CA1104957A - Pseudomonic acid c from pseudomonas fluorescens - Google Patents
Pseudomonic acid c from pseudomonas fluorescensInfo
- Publication number
- CA1104957A CA1104957A CA315,801A CA315801A CA1104957A CA 1104957 A CA1104957 A CA 1104957A CA 315801 A CA315801 A CA 315801A CA 1104957 A CA1104957 A CA 1104957A
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- Prior art keywords
- pseudomonic acid
- ester
- salt
- acid
- pseudomonate
- Prior art date
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Abstract
ABSTRACT
A compound of formula (II), named pseudomonic acid C or a salt or ester thereof:
A compound of formula (II), named pseudomonic acid C or a salt or ester thereof:
Description
;'7 ANTIBACTERIAL COMPOUNDS
.
This invention relates to antibacterial compounds and in particular to a novel antibacterial compound produced by the bacterium Pseudomonas fluorescens, together with salts and esters of the compound.
British Patent No. 1,395,907 describes and claims :
a process for the isolation of antibacterial compounds rom the bacterium Pseudomonas fluorescens, one such compound being called pseudomonic acid o formula (I) OH
~ C02~CHz)8C02 ~:~ OH
which will be referred -to herein as "pseudomonic acid A", It has now been found that~a further antibacterial compound:can be isolated from Pseudomonas fluorescens and this:~compound can also be prepared from pseudomonlc ac:id A.
15 : ~ ~Acc:ordingly the~present invention provides a compound:of formula (II) or a pharmaceutically accept- -able salt or ester thereof:
. .
OH
CH3 HO ~ ~ ~ ~ CO2(CH2)8c02 CH~ ~ CH3 (II) OH
The compound (II) will be ref~erred to herein as "pseudomonic acid C". It is believed that the compound has the absolute steriochemistry as shown in formula (II`I) OH
CH3 HO ~ ~
CH l l l C2(CH2)8C2H
.~` ~ CH3 ~ ~III) OH
both double bonds being in the trans - or E conigura tion.
Suitable non-toxic sal-ts of pseudomonic acid C
include metal salts, e.g. aluminium, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, and ammonium or substitu-ted ammonium salts for example those with lower alkylamino such as triéthylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine, bis-(2-hydrox-ethyl)-amine or tri-(2-hydroxyethyl)-amine, cyclo-alkylamines such as bicyclohexylamine, or with procaine, dibenzylamine, N,N-dibenzylethylene-diamine, l-ephen-amine, N-ethylpiperidine, N-benzyl-~ phenethyl-amine, dehydroabietylamine, N,N'-bis-dehydroabietylethylene~
diamine, or bases of the pyridine type such as pyridine, collidine or quinoline. ~ -Preferred salts are alkali metal salts. Suit-able esters incluùe: ;
~'''' ~ . :
, ~, .
(a~ Cl_20 alkyl, C2 8 alkenyl or C2 8 alkynyl each of which may be optionally substituted by C3_7 cycloalkyl, halogen, carboxy, Cl 6 alkoxycarbonyl, carbamoyl, aryl, heterocyclylg hydroxy, Cl 16 alkanoyloxy, amino, mono-s and di (Cl_6) alkylam (b) C3 ~ cycloalkyl optionally substituted with Cl 6 alkyl;
(c) aryl;
(d) heterocyclyl.
The term laryl' includes phenyl, and naphthyl optionally substituted with up to five halogen, Cl 6 alkyl, Cl 6 alkoxy, halo(Cl 6)alkyl 7 hydroxy, amino, carboxy, Cl 6 alkoxycarbonyl, or Cl 6 alkoxycarbonyl(C
(Cl_6)alkyl groups~
The term ~heterocyclyl' includes single or fused rings comprising up to four hetero atoms in the ring selected from oxygen, nitrogen sulphur and optiona:Lly substituted with up to three halogen, Cl 6 alkyl, Cl 6 alkoxy, halo(Cl 6)alkyl, hydroxy, amino, carboxy, 20 Cl 6 alkoxycarbonyl, Cl_6alkoxy-carbonyl(C1 6)alkyl, aryl or oxo groups.
One class of ester groups comprise allcyl, aryl, and aralkyl groups, any of which may be substituted with a hydroxy, amino or halogen group. For e~ample 25 the ester group may be a Cl 6 alkyl group in particular, methyl, ethyl, n or iso propyl, n, sec-, iso or tert-butyl; a halo-(Cl 6)-alkyl group such as tri~luoro-methyl, 2,2,2-trichloroethyl; an aminoalkyl group such as aminomethyl, 2-aminoethyl; hydroxymethyl, hyclroxy-30 ethyl; phenyl; substituted phenyl, or a benzyl group.
Preferred esters are Cl 6 alkyl esters.
Pseudomonic acid C, its salts and esters have antibacterial activity. They have particularly high activity against Haemphilus in~luenzae, _ isserla 35 gonorrhoeae and Mycoplosma sp, and are therefore of value in the treatment o~ respiratory and venereal diseases, and of mycoplasma-induced human ancl -.~ . ~- .
.: . - . . , . ' : . .
: . .
. . . :. -. . ~,:
veterinary diseases. Furthermore, the compounds of this invention have -the advan-tage over pseudomonic acid A of being stable to acidic conditions, and more stable to alkaline conditions.
In humans the infections which pseudomonic acid C
its salts and esters may be particularly useful agains-t include venereal disease. Because it is not a ~-lactam antibiotic it is effective against ~-lactamase-produc-ing strains of N. gonorrhoeae, against which standard treatments such as penicillin and cephalosporin anti-biotics would not be useful. Pseudomonic acid C may also be effective in the treatment of respiratory infections such as chronic bronchitis, and bacterial meningitis; non-specific urethritis and pneumonia. In animals it may be employed generally as a growth promoter, or for the ~reatment of mastitis in cattle and for treatment of mycoplasma infections in animc~ls such as turkeys, chickens and ~igs.
This invention also provides a pharmaceu-tical or veterinary composition which comprises pseudomonic acid C, or a salt or ester thereof together with a pharma ceutically or veterinary acceptable caxrier or excipi-ent.
The compositions may be formulated for administra tion by any route, and would depend on the disease being treated. The compositions may be in the form of table-ts~ capsules, powders~ granules~ lozenges, or liquid preparations, such as oral or sterile parenteral `
solutions or suspensions.
Tablets and capsules for oral adrninistration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica;
, , , , - :
-, . , . , . , . .
disintegrants, for example po~ato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical prac-tice. Oxal liquid prep-arations may be in the form OI ~ Ior example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a clry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain con-10 ventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, ylucose syrup, gelatin hydrogenated edible fats, emulsifying agents, for examplè lecithin, sorbitan monooleate, or acacia; non a~ueous vehicles (which may include edible 15 oils), for example almond oil 9 fracti.onated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl ~-hydroxybenzoate or sor'bic acid, and if desired convention flavouring or colouring agents.
Suppositories will contain conventional suppos-itory bases, e.g. cocoa-butter or other glyceride.
~or parenteral administration, fluid unit dosage forms are prepared utilizing'the compound and a sterile vehicle, water being preferred. The compound~ depend-25 ing on t'he vehicle and concentration used, can be either suspended or dissolved in the vehicle. In pre-parlng solutions the compouncl can be dissolved in water for injection and filter sterilized before fill-ing into a suitable vial or ampoule and sealing.
30 Advantageously, adjuvants such as a local anesthetic, p.reservative and buffering agents can be dissolved in the vehicle. To enhance the stability the composition can be frozen after filling into the vial and water removed under vacuum. The dry lypophilized powder is 35 then sealed in the vial. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of , : . ............ :. .
: . - . .:
':,: . . ~. ,: , ,... .~ .. ' . . ' being dissolved and sterilization cannot be accomplished by filtration. The compound can be s-terilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
The compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by ~eight, of the active material, depending on the method of administration.
Where the compositions comprise dosage units, each unit will preferably contain from 50-5~0 mg., of the active ingredient. The dosage as employed for adult human treatment will preferably range from 100 mg to 3 g., per day, for instance 250 mg-2 g., per day, depending on the route and frequency of adminis-tration.
Alternatively pseudomonic acid C or a sal-t or ester may be adrninistered as part of the total dietary intake. In this case the amount of compound employed may be less than 1% by weight of the diet and in pre-ferably no more than 0.5% by weight. The diet for ani-mals may consist of normal foodstuffs to which the com-pound may be added or it may be added to a premix.
The present invention also provides a process for the preparation of pseudomonic acid C or a salt or ester thereof which process comprises reacting pseudomonic acid A or a salt or ester thereof with a r~agent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(ij forming a sal-t of the pseudomonic acid C produced;
(ii~ esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic ~cid C.
A number of reagents for converting an epoxide to an olefin are known in the literature, and the part-icular reagent of choice for the process of the pre-sent invention is a matter of trial and error. Some .. : . . . .
'. , ' . ' :
: . ~
-such reagents are more suitable than others for this purpose. Some generally applicable methods are as follows:
(a) Potassium selenocyanate in methanol/water;
(see JCS Chem. CommO, 19/5, 1~16; J~S 1949~ 278j ~b) Lower valent tungsten halides; for example WC16/
butyl lithium (see J~Amer.Chem.SocO 1972,94,6538) (c) Ph3P = Se/-trifluroacetic acid;
(see JCS Chem. Comm. 1973, 253) ~O (d) Trifluoroacetyl iodide/sodium iodide;
(see J.Org.Chem., 1978~43,1841).
Other methods are described in the followiny references:
J. ~mer. Chem. Soc., 1973, 95, 2697.
Tet. Letts (17) 1976, 1395.
Ber. 1955, 88~ 165~.
J, Org. Chem., 1958t 22, 1118.
It has been ~ouncl that one convenient method is the use of potassium selenocyanate.
Suitable so]vents for use with potassium selen-ocyanate include mixtures of water with alkanols, in particular Cl-C20 alkanols. It has been found that higher yields of the compound of formula (II) are achieved if an alcohol is employed with a large, in particular branched or cyclic, alkyl group. Specific alcohols include iso-hexyl alcohol, tert-amyl alcohol and cyclohexyl alcohol. The reaction is generally performed at elevated temperatures, suitably at about the boiling point o the solvent employed. The time for which the reaction is performed depends on the temp~-erature of the reaction, and therefore on the solvent.
Generally a time of from 2-9 days is suitable.
Another suitable method for converting the epoxide of pseudomonic acid A, or a salt or ester thereof into an olefin, comprises treatment with trifluoroacetyl iodide and sodium iodide. ~he trifluoroacetyl iodide may be prepared in situ from trifluoroacetic anhydride.
The reaction is suitably conducted at ambient ,;' . . . . . . .
. . . :. .
. . . ' , ' ' ' . - . :
. .
temperature for ~rom about 10 to 36 hours, suitably about 24 hours.
When the free acid or salt of pseudomonic acid C is required it may be convenient to employ an ester of pseudomonic acid A for the above process9 which ester is a carboxyl-protecting group. Suitable carb-oxyl-protecting groups would depend on the reaction condi-tions for de epoxidation and include the 2,2,2-trichloro-ethyl ester, (which may be removed with zinc in a lower alcohol, espcially methanol) phenyl, penta-chlorophenyl, benzyl, and t-butyl ester groups. Other suitable carboxyl-protecting are silyl groups. In this case the carboxylic acid is reac-ted with a silyl-ating agent such as a halosilane or a silazane. A
preferred silylating agent is N,O-bis(trimethylsilyl) acetamide, which produces the trimethyl-silyl deriva-tive of the acid.
