AU650679B2 - Xanthines - Google Patents

Description

OPI DATE 15/04/92 AOJP DATE 28/05/92 APPLN. ID 86458 91 PCT NUMBER PCT/GB91/01634 TREATY (PCT)

INTERNA

(51) International Patent Classification 5 (11) International Publication Number: WO 92/05176 C07D 473/06, A61K 31/52 Al (43) International Publication Date: 2 April 1992 (02.04.92) (21) International Application Number: PCT/GB91/01634 (74) Agent: RUTTER, Keith; SmithKline Beecham, Corporate Patents, Great Burgh, Yew Tree Bottom Road, Epsom, (22) International Filing Date: 23 September 1991 (23.09.91) Surrey KT18 5XQ (GB).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 9020921.4 26 September 1990 (26.09.90) GB pean patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (71) Applicant (for all designated States except US): BEECHAM (European patent), IT (European patent), JP, KR, LU GROUP PLC [GB/GB]; SB- House-Gre at West-Read, (European patent), NL (European patent), SE (Euro- Brentfrd, Middlesex T.W8 .S DD pean patent), US.

(72) Inventors; and Inventors/Applicants (for US only BUCKLE, Derek, Ri- Published chard [GB/GB]; SMITH, D)avid, Glynn [GB/GB]; With international search report.

FENWICK, Ashley, Edward GB/GB]; SmithKline Beecham Pharmaceuticals, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey KTI8 5XQ 5 f (54) Title: XANTHINES a i -TOr.0tEiP UoCrJ -o 104 rjj (54) Title: XANTHINES 1 0 A R An

N\

NJ1 N O N N 12

R

CO.R

3

R

(57) Abstract A compound of formula or, if appropriate, a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, wherein R I and R 2 each independently represent a moiety of formula -(CH 2 wherein m represents zero or an integer 1, 2 or 3 and A represents a substituted or unsubstituted cyclic hydrocarbon radical; R 3 represents substituted or unsubstituted aryl or a substituted alkyl group; R 4 represents hydrogen or a group -CO.R 5 wherein R 5 represents substituted or unsubstituted alkyl or substituted or unsubstituted aryl; or R 3 together with R 5 represents a substituted or unsubstituted C 2 3 polymethylene chain; and A l represents hydrogen or substituted or unsubstituted alkyl; a process for preparing such a compound, a pharmaceutical composition containing such a compound and the use of such a compound or composition in medicine.

WO 92/05176 PCT/GB9I/01634 -1- Xanthines.

The present invention relates to certain novel compounds having pharmacological activity, to a process for the preparation of such compounds, to pharmaceutical compositions containing such compounds and to the use of such compounds and compositions in medicine.

Molecular Pharmacology, Volume 6, No. 6, 1970, p.597-603 discloses 1,3-dimethyl-8-nitro-xanthine. This compound is disclosed as having lipolytic activity. Ann Chim, 4.7, 362-365 (1957) discloses 1,3dimethyl-8-amino-xanthine and a process by which it may be prepared.

No pharmacological utility is disclosed for this compound. Drug Res. 27(1) Nr 19, 1977, pages 4-14, Van K.H. Klingler discloses certain 1,3-dimethyl- 8-substituted xanthines as intermediates solely in the synthesis of phenylethyl aminoalkyl xanthines. Drug Res. 31 Nr. 12, 1981, R.G.

Werner et al, pages 2044-2048 discloses certain 1,3-dimethyl-8-substituted xanthines. No pharmacological activity is disclosed for these compounds.

European Patent Application, Publication Number 0369744 also discloses certain 1,3- or 1,3,7- 8-H cycloalkylalkylene xanthines, for use inter alia as bronchodilators in the treatment of asthma.

European Patent Application, Publication Number 0389282 also discloses certain 1,3-cycloalkylalkylene 8-substituted xanthines for use inter alia in the treatment or prophylaxis of disorders associated with increased numbers of eosinophils.

It has now surprisingly been discovered that a novel series of substituted xanthines are indicated to be particularly effective as inhibitors of induced blcod eosinophilia and that they are therefore potentially of particular use in the treatment and/or prophylaxds of disorders associated with increased numbers of eosinophils, such as asthma, and allergic disorders associated with atopy, such as urticaria, eczema and rhinitis.

In addition these compounds show activity as phosphodiesterase inhibitors: These compounds are indicated to have bronchodilator activity and thus to WO 92/05176 PCT/GB91/01634 -2be of potential use in the treatment of disorders of the respiratory tract, such as reversible airways obstruction and asthma.

These compounds have a protective effect against the consequences of cerebral metabolic inhibition. The said compounds improve data acquisition or retrieval following transient forebrain ischaemia and are therefore useful in the treatment of cerebral vascular and neuronal degenerative disorders associated with learning, memory and cognitive dysfunctions including cerebral senility, multi-infarct dementia, senile dementia of the Alzheimer type, age associated memory impairment and certain disorders associated with Parkinson's disease.

These compounds are also indicated to have neuroprotectant activity.

They are therefore useful in the prophylaxis of disorders associated with neuronal degeneration resulting from ischaemic events, including cerebral ischaemia due to cardiac arrest, stroke and also after cerebral ischaemic events such as those resulting from surgery and/or during childbirth. In addition treatment with the compound is indicated to be of benefit for the treatment of functional disorders resulting from disturbed brain function following ischaemia.

These compounds are also active in increasing the oxygen tension in ischaemic skeletal muscle. This property results in an increase in the nutritional blood flow through ischaemic skeletal muscle which in turn indicates that the compounds of the invention are of potential use as agents for the treatment of peripheral vascular disease such as intermittent claudication.

These compounds are also of potential use in the treatment of proliferative skin disease in human or non-human mammals.

In addition these compounds may also have potential as inhibitors of the production of tumour necrosis factor (TNF) and hence have pote iia for the treatment of human immunodeficiency virus (HIV), acute immune deficiency syndrome (AIDS), rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, WNO 92/05176 PCT/GB91/01634 -3pulmonary inflammatory disease, bone resorption diseases, reperfusion injury, graft vs. host reaction, fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia secondary to AIDS, keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.

Accordingly, the invention provides a compound of formula 1 o 1 R A 3 NkN N

R

R

(I)

or, if appropriate, a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof; wherein R 1 and R 2 each independently represent a moiety of formula -(CH2)m-A (a) wherein m represents zero or an integer 1, 2 or 3 and A represents a substituted or unsubstituted cyclic hydrocarbon radical;

R

3 represents substituted or unsubstituted aryl or a substituted alkyl group;

R

4 represents hydrogen or a group -CO.R 5 wherein R 5 represents substituted or unsubstituted alkyl or substituted or unsubstituted aryl; or

R

3 together with R 5 represents a substituted or unsubstituted C2-3 polymethylene chain; and

A

1 represents hydrogen or substituted or unsubstituted alkyl.

Suitably, A is unsubstituted. Favourably, A represents a substituted or unsubstituted C3-8 cycloalkyl group, especially a C 3 6 cycloalkyl group.

In particular, A represents a substituted or, preferably, unsubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.

WO 92/05176 PCT/GB91/01634 -4- Favourably, A represents a cyclopropyl group or a cyclobutyl group.

Preferably, A represents a cyclopropyl group.

Suitably R 3 represent a substituted alkyl group. In particular an alkyl group substituted by a carboxy group. Favourably, R 3 represents a terminally substituted alkyl group, especially a terminally substituted ethyl or propyl group.

