AU3974700A

AU3974700A - Derivatives of pyrimido(6,1-a)isoquinolin-4-one

Info

Publication number: AU3974700A
Application number: AU39747/00A
Authority: AU
Inventors: David Jack; Alexander William Oxford
Original assignee: Vernalis PLC
Current assignee: Ligand UK Ltd
Priority date: 1999-03-31
Filing date: 2000-03-29
Publication date: 2000-10-16
Also published as: NZ514157A; NO20014729D0; MXPA01009847A; WO2000058309A1; CA2368460A1; CN1348452A; BR0009449A; JP2002543046A; EP1165556A1; NO20014729L

Description

wuV uui~/u9 PCT/GB00/01195 1 Derivatives of pvrimido[6. I -a]isoquinolin-4-one The present invention relates to derivatives of pyrimido[6,1-a]isoquinolin-4-one and their application as inhibitors of phosphodiesterase (PDE) isoenzymes. More 5 particularly the invention relates to 2-phenoxy derivatives of pyrimido[6,1 a]isoquinolin-4-one and their use in medicine for example as bronchodilators with anti-inflammatory properties. In all cells where cyclic AMP (cAMP) is present as a secondary messenger, 10 intracellular concentrations of cAMP are regulated by the two processes involved in its formation and degradation. Stimulation of membrane bound receptors on the external surface of the cells (e.g. by -adrenoceptor agonists) results in activation of adenylyl cyclase to generate cAMP from ATP. Phosphodiesterases present in the cell serve to reduce the concentration of cAMP by hydrolysing it to adenosine monophosphate 15 (AMP). In a disease such as asthma, the principal cells involved in the associated bronchoconstriction and inflammatory processes are subject to inhibitory control by cAMP. Inhibitors of type III phosphodiesterase raise intracellular levels of cAMP, 20 leading to relaxation of bronchial smooth muscle, whereas inhibitors of type IV phosphodiesterase inhibit the release of damaging mediators from pro-inflammatory cells. Thus, in principle, a combined PDE III/IV inhibitor should have the desirable effects of a P3-adrenoceptor agonist plus an inhaled anti-inflammatory steroid which are currently the mainstay of treatment in severe asthma. Moreover, a combined PDE 25 III/IV inhibitor given by inhalation should achieve beneficial effects similar to a 3 agonist plus inhaled steroid and should be an unusually effective treatment of asthma and other respiratory disorders without the undesirable glucocorticoid effects of the steroid such as osteoporosis and the stunting of growth.

VV uua.uy PCT/GB00/01195 2 The potential adverse effects of a PDE III/IV inhibitor (e.g. nausea and vomiting, gastric acid secretion, cardiovascular effects such as increased cardiac contractility, vasodilation and potential arrhythmogenic activity) should be avoidable with a compound that is delivered directly to the lungs by inhalation. It is desirable that the 5 substance is long acting, non irritant and has a taste which is not so unpleasant as to have any adverse effect on patient compliance. An example of a pyrimido[6,1-a]isoquinolin-4-one derivative with PDE III/IV inhibitory activity and known to possess antihypertensive vasodilator activity is 10 trequinsin (9,10-dimethoxy-3-methyl- 2 -mesitylimino-2,3,6,7-tetrahydro-4H pyrimido[6,1-a]isoquinolin-4-one), which is described by De Souza et al., J Med. Chemn. 27 1470-1480 (1984) and in GB-A-1597717. As described by De Souza et al. and in GB-A-1597717, trequinsin has valuable 15 pharmacological properties, and can be administered to human subjects suffering from, for example, respiratory disorders. However, it is unsuitable for administration by inhalation because of its bitter taste and in vitro data indicate its persistence of action is less than desirable. 20 It has now been found that it is possible to design certain pyrimido[6,1-a]isoquinolin 4-one derivatives which are PDE inhibitors, which have a longer duration of action relative to trequinsin and other useful properties, such as improved taste. According to a first aspect of the present invention there is provided a compound of 25 general formula I: Vo uu/auvy PCT/GB00/01195 3

R

1 0 X R 5

R

2 0 N

R

6 N R 0 9 8 R R wherein 5 each of R 1 and R 2 independently represents a C 1

-

6 alkyl or C 2

-

7 acyl group; X represents OCH 2 or a group CR 3

R

4 , wherein each of R 3 and R 4 independently represents a hydrogen atom or a C 1

-

3 alkyl group;

R

5 represents a hydrogen atom or a C1- 3 alkyl, C 2

.-

3 alkenyl or C 2

.-

3 alkynyl group;

R

6 represents a hydrogen atom or a C 1

-

6 alkyl, C 2

-

6 alkenyl, C 2

-

6 alkynyl, amino, C 1 -6 10 alkylamino, di(CI.- 6 ) alkylamino or C 2

-

7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1

.

6 alkyl, C 2

-

6 alkenyl, C 2 -6 alkynyl, C 2 -7 acyl, C1- 6 alkylthio, C 1 -6 alkoxy or C 3

-

6 cycloalkyl group;

R

9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI- 6 alkyl, C 2 15 6 alkenyl, C 2

-

6 alkynyl, C 2

-

7 acyl, C 1-6 alkylthio, C1- 6 alkoxy or C 3

-

6 cycloalkyl group; or a salt thereof; excluding the compound 6,7-dihydro-2-phenoxy-9,10-dimethoxy-4H pyrimido[6,1 a]isoquinolin-4-one. 20 The compound 6,7-dihydro-2-phenoxy-9,10-dimethoxy-4H pyrimido[6,1a]isoquinolin-4-one, which is excluded from the scope of the first aspect of the present invention, is compound "8f" in De Souza et al, cited above. 6,7-dihydrowU uua.uy PCT/GB00/01195 4 2-phenoxy-9,10-dimethoxy-4H-pyrimido[6,1 a]isoquinolin-4-one is described in De Souza et al only as being a poor hypotensive agent and is not described as having bronchodilatory activity. 5 As used herein the term "halogen" or its abbreviation "halo" means fluoro, chloro, bromo or iodo. As used herein the term "C 1 -6 alkyl" refers to straight chain or branched chain alkyl groups having from one to six carbon atoms. Illustrative of such alkyl groups are 10 methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl. C 1

-

4 alkyl groups are preferred. As used herein the term "C 2

-

3 alkenyl" refers to straight chain or branched chain hydrocarbon groups having from two to three carbon atoms and having in addition one 15 double bond, of either E or Z stereochemistry where applicable. This term would include for example, vinyl and 1-propenyl. As used herein the term "C 2

-

3 alkynyl" refers to straight chain hydrocarbon groups having from two to three carbon atoms and having in addition one triple bond. This 20 term would include for example, ethynyl and 1-propynyl. As used herein the term "C 2

-

6 alkenyl" refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one double bond, of either E or Z stereochemistry where applicable. This term would 25 include for example, vinyl, 1-propenyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

C

2

.

3 alkenyl groups are preferred. As used herein the term "C2.

6 alkynyl" refers to straight chain or branched chain hydrocarbon groups having from two to six carbon atoms and having in addition one wu uu/.35iuy PCT/GB00/01195 5 triple bond. This term would include for example, ethynyl, 1-propynyl, 1- and 2 butynyl, 2-methyl-2-propynyl, 2-pentanyl, 3-pentanyl, 4-pentanyl, 2-hexanyl, 3 hexanyl, 4-hexanyl and 5-hexanyl. C 2

-

3 alkynyl groups are preferred. 5 As used herein the term "C 1

-

6 alkoxy" refers to straight chain or branched chain alkoxy groups having from one to six carbon atoms. Illustrative of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, neopentoxy and hexoxy. C1- 4 alkoxy groups are preferred. 10 As used herein the term "C 2

-

7 acyl" refers to straight chain or branched chain acyl groups having from two to seven carbon atoms. Illustrative of such acyl groups are acetyl, propionyl (or propiono or propanoyl), isopropionyl (or isopropiono or isopropanoyl), butyryl (or butanoyl), isobutyryl (or isobutanoyl), pentanoyl (or valeryl), hexanoyl (or capronyl) and heptanoyl. 15 As used herein the term "C 2

-

7 acyloxy" refers to straight chain or branched chain acyloxy groups having from two to seven carbon atoms. Illustrative of such acyloxy groups are acetyloxy, propionyl (or propiono or propanoyl)oxy, isopropionyl (or isopropiono or isopropanoyl)oxy, butyryl (or butanoyl)oxy, isobutyryl (or 20 isobutanoyl)oxy, pentanoyl (or valeryl)oxy, hexanoyl (or capronyl)oxy and heptanoyloxy. C2- 4 acyloxy groups are preferred. As used herein the term "C 3

-

6 cycloalkyl" refers to an alicyclic group having from three to six carbon atoms. Illustrative of such cycloalkyl groups are cyclopropyl, 25 cyclobutyl, cyclopentyl and cyclohexyl. Cyclopentyl and cyclohexyl groups are preferred. As used herein the term "C 1

.

6 alkylthio" refers to straight chain or branched chain alkylthio groups having from one to six carbon atoms. Illustrative of such alkylthio " %-" '/oo.ne PCT/GB00/01195 6 groups are methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec butylthio, tert-butylthio, pentylthio, neopentylthio and hexylthio. C- 4 alkylthio groups are preferred. 5 As used herein the term "CI.

6 alkylamino" refers to straight chain or branched chain alkylamino groups having from one to six carbon atoms. Illustrative of such alkylamino groups are methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, neopentylamino and hexylamino. C 1

.

4 alkylamino groups are preferred. 10 As used herein, the term "di(C- 1

.

