CA1125204A - Antibacterial agents from streptomycete - Google Patents
Antibacterial agents from streptomyceteInfo
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- CA1125204A CA1125204A CA300,966A CA300966A CA1125204A CA 1125204 A CA1125204 A CA 1125204A CA 300966 A CA300966 A CA 300966A CA 1125204 A CA1125204 A CA 1125204A
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Abstract
ABSTRACT OF THE INVENTION
It has been found that novel antibiotics can be obtained by the fermentation of strains of Streptomyces olivaceus. These antibiotics have a wide spectrum of activity and when pure are extremely potent inhibitors of the growth of many gram-positive and gram-negative bacteria.
They can also enhance the effectiveness of penicillins and cephalosporins against certain .beta.-lactamase producing bacteria. These novel antibiotics are of the formula (I) and (II):
(I)
It has been found that novel antibiotics can be obtained by the fermentation of strains of Streptomyces olivaceus. These antibiotics have a wide spectrum of activity and when pure are extremely potent inhibitors of the growth of many gram-positive and gram-negative bacteria.
They can also enhance the effectiveness of penicillins and cephalosporins against certain .beta.-lactamase producing bacteria. These novel antibiotics are of the formula (I) and (II):
(I)
Description
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BACKGROUND TO THE INVENTION
British Patent Specifications Nos. 1467413, 1489235 and 1483142 discloses that fermentation of Streptomyces olivaceus can lead to the preparation of antibiotics named MM4550, MM13902 and MM17880 which have the formula (III), (IV) and (V) respectively:
H03S0 ~ S-C=C-NH-C0-CH3 (III) 3 , .
35 ~ S-C=C-NH-C0-CH3 (IV) H C H H
3 , , H0350 ~ S-cH2-cH2-NH-cO-cH3 (V) None of these British Specificati~s contained any suggestion that a further antibiotic could be obtained from the fermentation broth of Streptomyces olivaceus. Further antiblotics have now been found.
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DESCRIPTION OF THE INVENTION
-The present invention provides the compounds of the formula (I) and (II):
HO ~ . H
~ ¦ ~ S-C=C-NH-CO-CH3 (II) CH3 ,H
HO I / ~ 2 2 C 3 (I) ~ ~ .
and their salts.
Most suitably the compounds of the formulae (I) and (II) are in the form of a salt since it appears that salts of the compounds o~ formulae (I) and (II) are more stable than the parent acids.
Suitable salts of the compounds of the~formula (I) and tII) include the pharmaceutically acceptable alkali and alkaline earth metal salts such as the sodium, potassium and ealcium salts and pharmaceutically acceptable~addition salts with nitrogenous bases such as the ammonium, trimethylamine, ~ --.
dimethylamlne, pyrrolidine and llke salts.
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Particularly suitable salts of the compounds of the formulae ~I) and (II) include their sodium and potassium salts.
A preferred compound of this invention is the sodium salt of a compound of the formula (I). A second preferred compound of this invention is the sodium salt of a compound of the formula (II~.
Since the compounds of the formulae (I) and (II) and their salts are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 90% pure and yet more preferably at least 95% pure. Impure preparations of the compounds of the formulae (I) and (II) and their salts may be used for preparing the more pure forms used in the pharmaceutical compositions, these less pure preparatians of the compounds of the formulae ~I) and (II) and their salts should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or (II) or their salts. These less pure preparations most usefully comprise a salt of the compound of the formula ~Ij and a salt of the compound of the formula ~II). (% are on a wt/wt basis.) It is generally preferred that the substantially pure salts of the compound of the formula (I) or the formula (II~, are not contaminated by substantial amounts of other anti-bacterial agents such as salts of the compounds of the formula ~ 5 ~ ~
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BACKGROUND TO THE INVENTION
British Patent Specifications Nos. 1467413, 1489235 and 1483142 discloses that fermentation of Streptomyces olivaceus can lead to the preparation of antibiotics named MM4550, MM13902 and MM17880 which have the formula (III), (IV) and (V) respectively:
H03S0 ~ S-C=C-NH-C0-CH3 (III) 3 , .
35 ~ S-C=C-NH-C0-CH3 (IV) H C H H
3 , , H0350 ~ S-cH2-cH2-NH-cO-cH3 (V) None of these British Specificati~s contained any suggestion that a further antibiotic could be obtained from the fermentation broth of Streptomyces olivaceus. Further antiblotics have now been found.
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DESCRIPTION OF THE INVENTION
-The present invention provides the compounds of the formula (I) and (II):
HO ~ . H
~ ¦ ~ S-C=C-NH-CO-CH3 (II) CH3 ,H
HO I / ~ 2 2 C 3 (I) ~ ~ .
and their salts.
Most suitably the compounds of the formulae (I) and (II) are in the form of a salt since it appears that salts of the compounds o~ formulae (I) and (II) are more stable than the parent acids.
Suitable salts of the compounds of the~formula (I) and tII) include the pharmaceutically acceptable alkali and alkaline earth metal salts such as the sodium, potassium and ealcium salts and pharmaceutically acceptable~addition salts with nitrogenous bases such as the ammonium, trimethylamine, ~ --.
dimethylamlne, pyrrolidine and llke salts.
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Particularly suitable salts of the compounds of the formulae ~I) and (II) include their sodium and potassium salts.
A preferred compound of this invention is the sodium salt of a compound of the formula (I). A second preferred compound of this invention is the sodium salt of a compound of the formula (II~.
Since the compounds of the formulae (I) and (II) and their salts are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 90% pure and yet more preferably at least 95% pure. Impure preparations of the compounds of the formulae (I) and (II) and their salts may be used for preparing the more pure forms used in the pharmaceutical compositions, these less pure preparatians of the compounds of the formulae ~I) and (II) and their salts should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or (II) or their salts. These less pure preparations most usefully comprise a salt of the compound of the formula ~Ij and a salt of the compound of the formula ~II). (% are on a wt/wt basis.) It is generally preferred that the substantially pure salts of the compound of the formula (I) or the formula (II~, are not contaminated by substantial amounts of other anti-bacterial agents such as salts of the compounds of the formula ~ 5 ~ ~
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(III), (IV) and (V), derived from the fermentation broth.
The compounds of the formulae (I~ and (II) exist as cis- a~d trans-forms about the ~-lactam ring. These forms may be represented by formulae (Ia), (Ib), (IIa) and (IIb) thus:
H3C ,H H
HO ~ S-CH2-CH2-NH-CO-CH3 ~Ia) O . 2 H C H H
HO ~ I ~ S-CH2-CH2-NH-CO-CH3 (Ib)
(III), (IV) and (V), derived from the fermentation broth.
The compounds of the formulae (I~ and (II) exist as cis- a~d trans-forms about the ~-lactam ring. These forms may be represented by formulae (Ia), (Ib), (IIa) and (IIb) thus:
H3C ,H H
HO ~ S-CH2-CH2-NH-CO-CH3 ~Ia) O . 2 H C H H
HO ~ I ~ S-CH2-CH2-NH-CO-CH3 (Ib)
3 , ' H
H ~ S-C=C-NH-CO-CH3 (IIa) HO ~ C=C-n3-CU-CH3 ~IIb~
O : C02H
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It will be realised that the preceding compound~
~ay be named as follows:
Ia (5R,6R)-3-(2-acetamidoethylthio)-6-[(S)-l-hydroxyethyl]
-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylic acid.
Ib (5R,6S)-3-(2-acetamidoethylthio)~6-[(S)~l-hydroxyethyl]
-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
IIa (5R,6R~-3-[(E-2~acetamidoethenylthio~-6-[(S)-l-hydroxy-ethyl]-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylic acid.
IIb (5R,6S)-3-4E 2-acetamidoethenylthioD-6-~(S)-l-hydroxy-ethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid Both cis- and trans- isomers of the compounds of the 15 formulae tI) and (II) have useful antibacterial and ~-lactamase inhibiting properties and so this invention extends to the isolated compounds of the formulae ~Ia) and (b) as well as to mixtures thereof and to the isolated compounds of the formulae (IIa) and (IIb~ as well as mixtures thereof.
Naturally isolated cis- and trans-~forms~will most suitably be in the forms of substantially pure pharmaceutically acceptable~salts as described above; that lS they should be at :: : : : : ~ :
~ least 50% pure, more suitably at least 75~ pure, preferably .
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90~ pure and most preferably at least 95% pure. ~'urthermore it is preferred to use one o~ the aforementioned compounds when substantially free of its 6-position isomer ~hat is (Ia) free of (Ib), (IIa) free of (IIb?,(Ib) free of (Ia) or (IIb) free of (Ib)]. In general such compounds should not contain more than 5% of its 6-position isomer and preferably not more than 1~ of its ~~position isomer.(Iligh pressure liquid chromatography may be used to monitor purities.) A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ia) having a molar extinction coefficient (in water at neutral pH)of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 2g8 nm).
A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ib) having a molar extinction coefficient (in water at neutral pH) of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 301 nm).
A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (IIa) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for UV absorption maximum at about 308 nm).
~ favoured aspect of this invention provides an alkali metal salt of the compound of the formula (IIb) having a molar extinction coefficient (in water at neutral pH~ of not less than 13000 (preferably not less than 13500) (for UV absorption on maximum at about 308 nm).
Preferably the preceding alkali metal salts are the sodium salts.
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The present invention also provides a pharmaceutical composition which comprises a compound of the formula (I) or (II) or a salt thereof and a pharmaceutically acceptable carrier.
The compositions of this invention will generally utilise a pharmaceutically acceptable salt of a compound of the formula (I) or (II), for example a sodium or potassium salt.
The pharmaceutical compositions of this invention may be adapted for oral or parenteral administration. Suitably the compositions are provded as dosage forms which contain from 50 to 500 mg of a compound of the formula (I) or (II) or its salt, for example about 100, 150, 200 or 250 mgs.
Most usually the composition will be adapted for administration by injection.
These compositions may contain diluents, binders disintegrants, lubricants or other conventional excipient and may be fabricated by conventional methods of mixing, filling and the like.
The compositions may take the form of tablets, capsules, vials or other similar forms.
If desired the composition may advantageously conta:in a penicillin or cephalosporin. In such instances the ratio ;;
of synergyst (i.e. the compound of the invention) (preferably as a salt) to penicillin or cephalosporln is~ usual~ly~from 2:1 to 1:12, more usually from 1:1 to 1:5, for example~].:2l 1:3, or 1:4 wt/wt.
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Particularly suitable penicillins for inclusion in the compositions include ampicillin, amoxycillin, carbenicillin, ticarcillin and their pro-drugs. When adapted for injection such compounds are generally presented in the form of their sodium salts.
Particularly suitable cephalosporins for inclusions in the compositions include cephaloridin and cephazolin.
Preferred penicillins for inclusion in the compositions include ampicillin trihydrate, amoxycillin trihydrate, sodium ampicillin and sodium amoxycillin.
Preferred cephalosporins for inclusion in the compositions of this invention include cephaloridin and sodium cephazolin.
The compound of the formula (I) or (II) or its salt may be an isolated compound of the formula (Ia), (Ib) or (IIa), (IIb) or their salts or mixtures of the compounds of the formula (Ia) and (Ib) or (IIa) and (IIb) or their salts.
However it is preferred to use a compound of one of the preceding formulae free from its isomer. Said compound is generally in the form of a pharmaceutically acceptable salt such as the sodium salt.
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The present invention also provides a process for the preparation of a compound of the formula (I) or ( II ) or its salt which process comprises cultivating a produclng strain of Streptomyces olivaceus or Streptomyces ~edanesis until a substantial quanity of a compound of the formula (I) or ~II) or its salt is produced and thereafter recovering a compound of the formula (I) or (II) or its salt from the cultivation medium.
When used herein, the term "Streptomyces olivaceus"
is defined according to the classification of H~tter R
(in Systematic der Streptolyceten, S~ Korger, Basle, Pages 8-32). Note that on this definition S~reptom~ces fulvovoridis, Streptomyces flavus and Streptomyces flavovirens may be regarded as being synonymous with Streptomyces olivaceus.
Suitable strains include those described in British Patent Specification No. 1467413.
A preferred organism for use in this process is Streptomyces olivaceus ATCC 31126 or a high yielding mutant thereof.
A further preferred organism for use in this process is Streptomyces olivaceus ATCC 31365 or a high yielding mutant ` thereof.
As previously indicated the recovered material should be at least 1~ pure, more suitably 5% pure, yet more suitably at least 50% pure, preferably at least 75~ pure and more preferably at least 90~ pure~ for example at least 95~ pure.
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When used herein the term "cultivation" means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid medium but in general it is preferable to use a liquid medium. General cultivation conditions for the growth of Streptom~ces olivaceus are as described in British Patent Specification No. 1467413. General conditions for the growth of Streptomyces gedanensis are similar.
The process of this invention may be adapted to provide a compound of the formula (I) or its salt, a compound of the formula (II) or its salt or a compound of the formula (I) or its salt together with a compound of the formula (II) or its salt.
Normally the process is adapted to the preparation of a salt rather than the parent acid.
It is preferable that the cultivation medium does not contain added sulphate since this often leads to the prepara-tion of MM4550, MM13902 and MM17880 at the expense of the production of the compounds of the formula (I) and (II) and their salts.
The compounds of the formula (I) and (II) in the form of thelr salts may be obtained from the culture filtrate by (a) contacting the filtrate with carbon until the antibiotic activity is absorbed thereon, (b) eluting the antibiotic activity from the carbon using aqueous acetone, (c) combining ~ .. . .
the fractions containing ~-lactamase inhibitory fractions, (d) evaporating the acetone and much of the water to yield a more concentrated aqueous solution, (e) applying the solution to an anion exchange column, and (f) eluting the ~-lactamase inhibitory metabolites therefrom with a solution of an electrolyte buffered to approximate neutrality collecting the fractions containing the compound of the formula (I) or (II) in salt form, (g) applying the resulting solution to a resin which separates the inorganic materials from the compounds (I) and ~I)and (h) isolating the solid preparation of the salt of a compound of the formula (I) or (II) from the resulting solution.
The compounds of the formula (I) and (II) in the form of their salts may also be obtained from the culture filtrate by (1) contacting the clarified cultivation broth with a strongly basic acrylic based anion-exchange resin until the antibiotic activity is absorbed thereon, (2) eluting the antibiotic activity from the resin using an aqueous solution of a buffer optionally also containing a salt, (3) combine the fractions with ~-lactamase inhibitory activity, (4) apply the combined fractions to a XAD-4 column, (5) elute with aqueous isopropanol, (6) combine the fractions with ~-lactamase inhibitory activity, ~7) remove the isopropanol and concentrate the solution by evaporation, (8) apply the solution to an anion exchange resin and proceed as in steps (f), (g) and (h) outlined above.
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It is generally preferred to use an acrylic based strongly basic (type 1) anion exchange resin (in the form of an acid addition salt normally the hydrochloride) such as ,$
Amberlite IRA 458 (which may be obtained for example from Rohm and Haas for example at Lennig House, 2 Massons Avenue, Croydon, U.K.). An advantage of such a resin is that it allows the salt of the compounds of the formula (I) and (II) to be eluted successively by using an aqueous salt solution, for example a buffered solution of a chloride such as sodium chloride or the like. If the less favoured strongly basic resins having a polystyrene/divinyl benzene matrix are employed it is generally necessary to elute with an aqueous lower alkanolic solution of a salt (for example a chloride such as sodium chloride) in order to obtain satisfactory lS recoveries and such solvents can lead to a less pure preparation of the desired materials.
(This process variant differs from the carbon absorption process in that the salts of MM4550, MM13902 and MM17880 are separated from the salts of the compounds of the formula (I) and (II) at the first elution stage.) The process of this invention differs funda~entally from the previously disclosed process in that the fractions selected for further processing at stage (f) are those containing the salt of a compound of the formula (I) and (II) substantially free of other antibiotics.
The free acids of the formulae (I) and (XI) may be ~e~na~K
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obtained by careful acidification of a salt of the compound of the formula (I) or (II) respectively followed by rapid extraction into a water immiscible organic solvent followed by recovery of the acid from solution.
In the processes of this invention it is frequently most con~enient to work with an alkali metal salt, of the compound of the formulae (I) and/or (II) such as the lithium, sodium or potassium salts and of these the sodium salt is favoured. It is possible to prepare other salts by the extraction process but it is usually more suitable to first form the purified alkali metal salt especially the sodium salt and then convert this to an alternative salt, for example ky passing through a bed of cation exchange resin in the form of the alternative salt. Thus in this description other electrolytes (such as the lithium, potassium or other salt) can be substituted for the described sodium salts but in general it is preferred to work with the sodium salt. Similarly salts other than chloride (for example bromide, nitrate or the like) may be employed although in general it is preferred to work with a chloride.
A preferred method of chromatographic purification (steps f and g) uses an aqueous solution of a sodium salt buffered to approximate neutrality in conjunction with a basic ion-exchange resin. Thus an aqueous solution of sodium chloride ~or other similar salt) buffered to about pH 7 with a conventional buffer such as a phosphate buffer may be used in conjunction with support resins which contain . ~
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secondary or tertiary amino groups or quaternary amino groups. Suitable supports include basic ion-exchange celluloses and basic ion-exchange cross-linked dextrans such as DEAE cellulose, DEAE Sephadex, QAE-Sephadex and equivalent agents.
A related suitable method of chromatographic purification (steps f and g) uses a sol~-ent system comprising a mixture of water and small quantities of a water immiscible organic solvent such as a lower alkanol (i.e. Cl 4 alkanol) in conjunction with an inert support material such as silica gel or cellulose. Suitable solvent systems include aqueous isopropanol, aqueous n-butanol and the like. For example a very roughly 1:4 mixture of water and isopropanol may be used in combination with a cellulose support.
The product of the preceding proceedure frequently contains a high proportion of sodium chloride so that it is beneficial to de-salt the pooled solutions. De-salting may be effected by pas~ing the solution through a bed of lipophilic material onto which the antibiotic is adsorbed but which does not absorb the sodium chloride. Suitable materials include polystyrene based polymeric absorbants such as Amberlite XAD-4,Diaion HP20 and the like. The produce of the preceding process may also be desalted by chromatography on suitable ;`
gel filtration agents such as cross-linked dextrans such as ,~
Sephadex G10 and G15 and polyacrylamide gels such as Biogel P2. The antibiotic may be eluted from such materials using water, aqueous methanol or the like.
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, The columns are eluted at such a rate as to allow s~paration of the antibiotics into distinct fractions.
In general distinct zones can be eluted from these columns;these contain di-sodium MM4550, di-sodium MM13902, di-sodium MM17880, the sodium salts of the compounds of the formula (I) and the sodium salts of the compounds of the formula (II) eluting close to the sodium sal~ of the compound of the formula (I). In general the three di-sodium salts are fairly widely separated from the mono-sodium salts on anion exchange resins. If the column is not carefully monitored it may be that the mono-sodium salts are obtained in overlapping fractions. If this is 50 then either (a) this solution can be freeze dried to yield a useful impure complex containing the antibiotics which can be reworked later or (b) the solution per se can be re-chromatographed with careful monitoring of the eluant to ensure collection~
of the solution of sodium salt of a compound of the formula (I) free from the sodium salt of a compound of the formula (II) and/or the collection of the solution of the sodium salt of a compound of the formula (II) free from the sodium salt of a compound of the formula (I); these solutions may then be freeze dried or otherwise rendered solvent free.
The fractions chosen for collection will be those which show significant ~-lactamase inhibitory activity or anti-bacterial activity. Suitable methods of detecting ~-lactamase inhibitory activity include those of the aforementioned , - , : . .: .
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British Patents although any convenient method may be employed.
