CA1050981A - Intermediates for pteridine derivatives - Google Patents
Intermediates for pteridine derivativesInfo
- Publication number
- CA1050981A CA1050981A CA273,623A CA273623A CA1050981A CA 1050981 A CA1050981 A CA 1050981A CA 273623 A CA273623 A CA 273623A CA 1050981 A CA1050981 A CA 1050981A
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- hydroxy
- formula
- compound
- amino
- carbon atoms
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Abstract
ABSTRACT OF THE DISCLSOURE
Novel intermediates and their process of preparation are provided of fonmula (II)
Novel intermediates and their process of preparation are provided of fonmula (II)
Description
l~Sl)~8~L ~
. The present invention relates to intenmediates useful in the preparation of derivatives of pteridine, and their chemical synthesisO The specification also describes compo-sitions and pharmaceutical formulations comprising the pteridine derivatives in combinations which are useful. in the treabment of microbial infections.
This application is a divisional application of Canadian patent application SO ~. 177,817 filed July 31, 1973.
It is already established that the compounds 2-amino-4-hydroxy 6-hydroxymethyl-7,7-dimethyl 7,8-dihydro-pteridine and 2-amino-4-hydroxy-6-methyl-7,7-dimethyl-7,8-dihydro-pteridine or their tautomers or pharmaceutically acceptable 3alts thereof, have bacteriostatic activity, being particularly effective against Cl. E~ æ~ and Derm. dermatonomous, as disclosed in the specifications of Canadian patent 959,490 .C. S. Hamish Wood et al, issued December 17, 1974, and Canadian patént 997,760, C. S. Hamish Wood et al, issued September 28, 1976, (Belgian Patent No. 770,577).
It has now been found that the pteridines represented by the following formula (I) or their tautomers or pharma-ceutically acceptable salts thereof, : ~, O
R
~2N H
: ' , . .
:
:, , ~
~ , ~aso~
. wherein R is a lower alkyl group, optionally substituted with a hydroxy group, and Rl and R2 are the same or different and each is a lower alkyl group having together at least 3 carbon atoms or Rl and R2, together with the carbon atom in the pteridine ring structure, form a spirocycloalkyl ring system having 4 to 6 carbon atoms outside the pteri.dine ring structure, are also useful as antagonists of microbial metabolism . As used herein and throughout the specification, the term "lower alkyl group" refers to a straight or brancheci chain alkyl group which, unless otherwise specified, has 1 to 4 carbon atoms, In the above formula, preferred compounds are those wherein R is a hydroxyalkyl group, in particular a hydroxymethyl group. Moreover those compounds wherein Rl and R2, together with the carbon atom in the pteridine ring structure, form a spirocyclohexyl group, or especially those wherein Rl and R ~:
are both alkyl groups,in particular ethyl groups, are further preferred. Thus the compounds 2-~mino-4-hydroxy-6-hydroxymethyl 7,7-diethyl-7,8-dihydropteridine and less preferably 2-amino-4-hydroxy-6-hydroxymethyl-7-spirocyclohexyl-7,8-dihydropteridine have been found to be particularly:useful in.the treatment of microbial infections.
According to the present Lnvention, there are provided intermediates of formula;(II) useful in the preparation of the pteridines of formula ~
,, . ::
. ~ ~
: ~ :
. ~ ~
1~5~g~
According to the invention there is provided novel intermediates of formula (II) O
H~ ~ 2 ~ - R
112 ~ 2 (I~) .
or a tautomeric form thereof, wherein R is a lower alkyl or hydroxy-lower-alkyl group, and _ Rl and R2 are the same or different and each is a lower alkyl group having together at least 3 carbon atoms or Rl and R , together with the carbon atom to which they are attached form a spirocycloalkyl ring system having 4 to 6 carbon atoms, and Z is an oxime group.
According to another aspect of the invention there is provided a method of preparing-a compound of formula (II) a~ defined above comprising reacting a compound of formula (IV) R~
C C R (IV)
. The present invention relates to intenmediates useful in the preparation of derivatives of pteridine, and their chemical synthesisO The specification also describes compo-sitions and pharmaceutical formulations comprising the pteridine derivatives in combinations which are useful. in the treabment of microbial infections.
This application is a divisional application of Canadian patent application SO ~. 177,817 filed July 31, 1973.
It is already established that the compounds 2-amino-4-hydroxy 6-hydroxymethyl-7,7-dimethyl 7,8-dihydro-pteridine and 2-amino-4-hydroxy-6-methyl-7,7-dimethyl-7,8-dihydro-pteridine or their tautomers or pharmaceutically acceptable 3alts thereof, have bacteriostatic activity, being particularly effective against Cl. E~ æ~ and Derm. dermatonomous, as disclosed in the specifications of Canadian patent 959,490 .C. S. Hamish Wood et al, issued December 17, 1974, and Canadian patént 997,760, C. S. Hamish Wood et al, issued September 28, 1976, (Belgian Patent No. 770,577).
It has now been found that the pteridines represented by the following formula (I) or their tautomers or pharma-ceutically acceptable salts thereof, : ~, O
R
~2N H
: ' , . .
:
:, , ~
~ , ~aso~
. wherein R is a lower alkyl group, optionally substituted with a hydroxy group, and Rl and R2 are the same or different and each is a lower alkyl group having together at least 3 carbon atoms or Rl and R2, together with the carbon atom in the pteridine ring structure, form a spirocycloalkyl ring system having 4 to 6 carbon atoms outside the pteri.dine ring structure, are also useful as antagonists of microbial metabolism . As used herein and throughout the specification, the term "lower alkyl group" refers to a straight or brancheci chain alkyl group which, unless otherwise specified, has 1 to 4 carbon atoms, In the above formula, preferred compounds are those wherein R is a hydroxyalkyl group, in particular a hydroxymethyl group. Moreover those compounds wherein Rl and R2, together with the carbon atom in the pteridine ring structure, form a spirocyclohexyl group, or especially those wherein Rl and R ~:
are both alkyl groups,in particular ethyl groups, are further preferred. Thus the compounds 2-~mino-4-hydroxy-6-hydroxymethyl 7,7-diethyl-7,8-dihydropteridine and less preferably 2-amino-4-hydroxy-6-hydroxymethyl-7-spirocyclohexyl-7,8-dihydropteridine have been found to be particularly:useful in.the treatment of microbial infections.
According to the present Lnvention, there are provided intermediates of formula;(II) useful in the preparation of the pteridines of formula ~
,, . ::
. ~ ~
: ~ :
. ~ ~
1~5~g~
According to the invention there is provided novel intermediates of formula (II) O
H~ ~ 2 ~ - R
112 ~ 2 (I~) .
or a tautomeric form thereof, wherein R is a lower alkyl or hydroxy-lower-alkyl group, and _ Rl and R2 are the same or different and each is a lower alkyl group having together at least 3 carbon atoms or Rl and R , together with the carbon atom to which they are attached form a spirocycloalkyl ring system having 4 to 6 carbon atoms, and Z is an oxime group.
According to another aspect of the invention there is provided a method of preparing-a compound of formula (II) a~ defined above comprising reacting a compound of formula (IV) R~
C C R (IV)
2~ 1 or a salt thereof, wherein R, Rl and R2 are as defined above, with a 2-amino-4-halogeno-6-hydroxy-5-nitropyrimidine.
The above pteridine derivatlves (I) and their salts - inhibit one of the enzymes involved in the biosynthesls of di~
hydrofolic acid, namely hydroxynethyldihydropteridine pyro-phosphokinase, which is essential~to the growth of mlcro-organisms, for instance bacteria. They can thus be used in ~ . .
::
, : ~
11D5~9i3~
in in vitro pharmacological investigations in clinical and diagnostic tests establishing, for instance, the properties of bacteriaO When used as bacteriostats they may be present in a concentration of 50 to 500, in particular 110 to 180 mg of base/ml of the solutlon 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 ex~nple 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 de~cribed, .''.
. . ~ .
~ ' . .
, ' - -, . .
, ~:
_ .
:
~sa 9~
Tetrahydrofolate co-factors are ess.ential meta-bolites in all cells for the biosynthesis of purincs, thymidylic acid, serine and several other biologically important compoundsO Most of these co-f,actors are one-carbon adducts of tetrahydrofolic acid~ The ultimate source of these for higher animals and man is food, con-taining 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 Idihydrop~eridine' ~Pt), i.P. 2-amino-4-hydroxy-6-hydroxymethyl-7, ~-dihydropteridine ~HMPt) pyrophosphate ester, from it~s im~ediate precursor HMPt in the presence of the enzyme hydroxymethyldihydro-pteridine pyrophosphokinase tHMPPS). . Pt then condenses with p-aminobenzoic acid (pAB) ln the presence of the enzyme dihydropteroate synthetase to form dihydropteroic acid (DPtA). This intermediate further condenses with a glutamate to form dihydrofolic acid (DFA or 'folate') .
. which is then enzymatLcally. ~educed to provide the essen- ~
tial tetrahydrofolate in, for instance, bacteria and other micro-organisms, ~ .
The provision of the 'fola.e' from the basic bu~lding bloc~s~ .:pt~ridine,~pAB, andiglutamate, and~
' .
~ S ~
:
r ?
` . X113 . :~
'` l~S~9~1 the further conversion of this into t1le tetrahydro-folate is known to be inhibited in two different ways.
For instance sulphonamides displace p~B in the ahove -reaction scheme. Because of their close structural resemblance to pAB, sulphonamides or similar other 'competitors' enter the biosynthesis and prevent the form-ation of DPt~, and of DFA, and are therefore antimeta-bolites for the metabolite pAB. It is also known that compounds which are 'inhibitors' of the enzyme dihydro-folic acid reductase block the synthetic step leading to tetrahydrofolate. A considerable nun~er of pyrimidine derivatives show substan~ial anti-microbial propertieS on the basis of such blockage.
It was established later that such inhibitors may act synergistically with sulphonamides, L . e. there can he 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 20 ~ 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-.
r~
' , ' :
~, '.................. . .
.
,' ` Xl13 L()5~)983L
bolites to Pt could inhibi~ the biosynthesis of DP~A
(and DF~) (cf. ~itchings and Burchall Advanccs in Enzymolo~, 27, 417-468 (1965)) but compounds so far :
tested for the purpose have been disappoinking, being either inactive or too toxic or sometimes both ~cf. the compounds described in British Patent Nos.9~1.506 and 987,916)~
It has been established t~at, ~or 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 ~or the hydroxyl-. .
ation o 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 ~lith 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, a~ demonstxated for instance in chicks and rats~ These compounds on their own, not only inhibit the growth of microorganisns~ albeit to .
~ ~ 7 -- . .
? :.~
~5~8~L
a limited extent with certain bacteria, such as Sta~ylococcus aureus, Streptococcus pyo~enes, Streptococcus _aecalis, Escherichia coli, Salmonella typhi, Proteus vulqaris, Pseudomonas aeruqenosa, Pasteurella multocida among others, but have been found to act with a most remarkable synergistic effect when combined with a competitor of ~ aminobenzoic acid, i.e~ sulphonamides and similar compounds, or with selective inhibitors of dihydrofolic reductase, i.e.
pyrimidines and related compounds, or with a combination of L0 both of these types of anti-microbial agents. This potentiating effect of the compounds of formula (I) is the subject of co-pending Canadian Patent Application No. 148,837, James J.
~urchall et al, filed August 4, 1972.
In that application there is described and claimed a composition for testing or treating microbia:L
sy~tems or infections, comprising an effective potentiating amount of a compound of formula (I) in aombination with an effective amount of a competitor or inhibitor, or both, as Xerei~ aefined~ ~
The microbial infections again~st which these combinations are effective are protozoal or bacteria infections caused by those microorganisms which synthesise àt least a substantial part of their tetrahydrofolate co-factor requirements. More specifically these infecting .
- ~ , :
-, ~ ' , :
tl , ~ . ` ' ' . . : .
... . . ..
Xl13 ~L~50~
microor~anisms are those whi.ch adequately absorb the pharmaceutical combinations disclosed hereln and further are those in whicll these combinations have a synergistic effect in intcrfering with the de novo synthesis of the - 5 required tetr~hydrofolate co-factors. For example, the compositions described have been found to be use ful in the treatment of inections caused by Staphylococcus aureus, Pseudomonas aerugenosa and Pasteurella mu.ltocida.
- 1t has been found specifically thatl when compounds . .
io of formula (I) are combined with an amount of the competitor :
and/or the inhibitor which is not.ordinarily suf~icient to be effective as an antimicrobial agent in its own riyht, 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 ormula (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-~ery marked. Thus by;using an effective potentiating amount of a compound of formula (I~ together : with the competitor andJor the inhibitor, it~is now posslble to reduce si~nificantly the~amount of the competitor ~ and/~r ', .
.. . ~ . . . , .. _ . 0 ~:)5~98~
the inllib.itor requixed to inhibit the growt~? of these bact~ria.
