AU7187791A - Therapeutic agents - Google Patents

Description

THERAPEUTIC AGENTS

The present invention relates to novel therapeutic agents, and in particular to [1]benzopyrano[4,3-c]- pyrazoles, to processes for their preparation, to pharmaceutical compositions containing them and to their therapeutic activity as immunomodulatory agents.

The present invention relates to novel compounds of formula I

in which R₁ represents hydrogen, cyano, C_{2 -6} alkanoyl, C_{2 -6} alkoxycarbonyl, CONH₂, or R₁ represents C_{1 -6} alkyl or C_{2 -6} alkenyl both of which may be substituted bv cyano, halo, trifluoromethyl, hydroxy, benzoyl, C_{2 -6} alkanoyloxy, C_{3 -6} cycloalkoxycarbonyl, a 5-7 membered non-aromatic heterocyclic group containing 2 oxygen heteroatoms, CONR ₉R₉,, phenoxy or C_{1 -6} alkoxy, in which C_{1 -6} alkoxy may be further substituted by halo, hydroxy, C_{1 -6} alkoxy or C_2-6 alkanoyloxy;

R ₂ represents hydrogen or chloro;

R ₃ represents chloro or trifluoromethyl; or R₂ and R₃ are joined to form a fused benz ring;

R₄ represents hydrogen, C_{1 -6} alkyl, C_{1 -6} alkoxy or halo; R₉ and R₉, , which may be the same or different, represent hydrogen, C_{1 -6} alkyl or benzyl, or pharmaceutically acceptable salts thereof.

It will be understood that a group containing a chain of 3 or more carbon atoms may be straight or branched, for example propyl includes n-propyl and isopropyl, and butyl includes n-butyl, sec-butyl, isobutyl and tert-butyl. The term "halo" .includes fluoro, chloro or bromo. Preferably R₁ represents hydrogen, cyano, carbamoyl, C_{2 -4} alkanoyl (for example acetyl), C_{2 -4} alkoxycarbonyl (for example methoxycarbonyl or ethoxycarbonyl), or -CH₂R₁₀ in which R₁₀ represents hydrogen, cyano, trifluoro- methyl, halo, hydroxy, C _3-8 cycloalkoxycarbonyl

(preferably cyclobutoxycarbonyl), benzoyi, CONHR₉ (for example carbamoyl, methylcarbamoyl, ethylcarbamoyl or propylcarbamoyl) or a 5-7 membered non-aromatic heterocyclic group containing two oxygen heteroatoms (for example 1,3-dioxolan-2-yl or 1,3-dioxan-2-yl; or R₁₀ represents C_{1 -4} alkyl, (for example methyl, ethyl or propyl, more preferably methyl or ethyl) or C_{2 -4} alkenyl (for example vinyl) both optionally substituted with hydroxy, cyano, trifluoromethyl, halo (preferably chloro or bromo), C_{1 -6} alkoxy (preferably methoxy or ethoxy), C_{1 -6}alkoxy (C₁-₆)alkoxy (preferably 2-methoxyethoxy), C_{2 -6}alkanoyloxy(C_{1 -6})alkoxy

(preferably acetoxyethoxy), hydroxy (C_{1 -6} alkoxy)

(preferably 2-hydroxyethoxy), C_{2 -6} alkanoyloxy (preferably acetoxy or propionyloxy), phenoxy, benzoyi,

CONHR₉ (for example carbamoyl, methylcarbamoyl, ethylcarbamoyl or propylcarbamoyl) or a 5-7 membered non-aromatic heterocyclic group containing two oxygen heteroatoms (for example 1,3-dioxolan-2-yl or 1,3-dioxan-2-yl). In preferred compounds, R. represents (CH₂)_pJ in which J represents hydrogen, C_{1 -6} alkoxy, cyano, halo or carbamoyl, and p is 0, 1 or 2, particularly hydrogen, methyl, cyanomethyl, (CH₂)₂-halo, carbamoylmethyl or C_{1 -4} alkoxyethyl. Preferred substituents R₁ are hydrogen, methyl, ethyl, propyl, butyl, allyl,

2-methoxyethyl, 2-ethoxyethyl, 2-ethoxypropyl,

2-(2-methoxyethoxy) ethyl 2-(2-acetoxyethoxy) ethyl,

2-(2-hydroxyethoxy) ethyl

cyclobutyloxycarbonylmethyl,

carbamoyl, methylcarbamoylmethyl,

2-carbamoylethyl, 3-carbamoylpropyl

ethylcarbamoylmethyl, propylcarbamoylmethyl,

isopropylcarbamoylmethyl,

benzyl (methyl) carbamoylmethyl,

carbamoylmethyl, 2-hydroxyethyl, 3-hydroxypropyl

2-acetoxyethyl,

cyano, cyanomethyl, 2-cyanoethyl, 2-phenoxyethyl,

2-chloroethyl, 2-bromoethyl, 2,2,2-trifluoroethyl

2-propionyloxyethyl, 1,3-dioxolan-2-ylmethyl,

2-(1,3-dioxan-2-yl)ethyl,

2-(1,3-dioxolan-2-yl)ethyl,

acetyl, benzoylmethyl, ethoxycarbonyl.

In especially preferred compounds R₁ represents methyl or 2-ethoxyethyl.

In preferred compounds of formula I, R₂ represents hydrogen. In preferred compounds of formula I, R₃ represents chloro or trifluoromethyl, more preferably trifluoromethyl.

In preferred compounds of formula I, R₄ represents hydrogen, C_{1 -4} alkyl, C_{1 -4} alkoxy, fluoro, chloro, or bromo. More preferably R₄ represents hydrogen, methyl, methoxy or ethoxy most preferably hydrogen or methoxy.

Preferably R₉ represents hydrogen, methyl or ethyl or benzyl, especially hydrogen or methyl. Preferably R₉, represents hydrogen or benzyl.

Particular compounds of formula I are the compounds listed in Table A provided in the specific Examples of the invention, or pharmaceutically acceptable salts thereof. Compounds of formula I may contain one or more chiral centres and exist in different optically active forms. When compounds of formula I contain one chiral centre the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallisation; formation of diastereoisomeric derivatives which may be separated, for example, by crystallisation, gas-liguid or liquid chromatography; selective derivatisation of one enantiomer by reaction with an enantiomer-specific reagent, for example enzymatic oxidation or reduction; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, for example silica with a bound chiral ligand or in the presence of a chiral solvent. Alternatively, specific enantiomers may be synthesised by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

For example, the following compound may also exist in the R- or S- enantiomeric form: 4-(4-chlorophenyl)-2-(2-ethoxypropyl) [1]- benzopyrano[4,3-c]pyrazol-3(2H)-one.

When compounds of formula I contain more than one chiral centre, the compounds may exist in diastereoisomeric forms. The present invention includes each diastereoisomer and mixtures of the diastereoisomers. The diastereoisomers may be separated by methods known to those skilled in the art, for example by crystallisation or liquid chromatography. Certain compounds of formula I may exist in different tautomeric forms or as different geometric isomers.

Certain compounds of formula I may exist in more than one crystal form and the present invention includes each crystal form and mixtures thereof. Certain compounds of formula I may also exist in the form of solvates, for example hydrates, and the present invention includes each solvate and mixtures thereof. The present invention also includes pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I together with a pharmaceutically acceptable diluent or carrier.

As used" hereinafter, the term "active compound" denotes a [1]benzopyrano[4,3-c]pyrazole of formula I. In therapeutic use, the active compound may be administered orally, rectally, parenterally or topically, preferably orally or topically. Thus the therapeutic compositions of the present invention take the form of any of the known pharmaceutical compositions for oral, rectal, parenteral or topical administration. Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the art of pharmacy. The compositions of the invention may contain 0.1-90% by weight of active compound. The compositions of the invention are generally prepared in unit dosage form. The excipients used in the preparation of these compositions are the excipients known in the pharmacist's art. Compositions for oral administration are preferred compositions of the invention and these are known pharmaceutical forms for such administration, for example tablets, capsules, syrups and aqueous or oily suspensions. Tablets may be prepared by mixing the active compound with an inert diluent such as lactose or calcium phosphate in the presence of disintegrating agents, for example maize starch, and lubricating agents, for example magnesium stearate, and tableting the mixture by known methods. The tablets may be formulated in a manner known to those skilled in the art so as to give a sustained release of the compounds of the present invention. Such tablets may, if desired, be provided with enteric coatings by known methods, for example by the use of cellulose acetate phthalate. Similarly, capsules, for example hard or soft gelatin capsules, containing the active compound with or without added excipients, may be prepared by conventional means and, if desired, provided with enteric coatings in a known manner. The tablets and capsules may conveniently each contain 0.1 to 500 mg of the active compound. Other compositions for oral administration include, for example, aqueous suspensions containing the active compound in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxymethylcellulose, and oily suspensions containing a compound of the present invention in a suitable vegetable oil, for example arachis oil.

Compositions for topical administration are also preferred compositions of the invention. The pharmaceutically active compound may be dispersed in a pharmaceutically acceptable cream, ointment or gel. A suitable cream may be prepared by incorporating the active compound in a topical vehicle such as petrolatum and/or light liquid paraffin, dispersed in an aqueous medium using surfactants. An ointment may be prepared by mixing the active compound with a topical vehicle such as a mineral oil, petrolatum and/or a wax e.g. paraffin wax or beeswax. A gel may be prepared by mixing the active compound with a topical vehicle comprising a gelling agent e.g. basified Carbomer BP, in the presence of water. Topically administrable compositions may also comprise a matrix in which the pharmaceutically active compounds of the present invention are dispersed so that the compounds are held in contact with the skin in order to administer the compounds transdermally. A suitable transdermal composition may be prepared by mixing the pharmaceutically active compound with a topical vehicle, such as described above, together with a potential transdermal accelerant such as dimethyl sulphoxide or propylene glycol.