Prior to the above process of this invention, it may be desirable to protect the hydroxyl groups in pseudomonic acid A or its salt or ester. Although the reaction is possible without hydroxyl protection, in some cases higher yields of the pseudomonic acid C
derivative could be formed if the hydroxyl groups were protected. Such protecting groups must be removable under suitably mild conditions and suitable groups include silyl groups produced from a silylating agent as discussed above. Particularly suitable hydroxyl-protectlng groups include tri-methylsilyl, t-butyl-dimethylsilyl 7 methylthiomethyl. A preferred hydroxyl-protecting group is trimethylsilyl, as it is readily removed on completion o~ the reaction. Alternatively, for some de-epoxidation reactions it is possible to protect the hydroxyl groups with other ester radicals which may be removed by chemical or enzymatic means.
Examples include p-nitrobenzoate, methoxyaceta-te, phenoxyacetate, trifluoroacetate, each of which may be .
.. ~ , ~ . . ........................ ,... :: ::
-removed under mild basic conditions such as aqueous ammonia; or potassium carbonate in aqueous methanol.
It is also possible to protect the glycol moiety in pseudomonic acid A, and suitable reagents for form-lng such a hydroxyl-protecting group include compounds of formula (IV).
oR2 ., Rl C oR3 (IV) OR
wherein Rl is hydrogen or a Cl 6 alkyl group and R , R and R independently represent a Cl 6 alkyl group.
The group Rl may be for example hydrogen, methyl, ethyl, n- or iso-propyl. Most suitably, Rl represents hydrogen so tha-t the compound of formula (I~) is a trialkyl orthoformate. -Groups R2, R3, and R4 may be for example, methyl, ethyl, n- or iso-propyl, n-, _ -, sec- or tert-butyl. Preferably R2, R3, and R are all the same and each represents a methyl group.
Other glycol pro-tecting groups include those wherein the glycol moiety is converted to the structure:
o \ / ~a o / \Rb ' ' where Ra and Rb are hydrogen, Cl 6 alkyl, or phenyl.
Preferably R and R are both methyl, i.e. the group is the isopropylidene group. This group may be intro-duced onto pseudomonic acid A or its salt or ester by reaction with 2,2-dimethoxypropane, and removed by .
.: . :
.
~ .
' ' ' ' ': ' ' ': ' . . ~ , treatment with acetic acid.
The hydro~y-protecting group may be removed by a conventional method for the particular hydroxyl-protect~
ing group.
It may be such that it can be removed directly or alternatively, it may be converted into a different pro-tecting group which is then removable under ~lifferent conditions. This latter approach may be employed when a glycol protecting group derived from a compound (IV) is used; it is converted by acid to the group -OCOR
which is then removed.
When an ester of pseudomonic acid C is required, the esterification step, step (ii) above may be per~
formed by any conventional method, for exa~ple by reaction of the acid, or a salt thereof:-(a) with the appropriate halide, sulphate or alkane-sulphonate of the alcohol in the presence of a solvent such as acetone, dimethylsulphide or dimethvlsulphoxicle and calcium, or potassium carbonate or with the halide in the presence of hexamethyl phosphoramide; or (b) by phase transfer catalysis methods with the halide and/or sulphate of the alcohol in aqueous and/or organic solution in the presence of a quaternary ammon-ium salt such as tetrabutyl ammonium bisulphate or halide, or benxyltrime-thyl-ammonium halide; or (c) with a diazoalkane.
The hydrolysis of an ester of pseudomonic acid C
(step ~iii) above) may be chemical hydrolysis, for example by selective alkaline hydrolysis; or enzymic hydrolysis, for example by the use of Bakersl Yeast.
Also included within the scope of the present invention is a process for the preparation of pseudo-monic acid C or a salt or ester thereof which comprises yrowing Pseudomo_as fluorescens under aerobic conditions on or in a culture medium containing inoxganic salts and sources of asimilable carbon and nitxogen until the culture medium exhibits at least detectable antibacterial .: . :: : . - .
.. . , . . : . .. .:
- . . .
.
.
v`~
activity, acidifying the culture medium; extracting with an organic solvent for the active materials dissolved in the culture medium9 and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally there-after esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
In the above process, the cultivation step where Pseudomonas fluorescens is grown is conventional.
Any strain of this organism may to our knowledge be employed; one suitable public strain being Pseudomonas fluorescens NCIB 10586. (NCIB = National Collection of Industrial Bacteria).
After the fermentation is completed, the active rnater:ials, lncludlng pseudomonic acid C, are extracted from the acidified aqueous culture ~ledium into a suit-able solvent.
Preferably, the extraction procedure comprises extraction of the culture medium, after acidification, with an organic solvent; re-extraction of the organic extract with aqueous alkaline buffer solution; and finally re-extraction of the latter, after acidifica-tion, with organic solvent. Suitable solvents can be found by trial and error; examples include iso-butylmethyl ketone (IBMK), chloroform and preferably ethyl acetate.
The pseudomonic acid C may then be separated from other active materials produced in the ferment-ation, either directly (step (a) above) or by esterifying the mixture and separating the ester of pseudomonic acid C (step (b) above). When Pseudo- -monas flu~orescens NCIB 10586 is employed as the bact-erium, the majox material which is produced in . .
. : ;, . ..
. ' ,~
addition to pseudomonic acid C is pseudomonic acid A.
If this latter material is present in substantial quantities it is preferable to remove the majority by crystallisation of pseudomonic acid A9 optionally with seeding, from a suitable solvent for example diethyl ether.
If alternative (a) is carried out pseudomonic acid C may be separated directly by chromatography from the remaining mixture, either as the free acid itself or as a slat thereof. On chxomatography on silica gel, pseudomonic acid C is eluted slightly before pseudomonic acid A and the fractions can be identified accordingly.
The separated pseudomonic acid C or a salt thereof lS may be esterified by any of the methods described earlier in this specification.
If alternative (b) above is carried out the mixture of active materials is first esterified, preferably after removing the majority of the pseudo-monic acid A by crystallisation. Again any of the above described methods of esterification may be employed. It is convenient to form Cl 6 alkyl esters of the components in the mixture, preferably me-thyl esters.
The resultant mixture of esters may then be subjected to chromatography and the desired ester of pseudomonic acid C thereby separated. If the free acid or salt is required they may be produced by chemical or enzymatic hydrolysis of the separated ester.
The invention is illustrated in the following Examples.
' .
' :
. - . , r~ r~
Example 1 Methyl 10,11-D~oxypseudomonate_A (methyl pseudomonate _) from meth~ seudomonate A
A solution of methyl pseudomonate A (1.03 g ;
.
This invention relates to antibacterial compounds and in particular to a novel antibacterial compound produced by the bacterium Pseudomonas fluorescens, together with salts and esters of the compound.
British Patent No. 1,395,907 describes and claims :
a process for the isolation of antibacterial compounds rom the bacterium Pseudomonas fluorescens, one such compound being called pseudomonic acid o formula (I) OH
~ C02~CHz)8C02 ~:~ OH
which will be referred -to herein as "pseudomonic acid A", It has now been found that~a further antibacterial compound:can be isolated from Pseudomonas fluorescens and this:~compound can also be prepared from pseudomonlc ac:id A.
15 : ~ ~Acc:ordingly the~present invention provides a compound:of formula (II) or a pharmaceutically accept- -able salt or ester thereof:
. .
OH
CH3 HO ~ ~ ~ ~ CO2(CH2)8c02 CH~ ~ CH3 (II) OH
The compound (II) will be ref~erred to herein as "pseudomonic acid C". It is believed that the compound has the absolute steriochemistry as shown in formula (II`I) OH
CH3 HO ~ ~
CH l l l C2(CH2)8C2H
.~` ~ CH3 ~ ~III) OH
both double bonds being in the trans - or E conigura tion.
Suitable non-toxic sal-ts of pseudomonic acid C
include metal salts, e.g. aluminium, alkali metal salts such as sodium or potassium, alkaline earth metal salts such as calcium or magnesium, and ammonium or substitu-ted ammonium salts for example those with lower alkylamino such as triéthylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine, bis-(2-hydrox-ethyl)-amine or tri-(2-hydroxyethyl)-amine, cyclo-alkylamines such as bicyclohexylamine, or with procaine, dibenzylamine, N,N-dibenzylethylene-diamine, l-ephen-amine, N-ethylpiperidine, N-benzyl-~ phenethyl-amine, dehydroabietylamine, N,N'-bis-dehydroabietylethylene~
diamine, or bases of the pyridine type such as pyridine, collidine or quinoline. ~ -Preferred salts are alkali metal salts. Suit-able esters incluùe: ;
~'''' ~ . :
, ~, .
(a~ Cl_20 alkyl, C2 8 alkenyl or C2 8 alkynyl each of which may be optionally substituted by C3_7 cycloalkyl, halogen, carboxy, Cl 6 alkoxycarbonyl, carbamoyl, aryl, heterocyclylg hydroxy, Cl 16 alkanoyloxy, amino, mono-s and di (Cl_6) alkylam (b) C3 ~ cycloalkyl optionally substituted with Cl 6 alkyl;
(c) aryl;
(d) heterocyclyl.
The term laryl' includes phenyl, and naphthyl optionally substituted with up to five halogen, Cl 6 alkyl, Cl 6 alkoxy, halo(Cl 6)alkyl 7 hydroxy, amino, carboxy, Cl 6 alkoxycarbonyl, or Cl 6 alkoxycarbonyl(C
(Cl_6)alkyl groups~
The term ~heterocyclyl' includes single or fused rings comprising up to four hetero atoms in the ring selected from oxygen, nitrogen sulphur and optiona:Lly substituted with up to three halogen, Cl 6 alkyl, Cl 6 alkoxy, halo(Cl 6)alkyl, hydroxy, amino, carboxy, 20 Cl 6 alkoxycarbonyl, Cl_6alkoxy-carbonyl(C1 6)alkyl, aryl or oxo groups.
One class of ester groups comprise allcyl, aryl, and aralkyl groups, any of which may be substituted with a hydroxy, amino or halogen group. For e~ample 25 the ester group may be a Cl 6 alkyl group in particular, methyl, ethyl, n or iso propyl, n, sec-, iso or tert-butyl; a halo-(Cl 6)-alkyl group such as tri~luoro-methyl, 2,2,2-trichloroethyl; an aminoalkyl group such as aminomethyl, 2-aminoethyl; hydroxymethyl, hyclroxy-30 ethyl; phenyl; substituted phenyl, or a benzyl group.
Preferred esters are Cl 6 alkyl esters.
Pseudomonic acid C, its salts and esters have antibacterial activity. They have particularly high activity against Haemphilus in~luenzae, _ isserla 35 gonorrhoeae and Mycoplosma sp, and are therefore of value in the treatment o~ respiratory and venereal diseases, and of mycoplasma-induced human ancl -.~ . ~- .
.: . - . . , . ' : . .
: . .
. . . :. -. . ~,:
veterinary diseases. Furthermore, the compounds of this invention have -the advan-tage over pseudomonic acid A of being stable to acidic conditions, and more stable to alkaline conditions.
In humans the infections which pseudomonic acid C
its salts and esters may be particularly useful agains-t include venereal disease. Because it is not a ~-lactam antibiotic it is effective against ~-lactamase-produc-ing strains of N. gonorrhoeae, against which standard treatments such as penicillin and cephalosporin anti-biotics would not be useful. Pseudomonic acid C may also be effective in the treatment of respiratory infections such as chronic bronchitis, and bacterial meningitis; non-specific urethritis and pneumonia. In animals it may be employed generally as a growth promoter, or for the ~reatment of mastitis in cattle and for treatment of mycoplasma infections in animc~ls such as turkeys, chickens and ~igs.
This invention also provides a pharmaceu-tical or veterinary composition which comprises pseudomonic acid C, or a salt or ester thereof together with a pharma ceutically or veterinary acceptable caxrier or excipi-ent.
The compositions may be formulated for administra tion by any route, and would depend on the disease being treated. The compositions may be in the form of table-ts~ capsules, powders~ granules~ lozenges, or liquid preparations, such as oral or sterile parenteral `
solutions or suspensions.