6 ) alkylamino" refers to straight chain or branched chain di-alkylamino groups having from one to six carbon atoms in each of the alkyl groups. Illustrative of such dialkylamino groups are di-methylamino, di-ethylamino, di-propylamino, di-isopropylamino, di-butylamino, di-isobutylamino, di-sec 15 butylamino, di-tert-butylamino, di-pentylamino, di-neopentylamino and di hexylamino. Di(C 1 .4)alkylamino groups are preferred. As used herein, the term "C 2

-

7 acylamino" refers to straight chain or branched chain acylamino groups having from two to seven carbon atoms. Illustrative of such 20 acylamino groups are acetylamino, propionyl (or propiono or propanoyl)amino, isopropionyl (or isopropiono or isopropanoyl)amino, butyryl (or butanoyl)amino, isobutyryl (or isobutanoyl)amino, pentanoyl (or valeryl)amino, hexanoyl (or capronyl)amino and heptanoylamino. C 2

-

4 acylamino groups are preferred. 25 Where there is a substituent which renders a compound basic, for example when R 6 is an amino, alkylamino or dialkylamino group, addition of an acid results in a salt. The acid may be any suitable acid, and can be organic or inorganic.

vvu uuoo~u' PCT/GB00/01195 7 Preferred compounds of general formula I include those in which, independently or in any compatible combination: each of R' and R 2 represents a Cl- 6 alkyl, preferably a C1-4 alkyl, group; 5 R' and R 2 are the same as each other; each of R 3 and R 4 represents a hydrogen atom;

R

5 represents a hydrogen atom;

R

6 represents a hydrogen atom; each of R 7 and R 8 represents a CI-6 alkyl, preferably methyl, ethyl or isopropyl, group; 10 R 7 and R 8 are the same as each other;

R

9 represents a halogen atom or a methyl or acetyl group. Exemplary compounds include: 15 1. 6,7-Dihydro-2-(2,6-dimethylphenoxy)-9,10-dimethoxy-4H-pyrimido-[6,1 -a]iso quinolin-4-one; 2. 2-(2,6-diethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1-a]iso quinolin-4-one; 3. 6,7-Dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido-[6,1 20 a]isoquinolin-4-one; 4. 6,7-Dihydro-9, 1 0-dimethoxy-2-(2,4,6-trimethylphenoxy)-4H-pyrimido-[6,1 a]isoquinolin-4-one; 5. 2-(4-Chloro-2,6-dimethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido [6,1 -a]isoquinolin-4-one; 25 6. 2-(4-Bromo-2,6-dimethylphenoxy)-6,7-dihydro-9, 1 0-dimethoxy-4H-pyrimido [6,1-a]isoquinolin-4-one; 7. 6,7-Dihydro-9, 1 0-dimethoxy-2-(4-ethanoyl-2,6-dimethylphenoxy)-4H-pyrimido [6,1-a]isoquinolin-4-one; WO 00/58309 PCT/GB00/01195 8 8. 2

-(

2

,

6 -Dichlorophenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1 a]isoquinolin-4-one; 9. 9,10 O-Dimethoxy-2-(2-methylphenoxy)-6,7-dihydro-4H.-pyrimido[6,1 -a] isoquinolin-4-one; 5 10. 9,10-Dimethoxy-2-(2-isobutylphenoxy)-6,7-dihydro-4H-pyrimido[6,1 -a] isoquinolin-4-one; 11. 2

-(

2 -tert-butylphenoxy)-9 ,10-dimethoxy-6, 7 -dihydro-4H-pyrimido[6,1 -a] isoquinolin-4-one; 12. 9,10 O-Dimethoxy-2-(2-ethylphenoxy)-6,7-dihydro-4H-pyrimido[ 6 , 1-a]isoquinolin 10 4-one; 13. 2

-(

2 -Cyclopentylphenoxy)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a] isoquinolin-4-one. The compounds 2

-(

2 ,6-diethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H pyrimido 15 [6,1-a]isoquinolin-4-one and 6,7-Dihydro-2-(2, 6 -diisopropylphenoxy)-9,10 dimethoxy-4H-pyrimido-[6,1- a]isoquinolin-4-one are particularly preferred. Compounds of general formula I may be prepared by any suitable method known in the art and/or by the following process, which itself forms part of the invention. 20 According to a second aspect of the invention, there is provided a process for preparing a compound of general formula I as defined above, excluding the compound 6, 7 -dihydro-2-phenoxy-9,10-dimethoxy-4H-pyrimido[6,1 a]isoquinolin-4-one, the process comprising: 25 (a) reacting a compound of general formula II: WO 00/58309 PCT/GB00/01195 9

R

1 0 X R SN 0O R20 Re 6 - N LG II wherein R , R 2, R , R 6 and X are as defined for general formula I and LG represents a 5 leaving group, with a compound of general formula III OH

R

z R 9

R

8 III 10 wherein R 7, R and R 9 are as defined for general formula I; or (b) when X in general formula I represents a group CR 3

R

4 , wherein R 3 represents a hydrogen atom, R 4 represents a hydrogen atom or a C 1

-

3 alkyl group, and R 5 represents a hydrogen atom or a C1- 3 alkyl group, hydrogenating a compound of general formula 15 IX: wu uu/3juv PCT/GB00/01195 10 4 R R O RO 6 F O 0

R

7 R9 8 IX wherein R', R 2 , R 6 , R 7 , R 8 and R 9 are as defined for general formula I; and 5 (c) optionally converting a compound of general formula I so formed into another compound of general formula I. In compounds of general formula II, the leaving group LG may be chlorine, a thioalkyl group, preferably thiomethyl, or an alkylsulphonyl group, preferably methylsulphonyl. 10 Preferably it is chlorine. The reaction conditions of step (a) are generally such as to favour the reaction, which is a nucleophilic displacement which is preferably carried out in a suitable solvent such as dimethylformamide or isopropanol in the presence of a base such as potassium 15 carbonate. Suitable reaction conditions may be found in GB-A-1597717 and EP-A 0124893, which disclose the preparation of related compounds. The reaction of step (a) is generally applicable for producing compounds of general formula I where R 6 represents a hydrogen atom or a C 1 i-6 alkyl, C 2

-

6 alkenyl, C 2 -6 20 alkynyl, amino, C 1

-

6 alkylamino or C2- 7 acylamino group and R 1 to R 5 and R 7 to R 9 and X have the meanings given above.

Vn UU ZOuY PCT/GB00/01195 11 Compounds of general formula II where LG represents a chlorine atom may be prepared by reacting a compound of general formula IV or a compound of general formula V with phosphorous oxychloride, or by heating a compound of general 5 formula IV with phosphorous pentachloride : R'O6X RsN 2 N 0 NH R O 0 IV 10 R 1 0 RX NH 0 O V 15 wherein R 1 , R 2 , R 5 , R 6 and X are as defined for general formula I. Compounds of general formula II where LG represents a thioalkyl group may be prepared from compounds of general formula IV by heating with phosphorous pentasulphide in a solvent such as dioxan or pyridine to give initially the intermediate thio derivative of compound of formula IV which, on treatment with an alkylating agent such as an alkyl VV uui.ou' PCT/GB00/01195 12 iodide eg. methyl iodide, in a suitable solvent such as tetrahydrofuran or ethyl acetate, gives the thioalkyl compound. Oxidation of the thioalkyl compound with, for example, 3-chloroperbenzoic acid in a solvent such as methylene chloride, gives the alkylsulphone derivative. 5 Compounds of general formula III are substituted phenols which are either known in the art and available from commercial sources or may readily be prepared by methods known per se, for example from the correspondingly substituted aniline compound via hydrolysis of the diazonium salt. 10 Compounds of general formula IV may be prepared by reacting a compound of general formula V, wherein R 1 , R 2 , R 5 and R 6 are as defined for general formula I, with a cyclodehydrating agent such as phosphorous oxychloride, under less vigorous condition, ie lower temperatures, than those required to give compounds of the general 15 formula II where LG represents a chlorine atom. An alternative method has been described in NL-A-6,401,827 (Hoffmann-La Roche) which involves reacting the carbamoylmethylene-tetrahydroisoquinoline, formula VIII, with diethyl carbonate in ethanolic sodium ethoxide:

R

1 0 X.. R5 R 2NH R20N N H 2 O 20 VIII Compounds of general formula V may be prepared by reacting a compound of general formula VI vvu uu/3juy PCT/GB00/01195 13 O R1 0 H H 2/5 RO R VI 5 wherein R 1 , R 2 , R 5 and X are as defined for general formula I with R 6

CH(CO

2 Et) 2 , wherein R 6 is as defined for general formula I, and a strong base such as sodium ethoxide in hot ethanolic solution. Alternatively, the corresponding dimethyl ester can be employed in the presence of hot methanolic sodium methoxide. 10 Compounds of general formula VI may be prepared by reacting a compound of general formula VII

R

1 0 X TNH

R

2 0 2 VII 15 wherein R', R 2 , R 5 and X are as defined for general formula I, with urea by heating at 160 0 C. Alternatively, compounds of formula VII may be reacted with potassium cyanate in the presence of acetic acid in a suitable solvent such as ethanol. 20 Compounds of general formula VII are either known in the art or may readily be prepared by methods known per se. For example, the preparation of 1-(3,4 dimethoxyphenethyl)barbituric acid has been described by B. Lal et al. J.Med.Chem. 27 1470-1480 (1984).

VV uujoju, PCT/GB00/01195 14 Turning now to step (b), the reaction conditions of step (b) are generally to favour the hydrogenation reaction, and the reaction is generally carried out in a suitable solvent such as an alcohol, eg ethanol, with a noble metal catalyst such as palladium, 5 platinum, rhodium or nickel, at room temperature. The catalyst may be supported, for example on charcoal or alumina. Compounds of general formula IX may be prepared from a compound of general formula X: 4 R FO 0 6 H T 10 X wherein R , R 2 and R 6 are as defined for general formula I, and R 4 and R 5 independently represent a hydrogen atom or a C1.

3 alkyl group. The reactions are conducted as described above for converting a compound of general formula IV to a 15 compound of general formula I via compounds of general formula II and the preferred reaction conditions correspond accordingly. Compounds of general formula X may be prepared from compounds of general formula IV (wherein X represents a group CR 3

R

4 , wherein R represents a hydrogen 20 atom, R 4 represents a hydrogen atom or a C1- 3 alkyl group; and R' represents a hydrogen atom or a Ci.

3 alkyl group) by heating with a noble metal catalyst such as palladium, platinum, rhodium or nickel at a temperature of 300 to 350 0 C. The catalyst " U/'o',U PCT/GBOO/O1195 15 may be supported on charcoal or alumina and the reaction carried out in an inert solvent such as an aromatic hydrocarbon, egp-cymene. In optional step (c), a compound of general formula I may be converted into another 5 compound of general formula I. For example, compounds of general formula I where

R

6 represents NH 2 may be converted into compounds of general formula I where R 6 represents a C 1

-

6 alkylamino group by standard chemistry, such as by alkylation of a protected derivative such as an acyl or a p-toluenesulphonyl derivative followed by removal of the protecting group, such as by acid hydrolysis. Compounds of general 10 formula I where R 6 represents a di(CI_ 6 ) alkylamino group may be prepared by direct alkylation of the alkylamino derivative. Compounds of general formula I wherein R , R6 ,

R

7, R8 and/or R 9 represent a C 2

-

3 alkenyl, C2- 6 alkenyl, C 2

.

