DE2435619C3 - - Google Patents

Description

in which R ¹ can be a:

Glycine, D-alanine, J? -Alanine,
La-aminobutyric acid,

α-Aminoisobutyric acid, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine,
D-phenylglycine, L-phenylglycine,
D-phenylalanine, L-phenylalanine, L-tyrosine, O-benzyl-L-serine, S-benzyl-L-cysteine,
Sarcosine, LN-methylalanine or
N-phenylglycine resL

2. Tripeptides of the general formula (2) derived from tetaine

CH ₃

R ² -NH-CH C -NH-CH-COOH (2)

in which R ² can be a:

Glycine-, D-alanine-, L-alanine-.jS-alanine-,
L-ix-aminobutyric acid,
α-Aminoisobutyric acid, L-valine, L-norvaline, L-leucine, L-isoleucine, L-norleucine,
D-phenylglycine, L-phenylglycine,
D-phenylalanine, L-phenylalanine, L-tyrosine,
O-Benzyl-L-serine-, S-Benzyl-L-cysteine-,
Sarcosine, LN-methylalanine or
N-phenylglycine residue.

3. Process for the preparation of the compounds according to Claims 1 and 2, characterized in that that the amino group of desalanyl tetain or tetain in an aqueous organic Solvent at temperatures in the range of up to 28 ° G and at a pH ^ value between 7 and 9 with N'Carboxyanhydrides of the following * called aminocarboxylic acids or with hydrochlorides of acid chlorides mentioned below th aminocarboxylic acids acylated:

Glycine, D-alanine, L-aianiri, jS-alanine,
La-Aminobultersäure.a-Aminöisobutyric acid, The invention relates to dipeptides of the general formula (1) derived from desalanyl tetaine and prepared by acylation of desalanyl tetaine or tetaine with various aminocarboxylic acids,

R ¹ -NH-CH-COOH

CH,

tripeptides of the general formula (2) derived from tetaine,

R ² -NH-CH-C-NH -CH-COOH (2)

Il I

O CH,

and processes for their manufacture.
The antibiotic Tetain Krynski S., Borow-

4, ski E. et al. Bull.State Inst. Mar.Trop.Med., Gdansk, 3. 301, 1952. B ο r ο w s k i E., Przemys 1 Chemiczny 9, 503, 1953; Chem. Abstr. 51, 3092 h, 1957) is produced by the strain # -Bacillus pumilus and has a whole Has a number of interesting biological properties.

Above all, it is characterized by a very high antibacterial activity, its range of activity including numerous - both gram-positive and gram-negative as well as many other microorganisms.
A particularly valuable property of Tatein is its low toxicity for the animal organism. This is based on a selective mechanism of action that inhibits the synthesis of murein for the construction of the cell wall of microorganisms. Namely, the incorporation of L-alanine into a derivative

^ O of N-acetylmyrarnic acid. that is a forerunner of the Mureins is inhibited (Chmara H., Borowski E., Biochem. Biophys, Res. Cönimun., 52, 1381, 1973 and 55, 1147, 1973).

Investigations have shown that tetaine is a mixture of two isomeric compounds, the tetaine A and of tetain B, is. The two forms of tetain have similar biological properties and are enzymatically split into two amino acids,

c λ r \

XJ I Zl

namely in L-alanine and a desalanyl tetain called Fragment (Bo ro wski IL, Wojciechowska K., Roczniki Chemii 40, 1195, 1966, see also Chem. Abstr. 65, 20422 b, 1966).

Structural studies have shown that Tetain A is L-A) anyI- (2 ^ -epoxycyclohexanone-4) -L-alanine with the formula 3

CH ₃
H, N-CH-C-NH-CH-COOH (3)

Il I

O CH,

and that desalanyl tetain A is thus the (23-ox > c> clohexanon-4) -L-alanine with the formula 4

H, N-CH-COOH
CH ₂

is.

The exact structure of desalanyl tetain B has not yet been clarified, but it has been proven that it has a carbon structure is identical to that of desalanylteiain A and has the same functional groups

The two forms of tatain or desalanyl tetain can be obtained using an anion exchanger in formate form can be isolated in crude form using 70% methyl alcohol, wherein first the Α forms are eluted. Both fractions obtained are then twice on Sephadex® purified, first using a solution of magnesium chloride and the second time using water as the eluant to serve.