Tablets and capsules for oral adrninistration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica;
, , , , - :
-, . , . , . , . .
disintegrants, for example po~ato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical prac-tice. Oxal liquid prep-arations may be in the form OI ~ Ior example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a clry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain con-10 ventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, ylucose syrup, gelatin hydrogenated edible fats, emulsifying agents, for examplè lecithin, sorbitan monooleate, or acacia; non a~ueous vehicles (which may include edible 15 oils), for example almond oil 9 fracti.onated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl ~-hydroxybenzoate or sor'bic acid, and if desired convention flavouring or colouring agents.
Suppositories will contain conventional suppos-itory bases, e.g. cocoa-butter or other glyceride.
~or parenteral administration, fluid unit dosage forms are prepared utilizing'the compound and a sterile vehicle, water being preferred. The compound~ depend-25 ing on t'he vehicle and concentration used, can be either suspended or dissolved in the vehicle. In pre-parlng solutions the compouncl can be dissolved in water for injection and filter sterilized before fill-ing into a suitable vial or ampoule and sealing.
30 Advantageously, adjuvants such as a local anesthetic, p.reservative and buffering agents can be dissolved in the vehicle. To enhance the stability the composition can be frozen after filling into the vial and water removed under vacuum. The dry lypophilized powder is 35 then sealed in the vial. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of , : . ............ :. .
: . - . .:
':,: . . ~. ,: , ,... .~ .. ' . . ' being dissolved and sterilization cannot be accomplished by filtration. The compound can be s-terilized by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
The compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by ~eight, of the active material, depending on the method of administration.
Where the compositions comprise dosage units, each unit will preferably contain from 50-5~0 mg., of the active ingredient. The dosage as employed for adult human treatment will preferably range from 100 mg to 3 g., per day, for instance 250 mg-2 g., per day, depending on the route and frequency of adminis-tration.
Alternatively pseudomonic acid C or a sal-t or ester may be adrninistered as part of the total dietary intake. In this case the amount of compound employed may be less than 1% by weight of the diet and in pre-ferably no more than 0.5% by weight. The diet for ani-mals may consist of normal foodstuffs to which the com-pound may be added or it may be added to a premix.
The present invention also provides a process for the preparation of pseudomonic acid C or a salt or ester thereof which process comprises reacting pseudomonic acid A or a salt or ester thereof with a r~agent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(ij forming a sal-t of the pseudomonic acid C produced;
(ii~ esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic ~cid C.
A number of reagents for converting an epoxide to an olefin are known in the literature, and the part-icular reagent of choice for the process of the pre-sent invention is a matter of trial and error. Some .. : . . . .
'. , ' . ' :
: . ~
-such reagents are more suitable than others for this purpose. Some generally applicable methods are as follows:
(a) Potassium selenocyanate in methanol/water;
(see JCS Chem. CommO, 19/5, 1~16; J~S 1949~ 278j ~b) Lower valent tungsten halides; for example WC16/
butyl lithium (see J~Amer.Chem.SocO 1972,94,6538) (c) Ph3P = Se/-trifluroacetic acid;
(see JCS Chem. Comm. 1973, 253) ~O (d) Trifluoroacetyl iodide/sodium iodide;
(see J.Org.Chem., 1978~43,1841).
Other methods are described in the followiny references:
J. ~mer. Chem. Soc., 1973, 95, 2697.
Tet. Letts (17) 1976, 1395.
Ber. 1955, 88~ 165~.
J, Org. Chem., 1958t 22, 1118.
It has been ~ouncl that one convenient method is the use of potassium selenocyanate.
Suitable so]vents for use with potassium selen-ocyanate include mixtures of water with alkanols, in particular Cl-C20 alkanols. It has been found that higher yields of the compound of formula (II) are achieved if an alcohol is employed with a large, in particular branched or cyclic, alkyl group. Specific alcohols include iso-hexyl alcohol, tert-amyl alcohol and cyclohexyl alcohol. The reaction is generally performed at elevated temperatures, suitably at about the boiling point o the solvent employed. The time for which the reaction is performed depends on the temp~-erature of the reaction, and therefore on the solvent.
Generally a time of from 2-9 days is suitable.
Another suitable method for converting the epoxide of pseudomonic acid A, or a salt or ester thereof into an olefin, comprises treatment with trifluoroacetyl iodide and sodium iodide. ~he trifluoroacetyl iodide may be prepared in situ from trifluoroacetic anhydride.
The reaction is suitably conducted at ambient ,;' . . . . . . .
. . . :. .
. . . ' , ' ' ' . - . :
. .
temperature for ~rom about 10 to 36 hours, suitably about 24 hours.
When the free acid or salt of pseudomonic acid C is required it may be convenient to employ an ester of pseudomonic acid A for the above process9 which ester is a carboxyl-protecting group. Suitable carb-oxyl-protecting groups would depend on the reaction condi-tions for de epoxidation and include the 2,2,2-trichloro-ethyl ester, (which may be removed with zinc in a lower alcohol, espcially methanol) phenyl, penta-chlorophenyl, benzyl, and t-butyl ester groups. Other suitable carboxyl-protecting are silyl groups. In this case the carboxylic acid is reac-ted with a silyl-ating agent such as a halosilane or a silazane. A
preferred silylating agent is N,O-bis(trimethylsilyl) acetamide, which produces the trimethyl-silyl deriva-tive of the acid.
Prior to the above process of this invention, it may be desirable to protect the hydroxyl groups in pseudomonic acid A or its salt or ester. Although the reaction is possible without hydroxyl protection, in some cases higher yields of the pseudomonic acid C
derivative could be formed if the hydroxyl groups were protected. Such protecting groups must be removable under suitably mild conditions and suitable groups include silyl groups produced from a silylating agent as discussed above. Particularly suitable hydroxyl-protectlng groups include tri-methylsilyl, t-butyl-dimethylsilyl 7 methylthiomethyl. A preferred hydroxyl-protecting group is trimethylsilyl, as it is readily removed on completion o~ the reaction. Alternatively, for some de-epoxidation reactions it is possible to protect the hydroxyl groups with other ester radicals which may be removed by chemical or enzymatic means.
Examples include p-nitrobenzoate, methoxyaceta-te, phenoxyacetate, trifluoroacetate, each of which may be .
.. ~ , ~ . . ........................ ,... :: ::
-removed under mild basic conditions such as aqueous ammonia; or potassium carbonate in aqueous methanol.
It is also possible to protect the glycol moiety in pseudomonic acid A, and suitable reagents for form-lng such a hydroxyl-protecting group include compounds of formula (IV).
oR2 ., Rl C oR3 (IV) OR
wherein Rl is hydrogen or a Cl 6 alkyl group and R , R and R independently represent a Cl 6 alkyl group.
The group Rl may be for example hydrogen, methyl, ethyl, n- or iso-propyl. Most suitably, Rl represents hydrogen so tha-t the compound of formula (I~) is a trialkyl orthoformate. -Groups R2, R3, and R4 may be for example, methyl, ethyl, n- or iso-propyl, n-, _ -, sec- or tert-butyl. Preferably R2, R3, and R are all the same and each represents a methyl group.
Other glycol pro-tecting groups include those wherein the glycol moiety is converted to the structure:
o \ / ~a o / \Rb ' ' where Ra and Rb are hydrogen, Cl 6 alkyl, or phenyl.
Preferably R and R are both methyl, i.e. the group is the isopropylidene group. This group may be intro-duced onto pseudomonic acid A or its salt or ester by reaction with 2,2-dimethoxypropane, and removed by .
.: . :
.
~ .
' ' ' ' ': ' ' ': ' . . ~ , treatment with acetic acid.
The hydro~y-protecting group may be removed by a conventional method for the particular hydroxyl-protect~
ing group.
It may be such that it can be removed directly or alternatively, it may be converted into a different pro-tecting group which is then removable under ~lifferent conditions. This latter approach may be employed when a glycol protecting group derived from a compound (IV) is used; it is converted by acid to the group -OCOR
which is then removed.
When an ester of pseudomonic acid C is required, the esterification step, step (ii) above may be per~
formed by any conventional method, for exa~ple by reaction of the acid, or a salt thereof:-(a) with the appropriate halide, sulphate or alkane-sulphonate of the alcohol in the presence of a solvent such as acetone, dimethylsulphide or dimethvlsulphoxicle and calcium, or potassium carbonate or with the halide in the presence of hexamethyl phosphoramide; or (b) by phase transfer catalysis methods with the halide and/or sulphate of the alcohol in aqueous and/or organic solution in the presence of a quaternary ammon-ium salt such as tetrabutyl ammonium bisulphate or halide, or benxyltrime-thyl-ammonium halide; or (c) with a diazoalkane.
The hydrolysis of an ester of pseudomonic acid C
(step ~iii) above) may be chemical hydrolysis, for example by selective alkaline hydrolysis; or enzymic hydrolysis, for example by the use of Bakersl Yeast.
Also included within the scope of the present invention is a process for the preparation of pseudo-monic acid C or a salt or ester thereof which comprises yrowing Pseudomo_as fluorescens under aerobic conditions on or in a culture medium containing inoxganic salts and sources of asimilable carbon and nitxogen until the culture medium exhibits at least detectable antibacterial .: . :: : . - .
.. . , . . : . .. .:
- . . .
.
.
v`~
activity, acidifying the culture medium; extracting with an organic solvent for the active materials dissolved in the culture medium9 and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally there-after esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
In the above process, the cultivation step where Pseudomonas fluorescens is grown is conventional.
Any strain of this organism may to our knowledge be employed; one suitable public strain being Pseudomonas fluorescens NCIB 10586. (NCIB = National Collection of Industrial Bacteria).
After the fermentation is completed, the active rnater:ials, lncludlng pseudomonic acid C, are extracted from the acidified aqueous culture ~ledium into a suit-able solvent.
Preferably, the extraction procedure comprises extraction of the culture medium, after acidification, with an organic solvent; re-extraction of the organic extract with aqueous alkaline buffer solution; and finally re-extraction of the latter, after acidifica-tion, with organic solvent. Suitable solvents can be found by trial and error; examples include iso-butylmethyl ketone (IBMK), chloroform and preferably ethyl acetate.
The pseudomonic acid C may then be separated from other active materials produced in the ferment-ation, either directly (step (a) above) or by esterifying the mixture and separating the ester of pseudomonic acid C (step (b) above). When Pseudo- -monas flu~orescens NCIB 10586 is employed as the bact-erium, the majox material which is produced in . .
. : ;, . ..
. ' ,~
addition to pseudomonic acid C is pseudomonic acid A.
If this latter material is present in substantial quantities it is preferable to remove the majority by crystallisation of pseudomonic acid A9 optionally with seeding, from a suitable solvent for example diethyl ether.
If alternative (a) is carried out pseudomonic acid C may be separated directly by chromatography from the remaining mixture, either as the free acid itself or as a slat thereof. On chxomatography on silica gel, pseudomonic acid C is eluted slightly before pseudomonic acid A and the fractions can be identified accordingly.
The separated pseudomonic acid C or a salt thereof lS may be esterified by any of the methods described earlier in this specification.
If alternative (b) above is carried out the mixture of active materials is first esterified, preferably after removing the majority of the pseudo-monic acid A by crystallisation. Again any of the above described methods of esterification may be employed. It is convenient to form Cl 6 alkyl esters of the components in the mixture, preferably me-thyl esters.
The resultant mixture of esters may then be subjected to chromatography and the desired ester of pseudomonic acid C thereby separated. If the free acid or salt is required they may be produced by chemical or enzymatic hydrolysis of the separated ester.
The invention is illustrated in the following Examples.
' .
