USE OF XANTHINE OXIDASE INHIBITORS AS ANTI-ISCHAEMIC AGENTS

Abstract: The use of a compound of formula (I), in which R1 and R2 which may be the same or different are N or CH, X1 and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Z1 and Z2 which may be the same or different are hydrogen, hydroxy, keto (=O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z1 and X1 and Z2 and X2 form a second bond of a double bond at the 1,6 or 2,3 positions with the proviso that at least one of the groupings R1Z1X1R2Z2X2 and R1X1Z2 form a hydroxamate moiety (-N(OH)C(=O)- in which R1 and/or R2 is N, Z1 and/or Z2 is =O and X1 and/or X2 is OH or R1 is N, Z2 is =O and X1 is OH and B is a 5- or 6-membered ring of formula (II) or (III), in which R4, R5, R6, R7, R8, R9 and R10 which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (=O), and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or a salt thereof formed with a physiologically acceptable organic or inorganic acid, for the manufacture of a medicament for use as an anti-ischaemic agent.

Description: USE OF XANTHINE OXIDASE INHIBITORS AS ANTI-ISCHAEMIC AGENTS Background to the invention Field of the invention This invention relates to pharmaceutical compounds and to compositions containing them, being primarily concerned with substances of use as anti-ischaemic agents. Description of the prior art Free radicals are reactive substances produced constantly in the body during respiration, tissue turnover, tissue repair and in combatting infections. Under normal conditions, the antioxidant defence mechanisms operational in the body are able to minimise biological damage. However, there are situations, e.g. in certain disease processes, when free radical production overwhelms the natural defence mechanisms. A substantial amount of evidence has been accumulated which suggests that oxygen free radicals mediate ischaemia-reperfusion injury in a variety of tissues, including the heart, lung, kidney, gastrointestinal tract, brain and inflammatory joint disease such as rheumatoid arthritis (Korthius and Granger, 1986, in 'Physiology of Oxygen Radicals' Eds. Taylor, Matalos and Ward; Allen et al, 1989. Lancet ii. 282-283). Both direct and spin trapping EPR techniques have demonstrated mat a burst of oxygen free radical production occurs after reperfusion of ischaemic tissue (Blasig et al. 1986 Stud. Biophys. H£, 35-42; Arroyo et al, 1987 FEBS Lett.221 101-104: Zweier et al. 1987 PNAS USA £4, 1404-1407; Allen et al, 1989, Lancet ii, 282-283). Ischaemia-reperfusion injury is thought to involve a multicomponent process with a burst of free radical production occurring following reperfusion and a second event, possibly inflammatory damage, occurring later. Based on the protective effects of intravascularly administered selective enzyme inhibitors and radical scavenging agents/enzymes, it is widely believed that xanthine oxidase associated with endothelial cells is probably the main source for the production of oxygen radicals responsible for inducing tissue damage during the reperfusion phase. Treatment of ischaemia/reperfusion induced injury requires the development of compounds which suppress the harmful effects of oxygen radicals during both the reperfusion and inflammatory phases. An obvious approach would be the administration of agents that selectively block currently known pathways of radical generation and the release of inflammatory mediators. For example, peroxynitrite, formed by the reaction of nitric oxide and superoxide is thought to be one of the species responsible for bringing about the toxicity of the oxygen free radicals but inhibition of nitric oxide production may be deleterious because of its physiological role in regulating thrombogenicity and vasodilation. A more suitable target would be the inhibition of superoxide production. The above is a particularly attractive strategy, due to the possibility of selectively inhibiting xanthine oxidase. the enzyme involved in the first (reperfusion) stage of ischaemic injury. Due to the multifactorial nature of ischaemia induced injury, in addition to xanthine oxidase inhibitory activity, compounds with potent anti-oxidant activity are required to minimise free radical mediated damage to biological molecules during both the reperfusion and inflammatory phases in disease states wherein these symptoms manifest themselves. Finally compounds which bind redox active metals such as iron with high affinity such that the metal complex is not able to take part in redox cycling reactions will provide additional benefit. It has been a problem to find compounds which fulfil some or all these requirements. Summary of the invention It has surprisingly been found that compounds which are structurally analogous to natural substrates of the enzyme xanthine oxidase or which have inhibitory properties towards the enzyme xanthine oxidase, thus in both cases acting as xanthine oxidase inhibitors, fulfil some or all of the above requirements. Accordingly, the invention provides the use of a compound of formula I in which R] , R2 and R3 which may be the same or different are N or CH, X ] and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Z\ and Z2 which may be the same or different are hydrogen, hydroxy, keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z j and X j and Z2 and X2 form the second bond of a double bond