MXPA98008484A - New lantibiotic related with actagardine, procedure for its preparation and employment of the mi - Google Patents

Abstract

The present invention relates to a new lantibiotic with the formula NH2-R-actagardine, NH2-R meaning the remainder of the amino acid alanine, which is formed during fermentation by the microorganism Actinoplanes liguriae, DSM 11797 or Garbadian acinoplanes DSM 11796, to chemical derivatives of the lantibiotic, to a procedure for its preparation and to the use of lantibiotics as medicamen

Description

New lantibiotic related to actagardine, procedure for its preparation and use of it The present invention relates to a new lantibiotic related to actagardine, in particular a lantibiotic with the designation Ala ° -actagardine, to a process for its preparation, to chemical derivatives derived from the lantibiotic and to the use of lantibiotics as medicaments. A large number of lantibiotics have already been described. The lantibiotics are polycyclic peptide antibiotics that, as a characteristic feature, contain the amino acid lanthionine or methyl-alanthionine. They are microbially obtained natural substances which, as antibacterial active substances, find application in human therapy, as preservatives or as inhibitors of enzymes (G. Jung, Angew, Chem. Int. Ed. Engl., 1991, 30 , 1051-1068).

A large number of antibiotics are used therapeutically for the treatment of bacterial infectious diseases. However, pathogens become increasingly resistant to the drugs used, and there is even a great risk of so-called multiresistant germs that have not only become resistant against groups of individual antibiotics such as, for example, antibiotics of / 3- lactam or glycopeptics or macrolides, but at the same time carry several resistances. There are even pathogens that have become resistant to all commercially available antibiotics. Infectious diseases that are caused by these types of germs can no longer be treated therapeutically. Therefore, there is a great demand for new agents that can be used against resistant germs. Certainly, many thousands of antibiotics are described in the literature, most of them are too toxic to be used as medicines. Actagardine is a lantibiotic that has been described for the first time in 1977 by S. Somma et al. in Antimicrob. Agents Chemother. 11, 396-401. Its structure has been correctly clarified only recently (N. Zimmermann et al., Eur. J. Biochem, 1995, 228, 786-797). Surprisingly, it has been found that the strains Acti-nolannes liguriae and Actinoplanes garbadiensis are able to form in each case at least one new antibiotic, for example Ala0-actagardine, which is not only very effective antibacterially, but also well compatible. A material isolated from Actinoplanes liguriae was deposited at the Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg IB, 30124 Braunschweig, Germany (hereinafter "DSM"), according to the Budapest Treaty Rules on 24.09.97 under the following number: DSM 11797. An isolated material of Actino-plans garbadiensis was deposited in the DSM according to the Budapest Treaty Rules of 24.09.97 under the following number: DSM 11796. Correspondingly, the invention represents compounds of the formula I, R - Ala1 - Ser2 - Gly3 - Trp4 - Val5 - Ala6 - Abu7 - Leu8 - Abu9 lie 10 Glu1 1 - Ala 12 HOOC - Ala19 - Ala18 - Ala17 - He16 - Val15 wherein R means the remainder of an amino acid, as well as its physiologically compatible salts. R may be the remainder of a substituted or unsubstituted amino acid, wherein the amino group is in position or; to ? and has the D or L configuration. Particularly preferred are substituted or unsubstituted amino acids with the D or L configuration. Preferably, R means the remainder of a natural amino acid chosen from: Ala, Gly, Glu, Phe, Pro, Thr, Cys, Met, Trp, Tyr, Asn, Gln, Asp, His, Lie, Leu, Lys, Arg, Ser and Val. Particularly preferably, the amino acid means Ala, Lie, Lys, Phe, Val, Glu, Asp, His, Leu, Arg or Ser, and very particularly preferably, the amino acid means Ala0. R may also mean a substituted or unsubstituted diamino-alkyl acid residue such as, for example, 2,4-diaminobutyric acid (Dab). further, the invention relates to a compound of the empirical formula: C84H12: 3N2: L02SS4 (Ala ° -actagardine), obtainable by fermentation of Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796 or one of its variants and / or mutants, in a culture medium until the Ala ° -acctagardine compound accumulates in the culture broth and by subsequent isolation of the compound, as well as its pharmacologically compatible salts. The invention also relates to chemical derivatives that are derived from a compound of the empirical formula C84H12S) N:;? 02SS4 (Ala ° -actagardine), obtale by fermentation of Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796 or one of its variants and / or mutants, in a culture medium until the compound Ala ° -actagardine accumulates in the culture broth and by subsequent isolation of the compound and transformation into chemical derivatives, as well as its pharmacologically compatible salts. Preferred chemical derivatives are: lie0-, Lys0-, Phe0-, Val0-, Glu0-, Asp0-, His0-, Leu0-, Arg0- and Ser ° -actagardine. The transformation of Ala-actagardine into the chemical derivatives mentioned can be carried out according to methods known to those skilled in the art.

