IL44124A - Compositions containing 2-amino-4-hydroxy-7,8-dihydro pteridine derivatives for treating microbial infections - Google Patents

Description

- -ΐ3»»κ-2 ¾7 mt in D' san ο*Τ03η iD*B¾» 7»τ»ΐθ3 ητη' 8,7-'Βρΐ·η»η Compositions containing 2-amino-4-hydroxy-7,8 dihydro pteridine derivatives for treating microbial infections THE WELLCOME FOUEDATION LIMITED Cs- 42201 The present invention is. an improvement in or modification of that disclosed in Israel patent specification No. 40050. ' . / The above application relates, in one aspect, to a composition for testing or treating microbial systems or infections, comprising an effective potentiating amount of a microbial dihydropteridine antagonist, hereinafter called a 'potentiator' which itself inhibits hydroxymethyl dihydropteridine pyrophosphokinase (HMPPS) (an enzyme involved in the synthesis of tetrahydrofolate co-factors) and has a sufficiently lov; 'toxici '-.y , in combination with an effective amount of a competitor of para--aniino benzoic acid or an inhibitor of dihydrofolate reductase, or both.

In further and particular aspects the aforementioned application provides (1) a composition,, as hereinbefore defined, in which the potentiator is- a pteridine compound, such as a 2-ainino-4-hydroxy-7 , 8-aihydropteridine substituted on the 6th-position through a carbon atom and bisubstituted at the 7th position. In particular^ the composition comprises a compound of formula (A) 0 or tautomeric forms thereof,, wherein R is an alkyl group, optionally substituted with a hydroxy group, v/ith one or more halogen atoms or with an optionally substituted phenoxy group, 1 2 and R and R are the same or different and each is an alkyl 1 2 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, ompound has a substitution in the 7~th position (2) A compos ition f as specified under (l) , which comprises the compound of formula (A), wherein Ms an alkyl, a hydrc-yfa Iky 1 group or a phenoxyalkyl group, the alkyl group being preferably a methyl group, or R is a halogeno substituted alkyl group, preferably mono-o'r di--substituted especially with bromine substituent , (3) a composition, as hereinbefore defined in any aspect, comprising an effective potentiating cimount of a potentiator, in combination with an effective amount of both a competitor and an inhibitor; (.4) a pharmaceutical formulation containing any one of the above defined compositions in combination with a pharmaceutically acceptable carrier therefor; (5) a pharmaceutical formulation as defined under ( ) in the liquid form, for use in the inhibition of the production of dihydrofolic acid by microorganisms, under in vitro or in vivo conditions . (6) a pharmaceutical formulation, as hereinbefore defined, presented in the form of a kit of separately packaged potentiator inhibitor or competitor, with instructions to use them together in combination for the purpose of medical or veterinary treatment (7) an in vitro testing disc .: incorporating a pharmaceutical . formulation, as hereinbefore defined. (8). a pharmaceutical formulation for the testing or treatment of coccidial systems and infections, comprising an effective a compound of formula (A) an potentiating amount of , .in combination with effactive amount of a competitor or inhibitor o both, as hereinbefore defined. . (9) a' method of making a compositionor a pharmaceutical formulation, as hereinbefore defined, in which the effective amounts of the appropriate components are admixed and presented in 'combined form; (10) a method of treating and preventing microbial infections in animals comprising the administration to the non-human host of any one .of the compositions or pharmaceutical formulations hereinbefore defined. (11) a method of inhibiting the production of dihydrofolic acid by microorganisms which comprises contacting the microorganism with a composition or pharmaceutical formulation hereinbefore defined.

It has now been found that the novel compounds disclosed in Israel Patent Specification No. 44123 and British Patent Specification No's. 1 , 451, 043 and 1, 453 , 831 are also effective as potentiators. Thus these compounds exhibit a marked synergistic effect when combined with a competitor of para-amino benzoic acid e.g. sulphonamides and similar compounds, or with- a selective inhibitor of dihydrofolate reductase, or with a combination of both of these types' of antimicrobial agents. The theory underlying this synergistic effect is described in the specification of the afore-mentioned cognate application.

The novel compounds, many of which fall within the scope of formula (A) , have the general formula (I) (I) 1 ? wherein R is a chlorornethyl group and R and R are each .methyl groups or R is a 11 -bromoethyl group and R and R are each n-propyl groups. —' The present invention provides a composition for testing or treating microbial systems or infections, comprising an effective potentiating amount of a compound of formula (I) , 1 2 wherein R is a formyl group and R and R are the same or 1 2 different and each 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, or R is a chlorornethyl 1 ' 2 group and R and R are each methyl groups or R is a 1 2 1 ' -bromoethyl group and R and R are each n-propyl groups, in combination with an effective amount of a competitor or ! inhibitor, or both, as herein defined.