The fol.lowing Scheme shown preferred sequence for obtaining the compounds of the formulae (I) and (II) as their sodium salts. The sodium salts obtained in this manner can be further purified if desired by using the chromatographic procedures described hereinbefore.
Trituration of the salts of the compounds of the formulae (I) and tII) under an organic solvent such as moisture containing acetonitrile or acetone can aid in the removal of impurities.
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Cultu~e Filtrate Absorb onto Carbon ~ Elute with 20% aqueous acetone Combine Frartlons with~ -lactamase ~nhibitory Activity Remo~e Acetone and concentrate 1 by Evaporation Cellulose DE52 Anion Exchange Column , ,. :'.
Elute with Phosphate Bufer pH7 ~ ;~ ;:: `
Sodiu~ Salts of Sodium Salts of Di-Sodlum Salts of:
Compounds o~ Compounds of MM4550j MM 13302 and Formula (I) Formula (II) MM17880 ', ' ~ ' De-salted Solution De-salted Solution - ~
Freeze Dried Solid F~eeze D~ ed ~olld , '` ~
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Culture Flltrate Absorb onto Strongly Ba~ic Acrylic Anion ~xchange Resin ¦ Elute with Buffer Comblne Fractions wlth earlier eluting ~g-lactamase Inhibitory Actlvity De-salt on ~AD 4 1 Elute with Aqueous Isopropanol Combine Fractions with ~-lactamase Inhlbitory Actlvity ~.
Remove Isopropanol and . ~ concentrate by Evaporation Cellulose DE52 Anion Fxchange Resin : :
Elute with Phosphate Buffer pH7 \ .: ':
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Sodiu~ Salts of Compounds Sodium Salts of Compounds of of Formula (I) Formula (I~) ~ , De-salted Solution De-salted Solution : :
Freeze Dried Solid Freeze Dried Solld ,:':
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~ desired the salts of the compounds of the formula (I) and (II) prepared by the previous methods may be further subjected to chromatographic separation techniques to yield an isolated / salt of a compound of the formula (Ia), (Ib), (IIa) or (IIb).
Such processes are favoured aspects of this invention.
Normally the salt used in such a process will be a monovalent salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.
One suitable form of chromatography for the separation process is high pressure liquid chromatography(hplc?, for example using an aqueous ammonium formate buffered solution. Once fractions containing the desired compound are obtained a solid preparation may be obtained by freeze drying or the like.
Compounds of the formuIa (Ia) and (Ib) may be separated by column chromatography on supports such as acetylated cellulose eluting with alcohol/water mixtures. Compounds of the formula (IIa) and (IIb) may also be separated using similar chromatographic techniques.
Once fractions containing the desired compound are 20~ obtained a solid preparation may be obtained by freeze drying or the like. ;
- 21 -~
The present invention provides a process for the preparation of a salt of the compound of the formula (Ia) substantially free of the compound of the formula (Ib~ which comprises sub~ecting a mixture of said sal~s to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.
The present invention provides a process for the preparation of a salt of the compound of the formula (Ib) substantially free of the compound of the formula (Ia) which comprises subjecting a mixture of said salts to chromato- :
graphic separation on Diaion HP20 or a chromatographically equivalent resin~ ~
The present invention provides a process for the ~ :
preparation of a salt of the compound of the formula (IIa) substantially free of the compound of the formula (IIb) which comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.
The present invention provides a process for the preparation of a salt of the compound of the formula (IIb) substantially free of the compound of the formula (IIa) which comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin. :
The salts prepared by the preceding processes will normally be~monovalent salts~such as alkali metal salts, for example the lithium, sodium or potassium salt and:will :
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preferably be the sodium salt.
The salts prepared by the preceding process will not normally contain more than about 5% and more suitably not more than about 1% of the undesired isomer.
DiaiOn (which is a Registered Trade Mark) is a highly porous polymer manufactured by ~itsubishi Chemical Industries. It is not an ion exchange resin but is a synthetic adsorbant which has an extra large active surface area to which organic compounds may be effectively adsorhed.
Diàion HP20 is a styrene divinylbenzene copolymer in bead form having a macroreticular structure with a specific surface area of about 7.8 m2/g and a pore volume of 1.16 ml/g. Other details of this resin may be found in (HP series, October 1976, which is incorporated herein) ` the Diaion data sheet /(Mitsubishi Chemical Industries Ltd.
Offices may be found via 5-2 Marunouchi 2-chome, Chiyoda-ku, Tokyo, Japan; 277 Park Ave., New York, NY 10017 USA;
Ratinger Str. 45, 4 Duesseldorf, West Germany;~etc.) Resins chromatographically equivalent to Diaion HP20 be will also normally/chemically and physicalIy similar, that is they will generally be macroreticular resins based on styrene divinylbenzene copolymers and free of ionized gr~ups.
Most suitably the mixture of isomers applied to the resin will be of good purity and will be substantially free from other organic impurities although quantities of inorganic impurities (for example an alkali metal salt such as a chloride, for example sodium chloride) may be present.
Suitably the solvent employed will be water or water in admixture with a lower alkanol or similar miscible organic solvent.
Preferably the solvent used is water.
The desired material may now be obtained by removal of the solvent for example by evaporation, freeze drying or the like. Alternatively the solution may be rechromatographed directly on a suitable resin ~uch as Biogel P2 and/or Diaion HP20~ for further purification prior to removal of the solvent.
Water may be removed from aqueous solutions of salts of this invention by such processes as evaporation under vacuum to about 1/10 volume, makin~ up to the original volume with ethanol, reconcentration to about 1/10 volume under vacuum, making up to the original volume by adding toluene and evaporation to dryness in vacuùm. Residual solvents may be removed by storlng under high vacuum.
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Prcl~aration of Clarified ~roth A spore suspension of Stre~_omyces olivaceus ATCC 31126 was used to inoculate 100 ml of a seed sta~e medium COlltaine~ in a 500 ml Ehrlen~meyer flask closed with a foam plug. The seed medium was bean 2S' ~lucose a~d l`~o soya/flour made up in deionised water. (The soya bean flour was Arkasoy 50 supplied by the British Arkady Co. Ltd., 01d Tr2fford, Manchester, U,~.) The seed sta~re medium was ~ro~m for 48 hours on a rotary shaker at 26C. 5 ml portions of the seed stage medium were used to~inoculate 100 ml portions of the fermentation medium contained in 500 ml 3hrlencmeyer fla~ks closed with fc~ plu~s. The fermentation medium ~rhich was made up in deionised water had the followin~ composition:
So Glucose, 2.0 Soya bean flour 1.0 CaC03 0.02 CoC12 2 0.0001 The fermentation flasks were incubated at 26C on a rotary shaker for 72 hours. 20 flasks ~lere harvestcd and the resultant ~hole broth centrifuged at 2200 ~ for 10 minutes.
Use of Streptomyces olivaceus ATCC 31365 ln the above process i9 also favoured.
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D~:s~ ti~ll 2 Pre-paralion of Crude Antibiotics in Solu-tion 'rhe culture filtrate (1500 ml) obtaille~ art(~r c~ triu~ration ~a5 submitted to ~)urification usin~ a caxboll colwnn a. follo~ls:
A 2.5 ~ 37 cm Darco ~rarlulc~-^ carbon column was prepared in deionised ~ater, the column uas ~lashed successivcly ~ith 1 litre 2,~ ~aOH, 1 litre deiol~sed water, 1 litre lN HCl and 1 litre deionised water all at 15 ml/minute. The colu~l ~1a5 then washed ~/ith 0.05M pl~ 7 phosphate buffer until the pH of the eluant was 7Ø
'Mle culture filtrate (1500 ml) was r~l onto the carbon column at 15 ml/minute. The column was then eluted with acetone/water 1/4 at 15 ml/minute and 22 ml fractions were collected. Fractions were monitored for their ~-lactamase inhibitory ac-ti~,-ity a~ainst a preparation of RT~ enæyme (supplied by ~iicrobiolo~ical Research Establishment, Porton). Fractions showing the greates-t activity (~ - 22) were combined and evaporated under reduced~pressure to~remove acetone. The resulting - aqueous solution was stored deep frozen prior to subsequent work-up.
(The RT~I enæyme typlfles plasmid controlled ~-lactamases and may be replaced by otller such R-factor ~-lactamases if desired).
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Isolation of the Compounds of F_~mulae (I) and (Il) a8 their So~ium Salt~ Suostantiall~ Free of Salts of 1~ 5~0, I~I 13902 and i~ 178aO
The crude liquor obtained in ~escription 2 were evaporated under reduced pressure to approximately 2'j ml alld load~d onto a 3.~ x 27 cm D~AE cellulose ~ea~.ly basic anion e~chanse column (the DEAE cellul~e was D~52 ce~ lose supplied by ~natman Ltd., Sprin~field Mill, Maidstone, Kent) prepared in 0.025 M pH 7 phosphate buffer. The column was eluted ~lith 0.025 I~ pH 7 phosphate buffer at a rate of 8 ml/minute and 25 ml fractions were collected. ~he fractions were monitored for their ~-lactamase inhibitory activity against a preparation cf ~T~M
~-lactamase. The first two pec~ks of inhibitory activity were retained.
~he first band fractions (13 - 16) containing the salt of the compound o fo~ula (I) ~ere combined and freeza dried to yield a solid preparation containing the sodium salt of the ~ mpound of formula (I) substantially free of the salts of the di-basic a~tibio-tics.
The second band fractions (19 - 21) containing the salt of the compound of formula (II) were combined and reeze dried to yield a solid preparation containing the sodium salt o the compound of the formula (II) substantially free of the salts ofthe di-basic antibiotics.
(Freezc drying combined fractions 13 - 16 and 19 - 21 naturally leads to a preparation containing a mixture of the sodium salts ol`
the ~Itibiotics of the for~ulae (I) and (II). The di-sodium salts of ~14550,1~ 13902 and M~l 17880 eluted af~er the desired salts).
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F,xe~n~,le 2 Partial Purifica-tion of the Sodil~m Sa]tsof the Com~oun~ of the Fo~mlla ~) The freeze dried preparation of the ~alt of -the compound of Example 1 was dissolved in deionised wa-ter (10 ml) and sodium cllloride (1 g) added to the solution. This solution was run OlltO a 1.5 ~ 15 cm XAD-4 (supplied by Rohm & Haas) colu~m prep~red in deionised ~ater~ The column was eluted with water/n-propanol 4/1 at 2 ml/minute and ~r ml ~ractions were collected. ~ractiong were monitored for chloride by their reaction with ~N03 and for their ~-lactamaGe inhibitory activit~ against a preparation of RT~ lactc~mase. Fractions ~ith the greatest inhibitory activity and giving a negative reaction with ~ilver nitrate (fractions 6 - 13) ~rere combined and freeze dried to yield an amorphous solid (32.5 mg) containing the sodium salt of the compound of the formula (I).
The properti~s of this preparation were as follows:
(A) Chromato~raphic Properties (i) Chromatography on ~Ihatman D~81 Ion ~xchange Paper (a weakly basic anion exchange paper):
. , __ ~luant Rf of Sodium Salt _ _ 1. 0.05M pH 7 phoGphate buffer o.69 2. 0.05M pH 7 phosphate buffer 0 ~0 containing 0.2M NaCl .
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Solvent System I Rf of Sodi~ Salt ._____ _ . . _.
1. But~lol:Etha~ol:~ater 0 12 1 Top P~ase 4:1:5 .
¦ 2. Butanol:Pyri.dine :~latero . 42 (B) ~ h Volta~e Pa~er Flectro~horesis ~he electrophoresis was carried out on No. 2') ~aper in pyridine/acetic acid buf~er pH 5.3 at 5GOO volts for 15 minutes.
The ~ values ~or the sodium salt t~king benzyl penicillins as l.O-is also 1Ø
(C) Antibacterial Activit~
The antibacterial activity of the preparation using the microtitre method was determined as ~ollows:
Organism ¦ I/~C (~g/ml) Bacillus subtilis A C 4 Enterobacter cloacae Nn 1250 Bscherichia coli 1041~ 150 ~. coli JT 410 625 Klebsiella aerogenes A 312 Proteus mirabilis C977 62S
Pseudomonas aeru~inosa A> 2500 ~ Salmonella typhimurium CT10312 ~:
: 25 Serratia marcescens US39 625 Staph. aureus Russell 150 _,_ ~
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(D) ~'n~,~e In'~ ition Tlle enz~me inhibitory activity of the preparation against a series of ~-lactamase preparations is summarised below:
- , ..... . j .. . . . , ~-Lacta~ase Preparation 1~ Inhibition Concentration ~rom: I a-t 200~g/ml givin~ 50~' , ~ u,rr/ml Staph. ~ureus Russell 4o E. coli JT4 _ 95 Proteus mirabilis C~9 _ 112 Pseudomonas aeru~.grinosa Dal~leish ~ 170 Enterobacter cloacae P99 ~3 Pseudomonas aeru~inosa h 30 l~ebsiella aeFogenes E70 21 (i~lethod of ~elgian Patent No. 827926).
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Example 3 Partial Purlfication of ~he Sodium Salt of the Com~ounds of the Formula (II) The freeze dried preparation of the salt o~ the compound II of Example l was dissolved in deionised water (10 ml) and sodium chloride (l g) added to the solutlon, Thls solution was run onto a 1.5 x 15 cm XAD-4 (supplied by Rohm & Haas) column prepared in deionised water, The column was eluted wi~h water/~-propanol 4/1 at 2 ml/minute and 4 ml fractions were collected. Frac-tions were monitored for chloride by their reaction with AgN03 and for their ~-lactamase inhibitory activity against a preparation of R~EM ~-lactamase.
Fractions with the greatest inhibitory activity and giving a negative reaction with silver nitrate (fractions 7 - 13) were combined and freeze dried to yield an amorphous solid (32~5 mg) containing the sodium salt of the compound of the formula (II).
The properties of thls preparation were as follows:
(A~ _ Chromatographic Propertie~
(i) Using weekly basic anion exchange paper DE81 cellulose (~hatman~ the sodlum salt has an Rf of 0.54 when eluted with 0.05M pH 7 phosphate buffer.
(ii) Using Whatman* No. 1 paper: the sodium salt ha~ an Rf oP 0.20 when eluted with butanol/ethanol/water 4/1/5 top phase.
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(13) ~ii,r^ll Volt;,~ aller_,lccl;70~ 7(?~.is ~ he electrophoresis was earried ou-t on '~1atman No. 2u paper in pyr~ e/acetie acid buff~r pH 5.3 at 50()0 volts for 15 miluitos.
~le ~I value for the salt talcin~ benzyl penieillin as l.O is 0.95.
(C) ~ntibaoterial Aetivit~
The antibaeterial activity of the preparation using the micro-titre ~ethod was determined and the results are tabulated:
r --Organism I~IC ~,~/ml Baeillus subtilis A 250 Enterobaeter eloaeae ~ ~ lOOO
Eseherie11ia eoli 1o4la 250 Klebsiella ~-ero~enes A 5 Proteus mirabilis C977 500 Pseudomonas aer~inosa A~ ~ l()OO
Salmonella typhimurium CTlO250 Serratia marseens US39 ~ lOOO
Staph. aureus O.cford 500 Staph aureus Russell _ _ _ _ :
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( J~ ff"~ T~h ~ b i t i. c)n 1'3-Lactatnasc inhibito~,~ acti~ity ol` the preparation a~inst ran~e of enzyme preparations have been determined:
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I ~-L~ctc~as~ Pr~aration 1~-'. I,nhibi1;i.oll ¦ Concentra-tion I From:at 20~)~g/1nl j giYing 50,' Inllibition . . _ .~ " I Il/
Staph. aureus Russell _ 1 150 E. coli JT4 _ 72 ln Proteus mirabilis C~3~9 _ 62 Pseu(lomonas aeru~inosa j - 5' Dal~leish I
Enterobacter cloacae P99l - 10 Pseudomonas a.eruginosa Al ~9 Kleh~ n.- E70~ 13 _ : - 3-3 ':
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.~ Further l~u~ification of th~ Sodiiw~ Saltsof the Co~olu~dsof Eorrmlle (I) The preparation ob-tained in Exr~plc ~ isdissolvedin deionised water c~nd loaded onto ~ QAE Sephadex A25 column (Q~, Seplladex A25 is a stron~ly basic anion exchan~,er supplied b~ PharQlacia L-td.) prepared in deionised water. The column is eluted ~rith a sodi~m chloride concentration gradient from O to 0.1~ M NaCl in deionised water.
Fractions from the col-uQn are monitored for their ~-lact~Qase inhibitory cactivity acainst an R~ repara-tion and tho3e cho~lin~ the ~,reatest 1~ activity care coMbined. NaCl -to a final concentration of at least 5S3 is added to tho combined fractions. The reslllting solution is run onto anAmberlite XAD-column (Rohm & Haas Ltd,) and the column eluted wi-th n-propanol/water 1/4.
~ractions containin~ the desired salt as judged by their ~lactamase inhibitory activity are combined, evaporated under vacuo to remove orl~,anic solvent and freeze dried.
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' '~ . ' "' : ' ', ' ' , ' ~ , A Fllrthcr l'ur _ .~tion o the So~ Salts of 1;he Comlloun~of the FonQula (1) The combined fractions from the Q~E Sephadex chromatogTaphy of Exa[Q~le 4 may be desalted and fur-ther purified by chrom~to~Taphy on a Biogel P2 gel col~n (~io-Rad Laboratories Ltd., 27 Ilomesdale Road, 3rolnlcy, ~ent) as follows:
1'he freeze dried solid from the Q~E Sephadex column is dissolved in a small volu;ne of deionis~d water and run onto a ~io~el P2 column.
~e colu~n is eluted with 1~ aqueous butanol. ~ractions are monitored lo or their ~-lactamase inhibitory activity and for reaction with silver nitrate. Those giving a negative reaction to silver nitrate but giving suitable ~-lactamase inhibitory ac~ivity are combined and freeze dried.
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A I~lrther ~rification o~ the Soclium SaltcoL -the Comsoun(~ oî I~loLmula ~ ) The preparation obtaincd in ~xample 2 is dissolved in deionised t/ater ~Id applic(l to a col~mn of the stron~;ly basic anion exch~lge resin ~nberlite IRA 45~ (Rohm ~ Haas (~K) L-td., Le1~lin~ House, 2 Mason's Ave., Croydon, U.K.). The colwnn is prepared in 0.0511 pH 7 phosphate buffer and eluted with a sodiwn chloride ~adient in phosphate bufer. The elution is from 0.05i`1 pH 7 phosphate to 0.05M
p~I 7 phosphate containing l.OM NaCl. Fractions showin~ the ~reatest L3-lactamase i1~libitory activit~ are combined. N~Cl is added to the combined fr~ctions to gi~e a conc~ntration of at least 5',b. The resulting solution is run onto an Amberlite XAD-4 column prepared i.ndeionised water. ~he colwnn is eluted with n-~rop~nol/water l/4.
Fractions shot1ing the greatest ~3-lactamase inhibitory activity but givin~ a negative reaction with silver nitrate are combined and freeze dried.
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A ~nrther l~]rificatiorl of the So(liwn Sal-~ of thc Cor~ olirl-]~o;~ llorm;
The salt of the compound of the formula (I) may be further purified by chromato~L~aphy on a column of cellulose (Cellulose CC31 ~atman, Springfi~ld Mill, Maidstone, Kent, U.~.) as follo~s:
The impure solid containing the salt of the compound of tihe formula (I) is dissolved in a minimum of deionised water and n propanol added to about 5~o. The resultin~ solution is r~n on-to the cellulose column 2nd the colu~l eluted lJith n-propanol/~ater ~/l. The resulting fractions after dilution into deionised ~/ater are monitored for their ~-lact~mase inhibitor~ activity. ~ractions con-taining the desired salt are combined, evaporated under reduced pressure to remove solvent and free~e dried.
