IL44123A - 2-amino-4-hydroxy-7,8-dihydropteridine-6-carboxaldehyde derivatives their preparation and pharmaceutical compositions containing them - Google Patents

Abstract

Title compds are formula (I), where R' and R2 are 1-4C alkyl or CR1R2 is a 4-6 membered spiralkyl gp., and are prepd. e.g. by (a) mild oxidation (O2 and I2/H+) of the corresp. 6-hydroxymethyl deriv. or (b) reductive cyclisation of a 2-amino 4-(1,1'-dialkyl-3'-hydroxyacetonyl )amino 6-hydroxy 5-nitro-pyrimidine oxime. (I) inhibits hydroxymethyldihydropteridine pyrophosphokinase, and compsns. with dihydrofolate reductase inhibitors and/or p-aminobenzoic acid competitors are synergistic. [FR2215963A1]

Description

Novel 2-amino÷4-hydroxy~7»8-dihydropteridine-6-carboxaldehyde derivatives, their preparation and pharmaceutical compositions containing them THE WELLCOME FOUNDATION LIMITED 0. 42166 44123/3 The present invention relates to derivatives of pteridine, their chemical synthesis and pharmaceutical formulations containing them. The specification also describes compositions and pharmaceutical formulations comprising thes pteridlnes in combinations which are useful in the treatment of microbial infections.

It is already established that the compounds 2-amino-4-hydrQxy~6Hhydroxymeth l-7, 7-dlmethyl-7, 8 -dihydropteridine and 2~amino-4-hydroxy-6-methyl-7f 7 dimethyl--7 , 8-dihydro-pteridlne or their tautomers or pharmaceutically acceptable salts thereof, have bacteriostatic activity, being particularly effective against CI. perfringens and Perm, dermatonomous , as disclosed in th speci ications of British Patent No. 1303171 and Israel Patent Ilo. 37252 (Belgian Patent Ho. 770,577).

It is also known from J. Vitaminol (Kyoto) 1971, 17(1) , 1-4 (Chem. Mast, 75, 72963η (1971)) that 2-amino-4-hydroxy-6-carboay (or- ormyl) -7 ,8-dlhydroptenldine inhibit -4' ' bacterial growths (IC50 for E. coli B, 2.2 x 10 M and It has no been found that the novel pteridlnes in accordance with the following formula (I) or their tautomers or pharmaceutically acceptable salts thereof, being analogues of the pteridlnes described in the specifications of Israel Patent Ho* 42858 and British Patents Nos. 1,451,043 and 1,453,831 are also useful as antagonists of microbial metabolism. The novel compounds of formula (I) are more active against E. coli than the aforementioned compounds known from the J. Vitaminol publication by 1 to more than 2 orders of magnitude.

In formula (I) 0 1 2, R and R ' are the same or different and each is a lower alkyl 1 2 group or R and R , together with the carbon atom in the pteridine ring structure, form a spirocycloalk 1 ring system having 4 to 6 carbon atoms outside the pteridine ring structure.

As used herein and throughout the specification, the term "lower alkyl group" refers to a straight or branched chain alkyl group having .1 to 4 carbon atoms . 1 2 Those compounds of formula (I) wherein R and R are the same and are both lower alkyl groups, such as methyl groups and particularly ethyl groups are especially preferred. Thus the compounds 2-am.ino-- 4-hydroxy- 7 , 7-dimethyl-7 , 8-dihydropteridine- 6-carboxa.ldehyde and its , 7-diethyl analogue have been found to be particularly useful in the treatment of microbial infections.

According to the present invention, therefore, there are provided in one aspect the novel compounds of formula (I) and the salts thereof.

The above compounds and their salts inhibit one of the enzymes involved in the biosynthesis of di-hydrofolic acid, namely hydroxymethyIdihydropteridine pyrophosphokinase , which is essential to the growth of microorganisms, for instance bacteria. They can thus be used in in vitro pharmacological investigations in clinical and diagnostic tests establishing, for instance, the properties of bacteria. When used as bacteriostats they may be present in a concentration of 50 to 500, in particular 110 to I80,mg of base/ml of the solution in which the organism grows in the absence of a compound. A further use of the compounds, when in solution, is in the treatment of wounds, for example after surgery, to prevent the growth of bacteria. Moreover the compounds of formula (I) -and their salts manifest unexpectedly low toxicity in mammals or birds e.g. poultry, which makes them particularly suitable for application against microbial infections in such hosts _under circumstances hereinbelow described .

Tetrahydrofolate co-factors are essential metabolites in all cells for the biosynthesis of purines, thymidylic acid, serine and several other biologically important compounds. Most of these co-factors are one-carbon adducts of tetrahydrofolic acid. The ultimate source of these for higher animals and man is food, containing preformed folates usually in the form of vitamins.

In microorganisms, the co-factors are synthesised from simpler chemicals. Generally the bio-synthetic process first provides 'dihydropteridine'5 (Pt) , i.e. 2-a ino- -hydroxy- 6-hydroxymethy1-7 , 8-dihydropteridine (HMPt) pyrophosphate ester, from its immediate precursor HMPt in the presence of the enzyme hydroxymethyIdihydro-pteridine pyrophosphokinase (HMPPS) . Pt then, condenses with p-aminobenzoic acid (pAB) in the presence of the enzyme ciihydropteroate synthetase to form dihydropteroic acid (DPtA) . This intermediate further condenses wit a glutamate to form dihydrofolic acid (DFA or 'folate') which is then enzyma ically- reduced to provide the essential tetrahydrofolate in, for instance, bacteria and other rnicro-organisrns .