For the therapeutic use of tetaine it has proven to be a hindrance that its solutions do not are sufficiently stable that it is not easy to store and quickly loses activity

The invention is based on the object of compounds derived from desalanyl tetaine or tetaine indicate that with the same or improved biological activity compared to tetaine by a higher stability of their aqueous solutions and thus are characterized by better use properties.

This task is accomplished by means of acylation of desalanyl tetaine or tetaine with various, from the Claims apparent aminocarboxylic acid residues her ^ provided peptides with the general formulas (1) and (2) solved.

For the preparation of these new peptides a method was used which, due to the presence of the Λ, / 9-epoxycarbonyl grouping specially developed who ^ which, however, takes place using reaction times known per se

Tetaine (A and / or B) or desalanyl tetaine (A and / or B) are in an aqueous organic Solvent with N-carboxyanhydrides of aminocarboxylic acids or with hydrochlorides of Acid chlorides of aminocarboxylic acids at temperatures in the range of 0 ° -28 ° C at a pH of 7 to 9 acylated to the peptides according to the invention

Studies on the antibacterial effectiveness of the peptides according to the invention showed that aiese ίο the same effectiveness as tetain or even slightly improved effectiveness compared to tetain have, but are characterized by a considerably increased stability of their aqueous solutions, which for use in clinical practice is a considerable advantage over tetaine means

In order to explain the invention, reaction examples for the preparation of the invention are given below Peptides, a tabular listing of the peptides obtained and the results of the pharmacological and stability studies guided

Example I.

15 mg desalanyl tetaine in a lyophilized powder form are suspended in 1 ml of dry dimethylformamide. After stirring, 15 mg of D (-) - phenylglycyl chloride hydrochloride are added (D - (-) - a-aminophenyl acetic acid chloride hydrochloride) added After the reagents have been stirred for 1 hour, 5 ml of ether are added and the precipitate which has separated out is filtered off The main product obtained is D-phenylglycyl-desalanyltetaine. The mass spectrum shows a mass ion at 332, elemental analysis gave one N content of 8.21% (calculated: 8.429%).

Example II

15 mg desalanyl tetain A suspended in 0.1 ml Dimethylformamide are mixed with 10 mg of D - (-) - phenylclycyl-N-carboxyanhydride implemented. The pH is brought to approx. 9 with N-methylmorpholine. Stirring, the solvent is evaporated off in vacuo. The main product obtained is D-phenylglycyldesalanyltetaine, which showed a mass ion at 332 in the mass spectrum and had an N content of 8.25% (calc. 8.4290 / 0).

Example III

A solution of 1.3 mg glycyl-N-carboxyanhydride in 0.5 ml tetrahydrofuran is added to 3.7 mg desalanyl tetaine in 0.5 ml water. After the reaction for 20 hours at 0 ^° C., the mixture is evaporated off in vacuo. The main product obtained is crude glycyl desalanyl tetaine. which showed a mass ion at 256 and had an N content of 10.85% (calc. 10.933%).

Example IV

A solution of 2.4 mg of L-phenylalanine-N-carboxyanhydride is added to 3.7 mg of desalanyl tetaine in 0.5 ml of water in 0.5 ml of dimethylformamide. After a reaction time of 20 hours at 0 ° C, the Mixture evaporated in vacuo. The main product obtained is L-Phenylalanyldesalanyltetain, which is a Showed mass ion at 346 and had an N content of 8.01% (calc. 8.088%).

CAn

U 1 Z7

Example V

To 37 mg desalanyl tetaine in 5 ml Michaelis buffer / pH 8.2 / a solution of 21.2 mg of D-alanine-N-carboxyanhydride in 5 ml is added at 0 ° C. with vigorous stirring Tetrahydrofuran. After a reaction time of 20 hours at 0 ° C, the mixture is in vacuo evaporated and the resulting crude D-Alanyldesalanyltetain w ^ d on a silica gel chromatographic table 1

Column with chloroform / methanol / water 16: 4: 0.5 purified yield 68%.

The mass spectrum showed a mass ion at 270, the elemental analysis showed an N content of 10.25% (calc. 10.366%).

