Antibiotics

An active antibacterial substance from Pseudomonas fluoroescens bacterium designated pseudominic acid and as a co-product a minor amount of a compound having an additional OH group designated as pseudomonic acid I. These two substances obtained are separated and purified. Alkali metal salts and methyl esters can be prepared. The antibacterial activity of pseudomonic acid appears to reside in the free acid form which results from in vivo hydrolysis of the methyl ester which is crystalline.

This invention relates to an active antibiotic substance produced by the 
bacterium Pseudomonas fluorescens. 
It has been known for many years that the bacterium Pseudomonas fluorescens 
produces inhibitory substances, and a convenient review of the published 
work on the subject can be found in the book "Antibiotics" by H. W. 
Florey, E. B. Chain, N. G. Heatley, M. A. Jennings, A. G. Saunders, E. P. 
Abraham, and M. E. Florey, published by the Oxford University Press 
(1949), Volume 1. 
We have now succeeded in obtaining main and minor inhibitory substances 
which have been found to be essentially active pseudomonic acid with minor 
amounts of impurities. 
The present invention therefore provides an antibiotic substance in 
substantially pure form designated as pseudomonic acid, having the formula 
(I): 
##STR1## 
together with a minor amount of a closely related substance designated 
pseudomonic acid I having the formula (II): 
##STR2## 
The methyl ester of the compound of formula (I) may readily be 
crystallized and recovered. We have named the compound of formula (I) 
"pseudomonic acid" and the compound of formula (II) "pseudomonic acid I", 
and they will be referred to as such hereinafter. 
These compounds may be obtained and recovered together and then 
individually separated. Thus the present invention also provides, in 
substantially pure form, pseudomonic acid of formula (I) above, and the 
alkali metal salts and esters thereof, e.g. the sodium salts and methyl 
esters. 
Pseudomonic acid is conveniently obtained and characterized in the form of 
its crystalline methyl ester, which has the infra-red spectrum shown in 
FIG. 1 of the accompanying drawings and the proton magnetic resonance 
spectrum shown in FIG. 2 of the accompanying drawings. 
Pseudomonic acid of Formula (I) has antibacterial activity and its activity 
appears to be associated with the presence of the free carboxyl group. 
Thus the alkali metal salts of pseudomonic acid and the methyl and 
p-bromophenacyl esters of the carboxyl group --CO.sub.2 H are also active 
while in the salt and ester form. Hence the present invention also 
provides the alkali metal salts of pseudomonic acid and other derivatives 
of the free carboxyl group which are readily hydrolyzed to give the parent 
active free pseudomonic acid. 
It is believed from existing evidence that the compound represented by 
Formula (I) above exists as pseudomonic acid of trans-configuration at the 
double bond. 
The present invention also provide in substantially pure form an amount of 
pseudomonic acid I of Formula (II) above and alkali metal salts and lower 
alkyl esters thereof. 
Also included within the scope of the present invention is a process for 
the preparation and recovery of the compounds of Formula (I) and (II) in 
substantially pure form, which process comprises growing Pseudomonas 
fluorescens under aerobic conditions on or in a culture medium containing 
inorganic salts and sources of assimilable carbon and nitrogen until the 
culture medium exhibits at least detectable antibacterial activity, 
thereafter adding a source of barium ions to the culture medium and 
removing the resultant precipitated material therefrom, extracting the 
culture medium with an organic solvent for the active materials, dissolved 
in the culture medium, extracting the resultant organic solution with 
water at a pH between 7 and 9, evaporating the water to leave a solid 
residue. The active pseudomonic acid is recovered from the solid residue 
by re-crystallization or other accepted purification procedure. 
In the above-defined process, the cultivation step where Pseudomonas 
fluorescens is grown is conventional. All strains of this organism known 
to us produce pseudomonic acid to a greater or lesser extent, but one 
suitable public strain is Pseudomonas fluorescens N.C.I.B. 10586 
(NCIB=National Collection of Industrial Bacteria). 
However, we regard the next step, i.e. the addition of a source of barium 
ions to the culture medium, as being the step which enables practical 
separation of the components to be carried out efficiently. It appears 
that the major proportion of the active culture fluid is converted in this 
step to soluble barium salts (which in itself is surprising since most 
barium salts are water-insoluble) while the residual components which are 
primarily impurities are left behind as an insoluble precipitate. 
After removal of the precipitate the active substance is extracted from the 
aqueous solution with a suitable extractant solvent. Suitable solvents can 
be determined by simple trial and error, but we find that isobutylmethyl 
ketone (IBMK) is a good solvent. Other solvents include ether containing 
5% ethanol, and also chloroform (although these two are not as efficient 
as IBMK). 