A preferred example of'R 3 is the substituted alkyl group 2-carboxyethyl.

In another aspect, one example of R 3 is the substituted aryl group, o-carboxyphenyl.

In one aspect R 4 represents hydrogen.

In a further aspect R 4 represents -CO.R 5 wherein R 5 represents substituted or unsubstituted alkyl or substituted or unsubstituted aryl; or

R

3 together with R 5 represents a substituted or unsubstituted C 2 .3 polymethylene chain.

Suitable optional substituents for the C 2 -3 polymethylene chain include up to five, preferably up to three, of the substituents mentioned below in relation to the aryl group and, especially for the C 2 polymethylene chain, substituents of adjacent carbon atoms of the C2-3 polymethylene chain form a residue of a substituted or unsubstituted phenylene group.

When R 3 together with R 5 represent a substituted or unsubstituted polymethylene chain it is suitably a substituted or unsubstituted C 2 polymethylene chain for example -CH 2

CH

2 or a substituted or unsubstituted phenylene group.

When Al represents a substituted alkyl group it may be substituted as mentioned hereinafter in relation to alkyl groups.

Suitable substituted alkyl groups, represented by Al, include aralkyl groups wherein the aryl group may be substituted or unsubstituted.

WO 92/05176 PCI/GB91/01634 A suitable substituted aralkyl group represented by Al, is a substituted benzyl group, suitably a methoxybenzyl group, for example a 4methoxybenzyl group.

Preferably Al represents hydrogen.

Suitably, m represents zero or the integer 1.

Favourably, m represents 1.

Suitable pharmaceutically acceptable salts are pharmaceutically acceptable base salts and pharmaceutically acceptable acd addition salts.

Suitable pharmaceutically acceptable base salts of the compounds of formula include 7-N base salts including metal salts, such as alkali metal salts for example sodium salts, or organic amine salts such as that provided with ethylenediamine.

Suitable acid addition salts of the compounds of formula are the acid addition salts including pharmaceutically acceptable inorganic salts such as the sulphate, nitrate, phosphate, borate, hydrochloride and hydrobromide and pharmaceutically acceptable organic acid addition salts such as acetate, tartrate, maleate, citrate, succinate, benzoate, ascorbate, methanesulphonate, a-keto glutarate, a-glycerophosphate and glucose-1phosphate. Preferably the acid addition salt is a hydrochloride salt.

Pharmaceutically acceptable solvates include conventional solvates such as hydrates.

The pharmaceutically acceptable salts or solvates of the compounds of formula are prepared using conventional procedures.

When used herein the term 'cyclic hydrocarbon radical' includes single ring and fused ring, alicyclic hydrocarbons comprising up to 8 carbon atoms in each ring, suitably up to 6 carbon atoms, for example 3, 4, 5 or 6 carbon atoms.

Suitable optional substituents for any cyclic hydrocarbon radical includes a C 1 -6 alkyl group or a halogen atom.

WO 92/05176 PCT/GB91/01634 -6- When used herein the term 'aryl' includes phenyl and naphthyl optionally substituted with up to five, preferably up to three, groups selected from halogen, alkyl, phenyl, alkoxy, halo alkyl, hydroxy, amino, nitro, carboxy, alkoxycarbonyl, alkoxycarbonylalkyl,alkylcarbonyloxy, or alkylcarbonyl groups. Optional substituents for any phenylene group include up to three of the substituents mentioned in relation to the aryl group.

When used herein the term 'alkyl' whether used alone or when used as part of another group (for example as in an alkylcarbonyl group) includes straight and branched chain alkyl groups, containing from 1 to 12 carbon atoms, suitably 1 to 6 carbon atoms, for example methyl, ethylpropyl or butyl. Suitable optional substituents for any alkyl group include up to five, preferably up to three of the substituents mentioned above in relation to the aryl group.

When used herein the expression 'proliferative skin diseases' means benign and malignant proliferative skin diseases which are characterized by accelerated cell division in the epidermis, dermis or appendages thereto, associated with incomplete tissue differentiation. Such diseases include: psoriasis, atopic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, allergic contact dermatitis, basal and squamous cell carcinomas of the skin, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant sun induced keratosis, non-malignant keratosis, acne, and seborrheic dermatitis in humans and atopic dermatitis and mange in domesticated animals.

The compounds of formula are preferably in pharmaceutically acceptable form. By pharmaceutically acceptable form is meant, inter alia, of a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels. A pharmaceutically acceptable level of purity will generally be at least 50% excluding normal pharmaceutical additives, preferably 75%, more preferably 90% and still more preferably The invention further provides a process for the preparation of a compound of formula or where appropriate a pharmaceutically WO 92/05176 PCT/GB91/01634 -7acceptable salt thereof or a pharmaceutically acceptable solvate thereof, which process coraprises: a) for compounds of formula wherein R 3 is substituted or unsubstituted aryl or substituted alkyl and R 4 is hydrogen or -CO.R wherein R 5 is substituted or unsubstituted alkyl or substituted or unsubstituted aryl, by reacting a compound of formula (II):

N

l a R 2 N N H 0 N N 2 a

(II)

wherein R l a represents R 1 as defined in relation to formula or a group convertible to R 1 and R 2 a represents R 2 as defined in relation to formula or a group convertible thereto, A 2 represents Al as defined in relation to formula or a group convertible thereto and R 6 represents hydrogen, a group -CO.R 5 a, wherein R 5 a represents unsubstituted alkyl, or a group -COR 5 b, wherein R5b is substituted alkyl or substituted or unsubstituted aryl, with a compound cf formula (III):

R

7

.CO.L

1 (In) wherein, when R 6 in compound is hydrogen or a group CO.R5a then

R

7 represents substituted alkyl or substituted or unsubstituted aryl, or when R 6 is a group -CO.R 5 b then R 7 represents substituted or unsubstituted aryl or substituted or unsubstituted alkyl, and L represents a leaving group; or b) for compounds of formula wherein R 3 together with R represent a substituted or unsubstituted C2-3 polymethylene chain, by cyclising a compound of formula (IV): Rla 0 A 2 2

L

SH

Z

0 N 12a (I)

R

WO 92/05176 PCT/GB91/01634 -8wherein R l a

R

2 a and A 2 are as defined in relation to formula Z represents the substituted or unsubstituted C2- 3 polymethylene chain as defined in relation to formula or a protected form thereof, and L 2 represents a leaving group; and thereafter, if required carrying out one or more of the following optional steps: converting any group R 1 1 to R 1 and/or R 2 a to R 2 and/or A 2 to A 1 (ii) converting a compound of formula into a further compound of formula (iii) converting a compound of formula into a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof.

A suitable leaving group L 1 is a halo atom for example a bromine or chlorine atom.

Suitably, L 2 represents a halo atom, for example a bromine or chlorine atom, or a hydroxyl group.

Favourably, L 2 represents a hydroxyl group.

A compound of formula (II) wherein R 6 represents -CO.R5a wherein R 5 a is unsubstituted alkyl may be prepared by reacting a compound of formula (II) wherein R 6 is hydrogen with an appropriate compound of abovedefined formula (II).

The reaction between compounds of formulae (II) and may be carried out using conventional acylation conditions, for example in an aprotic solvent, such as dimethylformamide or tetrahydrofuran, at any temperature providing a suitable rate of formation of the required product, for example in the range of from 0oC to 100oC, conveniently at ambient temperature.

