WO1993003036A1

WO1993003036A1 - BENZOPYRANO[4,3-c]PYRAZOLES, THEIR PREPARATION AND THEIR USE AS IMMUNOMODULATORS

Info

Publication number: WO1993003036A1
Application number: PCT/EP1992/001639
Authority: WO
Inventors: Roger Bernard Titman; Michael Henry Hockley
Original assignee: The Boots Company Plc
Priority date: 1991-07-27
Filing date: 1992-07-21
Publication date: 1993-02-18
Also published as: GB9116241D0; IL102581A0; PT100732A; MX9204269A; ZA925577B; AU2345092A; IE922217A1

Abstract

Compounds of formula (I), wherein R1 represents hydrogen or methyl; R2 represents hydrogen, C2-6 alkoxycarbonyl or C2-6 alkanoyl; R3 represents hydrogen, halo, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy or S(O)mY1; R4 represents hydrogen, halo, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy or S(O)mY1; or R3 and R4 together with the carbon atoms to which they are attached, represent a fused benz ring which may be substituted by one or more groups selected from C1-6 alkyl, C1-6 alkoxy, S(O)mY1, halo or trifluoromethyl; R5 represents hydrogen, halo, trifluoromethyl, C1-6 alkyl, C1-6 alkoxy, hydroxy or C2-6 alkanoyloxy; Y1 represents C1-6 alkyl; m is 0, 1 or 2, or a pharmaceutically acceptable salt thereof for use as immunomodulatory agents.

Description

BENZOPYRANO[4,3-c]PYRAZOLES, THEIR PREPARATION AND THEIR USE AS IMMUNOMODULATORS

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 imrhunomodulatory agents.

European Patent Application 354693 relates to compounds of structure A

wherein R₁ represents hydrogen or together with R₂ represents a bond; R₂ together with either one of R₁ and

R₃ represents a bond; R₃ together with either one of R₂ and R₄ represents a bond; R₄ represents hydrogen or together with R₃ represents a bond; R₅ represents hydrogen or methyl; R₆ represents hydrogen, halo, a C_2-6 alkanoyl group, a C_2-6 alkoxycarbonyl group, a C_1-6 alkylthio group, a C_1-6 alkylsulphinyl group, a C_1-6 alkylsulphonyl group, carbamoyl, carboxy, or R₅ and R₆ together with the carbon atom to which they are attached represent a cyclopropyl group; R₇ represents hydrogen, halo, trifluoromethyl, methoxy, a C_1-6 alkyl group, a C_1-6 alkyithio group or a C_1-6 alkylsulphinyl group; R₈ represents hydrogen, halo or trifluoromethyl; R₉ and R₁₀, which may be the same or different, each represent halo; or R₉ represents hydrogen and R₁₀ represents hydrogen, halo, trifluoromethyl, hydroxy, nitro, a C_{2- 6} alkanoyloxy group, a C_{1- 6} alkyl group or a C_{2- 6} alkoxy group, which have immunomodulatory activity.

The present invention provides novel compounds of formula 1

wherein R₁ represents hydrogen or methyl; R₂ represents hydrogen, C_2-6 alkoxycarbonyl or C_2-6 alkanoyl;

R₃ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_{1- 6} alkoxy or S(O)_my_1;

R₄ represents hydrogen, halo, trifluoromethyl,

C_1-6 alkyl, C_1-6 alkoxy or S(O)_my_1; or R₃ and R₄, together with the carbon atoms to which they are attached, represent a fused benz ring which may be substituted by one or more groups selected from C_1-6 alkyl, C_1-6 alkoxy, S(O)_my₁, halo or trifluoromethyl; R₅ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy, hydroxy or C_2-6 alkanoyloxy;

Y₁ represents C_1-6 alkyl;

m is 0, 1 or 2; or pharmaceutically acceptable salts thereof, which have immunomodulatory activity.

Compounds of formula l may be considered to possess therapeutic advantages over compounds known in the art.

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 length of the carbon chain is specified for certain substituents, for example C_2-6 alkoxycarbonyl refers to an alkoxycarbonyl group having from two to six carbon atoms. The term "halo" covers fluoro, chloro or bromo.