at the 1 ,6 or 2,3 positions, with the proviso that at least one of the groupings Rj Zj Xi , K ^l ^-l md R l x l z 2 f orrn a hydroxamate moiety (- N(OH)C(= O)-) in which R j and/or R2 is N, Z j and/or Z2 is = O and Xj and/or X2 is OH or R ] is N, Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R5, R , R7, Rg, R9 and Rj Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O) and an optionally substituted alkyl. alkenyl. alkynyl, cycloalkyl, aryl or heterocyclyl group, or a salt thereof formed with a physiologically acceptable organic or inorganic acid, for the manufacture of a medicament for use as an anti-ischaemic agent. It will be appreciated that when Z\ is = O and X\ is H or when Z2 is = O and X2 is H then the grouping -C(=O)-CH2- or -C(= O)-NH- can exist in the tautomeric form -C(OH)=CH- or C(OH)=N- respectively. Description of the preferred embodiments In the above general formula, whenever the compounds of the invention contain an alkyl, alkenyl or alkynyl substituent group, this may be linear or branched and may contain up to 10 and more preferably up to 4 carbon atoms. A cycloalkyl (referring to a cyclic hydrocarbyl group) group may contain from 3 to 8, preferably 3 to 6, carbon atoms. An aryl group refers to any aromatic hydrocarbon group, especially a phenyl, benzyl or naphthyl group. A heterocyclyl group may be any saturated or unsaturated cyclic group containing at least one heteroatom, 5- and 6-membered rings being especially preferred. When any of the foregoing substituents are designated as being optionally substituted, the substituent groups which are optionally present include, for example, halogen atoms, hydroxyl. alkyl, alkoxy, amino. alkylamino, dialkylamino, formyl, alkoxycarbonyl, carboxyl, alkanoyl, carbamoyl, alkylamido, phenyl, cyano, nitro and phenoxy groups. When any of the foregoing substituents represents or contains an alkyl substituent group, this may be linear or branched and may contain up to 4 carbon atoms. Regarding ring A. there may be two hydroxamate moieties at positions 1 - 6 or 2- 3 of the ring present or more preferably only one hydroxamate moiety at positions 1 - 6 .2- 3 or 1-2 of the ring present. This is preferably at positions 1-6 or 2-3 of the ring. There may be one, two or three nitrogen atoms in ring A but preferably, there are two nitrogen atoms present in ring A, and one necessarily being part of the hydroxamate moiety. It is also possible that ring A contains only one nitrogen atom, that necessarily being part of the hydroxamate. X j is preferably H when R ] is N, and the hydroxamate moiety is present at positions 2-3 of ring A. If R ] is C, then X\ may be any of the substituents defined above but is most preferably H. When the hydroxamate moiety of ring A is at position 1-6 of the ring and R2 is N, X2 is preferably H. When R2 is C, X2 is any of the above substituents but preferably H. In all cases, ring A is preferably diketo, that is Z j and Z2 are both a keto group (= O). It is preferable that the compounds of the present invention differ from their 'natural' counterparts, i.e. they are not known xanthine oxidase inhibitors or substrates but analogues thereof. Ring A as defined above also encompasses any tautomeric forms which would be apparent to a person skilled in the art. Regarding ring B, when ring B is a 5 membered ring, the 5-membered ring is preferably pyrrole (containing IN), imidazole (containing 2N), pyrazole (containing 2N) and triazole (containing 3N). When ring B is a 6-membered ring, the 6 membered ring is preferably benzene, pyridine (containing IN) or pyrazine (containing 2N). Both ring B of formulas II and III may optionally be substituted in one or more positions with substituents as defined above. If substituted, preferred substituents are alkyl, hydroxyl or benzyl but more preferably, ring B is not substituted. Particularly preferred compounds of the present invention are analogues of the xanthine oxidase inhibitors allopurinol and oxypurinol and analogues of the xanthine oxidase substrates hypoxanthine and xanthine in which the hydrogen atom attached to the ring nitrogen at the 1 position in Formula I is replated by a hydroxyl group. Particularly preferred compounds however are those wherein B is a 6 membered pyrazine group. In these compounds preferably, ring A is diketo. A specifically preferred compound is, hereinafter referred to as SL301 is 2.6-dioxo-l-hydroxypyrazino-[2,3-d]- pteridine (in Formula 1, X j is OH, X2 is H, R j and R2 are N, R3 is CH, Zj and Z2 are keto, ring B is a pyrazine ring wherein R7 and RJ Q are N, Rg and R9 are CH). The structural formula of SL301 is shown below. H The compound has been described in EPO 240 859 B (Abbott Laboratories) as a compound which inhibits lipoxygenase and as such is useful as an anti-inflammatory agent. US 4,757,078 (Misra) discloses the same compound as an inhibitor of leukotriene production. Again, its potential use as an anti-allergy agent is discussed. There is no disclosure of the use of this compound as an anti-ischaemic agent. German patent number 2,356,690 discloses compounds of the formula wherein X is H, aryl, C } _4 alkyl, or C j _4 hydroxyalkyl for treatment of gout. Again, there is no disclosure of the use of these compounds as anti-ischaemic agents. Clark et al. J. Chem. Soc. 1968, 212, 919-922, discloses compounds having the following formula in