3 alkynyl or C2- 6 alkynyl group may be hydrogenated to give the corresponding compound with saturated bonds. The reaction conditions for the hydrogenation are as outlined above for step 15 (b). According to a third aspect, the present invention provides a composition comprising a compound of general formula I, including the excluded compound (6,7-dihydro-2 phenoxy-9,10-dimethoxy-4H-pyrimido[6,1a]isoquinolin-4-one), and a veterinarily or 20 pharmaceutically acceptable carrier or diluent. Preferably the composition is a pharmaceutical composition for human medicine. Compounds of the present invention are PDE inhibitors and thus possess valuable pharmacological properties, such as bronchodilator activity as demonstrated by the 25 inhibition of field-stimulated contraction of guinea-pig isolated trachea, and anti inflammatory activity as illustrated in studies on human mononuclear cells stimulated by PHA (phytohaemagglutinin). In vitro and in vivo data indicate the compounds have a long duration of action, as demonstrated by their persistent protective effects against histamine induced bronchospasm in the guinea-pig when inhaled directly into " 'co= PCT/GB00/01195 16 the lungs as a dry powder. The invention therefore also relates to acute, chronic or prophylactic treatment of patients suffering from respiratory disorders including, in particular, asthma, allergic asthma, hay fever, allergic rhinitis, bronchitis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), 5 and cystic fibrosis. They may also be used topically in skin disorders such as atopic dermatitis and psoriasis, or in ocular inflammation or any other disease including cerebral ischaemia or auto-immune diseases in which increasing intracellular concentrations of cAMP is considered beneficial. 10 One or more compounds as set out in the first aspect of the invention may be present in association with one or more non-toxic pharmaceutically and/or veterinarily acceptable carriers and/or diluents and/or adjuvants and/or propellants and, if desired, other active ingredients. Suitable carriers or diluents are known in the art (eg Handbook of Pharmaceutical Excipients (1994) 2 nd Edition, Eds. A. Wade/PJ Weller, 15 The Pharmaceutical Press, American Pharmaceutical Association). Preferably, the compounds and the compositions of the present invention are administered by inhalation, for example by aerosols or sprays which can disperse the pharmacological active ingredient in the form of a powder or in the form of a solution 20 or suspension. Pharmaceutical compositions with powder-dispersing properties usually contain, in addition to the active ingredient, a liquid propellant with a boiling point below room temperature and, if desired, adjuncts, such as liquid or solid non ionic or anionic surfactants and/or wetting agent to form a stable dispersion. Pharmaceutical compositions in which the pharmacological active ingredient is in 25 solution contain, in addition to this, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant, compressed air can also be use, it being possible for this to be produced as required by means of a suitable compression and expansion device. Pharmaceutical compositions vv u uoouoY PCT/GB00/01195 17 may also be delivered by breath activated inhalation devices. Dry powder compositions are preferred for administration by inhalation. According to a fourth aspect, the present invention provides a compound of general 5 formula I or a composition containing a compound of general formula I, including the excluded compound (6,7-dihydro-2-phenoxy-9,10-dimethoxy-4H pyrimido[6,la]isoquinolin-4-one), for use in medicine. Compounds of the present invention are useful as inhibitors of phosphodiesterase 10 isoenzymes. The compounds or compositions of the present invention may be used to prevent or treat any disease in which the compounds or compositions are useful, but particularly a disease in which raising the intracellular concentration of cAMP is desirable. Examples of diseases against which compounds are useful include respiratory disorders including, in particular, asthma, bronchitis, chronic obstructive 15 pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), allergic asthma, hay fever, allergic rhinitis, and cystic fibrosis. They may also be used topically in skin disorders such as atopic dermatitis or psoriasis, ocular inflammation, or any other disease including cerebral ischaemia or auto-immune diseases in which increasing intracellular concentrations of cAMP is considered beneficial. 20 This aspect of the invention is particularly relevant to the treatment of humans, but is also applicable to general veterinary industry, in particular domestic animals such as dogs and cats and farm animals such as horses, pigs, cattle, sheep, etc. 25 Dosage levels of the order of about 0.02 mg to about 200mg, to be taken up to three times daily, are useful in the treatment of the above-mentioned conditions. More particularly, a dosage range of about 0.2 mg to about 20 mg, taken up to three times daily, is effective. The particular dosage regime will however ultimately be determined by the attending physician and will take into consideration such factors as V UUowsoo PCT/GB00/01195 18 the medication being used, age, weight, severity of symptoms and/or severity of treatment being or to be applied, method of administration of the medication, adverse reactions and/or other contraindications. 5 The medication according to this aspect of the invention may be given to a patient together with other active agents, which may for example be a different compound of the present invention, or other compounds. Examples include 32-adrenoceptor agonists, glucocorticoid steroids, xanthine derivatives, antihistamine compounds, leukotriene antagonists, inhibitors of leukotriene synthesis and/or combinations 10 thereof. According to a fifth aspect, the present invention provides the use of a compound of general formula I, including the excluded compound (6,7-dihydro-2-phenoxy-9,10 dimethoxy-4H-pyrimido[6,1 a]isoquinolin-4-one), in the manufacture of an inhibitor of 15 a type III/IV phosphodiesterase isoenzyme. The invention encompasses the use of a compound of general formula I, including the excluded compound (6,7-dihydro-2 phenoxy-9,10-dimethoxy-4H-pyrimido[6,1 a]isoquinolin-4-one), in the manufacture of a bronchodilator and/or an anti-asthmatic medication and/or a medicament for the prevention or treatment of chronic obstructive pulmonary disease (COPD). 20 The invention also relates to a method for the treatment or prevention of a disease in a mammal where a phosphodiesterase isoenzyme inhibitor and/or a bronchodilator would be expected to be of benefit, which method comprises administering to said mammal an amount of an effective, non-toxic amount of a compound of general 25 formula I, including the excluded compound ( 6 ,7-dihydro-2-phenoxy-9,10-dimethoxy 4H-pyrimido[6,1a]isoquinolin-4-one). The invention encompasses a method of treating or preventing asthma and/or chronic obstructive pulmonary disease (COPD) in a mammal.

VV uu/oauY PCT/GB00/01195 19 Preferred features of each aspect of the invention apply to each other aspect of the invention, mutatis mutandis. The following examples, without being limiting, illustrate the invention, with 5 reference to the following figures: Figure 1, referred to in Example A(i) below, is a graph showing the effect of DMSO (0.05%) on cholinergic contractile response in superfused guinea pig trachea number of experiments (n) is 10); 10 Figure 2, referred to in Example A(i) below, is a graph showing the effect of 10 pM of the compound of Example 3 on the contraction of guinea pig trachea to electrical field stimulation over time (number of experiments (n) is 3), wherein the arrow denotes commencement of washout period; Figure 3, referred to in Example A(i) below, is a graph showing the effect of 10 pM of 15 the compound of Example 11 on cholinergic contractile response in superfused guinea-pig trachea (number of experiments (n) is 3), wherein the arrow denotes commencement of washout period; Figure 4, referred to in Example A(i) below, is a graph showing the effect of 10 p.M of the compound of Example 12 on cholinergic contractile response in superfused 20 guinea-pig trachea (number of experiments (n) is 3), wherein the arrow denotes commencement of washout period; Figure 5, referred to in Example A(i) below, is a graph showing the effect of 10 pM of the compound of Example 13 on cholinergic contractile response in superfused guinea-pig trachea (number of experiments (n) is 3), wherein the arrow denotes 25 commencement of washout period; Figure 6, referred to in Example A(ii) below, is a graph showing the effect of 10pM of the compound of Example 1 on the contraction of guinea pig trachea to electrical field stimulation over time (n=1), wherein the arrow denotes commencement of washout period; vv % Uw1juY PCT/GBOO/O1195 20 Figure 7, referred to in Example A(ii) below, is a graph showing the effect of 10ptM of the compound of Example 8 on the contraction of guinea pig trachea to electrical field stimulation over time (n=l), wherein the arrow denotes commencement of washout period; 5 Figure 8, referred to in Example A(ii) below, is a graph showing the effect of 10tM of the compound of Example 2 on contraction of guinea pig trachea to electrical field stimulation over time (n=1), wherein the arrow denotes commencement of washout period; Figure 9 referred to in Example A(i) below, is a graph showing the effect of 10tM of 10 the compound of Example 10 on contraction of guinea pig trachea to electrical field stimulation; Figure 10, referred to in Example B below, is a graph showing the effect of various compounds of the present invention against proliferation of human mononuclear cells stimulated by PHA, wherein each point represents the mean of six experiments, and 15 vertical lines represent standard error of the mean. Preparation 1: Synthesis of 2-Chloro-6.7-dihydro-9.10-Dimethoxv-4H-pyrimido-[6,1 a]isoquinolin-4-one 20 8 7 MeOC 9 6 6 MeO N 1 / Cl A mixture of 1-(3,4-dimethoxyphenyl) barbituric acid (70g, 0.24mol), prepared according to the method described in B. Lal et al. JMed.Chem. 27 1470-1480 (1984), " "',-,, PCT/GB00/01195 21 and phosphorus oxychloride (300ml, 3.22mol) was refluxed for 2.5h. The excess phosphorous oxychloride was removed by distillation (20mmHg) on warming. After cooling the residue was slurried in dioxan (100ml) and cautiously added to a vigorously stirred ice/water solution (11). Chloroform (11) was added and the resulting 5 mixture was basified with 30% sodium hydroxide solution. The organic layer was separated and the aqueous phase further extracted with chloroform (2x750ml). The combined organic extracts were washed with water (1.51), dried over magnesium sulphate and concentrated in vacuo to leave a gummy material (90g). This was stirred in methanol for a few minutes, filtered and washed with methanol (200ml), diethyl 10 ether (2x200ml) and dried in vacuo at 40 0 C to yield the title compound as a yellow/orange solid. 47g, 62% (300MHz, CDCl 3 ) 8 2.96(2H, t, C(7 ) H2); 3.96(6H, s, 2xOCH 3 ; 4.20(2H, t, C(6 )