In accordance with the above ther~fore, the term l'an effective amount" used in conjunction with the terms a dihydrofolic reductase 'inhibitor' and a para-amino-benzoic acid 'competitor' means either (a) an amount of the 'inhibitor' or 'competitor' which is e:Efective to a degree as an antimicrobial agent in its own rigllt but which a compound of is potentiated by the use of/formula (I) or ~b) an amount or 'competitor' of the 'inhibitor'/which is ineffective as an antimicrobial agent but which when combined with a compound of formula ~I) provides a composition whlch is an effective anti-microbial agent. An "efective 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 ._ , hiosynt~letic processes by such means could he termed as com-petitive antagonism in all three instances, and there might :
. ~
be potentiation between all three~types of agents. ThetermS'inhibitor'~ 'competitor', and 'potentiation' by a co~pound of formula (I) are arbitrary and should only serve as con-.
, ~ ~ 10 -, , ~ ~
. , : . , . .:, .. . . . .
~ , , :
.
~s~s~
., , venient names for the appropriate type of components in con~ination products described and claimed in the s~ecification of the a1~ore-~.entloned cognate application.
The inhibiting activity a~ainst IDMPPS of a selected S compound of ~ormula (I) can, for instance, be tested by triphosphate monitoring the transfer of the terminal phosphate of adenosine/
ATP-y-P to dihydropteridine. It was found that the concentrations required for SO~ inhibition of the form- -ation of Pt (IC50) in such tests ar~ ~ell coxrelated and ~lithin the margin of error obtained by other xelevant this tests in/respect, which measure the inhibition of either of the two enzymes involved in the ~ormation o~ HMPt and DPtA, Such inhibition may,for instance, be easily and simply carried out by incubating an extract of E. coli w1th pAB-7-C , A~P, Mg and dihydropteridine. The formation of the dihydropteroate-C can be quantitatively assayed a~ter separating the~ unreated pAB substrate, for instance by chromatography. It has been found that compo~nds . possessing in such tests an IC5~ value of about 1~0 -.
:
- :: :. . - . - .
~:, ` , , . . ~
98~
or less, usually below SO~M represent compounds exerting a useful potcntiating effect, provided their toxicity-in the appropriate vextebrates is acceptable~ Preferably the value. is 25~I or less, such as in the range bet~een 2 to 12~ Generally a value below 7~M 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 re~uirement, a thera-peutic indcx incorporates both the activity and toxicity values pertinont to the present ~.isciosur~ and could be used ~lth advantage for the selection of potent1ating compounds ~ -of formula (I). . . ~
. .
:
.: ' .
, . ~'' .. . . ... . . . . .
~113 o , .
The therapeutic index is de~ined as the ratio of the maximum toleratcd dosc to the minimum ef~ective dose and in most cases is prefcrably greater than 10, suitably a~ least 5 and in exceptlonal circumstances at ~east a~out 3 for humans~ but possibly as low as ~ for animals.
klthough the art is a~are of r.lany compounds which are known competitors of para-aminobenzoic acid and are antimicrobials, the sulphux compounds which are disclosed as antimicrobial agents from ~he top of page ~94 to page 1007 of the Merck Index, 8th Edition, 19~8 are presented by way o~ example only.
Of the known compounds which are competitors, the following sulphonamide compounds (or pharmaceutically accept-lS able salts thereof) are preferred for the purpose described.
~ulphanilamid~, sulphadiazine, sulphamethisazole, sulphamethizole, sulphapyridine, sulphathiazole, sulph-amerazine, sulphamethazine, sulphlsoxazole, sulphadoxine, sulphasomidine, sulphachlor~yridazine, 2-t~ aminobenzene)-sulphonamido-3-methoxypyrazine(Kelfizina), ~-amino-~-toiue-nesulphonamide, 5-sulphanilamido-2,4-aimethyl pyrimidine, ::: :
4-[N'-~cetyl sulphanilamido) -5,6-dimethoxy pyrimidine, , ~ 13 - -"
:' : ~ ~, , :
, , , , , , " ,, " , , , , , , , .... ... . ~
. X113 ~5098~L -
The above pteridine derivatlves (I) and their salts - inhibit one of the enzymes involved in the biosynthesls of di~
hydrofolic acid, namely hydroxynethyldihydropteridine pyro-phosphokinase, which is essential~to the growth of mlcro-organisms, for instance bacteria. They can thus be used in ~ . .
::
, : ~
11D5~9i3~
in in vitro pharmacological investigations in clinical and diagnostic tests establishing, for instance, the properties of bacteriaO When used as bacteriostats they may be present in a concentration of 50 to 500, in particular 110 to 180 mg of base/ml of the solutlon 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 ex~nple 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 de~cribed, .''.
. . ~ .
~ ' . .
, ' - -, . .
, ~:
_ .
:
~sa 9~
Tetrahydrofolate co-factors are ess.ential meta-bolites in all cells for the biosynthesis of purincs, thymidylic acid, serine and several other biologically important compoundsO Most of these co-f,actors are one-carbon adducts of tetrahydrofolic acid~ The ultimate source of these for higher animals and man is food, con-taining 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 Idihydrop~eridine' ~Pt), i.P. 2-amino-4-hydroxy-6-hydroxymethyl-7, ~-dihydropteridine ~HMPt) pyrophosphate ester, from it~s im~ediate precursor HMPt in the presence of the enzyme hydroxymethyldihydro-pteridine pyrophosphokinase tHMPPS). . Pt then condenses with p-aminobenzoic acid (pAB) ln the presence of the enzyme dihydropteroate synthetase to form dihydropteroic acid (DPtA). This intermediate further condenses with a glutamate to form dihydrofolic acid (DFA or 'folate') .
. which is then enzymatLcally. ~educed to provide the essen- ~
tial tetrahydrofolate in, for instance, bacteria and other micro-organisms, ~ .
The provision of the 'fola.e' from the basic bu~lding bloc~s~ .:pt~ridine,~pAB, andiglutamate, and~
' .
~ S ~
:
r ?
` . X113 . :~
'` l~S~9~1 the further conversion of this into t1le tetrahydro-folate is known to be inhibited in two different ways.
For instance sulphonamides displace p~B in the ahove -reaction scheme. Because of their close structural resemblance to pAB, sulphonamides or similar other 'competitors' enter the biosynthesis and prevent the form-ation of DPt~, and of DFA, and are therefore antimeta-bolites for the metabolite pAB. It is also known that compounds which are 'inhibitors' of the enzyme dihydro-folic acid reductase block the synthetic step leading to tetrahydrofolate. A considerable nun~er of pyrimidine derivatives show substan~ial anti-microbial propertieS on the basis of such blockage.
It was established later that such inhibitors may act synergistically with sulphonamides, L . e. there can he 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 20 ~ 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-.
r~
' , ' :
~, '.................. . .
.
,' ` Xl13 L()5~)983L
bolites to Pt could inhibi~ the biosynthesis of DP~A
(and DF~) (cf. ~itchings and Burchall Advanccs in Enzymolo~, 27, 417-468 (1965)) but compounds so far :
tested for the purpose have been disappoinking, being either inactive or too toxic or sometimes both ~cf. the compounds described in British Patent Nos.9~1.506 and 987,916)~
It has been established t~at, ~or 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 ~or the hydroxyl-. .
ation o 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 ~lith 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, a~ demonstxated for instance in chicks and rats~ These compounds on their own, not only inhibit the growth of microorganisns~ albeit to .
~ ~ 7 -- . .
? :.~
~5~8~L
a limited extent with certain bacteria, such as Sta~ylococcus aureus, Streptococcus pyo~enes, Streptococcus _aecalis, Escherichia coli, Salmonella typhi, Proteus vulqaris, Pseudomonas aeruqenosa, Pasteurella multocida among others, but have been found to act with a most remarkable synergistic effect when combined with a competitor of ~ aminobenzoic acid, i.e~ sulphonamides and similar compounds, or with selective inhibitors of dihydrofolic reductase, i.e.
pyrimidines and related compounds, or with a combination of L0 both of these types of anti-microbial agents. This potentiating effect of the compounds of formula (I) is the subject of co-pending Canadian Patent Application No. 148,837, James J.
~urchall et al, filed August 4, 1972.
In that application there is described and claimed a composition for testing or treating microbia:L
sy~tems or infections, comprising an effective potentiating amount of a compound of formula (I) in aombination with an effective amount of a competitor or inhibitor, or both, as Xerei~ aefined~ ~
The microbial infections again~st which these combinations are effective are protozoal or bacteria infections caused by those microorganisms which synthesise àt least a substantial part of their tetrahydrofolate co-factor requirements. More specifically these infecting .
- ~ , :
-, ~ ' , :
tl , ~ . ` ' ' . . : .
... . . ..
Xl13 ~L~50~
microor~anisms are those whi.ch adequately absorb the pharmaceutical combinations disclosed hereln and further are those in whicll these combinations have a synergistic effect in intcrfering with the de novo synthesis of the - 5 required tetr~hydrofolate co-factors. For example, the compositions described have been found to be use ful in the treatment of inections caused by Staphylococcus aureus, Pseudomonas aerugenosa and Pasteurella mu.ltocida.
- 1t has been found specifically thatl when compounds . .
io of formula (I) are combined with an amount of the competitor :
and/or the inhibitor which is not.ordinarily suf~icient to be effective as an antimicrobial agent in its own riyht, 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 ormula (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-~ery marked. Thus by;using an effective potentiating amount of a compound of formula (I~ together : with the competitor andJor the inhibitor, it~is now posslble to reduce si~nificantly the~amount of the competitor ~ and/~r ', .
.. . ~ . . . , .. _ . 0 ~:)5~98~
the inllib.itor requixed to inhibit the growt~? of these bact~ria.
In accordance with the above ther~fore, the term l'an effective amount" used in conjunction with the terms a dihydrofolic reductase 'inhibitor' and a para-amino-benzoic acid 'competitor' means either (a) an amount of the 'inhibitor' or 'competitor' which is e:Efective to a degree as an antimicrobial agent in its own rigllt but which a compound of is potentiated by the use of/formula (I) or ~b) an amount or 'competitor' of the 'inhibitor'/which is ineffective as an antimicrobial agent but which when combined with a compound of formula ~I) provides a composition whlch is an effective anti-microbial agent. An "efective 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 ._ , hiosynt~letic processes by such means could he termed as com-petitive antagonism in all three instances, and there might :
. ~
be potentiation between all three~types of agents. ThetermS'inhibitor'~ 'competitor', and 'potentiation' by a co~pound of formula (I) are arbitrary and should only serve as con-.
, ~ ~ 10 -, , ~ ~
. , : . , . .:, .. . . . .
~ , , :
.
~s~s~
., , venient names for the appropriate type of components in con~ination products described and claimed in the s~ecification of the a1~ore-~.entloned cognate application.
The inhibiting activity a~ainst IDMPPS of a selected S compound of ~ormula (I) can, for instance, be tested by triphosphate monitoring the transfer of the terminal phosphate of adenosine/
ATP-y-P to dihydropteridine. It was found that the concentrations required for SO~ inhibition of the form- -ation of Pt (IC50) in such tests ar~ ~ell coxrelated and ~lithin the margin of error obtained by other xelevant this tests in/respect, which measure the inhibition of either of the two enzymes involved in the ~ormation o~ HMPt and DPtA, Such inhibition may,for instance, be easily and simply carried out by incubating an extract of E. coli w1th pAB-7-C , A~P, Mg and dihydropteridine. The formation of the dihydropteroate-C can be quantitatively assayed a~ter separating the~ unreated pAB substrate, for instance by chromatography. It has been found that compo~nds . possessing in such tests an IC5~ value of about 1~0 -.
:
- :: :. . - . - .
~:, ` , , . . ~
98~
or less, usually below SO~M represent compounds exerting a useful potcntiating effect, provided their toxicity-in the appropriate vextebrates is acceptable~ Preferably the value. is 25~I or less, such as in the range bet~een 2 to 12~ Generally a value below 7~M 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 re~uirement, a thera-peutic indcx incorporates both the activity and toxicity values pertinont to the present ~.isciosur~ and could be used ~lth advantage for the selection of potent1ating compounds ~ -of formula (I). . . ~
. .
:
.: ' .
, . ~'' .. . . ... . . . . .
~113 o , .
The therapeutic index is de~ined as the ratio of the maximum toleratcd dosc to the minimum ef~ective dose and in most cases is prefcrably greater than 10, suitably a~ least 5 and in exceptlonal circumstances at ~east a~out 3 for humans~ but possibly as low as ~ for animals.
klthough the art is a~are of r.lany compounds which are known competitors of para-aminobenzoic acid and are antimicrobials, the sulphux compounds which are disclosed as antimicrobial agents from ~he top of page ~94 to page 1007 of the Merck Index, 8th Edition, 19~8 are presented by way o~ example only.
Of the known compounds which are competitors, the following sulphonamide compounds (or pharmaceutically accept-lS able salts thereof) are preferred for the purpose described.