The antibiotic Ala ° -actagardine differs from substances known in the literature by the structural formulas indicated. Some secondary components of actagar-dine have been described with actagardine (US patent 4,022,884 of May 10, 1976, as well as A. Malabarba et al., J. Antibiotics, 1985, 38, 1506 -1511), which, however, differ from the compound according to the invention by the polarity also in relation to the actagardine or by the amino acid composition or by the antimicrobial activity or by other physical properties. The strain Actinoplanes liguriae, DSM 11797 forms the actagardine as well as the by-products known from the literature in nutritional solutions containing glu- cose, starch or glycerol. Surprisingly, it has been found that the organism itself produces, in poorly digestible and hand-containing media, with very good yields the antibiotic NH2-R-actagardine according to the invention, where R represents the remainder of a natural amino acid, in particular alanine. , but not the compounds described, even actagardine only in trace amounts. In addition, the invention therefore relates to a process for the preparation of the compound of the formula I, characterized in that the microorganism Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis, DSM 11796 or one of its variants or mutants is cultured in a aqueous nutrient medium, a compound of formula I is isolated and purified and, if necessary, transformed into its pharmacologically compatible salts. Said method covers the cultivation of Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796, of their mutants and / or variants under aerobic conditions in culture media containing carbon and nitrogen sources, inorganic salts and trace elements. Preferably, the cultivation is carried out at a temperature between 20 and 35 ° C and at a pH between 4 and 10.

Furthermore, the invention relates to a process for the preparation of a compound of the formula I, characterized in that the actagardine compound is reacted with an amino acid. For example, an activated amino acid ester can be reacted with the terminal amino group of actagardine. A protective group, such as, for example, tert-butyl-xi-carbonyl (Boc-), is preferably attached to the amino nitrogen of the amino acid, in order to avoid reactions of the activated amino acid esters with themselves. Activated esters are, for example, the N-hydroxysuccinimide esters of the respective amino acids. The protecting group is separated and the reaction mixture is then purified.