H

2 ); 6.61(1H, s, C(1) H); 6.76(1H, s, Ar-H); 7.10(1H, s, Ar-H). 15 Preparation 2: 2.6-Diethylphenol To a solution of 2,6-diethylaniline (14.9g, 0.10mol) in glacial acetic acid (200ml) was added dropwise concentrated sulphuric acid (90ml) with stirring and cooling to 20 maintain temperature between 10 - 20 0 C. The solution was then stirred at 0 0 C while sodium nitrite (9.0g, 0.13mol) in water (50ml) was added dropwise at such a rate as to keep the temperature below 5 0 C. The mixture was stirred for a further 20 minutes at 0 0 C then an ice cold solution of urea (3.0g) in water (300ml) was added slowly to produce an amber solution. A mixture of concentrated sulphuric acid (150ml) and 25 water (600ml) was stirred and heated to gentle reflux while the ice cold solution of the diazonium salt was added dropwise maintaining a gentle reflux. It is important that the diazonium salt solution drops directly into the sulphuric acid solution without touching the sides of the flask. After the addition the mixture was stirred for a further 10 minutes at reflux then for 18h at room temperature. The dark red brown product wu iUU/:ZiU9 PCT/GB00/01195 22 was removed by filtration, washed with water and dissolved in dichloromethane. The solution was dried over magnesium sulphate and evaporated to obtain a solid which was purified by column chromatography (Silica gel; 2:1 petroleum ether 40 - 60 0 C : dichloromethane) to give the title compound as colourless crystals. 9.6g, 64% 5 mpt.: 38-39 0 C (60MHz, CDC1 3 ) 8 1.23(6H, t, CH 3 ); 2.63(4H, q, CH 2 ); 4.67 (1H, s, OH); 6.90 7.20(3H, m, Ar-H). 10 wu UU/iJU9 PCT/GB00/01195 23 Example 1: Synthesis of 6.7-Dihydro-2-(2.6-dimethylphenoxy)-9.10-dimethoxv-4H pyrimido-[6,1-a]isoquinolin-4-one 8 7 MeO N N 0 MeO 101 4 1 / N 3 2 0 5 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1 -a]isoquinolin-4-one (20g, 68.4mmol), prepared according to Preparation 1, and 2,6-dimethylphenol (obtained from Aldrich) (25.1g, 167mmol) were dissolved in anhydrous N,N-dimethyl formamide (160ml). Potassium carbonate (28.3g, 205mmol) was added and the 10 mixture heated to 90 0 C under nitrogen for 3h. The mixture was then cooled, added to water (600ml) and extracted with ethyl acetate (3x300ml). The combined organic extracts were washed with sodium bicarbonate (300ml), water (2x 500ml) and brine (300ml), dried over magnesium sulphate and concentrated in vacuo to give a gummy residue. This was triturated with ether (250ml), isolated by filtration and re 15 crystallised from methanol (300ml) to give the title compound as a pale yellow solid. 20g, 67% mpt. : 216-218 0 C. (300MHz, d 6 DMSO) 8 2.05(6H, s, 2x CH 3 ); 2.95(2H, t, C( 7 ) H 2 ); 3.85 (3H, s, 20 OCH 3 ); 3.90(3H, s, OCH 3 ); 4.05(2H, t, C( 6 ) H2); 6.95-7.15(5H, m, Ar-H); 7.55(1H, s, Ar-H).

vv uuoou PCT/GB00/01195 24 Example 2: Synthesis of 6 ,7-Dihydro-2-(2,6-diisopropvlphenoxy)- 9.10-dimethoxv 4H-pyvrimido-[6. I -a]isoquinolin-4-one 8 7 MeO 9 7 5 MeO 10 11 4 2 0 5 2-Chloro-6,7-dihydro-9, 1 0-dimethoxy-4H-pyrimido-[6, 1 -a]isoquinolin-4-one, prepared according to Preparation 1 ( 2 3.1g, 79mmol) and 2,6-diisopropylphenol (42.2g, 237mmol) were dissolved in anhydrous N,N-dimethyl formamide (160ml). Potassium carbonate (32.3g, 234mmol) was added and the mixture heated to 90 0 C 10 under nitrogen for 4.5h. The mixture was then cooled, added to water (800ml) and extracted with ethyl acetate (3x300ml). The combined organic extracts were washed with sodium bicarbonate (300ml), water (2x500ml) and brine (300ml), dried over magnesium sulphate and concentrated in vacuo. The residue was purified by column chromatography (Silica gel; dichloromethane then 2% methanol in dichloromethane. 15 The isolated product was re-crystallised from ethyl acetate (250ml) as a pale yellow solid. Residual ethyl acetate was removed by dissolving the compound in dichloromethane and concentrating in vacuo to give the title compound as a pale yellow solid. 20.5g, 52% mpt.: 123-126 0 C. 20 vv i uu/nnuy PCT/GB00/01195 25 (300MHz, CDC1 3 ) 8 1.15(12H, d, 4x CH-Me); 3.0(4H, m); 4.00(6H, s, 2x OCH 3 ); 4.25(2H, t, C( 6 ) H 2 ); 6.40(1H, s, C(1) H);6.65(1H, s, Ar-H); 7.20(4H, min). Example 3: Synthesis of 2-(2.6-diethylphenoxy)-6.7-dihydro-9,10-dimethoxv-4H 5 pyrimido-[6,1-a]isoquinolin-4-one MeO 9 7 5 MeO 10 1

/-N

3 2 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one, 10 prepared according to Preparation 1 (1.0g, 3.41mmol), and 2,6-diethylphenol (1.53g, 10.2mmol) were dissolved in propan-2-ol (125ml). Potassium carbonate (1.41g, 10.2mmol) was added and the mixture heated to reflux under nitrogen for 24h. The mixture was then cooled and the solid removed by filtration. The filtrate was evaporated in vacuo and the residue purified by column chromatography (Silica gel; 15 2:1 ethyl acetate : dichloromethane) to give the title compound as a pale yellow solid. 1.21g, 88% mpt. : 180- 181 0 C. HPLC: Area (%) 100.00 20 Column ODS (150 x 4.6mm) MP pH=4 KH2PO 4 / MeOH (50/50) FR 0.8 ml/min vV Vu uUiOc PCT/GB00/01195 26 RT 11.634 Detection 250nm (300MHz, CDCl 3 ) 6 1.21(6H, t, CH 3 ); 2.54(4H, q, CH 2 ); 2.99(2H, t, J= 6.5 Hz, C( 7 ) 5 H 2 ); 3.98(3H, s, OCH 3 ); 4.00 (3H, s, OCH 3 ); 4.22(2H, t, J= 6.5 Hz, C( 6 ) H 2 ); 6.42(1H, s, C(1) H); 6.79(1H, s, Ar-H); 7.10-7.19(3H, m, Ar-H); 7.22(1H, s, Ar-H). Example 4: Synthesis of 6.7-Dihydro-9.,10-dimethoxv-2-(2.4.6-trimethylphenoxy) 10 4H-pyrimido-[6,1 -a]isoquinolin-4-one 8 7 MeO 9 7 5 N O MeO 10 1 0 11 4 1 N3 2 0 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one, as 15 prepared in Preparation 1 (1.2g, 4.10Ommol), and 2 ,4,6-trimethylphenol (obtained from Aldrich) (1.68g, 12.34mmol) were dissolved in propan-2-ol (145ml). Potassium carbonate (1.70g, 12.30mmol) was added and the mixture heated under nitrogen for 16h. After cooling to room temperature, the mixture was filtered and the precipitate washed with acetone. The combined filtrates were evaporated in vacuo. The residue 20 was dissolved in dichloromethane (200ml) and washed with 10% sodium hydroxide (aq) (40ml) and then water (40ml). The organic layer was dried over magnesium sulphate, filtered and evaporated in vacuo to yield a brown oil. Column vv U UDO uY PCT/GB00/01195 27 chromatography (Silica gel: 4:1 ethyl acetate : petroleum ether 40-600 followed by 4:1 ethyl acetate : dichloromethane) gave a yellow solid. This material was washed with diethyl ether, collected by filtration and dried in vacuo to yield the title compound as a pale yellow solid. 0.56g, 35%. 5 mpt.: 230-231 0 C m/z: C 2 3

H

2 4 N20 4 requires M=392 found (M+1) = 393 HPLC: Area (%) 99.65 Column ODS (150 x 4.6mm) MP 0.2M NH 4 Ac / MeOH (30/70) 10 FR (ml/min) 0.7 RT (min) 13.006 Detection 250nm (300MHz, CDCl 3 ) 8 2.15(6H, s, 2x CH 3 ); 2.28(3H, s, CH 3 ); 3.0 (2H, t, C( 7 ) H 2 ); 15 3.98(6H, s, 2xOCH 3 ); 4.23(2H, t, C( 6 ) H 2 ); 6.42(1H, s, C(1) H); 6.78(1H, s, Ar-H); 6.86(2H, s, 2x Ar-H); 7.20(1H, s, Ar-H).

wr u owuu PCT/GB00/01195 28 Example 5: Synthesis of 2-( 4 -Chloro-2,.6-dimethylphenoxy)- 6,7-dihydro-9.10 dimethoxy-4H-pyrimido-[6.,1 -a]isoquinolin-4-one 8 MeO ~ 6 NO MeO 1 0 0 11 4 1 N3 2 0 CI 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1 -a]isoquinolin-4-one, prepared in Preparation 1, (1.5g, 5.13mmol) and 4-chloro-2,6-dimethylphenol (obtained from Lancaster Chemicals) (2.41g, 15.39mmol) were dissolved in propan-2 ol (180ml). Potassium carbonate (2.13g, 15.41mmol) was added and the mixture 10 heated under nitrogen for 16h. After cooling to room temperature, the mixture was filtered and the precipitate washed with acetone. The combined filtrates were evaporated in vacuo. The residue was dissolved in dichloromethane (200ml) and washed with 10% sodium hydroxide (aq) (45ml) and then water (45ml). The organic layer was dried over magnesium sulphate, filtered and evaporated in vacuo to yield an 15 orange oil. Column chromatography (Silica gel: 4:1 ethyl acetate : dichloromethane) gave a yellow solid. This material was washed with diethyl ether, collected by filtration and dried in vacuo to yield the title compound as a pale yellow powder. 1.78g, 84%. mpt. : 201 - 203 0 C. 20 m/z: C 2 2H 2 1 3 5 C1N 2 0 4 requires M=412 found (M+1) = 413 C2 2

H

2 1 3 7 C1N 2 0 4 requires M=414 found (M+1) = 415 HPLC: Area (%) 97.91 vvu UU/5JUY PCT/GB00/01195 29 Column ODS (250 x 4.6mm) MP 0.1M NH 4 Ac / MeCN (40/60) FR (ml/min) 0.7 RT (min) 12.466 5 Detection 250nm (300MHz, CDC1 3 ) 8 2.13(6H, s, 2xCH 3 ); 2.99 (2H, t, C( 7 ) H 2 ); 3.98(3H, s, OCH 3 ); 4.02(3H, s, OCH 3 ); 4.23(2H, t, C( 6 ) H 2 ); 6.43(1H, s, C(1) H); 6.78(1H, s, Ar-H); 7.04(2H, s, 2x Ar-H); 7.21(1H, s, Ar-H). 10 Example 6: Synthesis of 2

-(

4 -Bromo-2,6-dimethvlphenoxv)- 6,7-dihydro-9.10 dimethoxv-4H-pyrimido-[6.,1-a]isoquinolin-4-one 8 MeO 9 7 MeO 10 1 0 11 4 1 N3 2 0 Br 15 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1 -a]isoquinolin-4-one, prepared in Preparation 1, (1.5g, 5.13mmol) and 4-bromo-2,6-dimethylphenol (obtained from Lancaster Chemicals) (3.1g, 15.43mmol) were dissolved in propan-2 ol (180ml). Potassium carbonate (2.13g, 15.41mmol) was added and the mixture 20 heated under nitrogen for 16h. After cooling to room temperature, the mixture was filtered and the precipitate washed with acetone. The combined filtrates were evaporated in vacuo. The residue was dissolved in dichloromethane (200ml) and vV uw ouv PCT/GB00/01195 30 washed with 10% sodium hydroxide (aq) (45ml) and then water (45ml). The organic layer was dried over magnesium sulphate, filtered and evaporated in vacuo to yield an orange oil. Column chromatography (Silica gel: 4:1 ethyl acetate : dichloromethane) gave a yellow solid. This material was washed with diethyl ether, collected by 5 filtration and dried in vacuo to yield the title compound as a pale yellow powder 1.90g, 81%. mpt. : 232 - 234 0 C. m/z: C2 2