The provision of the 'folate' from the basic building blocks, i.e. pteridine, pAB , and glutamate, and. the further conversion of this into the tetrahydro-folate is known to be inhibited in two different ways.

For instance sulphonainides displace pAB in the above reaction scheme. Because of their close structural resemblance to pAB, sulphonamides- or similar other 'competitors' enter the biosynthesis and prevent the formation of DPtA, and of DFA, and are therefore antimetabolites for the metabolite pAB. It is also known that compounds which are 'inhibitors' of the enzyme dihyd.ro-folic acid reductase block the synthetic step leading to tetrahydrofolate . A considerable number of pyrimidine derivatives show substantial anti-microbial . proper ies' on the basis of such blockage.

It was established later that such inhibitors may act synergistically with sulphonamides, i.e. there can be a. sequential double blockade and a strong mutual potentiation of the anti-bacterial effects of the two materials. The range of anti-microbial action exerted by such combinations is considerably wider than that expected, from the activity of either drug, and organisms which are only marginally sensitive to the individual agents become very sensitive to the combinations.

It was also suggested hypothetically that antimeta- bolites to Pt could inhibit the biosynthesis of DPtA (and DFA) (cf. Hitchings and Burchall Advances in Enzyrno.lo y , 27, 417-468 (.1965)) but compounds so far tested for the purpose have been disappointing, being either inactive or too toxic or sometimes both (cf. the compounds described in British Patent Nos.981,506 and 987,916).

It has been established that, for antimicrobial purposes, it is a prerequisite for the effective antag¬ onism of Pt that the compound should be an inhibitor of HMPPS without also acting as an antimetabolite to the di- hydropteridine that serves as a cofactor for the hydroxyl- ation of phenylalanine and tyrosine, precursors of the catecholamines, such as norepinephrine, that have important actions as regulators of cardiovascular systems, Such an antimetabolic effect could lead to prohibitive toxicity to avian or mammalian species, which are normally the hosts infected v.;ith the microbes.

It has now been found that the compounds of formula (I) and their salts fulfil the above requirements i.e. in¬ hibition of HMPPS combined with low toxicity. to host species, as demonstrated for instance in chicks and rats. These compound on their own, not only inhibit the growth of microorganisms albeit to a limited extent with certain bacteri , such as Staphylococcus aureus , Streptococcus pyogenes , Streptococcus faecalis , '\_.

Escherichia coll, S Imono .1 la ¾p_hi, Proteus vulgaris , Pseucomonas aeruqe;josa, l^sLeurella ^^itj^.^l among others, . but have been found to act w h a mos rem rk ble syner¬ g s c effect when combined with a competitor of . p-aminobenzoic acid, i.e. sulpho mides and similar com¬ pounds, or with selective inhibitors of dihydrofolic reductase, i.e. pyrimidines and related compounds, or with, a combination of both of these types of .anti-miorobial agents. This potentiating effect of the compounds of formula (I) forms part of the subject of Israel Patent Specification No. 44124 of even date herewith.

In that application there is described and claimed a composition for testing or treating microbial systems or infections, comprising an effective potentiating amount of a compound of formula (I) in combination with an effective amount of a competitor or inhibitor, or both, as; herein defined.

The microbial infections against which these combinations are effective are protozoal or bacteria infections caused by those microorganisms which synthesise at least a substantial part, of their tet hydro olate co- factor requirements. More specifically these .infecting microorganisms are those which adequately absorb the pharmaceutical combinations disclosed herein and further are those in which these combinations have a synergistic effect in interfering with the de_ novo synthesis of the required tetrahydrofolate co-factors. For example, the compositions described have been found to be use¬ ful in the treatment of infections caused by S aphylococcus aureus , Pseudomonas aerugi osa and Pasteurella multocida .

It has been found specifically that, when compounds of formula (I) are combined with an amount of the competitor J and/or the inhibitor which is not ordinarily sufficient to be effective as an antimicrobial agent in its own right, the combination of a compound of formula (I) with this normally ineffective amount of the competitor and/or the inhibitor provides a 'composition which in totality acts as an effec¬ tive antimicrobial agent. This 'is especially notable when the amount of the compound of formula (I) is so low that it has substantially _no microbial effect at the particular level, yet in the combination the potentiation is marked, in some instances very marked. Thus by using an effective potentiating amount of a compound of formula (I) together with the competitor and/or the inhibitor, it is now possible to reduce significantly the amount of the competitor and/or the inhibitor required to inhibit the growth of these bacteria.

In accordance with the above therefore, the term "an effective amount" used in conjunction with the terms a dihydrofolic reductase 'inhibitor' and a para-arnino- benzoic acid 'competitor' means either (a) an amount of the 'inhibitor' or 'competitor' which is effective to a degree as an antimicrobi l agent in its own right but which a compound of is potentiated by the use of/formula (I) or (b) an amount or 'competitor' of the 1 inhibitor ' ^which. is ineffective as an antimicrobial agent but which when combined with a compound of formula (I) provides a composition which is an effective anti¬ microbial agent. An "effective potentiating amount" means an amount of the compound of formula (I) which increases the activity of an inhibitor and/or a competitor so as to. provide an improved or adequate effectiveness for the whole combi ation.