In the reaction between compounds (II) and (III), the 8-amino group of compound (II) is suitably in an activated form, favorably in an anionic form such as a salted form, for example an alkali metal salted form.

WO 92/05176 PCT/GB91/01634 -9- Alternatively and preferably, the reaction between compounds of formulae (II) and (III) is carried out in an aprotic solvent, such as tetrahydrofuran, at a temperature in the range of 0OC to 100 0 C, in the presence of a base such as triethylamine.

The cyclisation of compound (IV) may be carried out under analogous conditions as appropriate to the reaction between compounds (II) and (III), a favoured aprotic solvent being tetrahydrofuran.

A compound of formula (II) wherein R 6 is hydrogen, may be prepared by reducing a compound of formula lRa 0 2 0 N

N

R A A 12a

R

(V)

wherein R l a

R

2 a and A 2 are as defined in relation to formula and thereafter if required converting R l 1 into R 1 and/or R 2 a into R 2 and/or

A

2 into Al.

The reduction of compound may be carried out by using any suitable, conventional reduction method, for example using tin powder and concentrated hydrochloric acid at ambient temperature or by using sodium dithionite in aqueous methanol at ambient temperature.

A compound of formula may be prepared by nitrating a compound of the above defined formula (VI): R1la 0 A 2

N

R

2 a

(VI)

WO 92/05176 PCT/G B91 /01 634 wherein R l a

R

2 a and A 2 are as defined in relation to formula _nd thereafter, if required, converting R l a into R 1 and/or R 2 a into R 2 and/or

A

2 into A 1 The nitration of compound (VI) may be carried out using any suitable, conventional nitrating agent, for example a nitric acid/acetic acid mixture in an inert solvent, such as dichloromethane, at any temperature providing a convenient rate of formation of the required product, conveniently at ambient temperature.

A compound of formula may be prepared by reacting a compound of formula (II) wherein R 6 is hydrogen, with a compound of formula (VII):

L

3

CO-

z

L

4 CO

(VII)

wherein Z is as defined in relation to formula L 3 and L 4 each independently represent leaving groups or L 3 together with L 4 represents an oxygen atom; and thereafter, if required, converting R l a into R 1 and/or

R

2 a into R 2 and/or A 2 into A 1 When L 3 and L 4 represent leaving groups each may represent the group

L

2 as hereinbefore defined.

The reaction between compounds of formulae (II) wherein R 6 is hydrogen, and (VII) wherein L 3 and L 4 independently represent leaving groups may be carried out under analogous conditions to that used in the cyclisation of the compounds of formula (IV).

The reaction between the compound of formula (II) wherein R 6 is hydrogen and the compound of formula (VII) wherein L 3 together with L 4 is an oxygen atom may be carried out in an aprotic solvent, such as tetrahydrcfuran at any temperature providing a suitable rate of formation of the required product, such as in the range of from 20 to 800C, suitably at 600C and preferably in the presence of a base such as triethylamine.

Favourably, the compound of formula (IV) is not isolated from the reaction WO 92/0S176. PCT/GB91/01634 11between compounds of formulae wherein R 6 is hydrogen, and (VII), but is converted in-situ into a compound of formula A compound of formula (VII), especially when L 3 together with L 4 is an oxygen atom, may also be reacted with a compound of formula (II), wherein R 6 is hydrogen, to provide a compound of formula wherein R 1 and R 2 are as defined above, R 4 is hydrogen and R 3 is a substituted or unsubstituted phenyl group having a carboxyl group attached ortho to the carbon atom bonding R 3 to the carbonyl of the N-CO.R 3 group.

Conversions of one compound of formula into another compound of formula includes: i) converting a compound of formula wherein A l represents hydrogen into a compound of formula wherein Al represents substituted or unsubstituted alkyl; or ii) hydrolysing compounds wherein R 3 together with R 5 represents a substituted or unsubstituted C2- 3 polymethylene chain, for example when R 3 together with R 5 represents -CH 2

CH

2 the resulting compound of formula is that wherein R 4 is hydrogen and R 3 is -CH 2

CH

2

-CO

2

H,

or when R 3 and R 5 together represent a phenylene group then the resulting compound of formula is that wherein R 4 is hydrogen and R 3 is o-carboxyphenyl.

Alkylation reaction may be effected by conventional alkylation methods, for example by treatment of the appropriate compound of formula with an alkyl halide of a compound of formula (VII) described below and wherein A 3 is A 1 In the hydrolysis (ii) hydrolysis of the apuropriate compound of formula (I) may be effected by any suitable hydrol. procedure, for example treatment with lithium hydroxide in aqL jus tetrahydrofuran at ambient temperature.

Suitable values for R l a R2a and A 2 include R 1

R

2 and A 1 respectively or nitrogen protecting groups such as benzyl groups.

WO 92/05176 PC/GB91/01634 -12- When R l a

R

2 a or A 2 represents other than R 1

R

2 or A 1 repectively, the abovementioned conversions of R l a into R1,R 2 a to R 2 and A 2 into A 1 may be carried out using the appropriate conventional procedure. For example when R l a

R

2 a, or A 2 represents a nitrogen protecting group, such as a benzyl group, the protecting group may be removed using the appropriate conventional procedure, such as catalytic hydrogenation, and the resulting product reacted with a compound of formula (VIII):

X-A

3

(VIII)

wherein A 3 represents R 1 or R 2 (for converting R l a into R 1 or R 2 a into

R

2 or A 3 represents A 1 (for converting A 2 into A 1 wherein R 1

R

2 and Al are as defined in relation to formula and X represents a leaving group, such as halide, for example bromide or iodide.

The protection of any reactive group or atom, such as the xanthine nitrogen atom may be carried out at any appropriate stage in the aforementioned process. Suitable protecting groups include those used conventionally in the art for the particular group or atom being protected, for example suitable protecting groups for the xanthine nitrogen atoms are benzyl groups.

In the circumstances when variable Al represents a benzyl group or a substituted benzyl group and R l a and/or R 2 a represents a nitrogen protecting group, the particular protecting groups chosen will be those which may be prepared and removed without affecting Al, examples of such protecting groups are trialkyl silyl groups such as t-butyl dimethyl silyl or trimethyl silyl groups.

Preferably for compounds of formula wherein Al represents benzyl or substituted benzyl then R l a is R 1 and R 2 a is R 2 Protecting groups may be prepared and removed using the appropriate conventional procedure: For example, N-benzyl protecting groups may be prepared by treating the appropriate compound of formula (II) with benzyl chloride in the presence of a base such as triethylamine, bases such as potassium t-butoxide may WO 92/05176 PCT/GB91/01634 -13also be used The N-benzyl protecting groups may be removed by catalytic hydrogenation over a suitable catalyst, such as palladium on activated charcoal, in a suitable solvent, such as ethanol conveniently at an elevated temperature, or by treatment with anhydrous aluminium chloride in dry benzene at ambient temperature. Trialkylsilyl protected nitrogen groups may be prepared by treating the appropriate compound with a trialkylsilyl halide, for example trimethylsilyl chloride, in the presence of a base such as potassium t-butoxide. The N-trialkylsilyl protecting group may be removed by mild basic hydrolysis or by treatment with a source of fluoride ions such as tetrabutylammoniumfluoride.