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L~ .~ ?i . ~ 3 A Fllri.llor l'urification o~ t,he So(liurr~ ltsor t,he Com!)ov~(38of ~hc FoImula ~ ) Thc process of Example 4 ma.y be used but replacin~ the s~arting material with the preparation obtained in Example 3.
Exam~le 9 A Further Purification o~ the Sodium Saltsof the Com~ounr7sof the Formula (II) The process of E~ample 5 may be used but replacin~ the startin~
material with the product of Example ~.
Ex~m~l,e 10 -~ ~urther ~lrification o~` the Sodium Saltsof the Com~oundsof the Formula (II) The process of Example.~ ma,y be used replacing the startin~
material ~ith the preparation obtained in Example 3.
Examl~le 11 A F~rther Purification of the Sodium Saltsof the Compoundsof the For!l.la (II) q'he process of Example 7 may be used but replacin~ -the startin~
material with the yreyaration obtainel in Example 3.
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Preparation of Crude ~ntibiotics fro~ Cu]ture ~`iltrate Culture filtrate (t30 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 cm column of Amberlite IRA 453 a strongly basic acrylic based anion exchan~re resin (~0~ & IIaas). The column waS eluted ~ith a sodium chloride concentration ~rradient.
The grradient was from 0 to 1.0 M NaCl in 0.05~i pII 7 pllosphate buffer at a flo~r rate o 2.5ml/minute and 5 ml fractions collected. Fractions were monitored for their ~3-lactar~ase inhibitory activity against a preparation of ~T~ laota~ase. Those fractions griving ~od inhibito~
activity and containin~ the sodium salts of the ompounds of the formulae (I) and (II) t~ ) were combined. (~he di-sodium salts of ~l 4550, ~I 13902 and ~ 17l3~'30 eluted starting at fraction 17).
To the combined fractions was added sodium chloride (3 ~r) ~ and the resultingr solution ~as rwl onto a 1.5 ~ 15 cm Amberlite XAD-4 colur~l (Ro}lm ~ Haas) prepared in deionised water. q'he column ~las eluted with n-propanol/water 114 at 3 ml/minute and 4 ml fractions were collected. ~ractions were monitored for tlleir ~-lactamase inhibitory activity and for reaction with ~SrI~o3 solution. q~hose fractions giving ~ood inhibitory activity and a negative reaction ~/ith silver nitrate were combined and freeze dried to give a partially purified preparation of the sodium salts of the compoullds of the formulae (I) r~nd (II).
I~is impure preparation ma~ be furthe~ purifie~d by the proce~ses described hereinbefore.
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Preparation of Crude ~n-tibiotics from Cultlue ~iltrate C~ture filtrate (~05 ml) prepared essentially as described in Description 1 was run onto a 1.5 Y. 15 cm column of ~berlite IRA 45'~
a strongly basic acrylic based anion exchan~e resin (noh~ & ~iaas).
The column was washed with deioni~ed water (100 ml) a-t 5 ml/minute and eluted with 0.025 I~ p~I 7 phosphate buffer at 5 ml/minute, 10 ml fractions were collected. Fractions were monitored for their ~-lactamase inhibitory activity a~ainst a preparation of R~ lactamase. Those 1~ ractions ~rivin~r good inhibitory activity and containin~ the sodium salts of the compounds of the formulae (I) and (II) (12 -32) were combined. The combined fractions t~ere freeze dried. The freeze dried solid ~as dis~olved in deionised water (20 ml), l~aC1 (2 g) `~'a9 added and the resulting solution was run onto a 1.5 x 15 cm Amberlite ~D-4 colu3ln (Rohm & Haas) prepared in deionised water. The column tJas eluted with n-propanol/water 1/4 at 2 ml/minute and 4 ml fractions were collected. Fractions uere monitored for their ~-lactamase inhibitory activity and for reaction with ~rN03 solution. 'nlose fractions (7 - 14) giving ~rood inhibitory activity and a ne~^ative reaction with silver nitrate t~ere combined and freeze dried to yield a partiaIly purified preparation o the salts of the compounds of the formulae (I) and (II). This impure preparation may be further purified by the pl~ocesses described hereinbefore.
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ample 1 ~1 Fermentation Conditions for 300L Fermentati_ A freeze dried ampoule of Streptomyces olivaceus (ATCC 313~5) was resuspended in lO ml of a sterile solution of the following composition:
Glucose 2~
Soya bean flour 16 pH 6.5 prepared in deionised water (The soya bean flour is Arkasoy '50' supplied by the British Arkady Co. Ltd., of Old Trafford, Manchester).
l ml of this suspension was used to inoculate lO0 ml of medium of the same composition contained in a 500 ml Ehrlenemeyer flask closed with a foam plug. After inoculation the flask was incubated on a rotary shaker at 28C for 30 hours. 5 ml portions of this seed culture was used to inoculate solid agar slants in Roux bottles of the following composition:
V8 Vegetable juice 20.0%
Bacto agar (Dlfco)' 2.5%
p~l 6.0 , , prepared in deionised water ,' , (The V8 vegetable juice is supplied by Campbell's Soups Ltd., ~ings Lynn, Norfolk, England and Bacto agar is supplied by Difco Laboratories, Detrolt, Michlgan, U.S.A.).
~r~d~ ~a~K
H ~ S-C=C-NH-CO-CH3 (IIa) HO ~ C=C-n3-CU-CH3 ~IIb~
O : C02H
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It will be realised that the preceding compound~
~ay be named as follows:
Ia (5R,6R)-3-(2-acetamidoethylthio)-6-[(S)-l-hydroxyethyl]
-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylic acid.
Ib (5R,6S)-3-(2-acetamidoethylthio)~6-[(S)~l-hydroxyethyl]
-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
IIa (5R,6R~-3-[(E-2~acetamidoethenylthio~-6-[(S)-l-hydroxy-ethyl]-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylic acid.
IIb (5R,6S)-3-4E 2-acetamidoethenylthioD-6-~(S)-l-hydroxy-ethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid Both cis- and trans- isomers of the compounds of the 15 formulae tI) and (II) have useful antibacterial and ~-lactamase inhibiting properties and so this invention extends to the isolated compounds of the formulae ~Ia) and (b) as well as to mixtures thereof and to the isolated compounds of the formulae (IIa) and (IIb~ as well as mixtures thereof.
Naturally isolated cis- and trans-~forms~will most suitably be in the forms of substantially pure pharmaceutically acceptable~salts as described above; that lS they should be at :: : : : : ~ :
~ least 50% pure, more suitably at least 75~ pure, preferably .
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90~ pure and most preferably at least 95% pure. ~'urthermore it is preferred to use one o~ the aforementioned compounds when substantially free of its 6-position isomer ~hat is (Ia) free of (Ib), (IIa) free of (IIb?,(Ib) free of (Ia) or (IIb) free of (Ib)]. In general such compounds should not contain more than 5% of its 6-position isomer and preferably not more than 1~ of its ~~position isomer.(Iligh pressure liquid chromatography may be used to monitor purities.) A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ia) having a molar extinction coefficient (in water at neutral pH)of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 2g8 nm).
A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ib) having a molar extinction coefficient (in water at neutral pH) of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 301 nm).
A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (IIa) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for UV absorption maximum at about 308 nm).
~ favoured aspect of this invention provides an alkali metal salt of the compound of the formula (IIb) having a molar extinction coefficient (in water at neutral pH~ of not less than 13000 (preferably not less than 13500) (for UV absorption on maximum at about 308 nm).
Preferably the preceding alkali metal salts are the sodium salts.
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The present invention also provides a pharmaceutical composition which comprises a compound of the formula (I) or (II) or a salt thereof and a pharmaceutically acceptable carrier.
The compositions of this invention will generally utilise a pharmaceutically acceptable salt of a compound of the formula (I) or (II), for example a sodium or potassium salt.
The pharmaceutical compositions of this invention may be adapted for oral or parenteral administration. Suitably the compositions are provded as dosage forms which contain from 50 to 500 mg of a compound of the formula (I) or (II) or its salt, for example about 100, 150, 200 or 250 mgs.
Most usually the composition will be adapted for administration by injection.
These compositions may contain diluents, binders disintegrants, lubricants or other conventional excipient and may be fabricated by conventional methods of mixing, filling and the like.
The compositions may take the form of tablets, capsules, vials or other similar forms.
If desired the composition may advantageously conta:in a penicillin or cephalosporin. In such instances the ratio ;;
of synergyst (i.e. the compound of the invention) (preferably as a salt) to penicillin or cephalosporln is~ usual~ly~from 2:1 to 1:12, more usually from 1:1 to 1:5, for example~].:2l 1:3, or 1:4 wt/wt.
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Particularly suitable penicillins for inclusion in the compositions include ampicillin, amoxycillin, carbenicillin, ticarcillin and their pro-drugs. When adapted for injection such compounds are generally presented in the form of their sodium salts.
Particularly suitable cephalosporins for inclusions in the compositions include cephaloridin and cephazolin.
Preferred penicillins for inclusion in the compositions include ampicillin trihydrate, amoxycillin trihydrate, sodium ampicillin and sodium amoxycillin.
Preferred cephalosporins for inclusion in the compositions of this invention include cephaloridin and sodium cephazolin.
The compound of the formula (I) or (II) or its salt may be an isolated compound of the formula (Ia), (Ib) or (IIa), (IIb) or their salts or mixtures of the compounds of the formula (Ia) and (Ib) or (IIa) and (IIb) or their salts.
However it is preferred to use a compound of one of the preceding formulae free from its isomer. Said compound is generally in the form of a pharmaceutically acceptable salt such as the sodium salt.
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The present invention also provides a process for the preparation of a compound of the formula (I) or ( II ) or its salt which process comprises cultivating a produclng strain of Streptomyces olivaceus or Streptomyces ~edanesis until a substantial quanity of a compound of the formula (I) or ~II) or its salt is produced and thereafter recovering a compound of the formula (I) or (II) or its salt from the cultivation medium.
When used herein, the term "Streptomyces olivaceus"
is defined according to the classification of H~tter R
(in Systematic der Streptolyceten, S~ Korger, Basle, Pages 8-32). Note that on this definition S~reptom~ces fulvovoridis, Streptomyces flavus and Streptomyces flavovirens may be regarded as being synonymous with Streptomyces olivaceus.
Suitable strains include those described in British Patent Specification No. 1467413.
A preferred organism for use in this process is Streptomyces olivaceus ATCC 31126 or a high yielding mutant thereof.
A further preferred organism for use in this process is Streptomyces olivaceus ATCC 31365 or a high yielding mutant ` thereof.
As previously indicated the recovered material should be at least 1~ pure, more suitably 5% pure, yet more suitably at least 50% pure, preferably at least 75~ pure and more preferably at least 90~ pure~ for example at least 95~ pure.
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When used herein the term "cultivation" means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid medium but in general it is preferable to use a liquid medium. General cultivation conditions for the growth of Streptom~ces olivaceus are as described in British Patent Specification No. 1467413. General conditions for the growth of Streptomyces gedanensis are similar.
The process of this invention may be adapted to provide a compound of the formula (I) or its salt, a compound of the formula (II) or its salt or a compound of the formula (I) or its salt together with a compound of the formula (II) or its salt.
Normally the process is adapted to the preparation of a salt rather than the parent acid.
It is preferable that the cultivation medium does not contain added sulphate since this often leads to the prepara-tion of MM4550, MM13902 and MM17880 at the expense of the production of the compounds of the formula (I) and (II) and their salts.
The compounds of the formula (I) and (II) in the form of thelr salts may be obtained from the culture filtrate by (a) contacting the filtrate with carbon until the antibiotic activity is absorbed thereon, (b) eluting the antibiotic activity from the carbon using aqueous acetone, (c) combining ~ .. . .
the fractions containing ~-lactamase inhibitory fractions, (d) evaporating the acetone and much of the water to yield a more concentrated aqueous solution, (e) applying the solution to an anion exchange column, and (f) eluting the ~-lactamase inhibitory metabolites therefrom with a solution of an electrolyte buffered to approximate neutrality collecting the fractions containing the compound of the formula (I) or (II) in salt form, (g) applying the resulting solution to a resin which separates the inorganic materials from the compounds (I) and ~I)and (h) isolating the solid preparation of the salt of a compound of the formula (I) or (II) from the resulting solution.
The compounds of the formula (I) and (II) in the form of their salts may also be obtained from the culture filtrate by (1) contacting the clarified cultivation broth with a strongly basic acrylic based anion-exchange resin until the antibiotic activity is absorbed thereon, (2) eluting the antibiotic activity from the resin using an aqueous solution of a buffer optionally also containing a salt, (3) combine the fractions with ~-lactamase inhibitory activity, (4) apply the combined fractions to a XAD-4 column, (5) elute with aqueous isopropanol, (6) combine the fractions with ~-lactamase inhibitory activity, ~7) remove the isopropanol and concentrate the solution by evaporation, (8) apply the solution to an anion exchange resin and proceed as in steps (f), (g) and (h) outlined above.
.
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It is generally preferred to use an acrylic based strongly basic (type 1) anion exchange resin (in the form of an acid addition salt normally the hydrochloride) such as ,$
Amberlite IRA 458 (which may be obtained for example from Rohm and Haas for example at Lennig House, 2 Massons Avenue, Croydon, U.K.). An advantage of such a resin is that it allows the salt of the compounds of the formula (I) and (II) to be eluted successively by using an aqueous salt solution, for example a buffered solution of a chloride such as sodium chloride or the like. If the less favoured strongly basic resins having a polystyrene/divinyl benzene matrix are employed it is generally necessary to elute with an aqueous lower alkanolic solution of a salt (for example a chloride such as sodium chloride) in order to obtain satisfactory lS recoveries and such solvents can lead to a less pure preparation of the desired materials.
(This process variant differs from the carbon absorption process in that the salts of MM4550, MM13902 and MM17880 are separated from the salts of the compounds of the formula (I) and (II) at the first elution stage.) The process of this invention differs funda~entally from the previously disclosed process in that the fractions selected for further processing at stage (f) are those containing the salt of a compound of the formula (I) and (II) substantially free of other antibiotics.
The free acids of the formulae (I) and (XI) may be ~e~na~K
.. . .. , . . .,, , ~. . , . , . ~
obtained by careful acidification of a salt of the compound of the formula (I) or (II) respectively followed by rapid extraction into a water immiscible organic solvent followed by recovery of the acid from solution.
In the processes of this invention it is frequently most con~enient to work with an alkali metal salt, of the compound of the formulae (I) and/or (II) such as the lithium, sodium or potassium salts and of these the sodium salt is favoured. It is possible to prepare other salts by the extraction process but it is usually more suitable to first form the purified alkali metal salt especially the sodium salt and then convert this to an alternative salt, for example ky passing through a bed of cation exchange resin in the form of the alternative salt. Thus in this description other electrolytes (such as the lithium, potassium or other salt) can be substituted for the described sodium salts but in general it is preferred to work with the sodium salt. Similarly salts other than chloride (for example bromide, nitrate or the like) may be employed although in general it is preferred to work with a chloride.
A preferred method of chromatographic purification (steps f and g) uses an aqueous solution of a sodium salt buffered to approximate neutrality in conjunction with a basic ion-exchange resin. Thus an aqueous solution of sodium chloride ~or other similar salt) buffered to about pH 7 with a conventional buffer such as a phosphate buffer may be used in conjunction with support resins which contain . ~
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secondary or tertiary amino groups or quaternary amino groups. Suitable supports include basic ion-exchange celluloses and basic ion-exchange cross-linked dextrans such as DEAE cellulose, DEAE Sephadex, QAE-Sephadex and equivalent agents.
A related suitable method of chromatographic purification (steps f and g) uses a sol~-ent system comprising a mixture of water and small quantities of a water immiscible organic solvent such as a lower alkanol (i.e. Cl 4 alkanol) in conjunction with an inert support material such as silica gel or cellulose. Suitable solvent systems include aqueous isopropanol, aqueous n-butanol and the like. For example a very roughly 1:4 mixture of water and isopropanol may be used in combination with a cellulose support.
The product of the preceding proceedure frequently contains a high proportion of sodium chloride so that it is beneficial to de-salt the pooled solutions. De-salting may be effected by pas~ing the solution through a bed of lipophilic material onto which the antibiotic is adsorbed but which does not absorb the sodium chloride. Suitable materials include polystyrene based polymeric absorbants such as Amberlite XAD-4,Diaion HP20 and the like. The produce of the preceding process may also be desalted by chromatography on suitable ;`
gel filtration agents such as cross-linked dextrans such as ,~
Sephadex G10 and G15 and polyacrylamide gels such as Biogel P2. The antibiotic may be eluted from such materials using water, aqueous methanol or the like.
~" - .
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, The columns are eluted at such a rate as to allow s~paration of the antibiotics into distinct fractions.
In general distinct zones can be eluted from these columns;these contain di-sodium MM4550, di-sodium MM13902, di-sodium MM17880, the sodium salts of the compounds of the formula (I) and the sodium salts of the compounds of the formula (II) eluting close to the sodium sal~ of the compound of the formula (I). In general the three di-sodium salts are fairly widely separated from the mono-sodium salts on anion exchange resins. If the column is not carefully monitored it may be that the mono-sodium salts are obtained in overlapping fractions. If this is 50 then either (a) this solution can be freeze dried to yield a useful impure complex containing the antibiotics which can be reworked later or (b) the solution per se can be re-chromatographed with careful monitoring of the eluant to ensure collection~
of the solution of sodium salt of a compound of the formula (I) free from the sodium salt of a compound of the formula (II) and/or the collection of the solution of the sodium salt of a compound of the formula (II) free from the sodium salt of a compound of the formula (I); these solutions may then be freeze dried or otherwise rendered solvent free.
The fractions chosen for collection will be those which show significant ~-lactamase inhibitory activity or anti-bacterial activity. Suitable methods of detecting ~-lactamase inhibitory activity include those of the aforementioned , - , : . .: .
~5~
British Patents although any convenient method may be employed.
The fol.lowing Scheme shown preferred sequence for obtaining the compounds of the formulae (I) and (II) as their sodium salts. The sodium salts obtained in this manner can be further purified if desired by using the chromatographic procedures described hereinbefore.
Trituration of the salts of the compounds of the formulae (I) and tII) under an organic solvent such as moisture containing acetonitrile or acetone can aid in the removal of impurities.
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Cultu~e Filtrate Absorb onto Carbon ~ Elute with 20% aqueous acetone Combine Frartlons with~ -lactamase ~nhibitory Activity Remo~e Acetone and concentrate 1 by Evaporation Cellulose DE52 Anion Exchange Column , ,. :'.
Elute with Phosphate Bufer pH7 ~ ;~ ;:: `
Sodiu~ Salts of Sodium Salts of Di-Sodlum Salts of:
Compounds o~ Compounds of MM4550j MM 13302 and Formula (I) Formula (II) MM17880 ', ' ~ ' De-salted Solution De-salted Solution - ~
Freeze Dried Solid F~eeze D~ ed ~olld , '` ~
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Culture Flltrate Absorb onto Strongly Ba~ic Acrylic Anion ~xchange Resin ¦ Elute with Buffer Comblne Fractions wlth earlier eluting ~g-lactamase Inhibitory Actlvity De-salt on ~AD 4 1 Elute with Aqueous Isopropanol Combine Fractions with ~-lactamase Inhlbitory Actlvity ~.