It should be emphasised that the inhibition of the biosynthetic processes'by such means could be termed as com¬ petitive antagonism in all three instances, and there might be potentiation between all three types of agents. The terms ' inhibito ' , ' competitor ' , and 'potentiation' by a compound of formula (I) are arbitrary and should only serve as con- venient names for the. appropriate type of components in combination products described and claimed in the specification of the afore-mentioned cognate appl -cation , The inhibiting activity against HMPPS of a selected compound- of formula (I) can, for instance, be tested by triphosphate .monitoring the transfer of the terminal phosphate of adenosine/ 32 f t ΑΤΡ-γ-Ρ to dihydropteridine , It was found that the concentrations required for 50% inhibition of the form ~ "ation of Pt (ICr-) in such tests are well correlated and within the margin of error obtained by other relevant this tests in/respect, which measure .the inhibition of either of the tv/o enzymes involved in the formation of HMPt and DPtA. Such inhibition may, for instance, be easily and simply carried out by incubating an extract of E. coli with 14 1 · pAB-7-C , ATP, Mg and dihydropteridine . The formation J 4 of the dihydropteroate-C can be quantitatively assayed after separating the--unreactecJ pAB substrate, for instance ■ by chromatography.. It has been found that compounds possessing in such tests an IC value of about ΧΟ μΜ-- or less, usually below 50pM represent compounds exerting a useful potentiating effect, provided their toxicity · in the appropriate vertebrates is acceptable. Preferably the value is 25μΜ or less, such as in the range between 2 to Ι2)ίϋ. Generally a value below 7μΜ is desirable.

As explained above, for the purpose disclosed it is essential that the- compound of formula (I) should not have a prohibitive toxicity to the mammalian or avian hosts' cardiovascular systems. While low toxicity is therefore an essential requirement,' a therapeutic index Incorporates both the activity and toxicity values pertinent to the present disclosure' and could be us with advantage for the selection of potentiating compound of formula ( I ) .

The therapeutic index is defined as the ratio of the maximum tolerated dose to the minimum effective dose and in most cases is preferably greater than Ϊ0, suitably at least 5 and in exceptional circumstances at least about 3 for human¾> .but possibly as low as 2 for animals „ . Although the art is aware of many compounds which are. known competitors of para~aminoben.?.oic acid and are antimicrobials, the sulphur compounds which are disclosed as antimicrobial agents from the top of page 994 to page 1007 of the Merck Index, 8th Edition, 1968 are presented by way of example only.

Of the known compounds which are competitors, the following sulphonamide compounds (or pharmaceutically acceptable salts thereof) are preferred for the purpose described. nulphanilamide , sulphadiazine , sulphamethisazole , sulphamethizole , sulphapyridine , sulphathiazole , sulph-amerazine, sulphamethazine , sulphisoxazole , sulphadoxine , sulphasomidine , sulphachlorpyridazine , 2- (jo-aminobenzene) -sulphonamido-S-methoxypyra ine (Keifizina) r a~amino~p_-tolue~ nesulphona ide , 5-su.lphanilamido-2 , 4-dimethyl pyrimidine , 4-( '~acetyl sulphanilamido) -5 , 6-dimethoxy pyrimidine, 3~sulphani.lamido-4 , 5-dimethyl isoxazole , 4-sulphanilamido~5-methoxy-6~decyloxy pyrimidine , sulph- amonomethoxine , 4~_-- (8-hydroxy-quinilinyl-4-azo) -phenyl sulphanilarnido~5 , 6-dimethoxy pyrimidine , sulphadimetboxine sulphamethoxazole , sulphaquinoxaline , and p_- (2-methyl-8-hydroxy-quinoliny 1- (5 ) -azo) phenyl sulphanilainido-5 , 6-dimethoxy pyrimidine. Examples of a non-sulphonamide type of competitor are p-am'ino salicylic acid (PAS) and Ρ,ρ'-diaminodiphenylsulphone .

Similarly, although many compound.s are knov;n which inhibit dihydr'ofolic reductase and act as antimicrobial agents, the compounds disclosed in the following patents are presented by way of example of compounds suitable .for use for the purpose disclosed.

U.S. Patent Nos. 2 ,658/897 ; 2 767, 183* 3,021,332·, 2 ,937 ,284·, 3,322,765; 2,909 ,522: 2,624,732; 2 ,579 ,259 ; 2 ,945,859 ; 2 ,576,939; 2,926, 166·, 2,697 ,710 ; 2 ,749 , 345 ; and 2 ,749 , 344. .. __ · The following inhibitors (or pharmaceutically acceptable salts thereof) arc preferred for the combinations described.., however: 2 , 4-diamino- 6·- thyl-5- -chlorophenylpyrimidine (pyrimethamine) , 2 , 4-diamino-5~ (3 ' 1 , 5 ' -trimethoxybenzyl) -pyrimi.di.ne (trimethoprim) , 2 ,.4-diam.ino -5~ (314 ' -dimethoxy- benzyl) pyrimidinc (diaveridine ) , 2 , 4-diamino-5- (2 ' - isoprop 1- 1 -chlorophenoxy ) pyrimidine , 2 , 4~diamino-5-methy1-6- sec-butyIpy ido (2, 3-d) pyrimidine, 2 , 4-diamino- 5-metbyl---6~benzylpyrido (2, 3-d) pyrimidine, 2 , 4-dia.rnino~6- benzylpyrido (2 , 3-d) pyrimidine , 2 , 4~diarnino-5~6~tri ethy1-enequinazoline , 2 f 4-diamino-5 , 6-tetra!«ethylenequinazoline , 2 , 4-diamino-5-- (2 ' , 4 1 -trimethoxybenzyl) pyrimidine , 2 , 4-diamino-5~ -eth l-41 , 5~dimethoxybenzy1 ) pyrimidine , 2 , 4"diamine--5~ (21 - me hy1- ' , 5 ' -dimethoxybe z 1 ) pyrimidine.