Compounds of formulae (II) especially those wherein A 2 and/or R 6 are other than hydrogen, (IV) and especially those wherein A 2 is other than hydrogen are novel compounds and as such form part of the present invention. The compounds of formula (VI) wherein A 2 is other than hydrogen are novel compounds and accordingly form part of the present invention.

Compounds of formula (VI) wherein A 2 is hydrogen are known compounds and may be prepared according to methods disclosed in EP 0369744.

Compounds of formula (VII) and (VIII) are known compounds or are prepared according to methods used to prepare known compounds for example those disclosed in J. March, Advanced Organic Chemistry, 3rd Edition (1985), Wiley Interscience.

As mentioned above the compounds of the invention are indicated as having useful therapeutic properties: the present invention accordingly provides a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for use as an active therapeutic substance.

Thus the present invention provides a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for use in the treatment of and/or prophylaxis of disorders associated with increased numbers of eosinophils, such as asthma, and allergic disorders associated with atopy, such as urticaria, eczema and rhinitis.

WO 92/05176 PCT/GB91/01634 14- In a further aspect the present invention also provides a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for use as a phosphodiesterase inhibitor.

In a particular aspect, as indicated hereinbefore, the present invention provides a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for use in the treatment of disorders of the respiratory tract, such as reversible airways obstruction and asthma.

In a further particular aspect, the present invention provides a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for use in the treatments mentioned hereinbefore, such as cerebral vascular and neuronal denerative disorders associated with learning, memory and cognitive dysfunctions, peripheral vascular disease or proliferate skin disease or for the prophylaxis of disorders associated with neuronal degeneration resulting from ischaemic events or for the inhibition of the production of tumour necrosis factor in for example the treatment of human immunodeficiency virus.

A compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, may be administered p_ se or, preferably, as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier.

Accordingly, the present invention provides a pharmaceutical composition comprising a compound of formula or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, and a pharmaceutically acceptable carrier.

The active compound may be formulated for administration by any suitable route, the preferred route depending upon the disorder for which treatment is required, and is preferably in unit dosage form or in a form that a human patient may administer to himself in a single dosage.

Advantageously, the composition is suitable for oral, rectal, topical, WO 92/05176, PCT/GB91/01634 parenteral, intravenous or intramuscular administration or through the respiratory tract. Preparations may be designed to give slow release of the active ingredient.

The compositions of the invention may be in the form of tablets, capsules, sachets, vials, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations such as oral or sterile parenteral solutions or suspensions. Topical formulations are also envisaged where appropriate.

In order to obtain consistency of administration it is preferred that a composition of the invention is in the form of a unit dose.

Unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate.

The solid oral compositions may be prepared by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers.

Such operations are of course conventional in the art. The tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; WO 92/05176 PCT/GB91/01634 -16emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.

Compositions may also suitably be presented for administration to the respiratory tract as a snuff or an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case the particles of active compound suitably have diameters of less than 50 microns, such as from 0.1 to microns, preferably less than 10 microns, for example from 1 to microns, 1 to 5 microns or from 2 to 5 microns. Where appropriate, small amounts of other anti-asthmatics and bronchodilators, for example sympathomimetic amines such as isoprenaline, isoetharine, salbutamol, phenylephrine and ephedrine; corticosteroids such as prednisolone and adrenal stimulants such as ACTH may be included.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, and, depending on the concentration used, can be either suspended or dissolved in the vehicle.

In preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing.

Advantageously, adjuvants such as a local anaesthetic, a preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

WO 92/0D5176 PCT/GB91/01634 -17- The compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration.

Compounds of formula or if appropriate a pharmaceutically acceptable salt thereof, may also be administered as a topical formulation in combination with conventional topical excipients.

Topical formulations may be presented as, for instance, ointments, creams or lotions, impregnated dressings, gels, gel sticks, spray and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. The formulations may contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions.

Suitable cream, lotion, gel, stick, ointment, spray or aerosol formulations that may be used for compounds of formula or if appropriate a pharmaceutically acceptable salt thereof, are conventional formulations well known in the art, for example, as described in standard text books of pharmaceutics and cosmetics, such as Harry's Cosmeticology published by Leonard Hill Books, Remington's Pharmaceutical Sciences, and the British and US Pharmacopoeias.

Suitably, the compound of formula or if appropriate a pharmaceutically acceptable salt thereof, will comprise from about 0.5 to by weight of the formulation, favourably from about 1 to 10%, for example 2 to The dose of the compound used in the treatment of the invention will vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and the relative efficacy of the compound. However, as a general guide suitable unit doses may be 0.1 to 1000mg, such as 0.5 to 200, 0.5 to 100 or 0.5 to 10 mg, for example 0.5, 1, 2, 3, 4 or 5 mg; and such unit doses may be administered more than once a day, for example 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total daily dosage for a 70kg adult is in the range of about 0.1 to 1000 mg, that is in the range of about 0.001 to 20 mg/kg/day, such as 0.007 to 3, 0.007 to 1.4, 0.007 to 0.14 or 0.01 to 0.5 mg/kg/day, for example 0.01, 0.02, 0.04, 0.05, WO 92/05176 PCT/GB91/01634 -18- 0.06, 0.08, 0.1 or 0.2 mg/kg/day; and such therapy may extend for a number of weeks or months.

When used herein the term 'pharmaceutically acceptable' encompasses materials suitable for both human and veterinary use.

No toxicological effects have been established for the compounds of formula in the abovementioned dosage ranges.

The following pharmacological data and examples illustrate the invention.

The following preparations illustrate the preparation of intermediates to the novel compounds of formula WO 92/05176 WO 9205176PCI'/GB9I /01634 Examples 1 and 2 1. .3-Di(cvclouproTvlm ethyD- )8-(N-phth alimi do)xanthi ne and 1 .3-di(cvclonronvylmethvD-8-(2-carboxybenzoyl)amino ,canthine 0 H 0 0 N 2H.

O<N

NHCO

1 ~CO 2

H

(Ex. 1) (Ex.2) 8-Amino-i, 3-di(cyclopropylmethyl)xanthine (2 .7g, l0mmole), phthalic anhydride (1.48g,l0mmole)and triethylamine (3m1,2.2eq) were stirred together in THF(40m1)at 60 0 C for 48hr. After cooling the mixture was added to ethyl acetate (200m1) and the organic solution, washed with dilute HCl (230m-l), dried (MgSO 4 and the solvent removed under reduced pressure. Chromatography of the residue on silica (acetone/hexane gradient) gave 1 ,3-ci(cyclopropylmethyl)- 8-(N-phthalimido)xanthine (1.06g,26%) m.p.244-5 0

C,

vmax(KBr)1745(s),1705(s),1652(s)and 1504(s)cm-1; 8(CDCl 3 0.14-0.19 (2H, in), 0.22-0.29 0.32-0.54 1.11-1.19 1.34-1.44 3.77 (2H,d,J=7.OHz), 4.05 (2H,d,J=7.5Hz) 7.90 (2H,dd,J=5.5, 8.04 (2H,dd, J=5.5,3.OHz), 12.60 (1H, brs); in/e 405 267 281 377 (33); Found C, 62.28; H, 4.65; N, 17.35 C 21

H

19

N

5 0 4 requires C, 62.21; H, 4.72; N, 17.28%.

followed by 1 ,3-di(cylopropylmethyl)-8-(2-carboxybenzyol)aminoxanthine (0.79g, 19%) m.p. 185-80C v max (K.Br) 3428 1706(s), 1645(s), 1634(s) and 1535 (in) cm- 1 8 'd 6 -DMSO), 0.39-0.47 (8H,m), 1.26-1.38 3.86 (4H,t (overi...pping J=6.5 Hz), 5.86 (2H,brs), 7.53 (2H, dd, J=6.0, 3.Hz), 7.72 (2H, dd, J=6.0, 3.5 Hz), 10.91 (1H, brs); ni/e 185 93 276 424 Found C, 57.15; H, 5.12; N, 15.75. C 2 1

H

2 1

N

5 0 5

.H

2 0 requires C, 57.13; H, 5.25; N, 15.87%.