' :
. - . , r~ r~
Example 1 Methyl 10,11-D~oxypseudomonate_A (methyl pseudomonate _) from meth~ seudomonate A
A solution of methyl pseudomonate A (1.03 g ;
2 m.mole) and potassium selenocyanate (0.846 g ;
6 m.mole) in methanol-water 9 : 1 (30 ml) was heated under reflux for 7 days. The black precipitate of selenium was filtered off and the flltrate evaporated - to an oil. The latter was partitioned be-tween ethyl acetate and water and the organic phase separated, washed with sodium bicarbonate, brine, dried (MgS04) and evaporated to an oil. Chromatography on silica gel H (type 60) using a gradient of chloroform -to 4~0 methanol-chloroform afforded methyl 10,11 deoxypseudo-monate as an oil (0.129 g), tlc in chloroform-methanol 9 : 1 showed one component at Rf = 0.46 and a single Y P ~ max (C~IC13) 3400, 2900, 2850, 1720, 1~10, 1650, 1150 and 980 cm , Amax (EtOH) 221 nm (E 11,500), ~H(CDC13) 5.75 (IH, m, C2-H), 5.40 (2H, m, C10-H and Cll-H), 4.05 (2H, t, C97-CH2) 3.65 (3H, s, CH30), 2.21 (3H, broad s, C15-CH3), 1.21 (3H, d, C17-CH3) and 1.00 (3H, d, C14-CH3), ~c(CDCl3, 174.3 (Cl'), 166.8(Cl), 156.8(C3), 134.5 and 129.4 (clO and 11), 1~.7.6 (C2), 74.8 (C5), 71.2 (C13), 70.4 (C7), 2S 68.9 (C6), 64~8 (C16), 63.8 (C9'), 51.4 (CH30), 44.7 and 43.1 ~C4 and 12), 42.0 (C8), 34.1 (C27), 32.4 (C9), 29.1 (C41,5~ and 6'), 28.7 (C87), 26.0 (C7'), 24.9 (C3'), 20.4 'C14) 9 19.1 (C15) and 16.6 (C17), m/e (relative intensity) 499 (100%, M+~l by C~
:
,:
Example 2 Pseudomonic acid_C b~ fexmentation (a) fermen-tation Pseudomon _ fluorescens, strain NCIB lOS86 was cultured on an agar slope and flooded with sterile water. A sample was added to the following seed staye medium:
Oxoid*yeast extract 2% (w/v) Glucose O.ll lO Disodium hydrogen orthophosphate 0.2~
Potassium dyhydrogen orthophosphate 0.24 This was grown at 28C overnight and then used to inoc-ulate the :~ollowing production stage medium:
Corn steep liquor 0.3~o (w/v) 15 Glucose 2.0 Glycerol O.S
Am~nonium sulphate 0.2 Calcium carbonate 0.4 Potassium dyhydrogen orthophosphate 0.04 20 Disodium hydrogen orthophosphate 0.0~5 Manganese chloride R H20 0.0003 Potassium chloride 0.05 Magnesium sulphate 7 H20 0.0375 P2000 to minimise foaming.
The fermen-tation was carried out at 25C for 48 hours when production was essentially complete.
Aftèr removing the cells by centrifugation the super-natant was partitioned into ethyl acetate at pH 3. The ethyl acetate solution was dried ~MgSO~) evaporated to low volume, ether added and pseudomonic acid A allowed to crystallise.
(b) Isolation of Pseudomonlc_acid C
The mother liquors from the above crystallization * Trade Ma~k ~ ~ ' :
.; : . : . -.
_ 15 -were evaporated to an oil and chromatographed on silica gel preparative 20 x 20 cm thin layer chromato-graphy plates developed with chloroform : isopropanol :
acetic acid (80 : 20 : 0.5). The band above pseudomonic acid A of Rf 0.65 was removed and re-chromatographed as before to give pseudomonic acid C. [~ound: C, 6~.0;
H, 9.3%, C26H4408 requires C, 64.4; H9 9.2%], ~max (CHC13) 3430 (broad, 1710, 1650, 1220 (broad), 1153, 1110, 1050, and 977 cm 1, ~max (EtOH) 222 nm ( 14,100) ~H(CDC13) 5.69 (lH, m, C2-H), 5.4 (2H, m, C10-H), 4.65 (4H, broad), 4.01 (2H, t, C9'-CH2), 2.15 (3H, S5 C15-CH3), 1-12 (3H, d, C17-CH3; J = 6 Hz), 0.96 (3H, d, C14-CH3; J = 8 Hz), ~C (CDC13) 178.1 (Cl~), 166.9 (Cl), 156.9 (C3), 134.5 and 129.5 (C10 and Cll), 117.6 (C2), 74.9 (C5), 71.4 (C13), 70.4 (C7), 69.0 (C6), 64.9 ~C16), 63~9 (C9~), 44.7 (C12), 43.0 (C~), 41.9 (C8), 34.0 (C2'), 32.4 (C9), 28.9 (C4', 5' and 6'), 28.6 (C8~), 25.9 (C7'), 24.7 (C3'), 20.4 (C14), 19.2 (C15), 16.7 (C17).
Examele 3 Isolation o~ ~S~ Y~b~Y~ LY~a The residual oil from the mother liquors from Example 2(a) (ca 5 y) was dissolved in methanol (50 ml), diluted with water (50 ml) and the pH adjusted to 7 with aqueous sodium hydroxide. After evaporation to dryness a solution of the residue in dimethylformamide (50 ml), hexame-thylphosphoramide (5 drops) and methyl iodide (5 ml) was stirred overnight at room temperature.
The solution was evaporated to dryness and the residue partitioned between ethyl acetate (50 ml) and water (20 ml). The organic layer was washed with saturated brine, aqueous sodium bicarbonate, brine, dried (MyS04)~
and evaporated to an oil from which some methyl .
.
' .. .
- : .... ' . ' ' '7 pseudomonic A crystallised. The residual oil was chromatographed twice on silica (20 g then 15 g, type 60) eluting with gradient of O - ~% methanol -chloroform. ~ractions con-taining substantially pure methyl pseudomonate C (Rf 0.4~ silica tlc, cnloroform/
methanol 9 : 1, methylpseudomonate A Rf 0.42) were com-bined and chromatographed on silica (~ g, type 60) using gradient of O - 3% methanol - chloroform (distilled from phosphorus pentoxide). Fractions containing pure methyl pseudomonate C (by tlc) were combined and evap-orated to an oil (50 mgs) which was found to be spectro-scropically and chromatographically identical to methyl 10~11-deoxypseudomonate A obtained -in Example 1.
Example 4 Pseudomonic Acid C from meth~1 pseudomonate_C
OH
HO~ ~ O ~ ~ OH
~ ' O
OH
Methyl pseudomonate C (230 mgs) from Example 3 was dissolved in DM~ (25 ml) and diluted with 0.05 M
phosphate buffer (120 ml) then stirred with Bakers ~east (6 g) overnight. The mixture was filtered, evaporated to dryness and the residue dissolved in ethyl acetate (50 ml) and water (50 ml). The organic layer was washed with water and combined aqueous layers adjusted to pH
6 m.mole) in methanol-water 9 : 1 (30 ml) was heated under reflux for 7 days. The black precipitate of selenium was filtered off and the flltrate evaporated - to an oil. The latter was partitioned be-tween ethyl acetate and water and the organic phase separated, washed with sodium bicarbonate, brine, dried (MgS04) and evaporated to an oil. Chromatography on silica gel H (type 60) using a gradient of chloroform -to 4~0 methanol-chloroform afforded methyl 10,11 deoxypseudo-monate as an oil (0.129 g), tlc in chloroform-methanol 9 : 1 showed one component at Rf = 0.46 and a single Y P ~ max (C~IC13) 3400, 2900, 2850, 1720, 1~10, 1650, 1150 and 980 cm , Amax (EtOH) 221 nm (E 11,500), ~H(CDC13) 5.75 (IH, m, C2-H), 5.40 (2H, m, C10-H and Cll-H), 4.05 (2H, t, C97-CH2) 3.65 (3H, s, CH30), 2.21 (3H, broad s, C15-CH3), 1.21 (3H, d, C17-CH3) and 1.00 (3H, d, C14-CH3), ~c(CDCl3, 174.3 (Cl'), 166.8(Cl), 156.8(C3), 134.5 and 129.4 (clO and 11), 1~.7.6 (C2), 74.8 (C5), 71.2 (C13), 70.4 (C7), 2S 68.9 (C6), 64~8 (C16), 63.8 (C9'), 51.4 (CH30), 44.7 and 43.1 ~C4 and 12), 42.0 (C8), 34.1 (C27), 32.4 (C9), 29.1 (C41,5~ and 6'), 28.7 (C87), 26.0 (C7'), 24.9 (C3'), 20.4 'C14) 9 19.1 (C15) and 16.6 (C17), m/e (relative intensity) 499 (100%, M+~l by C~
:
,:
Example 2 Pseudomonic acid_C b~ fexmentation (a) fermen-tation Pseudomon _ fluorescens, strain NCIB lOS86 was cultured on an agar slope and flooded with sterile water. A sample was added to the following seed staye medium:
Oxoid*yeast extract 2% (w/v) Glucose O.ll lO Disodium hydrogen orthophosphate 0.2~
Potassium dyhydrogen orthophosphate 0.24 This was grown at 28C overnight and then used to inoc-ulate the :~ollowing production stage medium:
Corn steep liquor 0.3~o (w/v) 15 Glucose 2.0 Glycerol O.S
Am~nonium sulphate 0.2 Calcium carbonate 0.4 Potassium dyhydrogen orthophosphate 0.04 20 Disodium hydrogen orthophosphate 0.0~5 Manganese chloride R H20 0.0003 Potassium chloride 0.05 Magnesium sulphate 7 H20 0.0375 P2000 to minimise foaming.
The fermen-tation was carried out at 25C for 48 hours when production was essentially complete.
Aftèr removing the cells by centrifugation the super-natant was partitioned into ethyl acetate at pH 3. The ethyl acetate solution was dried ~MgSO~) evaporated to low volume, ether added and pseudomonic acid A allowed to crystallise.
(b) Isolation of Pseudomonlc_acid C
The mother liquors from the above crystallization * Trade Ma~k ~ ~ ' :
.; : . : . -.
_ 15 -were evaporated to an oil and chromatographed on silica gel preparative 20 x 20 cm thin layer chromato-graphy plates developed with chloroform : isopropanol :
acetic acid (80 : 20 : 0.5). The band above pseudomonic acid A of Rf 0.65 was removed and re-chromatographed as before to give pseudomonic acid C. [~ound: C, 6~.0;
H, 9.3%, C26H4408 requires C, 64.4; H9 9.2%], ~max (CHC13) 3430 (broad, 1710, 1650, 1220 (broad), 1153, 1110, 1050, and 977 cm 1, ~max (EtOH) 222 nm ( 14,100) ~H(CDC13) 5.69 (lH, m, C2-H), 5.4 (2H, m, C10-H), 4.65 (4H, broad), 4.01 (2H, t, C9'-CH2), 2.15 (3H, S5 C15-CH3), 1-12 (3H, d, C17-CH3; J = 6 Hz), 0.96 (3H, d, C14-CH3; J = 8 Hz), ~C (CDC13) 178.1 (Cl~), 166.9 (Cl), 156.9 (C3), 134.5 and 129.5 (C10 and Cll), 117.6 (C2), 74.9 (C5), 71.4 (C13), 70.4 (C7), 69.0 (C6), 64.9 ~C16), 63~9 (C9~), 44.7 (C12), 43.0 (C~), 41.9 (C8), 34.0 (C2'), 32.4 (C9), 28.9 (C4', 5' and 6'), 28.6 (C8~), 25.9 (C7'), 24.7 (C3'), 20.4 (C14), 19.2 (C15), 16.7 (C17).