Actinoplanes has a mycelium of orange substrate and no aerial mycelium. It forms the sporangia characteristic of Actinoplanes. The cell wall contains meso-diaminopimellic acid as well as glycine as characteristic amino acids and xylose as well as arabinose as sugars, which are characteristic traits for the genus Actinoplanes. Instead of the strain DSM 11797 or 11796, its mutants and variants can also be used, provided that they synthesize the compounds according to the invention. Mutants of this type can be produced in a manner known per se by physical means, for example irradiation such as with ultraviolet-ray or X-rays, or chemical mutagenesis such as, for example, ethylmethylanesulfonate (EMS); 2-hydroxy-4-methoxy-benzophenone (MOB) or N-methyl-N '-nitro-N-nitrosoguanidine (MNNG). The screening of mutants and variants producing the antibiotic according to the invention can be carried out by determining the biological activity of the active substance accumulated in the culture broth, for example by testing the antibacterial activity. As the preferred carbon source for aerobic fermentation, carbohydrates and sugar alcohols which are assimilable but difficult to digest are suitable, such as mannitol, isonisite, as well as natural products with carbohydrate content such as, for example, soy flour. Suitable nitrogenous substances are amino acids, peptides and proteins as well as their degradation products such as peptones or tryptons and, in addition, meat extracts, ground seeds, for example maize, wheat, beans, oats, soybeans or soybeans. cotton plant, residues from the distillation of the preparation of alcohols, meat meal or yeast extracts, but also ammonium and nitrate salts. The nutrient solution may contain in inorganic salts, for example, chlorides, carbonates, sulfates or phosphates of the alkali or alkaline earth metals, iron, zinc, cobalt and manganese. The formation of Alactactadina runs particularly well, for example, in a nutrient solution containing approximately 0.5 to 5% mannitol, preferably 1 to 3%, 0.5 to 5% soybean meal, preferably 1 to 3% and a solution of trace elements in a concentration of 0.1 to 0.5%, preferably 0.2 to 0.3%. The trace element solution contains CaCl2, Fe III citrate, MnS04, ZnCl2, CuS04, sodium tetraborate, CoCl2, and sodium molybdate. The cultivation is carried out under aerobic conditions, that is to say, for example, submerged with shaking or stirring in shaker flasks or fermentors, possibly with introduction of air or oxygen. The fermentation can be carried out, for example, in erect body bottles or round flasks of different volumes, in glass fermenters or V2A steel tanks. It can be carried out in a temperature range of about 20 to 35 ° C, preferably at about 25 to 30 ° C. The pH value should be between 4 and 10, advantageously between 5.5 and 8.5. The microorganism is grown under these conditions in general over a period of time of 20 to 300 hours, preferably 24 to 140 hours. Advantageously, the cultivation is carried out in several stages, that is to say first one or several previous cultures are prepared in a liquid nutrient medium which is then superinoculated in the production medium itself, the main culture, for example in the volume ratio 1:10 . The prior cultivation is obtained, for example, by superimposing a mycelium in a nutrient solution and allowing it to grow for approximately 20 to 120 hours, preferably 24 to 72 hours. The mycelium can be obtained, for example, by allowing the strain to grow for about 1 to 40 days, preferably 3 to 10 days in a solid or liquid nutrient medium, for example yeast-malt agar or oat flake agar. The course of the fermentation and the formation of the antibiotic according to the invention can be monitored in a manner corresponding to methods known to the person skilled in the art such as, for example, by assaying the biological activity in bioassays or by chromatographic methods such as chromatography. in thin layer (CD) or high performance liquid chromatography (HPLC). The antibiotic Ala ° -actagardine can occur both in the mycelium and in the culture filtrate, usually the main amount is found in the culture filtrate. Therefore, it is convenient to separate the fermentation solution by filtration or centrifugation. The filtrate is extracted as a solid phase with an adsorption resin. The mycelium is conveniently extracted with methanol or acetone, but other solvents can also be used. The extractions can be carried out over a wide range of pH's, however it is convenient to work in a neutral or weakly acidic medium, preferably between pH 3 and pH 7. The extracts can be concentrated, for example under vacuum, and dried. A method for the isolation of the antibiotic according to the invention is the distribution in solution in a manner known per se. Another method of purification is chromatography on adsorption resins such as, for example, in Diaion® HP-20 (Mitsubishi Casei Corp., Tokyo), in Amberlite® AD 7 (Rohm and Haas, USA), in Amberchrom ® CG, (Toso Haas, Fila-delfia, USA) or similar. In addition, numerous reverse phase supports, for example RP8 and RP18, are suitable as are generally known, for example, in the context of high pressure liquid chromatography (HPLC). Another possibility of purification for the antibiotic according to the invention consists in the use, in a manner known per se, of so-called normal phase chromatography supports such as, for example, silica gel or A1203 or others. An alternative isolation method is the use, in a manner known per se, of molecular sieves such as, for example, Fractogel® TSK HW-40, Sephadex® G-25 and others. In addition, it is also possible to obtain Alau-actagardine by crystallization from enriched material. Suitable for this purpose are, for example, organic solvents and their mixtures, free from water or with the addition of water. An additional process for the isolation and purification of the antibiotics according to the invention consists in the use of anion exchangers, preferably in the range of pH's from 4 to 10 and cation exchangers, preferably in the range of pH 's from 2 to 5. The use of buffer solutions to which portions of organic solvents have been added is particularly suitable for this purpose. Alactactadine, the chemical derivatives thereof mentioned as well as the obvious chemical equivalents thereof can be transformed, according to methods known to the person skilled in the art, into the corresponding pharmacologically compatible salts. Obvious chemical equivalents of the compounds according to the invention are compounds that have a low chemical difference, ie they have the same activity or are transformed under mild conditions in the compounds according to the invention. The mentioned equivalents include, for example, esters, amino derivatives, complexes or adducts of the or with the compounds according to the invention. Pharmacologically compatible salts of the compounds according to the invention are understood to mean both inorganic and organic salts as described in Remingtons Pharmaceutical Sciences (17th edition, page 1418 (1985)). Suitable salts include, in particular, alkali metal, ammonium, alkaline earth metal salts, salts with physiologically compatible amines and salts with inorganic or organic acids, such as, for example, HCl, HBr, H2SO4, maleic acid, fumaric acid. The physical-chemical as well as the spectroscopic properties of the antibiotics according to the invention can be summarized as follows: Ala ° -actagardine: Appearance: colorless substance soluble in methanol and water. Stable in a neutral and mildly alkaline medium, but unstable in strongly acidic and strongly alkaline solution. Empirical formula: C84H129N21025S4 Molecular weight: 1961.21 1H- and 13C-NMR: see Tables 1 and 2. UV maxima (log ¿): 280 nm (3.71), 288 nm (shoulder) Table 1: Spectroscopic data by ^ -RMN of Ala ° -actagardi-nas AminoHN Ha H / 3 H? Other acid Ala0 8,010 2,891 1 383 Ala1 8, 564 4, 727 3 397 2 603 Ser2 8.264 4, 350 3 651 OH: 5.102 Gly3 8,595 3,968 - 3,263 Trp4 8, 174 4,475 3, 311 H5: 7, 144 H6: 7, 554 H7: 6,983, H8: 7, 061, H9: 7,333, Indole: 10, 740 2. 978 Val5 7,454 4, 558 2, 048 0, 909 0, 856 Ala6 8,487 4, 704 2,578 2,960 Abu7 8,324 4, 557 3,596 1, 176 Leu8 7,636 4, 626 1,418 1,482 d 0.841 1,482? -Me: 0,860 Abu3 7, 604 4, 747 3 , 570 1, 207 He10 8, 378 3, 763 1, 605 1,063 d - faith: 0.860 i-3-Me: 0, 879 1, 642 Glu11 8,262 3, 690 2, 178 2, 319 Ala12 7,325 4,553 559 866 Gly13 8,140 3, 538 4, 167 Abu14 7,860 4, 381 336 1, 059 Val15 7, 778 4, 104 042 0, 875 d - Vfe: 0.833 Table 2: Spectrscopic data for 13 C-NMR of Ala ° -actagardi-nas AminoCO Ca Cß C? Other acids Ala0 169.51 48, 35 17.35 Ala1 169.27 50, -80 34.21 Ser2 170.48 55, -04 61.23 Gly3 168, 90 43,, 44 T Trrpp44 5 544 ,, -2 288 27, 62 110, 41, 123.28, 127, 08, 111.28, 120, 83, 136.00, 118, 10, 118.22 Waltz 171, 20 56, 73 31, 50 17,, 76 19, 02 Ala6 170, 14 53, 47 32, 79 Abu7 57, 83 43, 95 19,, 96 Leua 171, 45 51,, 01 41, 66 24 ,, 03 22.38 22, 62 Abu9 171, -59 55, 77 46, 38 20,, 10 He10 170, 96 60, 00 35, 71 24,, 75 11.59 ß- -Me:: 14, 70 Glu11 55, -86 24,, 49 30,, 98 173,75 Ala12 170, 78 55, 47 35, 26 Gly13 170, 03 44,, 18 Abu14 168, 33 55, -00 55,, 64 6,, 94 Val15 170,, 63 60,, 03 30,, 02 19,, 15 18,, 564 He16 170,, 66 59,, 64 35,, 69 24,, 55 10, 62 ß -Me:: 15, 53 Ala17 169,, 79 53,, 13 35,, 70 Ala18 171,, 52 48,, 89 15,, 34 Ala19 47,, 16 51,, 53 not assigned, 01, 169, -83 170,, 33 171,, 01 The amino acid analysis provides, in addition to the amino acids of actagardine [ 1 Ser, 2 Gly, 1 Trp, 2 Val, 1 Leu, 2 He, 1 Glu, 1 Ala, 1 lanthionine (Ala-S-Ala) and 3- / 3-methyl-lanthionine (Abu-S-Ala)] , another wing. In addition, it was found that the compound according to the invention exhibits strong antibacterial activities, Table 3 summarizes by way of example the minimum inhibitory concentrations (MICs) of Ala ° -actagardines.