WO 92/05176 WO 9205176PCT/GB91 /01634 20 Example 3 1 .3-Di(cvc] opronvlmethyl)-8-(N-succinimido)xanthine 0

H

1 -N 0;N 8-Amino-i ,3-di(cyclopropylmethyl)xanthine (2.7g, l0mmole), succinic anhydride (2.4g, 20mmole) and tri-etbhwlamine (3m1, 2.2eq) were stirred together in THF (40m1) at 60 0 C for 48hr. After cooling the solvent was removed under reduced pressure and the residue chromatographed on silica (MeOH/CHC1 3 1:20) to give 1,3-di(cyclopropyhnethyl)-8-CNsuccinimido)xanthine (2.46g, 70%) m.p. 235-60C, v max 1739 1703 1653 1605 and 1505 cm- 1 5 (CDC1 3 0.35-0.56 (8H,m), 1.22-1.44 3.00 3.92 (2H,d,J=7.OHz), 4.03 (2H,d,J=7.OHz), 12.25 (1H, br); ni/e 358 35(67), found 358.1518, C17Hj 9

N

5

O

4 requires 358.1516; Found C, 57.29, H, 5.19; N, 19.57; C 17

H

19

N,

5 0 4 requires C, 57.13; H, 5.36; N, 19,60%.

WO 92/05176, WO 9205176PCT/GB91/01634 21 Examile 4 1, .3-Di(cvcl opy- :nre thvl-8-Q carb oxyprop anovl) aminoxanthine 0

H

N

NHCO (CH 2 2 co 2

H

1 ,3-di(cyclopropylmeth34)-8-(N-succinimido)xanthine 18g, and lithium hydroxide monohydrate (0.042g, lminole) were stirred together in a water/THF mixture 3m1). After 0.5h at ambient temperature the solution was neutralized with dilute hydrochloric acid and filtered to give 1,3-dli(cyclopropylmethyl)-8-(3-carboxypropanoyl)aminoxanthine (0.19g, 100%) rn.p. 183-40C, v max (KBr) 3441 3051 1709 1700 1633 and 1523 cm-1; 8 W- 6

DMSO)

0.30-0.50 1.13-1.29 2.49-2.67 3.77 (2H,d,J=7.OHz), 3.83 (2H,d,J=7.0Hz), 11.77 (1H,br), 12.13 (2H, br); ni/e 35(100%), 358 276 376 found 376.2399, C 1 7

H

2 2

N

5 0 requires 376.1621.

WO 92/05176 PCT/GB91 /01634 22 Example 1 .3-Di(cyclopropylmethvl )-7-(4-methoxybenzl)-8-(Np~hthalimido)xanthine 0 0 0o N I

N

N >-NK)0

N

1, 3-Di(cycl opropylm ethyl)- 7-(4-methoxybenzyl)-8-(Nphthalimido)xanthine, rn.p. 190-192TC, was prepared in 82% yield from 1 3 -di,\cyclopropylmethyl)-7-(4-methoxybenzyl)xanthine in a similar manner to that of Example 1, 5 (CDC1 3 0.44-0.51 1.29-1.58 3.67 3.95 (2H,d,J=7.2Hz), 4.00 (2H,d,J=7.4Hz), 5.49 (2H,s), 6.66 (2H,d,J=8.8Hz), 7.07 (2H,d,J=8.8Hz) and 7.80-7.94 via (KBr) 1795 1742 Cs), 1702 1663 1513 and 724 Cm) cm- 1 nile 121 525 Found C, 66.08; H, 5.16; N, 13.12; C 2 9

H

2 7

N

5 0 requires C, 66.27; H, 5.18; N, 13.33%.

Example 6 8-(2-Carboxvbenzovlpmino)-l1.3-di(cvclopropy-lmethyvl)-7-(4methoxybenzyl )xanthirie 0 0 N -nN WO 92/05176Pr/B/O63 PCF/GB91/01634 23 8-(2-Carboxybenzoylamino)- 1,3-di(cyclopropylmethyl)-7-(4methoxybenzyl)xanthine, m.p. 165-6'C, was prepared in 90% yield from 1 ,3-di(cyclopropylmethyl)-7-(4-methoxybenzyl)-8-(N-phthalimido)xanthine in a similar manner to that of Example 4. 5 (CDCl 3 0.09-0.12 (2H,m), 0.25-0.26 0.37-0.49 0.91-0.96 (1H,na), 1.21-1.30 (1H,m), 3.67 (2H,d,J=7.4Hz), 3.78 3.92 (2H,d,J=7.2Hz), 5.89 (2H,brs), 6.86 (2H,d,J=8.5Hz), 7.32 (2H,d,J=8.5Hz), 7.53-7.91 (4H,nm) and 10.69 (1H,brs); v 3359 2950 1740 1695 1650 1603 1519 1459 (in) and 1232 (in) cm- 1 m/e (FAB,Na) 154 176 136 239 121 89 307 566 (MNa+,4); Found C, 63.68; H, 5.31; N, 12.78; C 29

H

2 9

N

5 0 6 requires C, 44.08; H, 5.38; N, 12.89%.

Example 7 8.(2-Carboxybenzoyj amino). 1 .3-di(cycloTropvylmethv1 methoxybenzyl )xanthine 0

H

N c

N

N

Carboxybenzoylamino)- 1,3-di(cyclopropylmethyl)-7-(4inethoxybenzyi)xanthine, in.p. 165-6"C, was prepared in 90% yield from 1 ,3-di(cyclopropylmethyl)-7-(4-methoxybenzyl)-8-(N-phthalimido)xanthine in a similar i1anner to that of Examnple 4. 8 (CDCl 3 0.09-0.12 (2H,m), 0.25-0.26 (2H,ni), 0.37-0.49 0.91-0.96 1.21-1.30 (1H,m), 3.67 (2H,d,J=7.4Hz), 3.78 3.92 (2H,d,J=7.2H-z), 5,89 (2H,brs), 6.86 (2H,d,J=8.5Hz), 7.32 (2H,d,J=8.5Hz), 7.53-7.91 (4H,m) and 10.69 (1Hbrs); v x (KB r) 3359(w), 2950 Mw, 1740 1695 1650 1603 1519 1459 (in) and 1232 (mn) cm 4 1; ni/e (FAB,Na) 154 176 136 239 121 89 307 566 (MNa+,4); Found C, 63.68; H, 5.31; N, 12.78; C 2 9

H

29

N

5 0 6 WO 92/05176 WO 9205176PCT/GB9I '/01634 24 requires C, 44.08; H, 5.38; N, 12.89%.