The organic solution is then extracted with water at an alkaline pH and the 
salts of the active materials are obtained by evaporating the water. If 
desired, the extraction with organic solvent followed by alkali can be 
repeated several times to ensure efficient extraction and purification of 
active material. 
The separation of inactive contaminations from the resulting salts can be 
achieved either by ion-exchange chromatography of the crude salts or by 
esterifying the salts at the carboxyl group --CO.sub.2 H and subjecting 
the esterified mixture to silica gel chromatography. When using 
ion-exchange methods we find that a polystyrene resin column eluted with a 
gradient of 0.01 N methanolic ammonia in 0.01 N aqueous ammonia is a 
suitable system. Using this system, a series of low molecular weight 
inactive acids are eluted first, followed by the active fraction (30%-60% 
elution). 
In a further aspect, the present invention provides a process for the 
preparation of pseudomonic acid, in substantially pure form which process 
comprises producing the active substance as in the previous aspect of the 
invention, esterifying the same to produce lower alkyl esters, separating 
out the pseudomonic acid ester and de-esterifying said ester as by 
hydrolysis or enzymatic cleavage, thereby producing the antibiotically 
active pseudomonic acid. 
In order to carry out the process aspect of this invention, the active 
substance is esterified, e.g. by conversion to the methyl ester, and the 
ester recovered. This may be achieved by thin layer chromatography in the 
conventional way, e.g. on silica gel developed by chloroform/isopropanol 
(9:1). This provides the ester of pseudomonic acid I, which is present in 
minor amount, and the antibiotically active main product pseudomonic acid 
methyl ester which can be recovered in crystallized form. 
De-esterification, when desired, will vary somewhat according to the 
particular ester involved. With the p-bromophenacyl ester of the 
carboxylic acid group --CO.sub.2 H, the method of Sheehan et al. J. Org. 
Chem (1964), Vol. 29, p. 2006 may be employed (i.e. treatment with sodium 
thiophenoxide).

The invention is illustrated by the following examples: 
EXAMPLE 1 
Production and recovery of Antibacterially active pseudomonic acid and 
Pseudomonic acid (I) 
Pseudomonas fluorescens, strain NCIB. 10586 was grown in submerged culture 
at 30.degree. C. in a medium containing 1% corn steep liquor and 0.5% 
glucose in a basic salts solution. The maximum yield of the antibiotic 
occurred after 24 hours and all of the detectable activity was in the 
culture fluid. After the addition of barium chloride (0.5%) the cells and 
precipitated non-active contaminant material were removed by 
centrifugation. The activity was progressively concentrated by 
partitioning into isobutylmethyl ketone (IBMK) (0.2 vol) at pH 4.5 water 
(0.8 vol) at pH 8.5, and then IBMK (0.25 vol) at pH 4.5 followed by 
evaporation to a small volume under reduced pressure. After a further 
partition into water at pH 8.5 and then adjustment to pH 7-8 the aqueous 
solution was freeze dried to give the sodium salt which could be stored at 
0.degree. C. for several months, without loss of activity. 
The antibiotic extract was stable within the range pH 4-9 at 37.degree. C. 
for 24 hours. Outside these limits rapid loss of activity occurred. The 
sodium salt showed a broad antibacterial spectrum against Gram positive 
and Gram negative bacteria, showed low toxicity and was bacteriostatic 
against S. aureus (N.C.T.C. 6571) and E. coli (M.R.E. 600). 
Further purification of the crude acid was effected by chromatography on 
Amberlite XAD-2 polystyrene resin with a linear gradient produced by 
adding 0.1 N methanolic ammonia, to 0.01 N aqueous ammonia. A series of 
low molecular weight acids was eluted first, followed by a fraction 
(30-60% elution) that possessed the major part of the antibacterial 
(biological) activity. 
EXAMPLE 2 
Purification of Pseudomonic acid and Pseudomonic Acid I 
The biologically active material produced in Example 1 upon methylation 
with diazomethane in ether showed two spots by thin layer chromatography 
corresponding to methyl pseudomonate as the major component and a minor 
amount of component methyl pseudomonate-I (ratio ca 9:1 by wt.). 
Methyl pseudomonate (ca 9 parts by wt.) was separated from methyl 
pseudomonate-I (ca. 1 part by wt.) by preparative layer silica gel 
(GF.sub.245) chromatography on development with chloroform/isopropanol 
(9:1). 50% by wt. of methyl pseudomonate was recovered from the impure 
residue by crystallization from benzene/petroleum ether to give colorless 
needles of m.p. 76.5.degree.-78.degree.. 