In preferred compounds of formula l, R₁ represents hydrogen. Preferably R₂ represents hydrogen, C_2-4 alkanoyl

(for example acetyl or propanoyl) or C_2-6 alkoxycarbonyl

(for example methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl or butoxycarbonyl). Most preferably R₂ represents hydrogen, acetyl or C_2-4 alkoxycarbonyl. Particularly preferred are compounds in which R₂ represents hydrogen, methoxycarbonyl, ethoxycarbonyl or propoxycarbonyl (namely n-propoxycarbonyl and isopropoxycarbonyl), especially hydrogen or ethoxycarbonyl. Suitably R₃ and R₄, which may be the same or different, represent hydrogen, halo, trifluoromethyl, C_1-6 alkyl, preferably C_1-4 alkyl (for example methyl, ethyl or propyl), C_1-6 alkylthio (for example methylthio or ethylthio), C_1-6 alkylsulphinyl (for example methylsulphinyl or ethylsulphinyl) or C_1-6 alkoxy (for example methoxy, ethoxy or propoxy).

In preferred compounds of formula l, R₃ represents hydrogen, halo, trifluoromethyl or C_1-6 alkylthio. More preferably R₃ represents hydrogen, fluoro, chloro, bromo or trifluoromethyl, especially hydrogen. In preferred compounds of formula l, R₄ represents hydrogen, halo, trifluoromethyl or C_1-6 alkylthio. More preferably R₄ represents hydrogen, fluoro, chloro, bromo or trifluoromethyl, especially hydrogen or trifluoromethyl, and most preferably trifluoromethyl. In further preferred compounds of formula l, R₃ and R₄, together with the carbon atoms to which they are attached, are joined to form a fused benz ring which may be substituted by one or more groups selected from C_1-4 alkyl (for example methyl, ethyl or propyl), C_1-4 alkoxy (for example methoxy, ethoxy or propoxy), halo or trifluoromethyl. Preferably the ring is unsubstituted or has a single substituent. The substitution may occur at any of positions 4-, 5-, 6- and/or 7- of the benzothiazole group. The substituent R₅ may be located at any position on the benz ring, namely in position 6-, 7-, 8- or 9- of the benz ring. Accordingly the substituents R₅ specified herein are to be considered as named at each of these positions. Suitably R₅ represents hydrogen, fluoro, trifluoromethyl, hydroxy, C_2-6 alkanoyloxy, C_1-6 alkyl (especially in position 8- or 9- of the benz ring), or C_1-6 alkoxy (especially in position 9- of the benz ring). As stated above R₅ may be located at the 6position, especially for the substituents 6-fluoro, 6hydroxy or 6-methoxy. The 7-position may also bear a substituent such as 7-fluoro or 7-hydroxy. In preferred compounds of formula l, R₅ is located at position 8- or 9- of the benz ring and represents hydrogen, fluoro, trifluoromethyl, hydroxy, C_2-6 alkanoyloxy (for example acetoxy, propanoyloxy, butanoyloxy or pentanoyloxy), C_2-4 alkyl (for example methyl, ethyl or propyl)- or C_2-4 alkoxy (for example methoxy or ethoxy), more preferably hydrogen, fluoro, hydroxy or C_2-6 alkanoyloxy. Most preferably R₅ represents hydrogen, fluoro, hydroxy or C_2-4 alkanoyloxy . In particularly preferred compounds

R ₅ represents hydrogen, fluoro or C_2-4 alkanoyloxy in position 8- of the benz ring or hydrogen, hydroxy or C_2-4 alkanoyloxy in position 9- of the benz ring.

Especially preferred are compounds in which R₅ represents hydrogen, 8-fluoro, 9-hydroxy or 9-acetoxy.

Preferred classes of compounds of formula l are those represented by formula lla

wherein R₁, R₂, R₃, R₄ and R₅ are as hereinbefore defined. In compounds of formula Ila, more preferably R₅ represents hydrogen, halo (for example fluoro or chloro), hydroxy or C_2-6 alkanoyloxy, especially hydrogen or fluoro. In compounds of formula llb, more preferably R₅ represents hydrogen, hydroxy, C_2-6 alkoxy or C_2-6 alkanoyloxy, especially hydrogen, hydroxy or C_2-4 alkanoyloxy.