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.

Preferably R is a formyl group and in these compounds 1 2 R and R are preferably the same and are both lower alkyl groups, especially methyl or ethyl groups.

The above compounds inhibit one of the enzymes involved in the biosynthesis of dihydrofolic acid , namely hydroxymethyl- dihydropteridinepyrophosphokinase , which is essential to the growth of microorganisms, for instance bacteria. They may therefore be considered for use by themselves against many Proteus vulgaris , Pseudomonas aerugenosa and Pasteurella multocida. Moreover combinations of such compounds with a competitor of para-amino benzoic acid and/or with an inhibitor of dihydrofolate reductase are effective against those protozoal or bacterial infections caused by those microorganisms which synthesise at least a part of their tet dihydrofolate co-factor requirements (see the specification of the afore-mentioned cognate application) . More specifically these infecting microorganisms are those which adequately absorb the '^ pharmaceutical combinations disclosed he-rein and further are those in which these combinations have a synergistic effect in interfering with the d_e ovo 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 Staphylococcus aureus , PGeudjamona s aeruginosa and P teurella multocjda .

It lias 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 b 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 sub tantially_.j'io microbial effect at the pa ticular level, yet in the combination the potentiation is marked, j.n some instances very marked. Thus by using an effective potentiating amoun 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/c the inhibitor required to inhib t the growth of these .In accordance with the above therefore, the term "an effective amount" used in conjunction with the terms a dihydrofolic reductase 'inhibitor' and a para-amino- benzoic acid 'compe i or' means either (a) an amount of the 'inhibitor' or 'competitor' which is effective to a degree as an antimicrobial agent in its own right but which a compound of is potentiated by the use of /formula (I) or (b) an amount or ' competitor 1 of the. ' nhibito '/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 combination.

• It should be emphasised that the inhibition of the biosynthetic processes by such means could be termed as com¬ petitive antagonism n all three instances, and there migh be potentiation between all three types of agents. The termC- ' inhibitor 1 , '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 com¬ bination products described and claimed in the specification of the afore-mentioned cognate application.

The inhibiting activity against HMPPS of a selected compound of formula (I) can, for instance, be tested by moni¬ toring the transfer of the terminal phosphate of adenosine 32 triphosphate ΛΤΡ-γ-Ρ to ' dihydropteridine 1 , as described in the specification of the afore-mentioned cognate application.

It was found that the concentrations required for 50% inhibi- tion of the formation of Pt (IC_ft) in such tests are well 50 correlated and within the margin of error obtained by other re¬ levant tests in this respect, which measure the inhibition of either of the two 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 pAB-7-C , -ATP , Mg and 'dihydropteridine'. The formation of 14 the dihydropteroate-C can. be- quantitatively assayed after separating the unreacted pAB substrate, for instance by chro¬ matography. It has been found that compounds possessing in such tests an IC^^ value of about ΙΟΟμΜ or less, usually below 50μΜ represent compounds exerting a useful potentiating effect, provided their toxicity in the appropriate vertebrates is accept able. Preferably the value is 25μΜ or less, such as in the range between 2 to 12μΜ. Generally a value below '7μΜ is desir- able.' The IC- value ·' given by 2-amino-4-hydroxy-7 , 7- 0 favourable. diethyl-7 , 8-dihydropteridine-6-carboxaldehyde is particular^ 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 used with advantage for the selection of potentiating compounds 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 10, suitably at least 5 and in exceptional circumstances at least about 3. for humans, but possibly as low as 2 for animals.

