-alkyl-3,5-dioxo-pyrazolidine derivatives with general formula: wherein either one of X or Y is phenyl and the other is phenyl, para-hydroxyphenyl, or a group of formula: or where X and Y, together with the nitrogen atoms to which they are attached, represent a 3-dimethylamino-7-methyl-[1,2,4]-benzotriazine ring system, and "Alk" represents an n-propyl or n-butyl group, as well as their acid addition salts with pharmacologically acceptable inorganic and organic acids, characterized in that a compound of general formula: wherein either X is phenyl and Y is phenyl or para-hydroxyphenyl, or where X and Y together with the nitrogen atoms to which they are bound, denotes a 3-dimethylamino-7-methyl-[1,2,4]-benzotriazine ring system fused with the pyrazolidine ring, and "Alk" is as above defined, is reacted with either (a) 2-acetoxy-benzoic acid chloride in the presence of a base to form the desired compound of formula X or an acid addition salt thereof, or (b) a mixed anhydride of general formula: wherein "Ayl" denotes an alkyl group of 1-20 carbon atoms, an aryl group of 6-20 carbon atoms, an alkaryl group of 7-20 carbon atoms or an aralkyl group of 7-20 carbon atoms; m is 0 or 1, and Q denotes a carbon atom C or when m is 0, a sulfinyl group S=0, forming the desired compound of formula X or an acid addition salt thereof, whereupon the desired compound is isolated, if necessary, by washing both with dilute acid, preferably dilute hydrochloric acid, and with dilute alkali, preferably dilute aqueous sodium carbonate or dilute aqueous sodium hydroxide, or (c) a solution in a water-immiscible solvent of 2-acetoxy-benzoic anhydride in the presence of a strong base such that the reaction mixture forms a tofase-reak ionic acid.tem which is stirred while forming the desired compound of formula X. 2. Process according to claim 1 in the preparation of 1,2-diphenyl-3-oxo-4-n-butyl-5-(2'-acetoxy-benzoyloxy)-pyrazolid-4-ene, characterized in that 2-acetoxy-benzoic acid chloride is reacted with 1,2-diphenyl-3,5-dioxo-4-n-butyl-pyrazolidine, and that the desired compound is isolated by washing both with dilute acid and with dilute alkali. 3. Process according to claim 1 for the preparation of 1-(2'-acetoxy-benzoyloxy)-2-n-propyl-3-oxo-5-dimethylamino-9-methyl-pyrazolid-2-eno-[1,2-a ][1,2,4]-benzotriazine, characterized in that 2-acetoxy-benzoic acid chloride is reacted with 5-dimethylamino-9-methyl-2-n-propyl-1H-pyrazolo-[1,2-a][1,2,4]-benzotriazine-1,3(2H )-dione, and that the desired compound is isolated by washing both with dilute acid and with dilute alkali.