Note: Descriptions are shown in the official language in which they were submitted.
~ 15669
il~3~
This invention is concerned generally with novel
compositions having antibacterial activity. More particularly,
it relates to compositions containing a 3-fluoro-D-alan;ne-
type compound in combination with a 3-fluoro-D-alanine auto-
antagonist-inhibitor, which compositions are characterized as
demonstrating, even at greatly elevated concentrations, the
highly effective antibacterial action of 3-fluoro-D-alanine-
type compounds, and are further characterized as showing a
remarkable synergistic action, the antibacterial potency of
the combination being substantially in excess of that con-
tributed by the individual components.
The 3-fluoro-D-alanine-type compounds, such as 3-
fluoro-D-alanine peY se and its deutero analogs, and lower
alkyl esters and pharmacologically acceptable salts thereof,
are valuable antibacterial agents showing effective activity
; in inhibiting the growth of pathogenic bacteria of both gram-
positive and gram-negative genera, including Streptococcus,
Escheriehia, StaphyZoeoeeus, Pseudomonas, DipZoeoecus~
~ZebsieZZa, Proteus, Myeobaeterium, Serratia, Vibrio and
PasteureZZa~ and in particular, bacteria belonging to the
species Eseheriehia eoZi, Proteus vuZgaris, Proteus mirabiZis,
Proteus morganii, Streptomyees pyogenes, Pseudomonas aerugi-
nosa, StaphyZoeoceus aureus and Aerobacter aerogenes.
The 3-fluoro-D-alanine-type compounds do, however,
possess the unusual characteristic of being autoantagonists at
high concentration, which characteristic is disadvantageous
where it is desired to employ the antibacterial at elevated
~'.
- 2 - 4~!
5669
lS~ 9~
dosages, or where high, autoantagonist concentrations of the
3-fluoro-D-alanine-type compound could result during therapy
as, for example, in patients with diminished renal function.
Indeed, since elevated dosages of 3-fluoro-D-alanine-type
compounds may be employed with safety, it is advantageous in
treatment of infections to utilize the 3-fluoro-D-alanine-type
compound at a sufficiently high dosage to provide effective
antibacterial action against the most resistant organism by
which the infection may be caused, at which high dosage the 3-
fluoro-D-alanine-type compound is an autoantagonist with
respect to more sensitive microorganisms which may be present.
It is now discovered, in accordance with the present
invention, that this autoantagonist action shown by 3-fluoro-
D-alanine-type compounds at elevated concentration can be
completely suppressed by certain substances which are herein-
after referred to as FDA-autoantagonist-inhibitors. As FDA-
autoantagonist-inhibitors, it is preferred to employ N-sub-
stituted cycloserine compositions which may be represented by
the following formula:
~0\
R-CH NH
HC C=0
I
NH
X-CH2-C-C-C-CH2-Y
Z O
wherein R is hydrogen or methyl, and X, Y and Z are hydrogen
or alkyl, and pharmacologically acceptable salts thereof, and
which may be referred to as D-4-(1'-methyl-3'-oxo-1'-butenyl
or alkyl-substituted-l'-methyl-3'-oxo-1'-butenyl)amino-3-
15669
i(~3~
isoxazolidinones, and their 5-methyl derivatives, as for ex-
ample D-4~ methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone,
D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-5-methyl-3-isoxazoli-
dinone, D-4-(1',2'-dimethyl-3'-oxo-1'-butenyl)amino-3-isoxazol-
idinone, D-4-(1',2'-dimethyl-3'-oxo-1'-butenyl)a~ino-5-methyl-
3-isoxazolidinone, D-4-(1'-ethyl-3'-oxo-1'-butenyl)amino-3-
isoxazolidinone, D-4-(1'-ethyl-3'-oxo-1'-butenyl)amino-5-
methyl-3-isoxazolidinone, D-4-(1'-ethyl-2'-methyl-3!-oxo-1'-
butenyl)amino-3-isoxazolidinone, D-4-(1'-ethyl-2'-methyl-3'-
oxo-1'-butenyl)amino-5-methyl-3-isoxazolidinone, D-4-(1'-ethyl-
3'-oxo-1'-pentenyl)amino-3-isoxazolidinone, D-4-(1'-ethyl-3'-
oxo-l'-pentenyl)amino-5-methyl-3-isoxaziolidinone, D-4-(1'-
ethyl-2'-methyl-3'-oxo-1'-pentenyl)amino-3-isoxazolidinone,
D-4-(1'-ethyl-2'-methyl-3'-oxo-1'-pentenyl)amino-5-methyl-3-
isoxazolidinone, and the likej pharmacologically acceptable
; salts of the foregoing such as alkali metal salts, preferably
the sodium and potassium salts; alkaline earth metal salts
preferably the calcium and magnesium saltsi ammonium saltsi
amine salts, preferably salts with triethylamine, diethylamine,
N-methyl-glucamine, diethanolamine, triethanolamine or 2-amino-
2-hydroxymethyl-1,3-propanediol, and the like, as for example,
the sodium salt of D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-
isoxazolidinone, the calcium salt of D-4-(1'-methyl-3'-oxo-1'-
butenyl)amino-3-isoxazolidinone, the sodium salt of D-4-(1'-
methyl-3'-oxo-1'-butenyl)amino-5-methyl-3-isoxazolidinone, and
the like. It is preferred to utilize these N-(l-methyl-3-oxo-
l-butenyl or alkyl-substituted-l-methyl-oxo-l-butenyl) deriva-
tives of cycloserine and methyl-cycloserine and their pharmaco-
logically acceptable salts as FDA-autoantagonist-inhibitors,
since these derivatives and, more particularly, D-4-(1'-methyl-
3'-oxo-1'-butenyl)amino-3-isoxazolidinone and D-4-(1',2'-
dimethyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone, and their
sodium and potassium salts, are remarkably stable in aqueous
solution; when administered clinically, they are extremely
effective in releasing cycloserine or methyl-cycloserine in
vivo and, at the same time, substantially avoiding the un-
wanted dimerization of the cycloserine or methyl-cycloserine.