~ulphanilamid~, sulphadiazine, sulphamethisazole, sulphamethizole, sulphapyridine, sulphathiazole, sulph-amerazine, sulphamethazine, sulphlsoxazole, sulphadoxine, sulphasomidine, sulphachlor~yridazine, 2-t~ aminobenzene)-sulphonamido-3-methoxypyrazine(Kelfizina), ~-amino-~-toiue-nesulphonamide, 5-sulphanilamido-2,4-aimethyl pyrimidine, ::: :
4-[N'-~cetyl sulphanilamido) -5,6-dimethoxy pyrimidine, , ~ 13 - -"
:' : ~ ~, , :
, , , , , , " ,, " , , , , , , , .... ... . ~
. X113 ~5098~L -
3 sulphanilamido-~,5-dimethyl iso~a~ole,
4-sulphanilamido-5-methoxy-6-decyloxy pyrimidine, sulph-amonometho~ine, 4-~-(8-hydroxy-quinilinyl.-4-azo)-phenyl . sulphanilami~o-S, 6-dimcthoxy pyrimidine, sulphadimethoxine, sulphamethoxazole, sulphaquinoxaline, and ~-(2 methyl-8-hydroxy-quinolinyl-15)-azo)phenyl sulphanilamido-5,6-di-methoxy pyrimidine Examples of a non-sulphonamide type of competitor are p-amino saiicylic acid (PAS~ and diaminodiphenylsulphone.
Similarly, although many compounds are known which inhibit dihydroolic 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;
2,937,28~; 3,322,765; 2,909,522: 2,624,732; 2,579,259; ~ ;
2,945,~59; 2,576,939, 2,926,166; 2,697,7100, 2,749,345;.
and 2,749,344.
. The following inhibitors tor~pharmaceutlcally accept-2Q able salts thereof) are preferred for the combinations : ~-.
descrlb~d., however~
~ 2~4-diamino-6-ethyl-5-~-chlorophenylpyrimidine (pyrimethamine), 2,4-diamino-5-(3'4',5'-trimethoxybenzyl ? -pyrimidine (trimethoprim~, 2~.4-diamino-5-(3'4'-dimethoxy-:
- 14 ~
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Xl13
Similarly, although many compounds are known which inhibit dihydroolic 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;
2,937,28~; 3,322,765; 2,909,522: 2,624,732; 2,579,259; ~ ;
2,945,~59; 2,576,939, 2,926,166; 2,697,7100, 2,749,345;.
and 2,749,344.
. The following inhibitors tor~pharmaceutlcally accept-2Q able salts thereof) are preferred for the combinations : ~-.
descrlb~d., however~
~ 2~4-diamino-6-ethyl-5-~-chlorophenylpyrimidine (pyrimethamine), 2,4-diamino-5-(3'4',5'-trimethoxybenzyl ? -pyrimidine (trimethoprim~, 2~.4-diamino-5-(3'4'-dimethoxy-:
- 14 ~
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Xl13
5~9~
ben~yl) pyrimidinc ~diaveridirle), 2,4 diamino-5~
isopropyl-~'-chlorophenoxy) pyrimidine, 2,4-diamino-5-methyl-G-scc-butylpyrido (2,3-d) pyrimidine, 2,4-diamino-5-methyl-6-benzylpyrido(2,3-d) pyrimidine, 2,4 diamino-6-benzylpyrido(2,3-d) pyrimidine, 2,4-diamino-5-G-trimethyl-enequinazoline, .
2,4-diamino-5,6-tetramethylenequinazoline, 2,9-diamino-5-t2',4'5'-trimethoxybenzyl) pyrimidine, 2,4-diamino-S-(2'-e-thyl-4',5-dimethoxybenzyl) pyrimidine, 2,4-diamino-5-(2'-meth~1-4',5'-dimethoxybcnzyl) pyrimidine.
However, the most preferred combinations include those cornbining a compound of formula (I), espec.iall~
that wherein R is a hydroxymethyl group and R and~R are both ethyl groups, with~sulphadiazine~ sulphamethoxazole, sulphadoxine or sulphaquinoxaline as competitors, or with trimethoprim, diaveridine or pyrimethamlne as inhibitors.
In view of possible Synergistic ad~antagcs of using certain competitors and inhibitors in combination against particular diseases, and the potentiating effect of compounds.of formula (I) on both of these types of~antlbacterial com-~
2~ pounds, it has been preferred to fornwlate 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 o~ sulphadiazine/
trimethoprim, suIphamethoxazole/trimethoprim, sulphadoxine/
25 ~ trimethoprim or sulphaquin ~ 1 5 I -.: :
.
~0~0981 oxalin~/diaveridine, each together with a compound of formula (I)J give improved effectiveness when com ~ared with tl~e componen-ts 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 presentcd in association with a carrier i.n ph~rmaceutical ~formulations suitable for parenteral,. topical, rectal or oral administration. The formulations for oral or ~ rectal administration ar~ advantageously presented in dis-crete units, such as tablets, capsules, cachets, ampoules or suppositories, each containlng 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 li~uid, or as an ointment or paste for topical administration. For parenteral use, the formulations in-: . . .
corporating an aqueous or non-aqueous liquid carrier must be sterile and be presented in sealed oontainers. The formulations may be_made by any of the kno~n methods and .. may include one or more of the following accessory ingre-. dients~
~ . diluents, solutes to render the~:solution iqotonic ~:
: - with the blood, buffers, flavouring, b1ndiny, dispersing,:
surface-active, thickening, lubricating and coating mate- :
rials, preservatives, bacteriostats,. antioxidants, ::
, ~ 6 - ~ ~
.
. . . . ..
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.
suppository and ointment bases, and any other acceptable excipients.
Formulations containing the compound of formula (I) in association with a competitor or an inhibitor may also be presented in the form of ~ kit, which comprises separately packaged units or dosages of these components with instruc-tions for use in a combined form. The instructions may also specify the manner of administration and indications for which the formula is sui.table. :.
m e compounds of formula (I), either for use alone or in association with a competitor and/or inhibitor, and al~o the competitors and inh.ibitors, may be presented in the form of their pharmaceutically a¢ceptable salts of a mineral or organic acid, for example hydrochloric acid, hydrobromic . :
.
' ' ~ .~ : ~ ' , ; ~
. ~ ' ~ . .' ' -.. 17 ~ ~ ~
. X113 acid, sulphuric acid, acetic acid, citric acid, tartaric acid, lactic acid, ma)eic acicl or salicylic acid, or, especially for the sulphonamidc competitor, of a base, such as sodium hydroxide~ potassium hydroY.ide, tetra-methyl ammonium hydroxide or ammonia.
the The ratios in wllich/therapeutically active com-pounds of formula (I) are utili~ed in the compositionC
descriked in this specification can be varied between wide limits. Depending on ~he nature and circumstance of use, the compositions may contain the compound of formula (I) with the competitor and/or the~inhibitor in a~pro-priate propor~ions and dosages For instance, in cases of uses in ivo it is oten desirable to maintain a certain proportion of componcnts in the blood serum or tissue fluids, prefexably for a prolonged period. Depending on the various absorption, discharge or decomposition rates of the components, the initial quantit1es~and proport1ons 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 paxticular human or animal disease must be adjusted according to the paxticular re~uirements o the reclpients of -the d1sease, the known activities of the competitor or inhibitor com-ponent againsL the causative organism, the half life and , ~ 18 - ~ ~
.
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~ X113 . ,, ~5~)9~
the toxicity of the components ln vi.vo, and othe'r practical requirements~
.
For example the composition or pharmaceutical about formulation may contain from/]. to'30 parts by ~le.ight, preferably 5 to 15 parts~ of the compound of formula (I), or an equivalen~ amount of a salt thereof, and 1 to 3Q
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 e~uivalent amount of a salt thereof.
10 , ' 'Dosage will vary depending upon the infecting `.
organism but under ordinary ciraumstances up to about , 60 mg/kg eacll of a compound oE formula (I) and comp~titor, and up to about 7.5 mg/kg of inhibitor, in combination, :
can be administered daily in several doses. The com-~
' ' position or pharmaceutical ~ormulation can be administered : .
to human pa~ients 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 pharmaceutica,l formulation comprising the com~
pound of formula (I) in combination with the competitor and/ -:
.: . : ~ , ;
~ ~ , ~113 ... . .
~05~9~ -or the inhibitor is also usable in solution for irrigating wounds, for cxample aftcr surgcry, so as to prevent the growth of bacteriaO For example, an anti-bacterial solution havin~ the following preferr~d con-centration of components may be used:
1-30 mg~ml of the compound of forr~ula (I), 1-30 mg/ml of the competitor and/or 0.03-1 mg/ml of the in-hibitor, injpharmaceutlcally acceptable so1vent, suitable for external use.
Th~ potentiating effect of compounds of formula (I) can be demonstrated and utilized in vitro relatively easily fvr research and practical purposes. Such possibil1ties include diagnosis and the identi~ication of the bactexial - , flora of individuals and the consequential selection of clinical treatment schedules.
The various combinations can be incorporated in porous discs Isuch as filter paper discsj or in Agar~Nutrient or other media for bacterial growth for dètermining sus-ceptibility. Those articles incorporating the compound of .
~ormula (I) with a competitor and/or an inhibitor compound :: . . .
may be distributed or sold to doctors, hospitals and clinlcs~
for the above purposes. A typical testing disc may be im-pregnated with a solution containing 5 to 5~ ~g/ml of a 2~ ~
.:
Xl~3 ,-~
~50g~3~
! para-aminobenzoic acid competitor, 0.5 to 5 ~g/ml of a dihydrofolic reductase inhibltor, and about lO to 100 ~g~ml of a compound of formula (I) in a medium csm-prisin~ a mixture of arl 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 accordiny to their , sensitivity and to their particular resistance for instance to a competitor when used alone, and such investigations involving a variety oE formulations as described herein also form the basis of determining the compositions of selected formulations for general treatment purposes.
The toxicity of compounds of formula (I) is generally con-lS siderably lower than that of the competitors or inhibitors commonly used, which may enable the clinician to maintain or increase the effec~iveness of the antibacterial activity of the formulation with a concurrent increase of the thera-peutic ratio or decrease in the toxic or side~effects of the medicament.
- ~ ' ~ - 21 ~ ~
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!. ` - .
In addition to the above,compo-lnds of formula (I~
have ~,een found to potentiate the activity of the afore-.
mentioned competitors an~/or inhibitors against infections with microorganisms in domestic an.imals~ including poultry, for example against Pasteurella multocida but especially _ against tlle protozoal disease coccidlosis. Such triple formulations comprising 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 posse~s an enhanced activity, being effective asainst all relevant Eimeria species- ca~sing this disease in poultry.
.
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S~981 - The compounds of formula (I) may be prepared by the reductive cyclisation of a compound of formula (II), 1; ~ ~ 2 _A ~ ~ 1 L12N / ~ 2 2 N ~2 (II) tI) wherein R, Rl and R2 are as defined above and Z is a ketonic oxygen atom or a protecting group therefor, such a~ a semicarbazone group or an oxime group ~compounds o~ this invention), prepared according to the procedures disclosed by Pfleiderer and Zondler (Chem. Ber. 99, 3008 (1966)) and the . ~
~pecificatio~s of Canadian Patent 959,490, C. S. Hamish Wood et al issued December 17, 1974, and o~ Canadian Patent 997,760, ~. S. Hamish Wood et al, issued Septèmber 28, 1976 (Belgian Patent No. 770,577) re~pectively.
The method described in the aforementioned Canadian Patent 997,760, however, is particularly preferred.
In this method, a compound RlR C = CHR ~VI~
wherein R, Rl and R2 are as defined above, undergoes an~
addition reaction with a n1trosy1 ha11de prepared in situ, ;-and the resulting~nitrosohalide (Vj~^~is converted to the~
oxime (IV) by reaction with ammonia solutionO Reacting the oxime ~IV) with a 2-amino-4-halogeno-6-hydroxy-5-nitro-pyrLmidine (III)~provides~the pyrimid1ne ketox1me (II) which is then reductively cyclised~
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`.
to give the pteridine (I) as shown in the following sequence.
Rl \ R~ NO Rl ~OH
C=CH. R NOHal C-CH. R NH3/ ~ C - C - R
R2 ~ R2 ~al MeOH RCH3~ Hal-(VI) (V) (IV3 .
HN ~ Z ~ ~ Rl 2 2 (III) (II) '.
~, - : :
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. Thc compound of formula (VI) where~n R is a hydroxy-alkyl group may in turn be prep~red from the ketone l 2 R R C=O (IX) by reacting this with a trialkylphosphono-ester (VIII) and reducing the ester (VII) so formed to ~ive .the alcohol (VI)~
/ 2~ .-H2.Co2Alk C-l~ C=CH.CO2Alk (IX) (VIII) (VII) .' ' ' ' I .
: LiAl~4 . ", ' ~ ~ .
, .
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: C=cH:cH2oH
2 "
R
(VI) If a pteridine having R and R as different substituents i5 required, then a racemlC mixture of two stereoisomers of the n1trosohalide (V~ will be obtain~d in view of the:asymmetric carbon atom present. Separation ~ , o the two isomers by conventional~techni~ues kno~Jn in the .
art a~:this stage may be advantageous. ~ ;
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The followin~ Examples illustrate the invention but are in no way intended ~o limit the scope of the invention.
. - Temperature are in deqrees CelsiusO
Exam~e A: reparation of 7 amino-~-hyc~roxy-6-hydro~y-methyl-7,7-diethyl-7,8-dihydropteridine.
(I,R-CH2 OH R =R =Ek) Example 1 .