In compounds of formula lla and llb, R₁ preferably represents hydrogen and R₂ preferably represents hydrogen or C_2-6 alkoxycarbonyl (for example methoxycarbonyl, ethoxycarbonyl or propoxycarbonyl). In such compounds R₃ preferably represents hydrogen and R₄ preferably represents halo or trifluoromethyl. More preferably R₃ represents hydrogen and R₄ represents trifluoromethyl.

Further preferred classes of compounds are those represented by formula llla

wherein R₁, R₂ and R₅ are as hereinbefore defined, R₈ represents C_1-6 alkyl, C_1-6 alkoxy, halo or trifluoromethyl and q is 0, 1 or 2, more preferably 0 or 1, most preferably 0. In compounds of formula llla, preferably R₁ represents hydrogen, R₂ represents hydrogen or C_2-6 alkoxycarbonyl and R₅ represents hydrogen, hydroxy, fluoro or C_2-6 alkanoyloxy and q is 0. In compounds of formula lllb, preferably R₁ represents hydrogen, R₂ represents hydrogen or C_2-6 alkoxycarbonyl and R₅ represents hydrogen, hydroxy or C_2-6 alkanoyloxy and q is 0. In compounds of formula llla, most preferably R₅ represents hydrogen or fluoro. In compounds of formula lllb, most preferably R₅ represents hydrogen, hydroxy or C_2-6 alkanoyloxy. Particularly preferred compounds of formula l are the compounds listed in Table A, including hydrates or solvates.

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 which may be separated, for example, by crystallisation; formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallisation, gas-liquid 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. It will be appreciated that where the active moiety is transformed by the separation procedures described above, a further step is required to convert the product to the active moiety. 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.

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.

Some compounds of formula I are bases and may form salts with inorganic or organic acids, for example hydrochloric acid or hydrobromic acid. Other compounds of formula I are acidic and may form salts with inorganic or organic bases, for example sodium hydroxide. It will be appreciated that such salts, provided they are pharmaceutically acceptable, may be used in therapy in place of the corresponding compounds of formula I. Such salts may be prepared for example by reacting the compound of formula I with a suitable acid or base in a conventional manner.

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 1 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 nontoxic suspending agent such as sodium carboxymethy- lcellulose, 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 semisynthetic 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 oxidising compounds of formula IV

or tautomers thereof, for example by reaction with chloranil.

Compounds of formula I may also be prepared by reacting compounds of formula V

or tautomers thereof, with compounds of formula VI

R₁R₂CHCR₉R₁₀ V I in which R₉ represents (OQ)₂ and R₁₀ represents OQ or NQ'₂; or R₉ represents (SQ)₂ and R₁₀ represents SQ or NQ'₂; or R₉ represents =NH and R₁₀ represents OQ or SQ; or R₉ represents =O and R₁₀ represents a leaving group for example an optionally substituted 1-imidazolyl group, in which Q and Q' represent C_1-4 alkyl or benzyl, for example by heating at 50-200°C. Compounds of formula I in which R₁ represents hydrogen and R₂ represents C_2-6 alkanoyl may also be prepared by reacting compounds of formula V, or tautomers thereof, with compounds of formula Vlla

or tautomers thereof, in which R₁₁ represents C_1-6 alkyl, R₁₂ and R₁₃ may be the same or different, and each represent C_1-6 alkyl or benzyl, for example by heating in an organic liquid for example xylene at a temperature between 50-200°C.

Compounds of formula I in which R₂ represents C_2-6 alkanoyl may also be prepared by reacting compounds of formula V, or tautomers thereof, with compounds of formula VIIb

or tautomers thereof, in which R₁₁ represents C_1-5 alkyl, R₁₂ and R₁₃ may be the same or different and each represents C_1-6 alkyl or benzyl, for example by heating in an organic liquid, for example xylene at a temperature between 50 and 250°C.