which Rj = R 2 = R3 = H Rj = CH 3 , R 2 = R3 = H R 2 = CH 3 , R } = R 3 = H R 3 = CH 3 , R ] = R 2 = H R j = H, R 2 = R 3 = CH 3 R 2 = H, Ri = R3 = CH 3 No therapeutic use of these compounds is suggested. Montgomery etal J. Med. Chem. 1972,11, 1334-1336 discloses compounds which have an anticancer effect. The compounds are prepared from intermediates which include wherein R = cyclopentyl, β-D-ribofuranosyl, β-D-arabinofuranosyl, or 2-deoxy- β-D-ribofuranosyl. Revankar et al. J. Med. Chem., IS. 1975, 721-726 discloses compounds as having anti-viral properties. The following is an intermediate in their preparation but is not disclosed as having any anti-viral activity itself. Teller et al, Cancer Research 2&, 1978, 2038-2042 describes oncogenicity of N- hydroxyxanthines. Compounds included in the discussion are 1-hydroxyxanthine and 3-hydroxyxanthine. Borbil et al. Rev. Roum. Biochim., 19781£, 3-8 discloses cytostatic compounds including the following: Bauer et al, J. Heterocyclyl Chemistry 1967, 4, 325-334 discloses compounds including the following: These compounds are described as being potential anti-metabolites. It will thus be appreciated that certain of the compounds of formula I are novel. Thus the invention extends these compounds per s£, processes for their preparation and their use in therapy. Thus according to a further aspect of the invention there is provided a compound of formula I in which R j , R2 and R3 which may be the same or different are N or CH, X j and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Zj and Z2 which may be the same or different are hydrogen, hydroxy keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Zj and X j and Z2 and X2 form the second bond of a double bond at the 1,6 or 2,3 positions with the proviso that at least one of the groupings Rj Z\ X R2 Z2 X2 and Rj Xj Z2 form a hydroxamate moiety (-N(OH)C(= O)-) in which R j and/or R2 is N, Zj and/or Z2 is = O and Xj and/or X2 is OH or R j is N j Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III ^ R Λ R3 \ ^ / II ^ Re in which R4, R5, R5, R7, Rg, R9 and R Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group with the proviso that the compound is not wherein X is hydrogen, aryl, C j _4 alkyl, or Cj.4 hydroxyalkyl or wherein R j = R 2 = R3 = H R j = CH 3 , R 2 = R3 = H R 2 = CH 3 , Rj = R 3 = H R 3 = CH 3 , R j = R 2 = H R j = H, R 2 = R 3 = CH 3 R 2 = H, Rj = R 3 = CH 3 or lopentyl or or or or any compound of Formula I wherein ring B is substituted by a natural or unnatural sugar molecule or a salt thereof formed with a physiologically acceptable organic or inorganic acid. The definitions regarding rings A and B have been given hereinbefore. The following are particularly preferred novel compounds of the invention. This also extends to their use in therapy and their formulation into a pharmaceutical composition. Regarding ring A, there may be two hydroxamate moieties at positions 1 , 6 or 2, 3 of the ring present or more preferably only one hydroxamate moiety at positions 1, 6, 2, 3 or 1 , 2 of the ring present. This is preferably at positions 1 , 6 or 2, 3 of the ring. There may be one, two or three nitrogen atoms in ring A but preferably, there are two nitrogen atoms present in ring A. and one necessarily being part of the hydroxamate moiety. It is also possible that ring A contains only one nitrogen atom, that necessarily being part of the hydroxamate. X j is preferably H when R j is N, and the hydroxamate moiety is present at positions 2, 3 of ring A. If R is C, then X j may be any of the substituents defined above but is most preferably H. When the hydroxamate moiety of ring A is at position 1,6 of the ring and R2 is N X2 is preferably H. When R2 is C, X2 is any of the above substituents but preferably H. In all cases, ring A is preferably diketo, that is Zj and Z2 are both a keto group (= O). Ring A as defined above also encompasses any tauto eric forms which would be apparent to a person skilled in the art. Regarding ring B, when ring B is a 5-membered ring, the 5-membered ring is preferably pyrrole (containing IN), imidazole (containing 2N), pyrazole (containing 2N) and triazole (containing 3N). When ring B is a 6-membered ring, the 6-membered ring is preferably benzene, pyridine (containing IN) or pyrazine (containing 2N). Both ring B of formulas II and III may optionally be substituted in one or more positions with substituents as defined above. If substituted, preferred substituents are alkyl. hydroxyl or benzyl but more preferably, ring B is not substituted. Particularly preferred compounds of the present invention are analogues of the xanthine oxidase inhibitors allopurinol and oxypurinol and analogues of the xanthine oxidase substrates hypoxanthine and xanthine in which the hydrogen atom attached to the ring nitrogen at the 1 position in Formula I is replated by a hydroxyl group. Particularly preferred compounds however are those wherein B is a 6-membered pyrazine group. In these compounds preferably, A is diketo. A specifically preferred compound is, SL301, 1 2,6-dioxo-l-hydroxypyrazino-[2,3-d]-pteridine (in Formula I, X j is OH. X2 is H, R j and R2 are N, R3 is CH, Zj and Z2 are keto, ring B is a pyrazine ring wherein R7 and RJQ are N, Rg and R9 are CH). Several methods are available for the preparation of the compounds of formula I. For example, hydroxamate-containing analogues