Ethyl 3-ethy].pent~3---c=te lv~l (R =R =Et) Sodium hydri.de ~6 g) was'placed in a flask with sodium dried benzene ~100 ml) and the flask was flushed with,oxygen-free d`ry nitrogenu: To this solution~was ad-. ded a slight excess of triethylphosphonoacetate (VIII) ~.
(Alk=Et) (61~7 g) over a period of 1.5 h and the temper-ature was maintained at ~15 during.the addition. The mixture was stirred at this temperature for an additional 1 h and then treated dropwise with pentan-3-olle (IX), ~ , , ~R =R =Et) (21.5 g). After addition of the ketone was ,.
.~complete the reaction mixture~was stirred at room temper-~ :
ature until the solid sodlum'~dlethyl phosphate had preci- ,~
:pitated (approx. 10 h). The mother liquor was decanted rom the sol1d,,which was washed..with~benzene (4 x 25 ml).
The benzene extracts were combined and evaporated in vacuo : ~ :
-, , . , X113 ~5~8~
! to give a pale yellow oil (29 g) which was distilled in vacuo to give ethyl 3-ethylpent-2-enoate tVII) (21.8 g, yie].d 56~) as a colourless oil~ b~p. 52-54/~
mm. HgO
Example 2(a) 3-~tllylpent-2-en-1-ol~(VI)tR -R -Et) Ethyl 3-ethylpent-2-enoate (VII) (42.3 g) in dry ether (409 ml) was treated dropwise with a 70~ solutlon ~in benzene) of a slight excess of sodium dihydro bis ethoxymethoxy aluminate (S.D.A.) (86.1 g~, the temperature being maintained at 0 until the addition of the reducin~
agen~ was conlplete. The reaction mixture was then stirred at room temperature for 6 h and the excess S.~.A. was des-troyed by the careful addition of water. ThP solid sodium aluminate which precipitated was flltered off and the filt-rate extract~d with ethyl acetate (4x50 ml)- The combined extracts were washed with brine, dried over sodium sulphate and the solvent removed. The resulting pale yel}ow oil (23 g) was distilled to give 3-ethylpent-2-en-1-ol (VI) . (18.5 g, yield 60~) as a colourless viscous oil b.p.
60/4 mm. ~g.
- Example 2(h) 3-Eth ~ ent-2-en-1-ol (VI) (Rl_R =Et) :
A slurry of lithium aluminium hydride (L.A.~,~
;
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.
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.
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(80 2 g) in dry ether was addecl drop~Jise to a solution of el:hyl 3-ethylp~nl:-2-enoate (VII~ (33. 7 y) i.n dry cther ~100 ml~ at 0 . After the addition of the LoA~ was com-plete the mixture was stirred at room tempera ture for 2 h .
The excess L.l~c H. was destroyed at 0 by adding a satu-rated solution of sodium sulphate. The solution was filtered and the filtrate extracted with ethyl acetate and worked up as described in Example 2 ta) to give the alcohol , ~VI) (19 g, yield 77~).
Example 3 ....... . 1 3-Chloro-3-ethyl~2-nitroso-pentan-1-o.l. ~V) (R=C~12OH~ R =R =Et) Concentrated hydrocl loric acid (23.ml) was adfled dropwise over 1. S h to a -mixture of 3-ethylpent-3 - . . .
.. .. .. : .
en-l~ol (VI) 123 g~ and amyl nitrite (22. 4 g) in glacial acetic acid (46 ml) at O (ice-salt bath). After the addition :., ' : ' . , ,:
of the acid was com~lete the mixture was stirred at this temperature for 30 min, then cooled in an acetc>ne-carbon dloxide bath for lS min when a whlte paste formed. The solid was f1ltercd oef ~ washed with water and cold methanol and recrystallised from benzene t o give the nitroschlori.de .t~ ~13 g, yield 36~6) as colourless crystals, m.p. 110 . ~ :
. , ~
.
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:
.
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-~~ X113 :. , ~05C~983L
Example 4 3-Amino-3-e~hyl-]-hydroxy-pentan-2-one oxime hydro-cnlori~ tTV) (R=CH QH; R =~ =Et) 3-Chloro-3-ethyl-2-nitroso-pentan-1-ol ~V) (10 g) was placed in a three-necked round-bottomed flas~ and treated with a saturated solution of ammonia in methanol. The flask was stoppered, each stopper being seeured with copper wire, and the mixture was stirred at room temperature for 2 days. A clear yellow solution was obtained. The solvent 1~ was removed in ~acuo at room temperature and the yellow oil o~tained was triturated with hot benzene and the benzene decan-ted. The residue was dissolved in ethanol and the insoluble ammonium chloride present was filtered off.
~ . .
The ethanol was removed in vacuo at~room temperature and , the residual yellow oil was treated with hot acetone to give a white solid which ~as filtered off, washed with acetone and recrystallised from butan-2-ol to give the ketoxime hydrochloride ~IV) (S g; yield 46~) as colourless ~eedles, M.Pt. 182~184 .
Example 5 - - 2-Amino-4-hvdroxY-651,1-d ~ ]-3-h~droxy-2-hydroxyimino-propylamino)-5-nitropyrimidlne tII) (R=cH~oNi~R =R =Ft)~
A suspension of 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine ~III);(Hal=Cl) (2.6 g) in dry ethanol ~S0 m1~ was tr~ated 29 _ : . ~
.
. . -. . . ,, ~ .~ .
.. ... ..... , , ~ . .... :, .
~ ~ X113 ~5~
with 3-amino-3-e~}lyl-1-hydroxy-~entan-2-one oxime hydro-chloride (IV) ~2.66 g~ and dry triethylamine (2,89 g) and the mixturc was refluxed for 8 h. The solution was filtered and the filtrate evaporated to dryncss in S vacuo at room temperature The yellow oil obtained ~as treated with cold water and the yellow solid which pre-cipitated was filtered off and washed with ~ater. Re-crystallisation ~rom water in the presence of charcoal gave the nitrop~rimidine oxime ~II) (1.3 g; yield 30~) as a fine white solid m.p. ~50 (decomp.).
~: , .
2-Amino-4-}l~ roxy-6-hydroxymethyl-7,7-diethyl-7,.8-di~dropteridine (I) (R-CH OH; R =R =Et) . . . . . 2 50dium dithionite was added portionwise to a warm soiution of 2-amino-4-hydroxy-6-(1,1-diethyl-3-hydroxy-~hydroxyimi~opropylamino)-5-nitropyrimidine (II) (450 mg~
in 0. lM sodium hydroxide until the colour changed from red to very pale yellow. A solid product was not obtained either on cooling or on adjusting the pll. In order to - separate the product ~rom inorganic material the solùtion was evaporated and the product extracted with ethanol and the inorganic material was::~iltered off . ThLs extraction ~ :
~ . .
was repeated and lhe combined extracts ~ere~evaporated to dryness in vacus. The residue~was dissolved in the ~ _ 30 ~
. , :
.
~: . . : -~ ~ X113 ; ~ , .
~63 5~9~3~
minimum ~uantity of water and placed on a column o~
Amberlite* (C.G.50) ion e~change resin (2.5 x 28 cm).
Elution with water gave two main fluorescen~ bands.
Evaporation of the solution containing the first band gave the 6-carboxaldehyde derlvati~e of the title compound (10 mg, yield 3%~ as a bright orange powder, whilst the second band gave the 7,8-dihydropteridine ~I) (160 mg, yield 44.5~) as a bright yellow powder, m.p. ~300 decomp.
Exam~le B: Preparatjon of 2-Amino-~-hydroxy-6-hydrox~-methyl-7-s~iroc~clohexyl-7,8-dihydropteridine(I) (R=CH2O~I; R R =spirocyclohexyl).
Example 1 ~
~ Eth~l_cyclohexylidenc acetate (VII~) (R R =spirocyclohexyl ?
Sodium dried benzene (200 ml~ was added to a flask containing sodium hydride (16 g) and the flask was flushed with oxygen-free dry nitrogen. To this mixture was aclded, over 1 h, a sligllt excess of triethylphosphono-acetate (VIII~ (Alk=Et) (16~.3 y) keeping the temperature at O. The/mixture was stirred for an-additional hour at O and then treated with cyclohexanone (IX) ~R R =spirocyclohexyl) (65.4 g) at~ the same~`temperature.
* trade mark - ~ - 3i -- ~ : : , . .
^ ~113 . ~ . ,' :
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After the addition of the cyclohexanone was complete t~0 min) the mixture was s~irred at room t~mperature for 3 h; stirring became di:Eficult after this time due to a gummy precipitate of sodium diethyl phosphatc.
The mixture was then heated at 60-65 for 15 ~in during which time it was stirred without difficulty. The mixture was cooled to 15 and the benzene solution was decanted and -the solid washed with benzene. The combined mother liquor and washings were evaporated to give a pale yellow oil which on distillation gave ethyl cyclohexylidene acetate (VII) (62 g; yield 55.4~) as a colourless oil,~
b.p; 86-88/2 mm~ ~g ,:
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ple 2(a) 2-~xclohexyli~ene ethanol (VI~(R R =spirocyclohexyl) A 70~ solution tin ~ell2ene) of sodium dihydrol~isethoxy-methoxy a].umlnate (lOOg) was added port:ionwise 'co ethyl cyclohexylidene acetate (VII) (58.8 g) in dry ether ~ (300 ml) at O . The reaction mixture was stirred for
ben~yl) pyrimidinc ~diaveridirle), 2,4 diamino-5~
isopropyl-~'-chlorophenoxy) pyrimidine, 2,4-diamino-5-methyl-G-scc-butylpyrido (2,3-d) pyrimidine, 2,4-diamino-5-methyl-6-benzylpyrido(2,3-d) pyrimidine, 2,4 diamino-6-benzylpyrido(2,3-d) pyrimidine, 2,4-diamino-5-G-trimethyl-enequinazoline, .
2,4-diamino-5,6-tetramethylenequinazoline, 2,9-diamino-5-t2',4'5'-trimethoxybenzyl) pyrimidine, 2,4-diamino-S-(2'-e-thyl-4',5-dimethoxybenzyl) pyrimidine, 2,4-diamino-5-(2'-meth~1-4',5'-dimethoxybcnzyl) pyrimidine.
However, the most preferred combinations include those cornbining a compound of formula (I), espec.iall~
that wherein R is a hydroxymethyl group and R and~R are both ethyl groups, with~sulphadiazine~ sulphamethoxazole, sulphadoxine or sulphaquinoxaline as competitors, or with trimethoprim, diaveridine or pyrimethamlne as inhibitors.
In view of possible Synergistic ad~antagcs of using certain competitors and inhibitors in combination against particular diseases, and the potentiating effect of compounds.of formula (I) on both of these types of~antlbacterial com-~
2~ pounds, it has been preferred to fornwlate 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 o~ sulphadiazine/
trimethoprim, suIphamethoxazole/trimethoprim, sulphadoxine/
25 ~ trimethoprim or sulphaquin ~ 1 5 I -.: :
.
~0~0981 oxalin~/diaveridine, each together with a compound of formula (I)J give improved effectiveness when com ~ared with tl~e componen-ts 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 presentcd in association with a carrier i.n ph~rmaceutical ~formulations suitable for parenteral,. topical, rectal or oral administration. The formulations for oral or ~ rectal administration ar~ advantageously presented in dis-crete units, such as tablets, capsules, cachets, ampoules or suppositories, each containlng 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 li~uid, or as an ointment or paste for topical administration. For parenteral use, the formulations in-: . . .
corporating an aqueous or non-aqueous liquid carrier must be sterile and be presented in sealed oontainers. The formulations may be_made by any of the kno~n methods and .. may include one or more of the following accessory ingre-. dients~
~ . diluents, solutes to render the~:solution iqotonic ~:
: - with the blood, buffers, flavouring, b1ndiny, dispersing,:
surface-active, thickening, lubricating and coating mate- :
rials, preservatives, bacteriostats,. antioxidants, ::
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.
. . . . ..
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.
suppository and ointment bases, and any other acceptable excipients.
Formulations containing the compound of formula (I) in association with a competitor or an inhibitor may also be presented in the form of ~ kit, which comprises separately packaged units or dosages of these components with instruc-tions for use in a combined form. The instructions may also specify the manner of administration and indications for which the formula is sui.table. :.
m e compounds of formula (I), either for use alone or in association with a competitor and/or inhibitor, and al~o the competitors and inh.ibitors, may be presented in the form of their pharmaceutically a¢ceptable salts of a mineral or organic acid, for example hydrochloric acid, hydrobromic . :
.