Examele 3 Isolation o~ ~S~ Y~b~Y~ LY~a The residual oil from the mother liquors from Example 2(a) (ca 5 y) was dissolved in methanol (50 ml), diluted with water (50 ml) and the pH adjusted to 7 with aqueous sodium hydroxide. After evaporation to dryness a solution of the residue in dimethylformamide (50 ml), hexame-thylphosphoramide (5 drops) and methyl iodide (5 ml) was stirred overnight at room temperature.
The solution was evaporated to dryness and the residue partitioned between ethyl acetate (50 ml) and water (20 ml). The organic layer was washed with saturated brine, aqueous sodium bicarbonate, brine, dried (MyS04)~
and evaporated to an oil from which some methyl .
.
' .. .
- : .... ' . ' ' '7 pseudomonic A crystallised. The residual oil was chromatographed twice on silica (20 g then 15 g, type 60) eluting with gradient of O - ~% methanol -chloroform. ~ractions con-taining substantially pure methyl pseudomonate C (Rf 0.4~ silica tlc, cnloroform/
methanol 9 : 1, methylpseudomonate A Rf 0.42) were com-bined and chromatographed on silica (~ g, type 60) using gradient of O - 3% methanol - chloroform (distilled from phosphorus pentoxide). Fractions containing pure methyl pseudomonate C (by tlc) were combined and evap-orated to an oil (50 mgs) which was found to be spectro-scropically and chromatographically identical to methyl 10~11-deoxypseudomonate A obtained -in Example 1.
Example 4 Pseudomonic Acid C from meth~1 pseudomonate_C
OH
HO~ ~ O ~ ~ OH
~ ' O
OH
Methyl pseudomonate C (230 mgs) from Example 3 was dissolved in DM~ (25 ml) and diluted with 0.05 M
phosphate buffer (120 ml) then stirred with Bakers ~east (6 g) overnight. The mixture was filtered, evaporated to dryness and the residue dissolved in ethyl acetate (50 ml) and water (50 ml). The organic layer was washed with water and combined aqueous layers adjusted to pH
3 (5M HC1) and extxacted several times with e-thyl acetate. After drying the combined extracts were evaporated to yield an oil. Chromatography on silica . ' .
:: : .: . ' " : ' ~. ~ :
_ 17 -gel H (type 60, 8 g) eluting with gradient 0 -to 6%
methanol - chloroform gave pseudomonic acid C (150 mgs, 67%) which was chromatographically and spectroscopically identical to ma-terial obtained in Example 2.
~ æ~
Methyl Pseudomonate C
(Alternative procedure to Example 3) Mother liquors (15 g) from Example 2 (a) were dissolved in acetone (150 ml), and stirred overnight with potassium carbonate (42 g) and methyl iodide (21 ml) at room temperature. The reaction rnixture was f;.ltered and the iltrate evaporated to dryness then taken up i.n ethyl acetate/water and worked up as in Exa~ple 3 to give methyl pseudomonate C.
The methyl ester may be hydrolysed as in Example 4.
Sodium Pseudomonate C
_ (Sodium 10,11-deoxypseudomonate A) A soluti.on of methyl pseudomonate C (0.330 g) in distilled tetra-hydrofuran (20 ml) and N/10 sodium hydroxide solutlon (20 ml) was stirred at room temper-ature for 2 hours. The tetrahydrofuran was removed ', in vacuo to give a turbid aqueous solution, which was washed with,ether-ethyl acetate to remove unreacted ester~ The aqueous layer was saturated with sodium chloride~ layered wlth ethyl acetate and-acidified with dilute hydrochloric acid to pH 1.5. The ethyl acetate layer was separated, washed with brine, dried (MgS04) ., .
.
- -' ' ' ' ' : ~
.
' '7 and evaporated to dryness. The pseudomonic acid C
obtained was suspended in water - tetrahydrofuran and N/10 sodium hydroxide solution added to pH 7.5. The resulting aqueous solution was evaporated to dryness in vacuo. The residue was dissolved in dry methanol (5 ml) filtered and excess dry ether added to the filtrate with stirring. ~he resulting white precipi-tate of sodium pseudomonate C was collected and dried in vacuo (0.153 g). The compound was homogeneous by thin layer chromatography and high pressure liquid chromatography, [~]D ~ 0.94 (c 1.0, MeOH), vmax (KBr) 3400, 2800, 1700, 1640, 1560, 1230, 1150, 975 cm~1, x (BtOH) 221 nm (~ 13,300)~ ~H((CD3)2SO)) 5-7 (lH, m, vinylic-~), 5.3 (2H, m, -CH=CH-), 3.95 (2H, 5, CH2-9~), 2.08 (3H, s, vinylic -CH3), 0.95 ~3H, d, secondary -CH3) and 0.88 (3H, d, secondary -CH3).
Example 7 Sodium P_eudomonate`~C
Methyl pseudomonate C (1 g) was dissolved in TH~
(50 ml)/water (50 ml) and the pH adjusted to 12 and maintained for 21~ hours. The solution was adjusted to pH7 and evaporated to dryness then redissolved in water (30 ml) and washed with ethylacetate. The aqueous fraction was then acidified to pH2 and extracted with ethyl acetate. After drying (MgS04) the combined extracts were evaporated i.n vacuo. The resultant oil (0.55 g) was treated with sodium bicarbonate (95 mgs, 1 eq) in water (20 ml)/methanol (20 ml) and evaporated to dryness. The sodium salt was dissolved in ethanol (minimum~ and added dropwise to ether ~300 ml) and the precipitate filtered off (0.58 g, 57%) ? vmax (KBr) 3380 (broad), 1700, 1642, 1560, 1225~ 1150 and 973 cm ... .- - ~.. . . .
'7 ~max (EtOH) 222 nm (~ 13,700), ~H(CH30D) 1.07 (3H, d9 J 7Hz~ C17-CH3), 1-18 (3H, d, J 7H~, C14-CH3), 1.44 (12H, m,-~CH2)6), 2.25 (3H, s, C15-CH3), 4-11 (2H~ 5, C9-C~I~), 5.5 (2H, m, }1-10, H-ll), 5.79 (lH, s, H-2), ~c (CD30D) 183.0 (Cl7), i68.4 (~1), 158.9 (C3), 135.7, 129.6 (C10, Cll), 118,2 (C2), 76.2 (C5)9 72.0 (C7), 71.
(C13), 69.8 (C6), 65.6 (C16), 64.8 (C9l), 45.2 (C12), 44.0, 43.6 (C4, C8), 39.3 (C2~), 33.6 (C9), 30.7, 30.4, 30.3, 29.8, 27.7, 27.1 (C37-C87), 20.3 (C14). 19.4 ~C15), 16.6 (C17) (~ound: C, 59.4; H, 8.4; Na, 4.9~C26H4308 Na.H20 requires C, 59.5; H, 8.6; Na, 4.4%).
Example 8 Pseudomonic acld C
Pseudomonic acid A (500 mgs) was dissolved in 2,2-dimethoxypropane (20 ml) and ethyl acetate (20 ml) then ~-toluene sulphonic acid (few crystals) added.
The solution was stirred for 1 hour then washed with brine and dried (MgS04). After evaporation of the sol-vent in vacuo, the residue was dissolved in water-methanol (1:1, 20 ml) and potassium bicarbonate (100 mgs, 1 eq) added. The solvents were removed in v~cuo and potassium selenocyanate (432 mgs, 3 eq), tert-amyl al-cohol-water (9:1, 15 ml) added and the reaction ~ refluxed for 4 days. Af~er filtering the solution was evaporated to dryness, water (20 ml) added and solution adjusted to pH2 (5M-HCl) under a layer of ethyl acetate (20 ml). The organic layer was separated and the aqueous layer further extracted with ethyl acetate (3 x 20 ml). The combined extracts were dried (MgS04) and evaporated to an oil which was redissolved in ~0% acetic acid (10 ml) and stirred at room temp~
exature overnight. The solution was evaporated to dryness and the residual oil chromatographed on silica -:: , ~ , ,, - ' : ' ' : :
. , . ~. , . .
:
~ t7 (10 g) eluting with 0 to 6% methanol-chloroform.
~ractions containing pure product (tlc) were combined and evaporated to give pseudomonic acid C (280 mgs, 58%).
Example 9 Pseudomonic acid C
Pseudomonic acid A (500 mgs) was dissolved in 2,2-dimethyoxypropane (50 ml) and treated with p-toluene sulphonic acid (few crystals). After 1 hour the solution was diluted with ethyl acetate, washed with brine and dried (MgS0~). The solution was evaporated in vacuo and the residue redissolved in water-methanol (1:1, 20 ml) and potassium bicarbonate (100 mgs, 1 eq) added. The solvents were removed in vacuo and potas-sium selenocyanate (432 mgs 7 3 eq) and iso-hexylalcohol-water (9:1, 15 ml) added and reaction refluxed for 4 days. After filtering, the reaction mixture was diluted with ethyl acetate (20 ml~ and extracted with aqueous sodium bicarbonate (3 x 20 ml). The aqueous extracts were combined and acidified with acetic acid under a layer o~ ethylacetate (20 ml). After stirring for ca.
1 hour the organic layer was separated and aqueous layer further extracted with ethylacetate (3 x 20 ml).
The combined extracts were washed with hrine, dried (MgS04) and evaporated to an oil`. Chromatography of the oil on silica (5 g) gave pure pseudomonic acid C
(215 mgs, 45%).
.: ~ , . . :
,~ . ' ... ' . : , .
,, . . . .: .
Example 10 Methyl pseudomonate C
Methyl pseudomonate A (5.14 g), potassium selen-ocyanate (4.32 9) in iso~hexyl alcohol - water (9:1, 150 ml) were refluxed for 3 days. The reaction mixture was filtered then evaporated to dryness and dissolved in ethyl acetate (50 ml) - brine (50 ml)0 The organic layer was separated, washed with brine (50 ml) then dried (MgS04). After evaporation of the solvents 1n vacuo, the xesidue was chromatographed on silica (80 g) eluting with 0 - 4% methanol-chloroform. Pure fractions were combined and evaporated to an oil which on stand-ing gave crystalline r~ethyl pseudomonate C, mp. ~L7-9C
(2.57 g, 52%) (~ound: C, 65.0; H, 9.5. C27H4608 req-uires C, 65.0; ~I, 9.3%).
Exam~le 11 Methyl_pseudomonate C and pseudomonic acid ~
Methyl pseudomonate A (514 mgs) was dissolved in 2,2-dimethoxypropane (20 ml) and a few crystals of p~
toluene sulphonic acid added. The solution was stirred for 1~ hour then ethyl acetate (20 ml) added and -the solution washed with brine then dried (MgS04). After evaporation of the solvents~ the acetonide was dissolved in tert-amyl alcohol-water (9:1, 15 ml), potasslum selenocyanate (432 mgs) added and reaction refluxed ~or 5 days. The solution was filtered, evaporated to dryness and the residue dissolved in ethyl acetate (20 ml)-brine (20 ml). The organic layer was separated and the aqueous layer further extracted with ethyl acetate (3 x 20 ml). The combined extracts were dried (MgS04) and evaporated to dryness. The crude product . ' . : .' ~ , -,. ' - ' .~
. ~ . . .