of known xanthine oxidase substrates or inhibitors such as hypoxanthine, xanthine, (substrates), allopurinol, oxypurinol (inhibitors) can be prepared by replacing the hydrogen atom attached to a ring nitrogen at the 1 position with a hydroxyl group. Other methods of preparing the compounds of the invention will be apparent to a person skilled in the art using processes known for the preparation of chemically related compounds. Accordingly, such processes form a further feature of this invention. According to a further aspect of the invention there is provided a compound of formula 1 in which R j , R2 and R3 which may be the same or different are N or CH, Xj and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Zj and Z2 which may be the same or different are hydrogen, hydroxy, keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z j and X j and Z2 and X2 form the second bond of a double bond at the 1,6 or 2,3 positions with the proviso that at least one of the groupings Rj Z Xj R2 Z2 X2 and RJXJZ2 form a hydroxamate moiety (-N(OH)C(= O)-) in which R j and or R 2 is N, Zj and/or Z 2 is = O and X j and/or X 2 is OH or R j is N, Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R5, R , R7, Rg, R9 and RJ Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O). or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group with the proviso that the compound is not wherein X is hydrogen, aryl C _4 alkyl, or Cj. 4 hydroxyalkyl or lopentyl or any compound of Formula I wherein ring B is substituted by a natural or unnatural sugar molecule or a salt thereof formed with a physiologically acceptable organic or inorganic acid for use in therapy. According to a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula I in which R j , R2 and R3 which may be the same or different are N or CH, X j and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Z j and Z2 which may be the same or different are hydrogen, hydroxy, keto (= O) or an optionally substituted alkyl, alkenyl. alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Zj and X j and Z2 and X2 form the second bond of a double bond at the 1,6 or 2,3 positions with the proviso that at least one of the groupings R j Z j Xj , R2 Z2 X2 and Rj Xj Z2 form a hydroxamate moiety (-N(OH)C(= O)-) in which R j and/or R2 is N, Zj and/or Z2 is = O and Xj and/or X2 is OH or R j is N, Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R 5 , Rβ, R 7 , R 8 . 9 ∞ d R 10 which ma y be me same or different ∞ CH or N with the proviso that B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O) and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group with the proviso that the compound is not X wherein X is hydrogen, aryl C j _4 alkyl, or C j . hydroxyalkyl or a ; salt thereof formed with a physiologically acceptable organic or inorganic acid together with a physiologically acceptable diluent or carrier. In general , compositions containing the compounds of formula II which are of particular interest are those in which the diluent or carrier excludes any liquid which is not sterile and pyrogen free. As indicated the compounds of formula I may be formulated as salts formed with physiologically acceptable inorganic or organic acids. These salts may be prepared by conventional methods and it is preferred to use methane sulphonic acid, isethionic acid, lactic acid, tartaric acid or another solubilising acid. The pharmaceutical composition of the invention may be in a form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for topical use. for example a cream, ointment, gel or aqueous or oily solution or suspension; for nasal use, for example a snuff, nasal spray or nasal drops; for vaginal or rectal use. for example a suppository; for administration by inhalation, for example as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oily solution or suspension. In general the above compositions may be prepared in a conventional manner using conventional excipients, using standard techniques well known to those skilled in the art of pharmacy. It will be appreciated that the dosage levels used may vary over quite a wide range depending upon the compound used and the severity of the ischoemic condition of the patient and the patient's body weight. However, without commitment to a rigid definition of dosages it may be stated that a daily dosage of the active constituent (estimated as the free base) is 50 - lOOOmg. More particularly, the daily dosage for a 70kg human administered parenterally will often be in the range from about 1 OOmg to about 500mg. but with the more active compounds it may be less than this (the dose being varied pro-rata for humans of different weight or other mammals). Where appropriate, the substances may also be compounded for oral administration in dosages which may be similar but may often be somewhat higher, so that the daily dose for a 70kg human may often be in a range from 100 - 500mg but may be somewhat less than this for the more active compounds. Such oral formulations may particularly take the form of tablets compounded in the presence of conventional solid carrier materials such as starch, lactose, dextrin and magnesium stearate, or of capsules or cachets. Suppositories, pessaries, aerosol and other formulations may also be employed. The compounds may be formulated in unit dosage form, i.e. in discrete portions each containing a unit dose, or