However, the most preferred combinations include j those combining a compound of formula (I) , especially those wherein and are the same and are both methyl or ethyl groups, with sulphadiazine, sulphamethoxazole , sulphadoxine or sulphaquinoxaline as competitors, or with trimethoprim, diaveridine or pyrimethamine as inhibitors.

In view of possible synergistic advantages of using certai competitors and inhibitors in combination against particular diseases, and the potentiating effect of compounds of formula (I) on both of these types of antibacterial coin- pounds, it has been preferred to formulate triple combi¬ nations, comprising a compound of formula (I) with one of the above-mentioned preferred competitors, and one of such inhibitors. For example, combinations of sulphadiazine/ trimethoprim, sulphamethoxazole/trimethoprim, sulphadoxine/ trimethoprim or sulphaquin- oxaline/diaveridine , each together with a compound of formul {I)f give improved effectiveness when com¬ pared with the components alone or with pairs of them.

The compounds of formula (I) either alone or to¬ gether with the competitor and/or the inhibitor, may be presented in association with a carrier in pharmaceutical formulations suitable for parenteral, topical, rectal or oral administration. The formulations for oral or rectal administration are advantageously presented in dis¬ crete units, such as tablets, capsules , .cachets , ampoules or suppositories, each containing ίΐ predetermined amount of each compound, but may also be presented as a powder, as granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an ointment or paste foa: topical administration. For parenteral use, . the formulations incorporating an aqueous or non-aqueous liquid carrier must be sterile and be presented in sealed containers. The formulations may be made by any of the .known methods and may include one or more of the following accessory ingre¬ dients! diluents, solutes to render the solution isotonic with the blood, buffers, flavouring, binding, dispersing, surface-active, thickening, lubricating and coating mate¬ rials, preservatives, bacteriostats , antioxidants, suppository and ointment bases, and any other accept¬ able excipients.

In another aspect of the present invention, there¬ fore, there is provided a pharmaceutical formulation com¬ prising a compound of formula (I) in combination with a pharmaceutically acceptable carrier. In yet another aspect the present invention provides a method of making a pharma¬ ceutical formulation b admixing the compound of formula (I) with a carrier by known techniques. The specification of the afore-mentioned cognate application further discloses and pharmaceutical defined , claims a/formulation comprising a composition, as hereinbefore- together with a carrier, and its method of preparation, by admixing--the composition with the carrier by known techniques.

Formulations containing the compound of formula (I) in ' association with a competitor or an inhibitor may also be pre¬ sented in the form of a kit, which comprises separately packaged units or dosages of these components with instructions for vise in a combined form. The instructions may also specify the manner of administration and indications for which the formula is suitable.

The compounds of formula (I) , either for use alone or in association with a competitor and/or inhibitor, and also the competitors and inhibitors, may be presented in the form of their pharmaceutically acceptable salts of a mineral or organic acid, for example hydrochloric acid, hydrobromic acid, sulphuric acid, acetic acid, citric acid, tartaric acid, lactic acid, maleic acid or salicylic acid, or, I especially for the sulphonamide competitor, of a base, such as sodium hydroxide, potassium hydroxide, tetra- methyl ammonium hydroxide or ammonia, the The ratios in which/ therapeutically active compounds of formula (I) are utilized in the compositions described in this specification can be varied between wide limits. Depending on the nature and circumstance of use, the compositions may contain the compound of formula (I) with the competitor and/or the inhibitor in appro- j priate proportions and dosages. For instance, in cases of uses in vivo it is often desireible to maintain a certain proportion of components in the blood serum or tissue fluids, preferably for a prolonged period, Depending on the various absorption, discharge or decomposition rates of the compone s, the initial quantities and proportions of the ingredients of the . formulation, can be different from that aimed at in the tissues in vivo. The formulations and do¬ sages recommended for the general treatment of a particular human or animal disease, must be adjusted according to the particular requirements of the recipients of the disease, the known activities of the competitor or inhibitor- com¬ ponent against the causative organism, the half life and the toxicity of the components in vivo , and other practical requirements ..

For example the compositio or pharmaceutical about formulation may contain from/I to 30 parts by weight, preferably 5 to 15 parts, of the compound of formula (I) , or an equivalent amount of a salt thereof, and 1 to 30 parts, preferably 5 to 15 parts, of a competitor, or an equivalent amount of a salt thereof, and/or one part of an inhibitor, or an equivalent amount of a salt thereof.

Dosage will vary depending upon the infecting organism but under ' ordinary circumstances up to about 60 mg/kg each of a compound of formula (I) and competitor, and up to about 7.5 mg/kg of inhibitor, in combination, can be admi istered daily in several doses. The com¬ position or pharmaceutical formulation can -be administered to human patients in unit dosage forms which contain up to 750 mg of the compound of formula (I) , and up to 750 mg of the. competitor and/or up to 25 mg of the inhibitor.

Preferably for adult dosages the amount of the compound of formula (I) would be about 200 mg, that of the competitor about 200 mg and/or that of the inhibitor about 25 mg.

The pharmciceutical' formulation comprising the com¬ pound of formula (I) in combination with the competitor and/ or the inhibitor is also usable in solution for ^ irrigating wounds, for example after surgery, so as to prevent the growth of bacteria. For example, an anti¬ bacterial solution having the following preferred con¬ centration of components may be used: 1-30 g/nil of the compound of formula (I) , 1-30 mg/ml of the competitor and/or 0.03-1 mg/ml of the in- a hibitor, in pharmaceutically acceptable solvent, suitable for external use.