:
~ 5 contained two major components (tlc) which were sep-arated by column chromatography o~ silica (10 g) elut-ing ~ith O - 8% methanol-chloro~orm. The first fraction was identified as methyl 6,7-0-isopropylidene pseu~o-~Gnate C (206 mgs, 38%), ~max (CHC:L3) 34503 1722, 1643 and 1220 cm 1, ~H~CDC13) 0.98 (3H, d, J 7Hz, C17-CH3), 1.13 (3H, d, J 7Hz, C14-CH3), 1.33 (15H, m, (CH2)6, acetonide CH3), 1.48 (3H, s, ace-tonide C_3), 2.18 (3H, s, C15-CH3), 3 63 (3H, s, OCH3), 4.05 (2H, 19 t, C91-CH2), 5.45 (2H, m, H-10, H-ll), 5.73 (lH9 s, H-2), ~c(CDCl3) 174.0 (Cl'), 166.6 (Cl), 156.2 (C3), 135.G9 128.9 (C10, Cll), 117.8 (C2), 108.7 (~ C ~ ), 76.5 (C5), 76.7 (C7), 74.3 (C6), 71.0 (C13), 66.5 (C16)~ 63-7 (C9~)s 51-3 (OCH3), 44.6 (C12), 44.1 (C4), 37.9 (C8), 34.2 (C2 9 ) ~ 34.1 (C9), 29-1 (C41~ 51~ 61)9 28.8 (C8l), 28.3, 26.3 (,,C ~MMee), 26.0 (C7'), 24.9 (C3')9 20.3 (C14), 19.1 (C15), 16.4 (Cl7), ~ e (relative int-ensity) 523 (6%), 494 (19), 436 (22), 369 (30), 306 ~22), 299 (20) (~ound: 523.3263~ M~-CH3 requires 523.3257), The second fraction was identified as 6,7-0-isopropylidene pseudomonic acid C (130 mgs, 2S%), ~ ax (CHC13) 2300-3600 (broad), 1702, 1642, 1220, 1152 and 1052 cm 1, ~H (CDC13) 0.98 (3H, d, J
7Hz, C17-CH3), 1.14 (3H, d, J 7Hz, C14-CH3), 1.33 (15H, m, ~CH2)6, acetonide CH3), 1.49 (3H, S9 acetonide CH3), 2.18 (3H, s, C15-CH3), 4-06 (2H~ t, C91-CH2)' 5.45 (2H, m, H-10, H-ll), 5.73 (lH, s, H-2), ~c (CDC13) 178.1 (Cl'), 166.7 (C~), 156.1 (C3)9 134.g, 129.0 (C10, Cll), 117.8 (C2), 108.7 (= C ~), 76.4 (C5), 75.6 (C7), 74.2 (C6), 71.2 (C13),66.4 (C16), 63.8 (C9~), 44.4 (C12), 44.0 (C4), 36-8 (C8), 34.0 ~C21), 33.7 (C9), 29.0 (C41~ 51, 6l), 28-7 (C81), 28.3, 26.2 (_~C--M )' 25.9 ~C7l), 24.7 (C3'), 20.1 (Cl~), 19.0 (C15), 16.4 (C17~, ~ e (relative intensity) 509 (7%), 480 (10), 422 (10), 404 (8), 394 (10), 387 (7~, 383 (8). (~ound:
509.3110. M+-CH3 requires 509.3108~. The acetonides , -' _ 23 -were quantitatively converted to methyl pseudomonate C
and pseudomonic acid C respectively with 80% acetic acid overnight.
Example 12 Methyl pseudomo a_e C
Sodium iodide (600 mgs, 4 eq) (dried at 110C/4 hours) was stirred in dry THF (1 ml) and dry acetoni-trile (1 ml) and trifluoroacetic anhydride (141 ~1, 1 eq) was added. After 5 minutes the yellow solution was cooled in ice and methyl pseudomonate A (514 mgs) added. After 5 minutes the ice bath was removed and the reaction stirred at room temperature for 24 hours.
The reaction mixture was diluted with aqueous sodium bisulph:ite arld extracted with ethyl ace~ate (4 x 25 ml).
The combined extracts were washed with brine then dried (MgS04) and evaporated to an oil then chromatographed on silica (5 g). Pure fractions (tlc, hplc) ~ere com-bined and evaporated to give desired product (63 mgs, 13%).
Isohexy~ pseudomonate_C
Methyl pseudomonate A (10 g) and po-tassium sel-enocyanate (4.32 g~ 1.5 eq) in 2-ethyl-n-butanol (iso-hexyl alcohol)-water (9:1, 150 ml) were refluxed for 2 days. The solution was filtered and evaporated then the residue redissolved in ethyl acetate~water. The organic layer was separated, washed with brine then dried (MgS04) and the solvent removed in vacuo. The crude product was chroFatographed on silica (100 g) . . :
- 24 _ elutin~ with 0 to 6% MeOH-CHC13. Fractions containing pure methyl pseudomonate C were combined and evaporated to an oil which crystallised on standing mp 47-9C
(2.2 g). Remaining fractions were combined and rechrom-atographed to yield a pure compound subsequently iden-tified as isohexyl pseudomonate C (1.0 g) vmax (CHC13) 3400 (broad), 1703, 1640, 1427, 1220, 1150, 1020 and 978 cm 1, ~H (CDC13) 0.94 (6H, t, (CH2CH3)2), 0-97 (3H, d, C17-CH3), 1.14 (3H, d, C14-CH3), 1~32 (12H~
m.-(CH2)6-), 2-19 (3H, s, C15-C_3), 3.95 (2H, d, OCH2CHEt2) 9 4.05 (2H, t, C9~-CH2), 5-45 (2H~ m~ H-10~
H-ll) 5-75 (lH, S9 H-2), ~c (CDC13) 174.0 (C1~3, 166.9 (C13, 157.2 (C3), 134.4, 129.0 (C10, Cll), 117.8 (C2), 75~0 (C5), 71.2 (C13), 70.4 (C7), 68.8 (C6), 6603 (0_~12CHEt2), 64.9 (C16), 63.8 (C9'), 44.4 (C12), 43.2 (C4), 42-1 (C8), 40.5 (OCH2C~IEt2), 34.4 (C2'), 32.5 (C9), 29.1 (C4', C597 C6l), 28.8 (C8'), 26.0 (C7l), 25-0 (C3~), 23-4 (CH2CH3) 20.3 (C14), 19.2 (C15), 16.4 (C17), 11.0 (CH2CH3).
:. . . ., :. .. . ..
.:
: . : .
, ,. .,, - : ., . . . : . -BIOLOGICAL DATA
(1) Antibacterial activity - human or~anisms Table 1 shows the antibacterial spectrum of sodium pseudomonate C and methyl pseudomonate C in terms of minimum inhibitory concentration (~g/ml) measured by serial dilution in nutrient agar con-taining 5% chocolated horse blood.
_ _ _ . ___ ORGANISM M.I~C. (~g/ml) __ _ ._ sodium methyl pseudo- pseudo-monate C monate C
E. coli NCTC 10418 >100 >100 E. coli ESS 1.0 2.5 P. mirabilis 889 >100 >100 K. aerogenes A >100 >100 Ps aeruginosa NCTC 10701>100 ~100 Pasteurella multocida 1633 1.0 2.5 Haemophilus influenzae Ql0.1 <0.2 Haeomophilus influenzae Wy 21 0.1 0.5 Neisseria flavescens 66330.2 0.5 Bacillus subtilis 0.02 <0.2 Corynebacterium xerosis 9755 ~100 ~100 Sarcina lutea ~100 ~100 Staph. aureus Oxford 0.1 <0.2 Staph. aureus Russell 0.2 0.5 Staph. aureus 1517 0.2 0.5 Strep. faecalis I 100 ~100 Strep. Pyogenes A 64/848 0.1 1.0 Strep. Pyogenes B 2788 2.5 1.0 Strep. Pyogenes C 2761 0.2 1.0 Strep. pneumoniae CN33 0.1 Ool __ __ _ _ .... . ~_ _ ., ~' ' , Y~ 7 (2) Anti-myco~asma activity Methyl pseudomonate C possess good antimyco plasma activity in vitro against mycoplasmas ~om human and veterinary sources, as shown in table 2.
Method (1) The minimum inhibitory concentrations ~MIC) of methyl pseudomonate C were determined in Microtitre plates, by a modification of the metabolic-inhibition test (Taylor-Robinson, 1967). The compounds were serially diluted in sterile de-ionised water to give a range of concentrations from 250-0.5 ~g/ml. Mycoplasma broth containing 1% (w/v~ of glucose and 0.005% (w/v) of phenon red, was added at a strength to compensate for its dilution by the aqueous drug solution. Appro~-imately 10~ colony forming units of mycoplasma were added to each concentration of dxug. Drug-free infected, non-infec-ted and pH control wells were included on each plate. Plates were sealed with cellophane tape and incubated at 37C for seven days. The MIC was the lowest concentration of compound that prevented a colour change in the mycoplasma broth, caused by the metabolism.
Reference ~_ .
Taylor-Robinson, 1967. Mycoplasmas of various hosts and their antibiotic sensitivities. Post. Grad. Med. J., 43 Suppl. CMarch~, 100.
. : , , ' '.,, :. .: .' ' ` .
' , ' ' .'., :.: . . , ~: ..
' '' ' ' ' . '....... ' :' -' - .'' ' ' . '' ' ' .. : ., . . :. , . - . .... . .
_ 27 -. _ ~_ MYCOPLASMA M.I.C. (~g/ml) __ _ __ M. gallisepticum S.6 62.5 M. synoviae 25204 ~0.5 M. pulmonis JB <0.5 M. suipneumoni~e L~ber <0.5 M. pneumoniae 427a 1.0 M. fermentans MW KL4 <0.5 Table 3 shows MIC values for sodium pseudomonate C and met'nyl pseudomonate C against further mycoplasma species determined in Friis~ broth using the micro-titer method.
ORGANISM M.I.C (~g/ml) . ~ , .
MethylSodium Pseudo-Pseudo-monate Cmonate C
M. suipneumoniae Str. 11 ~10 ~10 M. suipneumoniae J2206/183b >10 10 M. dispar H225 10 5.0 M. dispar NCTC 10125 50 2.5 M. pneumoniae 427a ~10 2.5 M. pneumoniae ATCC 15492 10 -M. fermentans MWKL4 0.039< 0.02 M. pulmonis JB 0.3120,039 . ~
.
.' ~ ,' . : ' .. " :, ' ~, ...... ... .
.. . . . . .
:: : .: . ' " : ' ~. ~ :
_ 17 -gel H (type 60, 8 g) eluting with gradient 0 -to 6%
methanol - chloroform gave pseudomonic acid C (150 mgs, 67%) which was chromatographically and spectroscopically identical to ma-terial obtained in Example 2.
~ æ~
Methyl Pseudomonate C
(Alternative procedure to Example 3) Mother liquors (15 g) from Example 2 (a) were dissolved in acetone (150 ml), and stirred overnight with potassium carbonate (42 g) and methyl iodide (21 ml) at room temperature. The reaction rnixture was f;.ltered and the iltrate evaporated to dryness then taken up i.n ethyl acetate/water and worked up as in Exa~ple 3 to give methyl pseudomonate C.
The methyl ester may be hydrolysed as in Example 4.
Sodium Pseudomonate C
_ (Sodium 10,11-deoxypseudomonate A) A soluti.on of methyl pseudomonate C (0.330 g) in distilled tetra-hydrofuran (20 ml) and N/10 sodium hydroxide solutlon (20 ml) was stirred at room temper-ature for 2 hours. The tetrahydrofuran was removed ', in vacuo to give a turbid aqueous solution, which was washed with,ether-ethyl acetate to remove unreacted ester~ The aqueous layer was saturated with sodium chloride~ layered wlth ethyl acetate and-acidified with dilute hydrochloric acid to pH 1.5. The ethyl acetate layer was separated, washed with brine, dried (MgS04) ., .
.
- -' ' ' ' ' : ~
.