Although the art is aware of many compounds which are known competitors of para-aminobenzoic 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 of this invention : sulphanilamide , sulphadiazine , sulphamethisazole , sulphamethizole , sulphapyridine , sulphathiazole , sulpha-merazine, sulphamethazine , sulphisoxazole , sulphadoxine , sulphasomidine , sulphachlorpyridazine ; 2- (p_-aminobenzene ) -sulphonamido-3-methoxypyrazine (Keifizina) , a-amino-jg-tolue-nesulphonamide , 5-sulphanilamido-2 , -dimethyl pyrimidine, 4- (Ν' -acetyl sulphanilamido) -5-6-dimethoxy pyrimidine, sulphamethopyrazine , sulphadimethoxide , 2-methyl-4-sulphani- i lamido-6-methoxy-3 (2H) -pyridazinone , 4-sulphanilamido-5-methoxy-6-chloropyrimidine , -sulphanilamido-5-butoxy-6-chloro pyrimidine, chlorosulphanamido-dihydrobenzothiadiazine dioxid 3-sulphani lamido-4 , 5-dimeth l isoxa ole , -sulpha ilamido-5-methoxy-6-dec l xy pyrimidine , sulpb-amonorncthoxin , 4-£- (8-hydroxy-quiniliriy1-4-azo) -phenyl sulphanilamido-5 , 6-dirncthoxy pyrimid ine , sulphadimethoxinc aulphamethoxazole , sulphaquinoxaline , and p_~ (2-methy1-8-hydroxy-qu.i.noliny1- (5 ) -azo) phenyl sulphanilamido-5 , 6-di-methoxy pyrimidine , Examples of a non-sulphonamide type of competitor are p-amino salicylic acid (PAS) and Ρ,ρ'-diaminodiphenylsulphone .

Similarly, although many compound*.; are known which inhibit dihydr'ofolio reductase and act as ant'irnicrobi 1 agents, the compounds disclosed in. the following patents are presented by way of example of .compounds suitable .for vise in this ' invention .

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 accept able salts thereof) are preferred for the purpose of this invention, however; 2 , 4—diamino— 6—ethy1— —_~ehlo ophenyIpyri idine (pyrimethamine) , 2 , -diamino-5- (3 '4 ' , 51 -trimethoxybenzyl) -•pyrimidine ( trime hoprim). , 2 ,.4-dd.ariino -5- ( 3 · 41 -dime thox - benzyl) pyrimidine (Oioveridinc) , 2 , 4-diamino~5- (2 ' - isopropy .1-4 ' -chlorophcnoxy ) pyr:i.]nidir.-2, 2 , 4.~dia]nino-5-methyl-6-soe-butylpyrido (2, 3-d) pyrimidine, 2 , 4-diamino- S-metbyl-G-benzylpyrido (2, 3-_d) py imidine, 2 , -diamino-G- bcn y!l pyrido (2 , 3-d) py imidine., 2 , 4-diamino- 5-6;- imethy1- enecjuinazoline , 2 t 4-diamino- 5 , 6~tetramethyler.equ:i.nazoline , 2 , 4--diamino-5- (2 ' , 4 51 -ti:ime hoxy en zy.1) pyrimidine , 2 , 4~diamino-5~ -ethyl-41 , 5~dimethoxybenzy 1) pyrimidine , 2 , 4 -diamino-LS- (21 ~ me hy - 4 ' , 5 ' -dimethoxybenzy 1 ) pyrimidine.

Hovjevcr, the o t preferred combinations include those combining a compound of formula (I), especially 1 2 those wherein R .is a formyl group and R and R are the same and are both lower alkyl groups, with sulphadiazine , sulphamethoxazole , sulphado ine or sulphaquinoxaline as compe itors, or with trimethoprim, diaveridinc or py ime hamine as inhibitors.

In view of possible synergistic advantages of using certain competitors and inhibitors in combina on against particular diseases, and the potentiating effect of compounds of formula (1) on both of these types of antibacterial com¬ pounds, it has been preferred to formulate triple combi¬ nations, comprising a compound of formula (J.) with one of the above-mentioned preferred competitors, and one of such inhibitors. For example, combinations of sulphadiazine/ tr im tho r im, sulph motho a:olc/tri.mo thoprim , <; Iphado i e/ trimethop im or sulphaquinoxaline/diaveridine have been shown to possess an improved effectiveness when compared with the components alone or with pairs of them.

The compounds (I) together 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 discrete units, such as tablets, capsules, cachets, ampoules or suppositories, each containing a 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 for 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 ingredients: diluents, solutes to render the solution isotonic with the blood, buffers, flavouring, binding, dispersing, surface-active, thickening, lubricating and coating materials, preservatives, bacteriostats , antioxidants, suppository and ointment bases, and any other acceptable excipients.

Formulations containing the potentiator in association with a competitor or an inhibitor may also be presented in the form of a kit, which comprises separately packaged units or dosages of these components with instructions for use in a combined form. The instructions may also specify the manner of administration and indications for which the formula is suitable.