H

2 1 7 9 BrN 2 0 4 requires M=456 found (M+l) = 457

C

2 2 H2, 1 8 1 BrN20 4 requires M=458 found (M+ 1) = 459 10 HPLC: Area (%) 99.77 Column ODS (150 x 4.6mm) MP 0.1M NH 4 Ac / MeCN (57/43) FR (ml/min) 1.0 RT (min) 11.565 15 Detection 250nm (300MHz, CDCl 3 ) 8 2.14(6H, s, 2xCH 3 ); 2.98(2H, t, C( 7 ) H 2 ); 3.97(3H, s, OCH 3 ); 3.99 (3H, s, OCH 3 ); 4.22(2H, t, C( 6 ) H 2 ); 6.43(1H, s, C( 1 ) H); 6.78(1H, s, Ar-H); 7.19(2H, s, 2x Ar-H); 7.21(1H, s, Ar-H). 20 " uu"o., PCT/GB00/01195 31 Example 7: Synthesis of 6,7-Dihydro-9. 1 0-dimethox-2-(4-ethanol-26 dimethylphenoxv)- 4H-pvrimido-[6.1 -a]isoquinolin-4-one 8 MeO 9 7 5 6 N O MeO 1 0 - N3 ,1 4 2 0 Me 0 5 6,7-Dihydro-2-chloro-9, 1 0-dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one, prepared in Preparation 1, (1.5g, 5.13mmol) and 2

,

6 -dimethyl-4-hydroxyacetophenone (obtained from Maybridge Chemicals) ( 2 .53g, 15.41mmol) were dissolved in propan 2-ol (180ml). Potassium carbonate (2.13g, 15.41mmol) was added and the mixture 10 heated under nitrogen for 23h. After cooling to room temperature, the mixture was filtered and the precipitate washed with acetone. The combined filtrates were evaporated in vacuo. The residue was dissolved in dichloromethane (200ml) and washed with water (2x 45ml). The organic layer was dried over magnesium sulphate, filtered and evaporated in vacuo to yield an orange oil. Column chromatography 15 (Silica gel: 4:1 ethyl acetate : dichloromethane) gave a yellow solid. This material was washed with diethyl ether, collected by filtration and dried in vacuo to yield the title compound as a pale yellow powder 1.81g, 84%. mpt. : 228- 230 0 C. m/z: C 24

H

24

N

2 0 5 requires M=420 found (M+1) = 421 20 HPLC: Area (%) 99.32 Column ODS vvo uu/o0vuy PCT/GB00/01195 32 MP 0.1M NH 4 Ac / MeCN (65/35) FR (ml/min) 1.0 RT (min) 13.840 Detection 250nm 5 (300MHz, CDC1 3 ) 8 2.25(6H, s, 2xCH 3 ); 2.60(3H, s, CH 3 CO); 3.02(2H, t, C(7 ) H2,); 3.99(3H, s, OCH 3 ); 4.01 (3H, s, OCH 3 ); 4.23(2H, t, C(6 ) H2); 6.47(1H, s, C(1) H); 6.78(1H, s, Ar-H); 7.21(1H, s, Ar-H); 7.68(2H, s, 2xAr-H). 10 Example 8: Synthesis of 2-(2.6-Dichlorophenoxy)- 6.,7-dihydro-9,10-dimethoxy-4H pyrimido-[6, I -a]isoquinolin-4-one 7 MeO 7 6 5 0 NO MeO 10 11 4 Cl 2 0 CI 15 2-Chloro-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1 -a]isoquinolin-4-one, prepared in Preparation 1 (25g, 85mmol) and 2,6-dichlorophenol (25.1g, 153mmol) were dissolved in anhydrous N,N-dimethyl formamide (150ml). Potassium carbonate (30g, 217mmol) was added and the mixture heated to 90'C under nitrogen for 2h. 20 The mixture was then cooled, partitioned between water (11) and ethyl acetate (500ml). The resulting precipitate was filtered, washed with ethyl acetate (100ml) and water (100ml), re-dissolved in dichloromethane (500ml) and washed with brine VV UU13a1 u PCT/GB00/01195 33 (200ml), dried over magnesium sulphate and concentrated in vacuo. The residue was re-crystallised from acetonitrile (600ml) to give the title compound as a pale yellow solid. 22g, 53% 5 mpt.: 240-242 0 C. (300MHz, d 6 DMSO) 8 2.05(2H, t, C(7 ) H2); 3.85(3H, s, OCH 3 ); 3.90 (3H, s,

OCH

3 ); 4.05(2H, t, C(6 )

H

2 ); 7.00(1H, s, C(1) H); 7.15(1H, s, Ar-H); 7.35(1H, t, Ar H); 7.65(1H, s, Ar-H). 10 Example 9: Synthesis of 9.10-Dimethoxy-2-(2-methylphenoxy)-6,7-dihydro-4H pyrimido[6. -a]isoquinolin-4-one 15 MeO N O MeO /N 0 2-Chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1) (1.5g, 20 5.13 mmol) and o-cresol (1.66g, 15.4 mmol) were dissolved in iso-propanol (180 ml). Potassium carbonate (2.13g, 15.4 mmol) was added and the mixture was heated at reflux, under nitrogen, for 18h. After cooling to room temperature, the mixture was IV uuu/ouv PCT/GB00/01195 34 filtered and the precipitate washed with acetone. The combined filtrates were evaporated in vacuo and the residue was dissolved in dichloromethane (220 ml), washed with 10% NaOH (40 ml), then with water (40 ml) and dried (MgSO4). Evaporation in vacuo gave a brown oil which was purified by column chromatography 5 on silica gel (EtOAc / petroleum ether 80:20). The title compound was obtained as a pale yellow solid, 0.56g, 35%. M.p.: 201-203 0 C m/z: C 2 1

H

20

N

2 0 4 requires M=364 found (M+I) = 365 10 HPLC: Area (%) 99.01 Column ODS (150 x 4.6 mm) MP 0.2M NH4OAc / MeOH (40/60) FR (ml/min) 0.8 RT (min) 14.976 15 Detection 235 nm 1 H NMR (300 MHz, CDC1 3 ): 8 2.22 (3H, s, ArCH 3 ), 2.97 (2H, t, CH 2 ), 3.98 (6H, s, 2xOCH 3 ), 4.21 (2H, t, CH2), 6.40 (1H, s, C=CH), 6.78 (1H, s, ArH), 7.04-7.25 (5H, m, 5xArH). 20 Example 10: Synthesis of 9.10-Dimethoxv-2-(2-isobutylphenoxv)-6,7-dihydro-4H pyrimido[6. 1-a] isoquinolin-4-one 2-Isobutyvlphenol 25 Potassium tert-butoxide (44.8g, 400mmol) was suspended in degassed heptane (1300ml) and butyllithium (258ml, 400mmol) added. The reaction mixture was allowed to stir at room temperature for 20 min and then O-cresol (10.8g, 100mmol) o ouwo.ou, PCT/GBOO/O1195 35 added followed by heating at reflux for 3 hours. The mixture was then cooled to room temperature and the precipitate allowed to settle. The excess solvent was decanted off and the residue washed with pentane. This process was repeated once more and the resultant precipitate suspended in THF (1500ml). This suspension was transferred, via 5 cannula, to a solution of isopropylbromide (10.33ml, 110mmol) in THF (500ml) and the reaction mixture stirred at room temperature overnight. The mixture was quenched by addition of water (50ml) and acidified with HCI (5M). The aqueous phase was separated and extracted with chloroform (3 x 100ml). The organic phases were combined, dried (MgSO 4 ), filtered and concentrated to give a red brown liquid. The 10 crude material was purified by flash column chromatography (90:10 petrol:EtOAc) to give the title compound as a yellow oil (7.63g, 51%). 9,10-Dimethoxv-2-(2-isobutylphenoxy)-6,7-dihydro-4H-pyrimido[6.1-a]isoquinolin 4-one 15 2-Isobutylphenol (1.24g, 8.16mmol) was dissolved in THF (40ml) and cooled to 78 0 C. Butyllithum (5.12ml, 8.16mmol) was then added and the reaction mixture stirred at -78 0 C for 1 hour and then slowly warmed to room temperature overnight. The reaction was quenched by addition of NH 4 C1 (40ml) and the mixture extracted 20 with EtOAc (3 x 50ml). The combined organic phases were washed with NH 4 C1 (50ml). This aqueous phase was combined with that from the previous washing and extracted with EtOAc (50ml). All the organic phases were now combined and washed with brine, dried (MgSO 4 ), filtered and concentrated to give a yellow oily solid. Ether was added to the residue and the resultant solid filtered off and washed with ice cold 25 ether. The residue was purified by flash column chromatography eluting with ethyl acetate to give the title compound as a white solid (0.5g, 18%).

r u Uowoau', PCT/GB00/01195 36 OH (i) KOtBu / BuLi / Heptane OH (ii) 'PrBr /THF OH OH MeO MeO N 0 N O MeO MeO N N BuLi /THF -800C C1 5 Example 11: Synthesis of 2-2-tert-butylphenoxr)-9,1 0-dimethoxy-6,7-dihydro-4H pvrimido[6.1 -a]isoquinolin-4-one MeO /N N MeO /N 0 2-Chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1.47g, 10 5.0 mmol) and 2-tert-butylphenol

(

2 .25g, 15.0 mmol) were dissolved in 2-propanol (180 ml). Potassium carbonate (2.07g, 15.0 mmol) was added and the mixture was stirred and heated to reflux, under nitrogen, for 6h. After cooling to room temperature wU uUa./u9 PCT/GB00/01195 37 the mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel [ethyl acetate/dichloromethane (3:2)] to provide the above compound (1.35g, 66%) as an off-white solid. 5 M.p.: 226-228 0 C M/z: C 24

H

26

N

2 0 4 requires M=406, found m/z [ES+] = 407 HPLC: Area (%) 99.68 Column S5 C8 (250 x 4.6 mm) MP pH 4 0.02M KH 2