Remove Isopropanol and . ~ concentrate by Evaporation Cellulose DE52 Anion Fxchange Resin : :
Elute with Phosphate Buffer pH7 \ .: ':
1~ ~~ ':
Sodiu~ Salts of Compounds Sodium Salts of Compounds of of Formula (I) Formula (I~) ~ , De-salted Solution De-salted Solution : :
Freeze Dried Solid Freeze Dried Solld ,:':
- 20 ~
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~ desired the salts of the compounds of the formula (I) and (II) prepared by the previous methods may be further subjected to chromatographic separation techniques to yield an isolated / salt of a compound of the formula (Ia), (Ib), (IIa) or (IIb).
Such processes are favoured aspects of this invention.
Normally the salt used in such a process will be a monovalent salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.
One suitable form of chromatography for the separation process is high pressure liquid chromatography(hplc?, for example using an aqueous ammonium formate buffered solution. Once fractions containing the desired compound are obtained a solid preparation may be obtained by freeze drying or the like.
Compounds of the formuIa (Ia) and (Ib) may be separated by column chromatography on supports such as acetylated cellulose eluting with alcohol/water mixtures. Compounds of the formula (IIa) and (IIb) may also be separated using similar chromatographic techniques.
Once fractions containing the desired compound are 20~ obtained a solid preparation may be obtained by freeze drying or the like. ;
- 21 -~
The present invention provides a process for the preparation of a salt of the compound of the formula (Ia) substantially free of the compound of the formula (Ib~ which comprises sub~ecting a mixture of said sal~s to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.
The present invention provides a process for the preparation of a salt of the compound of the formula (Ib) substantially free of the compound of the formula (Ia) which comprises subjecting a mixture of said salts to chromato- :
graphic separation on Diaion HP20 or a chromatographically equivalent resin~ ~
The present invention provides a process for the ~ :
preparation of a salt of the compound of the formula (IIa) substantially free of the compound of the formula (IIb) which comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.
The present invention provides a process for the preparation of a salt of the compound of the formula (IIb) substantially free of the compound of the formula (IIa) which comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin. :
The salts prepared by the preceding processes will normally be~monovalent salts~such as alkali metal salts, for example the lithium, sodium or potassium salt and:will :
:
- 22 - ~
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preferably be the sodium salt.
The salts prepared by the preceding process will not normally contain more than about 5% and more suitably not more than about 1% of the undesired isomer.
DiaiOn (which is a Registered Trade Mark) is a highly porous polymer manufactured by ~itsubishi Chemical Industries. It is not an ion exchange resin but is a synthetic adsorbant which has an extra large active surface area to which organic compounds may be effectively adsorhed.
Diàion HP20 is a styrene divinylbenzene copolymer in bead form having a macroreticular structure with a specific surface area of about 7.8 m2/g and a pore volume of 1.16 ml/g. Other details of this resin may be found in (HP series, October 1976, which is incorporated herein) ` the Diaion data sheet /(Mitsubishi Chemical Industries Ltd.
Offices may be found via 5-2 Marunouchi 2-chome, Chiyoda-ku, Tokyo, Japan; 277 Park Ave., New York, NY 10017 USA;
Ratinger Str. 45, 4 Duesseldorf, West Germany;~etc.) Resins chromatographically equivalent to Diaion HP20 be will also normally/chemically and physicalIy similar, that is they will generally be macroreticular resins based on styrene divinylbenzene copolymers and free of ionized gr~ups.
Most suitably the mixture of isomers applied to the resin will be of good purity and will be substantially free from other organic impurities although quantities of inorganic impurities (for example an alkali metal salt such as a chloride, for example sodium chloride) may be present.
Suitably the solvent employed will be water or water in admixture with a lower alkanol or similar miscible organic solvent.
Preferably the solvent used is water.
The desired material may now be obtained by removal of the solvent for example by evaporation, freeze drying or the like. Alternatively the solution may be rechromatographed directly on a suitable resin ~uch as Biogel P2 and/or Diaion HP20~ for further purification prior to removal of the solvent.
Water may be removed from aqueous solutions of salts of this invention by such processes as evaporation under vacuum to about 1/10 volume, makin~ up to the original volume with ethanol, reconcentration to about 1/10 volume under vacuum, making up to the original volume by adding toluene and evaporation to dryness in vacuùm. Residual solvents may be removed by storlng under high vacuum.
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Prcl~aration of Clarified ~roth A spore suspension of Stre~_omyces olivaceus ATCC 31126 was used to inoculate 100 ml of a seed sta~e medium COlltaine~ in a 500 ml Ehrlen~meyer flask closed with a foam plug. The seed medium was bean 2S' ~lucose a~d l`~o soya/flour made up in deionised water. (The soya bean flour was Arkasoy 50 supplied by the British Arkady Co. Ltd., 01d Tr2fford, Manchester, U,~.) The seed sta~re medium was ~ro~m for 48 hours on a rotary shaker at 26C. 5 ml portions of the seed stage medium were used to~inoculate 100 ml portions of the fermentation medium contained in 500 ml 3hrlencmeyer fla~ks closed with fc~ plu~s. The fermentation medium ~rhich was made up in deionised water had the followin~ composition:
So Glucose, 2.0 Soya bean flour 1.0 CaC03 0.02 CoC12 2 0.0001 The fermentation flasks were incubated at 26C on a rotary shaker for 72 hours. 20 flasks ~lere harvestcd and the resultant ~hole broth centrifuged at 2200 ~ for 10 minutes.
Use of Streptomyces olivaceus ATCC 31365 ln the above process i9 also favoured.
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D~:s~ ti~ll 2 Pre-paralion of Crude Antibiotics in Solu-tion 'rhe culture filtrate (1500 ml) obtaille~ art(~r c~ triu~ration ~a5 submitted to ~)urification usin~ a caxboll colwnn a. follo~ls:
A 2.5 ~ 37 cm Darco ~rarlulc~-^ carbon column was prepared in deionised ~ater, the column uas ~lashed successivcly ~ith 1 litre 2,~ ~aOH, 1 litre deiol~sed water, 1 litre lN HCl and 1 litre deionised water all at 15 ml/minute. The colu~l ~1a5 then washed ~/ith 0.05M pl~ 7 phosphate buffer until the pH of the eluant was 7Ø
'Mle culture filtrate (1500 ml) was r~l onto the carbon column at 15 ml/minute. The column was then eluted with acetone/water 1/4 at 15 ml/minute and 22 ml fractions were collected. Fractions were monitored for their ~-lactamase inhibitory ac-ti~,-ity a~ainst a preparation of RT~ enæyme (supplied by ~iicrobiolo~ical Research Establishment, Porton). Fractions showing the greates-t activity (~ - 22) were combined and evaporated under reduced~pressure to~remove acetone. The resulting - aqueous solution was stored deep frozen prior to subsequent work-up.
(The RT~I enæyme typlfles plasmid controlled ~-lactamases and may be replaced by otller such R-factor ~-lactamases if desired).
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Isolation of the Compounds of F_~mulae (I) and (Il) a8 their So~ium Salt~ Suostantiall~ Free of Salts of 1~ 5~0, I~I 13902 and i~ 178aO
The crude liquor obtained in ~escription 2 were evaporated under reduced pressure to approximately 2'j ml alld load~d onto a 3.~ x 27 cm D~AE cellulose ~ea~.ly basic anion e~chanse column (the DEAE cellul~e was D~52 ce~ lose supplied by ~natman Ltd., Sprin~field Mill, Maidstone, Kent) prepared in 0.025 M pH 7 phosphate buffer. The column was eluted ~lith 0.025 I~ pH 7 phosphate buffer at a rate of 8 ml/minute and 25 ml fractions were collected. ~he fractions were monitored for their ~-lactamase inhibitory activity against a preparation cf ~T~M
~-lactamase. The first two pec~ks of inhibitory activity were retained.
~he first band fractions (13 - 16) containing the salt of the compound o fo~ula (I) ~ere combined and freeza dried to yield a solid preparation containing the sodium salt of the ~ mpound of formula (I) substantially free of the salts of the di-basic a~tibio-tics.
The second band fractions (19 - 21) containing the salt of the compound of formula (II) were combined and reeze dried to yield a solid preparation containing the sodium salt o the compound of the formula (II) substantially free of the salts ofthe di-basic antibiotics.
(Freezc drying combined fractions 13 - 16 and 19 - 21 naturally leads to a preparation containing a mixture of the sodium salts ol`
the ~Itibiotics of the for~ulae (I) and (II). The di-sodium salts of ~14550,1~ 13902 and M~l 17880 eluted af~er the desired salts).
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F,xe~n~,le 2 Partial Purifica-tion of the Sodil~m Sa]tsof the Com~oun~ of the Fo~mlla ~) The freeze dried preparation of the ~alt of -the compound of Example 1 was dissolved in deionised wa-ter (10 ml) and sodium cllloride (1 g) added to the solution. This solution was run OlltO a 1.5 ~ 15 cm XAD-4 (supplied by Rohm & Haas) colu~m prep~red in deionised ~ater~ The column was eluted with water/n-propanol 4/1 at 2 ml/minute and ~r ml ~ractions were collected. ~ractiong were monitored for chloride by their reaction with ~N03 and for their ~-lactamaGe inhibitory activit~ against a preparation of RT~ lactc~mase. Fractions ~ith the greatest inhibitory activity and giving a negative reaction with ~ilver nitrate (fractions 6 - 13) ~rere combined and freeze dried to yield an amorphous solid (32.5 mg) containing the sodium salt of the compound of the formula (I).
The properti~s of this preparation were as follows:
(A) Chromato~raphic Properties (i) Chromatography on ~Ihatman D~81 Ion ~xchange Paper (a weakly basic anion exchange paper):
. , __ ~luant Rf of Sodium Salt _ _ 1. 0.05M pH 7 phoGphate buffer o.69 2. 0.05M pH 7 phosphate buffer 0 ~0 containing 0.2M NaCl .
_ .
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ro~.ato~ ?h~ on ~ atman ~o. 1 ~aper:
Solvent System I Rf of Sodi~ Salt ._____ _ . . _.
1. But~lol:Etha~ol:~ater 0 12 1 Top P~ase 4:1:5 .
¦ 2. Butanol:Pyri.dine :~latero . 42 (B) ~ h Volta~e Pa~er Flectro~horesis ~he electrophoresis was carried out on No. 2') ~aper in pyridine/acetic acid buf~er pH 5.3 at 5GOO volts for 15 minutes.
The ~ values ~or the sodium salt t~king benzyl penicillins as l.O-is also 1Ø
(C) Antibacterial Activit~
The antibacterial activity of the preparation using the microtitre method was determined as ~ollows:
Organism ¦ I/~C (~g/ml) Bacillus subtilis A C 4 Enterobacter cloacae Nn 1250 Bscherichia coli 1041~ 150 ~. coli JT 410 625 Klebsiella aerogenes A 312 Proteus mirabilis C977 62S
Pseudomonas aeru~inosa A> 2500 ~ Salmonella typhimurium CT10312 ~:
: 25 Serratia marcescens US39 625 Staph. aureus Russell 150 _,_ ~
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(D) ~'n~,~e In'~ ition Tlle enz~me inhibitory activity of the preparation against a series of ~-lactamase preparations is summarised below:
- , ..... . j .. . . . , ~-Lacta~ase Preparation 1~ Inhibition Concentration ~rom: I a-t 200~g/ml givin~ 50~' , ~ u,rr/ml Staph. ~ureus Russell 4o E. coli JT4 _ 95 Proteus mirabilis C~9 _ 112 Pseudomonas aeru~.grinosa Dal~leish ~ 170 Enterobacter cloacae P99 ~3 Pseudomonas aeru~inosa h 30 l~ebsiella aeFogenes E70 21 (i~lethod of ~elgian Patent No. 827926).
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Example 3 Partial Purlfication of ~he Sodium Salt of the Com~ounds of the Formula (II) The freeze dried preparation of the salt o~ the compound II of Example l was dissolved in deionised water (10 ml) and sodium chloride (l g) added to the solutlon, Thls solution was run onto a 1.5 x 15 cm XAD-4 (supplied by Rohm & Haas) column prepared in deionised water, The column was eluted wi~h water/~-propanol 4/1 at 2 ml/minute and 4 ml fractions were collected. Frac-tions were monitored for chloride by their reaction with AgN03 and for their ~-lactamase inhibitory activity against a preparation of R~EM ~-lactamase.
Fractions with the greatest inhibitory activity and giving a negative reaction with silver nitrate (fractions 7 - 13) were combined and freeze dried to yield an amorphous solid (32~5 mg) containing the sodium salt of the compound of the formula (II).
The properties of thls preparation were as follows:
(A~ _ Chromatographic Propertie~
(i) Using weekly basic anion exchange paper DE81 cellulose (~hatman~ the sodlum salt has an Rf of 0.54 when eluted with 0.05M pH 7 phosphate buffer.
(ii) Using Whatman* No. 1 paper: the sodium salt ha~ an Rf oP 0.20 when eluted with butanol/ethanol/water 4/1/5 top phase.
* Trade Mark ~ 31 "; :
(13) ~ii,r^ll Volt;,~ aller_,lccl;70~ 7(?~.is ~ he electrophoresis was earried ou-t on '~1atman No. 2u paper in pyr~ e/acetie acid buff~r pH 5.3 at 50()0 volts for 15 miluitos.
~le ~I value for the salt talcin~ benzyl penieillin as l.O is 0.95.
(C) ~ntibaoterial Aetivit~
The antibaeterial activity of the preparation using the micro-titre ~ethod was determined and the results are tabulated:
r --Organism I~IC ~,~/ml Baeillus subtilis A 250 Enterobaeter eloaeae ~ ~ lOOO
Eseherie11ia eoli 1o4la 250 Klebsiella ~-ero~enes A 5 Proteus mirabilis C977 500 Pseudomonas aer~inosa A~ ~ l()OO
Salmonella typhimurium CTlO250 Serratia marseens US39 ~ lOOO
Staph. aureus O.cford 500 Staph aureus Russell _ _ _ _ :
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( J~ ff"~ T~h ~ b i t i. c)n 1'3-Lactatnasc inhibito~,~ acti~ity ol` the preparation a~inst ran~e of enzyme preparations have been determined:
.. ... . .__ _ _ .......... . . _ I
I ~-L~ctc~as~ Pr~aration 1~-'. I,nhibi1;i.oll ¦ Concentra-tion I From:at 20~)~g/1nl j giYing 50,' Inllibition . . _ .~ " I Il/
Staph. aureus Russell _ 1 150 E. coli JT4 _ 72 ln Proteus mirabilis C~3~9 _ 62 Pseu(lomonas aeru~inosa j - 5' Dal~leish I
Enterobacter cloacae P99l - 10 Pseudomonas a.eruginosa Al ~9 Kleh~ n.- E70~ 13 _ : - 3-3 ':
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.~ Further l~u~ification of th~ Sodiiw~ Saltsof the Co~olu~dsof Eorrmlle (I) The preparation ob-tained in Exr~plc ~ isdissolvedin deionised water c~nd loaded onto ~ QAE Sephadex A25 column (Q~, Seplladex A25 is a stron~ly basic anion exchan~,er supplied b~ PharQlacia L-td.) prepared in deionised water. The column is eluted ~rith a sodi~m chloride concentration gradient from O to 0.1~ M NaCl in deionised water.
Fractions from the col-uQn are monitored for their ~-lact~Qase inhibitory cactivity acainst an R~ repara-tion and tho3e cho~lin~ the ~,reatest 1~ activity care coMbined. NaCl -to a final concentration of at least 5S3 is added to tho combined fractions. The reslllting solution is run onto anAmberlite XAD-column (Rohm & Haas Ltd,) and the column eluted wi-th n-propanol/water 1/4.
~ractions containin~ the desired salt as judged by their ~lactamase inhibitory activity are combined, evaporated under vacuo to remove orl~,anic solvent and freeze dried.
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' '~ . ' "' : ' ', ' ' , ' ~ , A Fllrthcr l'ur _ .~tion o the So~ Salts of 1;he Comlloun~of the FonQula (1) The combined fractions from the Q~E Sephadex chromatogTaphy of Exa[Q~le 4 may be desalted and fur-ther purified by chrom~to~Taphy on a Biogel P2 gel col~n (~io-Rad Laboratories Ltd., 27 Ilomesdale Road, 3rolnlcy, ~ent) as follows:
1'he freeze dried solid from the Q~E Sephadex column is dissolved in a small volu;ne of deionis~d water and run onto a ~io~el P2 column.
~e colu~n is eluted with 1~ aqueous butanol. ~ractions are monitored lo or their ~-lactamase inhibitory activity and for reaction with silver nitrate. Those giving a negative reaction to silver nitrate but giving suitable ~-lactamase inhibitory ac~ivity are combined and freeze dried.
T~lde ~1ark . - . ~ :, : . . .
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A I~lrther ~rification o~ the Soclium SaltcoL -the Comsoun(~ oî I~loLmula ~ ) The preparation obtaincd in ~xample 2 is dissolved in deionised t/ater ~Id applic(l to a col~mn of the stron~;ly basic anion exch~lge resin ~nberlite IRA 45~ (Rohm ~ Haas (~K) L-td., Le1~lin~ House, 2 Mason's Ave., Croydon, U.K.). The colwnn is prepared in 0.0511 pH 7 phosphate buffer and eluted with a sodiwn chloride ~adient in phosphate bufer. The elution is from 0.05i`1 pH 7 phosphate to 0.05M
p~I 7 phosphate containing l.OM NaCl. Fractions showin~ the ~reatest L3-lactamase i1~libitory activit~ are combined. N~Cl is added to the combined fr~ctions to gi~e a conc~ntration of at least 5',b. The resulting solution is run onto an Amberlite XAD-4 column prepared i.ndeionised water. ~he colwnn is eluted with n-~rop~nol/water l/4.
Fractions shot1ing the greatest ~3-lactamase inhibitory activity but givin~ a negative reaction with silver nitrate are combined and freeze dried.
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A ~nrther l~]rificatiorl of the So(liwn Sal-~ of thc Cor~ olirl-]~o;~ llorm;
The salt of the compound of the formula (I) may be further purified by chromato~L~aphy on a column of cellulose (Cellulose CC31 ~atman, Springfi~ld Mill, Maidstone, Kent, U.~.) as follo~s:
The impure solid containing the salt of the compound of tihe formula (I) is dissolved in a minimum of deionised water and n propanol added to about 5~o. The resultin~ solution is r~n on-to the cellulose column 2nd the colu~l eluted lJith n-propanol/~ater ~/l. The resulting fractions after dilution into deionised ~/ater are monitored for their ~-lact~mase inhibitor~ activity. ~ractions con-taining the desired salt are combined, evaporated under reduced pressure to remove solvent and free~e dried.
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- 37 ~
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L~ .~ ?i . ~ 3 A Fllri.llor l'urification o~ t,he So(liurr~ ltsor t,he Com!)ov~(38of ~hc FoImula ~ ) Thc process of Example 4 ma.y be used but replacin~ the s~arting material with the preparation obtained in Example 3.
Exam~le 9 A Further Purification o~ the Sodium Saltsof the Com~ounr7sof the Formula (II) The process of E~ample 5 may be used but replacin~ the startin~
material with the product of Example ~.
Ex~m~l,e 10 -~ ~urther ~lrification o~` the Sodium Saltsof the Com~oundsof the Formula (II) The process of Example.~ ma,y be used replacing the startin~
material ~ith the preparation obtained in Example 3.
Examl~le 11 A F~rther Purification of the Sodium Saltsof the Compoundsof the For!l.la (II) q'he process of Example 7 may be used but replacin~ -the startin~
material with the yreyaration obtainel in Example 3.
: - 38 - :
~ m,`^ 1~ f~
Preparation of Crude ~ntibiotics fro~ Cu]ture ~`iltrate Culture filtrate (t30 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 cm column of Amberlite IRA 453 a strongly basic acrylic based anion exchan~re resin (~0~ & IIaas). The column waS eluted ~ith a sodium chloride concentration ~rradient.