Any of the afore-mentioned compounds may be presented in the form of their pharmaceutically acceptable salts of a mineral or organic acid, for example hydrochloric acid, sulphuric acid, acetic acid, citric acid, tartaric acid, lactic acid, maleic acid or salicylic acid, or especially for the sulphonamide compe ito , of a base , such as sodium hydroxide, potassium hydroxide, t.etra- methyl ammonium hydroxide or ammonia. ' ' the The ratios in which/ therapeutically active com¬ pounds of formula (X) are utilized in the compositions according to he present invention 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 desirable to maintain a certain proportion of components in the blood -serum or tissue fluids, preferably for a proloncjed period. Depending on th various absorption, discharge or decomposition rates of the components, 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 trea ment of a particular human or animal disease must be ad usted 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 composition or pharrnaccut icnl about ormulation may contain from/1 to 30 parts by weight, preferably 5 to 15 parts of the compound of formula (I) , or an equivalent amount of a sa.lt 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 v ry depending upon the i fecting organism but under ordinary circumstances up to abou GO rng/kg each of a. compound of formula (I) and competitor, and up to about 7.5 mg/kg of inhibitor, in combination, can be administered dai-ly in several doses. The corn- position or pharmaceutical formulation can -be administered to human patients in unit dosage forms which con ain 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 pharmaceutical 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 grov.'tli of bacteria. For example, an anti¬ bacterial solution having the following preferred con¬ centration of components may be used: 1-30 mg/ml 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 vi'tro relat.ive.l.y easily or research and practical purposes. Such possibili ies include diagnosis · nd the identification 'of the bacterial flora of. individuals and the consequential selection of clinical treatment schedules.

The Veirious combinations can be incorporated iiv porous discs (such as filter paper discs) or in Agar Nutrient or other media for bacterial grov.'tli 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/ml of a par ci~aminoben¾oic acid competitor, 0.5 to L> yy/ynl of a dihydrofolic reductase inhibitor, and about 10 to 100 tig/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 formulations according to the present vention also form the basis of determining the compositions of selected formulations 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 medic ment. .- In addition to the anti-bacterial activity of the aforementioned competitors and inhibitors in man and in vitro , they have already been used against infections with microorganisms in domestic animals, including poultry, for example against Pasteurella multocida but especially against the protozoal disease coccidiosis. The compounds (I) can be combined with advantage with certain competitors and inhibitors which are active against coccidiosis to provide an effective triple combination, and in some instances the compounds of formula (I) may be active alone. The specification of co-pending cognate Patent Application No. 40050 describes the potentiation effect provided by the compounds of formula (A) and reference is directed thereto for a detailed consideration of coccidiosis and its treatment.

In particular it has been found, however, that combinations of sulphamethazine , sulphadiazine , sulphadimethoxine or especially sulphaquinoxaline , with diaveridine and a compound of formula (I) may provide triple formulations which are effective in lower concentrations that the sulphonamide or pyrirnidine alone and give increased control of the infection.

The compounds of the formulation against coccidiosis can preferably be presented alone or in combination as an additive to be mixed with the bird's food or drink, in the latter case .in the form of their acid addition salts.

Concentrations around 20 to 180 parts per million (p. p.m.) of the potentiator in the diet of the birds, in combination with 10 to 60 parts of diaveridine or sulphaqui oxaline may produce an improved effect. ' However triple combinations of of these components are preferred and, whilst a wide range/con¬ centrations may be. suitable, relative concentrations of 5 to 250 p. p.m. of potentiator, preferably 20 to ISOp.p.m., and about 10 to 100 p. p.m. of each of diaveridine and sulpha¬ quinoxaline, preferably 15 to 60 p. p.m., are employed . with advantage. 2-Amino- -hydroxy-7 , 7-dimetbyl-7 , 8-dihydropteridine- 6- carboxaldehyde has been found to be particularly effective in such compositions, even at concentrations as low as 20 p. p.m.

The synthesis of the compounds of formula (I) is described in the above-mentioned Israel Patent Specification o. 44123 and British Patent Speci ication os. 1,451,043 and 1,453,831. 44124/2 The synthesis of those compounds wherein R is a formyl group involves in essence the selective oxidation of the corresponding 6-hydroxyalkyl derivative, advantageously in the presence of an anti-oxidant to react with any by-products which could decompose the aldehyde once f.ormed. Alternatively the compounds can be px'oduced as by-products in the final reductive cyclisatxon preparation of the hydroxyalkyl derivatives, if the amount of added reducing agent is controlled.