Compositions of the invention suitable for rectal administration are known pharmaceutical forms for such administration, for example suppositories with semi-synthetic glycerides or polyethylene glycol bases.

Compositions of the invention suitable for parenteral administration are known pharmaceutical forms for such administration, for example sterile suspensions in aqueous and oily media or sterile solutions in a suitable solvent.

In some formulations it may be beneficial to use the compounds of the present invention in the form of particles of very small size, for example as obtained by fluid energy milling. In the compositions of the present invention the active compound may, if desired, be associated with other compatible pharmacologically active ingredients.

The compounds of formula I are indicated for use as immunomodulatory agents, and are generally immunosuppressants, but some compounds, in certain disease states, may exhibit immunostimulant activity.

The compounds according to the invention are useful in the treatment of diseases resulting from an aberrant immune reaction. Thus the pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I may be used to treat diseases with an immunological association for example tissue rejection, such as kidney rejection; autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus; cutaneous disorders, such as contact sensitivity, eczema and psoriasis; and neoplasia, such as melanoma.

In such treatment the amount of the compound of formula I administered per day will be such as to give a therapeutic effect and is generally in the range 0.1 to 2000 mg, preferably 1 to 500 mg. Accordingly, in another aspect, the present invention also includes a method of treating diseases with an immunological association, comprising the administration of a therapeutically effective amount of a compound of formula I. The therapeutic activity of compounds of formula I has been demonstrated by means of tests on standard laboratory animals, Such tests include, for example, the oral and parenteral administration of the compounds to BALB/c mice. Thus, compounds of formula I are useful as immunomodulatory agents. Whilst the precise amount of active compound administered will depend on a number of factors, for example the age of the patient, the severity of the condition and the past medical history and always lies within the sound discretion of the administering physician, a suitable dose for oral administration to mammals, including humans, is generally within the range 0.01-40 mg/kg/day, more usually 0.2-25 mg/kg/day given in single or divided doses. For parenteral administration, a suitable dose is generally within the range 0.001-4.0 mg/kg/day, more usually 0.005-1 mg/kg/day given in single or divided doses or by continuous infusion. A suitable preparation for topical administration generally contains the active ingredient within the range 0.01-20% by weight, more usually 0.05-5% by weight. Oral administration is preferred.

Processes for the preparation of compounds of formula I will now be described. These processes form a further aspect of the present invention.

Compounds of formula I may be prepared by reacting a compound of formula II,

or a tautomer thereof with a compound of formula III

in which R₁₄ represents (OQ)₂ or (SQ)₂ and R ₁₅ represents OQ or SQ or NQ'₂; R₁₄ represents = NH and R₁₅ represents OQ or SQ; or R₁₄ represents = 0 and R ₁₅ represents hydrogen, halo or 1-imidazolyl; and Q and Q represent a C_1-4 alkyl group or a benzyl group.

Compounds of formula I may be prepared by reacting compounds of formula IV

in which R₁₆ represents hydrogen, or a tautomer thereof, or in which R₁₆ represents a group COR₁₈ wherein R₁₈ represents hydrogen, a C_1-4 alkyl group, a benzyl group or a group R₅ and R₁₇ represents COR₅ in which R₅ represents IlIa

with a base e.g. piperidine in a suitable solvent e.g ethanol.

Compounds of formula I may be prepared by reacting compounds of formula IV in which R₁ and R₁₆ represent COR₁₈ and R₁₇ represents hydrogen with a carboxylic acid chloride for example a substituted benzoyl chloride in the presence of a base for example triethylamine followed by heating in the presence of a base for example piperidine. Compounds of formula I in which R₁ represents an alkyl group or alkenyl group substituted as herein described may be prepared by alkylating corresponding compounds of formula I in which R. represents hydrogen, for example by reaction with a compound of formula R₁X in which X represents halo in the presence of a base e.g. sodium hydride.

Compounds of formula I in which R₁ represents C_2-6 alkanoyl may be prepared by acylation of corresponding compounds of formula I in which R₁ represents hydrogen, for example by reaction with an acyl halide.

Compounds of formula I in which R₁ represents cyano may be prepared by cyanation of corresponding compounds of formula I in which R₁ represents hydrogen, for example by reaction with cyanogen bromide in the presence of a base, for example sodium hydride.

Compounds of formula I in which R₁ represents C_2-6 alkoxycarbonyl may be prepared by the substitution of corresponding compounds of formula I in which R. represents hydrogen with a C_2-6 alkoxycarbonyl group, for example by reaction with compounds of formula (C_2-6 alkoxycarbonyl) A where A is a leaving group, for example cyano or halo.

Compounds of formula I in which R₁ represents carbamoylalkyl, e.g. carbamoylmethyl, may be prepared by the hydrolysis of corresponding compounds of formula

I in which R₁ represents cyanoalkyl, e.g. cyanomethyl.

Compounds of formula I in which R₁ represents

CONHR₃₀ in which R₃₀ represents a readily hydrolysable group for example chlorosulphonyl, may be prepared by reacting corresponding compounds of formula I in which R₁ represents hydrogen with an isocyanate of formula R₃₀N=C=O. Compounds of formula I in which R₁ represents carbamoyl may be prepared by hydrolysis, for example acid hydrolysis, of corresponding compounds of formula I in which R₁ represents CONHR₃₀.

Compounds of formula I in which R₁ is substituted by a carboxylic amide group (CONR₉R₉,) may be prepared by reacting corresponding compounds of formula I substituted by a .group -COA where A represents a leaving group for example, hydroxyl, halo, C_1-4 alkoxy, aryloxy, arylmethoxy or C_1-6 acyloxy with a C_1-6 alcohol or amine (HNR₉R₉') respectively, for example at 0-250°C, optionally in the presence of an organic liquid which is preferably a solvent for the reactants and optionally in the presence of a catalyst for the reaction.

Compounds of formula I in which R. represents a group substituted by a hydroxyl group may be prepared by the hydrolysis of corresponding compounds of formula substituted by a C_2-6 alkanoyloxy group, for example acetoxy.

Compounds of formula I in which R₁ is substituted by a C_2-6 alkanoyloxy group may be prepared by acylation of corresponding compounds of formula I substituted by a hydroxy group.

Compounds of formula II may be prepared by reacting compounds of formula V

with a hydrazine of formula H₂NNHR₁.

Compounds of formula III wherein R₁₄ represents (OQ)₂ and R₁₅ represents OQ may be prepared for example a) by reacting compounds of formula R₅CX₃ in which X is halo with a sodium alkoxide of formula NaOQ in which Q is a C_1-4 alkyl group or a benzyl group, or b) by reacting compounds of formula R₅CN with an alcohol of formula QOH in the presence of an anhydrous acid, for example hydrogen chloride, to give compounds of formula R₅C(=NH) OQ as their acid salts, e.g. hydrochloride salts, which are then reacted with further alcohol of formula QOH.

Compounds of formula IV in which R₁₆ represents

COR₁₈ and R₁₇ represents COR₅ may be prepared by acylation of compounds of formula IV in which R₁₆ represents COR₁₈ and R₁₇ represents hydrogen, for example by reaction with an acid anhydride of formula (R₅CO)₂O in the presence of a salt (e.g. the sodium salt) of the corresponding acid or an acid halide e.g. of formula R₅COCl in the presence of a base e.g. triethylamine.

Compounds of formula IV in which R₁₆ represents COR₁₈ and R₁₇ represents hydrogen may be prepared by the acylation of compounds of formula II for example by reaction with an acid anhydride of formula (R₁₈CO)₂O in the presence of a salt (e.g. the sodium salt) of the corresponding acid.

Compounds of formula IV in which R₁₇ represents COR ₅ and R₁₆ represents hydrogen, or tautomers thereof, may be prepared by reacting a compound of formula IV in which R₁₆ represents COR₁₈ and R₁₇ represents COR₅ with a base e.g. piperidine in a suitable solvent e.g. ethanol. Compounds of formula IV in which R₁ and R₁₆ represents COR₁₈ in which R₁₈ is as hereinbefore defined, and R₁₇ represents hydrogen may be prepared by reacting compounds of formula II in which R₁ represents hydrogen with an acid anhydride of formula (R₁₈CO)₂O in the presence of a salt (e.g. the sodium salt) of the corresponding acid.

Compounds of formula V may be prepared by reacting compounds of formula VI

in which R₂₃ represents hydrogen with malonic acid in the presence of an acid chloride e.g. phosphoryl chloride and a Lewis acid e.g. zinc chloride.

Compounds of formula V may be prepared by reacting compounds of formula VI in which R₂₃ represents an acetyl group with a base, for example sodium hydride, and a dialkyl carbonate of formula (QO)₂CO in which Q represents a C_1-4 alkyl group or a benzyl group, e.g. dimethyl carbonate.

Compounds of formula V in which R. represents C_1-6 alkoxy may be prepared from compounds of formula VI in which R₄ represents halo e.g. fluoro, and R₂₃ represents an acetyl group with a base for example sodium hydride, and a dialkyl carbonate of formula (QO)₂CO in which Q represents a C_1-4 alkyl group or a benzyl group, e.g. dimethyl carbonate.