a multiple or sub-multiple of a unit dose of the active ingredient. The compounds of formula I described above find particular use for a wide range of ischaemic conditions where tissue has undergone damage caused by free radicals. Preferably however they find particular use for the treatment of reperfusion of the heart injury following myocardial infarction of the brain following a stroke. The invention is illustrated by the following Examples in which reference is made to the following Figures: Figure 1 : shows schematic methods of preparation of compounds of Formula I wherein ring B is pyrazole. Figures 2 and 3 : show schematic methods of preparation of compounds of Formula I wherein ring B is imidazole. Figure 4 : shows schematic methods of preparation of compounds of Formula I wherein ring B is pyrazine. Figure 5 : shows schematic methods of preparation of compounds of Formula I wherein ring B is triazole. Figure 6 : shows schematic methods of preparation of compounds of Formula I wherein ring B is pyridine. EXAMPLE 1 SYNTHESIS OF COMPOUNDS IN WHICH RING B IS PYRAZOLE These syntheses are shown schematically in Figure 1 a) Synthesis of 1. hvdroxy-6-oxypyrazolo-[3.4-d]-pyrimidine in the above general formula I, Rjand R2 are N, R3 is CH. Xj is OH, Zj is = O, X2 and Zo form a double bond, ring B is of formula II in which R5 and R are N and R4 is CH. (i) To a solution of hydroxylamine hydrochloride (9.82 mmol) and sodium hydroxide (24 mmol) in water, was added 3-amino-4-carbethoxy pyrazole (6.44 mmol). (ii) On completion of addition, the reaction mixture was stirred for 2 h at room temperature and then left to stand for 3 days. (iii) The mixture was then acidified with dilute HC 1 and refrigerated until precipitation occurred, (iv) The resultant hydroxamate was filtered, washed with ice-cold water and dried, (v) The hydroxamate (6.28 mmol), triethylorthoformate (37.5 mmol) and DMF (175 mmol) were heated with stirring at - 100°C for 10-15 mins, or until precipitation occurred, (vi) The reaction mixture was allowed to stand at room temperature for 18 h. (vii) The product was then filtered and recrystallized from water. b) Synthesis of 2.6-dioxo-l-hydroxypyrazolo-[3.4-d]-pyrimidine Formula I is as described in (a) above except that Z2 is keto (= O) and X-> is H. (i) 1.41 mmol of the hydroxamate (prepared as described above in (a)) in dry DMF (25.83 mmol) was added to a solution of 1,1-carbonyldiimidazole (1.425 mmol) in dry DMF (25.83 mmol). (ii) The reaction mixture was then stirred at - 100°C, under N2, for 2 h or until reaction was complete. (iii) The solvent was removed in vacuo and the resultant product recrystallized from water. (c) Synthesis of l-hvdroxy-6-oxypyrazolo-13.4-d]-9-methvl pvrimidine Formula I is as described in (a) above except that the N is position R is substituted with a methyl group. (i) A mixture of ethoxymethylenemalonitrile (43.4 mmol) and methyl hydrazine (45.6 mmol) in ethanol was refluxed for 17h. (ii) The resulting 1 -methyl-4-cyano-5-amino pyrazole precipitated out and was filtered and dried. (iii) The l-methyl-4-cyano-5-amino pyrazole was dissolved in 40% NaOH and the resulting solution refluxed for four days, (iv) The solution was acidified with dilute HC 1. (v) The solvent was then removed in vacuo and the resulting acid recrystallised from ethanol. (vi) The acid was converted to 1 -methyl-4-carbethoxy-5-amino pyrazole using SOC 12 and EtOH. (vii) Proceed as in 1 (a) steps i-viii. EXAMPLE 2 SYNTHESIS OF COMPOUNDS IN WHICH RING B IS IMIDAZOLE These syntheses are shown schematically in Figures 2 and 3. (a) Synthesis of 1 -hvdroxv-6-oxopurine. In formula I above, R j and R2 are N, R3 is CH, X is OH, Zj is keto, Z2 and X2 form a double bond, ring B is of formula II wherein R4 and Rβ are N and R5 is CH. (i) To a solution of oxime (lOg) in water (60ml) and saturated sodium bicarbonate (30ml) was added sodium dithionite (34g). (ii) After 30 mins, the mixture was extracted with chloroform (x 4) and dried (Na 2 SO ). (iii) The solvent was removed in vacuo to give the 2-amino-2-cyanoacetate as an oil which was used immediately. (iv) To a solution of the cyanoacetate (4g), chloroform (80ml). and ethanol (60ml). was added formamidine acetate (4g). (v) The mixture was refluxed under N 2 for 2 h, after which it was evaporated to dryness. (vi) The residue was taken up in chloroform ( 115ml) and extracted with water (40ml). The aqueous fraction was extracted with chloroform (3 x 20ml) and the combined organic extracts were dried (Na2SU4), filtered, and evaporated to dryness. (vii) The 4-amino-5-carbethoxy imidazole crystallized from ethyl acetate, (viii) A solution of the imidazole (8.16 mmol) in triethyl-orthoformate ( 15ml) and DMF (30ml) was refluxed for 4 h. (ix) The solvent was removed in vacuo and the resulting ethyl-forminidate-5- carbethoxyimidazole was recrystallized from ethanol. (x) Cyclization to the corresponding hydroxamate was achieved as previously described. (b) Synthesis of 3.8.9-trihvdro-l-hvdroxy-2.6 dioxopurine Formula I is the same as in (a) above except that Z2 is keto (= O) and the R^ N atom may be substituted by methyl, (i) 4,5-imidazoledicarboxylic acid (46.76 mmol) was suspended in ethanol (330ml) and cooled to - 5°C. (ii) SOC 12 (95ml) was added dropwise. (iii) The reaction mixture was refluxed for 5 h and then allowed to cool. (iv) On addition of