The synthesis of the 6-haloalkyl derivatives involves reacting a corresponding compound substituted in the 6-posi-tion with an appropriate alkyl, hydroxyalkyl or alkenyl group with a halogenating agent and isolating the compound so formed.

According to the present invention, therefore, there ( are provided in various aspects a) 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 an inhibitor, as hereinbefore defined, or both. i b) a composition as specified under (a) , which comprises a compound of formula (I)· wherein R is a formyl group. c) a composition as specified under (a) or (c) , which ' 1 2 comprises a compound of formula (I) wherein R and R are the same and are each lower alkyl groups ; and d) a composition, as hereinbefore defined in any aspect, comprising an effective potentiating amount of a compound of formula (I) in combination with an effective amount of both a competitor and an inhibitor: e) a pharmaceutical formulation containing any one of the above defined compositions in combination with a pharmaceutically acceptable carrier therefor: f) a pharmaceutical formulation as defined under (e) in the liquid form, for use in the inhibition of the production of di-hydrofolic acid by microorganisms, under in vitro or in vivo conditions . g a pharmaceutical formulation, as hereinbefore defined, presented in the form of a kit of separately packaged poterrEi- ' ator, inhibitor or competitor, with instructions to use them together in combination for the purpose of medical or veteri- nary treatment. h) * an in vitro testing disc incorporating a pharmaceutical formulation as hereinbefore defined .{ i) a method of making a composition or a pharmaceutical formulation, as hereinbefore defined, in "Which the effective amounts of the appropriate components are admixed and presented in a combined form: j) a method of treating and preventing microbial infections comprising the administration to the host, other than a human host of any one of the compositions or pharmaceutical formulations hereinbefore defined. k) a method of inhibiting the production of dihydrofolic acid by microorganisms which comprises contacting the microorganism with a composition or pharmaceutical formulation here¬ inbefore defined.

I) a pharmaceutical formulation for the testing or treatment of coccidial systems and infections, comprising an effective potentiating amount of a compound of formula (I), in combination with an effective amount of a competitor or an inhibitor, or both.

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

Example 1 "Screening tests" were performed to establish the effectiveness of the compounds of formula (I) in combination with a competitor and/or an inhibitor to determine the most useful levels of relative concentrations of each component in combatting or suppressing coccidiosis.

Groups of five, seven day old chicks, housed in small, heated cages with wire floors^, situated in. environmentally controlled . isolation rooms were infected orally with 100,000 sporulated oocysts of Eimeria tenella. The appropriate test compound of formula (I) in concentrations of 180,60 or 20 p. p.m. of diet in combination with 20 p. .m. each of diaveridine' and sulphaquinoxaline was incorporated into a small quantity of specially formulated laboratory ration deficient in vitamin K('LD 5 1 chick mash) as a 1/10 pre-mix, which was then mixed into the diet on a horizontal roller drum. Administration of the combination > to the chicks commenced one day prior to infection and continued for eight days , leaving some chicks untreated as controls. As a further control 20 p. p.m. each of diaveridine and sulphaquinoxaline without any potentiator were administered to some chicks.

On the final day of administration the amount of damage, marked by lesions in the caecal walls, was investigated in those chicks that died during the course of the experiment and in those sacrificed on the final day, and compared with the untreated controls as a basis for the determination of thera - ■ peutic activity. In addition the mortality rate and the percentage weight gains in the various groups were assessed.

Even if mortality occurred, effectiveness of the administered drug could be evaluated by reference to the increase in the weight gain and the reduction in the extension of lesions, as indicated by the lesion score.; The results of these screening tests are set forth in Table 1 showing the concentration of the various component compounds in- p. p.m. of the chick's diet, the mortality from coccidiosis, the percentage weight gain, the lesion score and. the activity. The caecal lesion index is calculated using a score system wherein :- 0 = no lesions 1 =: few haemorrhages, no thickening of caecal wall 2 = moderate haemorrhages, some thickening of caecal wall 3 = numerous haemorrhages with caecal cores present 4 = numerous haemorrhages, caeces enlarged with large cores and/or mortality during the experiment.

The caecal lesion index = (The total lesion score of the survivors )+ ( x no. of deat'hg) Original number of birds In the activity column, +++ , ++ , +, + and - relate respectively to very high, high, slight, doubtful and nil activity of the test drug against the infection. The test compounds of formula (I) are indicated by reference letters A-G, as explained in Table 2.