PO

4 / CH 3 CN (35/65) 10 RT (min) 9.175 FR (ml/min) 0.7 Detection 254 nm H NMR (300 MHz, CDCl 3 ): 5 1.39 (9H, s, CH(CH) 3 ), 2.97 (2H, t, CH 2 ), 3.97 (3H, 15 s, OCH 3 ), 3.98 (3H, s, OCH 3 ), 4.23 (2H, t, CH 2 ), 6.38 (1H, s, C=CH), 6.78 (1H, s, ArH), 7.05-7.27 (4H, m, 4xArH), 7.40 (1H, m, ArH). Example 12: Synthesis of 9,10-Dimethoxy-2-(2-ethylphenoxy)-6,7-dihydro-4H pyrimido[6. I -a]isoquinolin-4-one 20 MeO S N 0 MeO N 2-Chloro-9, 1 0-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a] isoquinolin-4-one (1.47g, 5.0 mmol) and 2-ethylphenol (1.22g, 10.0 mmol) were dissolved in 2-propanol (180 w u UU/owU5M PCT/GB00/01195 38 ml). Potassium carbonate (1.38g, 10.0 mmol) was added and the mixture was stirred and heated to reflux, under nitrogen, for 6h. After cooling to room temperature the mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel [ethyl acetate/dichloromethane (3:2)] to 5 provide the above compound (1.27g, 67%) as a pale yellow solid. M.p.: 167-169 0 C M/z: C 2 2

H

2 2

N

2 0 4 requires M=378, found m/z [ES+] = 379 HPLC: Area (%) 99.46 10 Column S5 C8 (250 x 4.6 mm) MP pH 4 0.02M KH 2

PO

4 / CH 3 CN (35/65) RT (min) 7.725 FR (ml/min) 0.7 Detection 254 nm 15 1H NMR (300 MHz, CDC1 3 ): 6 1.21 (3H, t, CH 2

CH

3 ), 2.59 (2H, q, CH 2

CH

3 ), 2.97 (2H, t, CH 2 ), 3.97 (6H, s, 2xOCH 3 ), 4.21 (2H, t, CH 2 ), 6.41 (1H, s, C=CH), 6.78 (1H, s, ArH), 7.05-7.28 (5H, m, 5xArH). 20 Example 13: Synthesis of 2-(2-Cyclopentvlphenoxy)-9,10-dimethoxy-6.7-dihvdro-4H pyrimido[6.1-a]-isoquinolin-4-one MeO S N 0 MeO N

O

vvu uunimu PCT/GB00/01195 39 2-Chloro-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (1.20g, 4.11 mmol) and 2 -cyclopentylphenol (1.0g, 6.17 mmol) were dissolved in 2 -propanol (150 ml). Potassium carbonate (1.14g, 8.23 mmol) was added and the mixture was 5 stirred and heated to reflux, under nitrogen, for 18h. After cooling to room temperature the mixture was filtered and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel [ethyl acetate/dichloromethane (3:2)] to provide the above compound (0.51g, 30%) as a pale yellow solid. 10 M.p.: 122-125 0 C M/z: C 2 5

H

26

N

2 0 4 requires M=418, found m/z [ES+] = 419 HPLC: Area (%) 99.43 Column S5 C8 (250 x 4.6 mm) MP pH 4 0.02M KH 2

PO

4 / CH 3 CN (35/65) 15 RT (min) 10.191 FR (ml/min) 0.7 Detection 254 nm IH NMR (300 MHz, CDCl 3 ): 6 1.57-2.05 (8H, m, cyclopentyl 4xCH 2 ), 2.97 (2H, t, 20 CH 2 ), 3.13 (1H, m, ArCH), 3.97 (6H, s, 2xOCH 3 ), 4.21 (2H, t, CH 2 ), 6.38 (1H, s, C=CH), 6.78 (1H, s, ArH), 7.07 (1H, m, ArH), 7.18 (3H, m, 3xArH), 7.32 (1H, m, ArH). The pharmacological utility of the compounds of the present invention has been 25 demonstrated in studies using compounds previously synthesised as described in the above Examples. The results below serve to illustrate the generic application of the compounds of the present invention.

vvwu uu/aju PCT/GB00/01195 40 Example A: Efficacy of compounds of the invention against electrical-induce contraction of guinea-pig isolated trachea The efficacy of compounds of the invention was tested against electrical-induced 5 contraction of guinea-pig isolated trachea. The results demonstrate that the compounds of Examples 2, 3, 10, 11, 12 and 13 inhibit the contractile responses with a long duration of action. Example A (i) - Superfusion experiments 10 Method Superfusion of guinea-pig tracheal rings was performed according to a previously described method (Coleman et al. Pulmonary Pharmacology 9 107-117 (1996)). Briefly, guinea-pig tracheal preparations were cut into rings and then opened by sectioning the ring opposite the smooth muscle and suspended between two platinum 15 electrodes under Ig tension. The tissues were superfused at a rate of from 1 to 3.25 ml/min with Krebs-Henseleit solution at 37 0 C containing the cyclooxygenase inhibitor, indomethacin (5ptM) and bubbled with 95% 02 and 5% CO 2 . Tracheal preparations were allowed to equilibrate for 40 minutes before commencement of electrical stimulation, delivered as a 10 s train of square wave pulses at 3Hz, 0.1ms 20 duration and 20V (approx 400 mAmps) generated every 100 sec by means of physiological square wave-stimulator. The compounds of the present invention were superfused at a rate of 0.2 to 0.3 ml/min and contractile responses to electrical field stimulation were recorded on a Macintosh computer using MacLab software. The drug was prepared in DMSO and diluted in Krebs-Henseleit solution which yielded a 25 final superfusion concentration of 0.05 to 0.1% DMSO. Results The duration of action was determined and expressed as onset time (OTs 50 ) and recovery time (RTs 5 o). The time taken to reach 50% of the maximum inhibition of the Swuuo.uy PCT/GB00/01195 41 contractile response (onset time: OT) was determined and similarly, the time taken for a 50% reversal of the maximum inhibition of the contractile response (recovery time:

RT

50 ) following the termination of exposing the tissue to compound. These values are shown in Table 1. 5 As shown in Table 1, all the tested compounds caused a time dependent inhibition of the contractile response to electrical field stimulation in guinea-pig isolated trachea. The recovery of the contractile response to electrical field stimulation was slow, with the inhibitory responses not reversing 4 hours after cessation of administration of the 10 drug. The vehicle, DMSO, failed significantly to inhibit the contractile response to electrical field stimulation (Figure 1). Table 1. OT .and RT0values Compound OTs 50 (min) RT 50 Figure (min) 2

-(

2 ,6-diethylphenoxy)-6,7-dihydro-9,10- 17±1 >240 Fig 2 dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one (Example 3) 2 -(2-tert-butylphenoxy)-9,10-dimethoxy-6,7- 14.7±1.33 >300 Fig 3 dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (Example 11) 9,10 O-Dimethoxy-2-(2-ethylphenoxy)-6,7-dihydro- 9.7±0.6 263 Fig 4 4H-pyrimido[6,1-a]isoquinolin-4-one (Example 12) 2

-(

2 -Cyclopentylphenoxy)-9,10-dimethoxy-6,7- 18.6±1.5 >300 Fig 5 dihydro-4H-pyrimido[6,1-a]- isoquinolin-4-one (Example 13) ' 9,10 O-Dimethoxy-2-(2-isobutylphenoxy)-6,7- 17.5+1.25 >300 Fig 9 wu ouU/iu PCT/GB00/01195 42 dihydro-4H-pyrimido[6,1 -a] isoquinolin-4-one (Example 10) 6

,

7 -Dihydro-2-(2,6-diisopropylphenoxy)-9,10- 24.9+5.0 >240 Fig 8 dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one (Example 2) Trequinsin (9,10-dimethoxy-3-methyl-2- 14 146 mesitylimino-2,3,6,7-tetrahydro-4H-pyrimido[6,1 a]isoquinolin-4-one) (Each value is the mean and s.e. mean of three observations except where otherwise indicated, as "n") 5 Example A (ii) - Organ bath experiments Method Guinea-pig tracheal rings were suspended in organ baths under 1 g tension between two electrodes and subjected to electrical field stimulation (3Hz, 0.1ms duration and 20V (approx 400mAmps) generated every 100 sec by means of physiological square 10 wave-stimulator. The compounds of Examples 1, 2 and 8 were dissolved in DMSO containing Tween 80 (10%) and distilled water (0.01M), which were then added to the organ bath to give a final concentration of 10pM. Results 15 The results are shown in Figures 6 (for compound of Example 1), 7 (for compound of Example 8) and 8 (for compound of Example 2). All compounds caused complete inhibition of the contractile response to electrical field stimulation and the effect was maintained for more than 2-4 hours. 20 Example B: Efficacy of compounds of the invention against proliferation of human mononuclear cells stimulated by PHA wu uu/35iu9 PCT/GB00/01195 43 The effect of compounds of the invention against proliferation of human mononuclear cells stimulated by PHA was also investigated. Proliferation was significantly inhibited by these compounds, indicating that they possess anti-inflammatory activity. 5 The results below serve to illustrate the generic application of the novel compounds of the present invention. Method Normal healthy volunteers underwent phlebotomy and 25ml of blood was collected. 10 Mononuclear cells were separated and purified according to the method of Banner et al. (Banner et al. Br. J. Pharmacol. 116 3169-3174 (1995)). Human peripheral mononuclear cells (100,000 per well) were stimulated for 24h with phytohaemagglutinin (PHA, 2pg/ml) in the absence or presence of the compounds of Examples 1, 2 and 8 (0.001-100pM) at 37 0 C in a 95% air, 5% CO 2 atmosphere. 15 Twenty four hours later, [ H]-thymidine (0.1 pCi) was added to each well and the cells were incubated for a further 24h period. Cells were then harvested onto glass fibre filters using a cell harvester (ICN Flow, Buckinghamshire) and counted in a scintillation counter. 20 Results All the compounds under test caused a concentration dependent inhibition of the proliferation of human mononuclear cells stimulated with PHA (n=6; Figure 9). The

IC

5 0 values and 95% confidence limits for these compounds are indicated in Table 2. The results are also shown in the graph of Figure 10. 25 Table 2: ICs values for various compounds against proliferation of human mononuclear cells stimulated with PHA (n=6 UU/jU PCT/GB00/01195 44 Compound

IC

50 so 6

,

7 -Dihydro-2-(2,6-dimethylphenoxy)-9,10- 5.13 iM dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one (2.88-9.14) (Example 1) 6

,

7 -Dihydro-2-(2,6-diisopropylphenoxy)- 9,10- 2.90pM dimethoxy-4H-pyrimido-[6,1 -a]isoquinolin-4-one (1.56-5.41) (Example 2) 2

-(

2 ,6-Dichlorophenoxy)- 6,7-dihydro-9,10- 8.22p4M dimethoxy-4H-pyrimido-[6,1-a]isoquinolin-4-one (4.23-16.0) (Example 8) Example C 6

,

7 -Dihydro-2-(2,6-diisopropylphenoxy)- 9,10-dimethoxy-4H-pyrimido-[6,1 a]isoquinolin-4-one (compound of Example 2) has been shown to be a potent inhibitor 5 of phosphodiesterase (PDE) type 3 and 4 isozymes. The IC 50 value is shown below. PDE3 (nM) PDE4 (nM) (human platelet) (human neutrophil) Example 2 107 1195 10 Rolipram ND 6412 Cilostamide 89 ND Rolipram is a known PDE 4 inhibitor and cilostamide is a known PDE3 inhibitor ND - Not determined 15 Example D WU UU/SIJuy PCT/GB00/01195 45 6

,

7 -Dihydro-2-(2,6-diisopropylphenoxy)- 9,10-dimethoxy-4H-pyrimido-[6, 1 a]isoquinolin-4-one (compound of Example 2) was tested for effects on LPS induced TNF-c release from human monocytes. Results are below. 5 IC50 (nM) Compound 250 n=6 CDP 840 (PDE4 inhibitor) 92 n=6 Siguazodan (PDE3 inhibitor) >100pM 10 Example E: in vivo tests on 6

.

