DE2136067C3 - 3-fluoro-D-alanine, 3-fluoro-D-alanine-2-d, their salts and medicinal products containing these compounds - Google Patents

Description

10

15th

20th

the use of «rays or X-rays Useful as an initiator for free radicals, since these high-energy rays hit the walls of the reactor penetrate.

The method described above can be carried out according to the usual batch technique, or it can also be carried out in a continuous manner in one tubular reactor with or without packing, such as Raschig rings, saddles or the like be carried out through which the substrate or its solution and the fluorinating agent are pumped are, preferably in countercurrent, while irradiating to form free radicals.

A mercury-xenon arc lamp proved to be a suitable source of radiation for generating radicals. operated with a 1000 watt power supply became. The lamp was mounted on a projector that had a quartz condenser lens and a Heat filter (water) was fitted.

example 1
3-fluoro-D-alanine

In a solution of 1.822 g of D - (-) - alanine in 45 ml of liquid HF, 0.6 g of fluorooxytrifluoromethane gas was added initiated over a period of about 1 hour while the solution was magnetically stirred, in one Dry ice-acetone bath was cooled and irradiated with ultraviolet light. After another 80 minutes Ultraviolet irradiation were introduced a further 2 g of fluoroxy jo trifluoromethane gas, while the solution IV2 Hours and then another hour was irradiated with ultraviolet light.

The solvent was removed by bubbling a stream of nitrogen gas through it. The decrease v, was dissolved in ice-water, and a sample thereof was analyzed in an amino acid, with a 41% yield of 3-fluoro-D-alanine and 32% unreacted Ausgangsrnaterial yielded. For isolation, the mixture was chromatographed on a cation exchange resin (H + form) of a commercially available polystyrene resin with sulfonic acid residues in the core. 2N HCl was used for elution. Pure 3-fluoro-D-alanine was obtained from the appropriate fractions by evaporation in vacuo. 3-fluoro-D-alanine was released from the hydrochloride in a water-pyridine-isopropanol mixture; Mp 166-168 ° C (dec.); [«]? ', -9.3 ° (c, 2 in 1 n-HCl).

^r > o example 2

3-fluoro-D-alanine-2-d

Deuterium was incorporated in the α-position of D-alanine by exposing L-alanine to the action of alanine racemase in D2O, for which purpose a crude enzyme preparation from Staphylococcus aureus (E. Ito and J. L Strominger, J. Biol. Chem., 237, 2689 [1962]), which _{had been dialyzed exhaustively against buffered D 2} O, was used. After salt-free alanine was obtained from the reaction mixture by ion exchange processes, the D-alanine formed was removed from the L-alanine used by chemical N-acetylation and specific enzymatic deacetylation of the μ L-isomer with pig kidney acylase I according to JP Greenstein and M. Winitz , Chemistry of the Amino Acids, Volume 3, page 1831 (Wiley and Sons, New York,

1961) separated The D-alanine-2-d obtained after acid hydrolysis of the acetylated residue was 95% enriched in D, especially in the 2-position (determined by NMR) and contained less than 1% L-alanine, which is enzymatic with L-alanine: oxoglutarate transaminase was determined.

Using the method according to Example 1, but using an equivalent amount of the D-alanine-2-d produced as above instead of the D-alanine used there, 3-fluoro-D-alanine-2-d was obtained; Mp 169 ° C; [*]? -9.2 (c 2 in 1 n-HCl).

Formulation examples
A) Dry-filled capsule containing 330 mg of active

Contains component per capsule

3-fluorine-D-alanine

Lactose, enough for a filling

a capsule

capsule

330 mg per capsule

145 mg

475 mg

3-fluoro-D-alanine is crushed to a powder with a sieve grain size of 250 μm, and then is Lactose put through a sieve cloth with sieve openings of 250 μΐη through on the powder, and the combined ingredients are mixed for 10 minutes When a uniform mixture is obtained, the solids will be reduced to a particle size of less than Grind 10 μπι and then put into dry gelatin capsules filled.

B) oral liquid, 1000 ml each 100.0 g which contains 100 g active ingredient / 1 60.0 g 25.0 g 3-fluoro-D-alanine 25.0 g Sucrose 0.2 ml glucose 1000.0 ml citric acid Concentrated orange oil Purified water, on

The sucrose and glucose are dissolved in about 400 ml of water with heating. The solution is then chilled and citric acid and orange oil are added. The solution is made to a volume of about Brought 900 ml with water, and 3-fluoro-D-alanine is added. The solution is filtered and on a Brought a volume of 1000 ml.