' ' ~ .~ : ~ ' , ; ~
. ~ ' ~ . .' ' -.. 17 ~ ~ ~
. X113 acid, sulphuric acid, acetic acid, citric acid, tartaric acid, lactic acid, ma)eic acicl or salicylic acid, or, especially for the sulphonamidc competitor, of a base, such as sodium hydroxide~ potassium hydroY.ide, tetra-methyl ammonium hydroxide or ammonia.
the The ratios in wllich/therapeutically active com-pounds of formula (I) are utili~ed in the compositionC
descriked in this specification can be varied between wide limits. Depending on ~he nature and circumstance of use, the compositions may contain the compound of formula (I) with the competitor and/or the~inhibitor in a~pro-priate propor~ions and dosages For instance, in cases of uses in ivo it is oten desirable to maintain a certain proportion of componcnts in the blood serum or tissue fluids, prefexably for a prolonged period. Depending on the various absorption, discharge or decomposition rates of the components, the initial quantit1es~and proport1ons 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 paxticular human or animal disease must be adjusted according to the paxticular re~uirements o the reclpients of -the d1sease, the known activities of the competitor or inhibitor com-ponent againsL the causative organism, the half life and , ~ 18 - ~ ~
.
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the toxicity of the components ln vi.vo, and othe'r practical requirements~
.
For example the composition or pharmaceutical about formulation may contain from/]. to'30 parts by ~le.ight, preferably 5 to 15 parts~ of the compound of formula (I), or an equivalen~ amount of a salt thereof, and 1 to 3Q
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 e~uivalent amount of a salt thereof.
10 , ' 'Dosage will vary depending upon the infecting `.
organism but under ordinary ciraumstances up to about , 60 mg/kg eacll of a compound oE formula (I) and comp~titor, and up to about 7.5 mg/kg of inhibitor, in combination, :
can be administered daily in several doses. The com-~
' ' position or pharmaceutical ~ormulation can be administered : .
to human pa~ients 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 pharmaceutica,l formulation comprising the com~
pound of formula (I) in combination with the competitor and/ -:
.: . : ~ , ;
~ ~ , ~113 ... . .
~05~9~ -or the inhibitor is also usable in solution for irrigating wounds, for cxample aftcr surgcry, so as to prevent the growth of bacteriaO For example, an anti-bacterial solution havin~ the following preferr~d con-centration of components may be used:
1-30 mg~ml of the compound of forr~ula (I), 1-30 mg/ml of the competitor and/or 0.03-1 mg/ml of the in-hibitor, injpharmaceutlcally acceptable so1vent, suitable for external use.
Th~ potentiating effect of compounds of formula (I) can be demonstrated and utilized in vitro relatively easily fvr research and practical purposes. Such possibil1ties include diagnosis and the identi~ication of the bactexial - , flora of individuals and the consequential selection of clinical treatment schedules.
The various combinations can be incorporated in porous discs Isuch as filter paper discsj or in Agar~Nutrient or other media for bacterial growth for dètermining sus-ceptibility. Those articles incorporating the compound of .
~ormula (I) with a competitor and/or an inhibitor compound :: . . .
may be distributed or sold to doctors, hospitals and clinlcs~
for the above purposes. A typical testing disc may be im-pregnated with a solution containing 5 to 5~ ~g/ml of a 2~ ~
.:
Xl~3 ,-~
~50g~3~
! para-aminobenzoic acid competitor, 0.5 to 5 ~g/ml of a dihydrofolic reductase inhibltor, and about lO to 100 ~g~ml of a compound of formula (I) in a medium csm-prisin~ a mixture of arl 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 accordiny to their , sensitivity and to their particular resistance for instance to a competitor when used alone, and such investigations involving a variety oE formulations as described herein also form the basis of determining the compositions of selected formulations for general treatment purposes.
The toxicity of compounds of formula (I) is generally con-lS siderably lower than that of the competitors or inhibitors commonly used, which may enable the clinician to maintain or increase the effec~iveness of the antibacterial activity of the formulation with a concurrent increase of the thera-peutic ratio or decrease in the toxic or side~effects of the medicament.
- ~ ' ~ - 21 ~ ~
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In addition to the above,compo-lnds of formula (I~
have ~,een found to potentiate the activity of the afore-.
mentioned competitors an~/or inhibitors against infections with microorganisms in domestic an.imals~ including poultry, for example against Pasteurella multocida but especially _ against tlle protozoal disease coccidlosis. Such triple formulations comprising 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 posse~s an enhanced activity, being effective asainst all relevant Eimeria species- ca~sing this disease in poultry.
.
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S~981 - The compounds of formula (I) may be prepared by the reductive cyclisation of a compound of formula (II), 1; ~ ~ 2 _A ~ ~ 1 L12N / ~ 2 2 N ~2 (II) tI) wherein R, Rl and R2 are as defined above and Z is a ketonic oxygen atom or a protecting group therefor, such a~ a semicarbazone group or an oxime group ~compounds o~ this invention), prepared according to the procedures disclosed by Pfleiderer and Zondler (Chem. Ber. 99, 3008 (1966)) and the . ~
~pecificatio~s of Canadian Patent 959,490, C. S. Hamish Wood et al issued December 17, 1974, and o~ Canadian Patent 997,760, ~. S. Hamish Wood et al, issued Septèmber 28, 1976 (Belgian Patent No. 770,577) re~pectively.
The method described in the aforementioned Canadian Patent 997,760, however, is particularly preferred.
In this method, a compound RlR C = CHR ~VI~
wherein R, Rl and R2 are as defined above, undergoes an~
addition reaction with a n1trosy1 ha11de prepared in situ, ;-and the resulting~nitrosohalide (Vj~^~is converted to the~
oxime (IV) by reaction with ammonia solutionO Reacting the oxime ~IV) with a 2-amino-4-halogeno-6-hydroxy-5-nitro-pyrLmidine (III)~provides~the pyrimid1ne ketox1me (II) which is then reductively cyclised~
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to give the pteridine (I) as shown in the following sequence.
Rl \ R~ NO Rl ~OH
C=CH. R NOHal C-CH. R NH3/ ~ C - C - R
R2 ~ R2 ~al MeOH RCH3~ Hal-(VI) (V) (IV3 .
HN ~ Z ~ ~ Rl 2 2 (III) (II) '.
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. Thc compound of formula (VI) where~n R is a hydroxy-alkyl group may in turn be prep~red from the ketone l 2 R R C=O (IX) by reacting this with a trialkylphosphono-ester (VIII) and reducing the ester (VII) so formed to ~ive .the alcohol (VI)~
/ 2~ .-H2.Co2Alk C-l~ C=CH.CO2Alk (IX) (VIII) (VII) .' ' ' ' I .
: LiAl~4 . ", ' ~ ~ .
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: C=cH:cH2oH
2 "
R
(VI) If a pteridine having R and R as different substituents i5 required, then a racemlC mixture of two stereoisomers of the n1trosohalide (V~ will be obtain~d in view of the:asymmetric carbon atom present. Separation ~ , o the two isomers by conventional~techni~ues kno~Jn in the .
art a~:this stage may be advantageous. ~ ;
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The followin~ Examples illustrate the invention but are in no way intended ~o limit the scope of the invention.
. - Temperature are in deqrees CelsiusO
Exam~e A: reparation of 7 amino-~-hyc~roxy-6-hydro~y-methyl-7,7-diethyl-7,8-dihydropteridine.
(I,R-CH2 OH R =R =Ek) Example 1 .
Ethyl 3-ethy].pent~3---c=te lv~l (R =R =Et) Sodium hydri.de ~6 g) was'placed in a flask with sodium dried benzene ~100 ml) and the flask was flushed with,oxygen-free d`ry nitrogenu: To this solution~was ad-. ded a slight excess of triethylphosphonoacetate (VIII) ~.
(Alk=Et) (61~7 g) over a period of 1.5 h and the temper-ature was maintained at ~15 during.the addition. The mixture was stirred at this temperature for an additional 1 h and then treated dropwise with pentan-3-olle (IX), ~ , , ~R =R =Et) (21.5 g). After addition of the ketone was ,.
.~complete the reaction mixture~was stirred at room temper-~ :
ature until the solid sodlum'~dlethyl phosphate had preci- ,~
:pitated (approx. 10 h). The mother liquor was decanted rom the sol1d,,which was washed..with~benzene (4 x 25 ml).
The benzene extracts were combined and evaporated in vacuo : ~ :
-, , . , X113 ~5~8~
! to give a pale yellow oil (29 g) which was distilled in vacuo to give ethyl 3-ethylpent-2-enoate tVII) (21.8 g, yie].d 56~) as a colourless oil~ b~p. 52-54/~
mm. HgO
Example 2(a) 3-~tllylpent-2-en-1-ol~(VI)tR -R -Et) Ethyl 3-ethylpent-2-enoate (VII) (42.3 g) in dry ether (409 ml) was treated dropwise with a 70~ solutlon ~in benzene) of a slight excess of sodium dihydro bis ethoxymethoxy aluminate (S.D.A.) (86.1 g~, the temperature being maintained at 0 until the addition of the reducin~
agen~ was conlplete. The reaction mixture was then stirred at room temperature for 6 h and the excess S.~.A. was des-troyed by the careful addition of water. ThP solid sodium aluminate which precipitated was flltered off and the filt-rate extract~d with ethyl acetate (4x50 ml)- The combined extracts were washed with brine, dried over sodium sulphate and the solvent removed. The resulting pale yel}ow oil (23 g) was distilled to give 3-ethylpent-2-en-1-ol (VI) . (18.5 g, yield 60~) as a colourless viscous oil b.p.
60/4 mm. ~g.
- Example 2(h) 3-Eth ~ ent-2-en-1-ol (VI) (Rl_R =Et) :
A slurry of lithium aluminium hydride (L.A.~,~
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(80 2 g) in dry ether was addecl drop~Jise to a solution of el:hyl 3-ethylp~nl:-2-enoate (VII~ (33. 7 y) i.n dry cther ~100 ml~ at 0 . After the addition of the LoA~ was com-plete the mixture was stirred at room tempera ture for 2 h .
The excess L.l~c H. was destroyed at 0 by adding a satu-rated solution of sodium sulphate. The solution was filtered and the filtrate extracted with ethyl acetate and worked up as described in Example 2 ta) to give the alcohol , ~VI) (19 g, yield 77~).
Example 3 ....... . 1 3-Chloro-3-ethyl~2-nitroso-pentan-1-o.l. ~V) (R=C~12OH~ R =R =Et) Concentrated hydrocl loric acid (23.ml) was adfled dropwise over 1. S h to a -mixture of 3-ethylpent-3 - . . .
.. .. .. : .
en-l~ol (VI) 123 g~ and amyl nitrite (22. 4 g) in glacial acetic acid (46 ml) at O (ice-salt bath). After the addition :., ' : ' . , ,:
of the acid was com~lete the mixture was stirred at this temperature for 30 min, then cooled in an acetc>ne-carbon dloxide bath for lS min when a whlte paste formed. The solid was f1ltercd oef ~ washed with water and cold methanol and recrystallised from benzene t o give the nitroschlori.de .t~ ~13 g, yield 36~6) as colourless crystals, m.p. 110 . ~ :
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Example 4 3-Amino-3-e~hyl-]-hydroxy-pentan-2-one oxime hydro-cnlori~ tTV) (R=CH QH; R =~ =Et) 3-Chloro-3-ethyl-2-nitroso-pentan-1-ol ~V) (10 g) was placed in a three-necked round-bottomed flas~ and treated with a saturated solution of ammonia in methanol. The flask was stoppered, each stopper being seeured with copper wire, and the mixture was stirred at room temperature for 2 days. A clear yellow solution was obtained. The solvent 1~ was removed in ~acuo at room temperature and the yellow oil o~tained was triturated with hot benzene and the benzene decan-ted. The residue was dissolved in ethanol and the insoluble ammonium chloride present was filtered off.
~ . .
The ethanol was removed in vacuo at~room temperature and , the residual yellow oil was treated with hot acetone to give a white solid which ~as filtered off, washed with acetone and recrystallised from butan-2-ol to give the ketoxime hydrochloride ~IV) (S g; yield 46~) as colourless ~eedles, M.Pt. 182~184 .
Example 5 - - 2-Amino-4-hvdroxY-651,1-d ~ ]-3-h~droxy-2-hydroxyimino-propylamino)-5-nitropyrimidlne tII) (R=cH~oNi~R =R =Ft)~
A suspension of 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine ~III);(Hal=Cl) (2.6 g) in dry ethanol ~S0 m1~ was tr~ated 29 _ : . ~
.
. . -. . . ,, ~ .~ .
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with 3-amino-3-e~}lyl-1-hydroxy-~entan-2-one oxime hydro-chloride (IV) ~2.66 g~ and dry triethylamine (2,89 g) and the mixturc was refluxed for 8 h. The solution was filtered and the filtrate evaporated to dryncss in S vacuo at room temperature The yellow oil obtained ~as treated with cold water and the yellow solid which pre-cipitated was filtered off and washed with ~ater. Re-crystallisation ~rom water in the presence of charcoal gave the nitrop~rimidine oxime ~II) (1.3 g; yield 30~) as a fine white solid m.p. ~50 (decomp.).
~: , .