7 -Dihydro-2-(2,6-diisopropylphenoxy)- 9.10-dimethoxv 4H-pyrimido-[6.,1-a]isoguinolin-4-one (compound of Example 2) 1. The above compound was tested in a model of histamine induced bronchospasm. Conscious guinea-pigs were exposed to dry powder (micronised) of the compound. 15 The drug was blended with lactose so that the final concentration was 0.25, 2.5 and 25%. At various times after exposure to drug the animals were anaesthetised and challenged with varying doses of histamine. Total airway resistance and mean arterial blood pressure were recorded. Exposure to dry powder (25%) provided significant protection against histamine induced bronchospasm without changing mean arterial 20 blood pressure. 2. Intravenous administration of the compound of Example 2 (0.1 to 300tg/kg) to urethane anaesthetised guinea-pigs produced a dose dependant reduction in mean arterial blood pressure. The compound was dissolved in DMSO then diluted with 25 saline (there was evidence that the compound had come out of solution). 3. In a model of antigen induced eosinophilia in the ovalbumin sensitised guinea-pig, the compound of Example 2 (10mg/kg) administered orally 1 hour prior to antigen VV uuwrouY PCT/GB00/01195 46 challenge, significantly inhibited the recruitment of eosinophils to the lungs following antigen challenge (aerosol) in sensitised guinea-pigs. Example F: Taste of compounds 5 For pharmaceutical compounds which are administered orally, how the compounds taste is a very important factor in ensuring patient compliance. Unexpectedly, the compounds of the present invention are substantially tasteless. They are therefore particularly suitable for oral administration, for example as dry powder to be inhaled. 10 Method Small amounts of all the compounds of the above Examples 1 to 13, trequinsin (9,10 dimethoxy-3-methyl-2-mesitylimino-2,3,6,7-tetrahydro-4H-pyrimido[6,1 a]isoquinolin-4-one) and desmethyl trequinsin (9,10-dimethoxy-2-mesitylimino 15 2

,

3

,

6

,

7 -tetrahydro-4H-pyrimido[6,1-a]isoquinolin-4-one) were placed on the tip of the tongue of an informed, healthy male volunteer and the taste of each compound was assessed. Results 20 All the compounds of Examples 1 to 13 were not bitter, and therefore have significantly improved taste compared wth trequinsin or desmethyl trequinsin, which are both very bitter.

Claims

1. A compound of general formula I: ROr R R N R 2 0 . R 6 R 7 5 R 9 R 8 wherein each of R' and R 2 independently represents a C 1 - 6 alkyl or C 2 - 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R 3 and R 4 independently 10 represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 - 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a C 1 . 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, C 1 -6 alkylamino, di(C 1 -. 6 ) alkylamino or C 2 - 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, 15 trifluoromethyl, C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 .- 6 alkoxy or C 3 -6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C1- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 - 6 alkoxy or C 3 - 6 cycloalkyl group; or a salt thereof; 20 excluding the compound 6,7-dihydro-2-phenoxy-9,10-dimethoxy-4H pyrimido[6,1 a]isoquinolin-4-one. o uUwDOa.uY PCT/GB00/01195 48

2. A compound as claimed in claim 1 wherein, independently or in any compatible combination, 5 each of R 1 and R 2 represents a C 1 - 6 alkyl, preferably a C 1 - 4 alkyl, group; R and R 2 are the same as each other; each of R 3 and R 4 represents a hydrogen atom; R 5 represents a hydrogen atom; R 6 represents a hydrogen atom; 10 each of R 7 and R 8 represents a C 1 - 6 alkyl, preferably methyl, ethyl or isopropyl, group; R 7 and R 8 are the same as each other; R 9 represents a halogen atom or a methyl or acetyl group.

3. 6,7-Dihydro-2-(2,6-dimethylphenoxy)-9,10-dimethoxy-4H-pyrimido-[6,1-a]iso 15 quinolin-4-one.

4. 2-(2,6-Diethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6,1-a]iso quinolin-4-one. 20 5. 6,7-Dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido-[6,1 a]isoquinolin-4-one.

6. 6,7-Dihydro-9,10-dimethoxy-2-(2, 4 ,6-trimethylphenoxy)-4H-pyrimido-[6,1 a]isoquinolin-4-one. 25

7. 2-(4-Chloro-2,6-dimethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido [6,1-a]isoquinolin-4-one. "v uu/o.u= PCT/GB00/01195 49

8. 2-(4-Bromo-2,6-dimethylphenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido [6,1-a]isoquinolin-4-one.

9. 6,7-Dihydro-9,10-dimethoxy-2-(4-ethanoyl-2,6-dimethylphenoxy)-4H-pyrimido 5 [6,1 -a]isoquinolin-4-one.

10. 2-(2,6-Dichlorophenoxy)-6,7-dihydro-9,10-dimethoxy-4H-pyrimido-[6, 1 a]isoquinolin-4-one. 10 11. 9,10 O-Dimethoxy-2-(2-methylphenoxy)-6,7-dihydro-4H-pyrimido[6,1 a]isoquinolin-4-one

12. 9,10 O-Dimethoxy-2-(2-isobutylphenoxy)-6,7-dihydro-4H-pyrimido[6, 1-a] isoquinolin-4-one. 15

13. 2 -(2-tert-butylphenoxy)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a] isoquinolin-4-one.

14. 9,10-Dimethoxy-2-(2-ethylphenoxy)-6,7-dihydro-4H-pyrimido[6, 1-a]isoquinolin 20 4-one.

15. 2 -( 2 -Cyclopentylphenoxy)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6, 1-a] isoquinolin-4-one. 25 16. A process for preparing a compound of general formula I as defined in claim 1, excluding the compound 6,7-dihydro-2-phenoxy-9,10-dimethoxy-4H pyrimido[6, a]isoquinolin-4-one, the process comprising: (a) reacting a compound of general formula II: vv" uuu.UY PCT/GB00/01195 50 R1 0 4A R5 ROX Rs R 2 0 N 0 R6 N LG II 5 wherein R , R 2, R 5 , R 6 and X are as defined for general formula I and LG represents a leaving group, with a compound of general formula III R 7 OH R9 R8 R 'R a 10 III wherein R 7 , R 8 and R 9 are as defined for general formula I; or (b) when X in general formula I represents a group CR 3 R 4 , wherein R 3 represents a 15 hydrogen atom, R 4 represents a hydrogen atom or a C 1 . 3 alkyl group, and R 5 represents a hydrogen atom or a C 1 . 3 alkyl group, hydrogenating a compound of general formula IX: " v o PCT/GB00/01195 51 4 R RO I R 5 /O O0 R6 R R 7 R 9 8 IX wherein R 1 , R 2 , R 6 , R , R and R 9 are as defined for general formula I; and 5 (c) optionally converting a compound of general formula I so formed into another compound of general formula I.

17. A process as claimed in claim 16, wherein LG in general formula II represents a chlorine atom. 10

18. A process as claimed in claim 16 or claim 17, wherein step (a) is carried out in a suitable solvent such as dimethylformamide or isopropanol in the presence of a base such as potassium carbonate. 15 19. A process as claimed in claims 16, 17 or 18 wherein the compound of general formula I is as defined in any of claim 1 to 15.

20. A composition comprising a compound of general formula I: 20 , uwooe' PCT/GBOO/O1195 52 R R 9 8 R R 0I R 9) R 8 wherein each of R' and R 2 independently represents a CI- 6 alkyl or C 2 -7 acyl group; 5 X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a CI- 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a C.- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, Cl-6 alkylamino, di(Ci. 6 ) alkylamino or C 2 - 7 acylamino group; 10 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 I-6 alkylthio, C 1 .- 6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C.- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 - 6 alkoxy or C 3 - 6 cycloalkyl group; 15 or a salt thereof; and a veterinarily or pharmaceutically acceptable carrier or diluent.

21. A composition as claimed in claim 20, further comprising an active agent such as a 32-adrenoceptor agonist or a glucocorticoid steroid. 20

22. A composition as claimed in claim 20 or claim 21, wherein the composition is a pharmaceutical composition for human medicine. .VV uu/0oo PCT/GB00/01195 53

23. A composition as claimed in claim 20, 21 or 22, adapted for administration by aerosol. 5 24. A composition as claimed in any of claims 20 to 23, wherein the compound is as defined in any of claims 1 to 15.

25. A compound of general formula I 2 N O R 7 OR 0 10 R R 8 wherein each of R' and R 2 independently represents a C I- 6 alkyl or C2- 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R 3 and R 4 independently 15 represents a hydrogen atom or a C 1 - 3 alkyl group; R represents a hydrogen atom or a C 1 - 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a CI- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, CI-6 alkylamino, di(CI- 6 ) alkylamino or C2- 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, 20 trifluoromethyl, C1.6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C2- 7 acyl, C 1 - 6 alkylthio, C 1 .- 6 alkoxy or C 3 - 6 cycloalkyl group; Svvu uo/uY PCT/GB00/01195 54 R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI.-6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C1-6 alkoxy or C 3 - 6 cycloalkyl group; or a salt thereof; for use in medicine. 5

26. A compound of general formula I: R2o 6 N 0O R NO R 7 ROO 0 98 R R 8 wherein 10 each of R 1 and R 2 independently represents a Cl- 6 alkyl or C 2 . 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R 3 and R 4 independently represents a hydrogen atom or a C 1 . 3 alkyl group; R 5 represents a hydrogen atom or a C 1 . 3 alkyl, C 2 .- 3 alkenyl or C 2 - 3 alkynyl group; 15 R 6 represents a hydrogen atom or a C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, C 1 .- 6 alkylamino, di(C 1 - 6 ) alkylamino or C 2 - 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 -6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, Cl- 6 alkylthio, C 1 -6 alkoxy or C 3 - 6 cycloalkyl group; 20 R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C1- 6 alkylthio, C1- 6 alkoxy or C 3 - 6 cycloalkyl group; or a salt thereof; vv v U/ Ou" PCT/GB00/01195 55 for use as an inhibitor of a phosphodiesterase isoenzyme.