dry diethyl ether turbidity occurred and the mixture was refrigerated until precipitation was complete. (v) Resulting diethyl 4.5-imidazoledicarboxylate was then filtered and dried. (vi) A solution of the dicarboxylate (15.1 1 mmol) and sodium ethoxide (19.71 mmol) in ethanol (160ml) was treated with methyl iodide (22.67 mmol). (vii) The reaction mixture was then refluxed for 4 h, after which the solvent was removed in vacuo and the residue treated with 5% Na2CO3- (viii) The product was extracted into chloroform, dried (Na2SO4), and filtered. (ix) Removal of the solvent in vacuo and subsequent distillation furnished diethyl 1 - methyl-4,5-imidazoledicarboxylate (b.p - 148-150°C, 0.25 m Hg). (x) To a suspension of KOH (56.28 mmol) in pyridine (25ml) was added an ice-cold solution of hydroxylamine hydrochloride (32.78 mmol) in pyridine (25ml). (xi) The dicarboxylate (14.07 mmol) was added and stirring continued for 2.5 h at 0-5°C. (xii) Stirring was then continued at room temperature for 18 h. after which the resulting solids were filtered and dried. (xiii) The salt formed (15.55 mmol) was suspended in THF (25ml) and a solution of benzenesulfonyl chloride (13.06 mmol) in THF (7ml) was added as such a rate as to maintain the temperature at 20-22°C. (xiv) Stirring was continued for 30min and sodium acetate trihydrate (6.13 mmol) added. (xv) The reaction mixture was then stirred for 2 h and the solids were filtered, and washed with THF (3 x 10ml). (xvi) The THF was removed in vacuo and the residue partitioned between water (30ml) and petroleum ether ( 16ml). (xvii) The resulting precipitate, the l-benzenesulfonyloxy-7-methylxanthine, was collected and recrystallized from ethanol. (xix) The above compound was dissolved in sodium hydroxide solution, which was then refluxed until it became clear. (xx) The solution was cooled, and acidified with cone. HC 1. (xxi) The reaction mixture was kept in an ice-bath until precipitation occurred, and the resulting 1 -hydroxy-7-methylxanthine was recrystallized from water. EXAMPLE 3 SYNTHESIS OF COMPOUNDS IN WHICH RING B IS PYRAZINE These syntheses are shown schematically in Figure 4. a) Synthesis of l-hvdroxv-6-oxvpvrazino-[2.3-d " |-pterine In formula I, R j are R2 and N, R3 is CH, Xj is OH, Z j is keto, Z2 and X2 form a double bond, ring B is of formula III wherein R7 and RJ Q are N and Rg and R9 are CH. (i) 3-aminopyrazine-2-carboxylic acid (14.37 mmol) was suspended in ethanol (50ml) and cooled to -5°C. (ii) SOCI2 (20 mmol) was added dropwise. (iii) The reaction mixture was refluxed for 5 h and then filtered hot. (iv) On cooling precipitation occurred to give ethyl 3-aminopyrazine-2-carboxylate. (v) The ester was then treated with NaOH and NH2OH.HC 1 as previously described to give the hydroxamate which was subsequently cyclized using triethylorthoformate and DMF; the mixture being stirred at - 160°. (b) Synthesis of 2.6-dioxo-l-hvdroxvpvrazino- 2.3-d1 nteridine Formula I is as described above except Z2 is keto and X2 is H. The diketo-pterine compound was prepared using the procedure described for 1 - hydroxy-7-methylxanthine in Example 1(c) above. EXAMPLE 4 SYNTHESIS OF COMPOUNDS IN WHICH RING B IS TRIAZOLE This synthesis is shown schematically in Figure 5. (a) Synthesis of 9-benzvl-2.6-dioxo-l-hvdroxvtriazalo-r3.4-d1 azapurine In formula I, R j and R2 are N, R3 is CH, Z j is keto, Xj is OH, Z2 is keto, X2 is H, ring B is of formula II in which R4, R5. and Rβ are N and R^ is substituted by benzyl. (i) Benzyl chloride (158 mmol) was added to a suspension of sodium azide (230 mmol) in ethanol (45ml). (ii) The reaction mixture was refluxed for 6 h and then allowed to cool, (iii) On the addition of water, the benzyl azide separated out as an oil which was distilled (~ 200° / 760mmHg) and dried over CaC^. (iv) To a solution of acetylenedicarboxylic acid (25.7 mmol) in acetone (10ml) was added benzyl azide (35.6 mmol). (v) The solution heated up rapidly to reflux and external cooling was required, (vi) The product began to crystallize almost immediately, and after being allowed to stand over night was filtered. (vii) The remainder of the procedure is as described for 1 -hydroxy-7-methylxanthine. EXAMPLE 5 SYNTHESIS OF COMPOUNDS IN WHICH RING B IS PYRIDINE This synthesis is shown schematically in Figure 6. (a) Synthesis of 2.6-dioxo-l -hvdroxvpvrido-.2.3-dlpvrimidine In formula I, R and R2 are N, R3 is CH, Z j and Z2 are keto, X is OH, X2 is H, ring B is of formula III in which RJ Q is N, R7, Rg, are CH. (i) The procedure is as described for l-hydroxy-7-methylxanthine in example 1(c) above. EXAMPLE 6: List of specific Compounds In this and the following examples, the following compounds are referred to by reference number. H XANTHINE OXIDASE INHIBITION ASSAY Superoxide produced from the oxidation of varying concentrations of xanthine to uric acid in the presence of xanthine oxidase (5 x 10 '3 U/ml) was quantified by the superoxide dismutase inhibitable reduction of cvtochro e c oxidase (10 μM) at 550nm with respect to time. Enzyme inhibition was determined at varying concentrations of the inhibitors. Results obtained are expressed as percentage inhibition of initial rate of reaction in comparison to controls and are shown in Table 1-4 below. IΔBLE [Xanthine] = gQμM % Inhibition % S.D. Inhibitor Cone. (μM) 1 μM 5 μM 10 μM 50 μM Allopurinol 13.83% 3.21 22.90% 3.21 20.63% 6.41 59.18% 11.11 Oxypurinol 2.26% 0.91 24.81% 2.21 32.33% 10.64 67.42% 9.38 SL300 N.A.