2-Amino-4-}l~ roxy-6-hydroxymethyl-7,7-diethyl-7,.8-di~dropteridine (I) (R-CH OH; R =R =Et) . . . . . 2 50dium dithionite was added portionwise to a warm soiution of 2-amino-4-hydroxy-6-(1,1-diethyl-3-hydroxy-~hydroxyimi~opropylamino)-5-nitropyrimidine (II) (450 mg~
in 0. lM sodium hydroxide until the colour changed from red to very pale yellow. A solid product was not obtained either on cooling or on adjusting the pll. In order to - separate the product ~rom inorganic material the solùtion was evaporated and the product extracted with ethanol and the inorganic material was::~iltered off . ThLs extraction ~ :
~ . .
was repeated and lhe combined extracts ~ere~evaporated to dryness in vacus. The residue~was dissolved in the ~ _ 30 ~
. , :
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~63 5~9~3~
minimum ~uantity of water and placed on a column o~
Amberlite* (C.G.50) ion e~change resin (2.5 x 28 cm).
Elution with water gave two main fluorescen~ bands.
Evaporation of the solution containing the first band gave the 6-carboxaldehyde derlvati~e of the title compound (10 mg, yield 3%~ as a bright orange powder, whilst the second band gave the 7,8-dihydropteridine ~I) (160 mg, yield 44.5~) as a bright yellow powder, m.p. ~300 decomp.
Exam~le B: Preparatjon of 2-Amino-~-hydroxy-6-hydrox~-methyl-7-s~iroc~clohexyl-7,8-dihydropteridine(I) (R=CH2O~I; R R =spirocyclohexyl).
Example 1 ~
~ Eth~l_cyclohexylidenc acetate (VII~) (R R =spirocyclohexyl ?
Sodium dried benzene (200 ml~ was added to a flask containing sodium hydride (16 g) and the flask was flushed with oxygen-free dry nitrogen. To this mixture was aclded, over 1 h, a sligllt excess of triethylphosphono-acetate (VIII~ (Alk=Et) (16~.3 y) keeping the temperature at O. The/mixture was stirred for an-additional hour at O and then treated with cyclohexanone (IX) ~R R =spirocyclohexyl) (65.4 g) at~ the same~`temperature.
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After the addition of the cyclohexanone was complete t~0 min) the mixture was s~irred at room t~mperature for 3 h; stirring became di:Eficult after this time due to a gummy precipitate of sodium diethyl phosphatc.
The mixture was then heated at 60-65 for 15 ~in during which time it was stirred without difficulty. The mixture was cooled to 15 and the benzene solution was decanted and -the solid washed with benzene. The combined mother liquor and washings were evaporated to give a pale yellow oil which on distillation gave ethyl cyclohexylidene acetate (VII) (62 g; yield 55.4~) as a colourless oil,~
b.p; 86-88/2 mm~ ~g ,:
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ple 2(a) 2-~xclohexyli~ene ethanol (VI~(R R =spirocyclohexyl) A 70~ solution tin ~ell2ene) of sodium dihydrol~isethoxy-methoxy a].umlnate (lOOg) was added port:ionwise 'co ethyl cyclohexylidene acetate (VII) (58.8 g) in dry ether ~ (300 ml) at O . The reaction mixture was stirred for
6 h at room temperature and the excess reducing agent was destroyed by the additlon of waterO The ~.olid sodium aluminate was filtered off and the filtrate extracted with ethyl acetate ~4 x 50 ml~. The combined extracts were washed with brine, dried over sodium sulphate and the solvent evaporated in vacuo. A pale ~ellow oil was obtained which on distillation gave 2-cyclohexylidene ethanol (VI~ (31 g; yield 70~) as a colourless oil~ b.p.
80/ 2mm Hg.
Example 2(b) 2-Cyclohexylidene ethanol_tVI) (RlR =splrocyclohexyl) A solution of ethyl cyclohexylidene acetate (VII) (60 g) in dry ether (300ml) was cooled to O and treated por-tionwise with a slurry of lithlum aluminium hydride ~15 ~ in dry ether (150 ml), the temperature being kept~
~-below 5 during the addition. The reaction mixture was .
stirred fox 15 mln at this tempera~ure ~and for an additional 20 min at room ten~perature. The excess hydride was des-33 - - :
. .
, .
: , :
' ~113 .
troyed with saturated sodium sulphate and the eth,ereal solution worked up as'a~ove to give the alcohol (VI) (23 ~; yield 51%) as a colourless oil.
Example 3~ ' 3-Chloro-2-nitroso-3-~pirocyc_ohexylprop~n-l-ol (V) (R=CII2OI~ R =spirocyclohexyl). ,~
2 Cyclohexylidene ethanol (VI) t23 g) was dissolved in glacial acetic acid ~76 ml). Amyl nitrite (21.5 g~
was added and the mixture was cooled in an ice-salt bath. The cooled solution ~las treated dropwise with cold concen~r~ted hydrochloric acid (23 ml) with stirring. ~ , , After the additlon of the acid was complete the reaction mixture was stirred at the,same temperature for 30 min, , '~
ollowed by cooling in an acetone-carbon dioxide bath for 10 min. The buff-coloured solid ~as filtered off, ,~
washed with cold methanol and recrystallised from acetone to give the ~i~r~ (V) (15 g; yield ~3%) as colourless needles, m.p. 130 .
. .
, Example ~
3-Amino-l-hydroxy-3-spirocyclohexy~propan-2-one oxime hydrochloride (IV) (R=CH2OH;~R~R =splrocyclohexyl - :
A solution of methanol saturated,wlth ammonia was added to 3-chloro 2-nitroso-3-spirocyc1Ohexylpropan-l-ol (V?~ (14.5 g) in a ti~htly s~cured stoppered~flask and the mixture was , ' ~
. .
1~5~
stirred for three days at room temperatureO The reaction mix-- ture ~was thcn re1uxed for 1~5 h in an atmosphere of ammonia, cooled and ~i3tercdO The solvent was removed and the residual yellow oil washed with hot benzene and decanted.
The solid was recrystallised from ethanol yiving the ox~me hy~rochlorid~ (IV) (7.8 g; yield 50~as colourless crystals, M,Pt. 197 .
Example 5 2- mino-4-hydroxy-6-(3-h~droxy-2-11ydrox~imino~ piro-c~clohexylpropylamino)-5-nitropyrimidine (II) ~R=CH2OH; R R aspirocyclohexyl) A suspension of 2-amino-4 chloro-6-hydroxy-5~nitropyrimidin~
(III) ~Hal-Cl) ~2.3 g) in dry ethanol was treated with 3-amino-1-hydroxy-3-spirocyclohexylpropan-2-one oxime lS hydrochloride (IV) (2.Sg) and dry triethylamine (2.7 g) and the mixture was refluxed for 7 h. The reaction miY~ture was filtered and the solid washed with hot ethanol. The solvent was r~moved from the filtrate and the resulting yellow oil was triturated with cold water giviny a yellow solld which on recrystallisati~on from water in the presence of cbarcoal ga~e the nit~opyrimidlne ~II) (1.85 g; yield -47~4~) as an off-white powder, m.pt ~300 (decomp.).
, , . Xl13 ' S~983~
Example 6 2-Amino~ }lydroxy-6-hydroxymethyl-7-spirocyclohexyl-
80/ 2mm Hg.
Example 2(b) 2-Cyclohexylidene ethanol_tVI) (RlR =splrocyclohexyl) A solution of ethyl cyclohexylidene acetate (VII) (60 g) in dry ether (300ml) was cooled to O and treated por-tionwise with a slurry of lithlum aluminium hydride ~15 ~ in dry ether (150 ml), the temperature being kept~
~-below 5 during the addition. The reaction mixture was .
stirred fox 15 mln at this tempera~ure ~and for an additional 20 min at room ten~perature. The excess hydride was des-33 - - :
. .
, .
: , :
' ~113 .
troyed with saturated sodium sulphate and the eth,ereal solution worked up as'a~ove to give the alcohol (VI) (23 ~; yield 51%) as a colourless oil.
Example 3~ ' 3-Chloro-2-nitroso-3-~pirocyc_ohexylprop~n-l-ol (V) (R=CII2OI~ R =spirocyclohexyl). ,~
2 Cyclohexylidene ethanol (VI) t23 g) was dissolved in glacial acetic acid ~76 ml). Amyl nitrite (21.5 g~
was added and the mixture was cooled in an ice-salt bath. The cooled solution ~las treated dropwise with cold concen~r~ted hydrochloric acid (23 ml) with stirring. ~ , , After the additlon of the acid was complete the reaction mixture was stirred at the,same temperature for 30 min, , '~
ollowed by cooling in an acetone-carbon dioxide bath for 10 min. The buff-coloured solid ~as filtered off, ,~
washed with cold methanol and recrystallised from acetone to give the ~i~r~ (V) (15 g; yield ~3%) as colourless needles, m.p. 130 .
. .
, Example ~
3-Amino-l-hydroxy-3-spirocyclohexy~propan-2-one oxime hydrochloride (IV) (R=CH2OH;~R~R =splrocyclohexyl - :
A solution of methanol saturated,wlth ammonia was added to 3-chloro 2-nitroso-3-spirocyc1Ohexylpropan-l-ol (V?~ (14.5 g) in a ti~htly s~cured stoppered~flask and the mixture was , ' ~
. .
1~5~
stirred for three days at room temperatureO The reaction mix-- ture ~was thcn re1uxed for 1~5 h in an atmosphere of ammonia, cooled and ~i3tercdO The solvent was removed and the residual yellow oil washed with hot benzene and decanted.
The solid was recrystallised from ethanol yiving the ox~me hy~rochlorid~ (IV) (7.8 g; yield 50~as colourless crystals, M,Pt. 197 .
Example 5 2- mino-4-hydroxy-6-(3-h~droxy-2-11ydrox~imino~ piro-c~clohexylpropylamino)-5-nitropyrimidine (II) ~R=CH2OH; R R aspirocyclohexyl) A suspension of 2-amino-4 chloro-6-hydroxy-5~nitropyrimidin~
(III) ~Hal-Cl) ~2.3 g) in dry ethanol was treated with 3-amino-1-hydroxy-3-spirocyclohexylpropan-2-one oxime lS hydrochloride (IV) (2.Sg) and dry triethylamine (2.7 g) and the mixture was refluxed for 7 h. The reaction miY~ture was filtered and the solid washed with hot ethanol. The solvent was r~moved from the filtrate and the resulting yellow oil was triturated with cold water giviny a yellow solld which on recrystallisati~on from water in the presence of cbarcoal ga~e the nit~opyrimidlne ~II) (1.85 g; yield -47~4~) as an off-white powder, m.pt ~300 (decomp.).
, , . Xl13 ' S~983~
Example 6 2-Amino~ }lydroxy-6-hydroxymethyl-7-spirocyclohexyl-
7,8-dihydroptcridi.ne (I~ (R=CIl20ll; (R R =spirocyclohexyl).
2-Amino-4-hydroxy-6-(3-hydroxy-2-hydroxyimino-l-spiro-cyclohexylprop~lamino~-5-nitropyrimidine (II) (500 mg) was dissolved in the minimum of O,lM sodium hydro~i~e by warming on the steam bath. Sodium dithionite was added portionwise until an almost colourless solution was obtained~ On cooling the dihydropteridine separated and was filtered off and purified by dissolving in 2M EICl and reprecipitated by the addition of 0.88 al~monia to pH8, On standing the dihydropteridine (I) ~150 mg; yield 38~) was obtained as a pale yellow crystalline solid, m.p. ~300 (decomp.). ~
.- . ;
, ~ -~ ' . 36 ` ~ ` ' - . .
.
. ~ .
~5~
Example C
Potential pteridinc anta~onists of formula (I) may be tested by investigating the inhibitory effect they : -imposc on the enzymes responsible for the biosynthesis of dil~ydropteroic acid (DPt~), namely hydroxymethyl-di~ydropteridine pyrophosphokinase (HMPPS), and dihydro~
pteroate synthetase, hereinafter referred to as 'synthetase'.
In the following reaction equations the compounds are referred to by their abbreviated forms defined on pa~e S of tlle specification.
.
1. ~IMPPS
HMFt ~ ATP ~ Pt + AMP
.
2. Synthetase ~~
Pt ~ pAB g DPtA + pyrophosphate }S (a) An assay for HMPPS was develope`d in which the transfer of the terminal phosphate of ATP-y-P to Pt could be monitored and correlat~d with the amo~mt o inhibition of ~MPPS by the compound under test. ~ :
The oompound of formula (I) whlch was under~test was incorporated into various formulations~comprising metabolites -. . .
a~d enzymes contained in test tubes, as indicated in TABLE 1.
: The components of the mixtùre~were as follows:-I-2-amino-4-hydroxy-6-hydroxymcthyl-7,8-dihydropteridine ~Pt~ in a conccntratisn of 800 ~M~i.e. micromolar;
37 ~~
.
~' j , . ' ~11~
: ~o~9~ :
II-a source of ~ rs, o~tained from an extr~ct o~ ~. coli ¦RecJistered Tra(~e Mark) ~nd separated from 'synthetase' on Sephaclex G-lOO,~ccord-, ing to the method of Richey and Brown in J.Biol.Chem.244, 1582-1592 (1969) -5 III -3m~1 ATP _y_p32 IV~-0.10 M Arrp neutralised (unlabelled).
V -0.02M ~gC12~H20.