Elemental analysis indicated the formula C.sub.27 H.sub.46 O.sub.9 (Found: 
C, 6.28; H, 8.9. C.sub.27 H.sub.46 O.sub.9 required C, 63.0; H, 9.0%), and 
the ester is optically active ([.alpha.].sub.D.sup.24 -9.degree.) (C, 1.5 
in chloroform). Analysis of the oily p-bromophenacyl ester indicated a 
formula C.sub.34 H.sub.49 BrO.sub.10 (Found: C, 58.1; H, 6.9 C.sub.34 
H.sub.49 BrO.sub.10 requires C, 58.5; H, 7.0%). Hence the formula of the 
parent monocarboxylic acid, pseudomonic acid, is C.sub.26 H.sub.44 
O.sub.9. Further support for this derived from the mass spectrum of the 
methyl ester which showed the expected molecular ion at m/e 514. The 
infra-red spectrum of the methyl ester (FIG. 1) showed .nu.max. 
(CCl.sub.4) 3440 (hydroxyl), 1740 (ester), 1715 and 1650 cm.sup.-1 
(.alpha.,.beta.-unsaturated ester). The u.v. spectrum (.lambda.max (EtOH) 
221.5 nm (.epsilon.13,400) confirms the presence of the 
.alpha.,.beta.-unsaturated ester linkage. The NMR spectrum (FIG. 2) showed 
the presence of two secondary methyl groups (.gamma.9.09, 8.81), an 
olefinic methyl group (.gamma.6.40) and an olefinic proton (.gamma.4.32). 
Acetylation of the methyl ester with pyridine/acetic anhydride affords a 
triacetate C.sub.33 H.sub.52 O.sub.12, which absorbs 1 mole of hydrogen 
giving a dihydro derivative C.sub.33 H.sub.54 O.sub.12 on catalytic 
hydrogenation. Reduction of the methyl ester with LiAlH.sub.4 in 
tetrahydrofuran afforded 1,9-dihydroxynonanoate m.p. 46.degree. 
(bis-phenylcarbamate derivative m.p. 168.degree.-9.degree.). Treatment of 
the p-bromophenacyl ester with KMnO.sub.4 NaIO.sub.4 gave 
p-bromophenacyl-9-hydroxynonanoate, C.sub.17 H.sub.23 BrO.sub.4, m.p. 
77.5.degree.-78.degree. (Found: C, 55.1; H, 6.4. C.sub.12 H.sub.23 
BrO.sub.4 requires C, 55.0; H, 6.2%). Mild base hydrolysis of the methyl 
ester yielded methyl 9-hydroxynonanoate (iol) C.sub.10 H.sub.20 O.sub.3. 
Further confirmation of the presence of the 9-hydroxynonanoate unit in 
pseudomonic acid is provided by the mass spectrum of the methyl ester. 
Mass measurement of the fragment at m/e 327 gave 327.18059 (C.sub.17 
H.sub.27 O.sub.6 requires 327.18059 corresponding to the loss of 
--O(CH.sub.2).sub.8 CO.sub.2 CH.sub.3 from the molecular ion. 
EXAMPLE 3 
The following is a summary of further observations which lead us to 
postulate structures for pseudomonic acid and pseudomonic acid I. 
(a) The presence of the C.sub.9 unit in pseudomonic acid is confirmed by 
the reactions described in Example 2. 
(b) 
(i) Attachment of C.sub.9 unit to the rest of molecule. 
That the C.sub.9 unit is attached to the rest of the molecule through an 
.alpha.,.beta.-unsaturated ester linkage to which is attached a --CH.sub.3 
group (n.m.r chemical shift in methyl pseudomonate and certain 
derivatives) was proved by the following observations: 
Treatment ofa hydroxyl protected derivative of methyl pseudomonate with (a) 
osmium tetroxide in pyridine, (b) aqueous sodium metabisulphate and (c) 
sodium periodate in aqueous ethanol gave a compound of formula: 
EQU OCH.CO.sub.2 CH.sub.2 (CH.sub.2).sub.6 CH.sub.2 CO.sub.2 CH.sub.3 
(characterized by analysis, nuclear magnetic resonance, and infra-red 
spectra; semi-carbazone derivative m.p 164.degree.-165.6.degree.) and also 
a nucleus methyl ketone derivative. This also proves that the --CH.sub.3 
group is attached to the .beta.-carbon of the .alpha.,.beta.-unsaturated 
ester system. 