The results in Table 1 indicate that all the test compounds potentiated the activity of a combination of sulphaquinoxaline and diaveridine, in some cases by as much as 3 to 4 fold, giving better control of the infection, a marked reduction in caecal lesions and restorations of the weight of the birds to their pre-infection levels, although mortality was not prevented in every instance. It is interesting to note that whilst generally a poor percentage weight gain was associated with a high lesion score, there appeared to be no simple mathematical correlation between the two sets of figures, suggesting that these two factors may not go completely hand-in-hand.

As expected in the majority of cases a reduction in activity was observed as the concentration of test compound was lowered but its inclusion in combinations even at the 20 p.p.m. showed a marked improvement in the activity in most instances.) The compound 2-amino-4-hydroxy-6-formyl-7 , 7-dimeth.yl-7 , 8-dihydropteridine was most active of those compounds tested A 414 at the 180 p. .m. level. It will be seen that the degree of activity was greater in each case when R 2 and R were the same and both methyl groups.

Example 2 The ability of certain compounds of formula (I) to act as pteridine antagonists was tested by inves igating the in¬ hibitory effect they imposed on the enzymes responsible for the biosynthesis of dihydropteroic acid (DPtA) , namely hydro- xymethyldihydropteridine pyrophosphokinase (HMPPS)' and di- hydropteroate synthetase, hereinafter referred to as 'synthetase'. 1) HMPPS 2-Amino-4-hydroxy-6-hydroxymethy1-7 , 8-dihydropteridine (ATP) Mg2+ adenosine triphosphate^ > 2-amino- 4-hydroxy- 6-pyrophos- phomethy1-7 , 8-dihydropteridine (Pt)+ adenosine monophosphate.

(AMP) 2) Synthetase 2+ Mg..

Pt + p-a i obehzoic acid (pAB) > DPtA + pyrophosphate- An assay for HMPPS was developed in which the transfer of 32 the terminal phosphate of ATP-^-P to Pt could be monitored and correlated with the amount of inhibition of HMPPS by the compound under test. The procedure for this assay is described in detail in Example 1 of the specification of the afore-mentioned cognate application, together with a description of a method for the determination of the activity of a test compound against 'synthetase' by monitoring the formation of dihydropteroate c14.

Those compounds which, as a result of these tests, were found to give 50% inhibition at' a concentration of ΙΟΟμΜ or' less, are those which exert a useful potentiating effect and may therefore be included with advantage in the compositions of the present invention. 2-Amino-4-hydroxy-7 , 7-diethyl-7 8-dihydropteridine- 6-carbox-aldehyde was found to be the preferred potentiator according to these tests with an IC value of 5.5 μΜ whilst its 50 7-7-dimeth l' iJialogue had an IC,-n value of 17y .

Example 3 In this experiment inhibitor zone data were determi'jgd to evaluate the synergistic activity. of 2-amino-4-hydroxy- -6-carboxaldehyde -7 , 7-diethyl-7 , 8-dihydropteridine, on its combination with trimethoprim (TMP) and/or sulphamethoxazole (SMX) against Staphylococcus aureus and Pseudomonas aeruginosa .

The pteridine was included in a soya peptone medium of low thymidine content (Wellcotest Sensitivity Test Agar) con- a tained in,Petri-dish and the other component (s) added to the well resulting from the removal of a small plug from the medium. The surface of the medium was inoculated with the test organism and then incubated.

The amount of zone inhibition is shown in Table 3, wherein the numbers represent the complete zone inhibition (i.e. the number of centimeters from the edge of the well after about 6 x magnification) and the figures in parenthesis include the zones of partial inhibition.

Example 4 Tablet Formulation ' 1 2 Compound (I) (R = CHBrCH^R = R = n - Pr) pure lOOmg Trimethoprim (pure) 25mg Sulfaguanidine (B.P.C.) lOOmg + cornstarch, lactose, gelatin, talcum and magnesium stearate. i Preparation - the above constituents were mixed together using known methods of pharmacy to form a granulation which was then compressed into tablets.

Example 5 Tablet Formulation 1 2 Compound (I) (R = CHOji'.R = R = Et) pure 150mg "Pyrema.thimine" (Pyrimethamine) B.P. 15mg which was then prepared to fqrm a tablet as in Example 1.

Example 6 Tablet Formulation 1 2 Compound (I) (R = CHO R = R = Me) pure 175rng Sulfanilamide B.P.C. ISOmg which was then prepared to form a tablet as in Example 1.

