DE2136067B2 - 3-fluoro-D-alanine, 3-fluoro-D-alanine-2-d, their salts and medicinal products containing these compounds - Google Patents

Description

The use of -rays or X-rays as initiators for free radicals is expedient, since these high-energy rays penetrate the walls of the reactor.

The method described above can be carried out using the usual batch-wise technique, or it can also be carried out in a continuous manner in a tubular reactor with or without packing, such as For example, Raschig rings, saddles or the like can be carried out through which the substrate or in whose solution and the fluorinating agent are pumped, preferably in countercurrent, while for Formation of free radicals is irradiated.

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

example 1
3-fluoro-D-alanine

To a solution of 1.822 g of D (-) - alanine in 45 ml of liquid HF, 0.6 g of fluorooxytrifluoromethane gas 2> was bubbled over a period of about 1 hour while the solution was stirred magnetically, cooled in a dry ice-acetone bath and by ultraviolet light has been irradiated. After 80minütige! · Another ultraviolet radiation further 2 g Fluoroxytrifluormethangas were initiated while the solution I ^1/2 hours and then for another hour was irradiated with ultraviolet light.

The solvent was removed by bubbling a stream of nitrogen gas through it. The residue was dissolved in ice water and a sample thereof was analyzed in an amino acid analyzer to find a 41% yield of 3-fluoro-D-alanine and 32% unreacted starting material. 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 4ϊ 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 1N HCl).

V)

Example 2 3-fluoro-D-alanine-2-d

Deuterium was incorporated in the «position of D-alanine, L-alanine being exposed to the action of ■■ >> Alanine racemase in D2O was exposed to, too for this purpose a crude enzyme preparation from Staphylococcus aureus (E. Ito and J. L Strominger, J. Biol. Chem., 237, 2689 [1962]), which is exhaustive against buffered D2O dialyzed was applied. After wi Recovery of salt-free alanine from the reaction mixture by ion exchange methods was the D-alanine formed from the L-alanine used by chemical N-acetylation and specific enzymatic deacetylation of the 6ϊ L-isomers with pig kidney acylase I according to J. P. 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 was determined enzymatically with L-alanine: oxoglutarate transaminase.

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-fluorine-D-alanine-2-d was obtained; Mp 169 ° C; [«];» - 9.2 (c 2 in 1N HCl).

Formulation examples

A) Dry-filled capsule containing 330 mg of active
Contains component per capsule

Per capsule

3-fluoro-D-alanine 330 mg

Lactose, enough for a filling
one capsule i45 mg

Capsule 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 μm on Jas powder, and the combined ingredients are mixed for 10 minutes. After a uniform mixture is obtained, the Grind solids to a particle size of less than 10 μm and then put into dry gelatin capsules filled.

B) oral liquid, 1000 ml each 100.0 g which contains 100 g active ingredient / l 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 will 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-fluoro-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 injected intraperetoneally with 0.5 ml of an appropriate dilution of a 16 hour culture broth containing the indicated pathogens. (The severity of the infection is represented in each FaN as the multiple of the dilution of the culture solution which was lethal to only 50% of the untreated herd, the LD50 dose.) The therapeutic agent was then immediately added in a 0.5 ml volume to a the routes indicated: administered either subcutaneously on the dorsal surface (sc), intraperitoneally (Lp.) or by administration (oral, po). In the case of mice caused by Streptococcus or Diplococcus organisms

were infected, a second dose of therapeutic agent was also 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 herd during a period of 7 days after infection and protect treatment (deaths among the

Therapeutic Efficacy (ED ₅₀ ), 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 are as follows: 3FDA = 3-fluoro-D-alanine; CYC = D-cycloserine; TET = tetracycline; CLM = chloramphenicol.

organism Application 3FDA CYCLO TtT CLM art Escherichia coli 2017 i.p. 0.331 0.008 0.040 7 to 10 LD _W s.c. 0.361 2.50 0.044 0.52 p.o. 0.377 2.36 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 0.4 4U Pseudomonas aeruginosa 2616 s.c. 0.897 - 2.4 7.66 9 LD ₅₀ Streptococcus pyogenes 3009 s.c. (x2) 0.023 1.25 0.02 OJ: 9LD ₅₀ Staphylococcus aureus 2949 s.c. 0.538 0.726 0.046 1.5 3 LD ₅₀ Diplococcus pneumoniae 1-37 S.C. (x2) 0.104 > 10 0.084 i.780 17 LD ₅₀ Salmonella schottmuellerie 3010 p.o. 0.294 2.5 1,217 0.440 14 LD ₅₀

3-fluoro-D-alanine shows a clear superiority of the Effective in treating a wide range of bacterial infections. Especially with the practical one Orally, 3-fluoro-D-alanine has an activity similar to that of the well-known antibiotic chloramphenicol and tetracycline is equal to or greater than.