In addition, following the procedures given in Examples I to V, the following in Table 1 were obtained specified dipeptides of the general formula 1 derived from desalanyl tetaine:

R '

1. jS-alanyl,

2. L-oc-aminobutyryl,

3. a-aminoisobutyryl,

4. L-VaIyI-.

5. L-norvalyl,

6. L-leucyl,

7. L-isoleucyl-,

8. L-norleucyl-,

9. L-phenylglycyl-,

10. D-phenylalanyl,

11. L-tyrosyl,

12. O-Benzyl-L-seryl-,

13. S-Benzyl-L-cysteinyl-,

14. Sarcosyl,

15. L-N-methylalanyl,

16. N-phenylglycyl-,

Production according to ex.

Mass spectrum

III
IV
III
III
III
IV
IV
IV
IV
IV
III
IV
IV
III
III
IV

270
284
284
2S8
298
312
312
312
332
346
362
372
392
270
284
332

N analysis (%)
ber.

10366 10.15 9,854 9.72 9,854 9.67 939 9.19 939 9.25 8369 BS2 8,969 8.85 8,969 8.71 8,429 821 8,088 8.05 7,731 7.62 7,443 7.25 7.138 7.02 10366 10.31 9,854 9.76 8,429 8.27

Example VI

Add to 10 mg Tetain B lyophilisate in 1 ml water a solution of 3 mg of L-alanine-N-carboxyanhydride in 1 ml of tetrahydrofuran. After 20 hours Reaction at 0 ° C, the mixture is evaporated in vacuo. The main product obtained is L-Alanyltetain B with a mass ion at 341 and an N content of 12.10% (calc. 12312%).

Example VII

To 10 mg of tetaine A lyophiluate in 3 ml of dimethylformamide a solution of D-phenylglycyl-N-carboxyanhydride is added (D-a-aminophenylacetic acid-N-carboxyanhydride) in 1 ml of dimethylformamide. After a reaction time of 20 hours at 0 ° C, the Mixture evaporated in vacuo. The main product obtained is D-phenylglycyltetaine A with a bulk ion at 405 and an N content of 10.22% (calc. 10.417%).

Example VIII

-to To lOmgTetain-Lyophüisal in 1 ml Michaelis buffer / pH 8.2 / a solution of 4.8 mg of L-phenylalanine-N-carboxyanhydride is added in 1 ml of tetrahydrofuran. After a reaction time of 20 hours at 0 ° C, the Mixture evaporated in vacuo. As the main product, L-phenylalanyl tetaine is obtained with a bulk ion at 417 and an N content of 10.01% (calc. 10.067%). According to those given in Examples VI to VIII Procedures were also given in Table 2 as follows, derived from tetaine Tripeptides of the general formula 2 prepared.

Table 2 Manufacturing Crowds N analysis (%) found R2 according to ex. spectrum ber. 12.81 VI 327 12-38 1230 1. Glycyl, VII 341 12312 1230 2. D-alanyl, VIII 312 12312 11.69 3. 0-alanyl, viii 355 11,825 II.82 4. L-a-aminobutyryl, VIII 355 11,825 11.25 5. a-'AminoisobutyryU, VIII 369 11376 11.19 6. L-VaIyU, VIII 369 11376 10.76 1. L-norvalyl, VIII 383 1036 10.70 8. L-leucyl, Viii 383 1036 10.69 9. L-isoleucyl, VIII 383 1036 1038 10. L-Norleueyl-, VI 403 10,417 ti. L-phenylglycyl-,

continuation

Production according to ex.

Mass Spectrum

N analysis (%) bef. found

12. D-phenylalanyl, VI 417 10,067 9.98 13. L-tyrosyl, VIII 433 9,695 9.59 14. O-BenzyNL-seryl-, VII 431 9.74 9.49 15. S-BenzyUL-cysteinyl-, VH 463 9,066 8.98 16. Sarcosyl, VIII 341 12,312 12.12 17. L-N-methylalanyl, VIII 355 11,825 11.62 18. N-phenylglycyl-, VII 417 10,417 10.23

The compounds obtained all had bands in their IR spectra at wavenumbers of 3300 (breil),

1A7C 7/1

1390 cm ' ¹ on. The argentometric titration proved the presence of an atom of oxirane oxygen in the molecule, ie the presence of the epoxy group, for all compounds, the purity of the compounds obtained being confirmed at the same time. The antibacterial activity of the compounds according to the invention was determined against the model microorganisms Candida albicans, Staphylococcus aureus, Shigella shigae and Escherichia coli using the standard Zy-Iinder method. In the experiments given below, series of differently diluted solutions of the stated compounds according to the invention and Shigella shigae as the test microorganism were used.