Pharmacological studies and comparisons Effectiveness of 3-Fiuor-D-alanine in comparison

with other antibiotics
in the treatment of infected mice

Female CD.-1 mice with a mean weight of 21.0 g were given 0.5 ml of a suitable Dilution of a dissolving culture broth containing the specified pathogens, injected intraperetoneally. (The strength of the infection is represented in each case as a multiple of the dilution of the culture solution, which was lethal for only 50% of the untreated herd, the LDw dose.) The therapeutic was then immediately in a 0.5 ml volume in one of the ways indicated: either subcutaneously by the Administered dorsal surface (see c), intraperitoneally (i.p.) or by administration (oral, p.o.). In the case of mice that by Streptococcus or Diplococcus organisms

were infected, a second dose was also taken Therapeutic administered 6 hours after infection The results of these experiments are reported as the statistically interpolated dose that is necessary to remove 50% of the infected population during a period of 7 days after infection and protect treatment (deaths among the

Therapeutic effectiveness (EDsoj, mg

Treatment failures and not, with drugs treated comparisons occur in all cases within the first three days). The abbreviations used for the Compounds subject to comparison were used as follows: 3FDA = 3-fluorine-D-alanine; CYC = D-cycloserine; TET = tetracycline; CLM = chloramphenicol

organism Application
art 3FDA CYCLO TET CLM Escherichia coli 2017 i.p. 0331 0.008 0.040 7 to 10 LDso S.C. 0.361 2.50 0.044 0.52 p.o. 0377 236 0.600 0.446 Klebsieila pneumoniae "B" S.C. 0.162 0.332 0.190 0.340 10 to 30 LD ₅₀ p.o. 0.109 1.51 1.42 0.440 Pseudomonas aeruginosa 2616 S.C. 0.897 - 2.4 7.66 9LD ₅₀ Streptococcus pyogenes 3009 S.C. (x2) 0.023 1.25 0.02 0.71 9LD ₅₀ Staphylococcus aureus 2949 S.C. 0.538 0.726 0.046 1.5 3LD ₅₀ Diplococcus pneumoniae 1-37 S.C. (x2) 0.104 > 10 0.084 1,780 17 LD ₅₀ Salmonella schottmuellerie 3010 p.o. 0.294 2.5 1,217 0.440 14LD ₅₀

3-Fluoro-D-alanine shows a clear superiority in effectiveness for treating a broad spectrum bacterial infections. Especially in the practical oral route, 3-fluoro-D-alanine gives an effectiveness that of the well-known antibiotics chloramphenicol and tetracycline is equal to or above goes out

Comparison of safety and therapeutic effectiveness

of 3-fluoro-L-alanine (3-FLA) and
3-fluoro-D-alanine (3-FDA) in mice

Groups of 5 female CD.-1 mice with a mean weight of 21 g served as either uninfected control animals or as animals for antibacterial therapy with subsequent intraperitoneal therapy Injection of 0.5 ml of a diluted solution culture of Escherichia coli 2017, which gives a value of 7 LD50 of bacterial pathogens represents Das Therapeutikur.i was administered in the indicated dosages in a 0.5 ml volume by the oral route in each Case administered

Below the maximum tolerated dose, 3-FLA has no therapeutic effect. During moderate amounts completely protect against 3-FDA, show higher values in vivo, as in the in-vitro test, Signs of auto-antagonism.

Influence of the concentration gradient

of 3-FDA and 3-FLA on bacterial growth

(Explanation of the autoantagonism)

The surface of a 2 mm thick nutrient agar layer in a Petri dish was ^{coated with 10 4} organisms / cm ^{2 of} a culture of Escherichia coli 2017, and then paper disks 7 mm in diameter bearing the indicated amounts of 3-FDA or 3-FLA were placed thereon. After incubation at 37 ° C. for 16 hours, the circular zones, which are free of bacterial growth and which surround the disks, were measured. Only in the case of 3-FDA, and there only with higher drug values, was an on the paper disk

so adhering growth ring observed to which a Connected ring made of bacteria-free agar. The inner one

dose % Survivors of the group infected mice 3-FDA rich of "V11W | O
by the diffusing one 60 μg * lMkB UIlUlI (IIfV
drug Autoanta 3-FDA Autoanta not infected 0 educated 1 ! Concentration gradient will than the zone gonism Inhi gonism Control animals 3-FLA 0
20th 55 of autoantagonism bation mg 3-FLA 3-FDA 0 100 25th 0 0 0 100 100 0
0 80 Crowd on
the disc 65 50 Diameter of the zones around the disks,
mm 0 30th 11th 0.078
0312 100 100 0 100 3-FLA 0 33 13th 1,250 100! -M) 0 _ 200 Inhi 0 35 15th 5 0 100 400 bation 0 38 17th 20th - 100 _ 3-FDA 41 40 - 100 ι factor 24 opposite to 28 The tolerance of mice exceeds that of 3-FLA by one 32 of at least 10. 34 37

Claims (3)