Certain intermediate compounds of formulae II, III, IV, V, and VI are believed to be novel compounds. All novel compounds herein are claimed as a further aspect of the invention.

This invention is illustrated by the following non-limitative Examples. In the Examples parts and percentages are by weight and compositions of mixed solvents are given by volume. Characterisation was by elemental analysis and one or more of the following spectroscopic techniques: nuclear magnetic resonance, infra-red and mass spectrosσopy.

Example 1 a) 2'-Fluoro-6'-hydroxyacetophenone (30 g) was added drσpwise over 30 minutes to a stirred suspension of sodium hydride (17.1 g, 60% dispersion in mineral oil) in dry toluene (400 ml) which was boiling under reflux under nitrogen. After boiling for a further 20 minutes heating was continued while diethyl carbonate (50.8 g) was added dropwise over 10 minutes. The resulting mixture was stirred and heated under reflux for 20 hours. The reaction mixture was cooled to ambient temperature and water (150 ml) added dropwise. After separation, the aqueous phase was washed with ether and then acidified with concentrated hydrochloric acid. The precipitate was collected by filtration and washed with water to give, after drying and recrystallisation from ethyl acetate, 5-ethoxy-4-hydroxycoumarin, m.p. 110-111°C. Example 2

A stirred mixture of 4-hydroxycoumarin (10.0 g), ethylhydrazine oxalate (14.25 g) and triethylamine

(24.0 ml) in dry toluene (50 ml) was heated under reflux for 3 hours with removal of water formed in the reaction. The solid collected after filtration was partitioned between water and dichloromethane. The organic layer was dried and evaporated to give a solid which was recrystallised from butanol to give 1-ethyl-3-(2-hydroxyphenyl)-2-pyrazolin-5-one, m.p.

147-150°C.

Example 3

Hydrazine (32.3 g) was added dropwise during 0.25 hours to a stirred solution of 4-hydroxycoumarin (50.0 g) in absolute alcohol (1.2 1) and the reaction mixture was heated under reflux with stirring for 20 hours. The solution was cooled and acidified with glacial acetic acid (154 ml) and the ethanol was evaporated off under reduced pressure to leave an oil which was then poured into water (1.5 1). The resulting solid was filtered off, washed with water, dried and recrystallised from isopropanol to give 3-(2-hydroxyphenyl)-2-pyrazolin-5-one, m.p. 205-207°C.

Examples 4 to 6 In a similar manner to that described in Example 3, a compound of formula II was prepared by reacting a compound of formula V, with a hydrazine of formula H₂NNHR₁ , as summarized in Table A below in which substituents R₄ and R₁ are given.

The compounds of formula II prepared in Examples 4-6were as follows:

Example 4

3-(2-hydroxyphenyl)-1-methyl-2-pyrazolin-5-one Example 5

3-(2-hydroxy-6-methoxyphenyl)-1-methyl-2-pyrazolin-5- one

Example 6

3-(2-hydroxy-6-methylphenyl)-1-methyl-2-pyrazolin-5-one Example 7

A solution of 4-trifluoromethylbenzonitrile (25 g) in dry ether (120 ml) and dry methanol (6 ml) was cooled at 0-5°C and saturated with hydrogen chloride gas. The reaction mixture was allowed to stand overnight in a melting ice bath and the imidate salt which precipitated was collected by filtration, washed with ether and dried in a desiccator. This solid was added to AR methanol (78.3 ml) with stirring and the mixture stirred at ambient temperature for 5 days. The reaction mixture was filtered and the filtrate was evaporated under reduced pressure to give trimethyl ortho(4-trifluoromethylbenzoate) as an oil which was used without further purification.

Example 8 a) A mixture of 4-hydroxy-5-methoxycoumarin

(4.0 g) and hydrazine hydrate (3.0 ml) in industrial methylated spirit (84 ml) was boiled under reflux for

4.75 hours. On cooling, glacial acetic acid (13.3 ml) was added and the alcohol removed under reduced pressure. The residue was added to water and a gum separated which solidified on the addition of a small amount of dichloromethane. The solid was collected by filtration to give 3-(2-hydroxy-6-methoxyphenyl)-2- pyrazolin-5-one, m.p. 208-210°C. b) A mixture of 3-(2-hydroxy-6-methoxyphenyl)- 2-pyrazolin-5-one (2.06 g) and 1 -(4-trifluoromethylbenzoyl) imidazole (5.04 g) in dry xylene (50 ml) was stirred and heated under reflux under nitrogen for 0.5 hours then cooled to ambient temperature. The solid formed was collected by filtration and then dissolved in boiling industrial methylated spirit/dichloromethane (1:2). Dichloromethane was boiled off until a solid separated. The mixture was cooled to ambient temperature and filtered to give 9-methoxy-4-(4-trifluoromethylphenyl)[1]benzopyrano- [4,3-c]pyrazol-3(2H)-one, m.p. >280°C (with

decomposition). c) Sodium hydride (0.114 g, 60% dispersion in mineral oil) was added to a suspension of 9-methoxy-4- (4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol- 3(2H)-one (0.94 g) in dry N,N-dimethylformamide (55 ml). The mixture was stirred at ambient temperature for 10 minutes, then 2-bromoethyl ethyl ether (0.36 ml, 90%) was added dropwise followed by stirring at ambient temperature for 16h. The mixture was added to water containing a small amount of petroleum ether (b.p. 62-68°C). The mixture was acidified with 2M hydrochloric acid and filtered to give 2-(2-ethoxyethyl)-9-methoxy-4-(4-trifluoromethyl- phenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one,

m.p. 164-168°C.

Example 9 a) A mixture of 5-ethoxy-4-hydroxycoumarin (5.0 g) (Example 1) and methylhydrazine (2.2 g) in industrial methylated spirit (60 ml) was heated under reflux for 20 hours. On cooling, the solvent was evaporated off under reduced pressure to give the crude product. Methanol (50 ml) and concentrated hydrochloric acid (20 ml) were added to this crude product and the solution obtained was heated under reflux for 2 hours. After evaporating off the solvents under reduced pressure, the residue was triturated with water and the solid obtained collected by filtration and dried to give 3-(2-hydroxy-6-ethoxyphenyl)-1-methyl-2-pyrazolin- 5-one. b) The solid obtained in (a) above (3.2 g) and trimethyl ortho (4-chlorobenzoate) (9.0 g) were heated together at 130-140°C for 45 minutes. The mixture was cooled to ambient temperature and hexane (50 ml) was added. The supernatant liquid was decanted off and ether (80 ml) added to the residual oil to precipitate a solid. The solid was collected by filtration, washed with ether, dried and then recrystallised from propan- 2-ol to give 4-(4-chlorophenyl)-9-ethoxy-2-methyl[1]- benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 197-199°C.

Example 10 a) A mixture of 4-hydroxycoumarin (3.6 g) and a 70% aqueous solution of 2,2,2-trifluoroethylhydrazine (5.0 g) in toluene (27 ml) was heated under reflux for 4 hours with removal of water. The solid collected after cooling and filtration was extracted with hot dichloromethane, filtered and the filtrate was evaporated. The solid product was recrystallised from toluene and then from dichloromethane to give 3-(2- hydroxyphenyl)-1-(2,2,2-trifluoroethyl)-2-pyrazolin-5- one, m.p. 163-165°C. b) The solid obtained in (a) above (1.2 g) and trimethyl ortho(4-chlorobenzoate) (3.0 g) were heated together at 125°C for 10 minutes. The mixture was cooled, diluted with ether (10 ml) and the solid obtained was recrystallised from a mixture of N,N- dimethylformamide and methanol to give 4-(4-chlorophenyl)-2-(2,2,2-trifluoroethyl) [1]benzpyrano[4,3-c]- pyrazol-3(2H)-one, m.p. 214-215°C (decomposed).

Example 11 a) A mixture of 3-(2-hydroxyphenyl)-1-methyl-2- pyrazolin-5-one (3.8 g), prepared as in Example 4, anhydrous sodium acetate (1.64 g) and acetic anhydride (30 ml) was stirred at ambient temperature for 1.5 hours. The reaction mixture was added to water (10 volumes) and extracted with ethyl acetate. The combined organic extracts were washed with water, dried and evaporated. The semi-crystalline residue was triturated with petroleum ether (b.p. 60-80°C) and the solid collected by filtration and dried to give 3-(2-hydroxyphenyl)-1-methyl-5-pyrazolyl acetate, m.p. 113-116°C. b) A solution of 3,4-dichlorobenzoyl chloride (4.4 g) in tetrahydrofuran (80 ml) was added over 10 minutes to a stirred solution of 3-(2-hydroxyphenyl)-1-methyl-5- pyrazolyl acetate (4.5 g) in tetrahydrofuran (100 ml) and triethylamine (3 ml) at 0-5°C. The mixture was stirred for 18 hours at ambient temperature and then more 3,4-dichlorobenzoyl chloride (4.4 g) in tetrahydrofuran (80 ml) and triethylamine (3 ml) was added. After stirring for a further 24 hours, the reaction mixture was poured into water (1 1) and extracted with ethyl acetate. The combined organic extracts were dried and then concentrated until precipitation occurred. After filtration to remove 3,4-dichlorobenzoic acid, the filtrate was evaporated under reduced pressure and the residue crystallised from ethyl acetate to give 2-(5-acetoxy-1-methyl-3- pyrazolyl)phenyl 3,4-dichlorobenzoate, m.p. 123°C. c) 2-(5-Acetoxy-1-methyl-3-pyrazolyl)phenyl 3,4- dichlorobenzoate (1.0 g) and piperidine (0.27 ml) were heated in ethanol (10 ml) under reflux for 2 hours. The reaction mixture was cooled to 0°C. The product was collected by filtration and recrystallised from ethanol to give 4-(3,4-dichlorophenyl)-2-methyl[1] benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 193-194°C.