27. A compound of general formula I: R 1 0, X, R 5 R N O R R 2 0 N 0 R61 \ R7 5 R 9 R wherein each of R' and R 2 independently represents a CI- 6 alkyl or C 2 - 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R 3 and R 4 independently 10 represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 .- 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a CI.-6 alkyl, C 2 - 6 alkenyl, C 2 .- 6 alkynyl, amino, C 1 -6 alkylamino, di(Cl. 6 ) alkylamino or C 2 .- 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, 15 trifluoromethyl, C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, Ci- 6 alkylthio, CI.-6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI.-6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C1- 6 alkylthio, C 1 - 6 alkoxy or C 3 - 6 cycloalkyl group; or a salt thereof; 20 for use in the prevention or treatment of a disease in which raising the intracellular concentration of cAMP is desirable. Vo uuwaaju PCT/GB00/01195 56

28. A compound of general formula I: RO X N 0 7 NO 0 R 7 " .N R R wherein 5 each of R 1 and R 2 independently represents a C 1 - 6 alkyl or C 2 .- 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a CI.- 3 alkyl, C 2 .- 3 alkenyl or C2- 3 alkynyl group; 10 R 6 represents a hydrogen atom or a Cl- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, C 1 -. 6 alkylamino, di(C.- 6 ) alkylamino or C 2 - 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 aikynyl, C2- 7 acyl, C 1 - 6 alkylthio, C 1 .- 6 alkoxy or C 3 - 6 cycloalkyl group; 15 R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 .- 6 alkyl, C 2 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, CI.-6 alkylthio, C 1 - 6 alkoxy or C 3 -6 cycloalkyl group; or a salt thereof; for use in the prevention or treatment of asthma. 20 29. A compound of general formula I: )v V UU/0o u PCT/GB00/01195 57 RO x R5 2 N 0 R20 / " R 7 0 R 9 CR8 I wherein each of R' and R 2 independently represents a CI- 6 alkyl or C 2 . 7 acyl group; 5 X represents OCH 2 or a group CR R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 - 3 alkyl, C 2 .- 3 alkenyl or C 2 - 3 alkynyl group; R ° represents a hydrogen atom or a C 1 - 6 alkyl, C 2 -6 alkenyl, C 2 -6 alkynyl, amino, Cl-6 alkylamino, di(Cl.-6) alkylamino or C 2 .- 7 acylamino group; 10 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 -6 alkenyl, C 2 -6 alkynyl, C 2 - 7 acyl, CI- 6 alkylthio, Cl- 6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl-6 alkyl, C 2 6 alkenyl, C 2 -6 alkynyl, C 2 - 7 acyl, C 1 l- 6 alkylthio, C1-6 alkoxy or C 3 -6 cycloalkyl group; 15 or a salt thereof; for use in the prevention or treatment of chronic obstructive pulmonary disease (COPD).

30. A compound as claimed in any of claims 25 to 29 wherein the compound is as 20 defined in any of claims 1 to 15.

31. The use of a compound of general formula I: vv UU/5 vY PCT/GB00/01195 58 R 1Oa A X- R5 R 1 0 \ X N., R2o Ix N 0 ROO R R 0 R 9)i R 8 wherein 5 each of R1 and R 2 independently represents a C1- 6 alkyl or C 2 - 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 .- 3 alkyl group; R 5 represents a hydrogen atom or a C 1 .- 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, Cl-6 10 alkylamino, di(Ci. 6 ) alkylamino or C 2 - 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 -6 alkoxy or C 3 -6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 .- 6 alkyl, C 2 . 15 6 alkenyl, C 2 - 6 alkynyl, C 2 . 7 acyl, Cl- 6 alkylthio, C 1 l- 6 alkoxy or C 3 - 6 cycloalkyl group; or a salt thereof; in the manufacture of an inhibitor of a type III/IV phosphodiesterase isoenzyme.

32. The use of a compound of general formula I: 20 " %- vuoJ"' PCT/GB00/01195 59 RO X Rs R N O R 6 7 R 0 R 9 R 8 wherein each of R' and R 2 independently represents a C 1 - 6 alkyl or C 2 - 7 acyl group; 5 X represents OCH 2 or a group CR R , wherein each of R 3 and R 4 independently represents a hydrogen atom or a C1- 3 alkyl group; R 5 represents a hydrogen atom or a CI- 3 alkyl, C 2 - 3 alkenyl or C 2 . 3 alkynyl group; R 6 represents a hydrogen atom or a CI-6 alkyl, C 2 -6 alkenyl, C 2 - 6 alkynyl, amino, C 1 - 6 alkylamino, di(C 1 -6) alkylamino or C 2 - 7 acylamino group; 10 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 -6 alkenyl, C 2 -6 alkynyl, C 2 - 7 acyl, CI- 6 alkylthio, Cl-6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 .- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 . 7 acyl, C 1 - 6 alkylthio, C 1 -6 alkoxy or C 3 - 6 cycloalkyl group; 15 or a salt thereof; in the manufacture of a bronchodilator.

33. The use of a compound of general formula I: Svu uuouvY PCT/GB00/01195 60 2 N O0 R 2 0N R 9 8 riy0 R R P 8 I wherein each of R' and R 2 independently represents a CI- 6 alkyl or C 2 - 7 acyl group; 5 X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R independently represents a hydrogen atom or a C 1 .- 3 alkyl group; R' represents a hydrogen atom or a CI. 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a CI- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, CI-6 alkylamino, di(CI. 6 ) alkylamino or C 2 - 7 acylamino group; 10 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, CI- 6 alkylthio, C 1 I-6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 . 7 acyl, C1- 6 alkylthio, Cl- 6 alkoxy or C 3 .- 6 cycloalkyl group; 15 or a salt thereof; in the manufacture of an anti-asthmatic.

34. The use of a compound of general formula I: vvo uu/auy PCT/GB00/01195 61 2 N 0 R20 R 6 R, 9 8 R 9 :R 8 wherein each of R' and R 2 independently represents a C 1 . 6 alkyl or C 2 - 7 acyl group; 5 X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R independently represents a hydrogen atom or a Cl- 3 alkyl group; R 5 represents a hydrogen atom or a C 1 - 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R 6 represents a hydrogen atom or a C 1 - 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, C 1 -6 alkylamino, di(Cl- 6 ) alkylamino or C 2 - 7 acylamino group; 10 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI-6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C1- 6 alkylthio, C 1 -6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 - 6 alkoxy or C 3 - 6 cycloalkyl group; 15 or a salt thereof; in the manufacture of a medicament for the prevention or treatment of chronic obstructive pulmonary disease (COPD).

35. The use as claimed in any of claims 31 to 34, wherein the compound is as defined 20 in any of claims 1 to 15. WU UU/ isJU PCT/GB00/01195 62

36. A method for the treatment or prevention of a disease in a mammal where a phosphodiesterase isoenzyme inhibitor and/or a bronchodilator would be expected to be of benefit, which method comprises administering to said mammal an effective, non-toxic amount of a compound of general formula I: 5 RO X Rs R 2 o N 0 0 R 7 98 R R 8 wherein 10 each of R I and R 2 independently represents a C 1 . 6 alkyl or C 2 - 7 acyl group; X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 .- 3 alkyl, C 2 - 3 alkenyl or C 2 . 3 alkynyl group; R 6 represents a hydrogen atom or a C1- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, C 1 -6 15 alkylamino, di(C 1 - 6 ) alkylamino or C 2 - 7 acylamino group; each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, CI- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, C 1 -6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 . 6 alkyl, C 2 20 6 alkenyl, C 2 - 6 alkynyl, C 2 . 7 acyl, C 1 .- 6 alkylthio, C.-6 alkoxy or C 3 . 6 cycloalkyl group; or a salt thereof wu UauwJU PCT/GB00/01195 63

37. A method for the treatment or prevention of asthma in a mammal, which method comprises administering to said mammal an effective, non-toxic amount of a compound of general formula I: 2 N 0 R 6 N RR 5 R 9 R wherein each of R1 and R 2 independently represents a CI- 6 alkyl or C 2 - 7 acyl group; 10 X represents OCH 2 or a group CR 3 R 4 , wherein each of R and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 .- 3 alkyl, C 2 - 3 alkenyl or C 2 - 3 alkynyl group; R6 represents a hydrogen atom or a Cl- 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, CI-6 alkylamino, di(Ci.- 6 ) alkylamino or C 2 - 7 acylamino group; 15 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C1- 6 alkyl, C 2 - 6 alkenyl, C 2 -6 alkynyl, C 2 - 7 acyl, Cl- 6 alkylthio, C 1 -6 alkoxy or C 3 - 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 . 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 -6 alkylthio, C1- 6 alkoxy or C 3 - 6 cycloalkyl group; 20 or a salt thereof. VV W VUwoouY PCT/GBOO/O1195 64

38. A method for the treatment or prevention of chronic obstructive pulmonary disease (COPD) in a mammal, which method comprises administering to said mammal an effective, non-toxic amount of a compound of general formula I: RO X Rs5 R N O 0 R 7 " ,N 5R 9R wherein each of R 1 and R 2 independently represents a Cl- 6 alkyl or C 2 -7 acyl group; 10 X represents OCH 2 or a group CR 3 R 4 , wherein each of R 3 and R 4 independently represents a hydrogen atom or a C 1 - 3 alkyl group; R 5 represents a hydrogen atom or a C 1 - 3 alkyl, C 2 - 3 alkenyl or C 2 .- 3 alkynyl group; R 6 represents a hydrogen atom or a C 1 -6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, amino, CI-6 alkylamino, di(Cl- 6 ) alkylamino or C 2 .- 7 acylamino group; 15 each of R 7 and R 8 independently represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, C 1 .- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 . 7 acyl, CI. 6 alkylthio, CI.-6 alkoxy or C 3 .- 6 cycloalkyl group; R 9 represents a hydrogen or halogen atom or a hydroxy, trifluoromethyl, Cl- 6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, C 2 - 7 acyl, C 1 - 6 alkylthio, CI- 6 alkoxy or C3- 6 cycloalkyl group; 20 or a salt thereof. WU UUo5I8U9 PCT/GB00/01195 65

39. A method as claimed in claim 36, 37 or 38, wherein the compound is as defined in any of claims 1 to 15.

40. A method as claimed in any of claims 36 to 39, wherein the compound is 5 administered by aerosol.

41. A method as claimed in any of claims 36 to 40, wherein the animal is a human.

42. A compound substantially as hereinbefore described in any of the examples. 10

43. A process substantially as hereinbefore described in any of the examples.