Antibacterial peptides

It has been found that dipeptides containing a 3-fluoro-D-alanine N-terminus are powerful antibacterials and produce a highly useful synergistic effect with antibiotics.

DETAILED DESCRIPTION OF THE INVENTION 
As micro-organisms become resistant to known antibiotics, continued effort 
is needed to find new compounds or combinations of compounds which 
effectively inhibit bacterial growth. 
It has now been found that a peptide of the formula 
##STR1## 
wherein the shown aminoacid is in the D-configuration, R is hydrogen or an 
easily removable protective group, R' is hydrogen or deuterium and R" is 
the imino moiety of an .alpha.-aminoacid in the L-configuration, the 
corresponding loweralkyl esters thereof, or nontoxic acid addition salts 
thereof, are useful antibacterials; they also represent powerful 
syngerists for D-cylcloserine and other antibiotics. 
The amino group substituent R particularly includes an acyl group of a 
lower fatty acid such as acetyl, propionyl, isobutyryl and the like. The 
above moiety R" particularly represents the known, protein-derived 
aminoacids, including glycine which, of course, does not have a chiral 
center. The definition also includes other aminoacids where the amino 
group is attached to the 2- or .alpha.-position of the acid. The 
protein-derived aminoacid may be represented by leucine, valine, 
norvaline, proline, serine, tyrosine, alanine, phenylalanine, threonine, 
methionine, glutamine, histidine, arginine, lysine and tryptophane. The 
new dipeptides have the unnatural sequence of a (deuterated) 
D-.beta.-fluoroalanine coupled to an L-aminoacid. Such a D-L sequence is 
usually restricted to the cell wall components of microorganisms and its 
antibacterial activity is completely unexpected. 
The new dipeptide can easily be synthesized by coupling, in well known 
fashion, the active ester of an N-protected .beta.-fluoro-D-alanine (or 
its deuterated analog) with an aminoacid in the L-configuration. Among the 
active esters, the hydroxysuccinimide, pentachlorophenyl, 4-nitrophenyl, 
2,4,5-trichlorophenyl, a fluorophenyl, N-hydroxyisobornyldicarboximide or 
similarly familiar esters of N-protected .beta.-fluoro-D-alanine can be 
used for the coupling reaction. The N.sup..alpha. -group and any sensitive 
functional group in the aminoacid moiety represented by R" above can be 
protected with the usual well-known groups that can subsequently be 
removed by a mild chemical reaction which does not affect the peptide bond 
formed. Among the groups frequently used as temporary protection are the 
carbobenzoxy (hereinafter identified as Z) or the tert. butoxycarbonyl for 
amino groups, particularly the N.sup..alpha. -group, while benzyl or other 
moieties can be used to protect the hydroxy groups in serine, tyrosine or 
hydroxyproline or the imidazole group of histidine. Hydrogenation will 
remove said benzyl group after the peptide coupling has been effected and 
treatment with hydrobromic acid or hydrofluoric acid will remove other 
protective groups used by the skilled artisan, without cleaving the 
peptide bond. The free acid can be converted into the desired alkyl ester 
in known fashion and/or the N.sup..alpha. -group can be acylated in known 
manner. The identical reaction sequence can also be used when the starting 
material is the 2-deuterated 3-fluoro-D-alanine.

In order to illustrate the preparation of the new peptide, reference is 
made to the following examples which, however, are not intended to limit 
this invention in any respect. In all examples, the optical rotations were 
taken at 25.degree. C. (unless shown differently) in 1 N HCl at the 
concentrations given. 
EXAMPLE 1 
A stirred solution of 965 mg. of N-carbobenzoxy-.beta.-fluoro-D-alanine and 
506 mg. of N-hydroxysuccinimide in 15 ml. of 1,2-dimethoxyethane (DME) was 
cooled in an ice bath and treated with 908 mg. of 
dicyclohexylcarbodiimide. After stirring for 2 hours at ice bath 
temperature, and 1 hour at room temperature, 10 ml. of DME was added and 
the slurry was filtered directly into a solution of 445 mg. of L-alanine 
and 840 mg. of sodium bicarbonate in 25 ml. of water. After stirring 
overnight, the solution was concentrated to a thick oil which was 
acidified with 1 N-hydrochloric acid. This mixture was stirred in an ice 
bath for 7 hours, producing a white, amorphous solid which was collected 
by filtration, washed with ice water and dried to give 850 mg. of 
N-carbobenzoxy-.beta.-fluoro-D-alanyl-L-alanine. 