Example 12 a) A solution of 3-(2-hydrσxyphenyl)-1-methyl-5- pyrazolyl acetate (2.32 g), prepared as in Example 11a, in dry tetrahydrofuran (50 ml) was treated successively with triethylamine (1.5 ml) and 2-naphthoyl chloride

(2.1 g) in dry tetrahydrofuran (10 ml) at 0-5°C. The mixture was stirred at ambient temperature for 20 hours and then more triethylamine (0.75 ml) and 2-naphthoyl chloride (1.05 g) were added and the mixture was stirred for another 20 hours. The reaction mixture was poured into water (10 volumes) and extracted with ethyl acetate. The combined organic extracts were washed with saturated sodium carbonate solution, then water and then dried. After evaporation under reduced pressure the residue was triturated with ether and the solid formed collected by filtration. b) Part of the crude solid of stage (a) (2.43 g) and piperidine (0.63 ml) were boiled under reflux in absolute ethanol (19 ml) for 30 minutes. On cooling, the mixture was filtered to give 2-(1-methyl-5-oxo- 4,5-dihydro-3-ρyrazolyl)phenyl 2-naphthoate, m.p. 207-210°C. c) The filtrate was boiled under reflux for a further hour. The mixture was concentrated and then cooled. The solid formed was collected by filtration to give 2-methyl-4-(2-naphthyl) [1]benzopyrano[4,3-c]pyrazol- 3(2H)-one, m.p. 159-161 °C.

Example 13

A stirred mixture of 3-(2-hydroxyphenyl)-2- pyrazolin-5-one (2.32 g) and triethyl ortho(4-chlorobenzoate) (10.2 g) was heated at 130-135 °C for 1 hour. The mixture was cooled to ambient temperature, diluted with ether and the resulting solid filtered off, washed and dried. The solid was recrystallised from industrial methylated spirit (charcoal used) to give 4-(4-chlorophenyl) [1]benzopyrano[4,3-c]pyrazol-3(2H)- one, m.p. 255-257°C.

Examples 14 to 18

In a similar manner to that described in Example 13, a compound of formula I was prepared by reacting a compound of formula II with a compound of formula III as summarized in Table B below in which substituents R₁ and R₄ in compound II are given. The compounds of formula III used were of the type R₅C (OQ)₃ in which R₂ represents hydrogen and R₃ and Q are as given in Table B.

Example 19

A mixture of 3-(2-hydroxyphenyl)-2-pyrazolin-5-one

(16 g) and 1-(4-chlorobenzoyl)imidazole (37.5 g) in xylene (200 ml) was stirred and heated under reflux for 3 hours. The mixture was allowed to cool to 70°C and then filtered to give 4-(4-chlorophenyl)[1]- benzopyrano[4,3-c]ρyrazol-3(2H)-one, m.p. 255-257°C.

Example 20

A mixture of trimethyl ortho(4-trifluoromethylbenzoate) (5.5 g), prepared as in Example 7, and 3-(2- hydroxyphenyl)-2-pyrazolin-5-one (1.94 g) was heated at 135-140°C for 4 hours. The mixture was cooled to 0°C and the solid formed filtered off and washed with ether. This solid was triturated with hot water and filtered to give 4-(4-trifluoromethylphenyl) [1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 271-273°C.

Example 21

A mixture of 3-(2-hydroxyphenyl)-2-pyrazolin-5-one (30.2 g), 1-(4-trifluoromethylbenzoyl)imidazole (86.5 g) and xylene (800 ml) was stirred and boiled under reflux for 4 hours under nitrogen. The reaction mixture was allowed to cool to 40°C and filtered to give 4-(4-trifluoromethylphenyl) [1]benzopyrano[4,3-c]- pyrazol-3(2H)-one, m.p. 270-272°C. Example 22 a) A mixture Of 3-(2-hydroxyphenyl)-2-pyrazolin-5-one (3.52 g) and sodium acetate (1.64 g) in acetic anhydride (30 ml) was stirred at ambient temperature for 3 hours. The mixture was poured into water and petroleum ether (b.p. 62-68°C, 10:1) and the solid collected by filtration to give crude 1-acetyl-3-(2- hydroxyphenyl)-5-pyrazolyl acetate, m.p. 62-64°C. b) A mixture of crude 1-acetyl-3-(2-hydroxyphenyl)-5-pyrazolyl acetate (1.53 g), triethylamine (2.1 ml) and dry tetrahydrofuran (25 ml) was stirred at 0-5°C while 4-trifluoromethylbenzoyl chloride (3.21 g) was added dropwise. The resultant mixture was stirred at ambient temperature for 64 hours and then poured into water. The product was extracted into ethyl acetate and the combined extracts were washed with water, saturated sodium bicarbonate solution and finally water. After drying, the solvent was evaporated off under reduced pressure. The residue was digested with hot propan-2-ol then cooled and filtered. The filtrate was evaporated to give crude 2-(5-acetoxy- 1-acetyl-3-pyrazolyl)phenyl 4-trifluoromethylbenzoate as an oil. c) The oil from stage (b) and piperidine (0.7 ml) were boiled under reflux in absolute ethanol (22 ml) for 1 hour. The mixture was cooled to 0-5°C and then filtered to give the crude product which was purified by preparative layer chromatography on silica, using dichloromethane/methanol (9:1) as the mobile phase, to give 4-(4-trifluoromethylphenyl) [1] benzopyrano- [4,3-c]ρyrazol-3(2H)-one, m.p. 268-272°C.

Example 23

A mixture of 3-(2-hydroxyphenyl)-2-pyrazolin-5-one (2.9 g) and potassium carbonate (3.2 g) in dry xylene (30 ml) was stirred and heated under reflux while 4-trifluoromethylbenzoyl chloride (6.9 g) was added dropwise over 15 minutes. The mixture was boiled and stirred for 2 hours then hot filtered. The solid collected was washed with toluene, water and finally ether to give 4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 259-263°C.

Example 24

3-(2-Hydroxyphenyl)-1-methyl-2-pyrazolin-5-one (Example 4) (1.90 g) was dissolved in warm toluene (100 ml) then cooled to ambient temperature and added over 5.25 hours to a stirred, boiling solution of 4-chlorobenzaldehyde (1.4 g) in dry toluene (50 ml) containing piperidine (1 ml), with removal of water formed in the reaction. The reaction mixture was evaporated under reduced pressure and the residue triturated with ether and recrystallised from propan- 2-ol to give 4-(4-chlorophenyl)-2-methyl[1]benzopyrano- [4,3-c]pyrazol-3(2H)-one, m.p. 204-205°C. Example 25

A mixture of 2-(2-chloroethoxy)ethyl acetate

(16.7 g), sodium bromide (103 g), N,N-dimethylformamide (200 ml) and dibromomethane (100 ml) was stirred and heated at 100°C for 48 hours. The mixture was poured into ether (300 ml) and water (200 ml) . The

Organic layer was separated, washed with water, dried and evaporated to give an oil. Distillation under reduced pressure gave 2-(2-bromoethoxy)ethyl acetate, b.p. 100-103°C (7mm Hg). Example 26

Sodium hydride (0.60 g, 50% dispersion in mineral oil) was added to a stirred solution of 4-(4-chlorophenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (3.70 g), prepared as in Example 13, in dry N,N-dimethylformamide (145 ml) under nitrogen. The mixture was stirred at ambient temperature for 5 minutes and then 2-bromoethyl acetate (1.38 ml) was added dropwise. The reaction mixture was stirred at ambient temperature for 16 hours and then poured into water and petrol (b.p. 60-80°: approx. 10:1 by volume). The mixture was cooled in an ice bath and the deposited solid filtered off and washed to give 2-[4-(4-chlorophenyl)-3-oxo-2,3-dihydro- [1]benzopyrano[4,3-c]pyrazol-2-yl]ethyl acetate, m.p. 126-129°C.

Examples 27 to 41 In a similar manner to Example 26, compounds of formula I were prepared by reacting 4-(4-chloro- phenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (A) (Example 13) with compounds of formula R,X, in which X is halo as summarized in Table C below.

Notes for Table C

(1) The reaction mixture was added to water (2 1), acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The combined extracts were washed with water, dried and concentrated until crystallisation occurred. On cooling, the product was obtained by filtration.

(2) The solid obtained was dissolved in a mixture of dichloromethane/ethanol and concentrated until crystallisation occurred. The product was collected by filtration.

(3) The quenched mixture was extracted with ethyl acetate. The combined extracts were washed with water, dried and concentrated until crystallisation occurred. The product was collected by filtration.

(4) The reaction mixture was added to water (10 volumes) and acidified with 2M hydrochloric acid. The solid formed was collected by filtration, dried and then dissolved in boiling industrial methylated spirit/dichloromethane, 1:2. The dichloromethane was removed and the solution cooled in an ice bath. The solid formed was collected by filtration.

(5) The reaction mixture was added to water (500 ml) and extracted with ethyl acetate. The combined extracts were washed with water, dried and evaporated. The solid obtained was recrystallised from propan-1-ol.