The grradient was from 0 to 1.0 M NaCl in 0.05~i pII 7 pllosphate buffer at a flo~r rate o 2.5ml/minute and 5 ml fractions collected. Fractions were monitored for their ~3-lactar~ase inhibitory activity against a preparation of ~T~ laota~ase. Those fractions griving ~od inhibito~
activity and containin~ the sodium salts of the ompounds of the formulae (I) and (II) t~ ) were combined. (~he di-sodium salts of ~l 4550, ~I 13902 and ~ 17l3~'30 eluted starting at fraction 17).
To the combined fractions was added sodium chloride (3 ~r) ~ and the resultingr solution ~as rwl onto a 1.5 ~ 15 cm Amberlite XAD-4 colur~l (Ro}lm ~ Haas) prepared in deionised water. q'he column ~las eluted with n-propanol/water 114 at 3 ml/minute and 4 ml fractions were collected. ~ractions were monitored for tlleir ~-lactamase inhibitory activity and for reaction with ~SrI~o3 solution. q~hose fractions giving ~ood inhibitory activity and a negative reaction ~/ith silver nitrate were combined and freeze dried to give a partially purified preparation of the sodium salts of the compoullds of the formulae (I) r~nd (II).
I~is impure preparation ma~ be furthe~ purifie~d by the proce~ses described hereinbefore.
: ~ ' :- ~ 39 ~ ~ ~
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Preparation of Crude ~n-tibiotics from Cultlue ~iltrate C~ture filtrate (~05 ml) prepared essentially as described in Description 1 was run onto a 1.5 Y. 15 cm column of ~berlite IRA 45'~
a strongly basic acrylic based anion exchan~e resin (noh~ & ~iaas).
The column was washed with deioni~ed water (100 ml) a-t 5 ml/minute and eluted with 0.025 I~ p~I 7 phosphate buffer at 5 ml/minute, 10 ml fractions were collected. Fractions were monitored for their ~-lactamase inhibitory activity a~ainst a preparation of R~ lactamase. Those 1~ ractions ~rivin~r good inhibitory activity and containin~ the sodium salts of the compounds of the formulae (I) and (II) (12 -32) were combined. The combined fractions t~ere freeze dried. The freeze dried solid ~as dis~olved in deionised water (20 ml), l~aC1 (2 g) `~'a9 added and the resulting solution was run onto a 1.5 x 15 cm Amberlite ~D-4 colu3ln (Rohm & Haas) prepared in deionised water. The column tJas eluted with n-propanol/water 1/4 at 2 ml/minute and 4 ml fractions were collected. Fractions uere monitored for their ~-lactamase inhibitory activity and for reaction with ~rN03 solution. 'nlose fractions (7 - 14) giving ~rood inhibitory activity and a ne~^ative reaction with silver nitrate t~ere combined and freeze dried to yield a partiaIly purified preparation o the salts of the compounds of the formulae (I) and (II). This impure preparation may be further purified by the pl~ocesses described hereinbefore.
~ - 40 , . -.: : : ::
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ample 1 ~1 Fermentation Conditions for 300L Fermentati_ A freeze dried ampoule of Streptomyces olivaceus (ATCC 313~5) was resuspended in lO ml of a sterile solution of the following composition:
Glucose 2~
Soya bean flour 16 pH 6.5 prepared in deionised water (The soya bean flour is Arkasoy '50' supplied by the British Arkady Co. Ltd., of Old Trafford, Manchester).
l ml of this suspension was used to inoculate lO0 ml of medium of the same composition contained in a 500 ml Ehrlenemeyer flask closed with a foam plug. After inoculation the flask was incubated on a rotary shaker at 28C for 30 hours. 5 ml portions of this seed culture was used to inoculate solid agar slants in Roux bottles of the following composition:
V8 Vegetable juice 20.0%
Bacto agar (Dlfco)' 2.5%
p~l 6.0 , , prepared in deionised water ,' , (The V8 vegetable juice is supplied by Campbell's Soups Ltd., ~ings Lynn, Norfolk, England and Bacto agar is supplied by Difco Laboratories, Detrolt, Michlgan, U.S.A.).
~r~d~ ~a~K
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l.ach Rou~ ~ottle was incubatc.i .~t 28 (' Lor 1 t`JCe~
After that time 100 ml sterile deionised water containing 0.1% Triton X (surfactant - Registered TradeMark) was added to one Roux bottle culture and the spores suspended by sha~ing. This spore suspension was added as inoculum to 75 L of sterilised seed stage medium in a stainless steel baffled fermen-ter. The composition of ~he r.ledium was:
Soya bean flour (Arkasoy 50) 1 Glucose 2%
Pluronic L81 antifoam 0.03%
Prepared in distilled water (Pluronic L81 was supplied by Ugine I<uhlmann Chemicals Ltd.).
The medium was steam sterilised in the fermenter for 20 minutes at 120C. The seed stage culture was stirred at 140 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75L/minute through an open ended sparger. The temperature was controlled at 28C and after incubation under these conditions for 48 hours 7.5L
of this seed culture was added as inoculum to l50L sterile fermentation medium in a 300L stainless steel fully baffled fermenter. The fermentation medium had the following co~positior:
:
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- -: . : :, ~ , , : : : : ~ : -: , . ~ : ,:
., :,.
,~ :
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~oya bcan flour (Ark~soy 50) 0.9'~
Glucose 2.0~o Chalk 0. 02-o CC12 6~12 O . 0001 o Pluronic L81 antifoam 0. 2o pH 6.0 before sterilisation Prepared in distilled water The fermentation stage medium was stirred at 3~10 rpm with an 8.5 inch turbine disc impeller. The temperature was controlled at 29C~ air was supplied at the rate of 50L/minute and the pE~ maintained at 6.5 - 7Ø
Fermenta~cns as above may be harvested at times ranging between 48 and 54 hours.
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E~ample 15 Fermentation Conditions for 2000L 1ermenta~ion The fermentation conditions up to ancl includincJ the seed stage were essentially as described in Example 14.
75L of this seed stage was used to inoculate 1500L of sterile fermentation medium contained in a 2000L fully baffled stainless steel fermenter. The férmentationm~d.ium was the same as that described in Example 14.
The fermentation was stirred with two 19" diameter turbine disc impellers at 106 rpm and air was supplied at a rate of 400L/minute. The temperature was maintained at 29C and the pH at 6.5 - 7.0, the fermenter was harvested at 48 hours.
. ~ , , ~ . .
, [.x~ 16 L
Isolation Procedure for the Preparation of Substantially Pure Sodium Salts of the Compounds of the Formulae (I) and 'II) 150L whole broth prepared essentially as described
. : : : .:.: : : :
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l.ach Rou~ ~ottle was incubatc.i .~t 28 (' Lor 1 t`JCe~
After that time 100 ml sterile deionised water containing 0.1% Triton X (surfactant - Registered TradeMark) was added to one Roux bottle culture and the spores suspended by sha~ing. This spore suspension was added as inoculum to 75 L of sterilised seed stage medium in a stainless steel baffled fermen-ter. The composition of ~he r.ledium was:
Soya bean flour (Arkasoy 50) 1 Glucose 2%
Pluronic L81 antifoam 0.03%
Prepared in distilled water (Pluronic L81 was supplied by Ugine I<uhlmann Chemicals Ltd.).
The medium was steam sterilised in the fermenter for 20 minutes at 120C. The seed stage culture was stirred at 140 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75L/minute through an open ended sparger. The temperature was controlled at 28C and after incubation under these conditions for 48 hours 7.5L
of this seed culture was added as inoculum to l50L sterile fermentation medium in a 300L stainless steel fully baffled fermenter. The fermentation medium had the following co~positior:
:
, ... , - :
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., :,.
,~ :
: .
~oya bcan flour (Ark~soy 50) 0.9'~
Glucose 2.0~o Chalk 0. 02-o CC12 6~12 O . 0001 o Pluronic L81 antifoam 0. 2o pH 6.0 before sterilisation Prepared in distilled water The fermentation stage medium was stirred at 3~10 rpm with an 8.5 inch turbine disc impeller. The temperature was controlled at 29C~ air was supplied at the rate of 50L/minute and the pE~ maintained at 6.5 - 7Ø
Fermenta~cns as above may be harvested at times ranging between 48 and 54 hours.
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E~ample 15 Fermentation Conditions for 2000L 1ermenta~ion The fermentation conditions up to ancl includincJ the seed stage were essentially as described in Example 14.
75L of this seed stage was used to inoculate 1500L of sterile fermentation medium contained in a 2000L fully baffled stainless steel fermenter. The férmentationm~d.ium was the same as that described in Example 14.
The fermentation was stirred with two 19" diameter turbine disc impellers at 106 rpm and air was supplied at a rate of 400L/minute. The temperature was maintained at 29C and the pH at 6.5 - 7.0, the fermenter was harvested at 48 hours.
. ~ , , ~ . .
, [.x~ 16 L
Isolation Procedure for the Preparation of Substantially Pure Sodium Salts of the Compounds of the Formulae (I) and 'II) 150L whole broth prepared essentially as described
5 in ~ample 14 were clarified on a continuous flow centrifuge (Sharples Super Centrifuge) at approx. 2.4L/minute.
120L of the clarified culture filtrate was percolated onto a 6" diameter column of the strongly basic anion exchange resin Amberlite IRA 458 (in chloride form) supplied by Rohm & ~laas Co., Philadelphia, Pa., U.S.A.) with a bed volume of 9.6L at a rate of 400ml/minute. ~The Amberlite I~A 458 column had been prèviously prepared in deionised water). After percolation of the culture filtrate -the column was washed with 12 a bed volume of water, then eluted with 0.21~ NaCl in 0.05M pH 7 sodium phosphate buffer at a rate of 230ml/minute. 2L fracticns were collected and those showing good inhibitory activity to a preparation of an RTE~1 mediated ~-lactamase preparation (fractions 2-ll) were combined. Sodium chloride was added to the combined fractions (46.75 g/L) to make the solution lM in respect to NaCl. The resulting solution was run onto a 4" diameter column of Amberlite XAD-4 (supplied by Rohm ~ ~laas Co.) with a bed volume of 4L and equilibrated in lM NaCI.
~:
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'll~e pe~col.ltioll ratc was 200ml/mi.l~ c . I llc col~ W;l -eluted witl~ I01. ~1c~iollised w~r ~ ?1~ waLer,~is~pl-op~ ol (1/l) both at lOOml/minute. Fractions showill~ goocl ~-lactamase inhibitory activity against an RTEM enzyme preparatiOn (3~10 and 13-17) were combined, evaporated at reduced pressure to remove isopropanol and freeze dried.
A 3.8 ~ 30 cm column of the weakly basic anion exchange Cellulose DE52 (in chloride form) (suE)plied by Whatman Ltd., Springfield Mill, Maidstone, Ken-t) was prepared in deionised water. The freeze dried solid from the desalting stage was dissolved in 300 ml deionised water and run onto the DE52 cellulose column at 6ml/minute.
The column was washed with deionised water (200 ml) and eluted with 0.025M pH 7 potassium phosphate buffer at 2.5ml/minute, 10 ml fractions were collect~ed. Fractions were monitored for their ~-lactamase inhibitory activity and the first two major peaks of activity, fractions 30-85 (containing the sodium salt of the compound of the formula (I)) and fractions 86-130 (containing the sodium salt of the compound of the formula ~II)) were combined separately.
Combined fractions (30-85) were run onto a 3.8 x 29 cm QAE Sephadex A25 (in chlorlde form) (supplied by Pharmacia Ltd., Uppsala, Sweden) column prepared in deionised water. The column was eluted with O~.lM NaC1 at 3ml/minute and 18 ml fract1ons were ccllected.
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l'ractions showil-~ good RTE~ ~-lac~alllase in~llhitory activity (58-68) were combined. 'l~o this solution (1~0 ml) was added 9 cJ sodium chloride and the resulting solution was run onto a 1.5 x 15 cm ~mberli-te xAr)-~ column. The column was eluted with deionised ~ater (l35 ml) then water/isopropanol (4/1) at 3ml/minute and 4.5 ml fractions were collec-ted. Fractions with UV spectra characteristic of the partially purified sodium saltsof the compouncs of the formula (I~ (UV maximum approx 297 m~l) (8-16 and 34-40) were combined and freeze dried to yield solids (53 mg and 42 mg respectively) with characteristic properties of the substantially pure sodium saltsof the compounds of the formula (I).
Fractions 86-130 from the cellul~e DE52 column were run onto a 3.8 x 29 cm QAE Sephadex A25 (in chloride form) column and the column eluted with O.lM NaCl at 3ml/minute, 18 ml fractions were collected. Fractions were monitored for their RTEM ~-lactamase inhlbitory activi~ty and those showing good activity (94-100) were combined (12G ml).
Sodium chloride (6 g) was added to the combined fractions and the resulting solution run onto a 1.5 x 15 cm Amberlite XAD-4 column. The column was eluted with deionised water (90 ml) then water/isopropanol (4/1) both at 3 ml/minute, and 4 ml fractions were collected.
Fractions with characteristlc~spectra of the impure sodium saltsof the compoundsof the formula (II) (maxlmum approx 307 m~) (10-16 and 26-32) were combined and freeze dried to yield solids (9.7mg and 21.7 mg respect,ively). These solids had properties consistent wlth substantially pure sodium salts 3Q Gf the~compoundsof the,formula (II).
~ ~-47 -- .~ ......... . ..... ~ . . :~ , . . .
, Example 17 Alternative Isolation Procedure for the Sodiu Salts of the Compounds of the Formulae (I) and ~
. .
Culture filtrate (120 L) containing the ~odium sal~s of the compounds of the form~lae (I) and (II) was processed by chromatography on Amberlite IRA 458 (ln chloride form) and desalting ~ Amberlite XAD-4 essentially as described ln Example 16 above.
The freeze dried ~olid ~rom the de3alting stage was dissolved in 300 ml deionlsed water and run onto a 3.8 x 25 cm ce~lulo~e DE52 column (in chloride form). The column was washed with deionised water ~200 ml) and eluted with 0,025M pH 7 potassium phosphate buffer at 6ml/minute and 20 ml fractlons were collected. Fractions showing good RT~M ~-lactamase inhibitory activity (350 ml) were combined and run onto a 3.8 x 30 cm ~ Sephadex A25 column, The column was eluted with 0.18M NaCl at 3 ml/minute and 20 ml fractions were collected.
Fractions were monitored for their UV æpectra and those showing characteristic adsorption of the sodium salts of the compounds of the formula (I) (50-56) and characteristic adsorptlon of the sodium salt of the compound of the formula (II~
(88-97) were comblned separately.
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'l'o the bulked lrilctic)ll~; (50-5(,) was .3~(~('Ci ';o(~
chloride (15 y) and the reslllting solution rurl on~o a 1.5 x 15 cm Amberlite XAD-4 column. The column was washed with deionised water (15 rnl) and elu-ted with water/n-propanol (4/1~ at 3ml/minute. 3 ml fractions were collected. Fractions with UV spectra characteristic of the purified sodium saltsof the compoundsof the formula (I) (9-15) were eombined, evaporated under reduced pressure to remove propanol and freeze dried to yield a solid (35 mg) wi~h properties consistent with the substantially pure sodium saltsof the compoun~ of the formula (I).
To the bulked Eraetions (88-97) from the QAE Sephadex eolumn, sodium chloride (24 g) was added and the solution percolated through an Amherlite XAD~4 column (1.5 x 15 cm).
The eolumn was eluted with water/n-propanol (~/1) after washing with deionised water (15 ml). Fractions with UV speetra eharaeteristie of the substantially pure sodium sal~ of the eompoundsof the formula (II) were combined Th2 eombined fractions were evaporated under redueed pressure to remove propanol and freeze dried to yield the substantially pure sodium sal~ of the eompoundsof the fol~ula (II) (-- mg).
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Alternative Isolation Procedure for the Sodium Sal s of the Compounds of the Formulae (I) and (II) _ Culture filtrate (105 L) was prepared essen-tially 5 as described in Example l~ and was processed on Amberlite IRA 458 and Amberlite XAD~4 as described in the same example.
The bulked fractions from the Amberlite XAD-9 column were evaporated to about half volume under reduced lO pressure to remove isopropanol and stored at 5C for approximately 65 hours. The resultin~ solution (800 ml) was run onto a 3.8 x 30 cm QAE Sephadex A25 column previously prepared in deionised water. The co]umn was eluted with 0.05M NaCl (800 ml) -then with O.lM NaCl bo-th at 15 4ml/minute and 20 ml fractions were collected. Fractions were monitored for their RTEM ~-lactamase inhibitory activity and by their UV spectra and those containing essentially the sodium saltsof the compoundsof the formula (I) (73~80) and the sodium saltsof the compounds of the ~rmu1a (II) (112-125) were combLncd sep,rately.
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C~mbined fractions (73-80) were evaporated under reduced pressure to approxi-mately 15 ml and run onto a 3,8 ~ 30 cm Biogel P2 (200.400 mesh~ (supplied by Bio Rad Laboratoriess 27 ~omesdale Road, Bromley, Kent) column previously prepared in deionised water contalning 1% butanol. The column was eluted at 3ml/minute with deionised water contalning 1% butanol and 6 ml fractions were collected. Fractions were monitored by thelr UV spectra and for reaction with silver nitrate. Fractions containing the sodium salts of the compounds of the formula (I) giving a negative reaction with AgN03 (29-39) were combined and freeze dried to yield a solid (72 mg) with prope~ties characteristlc of the 10 purlfied sodium salt of the compound of the formula (I).
Combined fractlons (112-125) were evaporated under reduced pressure to approximately 15 ml volume. This solution was chromatographed on a 3.8 x 30 cm Biogel P2 column (prepared as above). Ihe column was eluted with deionised water containing 1% butanol at 3ml/minute and 6 ml fractlons were collected. Fractions giving W spectra characteristic of the sodium salts of the compounds of the formula (II) but with a negative reac~ion for chloride were combined (43-48). The comb~ned fractions were free~e dried to yield a solid (27 mg) with properties characteris~ic of the substantially pure sodium salts of the compounds of the ormula (II).
~ ~ .
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],xalnplc 19 __ Alternative Isolation for the Sodlum Salts of the Compounds of the Formulae (I) and (I:L) 1500 L of whole brew prepared essentially as described in Example 15 was clarified by filtration using Dicalite 478 filter aid on a rotary pre-coat filter to yield 1400 L of culture filtra-te. This culture filtrate was percolated through a 12" diameter Amberlite IRA 458 co]umn (chloride form) (lO0 L bed volume) at an average percolation rate of 4L/minute.
The column was eluted at 1.6L/minute Witll 0.2M NaC1 in 0.05M pH 6.7 sodium phosphate buffer and 20L fractions were collected. Fractions showing good antibacterial activity when tested on Klebsiella aerogenes A (a variant of NCTC 418) (l-~) were combined. Sodium chloride was added to the bulked fractions to a final concentration of l.OM and the resulting solution run onto a 6" diameter column of Amberlite XAD-4 (22.4 L bed volume) at 1.2 L/minute. The AoberliteXAD-4 column was eluted with 20 L deionised water followed with water/iso-propanol (4/l) at 500 ml/minute and 8 L fractions were collected.
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, , l~ractions containin~3 the cleslrcd salts (2~ /er~ ~on~ c (2~ ~) and concentrated to 1l.4 L by evaporation. The concentrate was stored for approximately 65 hours (~t 5C after adjustment to p}l 7Ø 'L'his solution was further concentra-ted to 3.75 L zind the resulti~ solution run onto a 7.8 x 31 cm Cellulose DE52 column a-t 6 ml/minute.