Compounds of formula I in which R₅ represents C_2-6 alkanoyloxy may also be prepared by acylating compounds of formula I in which R₅ represents hydroxy by reaction with an acylating agent. The acylation reaction may be carried out by reacting the hydroxy compound of formula II with an acyl halide e.g. R₁₄COCl or an acid anhydride (R₁₄ CO)₂O in which R₁₄ represents C_1-5 alkyl in the presence of a base at a temperature in the range -10°C to 40°C. The acylation reaction may also be carried out by reacting the compound of formula I with a carboxylic acid R₁₄COOH in the presence of a dehydrating agent, for example dicyclohexylcarbodiimide, preferably in the presence of a base e.g. pyridine. Compounds of formula I in which R₅ represents hydroxy may be prepared by reacting compounds of formula I in which R₅ represents C_1-6 alkoxy with a Lewis acid, for example aluminium chloride or boron tribromide.

Compounds of formula I may also be prepared by reacting compounds of formula VIII

in which R₁₅ represents hydrogen, or tautomers thereof, or in which R₁₅ represents a group COR₁₇ wherein R₁₇ represents hydrogen, optionally substituted C_1-4 alkyl or benzyl and R₁₆ represents COCHR₁R₂, with a base e.g. piperidine in a suitable solvent e.g ethanol.

Compounds of formula I which are represented by formulae Ila/b and IIIa/b may be prepared as described with reference to the preparation of compounds of formula I above.

Compounds of formula IV may be prepared by reducing compounds of formula I for example by reaction with sodium borohydride. Compounds of formula lV may also be prepared by reacting compounds of formula lX

in which R₁₈ represents COOR₁₉ or carbamoyl and R₁₉ represents C_1-4 alkyl or benzyl with a hydrazine of formula X

for example, by heating at 50-250°C for example in acetic acid or in an inert organic liquid containing an acid catalyst, e.g. xylene containing p-toluenesulphonic acid. Compounds of formula V may be prepared by reacting compounds of formula Xl

in which R₂₀ represents hydrogen, with a hydrazine of formula X, for example by heating at 50-200°C in an organic liquid for example toluene. Preferably the compound of formula is used in excess of the stoichiometric amount .

Compounds of formula V in which R₅ represents hydroxyl may also be prepared by reacting compounds of formula V in which R₅ represents C_1-6 alkoxy with a

Lewis acid, for example aluminium chloride or boron tribromide.

Compounds of formula Vl in which R₉ represents (OQ)₂ and R₁₀ represents OQ may be prepared for example a) by reacting compounds of formula R₁R₂CH-CX₃ in which X is halo with a sodium alkoxide of formula NaOQ in which Q is C_1-4 alkyl or benzyl, or b) by reacting compounds of formula R₁R₂CH-CN with an alcohol of formula QOH in the presence of an anhydrous acid, for example hydrogen chloride, to give compounds of formula R₁R₂CH-C(=NH)OQ as their acid salts, e.g. hydrochloride salts, which are then reacted with further alcohol of formula QOH.

Compounds of formula Vl in which R₉ represents (SQ)₂ and R₁₀ represents SQ may be prepared for example from compounds of formula R₁R₂CH-COCl by reaction with thiols of formula QSH in which Q represents C_1-4 alkyl or benzyl in the presence of a Lewis acid, for example zinc chloride. Other compounds of formula Vl and compounds of formula Vlla may be prepared by methods known to those skilled in the art.

Compounds of formula Vllb or tautomers thereof may be prepared by the acylation of compounds of formula Xll

by reaction with an acyl chlori R₁₁-COCl, for example in the presence of pyridine in an inert solvent at a temperature in the range -10°C to 50°C.

Compounds of formula Vlll in which R₁₅ represents COR₁₇ and R₁₆ represents COCHR₁R₂) may be prepared by acylation of compounds of formula Vlll in which R₁₅ represents COR₁₇ and R₁₆ represents hydrogen, for example by reaction with an acid anhydride of formula (R₁R₂CHCO)₂O or an acid halide e.g. of formula R₁ R₂CHCOCl.

Compounds of formula Vlll in which R₁₅ represents COR₁₇ and R₁₆ represents hydrogen may be prepared by the acylation of compounds of formula V for example by reaction with an acid anhydride of formula (R₁₇CO)₂O in the presence of a salt (e.g. the sodium salt) of the corresponding acid (e.g. at ambient temperature for 2.5 hours).