Example 8

NN-B

NN

K> "N 8-t-Butvl amido- 1 3-di(cvcloporonvylmethvl )xanthine Potassium t-butoxide (1.12g l0mmole) was added to a stirred solution of 8-amino-i ,3-di(cylcopropylmethyl)xanthine (2.7g, l0mmole) in dimethyl formamide (40m1) at ambient temperature. After 2 h, a solution of chioromethyl-pivaloate (1,52g, l1inmole) in DMF (5m1) was slowly added and stirring continued overnight. The mixture was added to ethyl acetate (200m1) and was washed with dilute hydrochloric acid (50mi) and water After drying over MgSO 4 the solvent was removed under vacuum and the residue extracted with acetone. Chromatography of the extract on silica (hexane/acetone gradient) yielded:- 8-t-butylamino- 1,3di(cyclopropylmethyl)-7-poivaloyioxmymethyI xanthine (0.40g, m.p. 157- 8*C; 5 (CDCl 3 0.42-0.51 1.22 1.22-1.45 1.39 3.93 (2H,d,J=7.211z), 3.99 (2H~d,J=7.2Hz), 6.02 and 9.28 v (KBr) 1699 1647 1522 1456 1263 (mn) and 1092 (in) cm- 1 m/e (CI,NH 3 372 474 360 MH+ observed 474.2690, C 24

H

3 6

N

5 0 5 requires 474.2717; Found C, 60.77; H, 7.55; N, 14.65; C 24

H

3 5

N

5 0 requires C, 60.87; H, 7.45; N, 14.79%, followed by:- 8-t-butylamido-1,3-di(cyclopropylmethyl)xanthine, (0.52g, m.p. 178-9*C; 5 (CDCI 3 0.42-0.51 (8H,in), 1.27-1.57 (2H,in), 1.33 3.86 (2H,d,J=7.2Hz), 3.92 (2H,d,J=7.2Hz), 8.77 (1H,s) and 11.24 v .,(KIrO 1707 1649 1501 Cs) and 1157 (in) cm- 1 rne

(CI,NH

3 360 (MH+,100%) MH+ observed 360.2036, C 1 8

H

2 6

N

5 0 3 reqtu.ies 360.2036; Found C, 60.10; H, 7.17; N, 19.51; 0 18

H

2 5

N

5 0 3 requires C, 60.15; H, 7.01; N, 19.49%.

WO 92/05176 WO 920S~76PCT/GB9I /01634 25 83B enzamido- 1 .3-di(cvclonDroTovlmethvl)xpnthine N 0

N,

N N 0K> Sodium hydride (0.30g of a 60% dispersion in oil, 7.6mmole) was added to a suspension of 8 -amino- 1,3- di (cycl opropylmethyl)xanthine (1.Og,3.6mmole) in tetrahydrofuran (l0mi). After 0.5h benzoyl ch'Ioride (0.56g, 1.leq) was added and stirring continued for a further 16 h. The reaction mixture was poured into water (50n-l) and neutralised. The precipitate was collected by filtration, washed with benzene and dried for 16 h under vacuum to afford 8-benzamido-1,3di(cyclopropylmethyl)xanthine (1.2g, m.p. >250 0 C (CHCl 3 JMeOH); 8 (CDC1 3 0.37-0.55 1.23-1.42 3.88 (2H,d,J=7.lHz), 3.95 (2H,d,J=6.9Hz), 7.46-7.63 8.11-8.14 11.86 (1H,bs), and 12.13 (1li,s), v max (KBr) 3227 1709 1649 'L633 1583 Cs), 1493 1258 1095 703 (in) cm- 1 m/e (NCII) 330 (MIH+,100%) Found C, 63.14; H, 5.71; N, 18.37; C 20

H

2 1

N

5 0 3 requires C, 63.30; H, 5.58; N, 18.46%.

Procedure 1 1, .3-Di -cycl opropylm ethyl -8 -nitro xanthine 1,3-Di-cyclopropylmethyl xanthine (20g, 0.076mo1) was dissolved in acetic acid (33m1) and then treated with concentrated nitric acid (13.2g) at 8700.

After 1 hour, the mixture was cooled to 50C and the resulting yellow precipitate filtered off. The yellow crystals were dissolved in dichloroinethane and washed with water. The separated organic layer -was then dried over anhydrous sodium sulphate and concentrated in yM,2 The product crystallized from the concentrate to yield a yellow WO 92/05176 PCT/GB91/01634 26 crystalline product yield 12.2g, m.pt. 207 0 C (with decomposition).

.iH NMR (CD C1,2 ppm: 0.35-0.7 (in, 8H1), I 1 -1.7 (in, 2H), 3.95-4.2 (in, 4H), 9.0-11.0 (hr.

exchanges with D 2 0, 1HE).

Procedure 2 1. .3-Di -cvcl opropyl methyl- 8- amino xanthine 1,3-Di-cylop ropylmethyl-8-riitro xanthine (4g, 0,Ol4inol), suspended in 50m1 of concentrated hydrochloric acid,was treated with small portions of tin (8g) at room temperature. The mixture was then stirred at room temperature for two hours.

The resulting precipitate was filtered off and crystallised, from ethanol to give white crystals of the title product, yield 0.9g m.pt. 2810C.

In an alternative procedure, using sodium dithionite as reducing agent (in methanol-water mixture) The yield was 36%.

.iH NMR (CDC1~l 8: 0.3-0.6 1.0-1.6 (m,211), 3.7-4.0 5.75 (br,2H), 10.84 (hr.

exchianges with D 2 0, 1H).

Procedure 3 8-Amino-i .3-di(cvclooroTvlmethl)-7-(4-methoxvbenzvl)-xanthine WO 92/05176 WO 92/5176 Pcl'/GB91 /01634 27 Potassium t-butoxide(1.34g,l2mmole)was added to a solution of 8-am-ino-i ,3-di(cyclopropylmethyl)xanthine (2.7g, l0mmole)in DMF(25m1l)and the resulting mixture was stirred for O.5hr at ambient temperature. 4-Methoxybenzyl chloride(1.56g, 1.35m1, l0mmole) was added to the red sol4ution which lightened to an orange colour. After stirring for lhr at ambient temperature the mixture was added to ethyl acetate (200m1), washed with dilute hydrochloric acid(50m1),water(S5nil) and dried (MgSO 4 Removal of the solvent under reduced pressure gave a olid which was chromnatographed on silica. (hexane/acetone, gradient~to give 8-am-ino-i ,3-di(cyclopropylmethyl)-7-(4-methoxybenzyl) xanthine (2.55,64%) m.p.176 0 C, vmax (KBr) 3434(w), 1691(m),1639(s),1527(m) and 1456(m)cm- 1 5(CDCl 3 )0.43-0.53(8H,m), 1.26-1.35(2H,m),3.79(3H,s),3.89 (4H,t(overlapping J=7.0Hz), 4.55(2H,brs), 5.32(2H,s), 6.90(2H,d,J=9.OHz), 7.30(2H,d,J=9.OHz); mle 121(100%), 395(M+,20); Found C, 63.64; H, 6.36; N, 17.77. C 2 1

H

2 5

N

5 0 3 requires C, 63.78; H, 6.37; N, 17.71%.

WO 92/05176 PCT/GB91/01634 -28- PHARMACOLOGICAL DATA 1) Induction of blood eosinophilia and the effects of drugs, Animals Male Charles River Sprague Dawley rats weighing between 270 to 400g were used.

The method used was a modification of that described by Laycock et al (Int. Arch. Appl. Immunol, (1986). 81, 363).