VI - 0-1 ~ MgC12.6H20-VII- Source of HMPPS and 'synthetase' -VIII - the test compound in a concentration of 0.93xlO M
-IX-- 0.4mM pAB_cl4 As shown in T~BLE 1, tubes 1 to 9 all contain a source of HMPPS, labelled ATP and 0.02 M MgC12.6H20, tubes , 2 to 9 containing in addition HMPt and tubes ~ to 9 fur.ther -- containing the test compound. Control tub~s 10 tD 12 in- ~
clude a source of-both H~IPPS and synthetase, unlabelled ATP, O.lM MgC12.6H20 and labelled pAB, T~bes 1 to 9 containing the amounts of components shown in the Table, were filled up to 200~1 with distilled water, incub~ted for 60 minutes at 37 C and then chilled on ice. Dextrose (20~1 containing 7~.1 mg/ml) and hexo-kinase t5~1 containing 2000 units/ml~ were added~to the solution, which was then allowed to stand at room t~mperature for 15 minutes. aDarco-G-60'~(~Registered Trade Mark) (10 ~mg) ~:
.
was added to each tube and the contents mixed~ periodi.cally ~ o 38 - ~
:
. ~ , : , . ~. . . , ~
..
.
:
l l J
, 5~98~. ' for 10 minutes. The charcoal was removed through a ~Millipore ~P 250 2200'(Reg1stered Trade Mark) filter and the filter was washed wi~h three 10 ml portions of cold water. The charcoal and the filter were then S radioactively coun~edg ~ The radioactive count rom the contents of tubes 2 and 3 was taken as the maximum count, sinc:e these tubes con-tained no test compound and thus gave O~ enzyme inhibition.
The percentage inhibition produced by the contents of the remaining tubes could then be calculated by relating their radloactive count to the maximum, as determined above.
The contents of tubes 10 to 12 were chroma~ographically analysed as described under part (b), and used as controls, tu~es 10 and 11 containing no test compound (and hence giving O~ inhibition) being accorded the value of.100%. The per-centa~e inhibition ~xhibited by the contents of the tubes in part (bj of the experiment could then be calculated in relation to this, by comparing the respective chromato~rams.
(b) The activity of the test compound of formula (I) against 'synthetase' was determined as follows, by monitor-ing the~formation of dihydropteroate C
A pool of Pt~was prepared from~ATP neutralised (50 O.lM) , MgC12.6H2O~5O~l,O.l~S)~ dithiothreitol ~lOO~l~O.lM), ` t~is bu~er (lOO~l,O.~M,pH 8.3), HMPt (25~1,876~iM) and~l70~1 ~, :
, - of a so1ution containing HMPPS~ The~mixture was incubated ~ ~ 39 ~
..
~5~9~
ior 60 ~inutes at 37 C, chilled brief3y on ice and then dextrose (100~1 containing 72.lmg/ml) and hexokinase ~20~1 containing 2000 units/ml) were added at room tempera-ture to ~he solution, which was allowed to stand at this temperature for 15 minutes~
~ A solution of MgC12.6~2O (lO~l,O.lM), pAB-C
(10~1,0.4n~), dithiothrei~ol (20~1,0.1M) and tris buffcr (20~1,0.4M,pll8.3) was made in each of five test tubes and then 80~1 of the contents of the pool added to each, to-gether with synthetase and/or test compound of formula (I) as indicat~d in Table ~. IThe solution was then made Up to 200~1 with distilled water.
Two control test tubes were prepared, each conta~.ning . , . i ATP ~lO~l,O.iM), MgC12.6H2O~lO~l,O.lM), dithiothreitol IS (20~1,0.1M~ tris buffer ~20~1,0.4U,pH 8.3), pAB-C (10~1,.
0.4mM), and 2D~I of a solution containing HMPPS and 'synthetase' of ~nown activity. The test compound was added to the second of these two tubes up to a final concentration of lO M, and both tubes were made up with distilled water to 200 A}l seven tubes were then 1ncubated for 3~ minutes at 37 & , chilled on ice and then these, together with control tubes 10 to 12 from part ~a), were ~chromatographically analysed as follows.
: ~
~ 40 ~ ~
.
-~ :, ` Xl13 ~C~5~9~3~
.
lOO~l of the contents of each of the tubes was suo~ed onto Whatlllan no. 3MM chromatograplly paper (2x20 cm~
a~ the 'origin', tlle run desccnding in a S~renson bu~fer of potassium and sodium phosphates ~O.lM,pH 7.0~ or lO to 15 cm O Fxom the relative positions of the spots obtained from the contents of the different tubes, the various per-c~ntage inhibitions of synthetase could be evaluated by reference to control tubes lO and ll, which gave O~ inh:ibition.
Those compounds which, as result of these tests, were found to give 50~ inhibition at a concentration o~ lOO~M or less, aFe those wllicl cx~rt a useful potentiating effect, and ~ubject to their toxicity being favourable, may be included in the compositions ~,escribed in this specification.
- , , . : - : , ~ . , 2-Amino-4-hydroxy-6-hydroxymethyl-7,7-dlethyl-7,8- ~ :
dihydropteridine was found to give 50~ inhibition at a con-centra~ion of 2.1 ,~
.. ' ~.
. . , ' , ; . . , ., . ~ .
~, .
: . :
... . .
~ 41 ,' : . : ~ : . :
, . ~
~S~9~ .
Example D
In this ~xpcriment inllibitor z~ne data were deter mined to evaluate the synergistic activity of 2-amino-4-hydroxy-6-hydroxymethyl-7,7-diethyl-7,8-dihydropteridine on its combination with trimethoprim ~TMP) and~or sulpha-methoxazole (SMX) against Staph~lococcus ureus.
The pteridine was included in a soya p~ptone medium of low thymidine content (Wellcotest Sensitivity test agar) contained in a 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 o zone inhibition is shown in Table 3, whexein the numhers represent the complete æone inh1b1tion (i.e. the number of centlmeters from the edge of the well after about 6 x magnificatLon)and the figures in parenthesis include the zones o~ partial in-hibition.
The Results show that the pteridine shows synergism with TMP and SMX alone and multiple synergism with both against Staphylococcus aureus. ~ -:~ :
.
.
' :
, ~ :
' : ' ',' ' ' :.
~ ~ : .:. : ~ :
.
5a~9~
Examp]e E
Tablet Formul~tion Compoun~ of formula(IXR-Cll2O~l;R =R =Et)(pure) lOOmg Trimethoprim ~pure~ 25 mg Sulfaguanidine (B~P~Co ) 100 mg cornstarch, lactose, gelatin, tallc~n and magnesium stearate Preparation - the above constituentS were mixed together using known methods of pharmacy to form a granulation which was then c;ompressed into tabLets.
Example F
Tablet Formulation "Pyremathimine" ~Pyrimethamine) B P. 15 mg Compound of formula ~ R=CH2OH;R =R =Et~-(pure)150 mg 15 . which was then prepared to form a tablet as ln . Example-E.
Example G
.
Tablet Formulation . Sulfanil~mide B.P.Cc 150 mg .20 Compound of formula ~I)(R=CH2OH;R =R =Et)(pure)175 mg ~ -- : :
which was then prepared to form a tablet as in :: :
: Example E.
.
~ _ 43 _ : . :
.
" . . .
. _ ~ X113 ~................................................ . .
` ~S~9~1 ' '.
Ca~sul~ Formulation Trimethoprim (pure) 2¢my Compound of ~ormula (I)~R=CH20H;R =R =Et)~pure)100 mg Pre~aration: .
~. The compounds in granular form were blended together with lactose, cornstarch and magnesium stearate. The po~der was filled into a t~lo-piece, hard shell gelatin capsule using a capsulating machine.
~ E~e I
Irrigant Solution Compound of formula (I~(R=C~OH;R =R =Et)(pure) lmg/ml Trimethoprim (pure) : OD2mg/ml Solvent ~ :: water Example J
Irrigant Soluti.on ~ _ , Compound of formula (I)(R=CH20H;R -R =Et) 2mg/ml tpure) ~-amlno-~-toluenesulphonamide ~pure). 2mg/ml ~: ' ' : ' ~, ' _ ~4 T~ 1 . ' X~13 . . .
Exan~le K
' Solut.ion . _ Compound of formula (I) (R=~1120II;R =R --Et) 1~5 mg/ml ~pure) Diaveridine B. Vet C 0~5 mg/ml :
K~lfizina 1.0 mg/ml Solvent - water `
Exa~_e L :
T~blet Formulation ~ ~ . .
Compound o~ ormula (I) (R=CH20H;R =R =~t) 500 mg (pure) Microcrystalline cellulose 100 mg Starch ~ . :40 mg Magnesium stearate~ : 10 mg Methylhydroxyethylcellulose ~ 3 mg .
. :653 m~ _ :
lS The pteridine (I), microcrystal1ine cellulose and starch were granulated with a solutlon of the methylhydroxyethylcellulose in 50~ aqueous ethyl alcohol. The magnesium stearate was ~ added to the dried granules, and the whole then compressed.
'' ~ ' ~ . ' :
.
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~ ' O . I I ~ ~ U'o O O C~ O
. _ _ _ I ~ X X X X .
. . . u~ ~ ~r- o Ln o . . . ~1 . ~ 1 . . . . .
. Dl 1 1 1 1 1 1 1 1 1 . a .t- ~ ~ ____ ____ H _I .._ ~ 1 ~ .
. H L ~ _ l l l ~ ~¢¦0'~ ~ :
. . . E~V :~ I ~ + ~ + ~ I I ~
. . _ ~1~ .~ :' ~ ~.' a . _ __ . .
H O - = _ _ _ o = _ l l l O ~ ~ ~ :
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4 7 r - ; ~
-.
.
2-Amino-4-hydroxy-6-(3-hydroxy-2-hydroxyimino-l-spiro-cyclohexylprop~lamino~-5-nitropyrimidine (II) (500 mg) was dissolved in the minimum of O,lM sodium hydro~i~e by warming on the steam bath. Sodium dithionite was added portionwise until an almost colourless solution was obtained~ On cooling the dihydropteridine separated and was filtered off and purified by dissolving in 2M EICl and reprecipitated by the addition of 0.88 al~monia to pH8, On standing the dihydropteridine (I) ~150 mg; yield 38~) was obtained as a pale yellow crystalline solid, m.p. ~300 (decomp.). ~
.- . ;
, ~ -~ ' . 36 ` ~ ` ' - . .
.
. ~ .
~5~
Example C
Potential pteridinc anta~onists of formula (I) may be tested by investigating the inhibitory effect they : -imposc on the enzymes responsible for the biosynthesis of dil~ydropteroic acid (DPt~), namely hydroxymethyl-di~ydropteridine pyrophosphokinase (HMPPS), and dihydro~
pteroate synthetase, hereinafter referred to as 'synthetase'.
In the following reaction equations the compounds are referred to by their abbreviated forms defined on pa~e S of tlle specification.
.
1. ~IMPPS
HMFt ~ ATP ~ Pt + AMP
.
2. Synthetase ~~
Pt ~ pAB g DPtA + pyrophosphate }S (a) An assay for HMPPS was develope`d in which the transfer of the terminal phosphate of ATP-y-P to Pt could be monitored and correlat~d with the amo~mt o inhibition of ~MPPS by the compound under test. ~ :
The oompound of formula (I) whlch was under~test was incorporated into various formulations~comprising metabolites -. . .
a~d enzymes contained in test tubes, as indicated in TABLE 1.
: The components of the mixtùre~were as follows:-I-2-amino-4-hydroxy-6-hydroxymcthyl-7,8-dihydropteridine ~Pt~ in a conccntratisn of 800 ~M~i.e. micromolar;
37 ~~
.
~' j , . ' ~11~
: ~o~9~ :
II-a source of ~ rs, o~tained from an extr~ct o~ ~. coli ¦RecJistered Tra(~e Mark) ~nd separated from 'synthetase' on Sephaclex G-lOO,~ccord-, ing to the method of Richey and Brown in J.Biol.Chem.244, 1582-1592 (1969) -5 III -3m~1 ATP _y_p32 IV~-0.10 M Arrp neutralised (unlabelled).
V -0.02M ~gC12~H20.
VI - 0-1 ~ MgC12.6H20-VII- Source of HMPPS and 'synthetase' -VIII - the test compound in a concentration of 0.93xlO M
-IX-- 0.4mM pAB_cl4 As shown in T~BLE 1, tubes 1 to 9 all contain a source of HMPPS, labelled ATP and 0.02 M MgC12.6H20, tubes , 2 to 9 containing in addition HMPt and tubes ~ to 9 fur.ther -- containing the test compound. Control tub~s 10 tD 12 in- ~
clude a source of-both H~IPPS and synthetase, unlabelled ATP, O.lM MgC12.6H20 and labelled pAB, T~bes 1 to 9 containing the amounts of components shown in the Table, were filled up to 200~1 with distilled water, incub~ted for 60 minutes at 37 C and then chilled on ice. Dextrose (20~1 containing 7~.1 mg/ml) and hexo-kinase t5~1 containing 2000 units/ml~ were added~to the solution, which was then allowed to stand at room t~mperature for 15 minutes. aDarco-G-60'~(~Registered Trade Mark) (10 ~mg) ~:
.
was added to each tube and the contents mixed~ periodi.cally ~ o 38 - ~
:
. ~ , : , . ~. . . , ~
..
.