Compounds of formula Vlll in which R₁₅ and R₁₆ are identical and represent COCHR₁R₂ , may be prepared by acylation of compounds of formula V for example by using an acid anhydride of formula (R₁R₂CHCO)₂O in the presence of a salt (e.g. the sodium salt) of the corresponding acid (e.g. at ambient temperature for 19 hours or under reflux for 0.5 hours). Compounds of formula Vlll in which R₁₆ represents COCHR₂R₂ and R₁₅ represents hydrogen, or tautomers thereof, may be prepared by reacting a compound of formula Vlll in which R₁₅ represents COR₁₇ and R₁₆ represents COCHR₁R₂ with a base e.g. piperidine in a suitable solvent e.g. ethanol.

Compounds of formula lX in which R₁₈ represents COOR₁₉ may be prepared by alkylating compounds of formula Xlll

for example by reaction with lithium dialkyl copper.

Compounds of formula lX in which R₁₈ represents carbamoyl may be prepared from compounds of formula 1X in which R₁₈ represents cyano by methods known to those skilled in the art, for example by reacting with methanol saturated with hydrogen chloride and then water.

Compounds of formula X may be made by methods known to those skilled in the art. One method is the reaction of compounds of formula XIV

B NHNH X IV

\

in which B represents hydrogen or an acyl group for example C_2-6 alkanoyl such as acetyl, with compounds of formula XV O

II

R₄CCHR₃X X V

in which X represents halo. The above reaction yields compounds of formula X as the hydrohalide salt, which may be basified to give compounds of formula X.

Compounds of formula Xl may be prepared by reacting compounds of formula XVl

in which R₂₁ 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 Xl may also be prepared by reacting compounds of formula XVl in which R₂₁ represents a group COR₂₂ in which R₂₂ represents C_1-5 alkyl, with a base, for example sodium hydride, followed by treatment with a dialkyl carbonate of formula (QO)₂CO in which Q represents C_1-4 alkyl or benzyl, e.g. dimethyl carbonate.

Compounds of formula XII to XVI may be prepared by methods known to those skilled in the art.

Certain intermediate compounds of formulae IV, V, VIII, X and salts thereof are believed to be novel compounds. All novel compounds herein are claimed as a further aspect of the invention. The invention is illustrated by the following nonlimitative 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 spectroscopy.

Example 1 a) A stirred mixture of 4-hydroxycoumarin (6.5 g) and 2-hydrazinobenzothiazole (10.0 g) in dry toluene (69 ml) was heated under reflux for 3.7 hours, with removal of the water formed in the reaction. The solid obtained on cooling was collected by filtration and digested into boiling industrial methylated spirit followed by hot filtration. On cooling the filtrate, the solid formed was collected by filtration to give 1-(2benzothiazolyl)-3-(2-hydroxyphenyl)-2-pyrazolin-5-one, m.p. 193-196°C. b) A stirred mixture of 1- (2-benzothiazolyl)-3-(2- hydroxyphenyl)-2-pyrazolin-5-one (1.0 g) and triethyl orthoacetate (1.7 ml) was heated at 130-135°C for 10 minutes. On cooling, ether was added and the solid formed was collected by filtration to give 2-(2benzothiazolyl)-4-methyl[1]benzopyrano[4,3-c]pyrazol3(2H)-one, m.p. 239-241°C (with shrinkage at 223°C).

Example 2

A stirred mixture of 1-(2-benzothiazolyl)-3-(2hydroxyphenyl)-2-pyrazolin-5-one, (1.0 g) (Example 1a) and triethyl ortho (2-ethoxycarbonyl) acetate (2.3 g) was heated at 130-135°C for 15 minutes. On cooling, ether was added and the solid formed was collected by filtration to give ethyl 2-(2-benzothiazolyl)-3-oxo-2,3 dihydro[1]benzopyrano[4,3-c]pyrazole-4-acetate, m.p. 185-189°C.