Example 7 Capsule Formulation 1 2 Compound of formula (I) (R = CH2C1; R = R = Me)pure lOOmg Trimethoprim (pure) 20mg Preparation 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.

Example 8 Irrigant Solution 1 2 Compound. (I) (R = ,CHO; -R , R = spirocyclopentyl pure lm9/ml Trimethoprim (pure) 0.2mg/ml Solvent water Example 9 Solution ' 1 2 Compound (I) (R = CH2C1; R =R =Me) pure 1.5mg/ml Diaveridine B.Vet.C 0.5mg/ml Kelfizina l.Omg/ml Solvent ··' water Example 10 Irrigant Solution ·— 1 2 Compound (I) (R = CHO> R = R = Me) pure 2mg/ml o-amino-£-toluenesulphonamide (pure) 2mg/ml Solvent water —1 T A B L E I See Table 2 T A B L E 2 Compound of formula Table 1 R R" R' CHO Me Me J TABLE 3

Claims (3)

1. 44124/2 CLAIMS : 1. A composition for testing or treating microbial systems or infections, comprising an' effective potentiating amount of a compound of formula (I) , wherein R is a formyl group and R and R are the same or 1 2 different and each 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, or R is a chloromethyl 1 2 group and R and R are each methyl groups or R is a 1 2 1 -bromoethyl group and R and R are each n-propyl groups, in combination with an effective amount of a competitor or inhibitor, or both, as hereinbefore defined.

2. A composition for testing or treating microbial systems or infections, comprising an effective potentiating amount of a compound of formula (I) H 44124/2 1 2 wherein R is a chloromethyl group and R and R are each me½&yl 1 2 groups or R is a 1 ' -bromoethy1 group and R and R are each n-propyl groups, in combination with an effective amount of a competitor or inhibitor, or both, as hereinbefore defined.

