Note: Descriptions are shown in the official language in which they were submitted.
~25;~
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:
H3C H H
H03S0 ~ S-C=C-NH-C0-CH3 (III)
0 C02H H
3 , .
35 ~ S-C=C-NH-C0-CH3 (IV)
O C02H
H C H H
3 , ,
H0350 ~ S-cH2-cH2-NH-cO-cH3 (V)
O C02H
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):
CH3 H
HO ~ . H
~ ¦ ~ S-C=C-NH-CO-CH3 (II)
OC02H
CH3 ,H
HO I / ~ 2 2 C 3 (I)
~ ~ .
OC02H
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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.
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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)
C2H
3 , ' H
H ~ S-C=C-NH-CO-CH3 (IIa)
C2H
HO ~ C=C-n3-CU-CH3 ~IIb~
O : C02H
:
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.
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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.
- 10 --
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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
- 12 -
~ .. . .
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.
.
- 13 -
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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
- 14 -
.. . .. , . . .,, , ~. . , . , . ~
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
- 15 -
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.:
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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.
~" - .
-~` Jr.~
- 16 -
,
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.
18 -
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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
I XAD4 XAD4
', ' ~ '
De-salted Solution De-salted Solution
- ~
Freeze Dried Solid F~eeze D~ ed ~olld
- 19 -
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:
.
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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~)
XAD4 XAD4
~ ,
De-salted Solution De-salted Solution : :
Freeze Dried Solid Freeze Dried Solld
,:':
- 20 ~
:
~ 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 - ~
5~ 3 L~
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.
- 23 -
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).
,
:
~- 26 -
.
,. : : ~ : ` .: : , . ~ ::
: ~
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).
'~
- 27 - ~ ~
:;: , : . ~ ,:
~ . , : :
: . ~ :
~ ~5~
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 .
_
.
28 -~
,- : ~
,
- , : :
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
_,_ ~
::
`
.-- . ' `
.
-: : :. : : :
,: . , ..... , . , : ..
. : .: :: ~ ~ ~ . . .
(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).
:
~ :~
.
,
:1` -
: :1
- ~ 30 ~
~:
1 ~
. . . ` -` : : ` :- ` :.~.`. :
: `: . ` ' : :` : : : :
: ` - : :: . ~, ` ` : . : ;
.
: ;.: -` ` .
5~
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 _ _ _ _
:
' ~- 32 ~
:- : : : :: :::" : : . :~
: ; : , , : :: : : :
5~
( 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
':
. . ~ . , " " . .
s~ L~
.~ 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.
- 34 -
-:
. .', :. "~: : ' . , ' ` ` ' ' ~ ` ' ' . '
' ;' ' ' ~ `.. ' ',
' , ,, ; ' ' ' .. , ~ . .', ' ' .
'. " ' ~ ~ , ' ' ' ' ' .
'
'~ . ' "' : ' ', ' '
, ' ~ ,
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
- 35 -
. - . ~ :, : . . .
.. . .. . , :
, , ! `
. .
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.
:
.
.: .. . , . - ~
, . . .
" .,
- . : . . . . .
.. ~ , . . . .
s~
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.
: :
- 37 ~
:
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 ~ ~ ~
, ~ - - ; , : , , . , ., . . :
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
,
. -.: : : ::
~ . , .
- : . . . ~
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
- 4 1 - ~ :
. : : : .:.: : : :
- - : . ~ . ~ , ., :
,
~ 5~ ~
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:
:
,
- 42 -
... , - :
- -: . : :, ~ ,
, : : : : ~ : -: , . ~ : ,:
., :,.
,~ :
: .
~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.
:; ' .
: ~.
~` :: : :
:
~ ' '
43~ ; ~
- : : : :
\
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.
- 44 -
. ~ , , ~ . .
,
[.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.
~:
:
- ~, ,
:
'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
- 48 -
~ .
: ~ . , . , :
: . : '
.
'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).
- 51 -
~ ~ .
. , ~ .
.
],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.
- 52 -
- : . .... , - ~ . . . ..
~ : : .: ~,~
,
,
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
- 55 -
:: , : : : ' : ' ' : . ': ' '' ' ''' '' ''' '' '
' ' . ' '
"~
. : '
'`, " "' ' ' ' ` ' ' ~ ,
' '
` " ' ~ " ' '
. '
,, ' ' ''
. ' . ' ~ ' ~ '
~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 ~
- 56 -
~' ~
.
' ', , ' ' . ' ' ';, ' ~ , '' `
.
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
XAD-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
.
.
- 58 -
.
, '
, ~ .
~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
- 60 -
: , . ~ ,........... , . ~: . .
: . ' ' ,. ' .
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
.
- 62 -
,, .. , ,,,, .. .... . ~ . .. . ..
- : - . ,. . -:
.
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
-- - 63 -
:
: -
, :' . ~ : ' '
- ~, 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
- 65 -
~`-
~ ~ " , ~ ; ., ~ : .
.... , " .. ;,. . : .:
,: ~ : ~ ;
,~, . ' '
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.
.
- 67
- . ~ -
'. ' ' . ~ ' ' ~ ` ' , ' ' . '
, . : , ,
: .'. . ' ' , . , ` ' ' ' : .
" ' ' ` ' . . :
~ ~ ~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
.
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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
.
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