(6) The reaction mixture was added to water and the solid formed collected by filtration and dried. This solid was triturated with acetone and filtered. The residue was triturated with boiling industrial methylated spirit then filtered and the residue dissolved in warm dichloromethane, treated with charcoal and filtered. The filtrate was evaporated and the residue triturated with petrol and filtered to give the product. (7) The reaction mixture was quenched into water

(300 ml) containing 2M hydrochloric acid (5 ml) and extracted with dichloromethane. The combined extracts were dried and evaporated. The residue was partitioned between ether and water, then separated. The aqueous layer was extracted with ether and the combined ether extracts dried and concentrated (to 10 ml). The solid formed was collected by filtration, washed with petroleum ether (b.p. 40-60°C) and dried. After recrystallisation from ethanol the product was purified by flash chromatography on silica using dichloromethane/methanol, 98:2 as the mobile phase. The eluted material was recrystallised from ethanol to give the product.

(8) The reaction mixture was partitioned between 5M hydrochloric acid (600 ml) and ether (300 ml) . The aqueous layer was separated off and washed with ether. The combined ether extracts were washed well with water, dried and evaporated. The residue was purified by flash chromatography on silica using dichloromethane/methanol, 97:3 as the mobile phase. The solid obtained was recrystallised twice from a mixture of ether and ethyl acetate to give the product.

(10) The reaction mixture was added to water and acidified with 5M hydrochloric acid. The solid was collected by filtration and recrystallised from N,N-dimethylformamide. (11) The reaction mixture was added to water. The solid obtained was collected by filtration and recrystallised from propan-2-ol.

(12) 60% dispersion of sodium hydride in mineral oil used.

(13) The reaction mixture was added to water (500 ml) and extracted with ether. The combined extracts were washed with water, dried and evaporated. The solid obtained was recrystallised from ethanol. (14) The reaction mixture was added to water (2 1), acidified with 5M- hydrochloric acid and extracted with ethyl acetate. The combined extracts were washed with water, dried and evaporated. The oil obtained was crystallised from ether. Example 42

In a similar manner to Example 26, a mixture of sodium hydride (0.74 g, 60% dispersion in mineral oil), dry N,N-dimethylformamide (180 ml), 4-(4-chlorophenyl) [1]benzoρyrano[4,3-c]pyrazol-3(2H)-one

(Example 13) (5.0 g) and 2-bromomethyl-1,3-dioxolane

(1.97 ml) was stirred at ambient temperature for 16 hours. More sodium hydride (0.74 g) was added in portions followed by more 2-bromomethyl-1,3-dioxo,ane

(1.97 ml). The mixture was stirred at ambient temperature for 24 hours and then warmed at 70-80°C for 1 hour. The mixture was cooled to ambient temperature and then added to water. The solid formed was collected by filtration, dried and recrystallised from ethanol/dichloromethane to give 4-(4-chlorophenyl)- 2-(1,3-dioxolan-2-ylmethyl)[1]benzopyrano[4,3-c]- pyrazol-3(2H)-one, m.p. 230-231°C. Example 43

In a similar manner to Example 26, a mixture of sodium hydride (0.44 g, 60% dispersion in mineral oil), dry N,N-dimethylformamide (100 ml), 4-(4-chloro- phenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (3.0 g) (Example 13 ) and 1-bromo-2-ethoxypropane (2.9 g) was stirred at ambient temperature for 16 hours. The mixture was warmed at about 60°C for 1.5 hours then cooled to ambient temperature. The mixture was added to water and the product extracted into ether. The combined ether extracts were dried and evaporated. The residue was separated by flash chromatography on silica, using ethyl acetate/petroleum ether (b.p. 80-100°) (1:3) as the mobile phase, to give a red solid which was recrystallised from ethanol to give 4-(4-chlorophenyl)-2-(2-ethoxypropyl)[1]benzopyrano- [4,3-c]pyrazol-3(2H)-one, m.p. 118-119°C.

Example 44

In a similar manner to Example 26, a mixture of sodium hydride (1.82 g, 60% dispersion in mineral oil), dry N,N-dimethylformamide (450 ml), 4-(4- trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol- 3(2H)-one (15.0 g) (Example 20) and 2-bromoethyl acetate (7.62 g) was stirred at ambient temperature for 48 hours. The mixture was added to water. The solid formed was filtered off and dissolved in ethyl acetate which was washed with water. The residue obtained, after removal of the ethyl acetate under reduced pressure, was recrystallised from propan-2-ol to give 2-[3-oxo-4-(4-trifluoromethylphenyl)-2,3-dihydro[1]- benzopyrano[4,3-c]pyrazol-2-yl]ethyl acetate,

m.p. 134-136°C. Example 45

In a similar manner to Example 26, a mixture of sodium hydride (0.74 g, 60% dispersion in mineral oil), dry N,N-dimethylformamide (150 ml), 4-(4-chloro- phenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (5.0 g) and 2-(2-bromoethyl)-1,3-dioxane (2.52 ml) was stirred at ambient temperature for 42 hours. The reaction mixture was added to water. The product was extracted into ether and the extracts washed with water. Evaporation of the ether left a solid residue which was recrystallised from ethanol to give 4-(4-chlorophenyl)- 2-[2-(1,3-dioxan-2-yl)ethyl][1]benzopyrano[4,3-c]- pyrazol-3(2H)-one, m.p. 157-163°C.

Example 46 A mixture of 2-[3-oxo-4-(4-trifluoromethylphenyl)- 2,3-dihydro[1]benzopyrano[4,3-c]pyrazol-2-yl]ethyl acetate (17.93 g) (Example 44), 2M hydrochloric acid (22 ml) and industrial methylated spirit (750 ml) was stirred and boiled under reflux for 18 hours . The mixture was cooled to 0°C and the solid formed collected by filtration to give 2-(2-hydroxyethyl)- 4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]- pyrazol-3(2H)-one, m.p. 199-201 °C.

Example 47 Dilute hydrochloric acid (2 ml) was added to a stirred suspension of 2-[4-(4-chlorophenyl)-3-oxo-2,3- dihydro[1]benzopyrano[4,3-c]pyrazol-2-yl]ethyl acetate

(1.53 g), prepared as in Example 26, in industrial methylated spirit (80 ml) at ambient temperature. The mixture was heated under reflux for 6.5 hours. The solution was allowed to cool and was kept at ambient temperature for 16 hours. The resulting crystals were filtered off. and washed with industrial methylated spirit to give 4-(4-chlorophenyl)-2-(2-hydroxyethyl)- [1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 179-183°C.

Example 48 A mixture of 3-[4-(4-chlorophenyl)-3-oxo-2,3- dihydro[1]benzopyrano[4,3-c]pyrazol-2-yl]propyl acetate (1.5 g) (Example 41), 2M hydrochloric acid (1.9 ml) and industrial methylated spirit (75 ml) was boiled under reflux for 5 hours. The mixture was cooled to room temperature and the solid formed collected by filtration to give 4-(4-chlorophenyl)-2-(3-hydroxy- propyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 166-168°C.

Example 49 A mixture of 2-(2-[4-(4-chlorophenyl)-3-oxo-2,3- dihydrofl]benzopyrano[4,3-c]pyrazol-2-yl]ethoxyj ethyl acetate (11 g) (Example 39), 5M hydrochloric acid (100 ml) and water (100 ml) was stirred and boiled under reflux for 24 hours. The mixture was evaporated and the solid obtained was recrystallised from propan- 2-ol (100 ml) to give 4-(4-chlorophenyl)-2-[2-(2- hydroxyethoxy)ethyl][1]benzopyrano[4,3-c]pyrazol-3 (2H)- one, m.p. 110-112°C.

Example 50 A mixture of 4-(4-chlorophenyl)-3-oxo-2,3-dihydro-

[1]benzopyrano[4,3-c]pyrazole-2-acetonitrile (0.68 g), prepared as in Example 28, and concentrated sulphuric acid (6 ml) was heated at 120°C for 0.5 hour. The reaction mixture was poured on to ice (10 volumes) and the solid product was collected, washed and dried to give 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2-acetamide, m.p. 279-283°C.

Example 51

In a similar manner to Example 50, a mixture of 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano-

[4,3-c]pyrazole-2-propionitrile (1.0 g), prepared in

Example 32, and concentrated sulphuric acid (10 ml) was heated at 120-125 °C for 15 minutes to give, after recrystallisation from industrial methylated spirit, 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano-

[4,3-c]pyrazole-2-propionamide, m.p. 264-266°C.

Example 52 a) A stirred suspension of 4-(4-chlorσphenyl)-3- oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2-acetic acid (2.16 g), prepared as in Example 31, in dichloromethane (50 ml) was treated with dry N,N-dimethylformamide (0.24 ml) and then thionyl chloride (1.51 ml). The mixture was stirred at ambient temperature for 16 hours and then evaporated under reduced pressure. The residue was triturated with dry ether and the solid collected by filtration to give 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2-acetyl chloride. b) A portion of this acid chloride (1.08 g) in dry tetrahydrofuran (100 ml) was treated dropwise with an aqueous solution of ethylamine (0.43 ml, 70% w/w). The mixture was stirred at 0-5°C for 1.5 hours and then filtered. The filtrate was diluted with ether (100 ml), cooled in an ice-bath for 3 hours and the solid filtered off. On standing for several days a second crop of solid was obtained. The two combined crops were washed with water and then petroleum ether (b . p . 60-80 °C) to give 4- ( 4-chlorophenyl) -N-ethyl-3- oxo-2 , 3-dihydro [ 1 ] benzopyrano [4 , 3-c] pyrazole-2- acetamide , m. p . 238-239 °C .