The potentiating effect of compounds of formula (I) can be demonstrated and utilized In v ro relatively easily for research and practical purposes. Such possibilities include diagnosis and the identification 'of the bacterial flora of individuals and the consequential selection of clinical treatment schedules.

The- various combinations can be incorporated in porous discs (such as filter paper discs) or in Agar Nutrient' or other media for bacterial growth for determining sus¬ ceptibility. Those articles incorporating the compound of formula (I) with a competitor and/or an inhibitor compound may be distributed or sold to doctors, hospitals and clinics for the above, purposes. A typical testing disc may be im¬ pregnated with a solution containing 5 to 50 yg/rril of a para-aminobenzoic acid competitor, 0.5 to 5 yg/ml of a dihydrofo.lic reductase inhibitor, and about .10 to 100 ]jg/ml of a compound of formula (I) in a medium comprising a mixture of an aqueous infusion and papain digest of horse muscle.

Furthermore, such pharmacological tests involving potentiated competitors or inhibitors may also be useful for the characterisation of bacteria according to their sensitivity and to their particular resistance for instance to a competitor when used alone, and such investigations involving a variety of fo mulations as- described herein also form the basis of determining the compositions of selected formu3.at.ions for general treatment purposes. The toxicity of compounds of formula (I) is generally considerably lower than that of the competitors or inhibitors -commonly used, which may enable the clinician to maintain or increase the effectiveness of the antibacterial activity of the formulation with a concurrent increase of the therapeutic ratio or decrease in the toxic or side-effects of the medicament.

In addition to the above compounds of formula (I) have been found to potentiate the activity of the aforementioned competitors and/or inhibitors against infections with microorganisms in domestic animals f including poultry, for example against steu^ella multocida but especially against the protozoal disease coccidiosis. Such triple ormulations comprisi g a compound of formula (I) together with a compound such as sulphaquinoxaline and an inhibitor such as diaveridine are effective in lower concentrations than the competitor or inhibitor components alone and possess an enhanced activity, being effective against several Eimeria species, which cause this disease in poultry. 2-Amino~4-hydroxy-7 , 7~dimethyl - 7 , 8~dihydropteridine-6-car-boxaldehyde has been found to be particularly effective in such compositions, even at concentrations as low as 20 parts per million (pprn.).

The compounds of formula (I) may be prepared , for example, by selective oxidation of the ' correspond ing 6- hyd.roxya.lkyl derivative of formula (II) ,' even mild condi¬ tions being -successful. The oxidation may be carried out, for instance, in an acid medium, such as aqueous hydro¬ chloric acid, formic acid or sulphurous acid, with an oxidising agent, such as air, oxygen or iodine, advantageously in the presence of an anti-oxidant , such as sulphur dioxide or ascorbic acid, to react with any hydrogen peroxide formed which could decompose the aldehyde as soon as it has been formed, thus reducing the potential yield, of product.

(II) The desired compounds of formula (I) may also be formed d ing the f nal stage in the preparation of the corresponding compounds of formula .(II) , for example as a by- roduct, in the reductive c cl isa ion of the corresponding pyrimidine keto- xiine of formula (III) by a slight modification of the procedur described in the specif cation of Israel Patent No. 37252, wherein a controlled amount of re¬ ducing ag nt e.g. sodium dithionite, is added to limit the re¬ ducing po .rit.ial of the reaction mixture. The aldehyde derivative may then be separated from the compound of forma (II) by ion exchange methods. 0 (III) The compounds of formula (II) may be prepared following the procedures described in the specifications of British Patent No. 1303171 and Israel Patents numbers 37252 and 42858.

According to the present invention in further aspects there are also provided 1. The methods described herein for preparing any of the compounds of formula (I) comprising oxidation of the corresponding compound of formula (II) . 2. The methods described herein for preparing any of the compounds of formula (I) , comprising controlled redvictive cyc.lisation of the corresponding compound of formula (III) and separation from the corresponding compound of formula (II) . 3. Compounds of formula (I) , whenever prepared by a method as defined, under (1) or (2) . 4. A pharmaceutical formulation comprising a compound of formula (I) or a salt thereof in combination with a pharina- \ ceutically acceptable carrier., whenever prepared by the method herein described.

The following examples illustrate the invention but are in no way intended to limit the scope of the invention.

Temperatures are in degrees Celsius.

Exam le^l ^-Amin ~^~hyd^xy~_7 , 7-dimethyl -_7 , 8-di.hydropteridlne- 1 2 6 carboxaldeh^e_. _ (I) _ (R = R ~ Me) A suspensio of 2~amino-4~hydro.xy~6-hydroxymethyl-7 , 7-dimethy.1.-7 , 8-dihydropteridine (II) (Ri=R2=Me) (0.449g ) in 2M hydrochloric acid (5ml ) was stirred in air overnight at room temperature. The dark-red mixture was put on to an ion exchange resin (Amberlite CG-50, H+ form) and the yellow product eluted with water, the initial acid fraction being discarded. The eluate was concentrated, to a small volume in vacuo and the precipitate was collected by filtration to give the crude title compound as yellow crystals which darkened cibove 240°. (Yield 0.13g') The product was further puri¬ fied by dissolving it in water/methanol and by concentrating the solution slowly until the crystals separated out, these darkening when heated above 260°.