Patent claims: 1. 3-Fluoro-D-alanine and its salts. 2. 3-Fluoro-D-alanine-2-d and its salts. 3. Medicament, characterized by a content of a compound according to claim 1 or 2. The invention relates to 3-fluoro-D-alanine and its salts, 3-fluoro-D-alanine-2-d and its salts as well Medicaments containing one of the above compounds. The racemic compound 3-fluoro-D, L-alanine was described by Lettre et al in Ann. Chem, 708, pages 75 to 85 (1967). However, the optical Isomers have not yet been described, and the antibacterial effectiveness of the racemate has not been investigated It has now been found that the optical isomers of 3-fluoroalanine have an antibacterial effect in vitro. It has surprisingly been shown that the inhibiting effectiveness of 3-fluoro-D-alanine only is to be determined by specific antibacterial test methods in vitro, the antibacterial effectiveness of the optical isomers is comparable in vitro. However, the isomers differ considerably in vivo. That The D-isomer also has antibacterial properties, while the L-isomer has no protective properties seems to have bacterially infected organisms and is lethal even at low doses The in vitro antibacterial study is carried out by the unusual autoantagonistic property of 3-fluoro-D-alanine, determined at high concentrations. In standard antibacterial examinations, the efficacies are of a very small order of magnitude register and thus where relatively high concentrations of test compound are applied, therefore the effectiveness of 3-fluoro-D-alanine is lacking. This autoantagonistic property is not detrimental to usability, since it is only for example in vivo at dosages greater than about ten times the mean effective healing level occurs. While 3-fluoro-L-alanine quickly and completely with If converted into toxic effects, 3-fluoro-D-alanine is a much slower breakdown in vivo subject. Possibly D-amino acid oxidase acts as an enzyme that is abundant in humans and is present in rats, but to a lesser extent in the mouse and certain herbivorous ones Creature. However, neither does the metabolic rate affect the blood level of the therapeutic agent can affect nor the by-products of this metabolism act of using the compound contrary to the dosage level suggested for humans or animals. In the context of the invention it has been shown that the deutero analog 3-fluoro-D-alanine-2-d compared to the Attack by D-amino acid oxidase is noticeably less sensitive, however its antibacterial effectiveness of 3-fluoro-D-alaniiB retains. The in vivo effectiveness is increased and supported. The compounds according to the invention and their salts show in comparison with known antibacterial Means a distinct superiority. They are useful as antibacterial agents to inhibit growth from pathogenic bacteria of both gram-positive and gram-negative genera, for example Streptococcus, Escherichia, Staphylococcus, Salmonella, Pseudomonas, Diplococcus, Klebsiella, Proteus, Mycobacterium, Vibrio, Pasteurella and Serratia and their antibiotic-resistant strains. examples for such pathogens are Escherichia coli, Salmonella schottmuelleri, Proteus vulgaris, Proteus mirabilis, ίο Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. The compounds according to the invention and their salts can therefore be used as atiseptic Preparations for removing sensitive organisms from pharmaceutical, dental and medical Facilities are used and can also be used in other areas that the Infection by such organisms. However, they can also be used to treat diseases caused by the above organisms in humans and animals, and can be used in a variety of therapeutic doses in common carriers for oral use, for example in the form of a capsule or tablet or in a liquid solution or suspension administered. Suitable preparations can include customary diluents, granulating agents, preservatives, Contain binders, flavorings and coating agents. The dose of the products can be vary over a wide range; for example For adults weighing 70 kg, the doses range from about 250 mg to about 500 mg of the active ingredient two to four times a day. A growing animal with feed allotment should receive about 100 g per 907 kg of complete feed with growth-enabling therapy. The compounds of the invention can also be administered parenterally by injection into a sterile drug carrier.
The fluorinated alanine derivatives according to the invention are prepared as described in DE-AS 2136 008. D-alanine or the corresponding .deuterated D-alanine is used as a substrate with fluoroxytrifluoromethane under the influence of light, including ultraviolet light, ionizing radiation, 4ΐ treated as for example «rays or microwaves According to the preferred procedure, the substrate is in a suitable solvent which is inert to the fluorination reaction, such as liquid hydrogen fluoride, fluorosulfone > o acid, trifluoroacetic acid or sulfuric acid, dissolved that Solution is irradiated and the required amount of the fluorooxytrifluoromethane is added with vigorous stirring and while maintaining the temperature, slowly added to the reaction mixture and stirring and the irradiations are continued until the completion of the reaction. Because of the low boiling point of the reagents, it is convenient, the reaction at temperatures as low as - perform 80 ⁰ C, the reaction at atmos- bo spherical pressure progresses. A suitable reaction vessel for reactions at atmospheric pressure is one made from a metal rod manufactured vessel equipped with an ultraviolet-transparent window. The reaction can too b5 in a pressure vessel, or a steel bomb with a • Platinum lining are carried out, in which case higher temperatures, e.g. B. up to about 100 ° C can be used. In this case it is