3. A composition for testing or treating microbial systems or infections, comprising an effective potentiating amount of a compound of formula (I) , wherein R is a formyl group and R and R are the same or — 1 2 different and each is a lower alkyl group or R and R , to¬ gether 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, in combination with an effective amount of a competitor or an inhibitor, or both, as hereinbefore defined. 44124/2 4» A composition as claimed in any one of claims wherein R 1 and R2 are the same and both lower alkyl groups^. 5. A composition as claimed in claim 4, wherein R 1 and R2 are the same and both methyl groups. 6. A composition as claimed in claim , wherein R 1 and R2 are the same and both ethyl groups. 7» A composition as claimed in any one of claims 3, 4 and 5, wherein the compound of formula (I) is 2-amino-4-hydroxy-7 , 7-dimethy1-7 ,8-dihydropteridine-6-carboxaldehyd o 8. A composition as claimed in any one of claims 3, 4 and 6, wherein the compound of formula (I) is 2-amino- -hydroxy-7,7-diethyl-7 , 8-dihydropteridine-6-carboxaldehyde. 9o A composition as claimed in claim 2, wherein the compound of formula (I) is 2-amino-4-hydroxy-6-chloromethyl-7 ,7-dimethyl-7,8-dihydropteridine. 10. A composition as claimed in claim 2, wherein the compound of formula (I) is 2-amino-4-hydroxy-6-l°-bromoethyl-7 ,7-di-n-prop l-7, 8-dihydropteridine. 11. A composition as claimed in any one of claims 2 to 10, comprising about 1 to 30 parts of each of the compound of formula (I) and the competitor, preferably 5 to 15 parts, or equivalent amounts of salts thereof. 12. A composition as claimed in any one of claims 2 to 10, comprising about 1 to 30 parts of the compound of formula (I) , preferably 5 to 15 parts, and 1 part of the inhibitor, or equivalent amounts of salts thereof. 13. A composition as claimed in any one of claims 2 to 12, comprising an effective potentiating amount of the compound of formula (I) in combination with an effective amount of both the competitor and the inhibitor. 44124/2 14. A composition as claimed in claim 13, comprising about 1 to 30 parts of each of the compound of formula (I) ^ and the competitor, preferably 5 to 15 parts, and one part of the inhibitor, or equivalent amounts of salts thereof. 15. A composition as claimed in any one of claims 11 to 14, in unit dosage form, comprising up to 750 mg , , pre¬ ferably about 200 mg,, of the compound of formula (I). 16.. A composition as claimed in any one of claims 11 and 13 to 15 , in unit dosage form, comprising up to 750 mg , preferably about 200 mg , of the competitor. 17. A composition as claimed in any one of claims 12 to 16 , in unit dosage form, comprising up to 25 mg of the in¬ hibitor . 18. A pharmaceutical formulation containing a composition as claimed in any one of claims 2 to 17 , in combination with a pharmaceutically acceptable carrier. 19. A pharmaceutical formulation as claimed in claim 18 in the liquid form, for use in the inhibition of the pro¬ duction of dihydrofolic acid by microorganisms , under in vitro or _in vivo conditions. 20. A pharmaceutical formulation as claimed in either of claims 18 and 19 for external use, comprising 1 to 30 mg/ml of each of the compound of formula (I) and the com¬ petitor in a pharmaceutically acceptable solvent. 44124/2 21. A' pharmaceutical formulation as claimed in either of claims 18 and 19 for external use, comprising 1 to 30 rag/rni? of the compound of formula (I) and 0.03 to 1 mg/ml of the in¬ hibitor, in a pharmaceutically acceptable solvent. 22. A pharmaceutical formulation as claimed in any one of claims 18 to 21 for external use, comprising 1 to 30 mg/ml of each of the compound of formula (I) and the competitor and 0.03 to 1 mg/ml of the inhibitor in a pharmaceutically accept able solvent. 23. An ij vitro testing disc incorporating a pharmaceutical formulation as claimed in either of claims 18 and 1 , com¬ prising about 10 to_100 μ9 πι1 of the compound of formula (I) , 5 to 50 g/ml of the competitor and 0.5 to 5 μg/ml of the inhibitor in combination with a carrier. 24. A testing disc as claimed in claim 23 , wherein the carrier is a medium comprising a mixture of an aqueous in¬ fusion and papain digest of horse muscle. 25. A pharmaceutical formulation as claimed in any one of claims 18 'to 22 presented in the form of a kit of separately packaged compound of formula (I), inhibitor or competitor, with instructions to use them together in combination for the purpose of medical or veterinary treatment. 26. A pharmaceutical formulation for the testing or treat¬ ment of coccidial systems and infections, comprising an effective potentiating amount of a Compound of formula (I) , as defined in any one of claims 2 to 10 in combination with an effective amount of a competitor or inhibitor, or both, as hereinbefore defined. 27. A pharmaceutical formulation as claimed in claim 26, wherein the competitor is sulphaquinoxaline and the inhibitor is diaveridine. 28. A pharmaceutical formulation as claimed in either of claims 26 and 27, wherein the compound of formula (I is 2~amino-4-hydroxy-7 , 7-dimethyl-7 , 8-dihydropteridine-6-carbox- aldehyde . 29. A pharmaceutical formulation as claimed in any one of claims 26 to 28, comprising about 0.002 to 0.018% w/w of the compound of formula_(I) and about 0.001 to 0.006% w/w of sulphaquinoxaline. 30. A pharmaceutical formulation as claimed in any one of claims 26 to 28, comprising about 0.002 to 0.018% w/w of the compound of formula (I) and about 0.001 to 0.006% w/w of diaveridine. 31. A pharmaceutical formulation as claimed in any one of claims 26 to 30, comprising about 0.0005 to 0.025% w/w of the compound of formula (I), preferably 0.002 to 0.018% w/w^ and about 0.001 to 0.01% w/w of each of sulphaquinoxaline and diaveridine, preferably 0.0015 to 0.006% w/w. 32. A method of making a composition or pharmaceutical formulation, as defined in any one of claims 2 to 31, in which the effective amounts of the appropriate components are admixed and presented in a combined form. 33. A method of treating and preventing microbial infections, comprising the administration to the host, other than a human host, of a composition or phramaceutical formulation, as defined in any one of claims 2 to 31. 34. A method as claimed in claim 33, in which up to about 60 mg/kg of each of the compound of formula (I) and the competitor are administered to the host daily in several doses. ■ 35. A method as claimed in claim 33/ in which up to about 60 mg/kg of the compound of formula (I) and up to about 7.5 mg/kg of the inhibitor are administered to the host daily in several doses. 36. A method as claimed in any one of claims 33 to 35, in which up to about 60 mg/kg of each of the compound of formula (I) and the competitor and up to about 7.5 mg/kg of the inhibitor are administered to the host daily in several doses. 37. A method of inhibiting the production of dihydrofolic acid by microorganisms, which comprises contacting the microorganism with a composition or pharmaceutical formulation, as defined in any one of claims 2 to 30.