ExamjDle_2 2-Amino- 4--hyd oxy- 7 , 7-dimethyI - 7,8-dihydro teridine- 1 2 6-carboxaldehyde . (I) (R - R = Me) A 1:2 mixture of oxygen and sulphur dioxide was passed through a stirred suspension of 2-amino-4-hydroxy-6-hydr oxy- 2 methyl-7 , 7~dimethyl~7 , 8-dihydropteridine (II) (R"=R =Me) (1.0 in saturated aqueous sulphurous acid (20ml ) over 1 {. hours at room temperature. The resulting suspension of almost colour- j less solid was then kept at 0° overnight, treated with sulphur dioxide for 5 minutes, and filtered, -//ashing the filter solid with ice-cold aqueous sulphurous acid, to give a (1.112g. ) . sparingly- soluble aldehyde/sulphurous acid adduct/_ Concentration of the. liquors in vacuo to about 10ml, followed by satu¬ ration with sulphur dioxide and storing at 0° for several days, yielded .■ further adduct (57mg ') . To isolate the aldehyde a suspension of the combined adduct in water (20ml ) was heated in a. bath at 70° and treated with a current of nitrogen until, after about 20 minutes, evolution of sulphur dioxide was complete; the restil ing suspension was then cooled to 0° and filtered to give the title compound (pure), ( Yield.0.748g .1.

Example 3 2-Arriino 4~hyd oxy~7 , 7-diethyl-7 , 8-dihydropteridine- 6- .1 2 carboxaIdehyde (I) (R = R = Et) Sodium dithionite was added portionwise to a warm solution of 2-amino-4- (15 , 1 ' -diethyl-3 ' -hydroxyacetonyl) amino-6-hydroxy- 1 2 5--nitro~pyrimidine oxi e (III) (R = R = Et) (4b0mg ) in 0.1M sodium hydroxide until the colour changed from red to very pale yellow. To isolate the product the solution was evaporated and the product extracted with etlianoi and the inorganic material removed by filtration. The extraction was repeated and the combined extracts were evaporated to dryness in vacuo » The residue was dissolved in the minimum quantity of water and placed on an ion exchange resin (Amber lite CG-50 , li' form).

Elution with water gave two main fluorescent bands. Evapora¬ tion of the solution containing the first band gave the title compound as a bright orange powder. T e second, band gave the corresponding 6-hydroxymethy 1 derivative of formula (II) (R1 = RZ = Et) as a bright yellow powder , m.p. >300° (decomp) ) This derivative was then oxidised to the 6~carboxaldeh.yde as described under Example 1.

Example 4 2-Amino- 4-hydroxy-7 t 7-diethyl-7 , 8-dihydropteridine-6- 1 2 carboxa1dehyde (I) (R = R - Et) A stirred suspension, of 2-amino-4-hydroxy-6-hyd.roxy~ 1 2 methyl- 7, 7~dieth l-7 , 8-dihydropteridi.ne (II) (R =--R'=Et) (l„lg ) in saturated aqueous sulphxirous acid (20 ml ·') was treated with oxygen and sulphur dioxide as described in Exampl 2. The combined adduct/ obtained as the solid residue after filtration and by concentration of the liquors iri va^uc^w s suspended in. water (20ml ) heated at 70° and treated with a current of nitrogen as described in Example 2. The suspension was cooled to 0° and filtered to give the title compound (pure Example 5 2-Amino~4~hydroxy"'7 , 7-di-n-propyI-7 , 8-dihydropter idine- 6- 1 ? carboxaIdehyde (I) (R = R" = n - Pr) The procedure of Example 2 was followed using, however , . as starting material 2--amino-4~hydroxy-6--hydroxymethyl-7 , 7- 1 2 di-n~ ropyl-7 , 8-dihydropte idine (II) (R = R = n - Pr) (l-3g ) which was suspended in saturated aqueous sulphurous acid (25ml ) . The title compound was then isolated in a pure J form from the aldehyde/sulphurous acid adduct as described in Example 2.

Example. 6 Potential pteridine antagonists of formula (I) may be tested by investigating the inhibitory effect they impose on the enzymes responsible for the biosynthesis of dihydropteroic acid (DPtA) , namely hydroxymethy1- dihydropteridine pyrophosphokinase (HMPPS) , and dihydro- ptoroate synthetase, hereinafter referred to as 'synthetase' In the following reaction equations the compounds are referred to by their abbreviated forms defined on page. 5 of the specification* 1. HMPPS 2. S nthetase . ■ ~ 2+ Ma .

Pt -t pAB r> DPtA -I- pyrophosphate (a) An assay for HMPPS was developed in which the transfer 32 of the terminal phosphate of ΑΤΡ-γ-Ρ to Pt could be monitored and correlated, with the amount of inhibition of HMPPS by the compound under test.

The compound of formula (I) which was under test v/as incorporated, into .various formulations comprising metabolite and enzymes contained in test tubes, as indicated in TABLE 1 The components of the mixture were as follows : - I-2·-amino- -byd.roxy- 6-hydro rn V -0.02M MgC'l .6H 0.

VI - 0.1 M MgCl .6H 0.

VII- Source of K PPS and 'synthetase' VIII - the test compound in a concentration of 0.93x10 ~" As shown in TABLE 1, tubes 1 to 9 all contain a source of HMPPS, labelled ATP and 0.02 M MgCl .611 0 , tubes 2 to 9 containing in addition HMPt and tubes 4 to 9 further containing the test compound.' Control tubes 10 to 12 in- · elude a source of both HMPPS and synthetase, unlabelled ATP, 0.1M MgCl .61-1 0 and labelled pAB.