* 1.84% 2.60 14.17% 5.02 16.01% 3.71 SL301 29.13% 2.21 31.75% 3.71 39.63% 3.72 63.25% 3.71 * N.A. - not active. Values are expressed as percentage inhibition of the initial rate of reaction with respect to control % standard deviation, n=3. XΔELE [Xanthine] = 25μM % Inhibition % S.D. Inhibitor Cone. (μM ) 1 μM 5 μM 10 μM 50 μM Allopurinol N.A.* 2.41% 3.40 14.09% 4.86 58.76% 8.42 Oxypurinol 2.26% 0.92 37.34% 9.38 42.36% 12.78 62.40% 16.24 SL300 13.75% 3.30 9.09% 1.02 16.08% 3.1 34.73% 3.30 SL301 37.06% 3.31 51.05% 2.76 58.04% 4.21 72.03% 2.51 * N.A. - not active. Values are expressed as percentage inhibition of the initial rate of reaction with respect to control % standard deviation, n=3. IΔBLEi [Xanthing] = 2gμM % Inhibition % S.D. Inhibitor Cone. (μM) 1 μM 5 μM 10 μM 50 μM Allopurinol 23.88% 9.82 64.57% 3.94 44.88% 6.34 68.50% 23.18 Oxypurinol 15.38% 3.21 46.15% 6.28 30.77% 12.56 43.59% 3.63 SL300 N.A.* 5.55% 3.93 11.11% 7.86 27.78% 7.85 SL301 25.00% 3.21 41.67% 2.52 58.33% 2.67 77.78% 3.93 * N.A. - not active. Values are expressed as percentage inhibition of the initial rate of reaction with respect to control % standard deviation, n=3. IΔBLEJ [Xanthine) = gμM % Inhibition % S.D. Inhibitor Cone. (μM) 1 μM 5 μM 10 μM 50μM Allopurinol 17.40% 4.17 29.20% 3.71 49.85% 11.04 73.45% 7.23 Oxypurinol 6.06% 4.29 33.33% 4.29 45.45% 7.42 48.48% 8.57 SL300 6.15% 4.58 12.62% 3.12 19.09% 4.58 38.51% 12.1 1 SL301 22.33% 4.51 41.75% 11.42 49.75% 9.13 70.87% 6.78 SL100, SL101. SL200, SL201 were found to be inactive. Values are expressed as percentage inhibition of the initial rate of reaction with respect to control % standard deviation, n=3. FURTHER XANTHINE OXIDASE INHIBITION STUDIES The inhibition of xanthine oxidase by allopurinol and SL301 was studied by steady- state kinetic analysis. The study was conducted at 37 β C using 30 mU/ml bovine xanthine oxidase in 50 mM phosphate buffer (ph 7.4). The initial rate of uric acid formation was determined spectrophotometrically (292 nm) in the presence of varying concentrations of xanthine (5, 7.5, 10, 15 and 20 μM) and the inhibitors (1-3 μM). Enzyme-inhibitor dissociation constant (K j ) values were calculated from the apparent 1/V maχ values obtained at different inhibitor concentrations. The average K j values obtained for allopurinol and SL301 were 2.4 and 0.7 μM respectively, both compounds were found to inhibit xanthine oxidase in a competitive manner. EXAMPLE 7: HYDROXYL RADICAL SCAVENGING ACTIVITY Antioxidant activity is based on the selective hydroxylation of NPG (N,N'-[5-nitro-l,3-phenylene] bisglutaramide) by hydroxyl radicals produced from peroxynitrous acid (ImM) by the addition of peroxynitrite stabilised in base to a mixture of NPG (ImM) and varying concentrations of the anti-ischaemic agents prepared in 0.2M phosphate buffer, pH 7. The solutions were analysed by RP-HPLC. NPS (50mM), an analogue of NPG, was used as an internal standard. Inhibition of hydroxylation of NPG is a direct measure of the hydroxyl radical scavenging ability of the inhibitors. Results are expressed as the percentage scavenging of the hydroxyl radical compared to controls and are shown in Table 5 below. IΔBLEi o %. Activity % S.D. Antioxidant Ratio 0.1 0.5 1.0 5.0 SL100 6.56% 7.08 37.70% 6.69 65.58% 5.35 100 SL101 8.25% 5.35 20.96% 5.44 39.71% 1 1.0 72.79% 0.26 SL200 25.13% 5.01 48.49% 5.45 69.54% 0.50 90.09% 0.15 SL300 N.A. 6.04% 3.20 26.58% 4.74 68.58% 3.50 SL301 9.80% 8.03 18.00% 5.35 45.90% 4.02 70.21% 0.46 * N.A. - not active. Values are expressed as percentage scavenging of the hydroxyl radicals % standard deviation, n=3. EXAMPLE S HEART ISCHAEMIA REPERFUSION STUDIES Adult male Wistar rats (240-320 g) were anaesthetised intraperitoneally (i.p.) with sodium pentobarbital (Sagatal; 60 mg/kg and heparinised (100 IU/100 g body weight) through the femoral vein, and the heart excised in cold Krebs buffer. By Langendorff technique, the hearts were perfused at a constant pressure (100 mm H2O) with Krebs bicarbonate buffer. The study was initiated by 30 min aerobic perfusion followed by a 50 min period of global ischaemia. achieved by clamping perfusion flow into the aorta, and finally by reperfusion over 60 min. A ballon was inserted into the left ventricle and inflated to a left ventricular end diastolic pressure (LVEDP) of 4 mm Hg and used to measure left ventricular developed pressure (LVDP) and heart rate (HR). In addition, the coronary effluent was collected over timed intervals as a measure of coronary flow (CF). Assessment of function was made 20 min after the start of the study and at 10 min intervals thereafter. SL301 treated hearts followed a similar protocol as above except that the drug was present in the reperfusion perfusate following the ischaemic insult. The results are shown in Table 6 below. TABLE 6 % recovery (± sem) of pre-ischaemic function after 60 min. of aerobic perfusion following ischaemia (50 min.) in control and SL301 (150 μM) perfused rat hearts LVDP HR CF