The D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone
and its pharmacologically acceptable salts are of particular
effectiveness in releasing cycloserine in the blood stream and
in the bladder, and are thus of value in providing FDA-auto-
antagonist-inhibitor action throughout the vascular system as
well as in the urinary tract.
The critical effectiveness of FDA-autoantagonist-
inhibitors is exemplified as follows: 3-fluoro-D-alanine per
se, at a concentration of 3.2 ~g/ml, completely represses the
growth of a virulent culture of Ese~eric~ia coZi~ (Strain
2017), however, at higher concentrations (i.e. at 50 ~g/ml)
extensive growth of the organism occurs characteristic of the
autoantagonist action of the 3-fluoro-D-alanine. When the 3-
fluoro-D-alanine, at this concentration of 50 ~g/ml, is com-
bined with an FDA-autoantagonist-inhibitor such as D-4-(1'-
methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone or its sodium
salt at 0.72 ~g/ml, growth of the Esc~eric~ia coZi is com-
pletely repressed. Surprisingly, the 0.72 ~g/ml D-4-(1'-methyl-
3'-oxo-1'-butenyl)amino-3-isoxazolidinone concentration, com-
15669
1 pletely effective as FnA-autoantagonist-inhibitor~
2 is less than 2% of the minimum concentration which is
3 itself able to completely repress the growth of the
4 Escherichia coli organisms.
This novel combination is conveniently
6 admministered for antibacterial effectivéness in the form
~ of a pharmaceutical composition containing the 3-fluoro-D-
- 8 alanine-type compound in admixture with the FDA-autoantagonist-
g inhibitor and a pharmacologically acceptable carrier. r~hen
a 3-fluoro-D-alanine-type compound is combined with an N-
11 substituted cycloserine-type compound, the ratio of the 3-
fluoro-D-alanine-type component to the N-substituted cyclo-
serine-type component in the combination ordinarily varies f
14 from about 1:120 to about 200:1 with ratios within the range
of about 1:4 to about 4:1 being preferred. As the 3-1uoro-
16 D-alanine-type component, it is ordinarily preferred to
17 utilize 3-fluoro-I)-alanine, 2-deutero-3-fluoro-D-alanine,
18 2,3-dideutero-3-fluoro-D-alanine, 2,3,-trideutero-3-fluoro-
19 D-alanine, a lower alkyl ester thereof such as the methyl
ester, or a pharmacologically acceptable salt such as the
21 sodium, calcium, or hydrochloride salt. As the cycloserine-
22 type component, it is ordinarily preferred to utilize N-(l-
23 methyl-3-oxo-1-butenyl or alkyl-substituted-1-methyl-3-oxo-
24 l-butenyl) derivative of cycloserine or cyclothreonine, or
pharmacologically acceptable salts thereof, and it is particu-
26 larly preferred to employ D-4-(1'-methyl-3'-oxo-1'-butenyl)
27 amino-3-isoxazolidinone, its sodium or potassium salts such as
2~ D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone
29 sodium salt-~lemihydrate, or D-4-)1,2'-dimethyl-3'-oxo-1'-
butenyl)amino-3-isoxazolidinone or its sodium or potassium salts.