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 either as uninfected control animals or as animals for antibacterial therapy with subsequent intraperitoneal injection of 0.5 ml of a diluted solution culture of Escherich'a coli 2017, which is a Represents a value of 7 LD ₅ o in bacterial pathogens. The therapeutic agent was the length is gebenn dosages erabreicht in a 0.5 ml volume by the oral route in each case%.

dose % Survivors of the - 3-FDA group iuse not infected 100 infected M; Control animals - 3 FD, mg 3-FLA 100 3-FLA 0 0 100 100 0 0 0.078 - 100 0 20th 0.312 100 100 0 100 1,250 100 100 0 80 5 0 0 - 20th - 40

Below the maximum tolerated dose, 3-FLA has no therapeutic effect. While moderate amounts of 3-FDA provide complete protection, higher values in vivo, as in the in vitro study, show signs of autoantagonism.

Influence of the concentration gradient

of 3-FDA and 3-FLA on bacterial growth

(Explanation of the autoantagonism)

α 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 containing the specified amounts of 3-FDA or

4j wore 3-FLA. After incubation at 37 löstündiger ^J C the free bacterial growth circular zones surrounding the discs were measured. Only in the case of 3-FDA, and there only with higher drug values, was an on the paper disk

3d adherent growth ring observed to which a Connected ring made of bacteria-free agar. The inner growth zone, right in the high concentration area of the concentration gradient formed by the diffusing drug is called the zone

v> of autoantagonism.

Amount on the diameter of the zones around the discs. of the disc mm

3-FLA

Inhi- Autoanta-

bation gonism

3-FDA

inhibition

Autcantagonism

The tolerance of mice to 3-FDA exceeds that to 3FLA by a factor of at least Ϊ0.

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Claims (3)

Patent claims: 1. 3-Fluoro-D-alanine and its salts. 2. 3-Fluoro-D-alanine-2-d and its salts. 3. Medicines, go-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, nor has the investigated antibacterial effectiveness of the racemate. 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 optical isomers is comparable in vitro. However, the isomers differ significantly 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. Lie in vitro antibacterial testing is characterized by the unusual autoantagonistic property of the 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, as 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 analogs 3-fluoro-D-alanine-2-d are markedly less pronounced compared to attack by D-amino acid oxidase is sensitive, but retains the antibacterial effectiveness of 3-fluoro-D-alanine. 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 as well their antibiotic resistant strains. Examples of 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 used by the above organisms > n humans and animals, and can do this in a variety of therapeutic ways Dosages in conventional carriers for oral use, for example in the form of a capsule or tablet or in a liquid solution or suspension : ϊ be administered. Suitable preparations can Common diluents, granulating agents, preservatives, Contain binders, flavorings and coating agents. The dose of the products can be vary over a wide range; for example jn are for the adult of 70 kg Dosages ranging from about 250 mg to about 500 mg of the active ingredient two to four times daily. A growing animal with feed should be given about 100 g per r. 907 kg of complete feed received. The compounds according to the invention can also be administered parenterally by injection in a sterile pharmaceutical carrier.
The fluorinated alanine derivatives of the invention 4π are as described in DE-AS 2136 008, manufactured. D-alanine or the correspondingly deuterated D-alanine is used as a substrate Fluoroxytrifluoromethane under the influence of light, including ultraviolet light, ionizing radiation, · »<Such as Λ-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 ii 'acid, trifluoroacetic acid or sulfuric acid, dissolved the 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 Vi and the irradiation are continued until the end of the Reaction continued. Because of the low boiling point of the reagents, it is useful to ^{carry out the reaction at temperatures of only -80 0} C, the reaction at atmo- as spherical pressure advances. 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 (• ■ Ι can be carried out in a pressure vessel or a steel bomb with a platinum lining, in which case higher temperatures, for example up to about 100 ^° C., can be used