Claims: CLAIMS The use of a compound of formula I in which R j and R2 which may be the same or different are N or CH, X j and X2 which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group and Zj and Z2 which may be the same or different are hydrogen, hydroxy, keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z j and X j and Z2 and X2 form a second bond of a double bond at the 1 ,6 or 2,3 positions with the proviso that at least one of the groupings R j Z j X j R2 Z2 XT and Rj Xj Z2 form a hydroxamate moiety (-N(OH)C(= O)- in which R j and or R is N, Zj and/or Z 2 is = O and Xj and or X 2 is OH or R j is N, Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R5, R-5, R7, Rg, R9 and R Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O), and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or a salt thereof formed with a physiologically acceptable organic or inorganic acid, for the manufacture of a medicament for use as an anti-ischaemic agent. 2. The use according to Claim 1 , wherein the compound of formula I contains one hydroxamate moiety in ring A. 3. The use according to Claim 2. wherein the one hydroxamate moiety is at position 2-3 of ring A. 4. The use according to Claim 1, 2 or 3 wherein there is only one nitrogen atom in ring A of the compound of formula 1. 5. The use according to any one of Claims 1 to 4, wherein the ring A in a compound of formula I is di-keto. 6. The use according to Claim 1 -5 wherein ring B is unsubstituted. 7. The use according to any one of Claims 1 to 6, wherein B is a 6 membered ring of formula III. 8. The use according to Claim 7, wherein R < , and R 7 are nitrogen. 9. The use according to Claim 7, wherein the compound of formula I is SL301. 10 A compound of formula I in which R j and R2 which may be the same or different are N or CH, X and Xo which may be the same or different are hydrogen, hydroxy, or an optionally substituted alkyl, same or different are hydrogen, hydroxy, keto (= O), or an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Z j and X j and Z2 and X2 form a second bond of a double bond at the 1 ,6 or 2,3 positions with the proviso that at least one of the groupings R j Z j Xj R2 Z2 X2 and Rj Xj Z2 form a hydroxamate moiety (-N(OH)C(= O)- in which R j and or R is N, Z j and/or Z 2 is = O and X j and/or X 2 is OH or R j is N, Z2 is = O and X j is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R5, R β , R-, Rg, R9 and RJ Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy. keto (= O) and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group with the proviso that the compound is not wherein X is aryl hydrogen, C j .4 alkyl, or C j .4 hydroxyalkyl wherein X is aryl hydrogen, C\__\ alkyl, or C j .4 hydroxyalkyl or or wherein R j =R 2 =R3=H R j =CH3,R 2 =R3=H R 2 =CH3,Rj =R3=H R 3 =CH 3 ,R j =R 2 =H R j =H, R 2 =R 3 =CH 3 R 2 =H, R =R 3 =CH 3 cyclopentyl or or a salt thereof formed with a physiologically acceptable organic or inorganic acid. 11. A compound according to Claim 10 which contains one hydroxamate moiety in 12. A compound according to Claim 11 wherein the hydroxamate moiety is at position 2,3 of ring A. 13. A compound according to Claim 10, 11 or 12 wherein ring A contains only one nitrogen atom. 14. A compound according to any of Claims 10-13 in which Ring A is diketo. 15. A compound according to any of Claims 10-14 wherein ring B is unsubstituted. 16. A compound according to any of Claims 10-15 wherein B is a 6-membered ring of Formula III. 17. A compound according to Claim 16 wherein R^ and R7 are nitrogen. 18. Compound SL301. 19. A compound of formula I in which R j and R~ which may be the same or different are N or CH, Xj and X2 which may be the same or different are hydrogen, hydroxy. or an optionally substituted alkyl, alkenyl. alkynyl, cycloalkyl, aryl or heterocyclyl group and Z and Z2 which may be the same or different are hydrogen, hydroxy, keto (= O), or an optionally substituted alkyl. alkenyl. alkynyl, cycloalkyl, aryl or heterocyclyl group or one of Zj and Xj and Z~ and X2 form a second bond of a double bond at the 1 ,6 or 2,3 positions with the proviso that at least one of the groupings Rj Zj Xj and R2 Z2 X2 and Rj X Z2 form a hydroxamate moiety (-N(OH)C(= O)- in which Rj and/or R 2 is N, Z j and/or Z 2 is = O and X j and/or X is OH or R is N, Z2 is = O and Xj is OH and B is a 5 or 6 membered ring of formula II or III in which R4, R5, R β , R7, Rg, R9 and RJ Q which may be the same or different are CH or N with the proviso that ring B cannot contain more than 3 ring members which are nitrogen and the ring B may optionally be substituted by one or more of hydroxy, keto (= O) and an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclyl group with the proviso that the compound is not wherein X is hydrogen, aryl C j _4 alkyl. or Cj- hydroxyalkyl lopentyl or a salt thereof formed with a physiologically acceptable organic or inorganic acid for use in therapy. 20. A pharmaceutical composition comprising a compound according to Claim 19 or a salt thereof formed with a physiologically acceptable organic or inorganic acid together with a physiologically acceptable diluent or carrier. 21. A pharmaceutical composition according to Claim 12. adapted for parenteral administration. 22. A pharmaceutical composition according to Claim 12, adapted for oral administration.

Inventors: LEWIS ANTHONY EDWARD, SINGH SURINDER, LEWIS, ANTHONY, EDWARD, SINGH, SURINDER
Applicants: BRITISH TECH GROUP, LEWIS ANTHONY EDWARD, SINGH SURINDER, BRITISH TECHNOLOGY GROUP LIMITED, LEWIS, ANTHONY, EDWARD, SINGH, SURINDER
Application year: 1995
Main classification: A61K31
IPC codes: A61K31, A61P9, C07D471, C07D473, C07D475, C07D487