The column was eluted with 0.025M p~l 7 potassium phosphate buffer at 4ml/min~te and 22 ml fractions collected.
Fractions were monitored for their RTEM ~-lactamase inhibitory activity, fractions ~0-l35 and 136-210 showing good activity were combined separately, The combined fractions 60-135 (1580 ml) were run onto a 4.8 x 25 cm QAE Sephadex A25 column at 6ml/minute.
This column was eluted with O.lM NaCl at 4ml/minute and 20 ml fractions were collected. Fractions were monitored by thelr UV spectra and those characteristlc of the sodium salts of the compounds ~ the formula (I) (50-70) were combined ready for subsequent ~?rocessin~.
` The combined fractlons (136-21G) from the Cellulose DE52 column were run onto a 4.8 x 26 cm QAE Sephadex A25 column ; at 6ml/minute. The column was eluted with O.lM NaCl at 4ml/minute and 20 ml fractions were col~lected. Fractions showing characteristic adsorptlon spectra of the~ partially purified sal~ of the compoundsof the formula (II) (gl-104) were combined. To the combined~fractions was added~sodium chloride (25 g) and the resulting solution run~onto a ` 2.4 x 32 cm ~mberlite XAD-4 column at 6ml/minute.
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~rhe column was washc~d witn appro~imatc:l.y 50 Ml dcioni.sed water ancl elutcd with water/ll-propanol (~/l) at 5ml/minute .llld 10 mL fract:iolls WCI-C c(~l..l.c~ l...~d. l'r.lctic)ns ~JiVill~ a nc~ativc silvcrnitrate rcact:ioll .for chloridc but with characteristic UV spectra of the sodium salts:
vf the compoundsof the formula (II) were combined (17-30).
The combined fractions were evaporated under reduced pressure to remove n-propanol and freeze dried to yield 585 mgc~ the substantially pure sodium sal-~ of the compoundsof the formula (II).
: - 54 -:
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Example 20 Properties of the Sodium Salts of the Compounds of the Formulae (I) and (II) 1. W Spectra:
The mi~ed sodium salts of ~he compounds of the formula (I~ has a characteristic maximum at approxlmately 297 m~4.
The mixed sodium salts of the compounds of the formula (II) has a characteristic maxlmum at Qpproximately 307 m~ (one of two~.
2. The sodium salts have characteristic ~ -lactam carbonyl adsorptions at 1750 cm in their IR 3pectra.
3. The in vltro antibacterlal acti~ity oE material prepared essentially as described hereinbefore was as follows:
Sodium Salt of ~heSodium Salt of the .
Compound of the Compound of the Organism Pormula tI) Formula (II) M~C (~g/ml~ MIC ~g/ml) . ~ .
Bacillus subtilis A 0.8 0.2 Enterobacter cloacae Nl 25.0 25.0 Escherlchia coli 104181.5 1~5 Klebsiella aerogenes A12.5 6.25 :
Proteus mlrabilis C9776.25 6.25 Pseudomonas aeruginosa A ~ 100 > 100 Salmonells typhimurium CT10 3.12 0.8 Serratia marcescens US39 50.0 25.0 Staphylococcus aureus Oxford 0.8 1.5 Staphylococcus aureus Russel~ 3.12 1.5 :: , : : : ' : ' ' : . ': ' '' ' ''' '' ''' '' ' ' ' . ' ' "~
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~,x~r~lplc 2_ Separation of the Salts of the Com~ounds of the ~ormulae (Ia) and (Ib) A substantially pure preparation of the mixed sodium salts of the compounds of the formulae (la) clnd (I~) were separated by high pressure liquid chromato~raplly (hplc) by the following procedure:
Column: 300 mm x 3.9 mm filled with 1~ bondapack C18 (Waters Associates, Milford, Massachusetts, USA).
~0 Solvent: 0.05M ammonium acetate, adjusted to prl 4.5 with acetic acid in 5O acetonitrile - 95% water.
Plow rate: 2.5 ml per minute.
Detection: UV absorbance at 295 nm.
Load: 50 ~1 of a solution of 1.6 m~ in 0.5 ml water.
The two sodium salts were resolved into two peaks with re-tention times of 3.45 and 4.45 minutes. The eluate for each peak was separately combined and neutralised to p~l 7 with diIute sodium hydroxide solution.
The separated combined solutions were evaporated to yield the desired solid salts of thecompounds of the formulae (Ia) and (Ib).
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1.~;.,",1l. l.c _ _ _ _ S a _ t n of the Salts of the ~ompounds of the Yormulae (IIa) and (IIb) -Using the same system as described in Example 21 the mixed sodium salts of the compounds of the formulae (IIa) and (IIb) were separated by high pressure liquid chromatography with retention times of 6.0 and 7.1 minutes.
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Example 23 CULTURE FILTRATE
ABSORB ONTO STRONGLY
BASIC ~ESIN AMBERLITE IRA 458 1 Elute wi~h 0.2M NaCl in buffer COMBINE FRACS. CONTAINING
Ia, Ib, IIa and IIb De~alt on XAD-4 Elute with isop~opanol/water EVAPORATE ~NDER REDUCED
PRESS~RE TO REMOVE PROPANOL
¦ Chromatograph on QAE Sephadex Ia, Ib IIa, IIb Desalt on 1 ~Desalt on ~AD-4 FREEZE DRY FRACTIONS FREEZE DRY FRACTIONS
CONTAINING Ia, Ib CONTAINING IIa, IIb ?
Chromatograph on ¦ - ¦Chramatograph `~
QAE Sephadex on HP20 ~:
FRACTION CONTAINING ~
Ia, Ib FRACTIONS FRACTIONS
CONTAINING IIa CONTAINING IIb Chromatograph .
on Diaion HP20 _ Chroma~ograph ::
I 1 ~ , on Biogel P2 , -CONTAINING IaCONTAINING Ib CONFT~RI~C IGNIIa CONTAINI ~G IIb Chroma~ograph Chromatograph on Biogel P2 ~ on HP20 and .
Freeze Dry FRACTIONS FRACTIONS . , CONTAINING IaCONTAINING Ib IIa IIb ~.
Chromatograph on HP20 and Freeze Dry :
: : Ia Ib .
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(In this Example reference to (Ia), (IIa), (Ib) and ~IIb) means reference to their sodium salts.) Spores of Streptomy_es olivaceus ATCC 31365 were prepared essentially as described in Example 14.
The spore suspension from one Roux bottle was used as inoculum for 75L of sterilised seed stage medium contained in a lOOL stainless steel baffled fermenter. The composition of the medium was:-Soya bean flour (Arkasoy 50) 1%
~lucose 2%
Pluronic L81 antifoam 0.03%
Prepared in distilled water.
The medium was steam sterilised in the fermenter for 20 minutes at 120C. The seed stage culture was stirred at i40 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75 L/~.inute; (through an open ended sparger). The temperature was controlled at 28 and after inc~ation for 48 hoursj 75L of the seed culture was added as inoculum to 1500L of fermentation medium contained in a 2000L fully baffled stainless steel fermenter.
The composition of the fermentation medium was:-Soya bean flour (Arkasoy 50) 2.0%
Glucose ~0.9~
Chalk 0.02%
CoCl2 6 2 0.0001%
- Pluronic L81 antifoam 0.2%
.
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5~
(Medium made u~ in distille~ water, p~I adjus~ed to
120L of the clarified culture filtrate was percolated onto a 6" diameter column of the strongly basic anion exchange resin Amberlite IRA 458 (in chloride form) supplied by Rohm & ~laas Co., Philadelphia, Pa., U.S.A.) with a bed volume of 9.6L at a rate of 400ml/minute. ~The Amberlite I~A 458 column had been prèviously prepared in deionised water). After percolation of the culture filtrate -the column was washed with 12 a bed volume of water, then eluted with 0.21~ NaCl in 0.05M pH 7 sodium phosphate buffer at a rate of 230ml/minute. 2L fracticns were collected and those showing good inhibitory activity to a preparation of an RTE~1 mediated ~-lactamase preparation (fractions 2-ll) were combined. Sodium chloride was added to the combined fractions (46.75 g/L) to make the solution lM in respect to NaCl. The resulting solution was run onto a 4" diameter column of Amberlite XAD-4 (supplied by Rohm ~ ~laas Co.) with a bed volume of 4L and equilibrated in lM NaCI.
~:
:
- ~, , :
'll~e pe~col.ltioll ratc was 200ml/mi.l~ c . I llc col~ W;l -eluted witl~ I01. ~1c~iollised w~r ~ ?1~ waLer,~is~pl-op~ ol (1/l) both at lOOml/minute. Fractions showill~ goocl ~-lactamase inhibitory activity against an RTEM enzyme preparatiOn (3~10 and 13-17) were combined, evaporated at reduced pressure to remove isopropanol and freeze dried.
A 3.8 ~ 30 cm column of the weakly basic anion exchange Cellulose DE52 (in chloride form) (suE)plied by Whatman Ltd., Springfield Mill, Maidstone, Ken-t) was prepared in deionised water. The freeze dried solid from the desalting stage was dissolved in 300 ml deionised water and run onto the DE52 cellulose column at 6ml/minute.
The column was washed with deionised water (200 ml) and eluted with 0.025M pH 7 potassium phosphate buffer at 2.5ml/minute, 10 ml fractions were collect~ed. Fractions were monitored for their ~-lactamase inhibitory activity and the first two major peaks of activity, fractions 30-85 (containing the sodium salt of the compound of the formula (I)) and fractions 86-130 (containing the sodium salt of the compound of the formula ~II)) were combined separately.
Combined fractions (30-85) were run onto a 3.8 x 29 cm QAE Sephadex A25 (in chlorlde form) (supplied by Pharmacia Ltd., Uppsala, Sweden) column prepared in deionised water. The column was eluted with O~.lM NaC1 at 3ml/minute and 18 ml fract1ons were ccllected.
: : : , .:
.
- 46 - ~
;2~
l'ractions showil-~ good RTE~ ~-lac~alllase in~llhitory activity (58-68) were combined. 'l~o this solution (1~0 ml) was added 9 cJ sodium chloride and the resulting solution was run onto a 1.5 x 15 cm ~mberli-te xAr)-~ column. The column was eluted with deionised ~ater (l35 ml) then water/isopropanol (4/1) at 3ml/minute and 4.5 ml fractions were collec-ted. Fractions with UV spectra characteristic of the partially purified sodium saltsof the compouncs of the formula (I~ (UV maximum approx 297 m~l) (8-16 and 34-40) were combined and freeze dried to yield solids (53 mg and 42 mg respectively) with characteristic properties of the substantially pure sodium saltsof the compounds of the formula (I).
Fractions 86-130 from the cellul~e DE52 column were run onto a 3.8 x 29 cm QAE Sephadex A25 (in chloride form) column and the column eluted with O.lM NaCl at 3ml/minute, 18 ml fractions were collected. Fractions were monitored for their RTEM ~-lactamase inhlbitory activi~ty and those showing good activity (94-100) were combined (12G ml).
Sodium chloride (6 g) was added to the combined fractions and the resulting solution run onto a 1.5 x 15 cm Amberlite XAD-4 column. The column was eluted with deionised water (90 ml) then water/isopropanol (4/1) both at 3 ml/minute, and 4 ml fractions were collected.
Fractions with characteristlc~spectra of the impure sodium saltsof the compoundsof the formula (II) (maxlmum approx 307 m~) (10-16 and 26-32) were combined and freeze dried to yield solids (9.7mg and 21.7 mg respect,ively). These solids had properties consistent wlth substantially pure sodium salts 3Q Gf the~compoundsof the,formula (II).
~ ~-47 -- .~ ......... . ..... ~ . . :~ , . . .
, Example 17 Alternative Isolation Procedure for the Sodiu Salts of the Compounds of the Formulae (I) and ~
. .
Culture filtrate (120 L) containing the ~odium sal~s of the compounds of the form~lae (I) and (II) was processed by chromatography on Amberlite IRA 458 (ln chloride form) and desalting ~ Amberlite XAD-4 essentially as described ln Example 16 above.
The freeze dried ~olid ~rom the de3alting stage was dissolved in 300 ml deionlsed water and run onto a 3.8 x 25 cm ce~lulo~e DE52 column (in chloride form). The column was washed with deionised water ~200 ml) and eluted with 0,025M pH 7 potassium phosphate buffer at 6ml/minute and 20 ml fractlons were collected. Fractions showing good RT~M ~-lactamase inhibitory activity (350 ml) were combined and run onto a 3.8 x 30 cm ~ Sephadex A25 column, The column was eluted with 0.18M NaCl at 3 ml/minute and 20 ml fractions were collected.
Fractions were monitored for their UV æpectra and those showing characteristic adsorption of the sodium salts of the compounds of the formula (I) (50-56) and characteristic adsorptlon of the sodium salt of the compound of the formula (II~
(88-97) were comblned separately.
~0 ~ .
: ~ . , . , :
: . : ' .
'l'o the bulked lrilctic)ll~; (50-5(,) was .3~(~('Ci ';o(~
chloride (15 y) and the reslllting solution rurl on~o a 1.5 x 15 cm Amberlite XAD-4 column. The column was washed with deionised water (15 rnl) and elu-ted with water/n-propanol (4/1~ at 3ml/minute. 3 ml fractions were collected. Fractions with UV spectra characteristic of the purified sodium saltsof the compoundsof the formula (I) (9-15) were eombined, evaporated under reduced pressure to remove propanol and freeze dried to yield a solid (35 mg) wi~h properties consistent with the substantially pure sodium saltsof the compoun~ of the formula (I).
To the bulked Eraetions (88-97) from the QAE Sephadex eolumn, sodium chloride (24 g) was added and the solution percolated through an Amherlite XAD~4 column (1.5 x 15 cm).
The eolumn was eluted with water/n-propanol (~/1) after washing with deionised water (15 ml). Fractions with UV speetra eharaeteristie of the substantially pure sodium sal~ of the eompoundsof the formula (II) were combined Th2 eombined fractions were evaporated under redueed pressure to remove propanol and freeze dried to yield the substantially pure sodium sal~ of the eompoundsof the fol~ula (II) (-- mg).
, - ' ' ` ~,', ~' ~,:, ; ' , ' ', ': ' , r~ ]. c :1 ~
Alternative Isolation Procedure for the Sodium Sal s of the Compounds of the Formulae (I) and (II) _ Culture filtrate (105 L) was prepared essen-tially 5 as described in Example l~ and was processed on Amberlite IRA 458 and Amberlite XAD~4 as described in the same example.
The bulked fractions from the Amberlite XAD-9 column were evaporated to about half volume under reduced lO pressure to remove isopropanol and stored at 5C for approximately 65 hours. The resultin~ solution (800 ml) was run onto a 3.8 x 30 cm QAE Sephadex A25 column previously prepared in deionised water. The co]umn was eluted with 0.05M NaCl (800 ml) -then with O.lM NaCl bo-th at 15 4ml/minute and 20 ml fractions were collected. Fractions were monitored for their RTEM ~-lactamase inhibitory activity and by their UV spectra and those containing essentially the sodium saltsof the compoundsof the formula (I) (73~80) and the sodium saltsof the compounds of the ~rmu1a (II) (112-125) were combLncd sep,rately.
:
~ - 50 -.
I; ~
-: :
-: . . . ,. :
.
~ .
', ' :' ~ ~
, . , ~ , . .
:, . . : .. .
C~mbined fractions (73-80) were evaporated under reduced pressure to approxi-mately 15 ml and run onto a 3,8 ~ 30 cm Biogel P2 (200.400 mesh~ (supplied by Bio Rad Laboratoriess 27 ~omesdale Road, Bromley, Kent) column previously prepared in deionised water contalning 1% butanol. The column was eluted at 3ml/minute with deionised water contalning 1% butanol and 6 ml fractions were collected. Fractions were monitored by thelr UV spectra and for reaction with silver nitrate. Fractions containing the sodium salts of the compounds of the formula (I) giving a negative reaction with AgN03 (29-39) were combined and freeze dried to yield a solid (72 mg) with prope~ties characteristlc of the 10 purlfied sodium salt of the compound of the formula (I).
Combined fractlons (112-125) were evaporated under reduced pressure to approximately 15 ml volume. This solution was chromatographed on a 3.8 x 30 cm Biogel P2 column (prepared as above). Ihe column was eluted with deionised water containing 1% butanol at 3ml/minute and 6 ml fractlons were collected. Fractions giving W spectra characteristic of the sodium salts of the compounds of the formula (II) but with a negative reac~ion for chloride were combined (43-48). The comb~ned fractions were free~e dried to yield a solid (27 mg) with properties characteris~ic of the substantially pure sodium salts of the compounds of the ormula (II).
~ ~ .
. , ~ .
.
],xalnplc 19 __ Alternative Isolation for the Sodlum Salts of the Compounds of the Formulae (I) and (I:L) 1500 L of whole brew prepared essentially as described in Example 15 was clarified by filtration using Dicalite 478 filter aid on a rotary pre-coat filter to yield 1400 L of culture filtra-te. This culture filtrate was percolated through a 12" diameter Amberlite IRA 458 co]umn (chloride form) (lO0 L bed volume) at an average percolation rate of 4L/minute.
The column was eluted at 1.6L/minute Witll 0.2M NaC1 in 0.05M pH 6.7 sodium phosphate buffer and 20L fractions were collected. Fractions showing good antibacterial activity when tested on Klebsiella aerogenes A (a variant of NCTC 418) (l-~) were combined. Sodium chloride was added to the bulked fractions to a final concentration of l.OM and the resulting solution run onto a 6" diameter column of Amberlite XAD-4 (22.4 L bed volume) at 1.2 L/minute. The AoberliteXAD-4 column was eluted with 20 L deionised water followed with water/iso-propanol (4/l) at 500 ml/minute and 8 L fractions were collected.
- : . .... , - ~ . . . ..
~ : : .: ~,~
, , l~ractions containin~3 the cleslrcd salts (2~ /er~ ~on~ c (2~ ~) and concentrated to 1l.4 L by evaporation. The concentrate was stored for approximately 65 hours (~t 5C after adjustment to p}l 7Ø 'L'his solution was further concentra-ted to 3.75 L zind the resulti~ solution run onto a 7.8 x 31 cm Cellulose DE52 column a-t 6 ml/minute.
The column was eluted with 0.025M p~l 7 potassium phosphate buffer at 4ml/min~te and 22 ml fractions collected.
Fractions were monitored for their RTEM ~-lactamase inhibitory activity, fractions ~0-l35 and 136-210 showing good activity were combined separately, The combined fractions 60-135 (1580 ml) were run onto a 4.8 x 25 cm QAE Sephadex A25 column at 6ml/minute.
This column was eluted with O.lM NaCl at 4ml/minute and 20 ml fractions were collected. Fractions were monitored by thelr UV spectra and those characteristlc of the sodium salts of the compounds ~ the formula (I) (50-70) were combined ready for subsequent ~?rocessin~.
` The combined fractlons (136-21G) from the Cellulose DE52 column were run onto a 4.8 x 26 cm QAE Sephadex A25 column ; at 6ml/minute. The column was eluted with O.lM NaCl at 4ml/minute and 20 ml fractions were col~lected. Fractions showing characteristic adsorptlon spectra of the~ partially purified sal~ of the compoundsof the formula (II) (gl-104) were combined. To the combined~fractions was added~sodium chloride (25 g) and the resulting solution run~onto a ` 2.4 x 32 cm ~mberlite XAD-4 column at 6ml/minute.
=. _ 53 _ : ' - ~ : :
` : ` :
: : :
, ~ ~
`: .~' ' -:. : . ` :
: .. . , : . . . .