Sephadex G200, particle size 40 to 120 micron, was suspended in isotonic saline at 0.5mg/ml, and stored for 48h at 40C. Iml of the suspension was given intravenously to rats on days 0,2 and 5. A control group received saline. The test compound was givenbefore the Sephadex on each occasion, with a contact time expected to give maximum activity at the time of the Sephadex administration. Blood was taken from the tail vein of the rats on day 7 for the determination of total and differential leucocyte counts.

A control group of at least 6 animals was included each time a compound was evaluated. The control group received Sephadex and the vehicle without test compound. The results in the drug treated animals were compared with the control group.

Total and differential leucocyte counts.

samples of blood, taken from the tail vein of the rats, were added to 10ml of Isoton II and, within 30min, Zaponin (3 drops) was added, to lyse the erythrocytes. Five minutes later the total cell count was determined using a Coulter Counter Model DN. Differential leucocyte counts were carried out by fixing and staining a blood smear on a microscopic slide with May-Grunwald and Giemsa stains. A minimum of 400 cells were counted on each slide.

WO 92/05176 PCT/G B91 /01 6.34 -29- Statistics Probability values were calculated using the Student's t test.

Results The effect of the test compound upon Sephadex induced eosinophilia in the rat is set out below. The test compound was given orally 30 minutes before each injection of Sephadex.

Test Compound Dose mg/kg (orally -30 mins) of Control Mean SEM (n=16 Vehicle dosed control sephadex i.v Negative control saline i.v.

100 13 14 1 49 13* 60± 12* Example 2 Example 3 Notes p< 0.05 p< 0.001 WO 92/05176 PC/GB91/01634 2) Inhibition of Phosphodiesterase Isolation of phosphodiesterases The Ca2+/calmodulin-stimulated PDE (PDE I, see Table 1 and Beavo and Reifsynder (1990) for nomenclature) was prepared from bovine cardiac ventricle. Following chromatography on a Mono Q column, the fractions showing stimulation of PDE activity by Ca 2 and calmodulin were pooled and further purified on a calmodulin-affinity column. cGMP-stimulated PDE (PDE II), cGMP-inhibited PDE (PDE III) and cAMP-specific PDE (PDE IV) were all isolated from guinea-pig cardiac ventricle. Initial chromatography on a 20 ml Mono Q column resolved PDE III from a peak that contained both PDE II and PDE IV. The latter were separately rechromatographod on a 1 ml Mono Q column. cGMP-selective PDE (PDE V) was obtained from porcine lung using chromatography on DEAEcellulose and Mono Q columns; a calmodulin-affinity column was used to remove residual PDE I activity.

Characteristics of phosphodiesterase isoenzymes With the exception of PDE II, which displayed positive cooperativity, all the preparations showed simple Michaelis-Menton kinetics (see Table 1).

PDE I The activity of this isoenzyme was stimulated by the Ca 2 calmodulin complex. The isoenzyme could hydrolyse both cAMP and cGMP, the latter was the preferred substrate.

PDE II The activity of this isoenzyme with cAMP as a substrate was stimulated by cGMP. The isoenzyme could hydrolyse both cAMP and cGMP, the latter was the preferred substrate under basal conditions. The activity of this isoenzyme was unaffected by the Ca 2 +-calmodulin complex.

PDEIII The activity of this isoenzyme with cAMP as a substrate was inhibited by cGMP. The isoenzyme could hydrolyse both cAMP and cGMP, the former was the preferred substrate. The activity of this isoenzyme was unaffected by the Ca 2 calmodulin complex.

WO 92/05176.

PCT/GB91/01634 -31- PDE IV This isoenzyme had high affinity for cAMP, the hydrolysis of which was not inhibited by cGMP. The activity of this isoenzyme was unaffected by the Ca2-calmodulin complex.

PDE V This isoenzyme had high affinity for cGMP. The activity of this isoenzyme was unaffected by the Ca 2 +-calmodulin complex.

Assay of phosphodiesterase activity PDE activity was assayed by the boronate column method as previously described (Reeves et. al., 1987). The enzymes were assayed by incubation at 37°C for 4-30 min. in 50 mM Tris, 5 mM MgC1 2 pH 7.5 with 3Hlabelled cyclic nucleotide (4 x 10 5 disintegrations min and 1 4 C-labelled nucleotide 5'-monophosphate (3 x 103 disintegrations min-1). The assay was stopped by boiling and the 3 H-labelled 5'-monophosphate product separated from substrate on boronate columns. The reaction mixture was diluted with 0.5 mL 100 mM HEPES [N-(2-hydroxyethyl)piperazine-N 1 -2-ethanesulfonic acid], 100 mM NaC1, pH 8.5, and applied to the column. The column was extensively washed with the same buffer, and the 5'-nucleotide eluted with 6 mL of 0.25 M acetic acid. The recovery of product as judged by 1 4 C-recovery was approximately 80%. All assays were linear with time of incubation and concentration of enzyme over the range used in these experiments.

IC

5 0 values (the concentration of inhibitor required for 50% inhibition of activity) were obtained by incubation of the isoenzyme using 1 mM cGMP as a substrate for PDE I (in the absence of Ca 2 and calmodulin), PDE II and PDE V and with 1 mM cAMP as a substrate for PDE III and PDE IV.

A range of inhibitor concentrations from 0.1 x IC 5 0 to 100 x IC 5 0 was used.

References BEAVO, J.A. and D.H. REIFSNYDER, Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends. Pharmacol. Sci. 11, 150-155 (1990).

WO 92/05176 WO 9205176PCF/GB9]/01634 32 REEVES B.K. LEIGH and P.J. ENGLAND, The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Biochem. J. 241, 535-541 (1987).

Table 1: Kinetic properties of phosphodiesterase isoenzymes Iso enzyme KM 4~M) cAMP cGMP Vmax cA2*P Vmax cGMP Ca 2 +/calmodulinstimulated cGMP-stimulated cGMP-inhibited cAMP-specific cGNIP-specific 45 0.5 14 0.1 1 n.d.

N.d.

a n.d.

enzyme displayed positive cooperativity Km >100 mM not determined, due to inability of PDE to hydrolyse one of the substrates.

RESULTS

Example PD IV

(PM)

Inhibition of: PDE V

IC

5 0 4(M) 0.05 0.2 3 13

Claims (11)

1. A compound of formula 1 0 1 R A 3 N 4 0 N N R 12 R2 (I) or, if appropriate, a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, wherein R 1 and R 2 each independently represent a moiety of formula -(CH2)m-A (a) wherein m represents zero or an integer 1, 2 or 3 and A represents a substituted or unsubstituted cyclic hydrocarbon radical; R 3 represents substituted or unsubstituted aryl or a substituted alkyl group; R 4 represents hydrogen or a group -CO.R 5 wherein R 5 represents substituted or unsubstituted alkyl or substituted or unsubstituted aryl; or R 3 together with R 5 represents a substituted or unsubstituted C 2 -3 polymethylene chain; and Al represents hydrogen or substituted or unsubstituted alkyl.

2. A compound according to claim 1, wherein A represents a substituted or unsubstituted C 3 8 cycloalkyl group.

3. A compound according to claim 1 or claim 2, wherein A represents an unsubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group.

4. A compound according to any one of claims 1 to 3, wherein A represents a cyclopropyl group. A compound according to any one of claims 1 to 4, wherein R 3 represents a substituted alkyl group. -34-

6. A compound according to any one of claims 1 to 5, wherein R 3 represents a terminally substituted ethyl or propyl group.