Example 53 In a similar manner to Example 52(a) 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano

[4,3-c]pyrazole-2-acetic acid (1.87 g) was converted into the acid chloride (1.89 g) and then suspended in dry tetrahydrofuran (172 ml) at 0-5°C. Aqueous methylamine (0.69 ml, 25/30% w/v) was added dropwise and the mixture stirred in a melting ice bath for. 2.5 hours. The solid was filtered off, washed with water and then ether to give 4-(4-chlorophenyl)-N- methyl-3-oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetamide, m.p. 244-250°C.

Example 54

A solution of propylamine (0.2 ml) in dry tetrahydrofuran (10 ml) was added dropwise to a mixture of 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]- benzopyrano[4,3-c]pyrazole-2-acetyl chloride (1.07 g) (Example 52a) in dry tetrahydrofuran (95 ml) with stirring at 0-5°C. The mixture was stirred at 0-5°C for 50 minutes and then more propylamine (0.1 ml) in dry tetrahydrofuran (5 ml) was added. This mixture was stirred at 0-5°C for 2.3 hours and then diluted with dry ether (95 ml) and stirred at 0-5°C for 1 hour. The mixture was filtered and the filtrate washed with water, dried and evaporated under reduced pressure. The residue was separated on a Florisil® column using dichloromethane and then dichloromethane/methanol (95:5) as the mobile phase. The solid obtained was triturated with ether to give 4-(4-chlorophenyl)-3- oxo-N-propyl-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetamide, m.p. 218-223°C.

Example 55

A stirred mixture of 4-(4-chlorophenyl) [1]benzo- pyrano[4,3-c]pyrazol-3(2H)-one (Example 13) (1.0 g) in toluene (20 ml) was treated with chlorosulphonyl isocyanate (0.53 g) at ambient temperature. The mixture was heated to 70°C and stirred . at this temperature .for 15 minutes. On cooling to 0°C, the mixture was filtered and the residue washed with ether then suspended in glacial acetic acid (6 ml) and water (3 ml). This mixture was heated at 60°C for 15 minutes and then filtered. The residue was washed with water, dried and recrystallised from dichloromethane/methanol, 4:1, to give 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]- benzopyrano[4,3-cjpyrazole-2-carboxamide,

m.p. 215-216°C.

Example 56

Acetyl chloride (0.86 ml) was added dropwise to a stirred mixture of 4-(4-chlorophenyl)[1]benzopyrano-

[4,3-c]pyrazol-3(2H)-one (Example 13) (3.0 g) and potassium carbonate (2.79 g) in AR acetone (20 ml) at ambient temperature. The mixture was stirred at ambient temperature for 42 hours. More potassium carbonate (6.98 g) was added followed by more acetyl chloride (3.6 ml) in AR acetone (15 ml). The mixture was stirred at ambient temperature for 18 hours. Morepotassium carbonate (2.79 g) and acetyl chloride

(1.44 ml) were added and the mixture stirred at ambient temperature for 2.25 hours. The mixture was then boiled under reflux for 1 hour, cooled to ambient temperature and then added to water. The solid formed was collected by filtration, dried and then recrystallised from acetonitrile to give 2-acetyl-4- (4-chlorophenyl) [1 ] benzopyrano [4 , 3-c] pyrazol-3(2H)-one, m.p . 224-226°C.

Example 57 A stirred mixture of 4-(4-chlorophenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (Example 13) (20 g) and ethyl chloroformate (200 ml) was boiled under reflux for 5 hours. More ethyl chloroformate (50 ml) was added and the mixture was stirred and boiled under reflux for 4 hours. The reaction mixture was cooled to ambient temperature and the solid was collected by filtration and recrystallised from acetonitrile. The crude product obtained was purified by flash column chromatography on silica using dichloromethane/methanol (9:1) as the mobile phase. Evaporation of the eluent gave a solid which was recrystallised from ethyl acetate to give ethyl 4-(4-chlorophenyl)-3-oxo-2,3- dihydro[1]benzopyrano[4,3-c]pyrazole-2-carboxylate, m.p. 187-188°C. Example 58

A stirred mixture of 4-(4-chlorophenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (Example 13) (5 g) and ethyl cyanoformate (50 ml) was boiled under reflux for 3.5 hours. The reaction mixture was cooled to ambient temperature, diluted with petroleum ether (30 ml), filtered and dried to give ethyl 4-(4-chlorophenyl)-3- oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2-carboxylate, m.p. 187-189°C. Example 59

A stirred suspension of 4-(4-chlorophenyl)-3-oxo- 2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2-acetyl chloride (0.97 g), prepared as in Example 52(a), in dry tetrahydrofuran (76 ml) was treated with a solution of cyclobutanol (0.7 g) in dry tetrahydrofuran (10 ml) followed by triethylamine (0.35 ml). The resulting mixture was stirred at ambient temperature for 2.67 hours and then filtered. The filtrate was diluted with ether, washed with water, dried and then evaporated under reduced pressure. The residue was purified by preparative layer chromatography on silica using dichloromethane/methanol (9:1) as the mobile phase. The fast running band was extracted with ethyl acetate. Evaporation under reduced pressure gave a gum which was triturated with ether/petroleum ether (b.p. 62-68°C, 1:1) and filtered to give cyclobutyl 4-(4-chlorophenyl)-3-oxo-2,3-dihydro[1]benzopyrano- [4,3-c]pyrazole-2-acetate, m.p. 135-138°C. Example 60

Sodium hydride (0.48 g, 60% dispersion in mineral oil) was added to a stirred solution of 4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (3.3 g), prepared as in Example 20, in dry N,N- dimethylformamide (60 ml) under nitrogen. The mixture was stirred at ambient temperature for 0.5 hours and then bromoacetonitrile (1.46 g) added in one portion. The mixture was stirred at ambient temperature for 18 hours and then added to iced water. The mixture was extracted with ethyl acetate and the combined extracts washed with water, dried and evaporated to give a solid which was recrystallised from propanol to give 3-oxo- 4-(4-trifluoromethylphenyl)-2,3-dihydro[1]benzopyrano- [4,3-c]pyrazole-2-acetonitrile, m.p. 215-217°C.

Example 61

In a similar manner to Example 60, 4-(4- trifluoromethylphenyl) [1]benzopyrano[4,3-c]pyrazol-

3(2H)-one (1.35 g), sodium hydride (0.21 g, 50% dispersion in mineral oil), 2-bromoethyl ethyl ether (0.77 g) and dry N,N-dimethylformamide (50 ml) were mixed and stirred at ambient temperature for 42 hours. The crude product obtained was purified by flash column chromatography on silica using ethyl acetate as the mobile phase to give, after recrystallisation from propan-2-ol, 2-(2-ethoxyethyl)-4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one,

m.p. 126-128°C.

Example 62

2-(2-Hydroxyethyl)-4-(4-trifluoromethylphenyl)- [1]benzopyrano[4,3-c]pyrazol-3(2H)-one (8.22 g) prepared as in Example 46 and 48% aqueous hydrobromic acid (500 ml) were stirred and heated under reflux for 28 hours. The reaction mixture was cooled to 0°C and the solid collected by filtration washed with water and then dried and recrystallised from acetonitrile to give 2-(2-bromoethyl)-4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 145-147°C.

Example 63 a) In a similar manner to Example 60, 4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (5 g), sodium hydride (0.7 g, 60% dispersion in mineral oil), ethyl 4-bromobutyrate (2.4 ml) and dry N,N- dimethylformamide (230 ml) were mixed and stirred at ambient temperature for 24 hours. The reaction mixture was added to water (2.3 1) and petrol (b.p. 60-80°C), acidified with 5M hydrochloric acid and the product formed was collected by filtration and dried to give ethyl 3-oxo-4-(4-trifluoromethylphenyl)-2,3-dihydro[1]- benzopyrano[4,3-c]pyrazole-2-butyrate, m.p. 112-115°C. b) A mixture of ethyl 3-oxo-4-(4-trifluoromethylphenyl)-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole-2- butyrate (3.0 g) and 5M hydrochloric acid (30 ml) was stirred and heated at 96-105°C for 5 hours. The reaction mixture was allowed to cool and was kept at ambient temperature for 24 hours. The solid obtained was collected by filtration, washed with water and then ether, and dried to give 3-oxo-4-(4-trifluoromethylphenyl)-2,3-dihydro[1]benzopyrano[4,3-c]- pyrazole-2-butyric acid, m.p. 197-200°C with shrinking from 189°C. c) A stirred suspension of 3-oxo-4-(4-trifluoro- methylphenyl)-2,3-dihydro[1]benzopyrano[4,3-c]pyrazol-

2-butyric acid (1.8 g) in dichloromethane (37 ml) was treated with N,N,-dimethylformamide (0.2 ml) and then thionyl chloride (1.2 ml). The mixture was stirred at ambient temperature for 24 hours and then evaporated under reduced pressure to give 3-oxo-4-(4-trifluoromethylphenyl)-2,3-dihydro[1]benzopyrano[4,3-c]pyrazole- 2-butyryl chloride. d) A solution of the acid chloride from stage (c) in dry tetrahydrofuran (88 ml) was stirred at 0.5°C and treated with concentrated aqueous ammonia solution (0.6 ml). The reaction mixture was stirred at 0-5°C for 1 hour. More concentrated ammonia solution (0.2 ml) was added and the mixture was stirred at 0-5 °C for 0.75 hour. The solid formed was collected by filtration, washed with tetrahydrofuran and water, and dried to give 3-oxo-4-(4-trifluoromethylphenyl)-2,3- dihydro[1]-benzopyrano[4,3-c]pyrazole-2-butyramide, m.p. 218-221°C. Example 64

4-(4-Chlorophenyl)-2-(2-hydroxyethyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one (28 g), prepared as in Example 47, and thionyl chloride (300 ml) were stirred and boiled under reflux for 3.5 hours. Excess thionyl chloride was distilled off under reduced pressure and the residue stirred with acetonitrile. The mixture was filtered and the solid collected was recrystallised from dichloromethane to give 2-(2-chloroethyl)-4-(4- chlorophenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 169-170°C.