Hydrogenation of this product in 100 ml. of methanol in the presence of 0.2 
g. of 5% Pd-on-carbon gave, after filtration and solvent evaporation, 278 
mg. of .beta.-fluoro-D-alanyl-L-alanine as an amorphous powder; 
[.alpha.].sup.20 +56.6.degree. (C, 1.1). 
EXAMPLE 2 
By substituting the L-alanine of Example 1 with 826 mg. of L-phenylalanine, 
the above process produces 290 mg. of 
.beta.-fluoro-D-alanyl-L-phenylalanine as an amorphous solid; 
[.alpha.].sup.20 +5.5.degree. (C, 0.2). 
EXAMPLE 3 
By substituting the L-alanine of Example 1 with 1.19 g. of 
.gamma.-benzylglutamic acid, the described procedure gives 480 mg. of 
.beta.F-D-Ala-L-Glu; [.alpha.].sub.D +32.degree. (C, 1.0). 
EXAMPLE 4 
In the fashion described above, 650 mg. of L-leucine is converted to 175 
mg. of F-D-Ala-L-Leu; [.alpha.].sub.D +29.degree. (C, 1.0) an an amorphous 
powder after passing it through a Sephadex.RTM. column using water as 
eluent. 
EXAMPLE 5 
The process of Example 1 used with 1.03 g. of 2-aminoisobutyric acid 
produced 120 mg. of .beta.F-D-Ala-Aibu; [.alpha.].sub.D +4.5.degree. (C, 
1.0). 
EXAMPLE 6 
The process of Example 4 used with 560 mg. of valine produced 100 mg. of 
.beta.F-D-Ala-L-Val, [.alpha.].sub.D +10.degree. (C, 0.5). 
When the L-aminoacids in the above examples are replaced by the 
corresponding known loweralkyl esters, i.e., leucyl methyl ester, valine 
butyl ester, and alanyl ethyl ester, the corresponding D-L-dipeptide 
loweralkyl esters are obtained in the same fashion. 
By using the deuterated .beta.F-D-Ala-2-d (made according to the method of 
Dolling et al., J. Org. Chem., 43, 1634 of 1978) in the process of Example 
1, .beta.F-D-Ala-2-d-L-Ala is obtained in similar yield. 
Other compounds of the above general description can easily be made by 
repeating Example 1 but using other protective groups for .beta.F-D-Ala or 
.beta.F-D-Ala-2-d, other active esters thereof or other L-aminoacids. For 
instance, if said L-aminoacid is isoleucine or .alpha.-aminocaproic acid, 
the corresponding compounds are obtained where R represents L-isoleucyl or 
L-.alpha.-aminocaproyl. Obviously, other amino acids carrying protected 
additional functional groups can be employed to make the dipeptides of the 
current invention. Particularly, (deuterated) .beta.-fluoro-D-alanyl- 
L-threonine, L-tryptophane, and -L-tyrosine can be made by the above 
route. In these and other instances, functional groups where present, can 
be temporarily protected in known fashion by benzyl, carbobenzyloxy, tert. 
butyl or other protective groups commonly used in the peptide art. 
EXAMPLE 7 
The above fluorinated dipeptides are prepared in sterile concentrated 
aqueous solutions. Serial dilutions are made to give a range of 
concentrations of the test substances. Samples of the dilutions are mixed 
with an appropriate sterile synthetic bacterial growth medium in test 
tubes. The tubes are then inoculated with an appropriate test organism and 
incubated at 35.degree.-37.degree. C. for 16-20 hours. Minimum inhibitory 
concentrations, i.e., that concentration which inhibits visible growth, 
are shown in Table I. Where the results are shown in parentheses, a higher 
concentrations did not always inhibit organism growth. The organisms used 
in this test were as follows: 
TABLE I 
______________________________________ 
I Staph. aureus Smith 
II Strep. faecalis 10541 
III Enterococcus 89 
IV Bacillus subtilis 9466 
V E. coli Juhl 
VI Kleb. pneumonise 8045 
VII Shigella sonnei 9290 
______________________________________ 
Example No. 