-- 6 --
15669
Pharmaceutically acceptable carriers for the afore-
said compositions include convenient vehicles adapted for oral
administration such as capsules, tablets, or liquid solutions
or suspensions, or the combination may be dissolved in a vehi-
cle adapted for administration by injection. Suitable formu-
lations for oral use may include diluents, granulating agents,
preservatives, binders, flavoring agents and coating agents
which are well known to those skilled in the art, and the
dosage of the products may be varied over a wide range. A
composition for oral use comprising the combination of 3-
fluoro-D-alanine-type compound and N-substituted-cycloserine-
type compGund is conveniently prepared by intermixing the
individual components in a dry, pulverulent state with gelatin,
starch, magnesium stearate and alginic acid, pressed into a
tablet. The 3-fluoro-D-alanine-type compound and the N-
substituted-cycloserine-type compound may also be incorporated
together in aqueous solution, and the solution evaporated to
provide the combination in the form of an intimate mixture of
the components.
Alternatively, in accordance with a further embodi-
ment of this invention, the components of the combination may
be separately administered, particularly where it is desired
for maintenance of blood levels of the more rapidly excreted
3-fluoro-D-alanine-type component.
15669
The N-(l-methyl-3-oxo-1-butenyl or alkyl-substituted-
l-methyl-3-oxo-1-butenyl) cycloserine or methyl-cycloserine 3-
FDA-autoantagonist inhibitors may be prepared by stirring
together, at room temperature for a period of about two days,
cycloserine or methyl-cycloserine with an excess of a 2,4-
pentanedione compound having the following formula:
O O
Il 11
X-CH2-C-CH-C-CH2-Y
wherein X, Y and Z are hydrogen or alkyl, such as 2,4-pentane-
dione, under which conditions, the cycloserine compound gradu-
ally goes into solution, with formation of the corresponding
N-substituted-cycloserine compound of the formula:
R C/ 0 ~ NH
HC C=0
7H
X-CH2-C= lC- ICI-CH2 Y
wherein R is hydrogen or methyl, and X, Y and Z are hydrogen
or alkyl, which, as the reaction proceeds, crystallizes from
the resulting reaction solution, and is recovered by fil-
tration, washed with ether and dried. For example, D-4-
(l'-methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone is con-
veniently prepared by stirring a mixture of about 3.0 g. of
D-4-amino-3-isoxazolidinone and 30 ml. of 2,4-pentanedione
in a dry atmosphere at approximately room temperature for
15669
~a~ lsl
1 about two days. The D-4-amino-3-isoxazolidinone gradually
2 goes into solution, and the reaction product, which
3 crystallizes from the reaction solution, is recovered by
4 Eil~ration, washed with three 20 ml.-portions of ether,
and dried at room temperature ln vacuo to give about 3~5 g.
6 o~ D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone.
7 The latter is convenlently converted to its
3 pharmacologically acceptahie salts, e.g. the sodium or
9 calcium salts as follows: 0.143 g. of D-4-(1'-methyl-3'-
oxo-1'-butenyl)amino-3-isoxazolidinone is dissolved in 0.8 ml.
11 of methanol,and 1~56 ml. of an O.OS molar solution of
12 sodium hydroxide in methanol, is added to give a pH of
13 approximately 7Ø The methanol is evaporated from the re-
14 sulting solution under a stream of nitrogen until crystals
form; the crystalline slurry is further evaporated to dryness
16 in vacuo. The residual material is washed with 2.5 ml. of
17 acetone, and redissolved in 0.55 ml. of methanol; the solution
18 i5 filtered and the filtrate diluted with 0.45 ml. of methanol.
19 To the resulting solution is added 4 ml. of ether, and the
crystalline precipitate which forms is recovered by filtration,
21 washed with ether and dried co give substantially pure D-4~
22 (1'-methyl-3'-oxo-1' butenyl)amino-3-isoxazolidinone sodium
23 salt-hemihydrate.
2~ rrhree grams of calcium o~ide ~re slurried in
40 ml. of water, and the slurry cooled to 0-5C. Five grams
26 of D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-isoxazolidinone
27 is added to the cooled slurry, the resulting mixture is heated
28 with slurry to room temperature alld stirred at that temperature
29 for 5 minutes. r~e mixture is filtered, and the insoluble
material is washed with 10 ml. of water. To the combined
15669
solution and washings (having a p~ of about 11.0 - 11.5) is
added sufficient D-4-(1'-methyl-3'-oxo-1'-butenyl)amino-3-
isoxazolidinone to give a final pH of about 9.5. Activated
carbon (0.5 9.) is added to the resulting solution, and the
mixture is stirred for about 15 minutes and filtered. The
filtered solution is diluted with six times its volume of
ethanol, and the crystalline precipitate which forms is
recovered by filtration, washed with ethanol and dried in va~uo
to give, in substantially pure form, the calcium salt of D-4-
(1'-methyl~3'-oxo-1'-butenyl)amino-3-isoxazolidinone.
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