:
l l J
, 5~98~. ' for 10 minutes. The charcoal was removed through a ~Millipore ~P 250 2200'(Reg1stered Trade Mark) filter and the filter was washed wi~h three 10 ml portions of cold water. The charcoal and the filter were then S radioactively coun~edg ~ The radioactive count rom the contents of tubes 2 and 3 was taken as the maximum count, sinc:e these tubes con-tained no test compound and thus gave O~ enzyme inhibition.
The percentage inhibition produced by the contents of the remaining tubes could then be calculated by relating their radloactive count to the maximum, as determined above.
The contents of tubes 10 to 12 were chroma~ographically analysed as described under part (b), and used as controls, tu~es 10 and 11 containing no test compound (and hence giving O~ inhibition) being accorded the value of.100%. The per-centa~e inhibition ~xhibited by the contents of the tubes in part (bj of the experiment could then be calculated in relation to this, by comparing the respective chromato~rams.
(b) The activity of the test compound of formula (I) against 'synthetase' was determined as follows, by monitor-ing the~formation of dihydropteroate C
A pool of Pt~was prepared from~ATP neutralised (50 O.lM) , MgC12.6H2O~5O~l,O.l~S)~ dithiothreitol ~lOO~l~O.lM), ` t~is bu~er (lOO~l,O.~M,pH 8.3), HMPt (25~1,876~iM) and~l70~1 ~, :
, - of a so1ution containing HMPPS~ The~mixture was incubated ~ ~ 39 ~
..
~5~9~
ior 60 ~inutes at 37 C, chilled brief3y on ice and then dextrose (100~1 containing 72.lmg/ml) and hexokinase ~20~1 containing 2000 units/ml) were added at room tempera-ture to ~he solution, which was allowed to stand at this temperature for 15 minutes~
~ A solution of MgC12.6~2O (lO~l,O.lM), pAB-C
(10~1,0.4n~), dithiothrei~ol (20~1,0.1M) and tris buffcr (20~1,0.4M,pll8.3) was made in each of five test tubes and then 80~1 of the contents of the pool added to each, to-gether with synthetase and/or test compound of formula (I) as indicat~d in Table ~. IThe solution was then made Up to 200~1 with distilled water.
Two control test tubes were prepared, each conta~.ning . , . i ATP ~lO~l,O.iM), MgC12.6H2O~lO~l,O.lM), dithiothreitol IS (20~1,0.1M~ tris buffer ~20~1,0.4U,pH 8.3), pAB-C (10~1,.
0.4mM), and 2D~I of a solution containing HMPPS and 'synthetase' of ~nown activity. The test compound was added to the second of these two tubes up to a final concentration of lO M, and both tubes were made up with distilled water to 200 A}l seven tubes were then 1ncubated for 3~ minutes at 37 & , chilled on ice and then these, together with control tubes 10 to 12 from part ~a), were ~chromatographically analysed as follows.
: ~
~ 40 ~ ~
.
-~ :, ` Xl13 ~C~5~9~3~
.
lOO~l of the contents of each of the tubes was suo~ed onto Whatlllan no. 3MM chromatograplly paper (2x20 cm~
a~ the 'origin', tlle run desccnding in a S~renson bu~fer of potassium and sodium phosphates ~O.lM,pH 7.0~ or lO to 15 cm O Fxom the relative positions of the spots obtained from the contents of the different tubes, the various per-c~ntage inhibitions of synthetase could be evaluated by reference to control tubes lO and ll, which gave O~ inh:ibition.
Those compounds which, as result of these tests, were found to give 50~ inhibition at a concentration o~ lOO~M or less, aFe those wllicl cx~rt a useful potentiating effect, and ~ubject to their toxicity being favourable, may be included in the compositions ~,escribed in this specification.
- , , . : - : , ~ . , 2-Amino-4-hydroxy-6-hydroxymethyl-7,7-dlethyl-7,8- ~ :
dihydropteridine was found to give 50~ inhibition at a con-centra~ion of 2.1 ,~
.. ' ~.
. . , ' , ; . . , ., . ~ .
~, .
: . :
... . .
~ 41 ,' : . : ~ : . :
, . ~
~S~9~ .
Example D
In this ~xpcriment inllibitor z~ne data were deter mined to evaluate the synergistic activity of 2-amino-4-hydroxy-6-hydroxymethyl-7,7-diethyl-7,8-dihydropteridine on its combination with trimethoprim ~TMP) and~or sulpha-methoxazole (SMX) against Staph~lococcus ureus.
The pteridine was included in a soya p~ptone medium of low thymidine content (Wellcotest Sensitivity test agar) contained in a 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 o zone inhibition is shown in Table 3, whexein the numhers represent the complete æone inh1b1tion (i.e. the number of centlmeters from the edge of the well after about 6 x magnificatLon)and the figures in parenthesis include the zones o~ partial in-hibition.
The Results show that the pteridine shows synergism with TMP and SMX alone and multiple synergism with both against Staphylococcus aureus. ~ -:~ :
.
.
' :
, ~ :
' : ' ',' ' ' :.
~ ~ : .:. : ~ :
.
5a~9~
Examp]e E
Tablet Formul~tion Compoun~ of formula(IXR-Cll2O~l;R =R =Et)(pure) lOOmg Trimethoprim ~pure~ 25 mg Sulfaguanidine (B~P~Co ) 100 mg cornstarch, lactose, gelatin, tallc~n and magnesium stearate Preparation - the above constituentS were mixed together using known methods of pharmacy to form a granulation which was then c;ompressed into tabLets.
Example F
Tablet Formulation "Pyremathimine" ~Pyrimethamine) B P. 15 mg Compound of formula ~ R=CH2OH;R =R =Et~-(pure)150 mg 15 . which was then prepared to form a tablet as ln . Example-E.
Example G
.
Tablet Formulation . Sulfanil~mide B.P.Cc 150 mg .20 Compound of formula ~I)(R=CH2OH;R =R =Et)(pure)175 mg ~ -- : :
which was then prepared to form a tablet as in :: :
: Example E.
.
~ _ 43 _ : . :
.
" . . .
. _ ~ X113 ~................................................ . .
` ~S~9~1 ' '.
Ca~sul~ Formulation Trimethoprim (pure) 2¢my Compound of ~ormula (I)~R=CH20H;R =R =Et)~pure)100 mg Pre~aration: .
~. The compounds in granular form were blended together with lactose, cornstarch and magnesium stearate. The po~der was filled into a t~lo-piece, hard shell gelatin capsule using a capsulating machine.
~ E~e I
Irrigant Solution Compound of formula (I~(R=C~OH;R =R =Et)(pure) lmg/ml Trimethoprim (pure) : OD2mg/ml Solvent ~ :: water Example J
Irrigant Soluti.on ~ _ , Compound of formula (I)(R=CH20H;R -R =Et) 2mg/ml tpure) ~-amlno-~-toluenesulphonamide ~pure). 2mg/ml ~: ' ' : ' ~, ' _ ~4 T~ 1 . ' X~13 . . .
Exan~le K
' Solut.ion . _ Compound of formula (I) (R=~1120II;R =R --Et) 1~5 mg/ml ~pure) Diaveridine B. Vet C 0~5 mg/ml :
K~lfizina 1.0 mg/ml Solvent - water `
Exa~_e L :
T~blet Formulation ~ ~ . .
Compound o~ ormula (I) (R=CH20H;R =R =~t) 500 mg (pure) Microcrystalline cellulose 100 mg Starch ~ . :40 mg Magnesium stearate~ : 10 mg Methylhydroxyethylcellulose ~ 3 mg .
. :653 m~ _ :
lS The pteridine (I), microcrystal1ine cellulose and starch were granulated with a solutlon of the methylhydroxyethylcellulose in 50~ aqueous ethyl alcohol. The magnesium stearate was ~ added to the dried granules, and the whole then compressed.
'' ~ ' ~ . ' :
.
..
- ~, , : -~ ~5 . , ~, ~ ~h~ ~:
~, .
~11;~
, ~5~9l3~
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. ~ ~ ~ ~~
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H O - - .
. :' 1. 1 1 1 1 1 1 1 1- . ~
. _ . . _ ... --~
1~
. ~1 . ~ . O t)11'1 L~ W ~ Ln . __ = _ _ _ _ = _ . ~. ~ l l l . ~
~ ' O . I I ~ ~ U'o O O C~ O
. _ _ _ I ~ X X X X .
. . . u~ ~ ~r- o Ln o . . . ~1 . ~ 1 . . . . .
. Dl 1 1 1 1 1 1 1 1 1 . a .t- ~ ~ ____ ____ H _I .._ ~ 1 ~ .
. H L ~ _ l l l ~ ~¢¦0'~ ~ :
. . . E~V :~ I ~ + ~ + ~ I I ~
. . _ ~1~ .~ :' ~ ~.' a . _ __ . .
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~ ~I _ ;1~ r~ I ~ ~ _ L _ 1 111: . .
. H, I n .. . . .
': :.. ~ . .~_ _ .
: . ' . ~ ~: ~':
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~ O O
: ~t . ,~ ~ ,~
_ ._ V
-;
- - : :
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.
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:. ~ :
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~ .
. :105098:~L
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.
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. ~, i .~ .: ~ . ~ .
p~ , U~ .- . ' U~ ..
~1 o I, o u~ C ¦ N , ~ O r I ~
, . ~ Pl ' O U). ' O ' U~ ~
~ E~ ,_ ~ I i ~1 ~) ' ".
-. ' ~ . ' . ' . , , , , ~ ' ~ ' ' ,' ' '.
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- - ~ ~C. _ ~ . ~ , . ..
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. . .. ..... . .
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.
Claims (9)
1. A method of preparing a compound of formula (II) (II) or a tautomeric form thereof, wherein R is a lower alkyl or hydroxy-lower-alkyl group, and R1 and R2 are the same or different and each is a lower alkyI group having together at least 3 carbon atoms or R1 and R2, together with the carbon atom to which they are attached form a spiro-cycloalkyl ring system having 4 to 6 carbon atoms, and Z is an oxime group, comprising reacting a compound of formula (IV) (IV) or a salt thereof, wherein R, R1 and R2 are as defined above, with a 2-amino-4-halogeno-6-hydroxy-5-nitropyrimidine,
2. A method according to claim 1, wherein R in said compound of formula (IV) is a hydroxy-lower-alkyl group.
3. A method according to claim l,wherein, in said compound of formula (IV), R is a hydroxy-lower-alkyl group of 1 to 4 carbon atoms, and R1 and R2 are each lower alkyl groups of 1 to 4 carbon atoms, having together at least 3 carbon atoms.
4. A method according to claim l wherein,in said com-pound of formula (IV), R is a hydroxy-lower-alkyl group of 1 to 4 carbon atoms and R1 and R2, together with the carbon atoms to which they are attached form a spirocycloalkyl ring system having 4 to 6 atoms.
5. A method according to claim 1, wherein, in said compound of formula (IV), R is a hydroxymethyl group, and R1 and R are both ethyl groups or together with the carbon atom to which they are attached, form a spirocyclohexyl ring.
6. A method according to claim 1 for preparing 2-amino-4-hydroxy-6(1,1-diethyl-3-hydroxy-2-hydroxyiminopropylamino)-5-nitropyrimidine comprising reacting 3-amino-3-ethyl-1-hydroxy-pentan-2-one oxime with 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine.
7. A method according to claim 1 for preparing 2-amino-4-hydroxy-6-(3-hydroxy-2-hydroxyimino-1-spirocyclohexylpropyl-amino)-5-nitropyrimidine comprising reacting 3-amino-1-hydroxy-3-spirocyclohexylpropan-2-one oxime with 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine.
8. A compound of formula (II), as defined in claim 1, or a tautomeric form thereof, whenever prepared by the method of claim 1 or by an obvious chemical equivalent.
9. A compound of formula (II), as defined in claim 1, or a tautomeric form thereof wherein R is a hydroxy-lower-alkyl group of 1 to 4 carbon atoms, and R1 and R2 are each lower alkyl groups of 1 to 4 carbon atoms, having together at least 3 carbon atoms, whenever prepared by the method of claim 3 or by an obvious chemical equivalent.
l0. 2-Amino-4-hydroxy-6(1,1-diethyl-3-hydroxy-2-hydroxy-iminopropylamino)-5-nitropyrimidine whenever prepared by the method of claim 6 or by an obvious chemical equivalent.
l0. 2-Amino-4-hydroxy-6(1,1-diethyl-3-hydroxy-2-hydroxy-iminopropylamino)-5-nitropyrimidine whenever prepared by the method of claim 6 or by an obvious chemical equivalent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB3581472A GB1454165A (en) | 1972-08-01 | 1972-08-01 | Biologically active compounds and compositions |
| CA177,817A CA1019736A (en) | 1972-08-01 | 1973-07-31 | Pteridine derivatives |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1050981A true CA1050981A (en) | 1979-03-20 |
Family
ID=25667324
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA273,623A Expired CA1050981A (en) | 1972-08-01 | 1977-03-10 | Intermediates for pteridine derivatives |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1050981A (en) |
-
1977
- 1977-03-10 CA CA273,623A patent/CA1050981A/en not_active Expired
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