1 2 3 4 5 6 
______________________________________ 
I (25) (3.1) 800 800 800 800 
II (1.56) 0.78 50 3.1 800 (6.2) 
III 200 25 25 25 25 6.2 
IV (.78) 3.1 3.1 6.2 (25) (3.1) 
V (.78) (6.2) (12.5) 
800 (12.5) 
800 
VI (12.5) (12.5) (25) 800 800 800 
VII (.39) (1.56) (12.5) 
800 (6.2) (25) 
______________________________________ 
EXAMPLE 8 
The in vivo activity of the fluorinated dipeptides and the fluorinated 
dipeptide-antibiotic combinations provided by the present invention were 
demonstrated as follows: 
Charles River mice weighing approximately 20 g. each, were infected 
intraperitoneally with 10-100 times the LD.sub.50 of the infecting 
organism. At predetermined intervals post-infection, e.g., 1 and 5 hours, 
mice were dosed subcutaneously with graded doses of the new dipeptide, 
antibiotic and a combination thereof. The number of mice surviving each 
treatment for 7 days post-infection was observed and the CD.sub.50 is 
calculated. The results using D-cycloserine as an example of the 
antiobiotic and representative fluorinated dipeptides are shown in Table 
II, using Staph. aureus (Smith) as the infecting organism. The values are 
listed in mg./kg. 
TABLE II 
______________________________________ 
Peptide + 
Example Peptide Cycloserine Cycloserine 
______________________________________ 
1 9.4 8.4 1.9 + 0.18 
2 75-150 25 4-9 + 0.4- 0.9 
______________________________________ 
The compounds of the present invention can be administered intramuscularly, 
orally, subcutaneously or intravenously. Sterile, liquid dosage forms can 
easily be prepared for parenteral administration by dissolving the above 
dipeptide in the form of a water-soluble, nontoxic salt in isotonic sodium 
chloride solutions containing optional buffers, stabilizers, and/or 
preservatives. Liquid oral dosage forms in the form of elixirs, syrups or 
suspensions can be made in standard fashion, also optionally containing 
the above additives together with coloring or flavoring agents. 
Solid dosage forms for oral administration include tablets, capsules, pills 
and wafers. For these dosage forms, the usual solid diluents are used 
where required. Capsules can be filled with undiluted powdered or 
granulated crystals of the new compounds. For tablets, the following 
standard procedure may be used: 
About one-half of 50 g. of cornstarch is milled together with 50 g. of the 
above dipeptide and 220 g. of calcium phosphate dibasic dihydrate. This 
blend is milled until homogenous and passed through a 40-mesh screen. The 
remaining portion of the cornstarch is granulated with water, heated and 
mixed with the above drug blend in a hot air oven at 50.degree. C. and 
sifted through a 16-mesh screen. These granules are then mixed with 16 g. 
of talcum powder, 4 g. of magnesium stearate and 0.8 g. of combined 
coloring and flavoring additives. The mixture is blended to homogeneity, 
passed through a 30-mesh screen and blended for another 15 minutes. This 
blend is compressed into tablets weighing approximately 350 mg. using a 
9/32" standard convex punch resulting in tablets of a hardness of 7-9 with 
each tablet containing 50 mg. of the drug. In a similar fashion, tablets 
weighing 600 mg. containing 250 mg. of drug can be prepared, preferably in 
a tableting machine producing bisected tablets. 
While the above examples are directed to the peptides and their esters, 
their acid addition salts can readily be prepared and used in the same 
known fashion. The nontoxic salts useful as antibacterials include 
primarily the hydrochloride, phosphate, sulfate, acetate, succinate and 
citrate. 
As will be seen from the above examples, the current dipeptides are 
antibacterially active in warmblooded animals. Against certain bacteria, 
the new dipeptides are powerful synergists for known antibacterials, 
enabling the use of the latter in quantities of only a small fraction of 
its curative dose. In particular, by combining the current dipeptide with 
a medicinally useful antibiotic in a weight ratio of 1:1 to 10:1, 
excellent antibacterial synergism is observed. While the demonstrated 
synergistic results above are based on the use of a specific antibiotic, 
it will be understood that other antiobiotics including pencillins such as 
carbenicillin, cephalosporins such as cephalothin, streptomycin, 
erythromycin, tetracyclin, etc. can be combined with the new peptides to 
obtain better results than with such antibiotics alone. The dipeptides can 
be used in the usual form, as the corresponding loweralkyl esters thereof 
and/or as nontoxic addition salts thereof. The dipeptides wherein R is a 
protective group are intermediates for the preparation of the dipeptides 
wherein R is hydrogen and in some instances have useful pharmacological 
effects as precursors for N.sup..alpha. -unprotected dipeptides or 
dipeptide esters of this invention.