' '7 and evaporated to dryness. The pseudomonic acid C
obtained was suspended in water - tetrahydrofuran and N/10 sodium hydroxide solution added to pH 7.5. The resulting aqueous solution was evaporated to dryness in vacuo. The residue was dissolved in dry methanol (5 ml) filtered and excess dry ether added to the filtrate with stirring. ~he resulting white precipi-tate of sodium pseudomonate C was collected and dried in vacuo (0.153 g). The compound was homogeneous by thin layer chromatography and high pressure liquid chromatography, [~]D ~ 0.94 (c 1.0, MeOH), vmax (KBr) 3400, 2800, 1700, 1640, 1560, 1230, 1150, 975 cm~1, x (BtOH) 221 nm (~ 13,300)~ ~H((CD3)2SO)) 5-7 (lH, m, vinylic-~), 5.3 (2H, m, -CH=CH-), 3.95 (2H, 5, CH2-9~), 2.08 (3H, s, vinylic -CH3), 0.95 ~3H, d, secondary -CH3) and 0.88 (3H, d, secondary -CH3).
Example 7 Sodium P_eudomonate`~C
Methyl pseudomonate C (1 g) was dissolved in TH~
(50 ml)/water (50 ml) and the pH adjusted to 12 and maintained for 21~ hours. The solution was adjusted to pH7 and evaporated to dryness then redissolved in water (30 ml) and washed with ethylacetate. The aqueous fraction was then acidified to pH2 and extracted with ethyl acetate. After drying (MgS04) the combined extracts were evaporated i.n vacuo. The resultant oil (0.55 g) was treated with sodium bicarbonate (95 mgs, 1 eq) in water (20 ml)/methanol (20 ml) and evaporated to dryness. The sodium salt was dissolved in ethanol (minimum~ and added dropwise to ether ~300 ml) and the precipitate filtered off (0.58 g, 57%) ? vmax (KBr) 3380 (broad), 1700, 1642, 1560, 1225~ 1150 and 973 cm ... .- - ~.. . . .
'7 ~max (EtOH) 222 nm (~ 13,700), ~H(CH30D) 1.07 (3H, d9 J 7Hz~ C17-CH3), 1-18 (3H, d, J 7H~, C14-CH3), 1.44 (12H, m,-~CH2)6), 2.25 (3H, s, C15-CH3), 4-11 (2H~ 5, C9-C~I~), 5.5 (2H, m, }1-10, H-ll), 5.79 (lH, s, H-2), ~c (CD30D) 183.0 (Cl7), i68.4 (~1), 158.9 (C3), 135.7, 129.6 (C10, Cll), 118,2 (C2), 76.2 (C5)9 72.0 (C7), 71.
(C13), 69.8 (C6), 65.6 (C16), 64.8 (C9l), 45.2 (C12), 44.0, 43.6 (C4, C8), 39.3 (C2~), 33.6 (C9), 30.7, 30.4, 30.3, 29.8, 27.7, 27.1 (C37-C87), 20.3 (C14). 19.4 ~C15), 16.6 (C17) (~ound: C, 59.4; H, 8.4; Na, 4.9~C26H4308 Na.H20 requires C, 59.5; H, 8.6; Na, 4.4%).
Example 8 Pseudomonic acld C
Pseudomonic acid A (500 mgs) was dissolved in 2,2-dimethoxypropane (20 ml) and ethyl acetate (20 ml) then ~-toluene sulphonic acid (few crystals) added.
The solution was stirred for 1 hour then washed with brine and dried (MgS04). After evaporation of the sol-vent in vacuo, the residue was dissolved in water-methanol (1:1, 20 ml) and potassium bicarbonate (100 mgs, 1 eq) added. The solvents were removed in v~cuo and potassium selenocyanate (432 mgs, 3 eq), tert-amyl al-cohol-water (9:1, 15 ml) added and the reaction ~ refluxed for 4 days. Af~er filtering the solution was evaporated to dryness, water (20 ml) added and solution adjusted to pH2 (5M-HCl) under a layer of ethyl acetate (20 ml). The organic layer was separated and the aqueous layer further extracted with ethyl acetate (3 x 20 ml). The combined extracts were dried (MgS04) and evaporated to an oil which was redissolved in ~0% acetic acid (10 ml) and stirred at room temp~
exature overnight. The solution was evaporated to dryness and the residual oil chromatographed on silica -:: , ~ , ,, - ' : ' ' : :
. , . ~. , . .
:
~ t7 (10 g) eluting with 0 to 6% methanol-chloroform.
~ractions containing pure product (tlc) were combined and evaporated to give pseudomonic acid C (280 mgs, 58%).
Example 9 Pseudomonic acid C
Pseudomonic acid A (500 mgs) was dissolved in 2,2-dimethyoxypropane (50 ml) and treated with p-toluene sulphonic acid (few crystals). After 1 hour the solution was diluted with ethyl acetate, washed with brine and dried (MgS0~). The solution was evaporated in vacuo and the residue redissolved in water-methanol (1:1, 20 ml) and potassium bicarbonate (100 mgs, 1 eq) added. The solvents were removed in vacuo and potas-sium selenocyanate (432 mgs 7 3 eq) and iso-hexylalcohol-water (9:1, 15 ml) added and reaction refluxed for 4 days. After filtering, the reaction mixture was diluted with ethyl acetate (20 ml~ and extracted with aqueous sodium bicarbonate (3 x 20 ml). The aqueous extracts were combined and acidified with acetic acid under a layer o~ ethylacetate (20 ml). After stirring for ca.
1 hour the organic layer was separated and aqueous layer further extracted with ethylacetate (3 x 20 ml).
The combined extracts were washed with hrine, dried (MgS04) and evaporated to an oil`. Chromatography of the oil on silica (5 g) gave pure pseudomonic acid C
(215 mgs, 45%).
.: ~ , . . :
,~ . ' ... ' . : , .
,, . . . .: .
Example 10 Methyl pseudomonate C
Methyl pseudomonate A (5.14 g), potassium selen-ocyanate (4.32 9) in iso~hexyl alcohol - water (9:1, 150 ml) were refluxed for 3 days. The reaction mixture was filtered then evaporated to dryness and dissolved in ethyl acetate (50 ml) - brine (50 ml)0 The organic layer was separated, washed with brine (50 ml) then dried (MgS04). After evaporation of the solvents 1n vacuo, the xesidue was chromatographed on silica (80 g) eluting with 0 - 4% methanol-chloroform. Pure fractions were combined and evaporated to an oil which on stand-ing gave crystalline r~ethyl pseudomonate C, mp. ~L7-9C
(2.57 g, 52%) (~ound: C, 65.0; H, 9.5. C27H4608 req-uires C, 65.0; ~I, 9.3%).
Exam~le 11 Methyl_pseudomonate C and pseudomonic acid ~
Methyl pseudomonate A (514 mgs) was dissolved in 2,2-dimethoxypropane (20 ml) and a few crystals of p~
toluene sulphonic acid added. The solution was stirred for 1~ hour then ethyl acetate (20 ml) added and -the solution washed with brine then dried (MgS04). After evaporation of the solvents~ the acetonide was dissolved in tert-amyl alcohol-water (9:1, 15 ml), potasslum selenocyanate (432 mgs) added and reaction refluxed ~or 5 days. The solution was filtered, evaporated to dryness and the residue dissolved in ethyl acetate (20 ml)-brine (20 ml). The organic layer was separated and the aqueous layer further extracted with ethyl acetate (3 x 20 ml). The combined extracts were dried (MgS04) and evaporated to dryness. The crude product . ' . : .' ~ , -,. ' - ' .~
. ~ . . .
:
~ 5 contained two major components (tlc) which were sep-arated by column chromatography o~ silica (10 g) elut-ing ~ith O - 8% methanol-chloro~orm. The first fraction was identified as methyl 6,7-0-isopropylidene pseu~o-~Gnate C (206 mgs, 38%), ~max (CHC:L3) 34503 1722, 1643 and 1220 cm 1, ~H~CDC13) 0.98 (3H, d, J 7Hz, C17-CH3), 1.13 (3H, d, J 7Hz, C14-CH3), 1.33 (15H, m, (CH2)6, acetonide CH3), 1.48 (3H, s, ace-tonide C_3), 2.18 (3H, s, C15-CH3), 3 63 (3H, s, OCH3), 4.05 (2H, 19 t, C91-CH2), 5.45 (2H, m, H-10, H-ll), 5.73 (lH9 s, H-2), ~c(CDCl3) 174.0 (Cl'), 166.6 (Cl), 156.2 (C3), 135.G9 128.9 (C10, Cll), 117.8 (C2), 108.7 (~ C ~ ), 76.5 (C5), 76.7 (C7), 74.3 (C6), 71.0 (C13), 66.5 (C16)~ 63-7 (C9~)s 51-3 (OCH3), 44.6 (C12), 44.1 (C4), 37.9 (C8), 34.2 (C2 9 ) ~ 34.1 (C9), 29-1 (C41~ 51~ 61)9 28.8 (C8l), 28.3, 26.3 (,,C ~MMee), 26.0 (C7'), 24.9 (C3')9 20.3 (C14), 19.1 (C15), 16.4 (Cl7), ~ e (relative int-ensity) 523 (6%), 494 (19), 436 (22), 369 (30), 306 ~22), 299 (20) (~ound: 523.3263~ M~-CH3 requires 523.3257), The second fraction was identified as 6,7-0-isopropylidene pseudomonic acid C (130 mgs, 2S%), ~ ax (CHC13) 2300-3600 (broad), 1702, 1642, 1220, 1152 and 1052 cm 1, ~H (CDC13) 0.98 (3H, d, J
7Hz, C17-CH3), 1.14 (3H, d, J 7Hz, C14-CH3), 1.33 (15H, m, ~CH2)6, acetonide CH3), 1.49 (3H, S9 acetonide CH3), 2.18 (3H, s, C15-CH3), 4-06 (2H~ t, C91-CH2)' 5.45 (2H, m, H-10, H-ll), 5.73 (lH, s, H-2), ~c (CDC13) 178.1 (Cl'), 166.7 (C~), 156.1 (C3)9 134.g, 129.0 (C10, Cll), 117.8 (C2), 108.7 (= C ~), 76.4 (C5), 75.6 (C7), 74.2 (C6), 71.2 (C13),66.4 (C16), 63.8 (C9~), 44.4 (C12), 44.0 (C4), 36-8 (C8), 34.0 ~C21), 33.7 (C9), 29.0 (C41~ 51, 6l), 28-7 (C81), 28.3, 26.2 (_~C--M )' 25.9 ~C7l), 24.7 (C3'), 20.1 (Cl~), 19.0 (C15), 16.4 (C17~, ~ e (relative intensity) 509 (7%), 480 (10), 422 (10), 404 (8), 394 (10), 387 (7~, 383 (8). (~ound:
509.3110. M+-CH3 requires 509.3108~. The acetonides , -' _ 23 -were quantitatively converted to methyl pseudomonate C
and pseudomonic acid C respectively with 80% acetic acid overnight.
Example 12 Methyl pseudomo a_e C
Sodium iodide (600 mgs, 4 eq) (dried at 110C/4 hours) was stirred in dry THF (1 ml) and dry acetoni-trile (1 ml) and trifluoroacetic anhydride (141 ~1, 1 eq) was added. After 5 minutes the yellow solution was cooled in ice and methyl pseudomonate A (514 mgs) added. After 5 minutes the ice bath was removed and the reaction stirred at room temperature for 24 hours.
The reaction mixture was diluted with aqueous sodium bisulph:ite arld extracted with ethyl ace~ate (4 x 25 ml).
The combined extracts were washed with brine then dried (MgS04) and evaporated to an oil then chromatographed on silica (5 g). Pure fractions (tlc, hplc) ~ere com-bined and evaporated to give desired product (63 mgs, 13%).