.: : ~ : , , : , 5~
~rhe column was washc~d witn appro~imatc:l.y 50 Ml dcioni.sed water ancl elutcd with water/ll-propanol (~/l) at 5ml/minute .llld 10 mL fract:iolls WCI-C c(~l..l.c~ l...~d. l'r.lctic)ns ~JiVill~ a nc~ativc silvcrnitrate rcact:ioll .for chloridc but with characteristic UV spectra of the sodium salts:
vf the compoundsof the formula (II) were combined (17-30).
The combined fractions were evaporated under reduced pressure to remove n-propanol and freeze dried to yield 585 mgc~ the substantially pure sodium sal-~ of the compoundsof the formula (II).
: - 54 -:
.. . .
' ' . ~ , ', !
~' ' '` '' '' " ' : ' ' , ~5~
Example 20 Properties of the Sodium Salts of the Compounds of the Formulae (I) and (II) 1. W Spectra:
The mi~ed sodium salts of ~he compounds of the formula (I~ has a characteristic maximum at approxlmately 297 m~4.
The mixed sodium salts of the compounds of the formula (II) has a characteristic maxlmum at Qpproximately 307 m~ (one of two~.
2. The sodium salts have characteristic ~ -lactam carbonyl adsorptions at 1750 cm in their IR 3pectra.
3. The in vltro antibacterlal acti~ity oE material prepared essentially as described hereinbefore was as follows:
Sodium Salt of ~heSodium Salt of the .
Compound of the Compound of the Organism Pormula tI) Formula (II) M~C (~g/ml~ MIC ~g/ml) . ~ .
Bacillus subtilis A 0.8 0.2 Enterobacter cloacae Nl 25.0 25.0 Escherlchia coli 104181.5 1~5 Klebsiella aerogenes A12.5 6.25 :
Proteus mlrabilis C9776.25 6.25 Pseudomonas aeruginosa A ~ 100 > 100 Salmonells typhimurium CT10 3.12 0.8 Serratia marcescens US39 50.0 25.0 Staphylococcus aureus Oxford 0.8 1.5 Staphylococcus aureus Russel~ 3.12 1.5 :: , : : : ' : ' ' : . ': ' '' ' ''' '' ''' '' ' ' ' . ' ' "~
. : ' '`, " "' ' ' ' ` ' ' ~ , ' ' ` " ' ~ " ' ' . ' ,, ' ' '' . ' . ' ~ ' ~ ' ~25~
~,x~r~lplc 2_ Separation of the Salts of the Com~ounds of the ~ormulae (Ia) and (Ib) A substantially pure preparation of the mixed sodium salts of the compounds of the formulae (la) clnd (I~) were separated by high pressure liquid chromato~raplly (hplc) by the following procedure:
Column: 300 mm x 3.9 mm filled with 1~ bondapack C18 (Waters Associates, Milford, Massachusetts, USA).
~0 Solvent: 0.05M ammonium acetate, adjusted to prl 4.5 with acetic acid in 5O acetonitrile - 95% water.
Plow rate: 2.5 ml per minute.
Detection: UV absorbance at 295 nm.
Load: 50 ~1 of a solution of 1.6 m~ in 0.5 ml water.
The two sodium salts were resolved into two peaks with re-tention times of 3.45 and 4.45 minutes. The eluate for each peak was separately combined and neutralised to p~l 7 with diIute sodium hydroxide solution.
The separated combined solutions were evaporated to yield the desired solid salts of thecompounds of the formulae (Ia) and (Ib).
~ Jr~e ~
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.
' ', , ' ' . ' ' ';, ' ~ , '' `
.
52~
1.~;.,",1l. l.c _ _ _ _ S a _ t n of the Salts of the ~ompounds of the Yormulae (IIa) and (IIb) -Using the same system as described in Example 21 the mixed sodium salts of the compounds of the formulae (IIa) and (IIb) were separated by high pressure liquid chromatography with retention times of 6.0 and 7.1 minutes.
~ - 57 -, .
.. . ., . ~ , .
- .' ~ ' '' ' .:
~5;2~
Example 23 CULTURE FILTRATE
ABSORB ONTO STRONGLY
BASIC ~ESIN AMBERLITE IRA 458 1 Elute wi~h 0.2M NaCl in buffer COMBINE FRACS. CONTAINING
Ia, Ib, IIa and IIb De~alt on XAD-4 Elute with isop~opanol/water EVAPORATE ~NDER REDUCED
PRESS~RE TO REMOVE PROPANOL
¦ Chromatograph on QAE Sephadex Ia, Ib IIa, IIb Desalt on 1 ~Desalt on ~AD-4 FREEZE DRY FRACTIONS FREEZE DRY FRACTIONS
CONTAINING Ia, Ib CONTAINING IIa, IIb ?
Chromatograph on ¦ - ¦Chramatograph `~
QAE Sephadex on HP20 ~:
FRACTION CONTAINING ~
Ia, Ib FRACTIONS FRACTIONS
CONTAINING IIa CONTAINING IIb Chromatograph .
on Diaion HP20 _ Chroma~ograph ::
I 1 ~ , on Biogel P2 , -CONTAINING IaCONTAINING Ib CONFT~RI~C IGNIIa CONTAINI ~G IIb Chroma~ograph Chromatograph on Biogel P2 ~ on HP20 and .
Freeze Dry FRACTIONS FRACTIONS . , CONTAINING IaCONTAINING Ib IIa IIb ~.
Chromatograph on HP20 and Freeze Dry :
: : Ia Ib .
.
.
, ' , ~ .
~5f~
(In this Example reference to (Ia), (IIa), (Ib) and ~IIb) means reference to their sodium salts.) Spores of Streptomy_es olivaceus ATCC 31365 were prepared essentially as described in Example 14.
The spore suspension from one Roux bottle was used as inoculum for 75L of sterilised seed stage medium contained in a lOOL stainless steel baffled fermenter. The composition of the medium was:-Soya bean flour (Arkasoy 50) 1%
~lucose 2%
Pluronic L81 antifoam 0.03%
Prepared in distilled water.
The medium was steam sterilised in the fermenter for 20 minutes at 120C. The seed stage culture was stirred at i40 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75 L/~.inute; (through an open ended sparger). The temperature was controlled at 28 and after inc~ation for 48 hoursj 75L of the seed culture was added as inoculum to 1500L of fermentation medium contained in a 2000L fully baffled stainless steel fermenter.
The composition of the fermentation medium was:-Soya bean flour (Arkasoy 50) 2.0%
Glucose ~0.9~
Chalk 0.02%
CoCl2 6 2 0.0001%
- Pluronic L81 antifoam 0.2%
.
`
.
.: ,.... .
, -.: - . . ~ :
: . . . .
. .
5~
(Medium made u~ in distille~ water, p~I adjus~ed to
6.0 before sterilisation.) The fermentation was stirred with two 19 inch diamete,r turbine disc impellers at 106 rpm and air was supplied at a rate of 4001~ninute. The temperature was maintained at 29C and the pH at 6.5-7.00 The fermenter was harvested at 48 hours~
Eurther culture ~iltrate suitable for the extraction of the compounds Ia, Ib, IIa and IIb was prepared as follows:
A spore suspension of S olivaceus prepared from two Roux bottles as described above was used to inoculate 1501 of sterilised seed stage medium contained in a 300 1 fully baffled stainless steel fermenter. The medium and growth conditions of this seed stage were essentially as described above~ After 48 hours 1501 of the seed stage were used to inoculate 30001 of the fermentation medium contained in a 5000 lfully baffled stainless steel fermenterO
The fermentation stage was continued under essentially similar conditions to that described above except that ~he fermentation was harvest~3d at 55 hours.
Whole brew from the 20001 and 50001 fermenters prepared as described above gave a combined volume of 472S L The brew was clarified by filtration on a rotary pre-coat vacuum filter, the comblned filtrates yielding 4200 1 of clarified brew. The clarified brew was percolated at 10 lh~inute Amberlite IRA458 thro~lgh columns of the strongly basLc anion exchanger resin /
(chloride form). The resin was washed with 60 ldeionised water then eluted with an aqueous solution of 0.~ NaCl : , . ~ ,........... , . ~: . .
: . ' ' ,. ' .
5'~
with 0.075M sodium phosphate pEI 6.7. Elution of Ia, Ib,IIa and IIb commenced when the conductivity of the eluant had reached that of O.lM NaCl and had been eluted when 4501 of eluant had been collected. Where necessary the presence of Ia, Ib, IIa and IIb was determined using an analytical high pressure liquid chromatography (hplc) system based on the preparative method described in Example 21.
To the combined eluates containing Ia, Ib, IIa and IIb from the IRA458 column was added Amberlite XAD-4 (90 kg damp weight). The mixture was adjusted to pH 6.0 using 50% hydrochloric acid and stirred gently at 5C for 1 hour.
The resin was then filtered off and washed with deionised water at 5C untll the conductivity of the washings was less than that of a 0.05M NaCl 601ution. The washed resin was slurried ln 421 of isopropanol/water (1/1) and 20~ w/v NaOH
added until the pH was steady at 7.5. The eluant was filtered off and retainedO The elution of the resln was repeated with a further portion of isopropanol/water (1/1) and finally ~he process was repeated using isopropano~/water (1/3). The eluates were combined (1301) and evaporated under reduced pressure to remove isopropanol. The resulting solution (67 was percolated at 15 ~hour onto a column of QAE Sephadex A25 (15 x 43 cm) p~e-equilibrated in O.lM NaCl. The column was of - 25 washed with 7.51/0.05M NaCl and eluted with O.lM NaCl at
Eurther culture ~iltrate suitable for the extraction of the compounds Ia, Ib, IIa and IIb was prepared as follows:
A spore suspension of S olivaceus prepared from two Roux bottles as described above was used to inoculate 1501 of sterilised seed stage medium contained in a 300 1 fully baffled stainless steel fermenter. The medium and growth conditions of this seed stage were essentially as described above~ After 48 hours 1501 of the seed stage were used to inoculate 30001 of the fermentation medium contained in a 5000 lfully baffled stainless steel fermenterO
The fermentation stage was continued under essentially similar conditions to that described above except that ~he fermentation was harvest~3d at 55 hours.
Whole brew from the 20001 and 50001 fermenters prepared as described above gave a combined volume of 472S L The brew was clarified by filtration on a rotary pre-coat vacuum filter, the comblned filtrates yielding 4200 1 of clarified brew. The clarified brew was percolated at 10 lh~inute Amberlite IRA458 thro~lgh columns of the strongly basLc anion exchanger resin /
(chloride form). The resin was washed with 60 ldeionised water then eluted with an aqueous solution of 0.~ NaCl : , . ~ ,........... , . ~: . .
: . ' ' ,. ' .
5'~
with 0.075M sodium phosphate pEI 6.7. Elution of Ia, Ib,IIa and IIb commenced when the conductivity of the eluant had reached that of O.lM NaCl and had been eluted when 4501 of eluant had been collected. Where necessary the presence of Ia, Ib, IIa and IIb was determined using an analytical high pressure liquid chromatography (hplc) system based on the preparative method described in Example 21.
To the combined eluates containing Ia, Ib, IIa and IIb from the IRA458 column was added Amberlite XAD-4 (90 kg damp weight). The mixture was adjusted to pH 6.0 using 50% hydrochloric acid and stirred gently at 5C for 1 hour.
The resin was then filtered off and washed with deionised water at 5C untll the conductivity of the washings was less than that of a 0.05M NaCl 601ution. The washed resin was slurried ln 421 of isopropanol/water (1/1) and 20~ w/v NaOH
added until the pH was steady at 7.5. The eluant was filtered off and retainedO The elution of the resln was repeated with a further portion of isopropanol/water (1/1) and finally ~he process was repeated using isopropano~/water (1/3). The eluates were combined (1301) and evaporated under reduced pressure to remove isopropanol. The resulting solution (67 was percolated at 15 ~hour onto a column of QAE Sephadex A25 (15 x 43 cm) p~e-equilibrated in O.lM NaCl. The column was of - 25 washed with 7.51/0.05M NaCl and eluted with O.lM NaCl at
7.8 l/hour and 500 m] fractions collected. Fractions were ` '' ~- - 61 -~, .
' . . ~; ~ ~ ' .
. ~ : , , , , . :
:- . : . : . . . :
' .
'` ~ , `'`' '' ' ' ' ' '` ' monitored for the presence of Ia, Ib, IIa and IIb by hplc.
Fractions containing Ia and Ib, which were eluted together, were combined (12.61) and those containing IIa and IIb which were eluted later were also combined (5.91).
To the combined fractions containing Ia and Ib was added 44.16 g/l NaCl. I'he resulting solution was percolated at 5.8 yhour through a column of Amberlite XAD-4 llO x 36 cm).
The column was washed with 3 ldeionised water at 2.9 l/hour then eluted with isopropanol/water 1/9. Eluant containing Ia and/or Ib at suitable levels as judged by hplc was collected after the conductivity of the eluant had fallen to a level equivalent to O.OlM NaCl. The eluate solution (7.7I) con-taining Ia and Ib was adjusted to pH 7.0 using 20% w/v NaOH, concentrated under reduced pressure and freeze dried to yield a solid (38.5 g).
The combined eluates from the QAE Sephadex A25 column containing IIa and IIb were treated similarly to yield a solid (13.7 g).
The freeze dried product (38.5 g) from the XAD~4 column containing Ia and Ib was dissolved in deionised water (50 ml) and run onto a QAE Sephadex A25 column (7.8 x 30 cm) prepared in deionised water. The column was washed with 41 of 0.05M -NaCl then eluted with 0.08M NaCl both at 8 ml/min. Approximately 20 ml fractions were collected from the commencement of the elution. The fractions wQre monitored for their UV spectra and those with spectra consistent with containing compounds Ia and Ib (130 - 170) were combined (9SO ml). The .
,, .. , ,,,, .. .... . ~ . .. . ..
- : - . ,. . -:
.
QAE Sephadex chromatography was carried out at 5C, A portion (450 ml) of the combined fractions containing Ia and Ib from the QAE Sephadex chromatography was taken and NaCl (23.8g) added. The resulting solution was run onto a Diaion HP20 column (408 x 62 cm) (Mitsubishi Chemicals Ltd., Agents Nippon Rensui Co., Fuji Bldg.~ 2-3 Marunouchi, 3-Chome, Ch-,yoda - ~u, Tokyo 100, Japan) at 12 ml/min. and approximately 20 ml. fractions were collected.
Fractions were monitored by their UV spectra and those con-taining Ia (58-74) and Ib (78~97) were combined separately, concentrated by evaporation under reduced pressure and freeze dried to yield solids 490 mg and 357 mg respectively.
The remaining combined eluates from the QAE column containing Ia and Ib were processed in a similar manner to yield solids 363 mg and 258 mg containing Ia and Ib respec-tively.
A portion (538 mg~ of solid containing Ia from the above process was dissolved in deionised water (25 ml) and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm).
The column was eluted with deionised water at 3 ml/mins. and 25 ml fractions were collected. The chromatography was carried out at 5 & . Fractions were monitored by their UV
spectra and those with spectra characteristic of highly puriied Ia ~37-42) were combined. The combined fractions were concentrated by evaporation under reduced pressure to approximately lO ml. The resulting solution was run onto a :
: -, :' . ~ : ' ' - ~, i -~5~
3. 0 x 50 cm Diaion HP20 (chromatographic grade) column. The column was eluted with deionised water at 5 ml/minute and 10 ml fractions collected. Fractions were monitored by their UV
spectra and those with spectra characteristic of highly purified Ia (50-62) were combined, concen~rated under reduced pressure and freeze dried to yield a solid (40 mg) of Ia.
A portion (5.8 mg) of the solids containing Ib from the HP20 chromatography was dissolved in approx. 25 ml deionised water and the resulting solution run onto a 7.8 x 40 cm Biogel P2 column. The column was eluted with deionised water at 3 ml/mm and 25 ml fractions collected. Fractions containing highly purified Ib(32-37) as judged by thelr UV spectra were combined. The combined fractions were evaporated under reduced pressure to approximately 10 ml and the resulting solution run onto a 2.4 x 40 cm Diaion HP 20 (chromatographic grade) column. The co]umn was eluted ~ -with deionised water at 5 ml/min and 10 ml fractions were collected. Fractions were monitored for their UV spectra ; and those containing highly purified Ib (35-48) were combined `~` 20 and freeze dried to yield a solid 53 mg of Ib.
A portion of the freeze dried solid (7 g) from the XAD-4 desalting stage containing IIa and IIb was dissolved in deionised water (50 ml) and run onto a 4.8 x 57 cm Diaion HP20 column. The column was eluted with deionised water at 10 ml/min and approximately 20 ml fractions were collected.
Fractions were monitored by their UV spectra and those con-taining IIa (49-63) and IIb (71-110) were combined separately ~_ - 64 -:. : ,. .
and freeze dried to yield solids 875 mg and 1.47 g respectively.
The remaining solid IIa and IIb from the ~.AD-4 stage was processed in a similar way to yield solids 860 mg and 1.44 g of IIa and IIb respectively.
A portion (860 mg) of IIa prepared above was dissolved in 25 ml deionised water and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected.
Fractions containing highly purified IIa as judged by their W spectra (42-50) were combined. The combined fractions were concentrated to approximately 10 ml by evaporation under reduced pressure and loaded onto a 2.8 x 40 cm Diaion HP20 (chromatographic grade)column. The column was eluted with deionised water and fractions were monitored by their UV
spectra and those with spectra characteristic of highly purified IIa (52-64) were combined. The combined fractions were concentrated by evaporation and freeze dried to yield a solid (95 mg) of IIa.
A portion (750 mg)of IIb prepared above was dissolved in approximately 25 ml deionised water and run onto a Biogel P2 column t200-400 mesh) (7~8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected.
Fractions containlng highly purified IIb as judged by their UV
spectra (47-55) were combined. The combined fractions were evaporated under re~uced pressure to approximately 10 ml and this solution loaded onto a 2.8 x 42 cm Diaion HP20 (chromatographic ~`-~ ~ " , ~ ; ., ~ : .
.... , " .. ;,. . : .:
,: ~ : ~ ;
,~, . ' ' grade) column. The column was eluted with deionised water at 5 ml/minute. The first 25 fractions were collected a5 5 ml and remaining fractions as 10 ml. Fractions were monitored by their UV spectra and those containing highly purified IIb (77-91) were combined and freeze dried -to yield a solid (105 mg) of IIb.
. - 66 -.
, .. , . , ~ ~ :
-~ ' `
Example 24 Pr~perties of the Sodium Salts of Compounds of the Formulae Ia, Ib, IIa and IIb.
The materials prepared essentially as described in Example 23 and being essentially pure have the following properties:
(1) UV spectra Ia Single maxima 298 nm (molar extinction ~=8,131)(see Fig.l) (see Fig. 1) Ib Single maxima 301 nm (molar extinction ~=7,930) (see Fig. 2) and IIa 2 maxima 228/308-309 (~=13,627) (see Fig. 3) and IIb 2 maxima 229 ~08-310 (~=13,933) (see Fig. 4) (2) The antibacterial activity of the sodium salts determined by the microtitre method is demonstrated in -Table A.
(3) The ~-lactamase inhibitory activity of the sodium salts is demonstrated in Table B.
(4) NMR spectra Ia See Fig. 5 for spectrum in D20 Ib See Fig. 6 for spectrum in D20 IIa See Fig. 7 for spectrum in D20 IIb See Fig. 8 for spectrum in D20 (5) The synergistic activity of the sodium salts is demonstrated in Table C.
(6) The sodium salts did not cause any obvious ~oxic effects in mice when administered in aqueous solution sub-cutaneously at 50 mg/kg.
.
- . ~ -'. ' ' . ~ ' ' ~ ` ' , ' ' . ' , . : , , : .'. . ' ' , . , ` ' ' ' : .