Tubes 1 to 9 containing the amounts of components shown in the Table, were filled, up . to 200 1 with distilled o water, incubated for 60 minutes at 37 C and then chilled on ice. Dextrose (20μ1 containing 72.1 mg/ l) and hexo- kinase (5μ1 containing 2000 units/ml) were added to the solution, which was then allowed to stand at room temperature for 15 minutes. Darco-G-60 ' (Registered Trade Mark) (10 mg) was added to each tube and the contents mixed periodically for 10 minutes. The charcoal was removed through a i 'Millipore A3? 250 2200 * (Registered Trade Mark) filter ^ and the filter was washed with three 10 ml portions of cold a er. The charco l and the ilter were then rad.io.active.ly counted „ The radioactive count from the contents of tubes 2 and 3 was taken as the maximum count, since these tubes contained no test compound and thus gave 0% enzyme inhibition. The percentage inhibition produced by the contents of the remaining tubes could then be calculated by relating their radioactive count to the maximum, as determined above.

The contents of tubes 10 to 12 were chromatographica1ly analysed as described under part (b) , and used as controls, tubes 10 and 11 containing no test compound (and hence giving 0% .inhibition) being accorded the value of .100%. The percentage inhibition exhibited by the contents of the tubes in part (b) o the experiment could then be calculated in relation to this, by comparing the respective chromatograms . (b) The activity of the test compound of formula (I) against 'synthetase' was determined by mo itoring the formation Gf dihydropteroate C"1^ as follows.

A pool of Pt was prepared from ATP neutralised (50μ1, .1M), MgCl -6H 0 (5θμ1,0.1M) , dithiothreitol (ΙΟΟμΙ,Ο.ΙΜ) , tris buffer (ΙΟΟμΙ ,0.4M,pH 8.3), 1-IMPt (25μ1,876μΜ) and Ι70μ1 of a solution containing HMPPS. The mixture was incubated for 60 minutes at 37 C , chilled briefly on ice and then dextro.se ( ΟΟμΙ containing 72. lmg/ml ) and hexokina.se (20μ1 containing 2000 units/ml) were added at room tempera¬ ture to the solution, which was allowed to stand at this temperature for 15 minutes. 1 4 A solution of MgCl2.GH 0 (ΙΟμΙ,Ο.ΐΜ), pAB-C " (ΙΟμΙ,Ο.4mM) , dithiothreitol ( 20μ.1 , 0. IM) and tris' buffer (20μ1 , 0.4M ,pH8.3) was made in each of five test tubes and. then. 80μ.1 of the contents of the pool added to each, to¬ gether wi h synthetase and/or test compound of formula (I) as indicated in Table 2e The solution was then made up to 200μ1 with dist lled water.

Two control test tubes were prepared, each containing ATP (ΙΟμΙ,Ο.ΐΜ), MgCl .6H 0 ( ΙΟμΙ ,0. IM) , dithiothreitol 14 (20μ1,Ο.ΐΜ) tris buffer (20μ1 ,0.4M,pH 8.3·), pAB-C (ΙΟμΙ, O.4mM) , and 20μ1 of a solution containing HMPPS and 1 synthetase 1 of known activity. 'Die test compound was added to the second of these two tubes up to a final concentration of 10 ~M, and both tubes were made up with distilled water to 200μ1.

All seven tubes were then incubated for 30 minutes at 37°C, chilled on ice and then these, together with control tubes 10 to 12 from part (a) , were chromatographically analysed as follows. ΙΟΟμΙ of the contents of each of the tubes was spotted onto Whatman no. 3MM chromatography paper (2x20 cm) at the 'origin', the run descending in a Sirenson buffer of potassium and sodium phosphates (Ο.ΙΜ,ρΗ 7.0). for 10 to 15 cm „ Fro.m the relative positions of the spots obtained, from the contents of the different tubes, the various percentage inhibitions of synthetase could be evaluated by reference to control tubes 10 and .11,. which gave 0¾ inhibitioi Those compounds which, as result of these tests, were found to give 50% inhibition at a concentration of 100/iM or less, are those which exert a useful potentiating effect, and subject to their toxicity being favourable, may be included in the compositions described in this specification . 2-Amino- -hyd oxy- 7 ,7- diethy. , 8-dihydropteridine~6-carboxaldehyde and its 7,7-dimethyl analogue were found to give 50¾ inhibition at concentrations of 5.5μΜ and 17 }M respectively.

Exain le^ 7 Tab].et. Fo mυ 1at.ion Co1pound of forrau 1a (I) (R"=R =Et) (pure) lOOmg Trimethoprim (pure) 25 mg Sulf gua.nid.ine (B.P.C.) 100 mg + cornstarch, lactose, gelatin, talcum and magnesium stearate Preparation. — the above constituents were mixed together using known methods of pharmacy to form a granulation which was then compressed into tablet.?.

Example '8 ab1et Formu1ation "Pyremathimine " (Pyrimethamine ) 15 mg Compound of formula (l)-. : (R"=R~=Me) - (pure) 150 ing was then prepared to form a tablet as in Exam le E Example 9' ab.1et Formu1ation Sulfanilamide B.P.C, 150 mg Compound of formula (i) Et) (pure ) 175 mg which was then prepared to form a tablet as in Example E.