Example 65

In the preparation of capsules, 10 parts by weight of active compound and 240 parts by weight of lactose are de-aggregated and blended. The mixture is filled into hard gelatin capsules, each capsule containing

10 mg active compound.

Example 66

In the preparation of capsules, 50 parts by weight of active compound, 300 parts by weight of lactose and 3 parts by weight of magnesium stearate are de-aggregated and blended. The mixture is filled into hard gelatin capsules, each capsule containing 50 mg of active ingredient. Example 67

Tablets are prepared from the following ingredients.

Parts by weight Active compound 10

Lactose 190

Maize starch 22

Polyvinylpyrrolidone 10

Magnesium stearate 3 The active compound, the lactose and some of the. starch are de-aggregated, blended and the resulting mixture is granulated with a solution of the polyvinylpyrrolidone in ethanol. The dry granulate is blended with magnesium stearate and the rest of the starch. The mixture is then compressed in a tableting machine to give tablets containing a) 10 mg, b) 100 mg and c) 500 mg of active compound.

Example 68

Tablets are prepared by the method of Example 67. The tablets are enteric coated in a conventional manner using a solution of 20% cellulose acetate phthalate and

3% diethyl phthalate in ethanol:dichloromethane (1:1).

Example 69

In the preparation of suppositories, 100 parts by weight of active compound is incorporated in 1300 parts by weight of semi-synthetic glycerides as the suppository base and the mixture formed into suppositories each contining 100 mg of active ingredient. Example 70

In the preparation of ointments the active compound is incorporated into the base by thorough homogenization until the drug is evenly distributed. The ointment is packed into 10 g amber jars with screwcapped lined lids.

Active compound 0.1 g

White soft paraffin to 10 g

The compounds of the invention are immunomodulatory agents, especially immunosuppressants and may show therapeutic activity at a dose of 200 mg/kg or lower. Preferred compounds of the invention show activity at 50 mg/kg or lower. The therapeutic activity of the preferred compounds of the present invention has been demonstrated by a cutaneous hypersensitivity test (CH test) in which the compounds are administered parenterally to BALB/c mice. This test was carried out in the following way.

Female BALB/c mice, weight range 16-24 g, were used in groups of eight. The abdomen of each mouse was shaved and 20 μl of a solution of a sensitising agent,

5% w/v 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one

(oxazolone) in acetone:ethanol (1:1 by volume), was applied to the shaved area. Immediately after sensitisation, the test compound in one of the dosages listed below was injected intraperitoneally as a suspension in 1.5% v/v sorbitan esters, under the trade name Tween 80, in sterile water (100 μ1). 100 μl of the same suspension was injected likewise every 24 hours for a further 7 days. The dosages used weee selected from the following values: 50, 30, 10, 3, 1, 0.3, 0.1, 0.03 or 0.01 mg/kg.

Two groups of at least eight BALB/c mice were used as a control simultaneously with each test in a similar manner to that described above except that no test compound was included in the daily injections.

On the seventh day after sensitisation, 10 μl of a solution of 1% w/v oxazolone in acetone: olive oil (3:1 by volume) was applied to one ear (the challenged ear) of each of the test mice and the control mice. (A more potent challenge dose of 1.5% w/v oxazolone in acetone:olive oil was employed in a few cases). After 24 hours the thickness of the challenged ear and the thickness of the non-challenged ear of the same animal were measured with an engineer's screw gauge micrometer. The difference in thickness between the challenged ear and the non-challenged ear in each animal is a measure of the response of that animal to oxazolone. A comparison between the response of mice treated with the test compound and mice treated with the control indicates the effectiveness of the test compound as an immunomodulatory agent. The compounds were considered to be active at a particular dose if a 20% or greater reduction in ear swelling, which was statistically significant (p <0.05) according to Dunnett's test, between treated and control groups was obtained in at least two out of three CH tests, (or, where more than three tests have been carried out, a majority of the tests) at that dose (see for example Int. Arch. Allergy, 38, p246-259 (1970)).

Each of the compounds of formula I illustrated in Table A below was active at 50 mg/kg in at least two out of three tests at 50 mg/kg unless indicated otherwise (see Notes following the Table). The minimum effective dose for each compound is given in Table A. The Example (Ex) number or numbers listed adjacent to each compound indicates the process or processes illustrating the preparation of that compound in the Examples.

Table A

Ex Compound Name Minimum

Effective Dose

(mg/kg)

9-methoxy-4-(4-trifluoromethylphenyl)- ≤3 8b [1]benzopyrano[4,3-c]pyrazol-3(2H)-one

2-(2-ethoxyethyl)-9-'methoxy-4-(4- 3

8c trifluoromethylphenyl) [1]benzopyrano- [4,3-c]pyrazol-3(2H)-one

4-(4-chlorophenyl)-9-ethoxy-2-methyl- 50 9 [1]benzopyrano[4,3-c]pyrazol-3(2H)- one 4-(4-chlorophenyl)-2-(2,2,2-trifluoro50 10 ethyl)[1]benzopyrano[4,3-c]pyrazol-3- (2H)-one

4-(3,4-dichlorophenyl)-2-methyl[1]- 50 11 benzopyrano[4,3-c]pyrazol-3(2H)-one

2-methyl-4-(2-naphthyl) [1]benzo- 50 12 pyrano[4,3-c]pyrazol-3(2H)-one

4-(4-chlorophenyl) [1]benzopyrano- 3

19 [4,3-c]ρyrazol-3(2H)-one

4-(4-chlorophenyl)-2-methyl[1]benzo1024 pyrano[4,3-c]pyrazol-3(2H)-one 4-(4-chlorophenyl)-2-ethyl[1]benzo^50

15 pyrano[4,3-c]pyrazol-3 (2H)-one Ex Compound Name Minimum

Effective Dose

(mg/kg) 4-(4-chlorophenyl)-9-methoxy-2-methyl- 50

16 [1] benzopyrano[4,3-c]pyrazol-3(2H)-one

4-(4-chlorophenyl)-2,9-dimethyl[1]- 50

17 benzopyrano[4,3-c]pyrazol-3(2H)-one

2-methyl-4-(4-trifluoromethylphenyl)- 3 18 [1] benzopyrano[4,3-c]pyrazol-3(2H)- one 23,20 4- (4-trifluoromethylphenyl)[1]benzo3

21,22 pyrano[4,3-c]pyrazol-3(2H)-one

2-[4-(4-chlorophenyl)-3-oxo-2,3- 50 26 dihydro[1]benzopyrano[4,3-c]pyrazol- 2-yl]ethyl acetate

4-(4-chlorophenyl)-2-(2-methoxyethyl) 10 27 [1] benzopyrano[4,3-c]pyrazol-3(2H)- one 4-(4-chlorophenyl)-3-oxo-2,3-dihydro ≤50

28 [1] benzopyrano[4,3-c]pyrazole-2- acetonitrile

29 4-(4-chlorophenyl)-2-(2-phenoxyethyl) 50

[1] benzopyrano[4,3-c]pyrazole-3(2H)- one

30 2-allyl-4-(4-chlorophenyl) [1]benzo50

pyrano[4,3-c]pyrazol-3(2H)-one Ex Comound Name Minimum

Effective Dose

(mg/kg)

32 4-(4-chlorophenyl)-3-oxo-2,3-dihydro- 50

[1]benzopyrano[4,3-c]pyrazole-2- propionitrile

33 4-(4-chϊorophenyl)-2-propyl[1]benzo50

pyrano[4,3-c]pyrazol-3(2H)-one 34 4-(4-chlorophenyl)-3-oxo-2,3-dihydro- ≤30 (a)

[1] benzopyrano[4,3-c]pyrazole-2- carbonitrile

35 4- (4-chlorophenyl) -2- (2-ethoxyethyl)

[1 ] benzopyrano [4 , 3-c] pyrazol-3(2H)- one

36 2-butyl-4- (4-chlorophenyl) [ 1 ]benzo50

pyrano [4 , 3-c] pyrazol-3(2H)-one

37 4-(4-chlorophenyl)-2-[2-(2-methoxy- 50

ethoxy)ethyl][1]benzopyrano[4,3-c]- pyrazol-3(2H)-one 8 4-(4-chlorophenyl)-3-oxo-2,3-dihydro- 50

[1]benzopyrano[4,3-c]pyrazole-2- acetophenone 9 2-(2-[4-(4-chlorophenyl)-3-oxo-2,3- 50

dihydro[1]benzopyrano[4,3-c]pyrazol- 2-yl]ethoxy)ethyl acetate Ex Compound Name Minimum