" ' ' ` ' . . :
~ ~ ~25~
TABLE A
MIC (~/ml) _ . __ _ _ Strain Ia Ib IIa IIb cillin ~!
. _ _ _ _ '.-B.subtilis 0.16 1.2~0.08 2.5 <3.0 Enterobacter 2 510 2 5 10 200 cloacae Nl .
E.coli 10418 0.32.5 0.3 5.0 <3.0 E.coli JT39 5.02.5 5.0 5.0 800 E.coli JT68 5.02.5 10 5.0 1600 E.coli JT410 0.65.0 0.6 5.0 200 Klebsiella A 2.55.0 1.2 5.0 100 Klebsiella E70 10 5.0 10 10 400 Klebsiella Ba95 40 10 40 10 >6400 ~ .
ProteuS mirabilis 0.6 10 0.6 10 <~3.0 C889 5.010 10 20 400 I5'30 2.510 2.5 20 100 Proteus vulgaris 5.0 10 10 20 400 Pseudomonas >160>160 >160 >160 1600 aeruginosa A
Salmonella CT10 0.3 2.5 0.3 5.0 ~0.3 Serratia US39 20 10 20 10 1600 Staph. Oxford 1.25 1.2 0.3 2.5 ~3.0 Staph. Russell 0.6 2.5 0.3 2.5 400 :;
Staph. Smith 0.62.5 0.6 2.5 ~3.0 Strep.faeoalis 1.25 20 1 2 20 <~3.0 .
~ 68 --- - - - :- . - ~ ~ . j, :. . . ~ . , TABLE B
. Iso (~g/ml~
Compound Entero- Ps.aerug Proteus E.coli Staph . C889 JT4 Russell IIa 0.02 3.0 0.04 >2.0 0.08 IIb 0.04 4.0 0.4 0.1 >2.0 Ia 0.02 4.0 0.1 >2.0 3.25 Ib 0 0- ~ ~- 0 0.28 >>2~0 :
TABLE C
, _ . MIC (~g/ml) Compound Staph aureus E.coll JT3g Russell :
Ampicillin alone 1000 2000 0.1 ~g/ml >10 >500 +(Ia) 1 ~g/ml _ 500 _ ~
0.1 ~gjml >10 500 : ~ ~:
+(Ib) 1 ~g/ml 10 31.2 -:
__ ::
0.1 ~g/ml 10 >500:
+(I~Ia) 1 ~g/ml ~ 500 0.1 ~g/ml >10 500 +(IIb) 1 ~g/ml _ 31.2 .
::
. .
. : , : . . : ,: . .:
. . , .,, :~, .: :
:, :.
: . . . .. .
.. . . ,:
' . . ~; ~ ~ ' .
. ~ : , , , , . :
:- . : . : . . . :
' .
'` ~ , `'`' '' ' ' ' ' '` ' monitored for the presence of Ia, Ib, IIa and IIb by hplc.
Fractions containing Ia and Ib, which were eluted together, were combined (12.61) and those containing IIa and IIb which were eluted later were also combined (5.91).
To the combined fractions containing Ia and Ib was added 44.16 g/l NaCl. I'he resulting solution was percolated at 5.8 yhour through a column of Amberlite XAD-4 llO x 36 cm).
The column was washed with 3 ldeionised water at 2.9 l/hour then eluted with isopropanol/water 1/9. Eluant containing Ia and/or Ib at suitable levels as judged by hplc was collected after the conductivity of the eluant had fallen to a level equivalent to O.OlM NaCl. The eluate solution (7.7I) con-taining Ia and Ib was adjusted to pH 7.0 using 20% w/v NaOH, concentrated under reduced pressure and freeze dried to yield a solid (38.5 g).
The combined eluates from the QAE Sephadex A25 column containing IIa and IIb were treated similarly to yield a solid (13.7 g).
The freeze dried product (38.5 g) from the XAD~4 column containing Ia and Ib was dissolved in deionised water (50 ml) and run onto a QAE Sephadex A25 column (7.8 x 30 cm) prepared in deionised water. The column was washed with 41 of 0.05M -NaCl then eluted with 0.08M NaCl both at 8 ml/min. Approximately 20 ml fractions were collected from the commencement of the elution. The fractions wQre monitored for their UV spectra and those with spectra consistent with containing compounds Ia and Ib (130 - 170) were combined (9SO ml). The .
,, .. , ,,,, .. .... . ~ . .. . ..
- : - . ,. . -:
.
QAE Sephadex chromatography was carried out at 5C, A portion (450 ml) of the combined fractions containing Ia and Ib from the QAE Sephadex chromatography was taken and NaCl (23.8g) added. The resulting solution was run onto a Diaion HP20 column (408 x 62 cm) (Mitsubishi Chemicals Ltd., Agents Nippon Rensui Co., Fuji Bldg.~ 2-3 Marunouchi, 3-Chome, Ch-,yoda - ~u, Tokyo 100, Japan) at 12 ml/min. and approximately 20 ml. fractions were collected.
Fractions were monitored by their UV spectra and those con-taining Ia (58-74) and Ib (78~97) were combined separately, concentrated by evaporation under reduced pressure and freeze dried to yield solids 490 mg and 357 mg respectively.
The remaining combined eluates from the QAE column containing Ia and Ib were processed in a similar manner to yield solids 363 mg and 258 mg containing Ia and Ib respec-tively.
A portion (538 mg~ of solid containing Ia from the above process was dissolved in deionised water (25 ml) and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm).
The column was eluted with deionised water at 3 ml/mins. and 25 ml fractions were collected. The chromatography was carried out at 5 & . Fractions were monitored by their UV
spectra and those with spectra characteristic of highly puriied Ia ~37-42) were combined. The combined fractions were concentrated by evaporation under reduced pressure to approximately lO ml. The resulting solution was run onto a :
: -, :' . ~ : ' ' - ~, i -~5~
3. 0 x 50 cm Diaion HP20 (chromatographic grade) column. The column was eluted with deionised water at 5 ml/minute and 10 ml fractions collected. Fractions were monitored by their UV
spectra and those with spectra characteristic of highly purified Ia (50-62) were combined, concen~rated under reduced pressure and freeze dried to yield a solid (40 mg) of Ia.
A portion (5.8 mg) of the solids containing Ib from the HP20 chromatography was dissolved in approx. 25 ml deionised water and the resulting solution run onto a 7.8 x 40 cm Biogel P2 column. The column was eluted with deionised water at 3 ml/mm and 25 ml fractions collected. Fractions containing highly purified Ib(32-37) as judged by thelr UV spectra were combined. The combined fractions were evaporated under reduced pressure to approximately 10 ml and the resulting solution run onto a 2.4 x 40 cm Diaion HP 20 (chromatographic grade) column. The co]umn was eluted ~ -with deionised water at 5 ml/min and 10 ml fractions were collected. Fractions were monitored for their UV spectra ; and those containing highly purified Ib (35-48) were combined `~` 20 and freeze dried to yield a solid 53 mg of Ib.
A portion of the freeze dried solid (7 g) from the XAD-4 desalting stage containing IIa and IIb was dissolved in deionised water (50 ml) and run onto a 4.8 x 57 cm Diaion HP20 column. The column was eluted with deionised water at 10 ml/min and approximately 20 ml fractions were collected.
Fractions were monitored by their UV spectra and those con-taining IIa (49-63) and IIb (71-110) were combined separately ~_ - 64 -:. : ,. .
and freeze dried to yield solids 875 mg and 1.47 g respectively.
The remaining solid IIa and IIb from the ~.AD-4 stage was processed in a similar way to yield solids 860 mg and 1.44 g of IIa and IIb respectively.
A portion (860 mg) of IIa prepared above was dissolved in 25 ml deionised water and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected.
Fractions containing highly purified IIa as judged by their W spectra (42-50) were combined. The combined fractions were concentrated to approximately 10 ml by evaporation under reduced pressure and loaded onto a 2.8 x 40 cm Diaion HP20 (chromatographic grade)column. The column was eluted with deionised water and fractions were monitored by their UV
spectra and those with spectra characteristic of highly purified IIa (52-64) were combined. The combined fractions were concentrated by evaporation and freeze dried to yield a solid (95 mg) of IIa.
A portion (750 mg)of IIb prepared above was dissolved in approximately 25 ml deionised water and run onto a Biogel P2 column t200-400 mesh) (7~8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected.
Fractions containlng highly purified IIb as judged by their UV
spectra (47-55) were combined. The combined fractions were evaporated under re~uced pressure to approximately 10 ml and this solution loaded onto a 2.8 x 42 cm Diaion HP20 (chromatographic ~`-~ ~ " , ~ ; ., ~ : .
.... , " .. ;,. . : .:
,: ~ : ~ ;
,~, . ' ' grade) column. The column was eluted with deionised water at 5 ml/minute. The first 25 fractions were collected a5 5 ml and remaining fractions as 10 ml. Fractions were monitored by their UV spectra and those containing highly purified IIb (77-91) were combined and freeze dried -to yield a solid (105 mg) of IIb.
. - 66 -.
, .. , . , ~ ~ :
-~ ' `
Example 24 Pr~perties of the Sodium Salts of Compounds of the Formulae Ia, Ib, IIa and IIb.
The materials prepared essentially as described in Example 23 and being essentially pure have the following properties:
(1) UV spectra Ia Single maxima 298 nm (molar extinction ~=8,131)(see Fig.l) (see Fig. 1) Ib Single maxima 301 nm (molar extinction ~=7,930) (see Fig. 2) and IIa 2 maxima 228/308-309 (~=13,627) (see Fig. 3) and IIb 2 maxima 229 ~08-310 (~=13,933) (see Fig. 4) (2) The antibacterial activity of the sodium salts determined by the microtitre method is demonstrated in -Table A.
(3) The ~-lactamase inhibitory activity of the sodium salts is demonstrated in Table B.
(4) NMR spectra Ia See Fig. 5 for spectrum in D20 Ib See Fig. 6 for spectrum in D20 IIa See Fig. 7 for spectrum in D20 IIb See Fig. 8 for spectrum in D20 (5) The synergistic activity of the sodium salts is demonstrated in Table C.
(6) The sodium salts did not cause any obvious ~oxic effects in mice when administered in aqueous solution sub-cutaneously at 50 mg/kg.
.
- . ~ -'. ' ' . ~ ' ' ~ ` ' , ' ' . ' , . : , , : .'. . ' ' , . , ` ' ' ' : .
" ' ' ` ' . . :
~ ~ ~25~
TABLE A
MIC (~/ml) _ . __ _ _ Strain Ia Ib IIa IIb cillin ~!
. _ _ _ _ '.-B.subtilis 0.16 1.2~0.08 2.5 <3.0 Enterobacter 2 510 2 5 10 200 cloacae Nl .
E.coli 10418 0.32.5 0.3 5.0 <3.0 E.coli JT39 5.02.5 5.0 5.0 800 E.coli JT68 5.02.5 10 5.0 1600 E.coli JT410 0.65.0 0.6 5.0 200 Klebsiella A 2.55.0 1.2 5.0 100 Klebsiella E70 10 5.0 10 10 400 Klebsiella Ba95 40 10 40 10 >6400 ~ .
ProteuS mirabilis 0.6 10 0.6 10 <~3.0 C889 5.010 10 20 400 I5'30 2.510 2.5 20 100 Proteus vulgaris 5.0 10 10 20 400 Pseudomonas >160>160 >160 >160 1600 aeruginosa A
Salmonella CT10 0.3 2.5 0.3 5.0 ~0.3 Serratia US39 20 10 20 10 1600 Staph. Oxford 1.25 1.2 0.3 2.5 ~3.0 Staph. Russell 0.6 2.5 0.3 2.5 400 :;
Staph. Smith 0.62.5 0.6 2.5 ~3.0 Strep.faeoalis 1.25 20 1 2 20 <~3.0 .
~ 68 --- - - - :- . - ~ ~ . j, :. . . ~ . , TABLE B
. Iso (~g/ml~
Compound Entero- Ps.aerug Proteus E.coli Staph . C889 JT4 Russell IIa 0.02 3.0 0.04 >2.0 0.08 IIb 0.04 4.0 0.4 0.1 >2.0 Ia 0.02 4.0 0.1 >2.0 3.25 Ib 0 0- ~ ~- 0 0.28 >>2~0 :
TABLE C
, _ . MIC (~g/ml) Compound Staph aureus E.coll JT3g Russell :
Ampicillin alone 1000 2000 0.1 ~g/ml >10 >500 +(Ia) 1 ~g/ml _ 500 _ ~
0.1 ~gjml >10 500 : ~ ~:
+(Ib) 1 ~g/ml 10 31.2 -:
__ ::
0.1 ~g/ml 10 >500:
+(I~Ia) 1 ~g/ml ~ 500 0.1 ~g/ml >10 500 +(IIb) 1 ~g/ml _ 31.2 .
::
. .
. : , : . . : ,: . .:
. . , .,, :~, .: :
:, :.
: . . . .. .
.. . . ,:
Claims (46)
1. A process for the preparation of a compound of formula (I) or (II) (I) (II) or a salt thereof which comprises cultivating a producing strain of Streptomyces olivaceus or Streptomyces gaddenensis until a substantial quantity of said compound or salt thereof is produced and thereafter recovering said compound of the formula (I) or (II) or salt thereof from the cultivation medium, said compound or said salt being a mixture of cis and trans isomers about the .beta.-lactam ring.
2. A process as claimed in claim 1 which utilizes Streptomyces olivaceus ATCC 31126 or a high yielding mutant thereof.
3. A process as claimed in claim 1 which utilizes Streptomyces olivaceus ATCC 31365 or a high yielding mutant thereof.
4. A process as claimed in claim 1, wherein the salts of the compound of formula (I) or (II) is a pharmaceutically acceptable salt thereof.
5. A process as claimed in claim 1, wherein the salt of the compound of formula (I) or (II) is an alkali metal or alkaline earth metal salt.
6. The process of claim 5, wherein the salt is the sodium salt.
7. The process of claim 5, wherein the salt is the potassium salt.
8. The process of claim 4, wherein the salt is at least 50%
pure.
pure.
9. The process of claim 4, wherein the salt is at least 75% pure.
10. The process of claim 4, wherein the salt is at least 90% pure.
11. The process of claim 1, wherein a mixture of salts of the isomers of compound of formula (I) is recovered and subjected to chromatographic separation on Diaion HP20 or chromatographically equivalent resin and the corresponding salt of the isomer (5R,6R)-3-(2-acetamidoethyl-thio)-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid recovered which is substantially free from the salt of the isomer (5R,6S)-3-(2-acetamidoethylthio)-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo-[3.2.0]hept-2-ene-2-carboxylic acid.
12. The process of claim 1, wherein a mixture of salts of the isomers of the compound of formula (I) is recovered and subjected to chromatographic separation on Diaion HP20 or chromatographically equivalent resin and the corresponding salt of the isomer (5R,6S)-3-(2-acetamidoethyl-thio)-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ena-2-carboxylic acid recovered which is substantially free from the salt of the isomer (5R,6R)-3-(2-acetamidoethylthio)-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
13. The process of clalm 1, wherein a mixture of salts of the isomers of the compound of formula (II) is recovered and subjected to chromacographic separation on DiaLon HP20 or chromatographically equivalent resin and the corresponding salt of the isomer (5R,6R)-3-[(E-2-acetamido-ethanylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3,2.0]hept-2-ene-2-carboxylic acid recovered which is substantially free of the salt of the isomer (5R,6S)-3-[(E-2-acetamidoethenylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
14. The process of claim l, wherein a mixture of salts of the isomers of the compound of formula (II) is recovered and subjected to chromatographic separation on Diaion HP20 or chromatographically equivalent resin and the corresponding salt of the isomer (5R,6S)-3-[(E-2-acetamido-ethenylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid recovered which is substantially free of the salt of the isomer (5R,6R)-3-[(E-2-acetamidoethenylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
15. The process of claim 11 or 12, wherein the salt is a pharmaceutically acceptable salt.
16. The process of claim 13 or 14, wherein the salt is a pharmaceutically acceptable salt.
17. The process of claim 11 or 12, wherein the salt is an alkali metal or alkaline earth metal salt.
18. The process of claim 13 or 14, wherein the salt is an alkali metal or alkaline earth metal salt.
19. The process of claim 11 or 12, wherein the salt is the sodium salt.
20. The process of claim 13 or 14, wherein the salt is the sodium salt.
21. The process of claim 11 or 12, wherein the salt is the potassium salt.
22. The process of claim 13 or 14, wherein the salt is the potassium salt.
23. The process of claim 11 or 12, wherein the salt is at least 50% pure.
24. The process of claim 13 or 14, wherein the salt is at least 50% pure.
25. The process of claim 11 or 12, wherein the salt is at least 75% pure.
26. The process of claim 13 or 14, wherein the salt is at least 75% pure.
27. The process of claim 11 or 12, wherein the salt is at least 90% pure.
28. The process of claim 13 or 14, wherein the salt is at least 90% pure.
29. The process of claim 11, wherein the recovered salt is an alkali metal salt and has a molar extinction coefficient when dissolved in water at neutral pH of not less than 7700.
30. The process of claim 29, wherein the molar extinction coefficient is not less than 7900.
31. The process of claim 29 or 30, wherein the salt is the sodium salt.
32. The process of claim 12, wherein the recovered salt is an alkali metal salt and has a molar extinction coefficient when dissolved in water at neutral pH of not less than 7700.
33. The process of claim 32, wherein the molar extinction coefficient is not less than 7900.
34. The process of claim 32 or 33, wherein the salt is the sodium salt.
35. The process of claim 13, wherein the recovered salt is an alkali metal salt and has a molar extinction coefficient when dissolved in water at neutral pH of not less than 13000.
36. The process of claim 35, wherein the molar extinction coefficient is not less than 13500.
37. The process of claim 35 or 36, wherein the salt is the sodium salt.
38. The process of claim 14, wherein the recovered salt is an alkali metal salt and has a molar extinction coefficient when dissolved in water at neutral pH of not less than 13000.
39. The process of claim 38, wherein the molar extinction coeffient is not less than 13500.
40. The process of claim 38 or 39, wherein the salt is the sodium salt.
41. A compound of formula (I) or (II) (I) (II) or a salt thereof when prepared by the process of claim 1, 2 or 3, or an obvious chemical equivalent.
42 A compound of formula (I) or (II) (I) (II) or a pharmaceutically acceptable salt thereof when prepared by the process of claim 4, or an obvious chemical equivalent.
43; A salt of (5R,6R)-3-(2-acetamidoethylthio)-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid when prepared by the process of claim 11 or an obvious chemical equivalent.
44. A salt of (5R,6S)-3-(2-acetamidoethylthio)-6-[(S)-1-hydroxyethyl]
7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid when prepared by the process of claim 12 or an obvious chemical equivalent.
7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid when prepared by the process of claim 12 or an obvious chemical equivalent.
45. A salt of (5R,6R)-3-[(E-2-acetamidoethenylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid when prepared by the process of claim 13 or an obvious chemical equivalent.
46. A salt of (5R,6S)-2-[(E-2-acetamldoethenylthio)]-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid when prepared by the process of claim 14 or an obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA300,966A CA1125204A (en) | 1978-04-12 | 1978-04-12 | Antibacterial agents from streptomycete |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA300,966A CA1125204A (en) | 1978-04-12 | 1978-04-12 | Antibacterial agents from streptomycete |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1125204A true CA1125204A (en) | 1982-06-08 |
Family
ID=4111214
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA300,966A Expired CA1125204A (en) | 1978-04-12 | 1978-04-12 | Antibacterial agents from streptomycete |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1125204A (en) |
-
1978
- 1978-04-12 CA CA300,966A patent/CA1125204A/en not_active Expired
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