Example 3 a) A stirred mixture of 6-fluoro-4-hydroxycoumarin (10 g) and 2-hydrazinobenzothiazole (6.9 g) in dry toluene (100 ml) was heated under reflux for 0.5 hours, with removal of the water formed in the reaction. The solid obtained on cooling was collected by filtration, digested with dichloromethane and filtered. The filtrate was extracted with aqueous sodium hydroxide solution (5M) and the extract was acidified with dilute hydrochloric acid. The resulting solid was collected by filtration, washed with water and isopropanol, and recrystallised from ethyl acetate to give 1-(2-benzothiazolyl)-3-(5-fluoro-2-hydroxyphenyl)-2-pyrazolin-5- one, m.p. 221-222°C. b) A stirred mixture of 1-(2-benzothiazolyl)-3-(5fluoro-2-hydroxyphenyl)-2-pyrazolin-5-one (0.8 g) and triethyl orthoacetate (7.2 ml) was heated at 140°C for 5 minutes. On cooling, the solid obtained was collected by filtration and washed with ether and then hot ethyl acetate. Recrystallisation from dichloromethane/ methanol gave 2-(2-benzothiazolyl)-8-fluoro-4- methyl[1]benzopyrano[4,3-c]pyrazol-3(2H)-one, m.p. 278- 280°C (with decomposition).

Example 4 a) A stirred mixture of 4,5-dihydroxycoumarin (7.2 g) and 2-hydrazinobenzothiazole (10 g) in dry toluene (69 ml) and p-toluenesulphonic acid (0.2 g) was heated under reflux for 5.5 hours, with removal of the water formed in the reaction. The solid obtained on cooling was collected by filtration and digested with boiling industrial methylated spirit. After hot filtration, the solid collected was partitioned between aqueous sodium hydroxide solution (5M) and ethyl acetate. The aqueous phase was acidified with concentrated hydrochloric acid and was digested with a mixture of boiling methanol and dichloromethane and the mixture hot filtered to give 1- (2-benzothiazolyl)-3-(2,6-dihydroxyphenyl)-2-pyrazolin- 5-one, m . p . 264-266°C (with decomposition ) . b) A stirred mixture of 1-(2-benzothiazolyl)-3-(2,6- dihydroxyphenyl)-2-pyrazolin-5-one (1.3 g) and triethyl orthoacetate (2.2 ml) was heated at 130-135°C for 15 minutes. A further portion of triethyl orthoacetate

(2.2 ml) was added to the reaction mixture and stirring was continued at 130-135°C for a further 15 minutes. On cooling, ether was added to the reaction mixture. After filtration the solid collected was suspended in boiling methanol and then dichloromethane was added. The mixture was heated under reflux and then hot filtered. The filtrate was concentrated and the solid product obtained was collected by filtration to give 2-(2- benzothiazolyl)-9-hydroxy-4-methyl[1]benzopyrano[4,3- c]pyrazol-3(2H)-one, m.p. 283-286°C (with decomposition).

Example 5 a) A stirred mixture of 1-(2-benzothiazolyl)-3-(2,6- dihydroxyphenyl)-2-pyrazolin-5-one (1.6 g) (Example 4a) in acetic anhydride (10 ml) was treated with sodium acetate (1.8 g). The reaction mixture was stirred at ambient temperature for 16 hours. A further portion of sodium acetate (0.9 g) was added and stirring was continued for a further 24 hours. The reaction mixture was then added to water (9 volumes) /petroleum ether (1 volume; bp 60-80°C) and the solid product obtained on standing was collected by filtration to give 1-(2- benzothiazolyl)-3-(2,6-diacetoxyphenyl)pyrazol-5-yl acetate, m.p. 159-162°C. b) A stirred mixture of 1-(2-benzothiarolyl)-3-(2,6- diacetoxyphenyl)pyrazol -5-yl acetate (1.9 g) in ethanol

(8 ml) was treated with piperidine (0.4 ml) and heated under reflux for 3 minutes. On cooling, the solid collected by filtration was purified by chromatography on Florisil (R) using dichloromethane and then 5% methanol in dichloromethane as the mobile phase to give, after trituration with ether, 2-(2-benzothiazolyl)-4- methyl-3-oxo-2,3-dihydro[1]benzopyrano[4,3-c]pyrazol-9- yl acetate, m.p. 246-251°C.