Effective

Dose

(mg/kg) 4-(4-chlorophenyl)-2-[2-(1,3-dioxolan- 50

40 2-yl)ethyl][1]benzopyrano[4,3-c]pyrazol- 3(2H)-one

4-(4-chϊorophenyl)-2-(1,3-dioxolan-2- 50

42 ylmethyl)[1]benzopyrano[4,3-c] pyrazol- 3(2H)-one

4-(4-chlorophenyl)-2-(2-ethoxypropyl)- 50

43 [1]benzopyrano[4,3-c]pyrazol-3(2H)-one

2-[3-oxo-4-(4-trifluoromethylphenyl)- 50

44 2,3-dihydro[1]benzopyrano[4,3-c]pyrazol- 2-yl]ethyl acetate

4-(4-chlorophenyl)-2-[2-(1,3-dioxan-2- 50

45 yl)ethyl][1]benzopyrano[4,3-c]pyrazol- 3(2H)-one

2-(2-hydroxyethyl)-4-(4-trifluoro50

46 methylphenyl)[1]benzopyrano[4,3-c]- pyrazol-3(2H)-one

4-(4-chlorophenyl)-2-(2-hydroxyethyl)- 50

47 [1]benzopyrano[4,3-c]pyrazol-3(2H)-one

4-(4-chlorophenyl)-2-(3-hydroxypropyl)- 50

48 [1]benzopyrano[4,3-c]pyrazol-3(2H)-one

4-(4-chlorophenyl)-2-[2-(2-hydroxy50

49 ethoxy)ethyl][1]benzopyrano[4,3-c]- pyrazol-3(2H)-one Ex Compound Name Minimum

Effective Dose

(mg/kg)

4-(4-chlorophenyl)-3-oxo-2,3-dihydro- 50 [1] benzopyrano[4,3-c]pyrazole- 2-acetamide

4-(4-chlorophenyl)-3-oxo-2,3-dihydro- 50

51 [1]benzopyrano[4,3-c]pyrazole-2- propionamide

4-(4-chlorophenyl)-N-ethyl-3-oxo-2,3- 50

52 dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetamide

4-(4-chlorophenyl)-N-methyl-3-oxo-2,3- 50

53 dihydro[1]benzopyrano[4,3-c] pyrazole- 2-acetamide

4-(4-chlorophenyl)-3-oxo-N-propyl-2,3- 50

54 dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetamide 4-(4-chlorophenyl)-3-oxo-2,3-dihydro- ≤50

55 [1]benzopyrano[4,3-c]pyrazole-2-carbox- amide

2-acetyl-4-(4-chlorophenyl)[1]benzo50

56 pyrano[4,3-c]pyrazol-3(2H)-one Ethyl 4-(4-chlorophenyl)-3-oxo-2,3- 50

58 dihydro[1]benzopyrano[4,3-c]pyrazole-2- carboxylate Ex Compound Name Minimum

Effective Dose

(mg/kg) cyclobutyl 4-(4-chlorophenyl)-3-oxo- 50

59 2,3-dihydro[1]benzopyrano[4,3-c]- pyrazole-2-acetate

3-oxo-4-(4-trifluoromethylphenyl)-2,3- ≤3

60 dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetonitrile

2-(2-ethoxyethyl)-4-(4-trifluoromethyl- 61 phenyl)[1]benzopyrano[4,3-c]pyrazol- 3(2H)-one

2-(2-bromoethyl)-4-(4-trifluorσmethyl- ≤3

62 phenyl)[1]benzopyrano[4,3-c]pyrazol- 3(2H)-one

3-oxo-4-(4-trifluoromethylphenyl)-2,3- ≤50

63 dihydro[1]benzopyrano[4,3-c]pyrazole- 2-butyramide 2-(2-chloroethyl)-4-(4-chlorophenyl)- ≤50

64 [1]benzopyrano[4,3-c]pyrazol-3(2H)-one

The following compounds were prepared in an

analogous manner and also found to be active in the above described CH test at 50 mg/kg.

2-[4-(4-chlorophenyl)-3-oxo-2,3-dihydro- [1]benzopyrano[4,3-c]pyrazol-2-yl]ethyl

propionate, m.p. 112-114°C. Ex Compound Name

4-(4-chlorophenyl)-N-isopropyl-3-oxo-

2,3-dihydro[1]benzopyrano[4,3-c]pyrazole- 2-acetamide, m.p. 238-241 (decomposition)

N-benzyl-4-(4-chlorophenyl)-N-methyl-3- oxo-2,3-dihydro[1]benzopyrano[4,3-c]- pyrazole-2-acetamide m.p. 182-183°C

Notes

(a) Active in each of two tests at 30 mg/kg.

The compounds of the present invention also show activity in a variety of other in-vivo screens, which show the utility of the compounds as immunomodulants, particularly in suppressing the immune response.

Administration of the compounds has been carried out orally, or parenterally. Some compounds have been found to be active in a test which determines their effects on humoral immunity by assaying the sera collected at the end of the oxazolone induced cutaneous hypersensitivity test described above (CH test) for changes in the amount of anti-oxazolone antibody produced, and a Graft versus Host test similar to that used by Smith S R, Terminelli C, Kipilman C T and Smith Y., J. Immunopharmacology 1981; 3 (2), 133-170.

For example, the compounds prepared in the

following Examples were also found to be active in the above-described antibody test after parenteral

administration at 50 mg/kg. A compound was deemed to be active, if at a dose of 50 mg/kg it caused a decrease in the relative serum anti-oxazolone antibody concentration determined by an enzyme linked

immunosorbent assay (ELISA) by a factor of 0.5 or greater calculated by the following formula:-

O.D. (C₁) - O.D. (T₁)

O.D. (C₁) - O.D. (C₂) where O.D.(C₁) is the optical density of the control serum at a dilution of 1/128

O.D. (C₂) is the optical density of the control serum at a dilution of 1/256

O.D. (T₁) is the optical density of the test serum at a dilution of 1/128

The control and test sera were diluted with phosphate buffered saline (pH 7.3) containing 0.05% v/v Tween 20 (trade name).

Compounds active in above test:

Examples: 8b, 8c, 10-15, 18, 20, 26, 27, 30, 33, 35, 37, 38, 40, 42, 44-47, 49, 52, 55-64.

Claims (12)

1. A compound of formula I

in which R₁ represents hydrogen, cyano, C_{2 -6} alkanoyl,

C_2-6 alkoxycarbonyl, CONH₂, or R₁ represents C_{1 -6} alkyl or C_2-6 alkenyl both of which may be substituted by cyano, halo, trifluoromethyl, hydroxy, benzoyi, C_{2 -6} alkanoyloxy, C_{3 -8} cycloalkoxycarbonyl, a 5-7 membered non-aromatic heterocyclic group containing 2 oxygen heteroatoms- CONR₉R₉,, phenoxy or C_1-6 alkoxy, in which C_{1 -6} alkoxy may be further substituted by halo, hydroxy, C_{1 -6} alkoxy or C_{2 -6} alkanoyloxy;

R₂ represents hydrogen or chloro;

R ₃ represents chloro or trifluoromethyl; or R₂ and R₃ are joined to form a fused benz ring;

R₄ represents hydrogen, C_{1 -6} alkyl, C_{1 -6} alkoxy or halo;

R₉ and R₉, , which may be the same or different. represent hydrogen, C_{1 -6} alkyl or benzyl.

2. A compound according to claim 1 in which R₂ represents hydrogen; R₃ represents chloro or trifluoromethyl; R₁ represents (CH₂)_pJ, J represents hydrogen, C_{1 -6} alkoxy, carbamoyl, cyano or halo; R₄ represents hydrogen or C_{1 -6} alkoxy and p is 0, 1 or 2.

3. A compound according to claim 2 in which R₁ represents hydrogen, methyl, cyanomethyl, (CH₂)₂halo, carbamoylmethyl or C_{1 -4} alkoxyethyl.

4. A compound according to claim 3 in which R₆ represents methyl or 2-ethoxyethyl.

5. A compound selected from:

2-(2-ethoxyethyl)-9-methoxy-4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one

2-methy1-4-(4-trifluoromethylphenyl)[1]benzopyrano[4,3-c]pyrazol-3(2H)-one 2-(2-ethoxyethyl)-4-(4-trifluoromethylphenyl)[1]- benzopyrano[4,3-c]pyrazol-3(2H)-one

6. A pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1-5 together with a pharmaccutical carrier.

7. A pharmaceutical composition according to claim 6 in unit dosage form.

8. A method of treating diseases with an immunological association comprising the administration of a therapeutically effective amount of a compound of formula I as claimed in any one of claims 1-5.

9. A compound of formula I as claimed in any one of claims 1-5 for use as an immunomodulatory agent.

10. A compound of formula I as claimed in any one of claims 1-5 in the manufacture of a medicament for use in the treatment of diseases with an immunomological association.

11. A process to prepare a compound of formula I as claimed in claim 1:- a) comprising the reaction of a compound of formula II

or a tautomer thereof , with a compound of formula III

in which R₁₄ represents (OQ)₂ or (SQ)₂ and R₁₅ represents OQ or SQ or NQ'₂; R₁₄ represents =NH; and R₁₅ represents OQ or SQ; or R₁₄ represents =O and R₁₅ represents hydrogen, halo or 1-imidazolyl; and Q and Q' represent a C_{1 -4} alkyl group or a benzyl group; b) comprising the reaction of compounds of formula IV

in which R₁₆ represents hydrogen, or a tautomer thereof, or in which R₁₆ represents a group COR₁₈ wherein in R₁₈ represents hydrogen, a C_{1 -4} alkyl group, a benzyl group or a group R₅ and R₁₇ represent COR₅ with a base;

12. A compound of formula II

in which R₁ and R₄ are as defined in claim 1