(ii) Confirmation of double bond 
Methyl pseudomonate and its triacetate derivative absorb 1 mole hydrogen 
giving the respective dihydro derivatives, on catalytic hydrogenation 
showing only end absorption in the ultra-violet spectrum. 
(iii) Stereochemistry around double bond 
That the double bond was trans aligned was derived from the literature 
values of chemical shifts (nuclear magnetic resonance) of cis and trans 
CH.sub.3 groups attached to double bonds of this type. 
(c) Nature of functionalities in rest of molecule 
(a) Proof of Glycol system 
(i) Methyl pseudomonate forms an acetonide derivative, characterized by 
analysis, nuclear magnetic resonance, infra-red and ultraviolet spectra. 
(ii) Treatment of methyl pseudomonate with sodium periodate in aqueous 
ethanol gave a dialdehyde as sole product. Hence a glycol system is 
present and must be in a ring. 
(iii) This was also confirmed by n.m.r. double resonance experiments on the 
triacetate and tribenzoate derivatives, 
(b) Proof of epoxide 
(i) The presence of the epoxide was inferred from chemical shifts, in the 
n.m.r. spectra of methyl pseudomonate and derivatives, of the two attached 
protons. This was confirmed by n.m.r. double resonance and indor 
experiments. 
(c) Part structure 
##STR3## 
was inferred from the chemical shifts of the relevant protons (n.m.r.) in 
methyl pseudomonate acetonide derivative and the oxidation product, methyl 
pseudomonate acetoxide ketone derivative, with part structure 
##STR4## 
(D) Using indor and double resonance techniques in the n.m.r. specta of 
the nucleus methyl ketone triacetate and tribenzoate derivatives, the 
structure of the nucleus methyl ketone was deduced and hence the structure 
of pseudomonic acid shown to be as formula (I) herein. 
PSEUDOMONIC ACID I 
The spectra of methyl pseudomonate I and its triacetate derivative (mass 
spectrum, ultra-violet, n.m.r. and infra-red) indicated its close 
relationship to methyl pseudomonate and that it possessed an additional 
hydroxyl group. 
The structure shown in formula (II) herein was deduced from n.m.r. double 
resonance experiments on the triacetate derivative of the nucleus methyl 
ketone. 
EXAMPLE 4 
Hydrolysis of p-bromophenacyl ester of impure pseudomonic acid 
To the p-bromophenacyl ester (234 mg) in dimethyl formamide (3 ml) was 
added sodium thiophenoxide (230 mg). After 30 min, excess ice-cold acetone 
was added and the reaction mixture kept 2 hr at 20.degree.. The impure 
sodium salt of the pseudomonic acid collected by centrifugation. The 
sodium salt was dissolved in water and the pH adjusted to pH 4.5 with dil. 
HCl. The free acid was extracted into ether containing 5% EtOH. The 
ethereal layer was washed with water, dried and evaporated to 15 ml in 
vacuo to give the impure free acid. 
Esterification of acid 
To the above impure acid an excess of ethereal diazomethane was added. 
After 1 hr the solvent was removed in vacuo and the product was purified 
by preparative layer chromatography on Kieselgel GF.sub.254 developing 
twice with 3% isopropanol in chloroform. Band R.sub.F 0.2-0.25 afforded 
the impure methyl ester of pseudomonic acid as an oil (180.3 mg), .nu.max 
(CHCl.sub.3) 3440 br. 1735, 1710, 1220, 1155, 1050 cm.sup.-1 : .lambda.max 
(EtOH) 220 nm (.epsilon.1600); .tau.9.09 3H, d (J 7 HZ); .tau.8.81 3H, d 
(J6.5 HZ); .tau.7.84, 3H, d (J 1 HZ) and .tau.4.32, 1 H br.s. 
The above methyl ester was identical (t.l.c., u.v., I.R. and N.M.R.) with 
the impure methyl ester prepared directly from the acidic extract of the 
fermentation culture fluid by treatment with ethereal diazomethane. 
EXAMPLE 5 
Procedure for salt formation 
This is typified by the formation of the sodium salt of pseudomonic acid as 
obtained following purification on the XAD-2 resin. The impure acid was 
dissolved in isobutyl methyl ketone and extracted into water by the 
gradual addition of dilute aqueous sodium hydroxide until the pH of the 
aqueous layer reached 7. The aqueous layer was lyophilized to give the 
sodium salt of the impure acid. The barium salt was similarly obtained.