Example 6 a) A stirred mixture of 1-acetylthiosemicarbazide (5 g), 3-bromo-1,1,1-trifluoropropan-2-one (7.2 g) and ethanol (50 ml) was heated under reflux for 3 hours. The mixture was evaporated to dryness, triturated with acetonitrile and filtered to give 4-trifluoromethyl-2- thiazolylhydrazine as the hydrobromide salt. The solid collected was dissolved in water and basified to pH 12 with aqueous ammonium hydroxide solution. After warming at 90°C to give a solution, followed by cooling, the solid obtained was 4-trifluoromethyl-2- thiazolylhydrazine, m.p. 118-119°C. b) A mixture of 4-trifluoromethyl-2-thiazolylhydrazine (2.1 g), p-toluenesulphonic acid (0.2 g) and 4-hydroxycoumarin (0.9 g) in xylene (50 ml) was heated under reflux for 6 hours, with removal of water formed in the reaction. After cooling and filtration, the filtrate was evaporated to dryness and the solid obtained was triturated with toluene. The mixture was then filtered and the solid collected was triturated with hydrochloric acid (5M) and washed with water to give 3-(2-hydroxyphenyl)-1-(4-trifluoromethyl-2- thiazolyl)-2-pyrazolin-5-one, m.p. 210-211°C. c) A stirred mixture of 3-(2-hydroxyphenyl)-1-(4- trifluoromethyl-2-thiazolyl)-2-pyrazolin-5-one (3.0 g) and triethyl orthoacetate (10 ml) was heated at 140°C for 40 minutes. On cooling, the reaction was quenched with ethyl acetate and the mixture filtered. The solid product collected was 4-methyl-2-(4-trifluoromethyl-2- thiazolyl) [1]benzopyrano[4,3-c]-pyrazol-3(2H)-one, m.p. 274-276°C.

Example 7 A stirred mixture of 3-(2-hydroxyphenyl)-1-(4- trifluoromethyl-2-thiazolyl)-2-pyrazolin-5-one (3.0 g) (Example 6b) and triethyl ortho (2-ethoxycarbonyl)- acetate (6.0 g) was heated at 140°C for 0.5 hours. On cooling, the reaction mixture was quenched with ethyl acetate and the solid formed was collected by filtration to give ethyl 3-oxo-2-(4-trifluoromethyl-2-thiazolyl)- 2,3-dihydro [1]benzopyrano [4,3- c]pyrazole-4-acetate, m.p. 172-173°C.

Example 8 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 9

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 10

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

c) 500 mg of active compound.

Example 11

Tablets are prepared by the method of Example 10. 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 12

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 containing 100 mg of active ingredient.

Example 13 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 screw-capped 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 acetonerolive oil (3:1 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 μl). 100 μl of the same suspension was injected likewise every 24 hours for a further 7 days. The dosages used were 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 (i.e the lowest dose of those used at which the compound is active as defined above) 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)

1 2-(2-benzothiazolyl)-4-methyl[1]benzo3

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

2,3-dihydro[1]benzopyrano[4,3-c]- pyrazole-4-acetate 3 2-(2-benzothiazolyl)-8-fluoro-4- ≤3*

methyl[1]benzopyrano[4,3-c]pyrazol- 3 (2H)-one 4 2-(2-benzothiazolyl)-9-hydroxy-4- ≤3*

methyl[1]benzopyrano[4,3-c]pyrazol- 3 (2H)-one 5 2-(2-benzothiazolyl)-4-methyl-3-oxo- ≤3*

2,3-dihydro[1]benzopyrano[4,3-c]- pyrazol-9-yl acetate 6 4-methyl-2-(4-trifluoromethyl-2- ≤3

thiazoly1)[1]benzopyrano[4,3-c]- pyrazol-3(2H)-one

7 Ethyl 3-oxo-2-(4-trifluoromethyl-2- 3

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

NOTES: * Active in each of 2 tests at 3mg/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 the doses described in Table A. A compound was deemed to be active if, at a dose of 50 mg/kg or lower, 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:-

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).

Activity of compounds in above test: Table B

Minimum Effective

Examples Dose (mg/kg)

1 ≤3

2 ≤3

4 ≤3

5 ≤3

6 ≤3

7 ≤3

Claims

1. A compound of formula I

wherein R₁ represents hydrogen or methyl;

R₂ represents hydrogen, C_2-6 alkoxycarbonyl or C_2-6 alkanoyl;

R₃ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy or S(O)_mY₁;

R₄ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy or S(O)_mY₁; or R₃ and R₄, ^.together with the carbon atoms to which they are attached, represent a fused benz ring which may be substituted by one or more groups selected from C_1-6 alkyl, C_1-6 alkoxy, S(O)_mY₁, halo or trifluoromethyl; R₅ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy, hydroxy or C_2-6 alkanoyloxy;

Y₁ represents C_1-6 alkyl; m is 0, 1 or 2, or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 wherein R₁ represents hydrogen and R₂ represents hydrogen or ethoxycarbonyl.