The test microorganism was routinely grown on Bacto Antibiotic Medium 3, and that Cylinder procedure was performed on Bacto Antibiotic Medium 1.

For tetaine and 3 representative compounds, the following values were obtained, which represent the concentrations represent the rriit one under standard conditions to create a growth inhibition zone 21 mm diameter are required.

Tested connection Concentration mcM / ml, soften the increment inhibition zone of 21 mm causes Tetaine 0.004 Phenylalanyldesalanyl tetaine 0.003 Alanyl tetaine 0.005 Phenylalanyl tetaine 0.005

With the help of a peptide permease is able to penetrate the cell wall of a bacterium. In the bacterial cell

The table shows that all of the above Compounds have about the same antibacterial effectiveness. This statement generally applies to the entire group of compounds according to the invention.

Detailed studies on the mechanism of action of tetain and desalanyl tetain derived peptides provided an explanation for this observed relative insensitivity of the antibacterial Effectiveness against changes in the amino acids or peptide residues bound to the desalanyl tetain. It turned out that the actual antibacterial effectiveness was due to the desalanyl tetam part of the molecule is due, while the residue bound to it causes the molecule under

erfr »liTl Aann Alt» nrfywron I icflio Cnallii

T r \ f> G Pf »ntMmrvlp.

küls, and the released desalanyl tetain can be used Develop antibacterial effectiveness. Since the di- and tripeptide carriers in the cell wall and the cytoplasmic Peptidases in the cell are enzymes with relatively low substance specificity, thus influenced a change in the amino acid or dipeptide residues bound to the desalanyl tetain is antibacterial Effectiveness of the desalanyltetainderival very little.

This fact allows it, while maintaining the antibacterial effectiveness more stable and therefore for the clinical practice of specifying more valuable etain-like compounds.

The stability of the compounds according to the invention was determined by using 1 percent as test solutions Solutions of the compounds to be tested in the incubator in the dark at opposite clinical practice under severe conditions at a temperature of 36 ° C. After two weeks of storage the activities of the samples were determined using paper chromatography and after the Cylinder method determined.

Paper chromatography was performed with two different solvent systems. In fact once with a solvent mixture t-ButanoI to formic acid to water = 69.5: 1: 29.5, as well as with n-butanol to acetic acid to water = 4: 1: 5. In some Experiments were also carried out using a mixed solvent n-butanol to pyridine to acetic acid to water = 6: 2: 3: 3 used.

The chromatograms were developed with ninhydrin, the activity of the spots was determined according to the standard method using also Shigella shigae. In the case of quantitative evaluations, the areas of the active spots of the stored samples were related to the areas of the active spots of a corresponding fresh sample. The primary purpose of the chromatographic investigation was to determine the number of degradation products.It was found that the compounds according to the invention are significantly more stable than tetaine, and the quantitative evaluation of the chromatograms provided results which corresponded to the more precise results of the cylinder test given below with sufficient accuracy.
After the cylinder test, in which the diameter of the growth inner zones were used to determine the concentrations of the active compounds in the solutions, the following results were obtained:

ίο

Compound tested% of initial stability activity after 2 weeks

Tetain 30.1.0

Phenylalanyldesal- 90 3.0

anyltetain

Alanyl tetaine 45 1.5

Phenylalanyl tetaine 60 2.0

These results show that the stability of the antibacterial efficacy is approximately identical aqueous solutions of the compound according to the invention means that they are for clinical use dung compared to tetaine is considerably increased, which are significantly more suitable than tetaine.

Claims (1)

Claims; 1, Dipeptides derived from desalanyl tetaine cjer general formula (1) L-valine, L-norvaline, L-leucine, L-isoleucine,
L-norleucine, D-phenylglycine, L-phenylglycine,
D-phenylalanine, L-phenylalanine, L-tyrosine,
O-benzyl-L-serine, S-benzyl-L-cysteine, sarcosine, LN-methylalanine or N-phenylglycine. R ¹ -NH-CH-CC) OH CH, (1) IO