7. A compound according to any one of claims 1 to 6, wherein R 3 is 2- carboxyethyl.

8. A compound according to any one of claims 1 to 4, wherein R 3 together with represents a substituted or unsubstituted C -3 polymethylene chain substituted with one, two or three selected from the list consisting of: halogen, alkyl, phenyl, alkoxy, halo alkyl, hydroxy, amino, nitro, carboxy, alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy and alkylcarbonyl groups or substituents of adjacent carbon atoms of the C 2 3 polymethylene chain form a residue of a substituted or unsubstituted phenylene group.

9. A compound according to any one of claims 1 to 8, wherein R 4 represents hydrogen, A compound according to claim 1, selected from the group consisting of: 1,3-di(cyclopropylmnethyl)-8-(N-phthalimido)xanthine; 1,3-di(cyclopropylmethyl)-8-(2-carboxybenzoyl)amino xanthine; 1,3-di(cyclopropylmethyl)-8-(N-succinimido)xanthine; 1,3-di(cyclopropylmethyl)-8-(3-carboxypropanoyl)amino xanthine; 1,3-di(cycloprop)-7-(4-methoxybenzyl)-8-(N-phthalimido)xanthine; 8-(2-carboxybenzoylamino)-1,3-di(cyclopropylmethyl)-7-(4-methoxybenzyl)xanthine; 8-t-butylamido-1,3-di(cyclopropylmethyl)xanthine; and 8-benzamido-1,3-di(cyclopropylnmethyl)xanthine; or if appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof. 940502,p:\oper\dab,86458.spe,34 WO 92/05176. PCT/GB91/01634 Ac r te\, \h 4@ 7. A process for the preparation of a compound of formula (Ikor where appropriate, a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, which process comprises: a) for compounds of formula wherein R 3 is substituted or unsubstituted aryl or substituted alkyl and R 4 is hydrogen or -CO.R wherein R 5 is substituted or unsubstituted alkyl or substituted or unsubstituted aryl, by reacting a compound of formula (II): Rla 0 R2a (II) wherein R l a represents R 1 as defined in relation to formula or a group convertible to R 1 and R 2 a represents R 2 as defined in relation to formula or a group convertible thereto, A 2 represents A 1 as defined in relation to formula or a group convertible thereto and R 6 represents hydrogen, a group -CO.R 5 a, wherein R 5 a represents unsubstituted alkyl, or a group -COR 5 b, wherein R 5 b is substituted alkyl or substituted or unsubstituted aryl, with a compound of formula (III): R 7 .CO.L 1 (III) wherein, when R 6 in compound (II) is hydrogen or a group CO.R 5 a then R 7 represents substituted alkyl or substituted or unsubstituted aryl, or when R 6 is a group -CO.R 5 b then R 7 represents substituted or unsubstituted aryl or substituted or unsubstituted alkyl, and L 1 rpresents a leaving group; or b) for compounds of formula wherein R3 together with R 5 represent a substituted or unsubstituted C 2 3 polymethylene chain, by cyclising a compound of formula (IV): 36 la A2 L -CO O N 2a R (IV) wherein R1 a R 2 a and A 2 are as defined in relation to formula Z represents the substituted or unsubstituted C 2 3 polymethylene chain as defined in relation to formula or a protected form thereof, and L 2 represents a leaving group; and thereafter, if required carrying out one or more of the following optional steps: converting any group R l a to R 1 and/or R 2 a to R 2 and/or A 2 and Al; (ii) converting a compound of formula into a further compound of formula (iii) converting a compound of formula into a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising a compound of formula as defined in claim 1; or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, and a pharmaceutically acceptable carrier.

13. A method for the treatment of and/or prophylaxis of disorders associated with increased numbers of eosinophils, and allergic disorders associated with atopy comprising administering a therapeutically effective amount of a compound of formula as defined in claim 1; or where appropriate a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof.

14. Use of a compound of formula as defined in claim 1; or where appropriate 940502,p:\oper\dab,86458.spe,36 -37- a pharmaceutically acceptable salt thereof; or a pharmaceutically acceptable solvate thereof, for the manufacture of a medicament. Compounds of formula processes for their preparation, pharmaceutical compositions containing them or methods involving them, substantially as hereinbefore described with reference to the Examples. DATED this 3rd day of May, 1994 Beecham Group p.l.c. By Its Patent Attorneys DAVIES COLLISON CAYVE 940S02,p:\opcr dab,86458.spc,3/ INTERNATIONAL SEARCH REPORT International Application No PCT/GB 91/01634 1. CLASSIFICATION OF SUBJECT MATTER-(if several classification symnbols apply, Indicate aiI)fi According to International Patent Classification [PC) or to both National Classification and IPC Int.C1. 5 C070473106; A61K31/52 IL FIELDS SEARCHED Minimum Documentation Swnrded 7 Classification System Classification Symnbols Int.Cl. 5 C07D Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields SearcheO III. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category 0 Citation of Document, I t with Indication, where appropriate, of the relevant passages 12 lilevant to Claim No.D A EPA,0 386 683 (POLl INDUSTRIA CHIMICA) 12 1-16 September 1990 *Complete document* A EP,A,O 258 191 (SANOOZ-PATENT-GMBH) 2 March 1988 1-16 *Complete document* P,A EP,A,U 389 282 (BEECHAM WUELFING) 26 September 1-16 1990 cited in the applicatiom *Complete document* *Special categories of cited documents 10 -T later document published after the International filing date ocuentdefiingthegenral tat oftheart hic Isnotor priority date and not In conflict with the application but A doumen defningthegeneal sate f te ar whih isDotcite to understand the prtnciple or theory underlying the considered to be of particular reee Inntion E' earlier document but published on or after the International 'r document of particular relevnce; the claimed Invention filing date nnot be considered liovel or canot be considered to docureent which may throw doubts oA priority cdax(s) or Involve an Inventive step which Is cited to establish the publicatIon date of another 'r document of particubir relevance; the claimed Invention citation or other specil reason (as specified) cannot be coosiderefl to Involve an inventive step when the 0' document rrilng to at. oral disclosure, use, exhiblbit document Is combloed with one or more other such doco- other means moots, such combiniation being obvicus to a person skilled Ill document published prior to the international filing date but In the art later than the priotity date claimed OW document Mmere o1' the same "aet family Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 02 JANUARY 1992 14 t-9 Intenatona Serchig AthoitySignature of Authorized Offlca EUROPEAN PATENT OFFICE E- LUYTEN H.W. 1 1 remn PCrIISA40jleZz I.-g46I0moy 19451 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION No. GB SA 9101634 51593 Tlis annex fists the patent family members relating to the patent documents cited in the above-mentioned international search report The inembers ame as contained in the European Patent Office EDP file on 'Me Eurvyean Patent Office is in no way liable for these Particulars which are merely given for the purpose of information. 02101/92 EP-A-0386683 12-09-90 3 P-A- 3200787 02-09-91 EP-A-0258191 02-03-88 AU-B- 600021 02-08-90 AU-A- 7744587 03-03-88 JP-A- 63063679 22-03-88 AT-B- 392968 25-07-91 WO-A- 8805775 11-08-88 EP-A-0389282 26-09-90 AU-A- 5208390 27-09-90 CN-A- 1047502 05-12-90 JP-A- 2273676 08-11-90 2 I I a h. I 0 For more details about this annel see Official Joirnal of the European Patent Office, No. 12)BZ