3. A compound according to either one of claims 1 and 2 wherein R₃ represents hydrogen, halo or trifluoromethyl.

4. A compound according to any one of claims 1 to 3 wherein R₄ represents hydrogen, halo or trifluoromethyl.

5. A compound according to either one of claims 1 and 2 wherein R₃ and R₄ together with the carbon atoms to which they are attached form a fused benz ring.

6. A compound according to any one of claims 1 to 5 wherein R₅ represents hydrogen, fluoro, hydroxy or C_2-6 alkanoyloxy in the 8- or 9- position.

7. A compound according to claim 6 wherein R₅ represents hydrogen, 8-fluoro, 9-hydroxy or 9-acetoxy.

8. A compound according to claim 1 selected from: 2-(2-benzothiazolyl)-4-methyl[1]benzopyrano[4,3-c]- pyrazol-3(2H)-one, and

ethyl 3-oxo-2-(4-trifluoromethyl-2-thiazolyl)-2,3- dihydro[1]benzopyrano[4,3-c]pyrazole-4-acetate.

9. A pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1 to 8 together with a pharmaceutically acceptable carrier.

10. A pharmaceutical composition according to claim 9 in unit dosage form.

11. A method of treating diseases with an immunological association which comprises administering a therapeutically effective amount of a compound of formula I as claimed in any one of claims 1 to 8 to a mammal in need thereof.

12. A method of modulating the immune system which comprises administering a therapeutically effective amount of a compound of formula I as claimed in claim 1 to a mammal in need thereof.

13. A compound of formula I according to any one of claims 1 to 8 for use an an immunomodulatory agent.

14. A compound of formula I according to any one of claims 1 to 8 in the manufacture of a medicament for use in the treatment of diseases with an immunological association.

15. A process to prepare a compound of formula I as defined in claim 1 comprising the oxidation of a compound of formula IV

or a tautomer thereof;

16. A process to prepare a compound of formula I as defined in claim 1 comprising reacting a compound of formula V

or a tautomer thereof with a compound of formula VI

R₁R₂CHCR₉R₁₀ V I

in which R₉ represents (OQ)₂ and R ₁₀ represents OQ or

NQ' ₂; or R₉ represents (SQ)₂ and R₁₀ represents SQ or

NQ'₂; or R₉ represents =NH and R₁₀ represents OQ or SQ; or R₉ represents =O and R₁₀ represents a leaving group; and Q and Q' represent C_1-4 alkyl or benzyl.

17. A process to prepare a compound of formula I as defined in claim 1 'comprising reacting a compound of formula V

or a tautomer thereof; with a) a compound of formula VIla

or a tautomer thereof, or b) a compound of formula VIlb

or a tautomer thereof, in which R₁₁ represents C_1-5 alkyl, R₁₂ and R₁₃ may be the same or different and each represents C_1-6 alkyl or benzyl.

18. A process to prepare a compound of formula I as defined in claim 1 comprising reacting a compound of formula VIII

in which R₁₅ represents hydrogen or a tautomer thereof or in which R₁₅ represents a group COR₁₇ wherein R₁₇ represents hydrogen, optionally substituted C_1-4 alkyl or benzyl and R₁₆ represents COCHR₁R₂, with a base.

19. A compound of formula V

or a tautomer thereof, wherein R₃ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy or S(O)_mY₁; R₄ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy or S(O)_mY₁ or R₃ and R ₄' together with the carbon atoms to which they are attached, represent a fused benz ring which may be substituted by one or more groups selected from C_1-6 alkyl, C_1-6 alkoxy, S(O)_mY₁, halo or trifluoromethyl; R₅ represents hydrogen, halo, trifluoromethyl, C_1-6 alkyl, C_1-6 alkoxy, hydroxy or C_1-6 alkanoyloxy; Y₁ represents C_1-6 alkyl; and m is 0, 1 or 2.