Example 10 Ca sule Formu ation Trime hoprim (pure ) 20my 2 Compound of formula = =E ) (pure) 100 rag e a a ion : The compounds in granular form were blended, together with lactose, cornstarch and magnesium stearate. The powder was filled into a two-piece, hard shell gelatin capsule using a capsulating machine.

E2^aj] le_ll I'rrigant Solution ' · Compound of formula ( R""=R- =Et) (pure) Img/ml TrimetIT.op im (pure ) 0.2mg/ml Solvent wat r Ϊrrigan t So1ution Compound of formula (I) (R1=R2=j:.ie) 2mg/ml (pure) -amino-p_~ to1ue esuIphonamide ( ure ) 2mg/ml 3b •le 13 Solu ion Compound of formula (I) = =Et) 1.5 mg/ml ure ) Diaveridine B. Vet C 0.5 mg/ml Keifizina 1.0 mg/ml Solvent water Example 14 Tablet Form·.x1ation Compound of formula (I) R --]¾«= 500 mg (pure ) Mic ocrysta11ine cellulose 100 mg Starch 40 mg Magnesium stearate 3.0 mg Methy h droxyethyIcellulo 3 mg 653 mg The pteridine (I) , microcrystalline cellulose and starch were granulated with a solution of the metliylhydroxyethy lcellulose in 50% aqueous ethyl alcohol. The magnesium stearate was added to the dried granules, and the whole then compressed.

TABLE 1. .1 - 37b - TABLE 2.

Test compound .

Tube No. Synthetase Final Concen% tration Inhibn. 1 2 + ■ 3 ' + 8.7 X 10 M 9 4 + 1 x 10-5M _6 0 5 ■ + 2.5 X 10 M 5 Controls "6 - .7 1 x 10 "5 M 83 ,

Claims (27)

X 119 What we claim is:- 1. A compound of formula (I), or a tautomeric form thereof," 1 2 wherein R and R are the same or different and -each

1. 2 is a lower alkyl group or R and R , together with the carbon atom in the pteridine ring structure, form a spirocycloalkyl ring system having 4 to 6 carbon atoms outside the pteridine ring structure.

2. „ A compound as claimed in claim 1, wherein R 1 and R2 are lower alkyl groups.

3. '3. A compound as claimed in claim 2, wherein R 1 and R2 are the same and are ethyl groups.

4. A compound as claimed in claim 2, wherein R 1 and R2 are the same and are methyl groups.

5. 2-amino-4-hydroxy- 7 , 7~diethyl-7 , 8-dihydropteridine-6- carboxaldehyde or a tautomer thereof.

6. 2-amino-4-hydroxy-7 j 7-dime hyl-7, 8-dihydropteridine-6- carboxaldehyde or a tautomer thereof.

7. A pharmaceutically acceptable salt of a compound of formula (I), as defined in any one of the preceding claims, or a tautomeric form thereof.

8. A salt as claimed in claim 7 of a base or a mineral or organic acid. - 38 - X 119

9. A pharmaceutical formulation of a compound of formula (I) or a salt thereof, as defined in any one of the preceding claims, in combination with a pharmaceutically acceptable carrier.

10. A pharmaceutical formulation as claimed in claim 9 in unit dosage form.

11. A pharmaceutical formulation as claimed in either of claims 9 and 10, substantially as herein described with reference to Example 14.

12. A method of making a pharmaceutical formulation, as ■ defined in any one of claims 9 to 11, by admixing the compound of formula (I) or a salt thereof with a carrier by known techniques.

13. A method as claimed in claim 12, substantially as herein described with reference to Example 14·

14. A pharmaceutical formulation as claimed in any one of claims 9 to 11, whenever prepared by a method as claimed in either of claims 12 and 13.

15. A method of preparing a compound of formula (i) or a salt thereof, as defined in any one of claims 1 to 8, comprising the selective oxidation of a compound of formula ( II ) , (II) wherein R and R are as defined in claim 1. - 39 - X 119

16. A method as claimed in claim 15, v/herein the oxidation with an oxidising agent is carried out in an acid ÷ medium.

17. A method as claimed in claim 16, wherein the oxidising agent is air, oxygen or iodine.

18. A method as claimed in either of claims 16 and 17, wherein the acid is aqueous hydrochloric acid, formic acid or sulphurous acid.

19. A method as claimed in any one of claims 15 to 18, wherein the oxidation is conducted in the presence of an anti-oxidant .

20. A method as claimed in claim 19, v/herein the antioxidant is sulphur dioxide or ascorbic acid.

21. A method of preparing a compound of formula (I) or a salt thereof, as defined in any one of claims 1 to 8, comprising the steps of controlled reduction of a compound of formula (III), (III) wherein R and R are as defined in claim 1, and separation of the compound of formula (I) from the compound of formula (ll). ^

22. A method as claimed in claim 21, wherein the reduction is effected with a controlled amount of reducing agent to limit the reducing potential of the mixture. - AC) X 119

23. A me ho.d as claimed in claim 22, wherein the reducing agent is sodium dithionite.

24. A method as claimed in any one of claims 21 to 23, wherein the compound of formula (I) is separated from the compound of formula (II) by ion exchange methods.

25. A method as claimed in any one of claims 15 to 20, substantially as described herein with reference to Examples 1, 2, 4 and 5·

26. A method as claimed in any one of claims 21 to 24, substantially as described herein with reference to Example 3.

27. A compound of formula (I) or a tautomeric form thereof or a salt thereof, as defined in any one of claims 1 to 8, whenever prepared by a method as claimed in any one of claims 15 to 20 or 21 to 24. - 41 -