Isohexy~ pseudomonate_C
Methyl pseudomonate A (10 g) and po-tassium sel-enocyanate (4.32 g~ 1.5 eq) in 2-ethyl-n-butanol (iso-hexyl alcohol)-water (9:1, 150 ml) were refluxed for 2 days. The solution was filtered and evaporated then the residue redissolved in ethyl acetate~water. The organic layer was separated, washed with brine then dried (MgS04) and the solvent removed in vacuo. The crude product was chroFatographed on silica (100 g) . . :
- 24 _ elutin~ with 0 to 6% MeOH-CHC13. Fractions containing pure methyl pseudomonate C were combined and evaporated to an oil which crystallised on standing mp 47-9C
(2.2 g). Remaining fractions were combined and rechrom-atographed to yield a pure compound subsequently iden-tified as isohexyl pseudomonate C (1.0 g) vmax (CHC13) 3400 (broad), 1703, 1640, 1427, 1220, 1150, 1020 and 978 cm 1, ~H (CDC13) 0.94 (6H, t, (CH2CH3)2), 0-97 (3H, d, C17-CH3), 1.14 (3H, d, C14-CH3), 1~32 (12H~
m.-(CH2)6-), 2-19 (3H, s, C15-C_3), 3.95 (2H, d, OCH2CHEt2) 9 4.05 (2H, t, C9~-CH2), 5-45 (2H~ m~ H-10~
H-ll) 5-75 (lH, S9 H-2), ~c (CDC13) 174.0 (C1~3, 166.9 (C13, 157.2 (C3), 134.4, 129.0 (C10, Cll), 117.8 (C2), 75~0 (C5), 71.2 (C13), 70.4 (C7), 68.8 (C6), 6603 (0_~12CHEt2), 64.9 (C16), 63.8 (C9'), 44.4 (C12), 43.2 (C4), 42-1 (C8), 40.5 (OCH2C~IEt2), 34.4 (C2'), 32.5 (C9), 29.1 (C4', C597 C6l), 28.8 (C8'), 26.0 (C7l), 25-0 (C3~), 23-4 (CH2CH3) 20.3 (C14), 19.2 (C15), 16.4 (C17), 11.0 (CH2CH3).
:. . . ., :. .. . ..
.:
: . : .
, ,. .,, - : ., . . . : . -BIOLOGICAL DATA
(1) Antibacterial activity - human or~anisms Table 1 shows the antibacterial spectrum of sodium pseudomonate C and methyl pseudomonate C in terms of minimum inhibitory concentration (~g/ml) measured by serial dilution in nutrient agar con-taining 5% chocolated horse blood.
_ _ _ . ___ ORGANISM M.I~C. (~g/ml) __ _ ._ sodium methyl pseudo- pseudo-monate C monate C
E. coli NCTC 10418 >100 >100 E. coli ESS 1.0 2.5 P. mirabilis 889 >100 >100 K. aerogenes A >100 >100 Ps aeruginosa NCTC 10701>100 ~100 Pasteurella multocida 1633 1.0 2.5 Haemophilus influenzae Ql0.1 <0.2 Haeomophilus influenzae Wy 21 0.1 0.5 Neisseria flavescens 66330.2 0.5 Bacillus subtilis 0.02 <0.2 Corynebacterium xerosis 9755 ~100 ~100 Sarcina lutea ~100 ~100 Staph. aureus Oxford 0.1 <0.2 Staph. aureus Russell 0.2 0.5 Staph. aureus 1517 0.2 0.5 Strep. faecalis I 100 ~100 Strep. Pyogenes A 64/848 0.1 1.0 Strep. Pyogenes B 2788 2.5 1.0 Strep. Pyogenes C 2761 0.2 1.0 Strep. pneumoniae CN33 0.1 Ool __ __ _ _ .... . ~_ _ ., ~' ' , Y~ 7 (2) Anti-myco~asma activity Methyl pseudomonate C possess good antimyco plasma activity in vitro against mycoplasmas ~om human and veterinary sources, as shown in table 2.
Method (1) The minimum inhibitory concentrations ~MIC) of methyl pseudomonate C were determined in Microtitre plates, by a modification of the metabolic-inhibition test (Taylor-Robinson, 1967). The compounds were serially diluted in sterile de-ionised water to give a range of concentrations from 250-0.5 ~g/ml. Mycoplasma broth containing 1% (w/v~ of glucose and 0.005% (w/v) of phenon red, was added at a strength to compensate for its dilution by the aqueous drug solution. Appro~-imately 10~ colony forming units of mycoplasma were added to each concentration of dxug. Drug-free infected, non-infec-ted and pH control wells were included on each plate. Plates were sealed with cellophane tape and incubated at 37C for seven days. The MIC was the lowest concentration of compound that prevented a colour change in the mycoplasma broth, caused by the metabolism.
Reference ~_ .
Taylor-Robinson, 1967. Mycoplasmas of various hosts and their antibiotic sensitivities. Post. Grad. Med. J., 43 Suppl. CMarch~, 100.
. : , , ' '.,, :. .: .' ' ` .
' , ' ' .'., :.: . . , ~: ..
' '' ' ' ' . '....... ' :' -' - .'' ' ' . '' ' ' .. : ., . . :. , . - . .... . .
_ 27 -. _ ~_ MYCOPLASMA M.I.C. (~g/ml) __ _ __ M. gallisepticum S.6 62.5 M. synoviae 25204 ~0.5 M. pulmonis JB <0.5 M. suipneumoni~e L~ber <0.5 M. pneumoniae 427a 1.0 M. fermentans MW KL4 <0.5 Table 3 shows MIC values for sodium pseudomonate C and met'nyl pseudomonate C against further mycoplasma species determined in Friis~ broth using the micro-titer method.
ORGANISM M.I.C (~g/ml) . ~ , .
MethylSodium Pseudo-Pseudo-monate Cmonate C
M. suipneumoniae Str. 11 ~10 ~10 M. suipneumoniae J2206/183b >10 10 M. dispar H225 10 5.0 M. dispar NCTC 10125 50 2.5 M. pneumoniae 427a ~10 2.5 M. pneumoniae ATCC 15492 10 -M. fermentans MWKL4 0.039< 0.02 M. pulmonis JB 0.3120,039 . ~
.
.' ~ ,' . : ' .. " :, ' ~, ...... ... .
.. . . . . .
Claims (22)
IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof:
(II) which process is selected from the group of processes comprising:
(1) reacting pseudomonic acid A or a salt or ester thereof with a reagent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(i) forming a salt of the pseudomonic acid C produced;
(ii) esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic acid C, (2) growing Pseudomonas fluorescens under aerobic conditions on or in a culture medium containing inorganic salts and sources of asimilable carbon and nitrogen until the culture medium exhibits at least detectable antibacterial activity acidifying the culture medium; extracting with an organic solvent for the active materials dissolved in the culture medium, and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally thereafter esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
(II) which process is selected from the group of processes comprising:
(1) reacting pseudomonic acid A or a salt or ester thereof with a reagent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(i) forming a salt of the pseudomonic acid C produced;
(ii) esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic acid C, (2) growing Pseudomonas fluorescens under aerobic conditions on or in a culture medium containing inorganic salts and sources of asimilable carbon and nitrogen until the culture medium exhibits at least detectable antibacterial activity acidifying the culture medium; extracting with an organic solvent for the active materials dissolved in the culture medium, and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally thereafter esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
2. A process for the preparation of a compound of formula (II) or a pharmaceutically acceptable salt or ester thereof:
(II) which process comprises reacting pseudomonic acid A or a salt or ester thereof with a reagent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(i) forming a salt of the pseudomonic acid C produced:
(ii) esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic acid C.
(II) which process comprises reacting pseudomonic acid A or a salt or ester thereof with a reagent which converts an epoxide to an olefin; and optionally carrying out one of the following steps:
(i) forming a salt of the pseudomonic acid C produced:
(ii) esterifying the pseudomonic acid C or salt thereof to produce an ester of pseudomonic acid C; or (iii) hydrolysing an ester of pseudomonic acid C.
3. A process as claimed in claim 2 wherein said reagent is potassium selenocyanate or trifluoroacetyl iodide.
4. A process as claimed in claim 3 where potassium selenocyanate is employed in a solvent comprising a mixture of water and a branched or cyclic alkyl alcohol containing from 6 to 20 carbon atoms.
5. A process as claimed in claim 1, wherein the hydroxyl groups in pseudomonic acid A or a salt or ester thereof are protected prior to the reaction with said reagent and removed subsequent to the reaction.
6. A process as claimed in claim 5 wherein the glycol moiety of pseudomonic acid or a salt or ester thereof is protected by conversion to a structure:
wherein Ra and Rb are hydrogen, C1-6 alkyl, or phenyl.
wherein Ra and Rb are hydrogen, C1-6 alkyl, or phenyl.
7. A process for the preparation of a compound of formula (II) or a salt or ester thereof which process comprises growing Pseudomonas fluorescens under aerobic conditions on or in a culture medium containing inorganic salts and sources of assimilable carbon and nitrogen until the culture medium exhibits at least detectable antibacterial activity, acidifying the culture medium;
extracting with an organic solvent for the active materials dissolved in the culture medium, and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally thereafter esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
extracting with an organic solvent for the active materials dissolved in the culture medium, and thereafter either:
(a) separating pseudomonic acid C or a salt thereof from any other active materials and optionally thereafter esterifying the separated acid; or (b) esterifying the active materials, separating an ester of pseudomonic acid C from esters of any other active materials and optionally thereafter hydrolysing the separated ester to form pseudomonic acid C or a salt thereof.
8. A compound of formula (II) or a pharmaceutically acceptable salt or ester thereof:
(II) when prepared by the process of any one of claims 1, 2 or 7 or by their obvious chemical equivalents.
(II) when prepared by the process of any one of claims 1, 2 or 7 or by their obvious chemical equivalents.
9. A process for preparing methyl 10, 11-deoxypseudomonate A comprising reacting methyl pseudomonate A and potassium selenocyanate in a methanol-water solvent.
10. Methyl 10, 11-deoxypseudomonate A when prepared by the process of claim 9 or by its obvious chemical equivalent.
11. Pseudomonic acid C when prepared by the process of claim 7 or by its obvious chemical equivalent.
12. Methyl pseudornonate C when prepared by the process of claim 9 or by its obvious chemical equivalent.
13. A process for preparing pseudomonic acid C comprising treating methyl pseudomonate C in phosphate buffered DMF with Bakers Yeast.
14. Pseudomonic Acid C when prepared by the process of claim 13 or by its obvious chemical equivalent.
15. A process for preparing sodium pseudomonate C comprising reacting pseudomonic acid C in one of a sodium hydroxide solution and a sodium bicarbonate solution.
16. Sodium pseudomonate C when prepared by the process of claim 15 or by its obvious chemical equivalent.
17. A process for preparing pseudomonic acid C comprising protecting the glycol group on pseudomonic acid A by reacting with 2, 2-dimethoxypropane reacting the resultant product with potassium selenocyanate, and then removing the glycol protecting group with acetic acid.
18. Pseudomonic acid C when prepared by the process of claim 17 or by its obvious chemical equivalent.
19. A process for preparing methyl pseudomonate C comprising adding methyl pseudomonate A to a solution containing sodium iodide and trifluoroacetyl iodide.
20. Methyl pseudomonate C when prepared by the process of claim 19 or by its obvious chemical equivalent.
21. A process for preparing isohexyl pseudomonate C comprising reacting methyl pseudomonate A and potassium selenocyanate in an isohexyl alcohol and water solvent.
22. Isohexyl pseudomonate C when prepared by the process of claim 21 or by its obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA315,801A CA1104957A (en) | 1978-11-03 | 1978-11-03 | Pseudomonic acid c from pseudomonas fluorescens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA315,801A CA1104957A (en) | 1978-11-03 | 1978-11-03 | Pseudomonic acid c from pseudomonas fluorescens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1104957A true CA1104957A (en) | 1981-07-14 |
Family
ID=4112883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA315,801A Expired CA1104957A (en) | 1978-11-03 | 1978-11-03 | Pseudomonic acid c from pseudomonas fluorescens |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1104957A (en) |
-
1978
- 1978-11-03 CA CA315,801A patent/CA1104957A/en not_active Expired
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