Table 3: In vitro activity of Ala0-actagardine against Gram-positive and anaerobic bacteria in the serial dilution test GERMEN CIM values of Ala0 -ac- tagardine (μg / ml) Staph. aureus SG 511 6, 25 Staph. aureus 285 6.25 Staph. aureus 503 3, 13 Staph. aureus FH 1982 12.5 Staph. aureus 701 E 12.5 Staph. aureus 707 E 12.5 Staph. aureus 9 Tüb. 6, 25 Staph. aureus 8236 6.25 S. epidermidis ZH2c 6.25 S. epidermidis 6098W 12.5 S. epidermidis 763 6.25 S. epidermidis 5747II 6.25 S. epidermidis 291 12.5 S. epidermidis 799 6.25 E. faecium Md8B 6, 25 E. faecium VR1 50 E. faecium VR2 50 S. pyogenes VR3 25 S. pyogenes 308A 6.25 S-pyogenes 77A 0. 195 Propionib. acnes 6919 1.0 Propionib. acnes 6922 1.0 Clostrid. tetani 9406 8, 0 Clostrid. perfringens 194 0.5 It is particularly noteworthy that the compound according to the invention not only exhibits approximately twice as good anti-bacterial activities against Gram-positive germs as actagardine, but, at the same time, does not present any kind of cross-resistance with customary antibiotics such as, for example, β-lactams (penicillins, cephalosporins), aminoglycosides (streptomycin), macrolides (erythromycin), quinolones (ciprofloxacin), sulfonamides or glycopeptides (vancomycin) and others. In addition, it should be noted the strong inhibitory effect on anaerobic organisms that can cause pertinacious infectious diseases, which even endanger life. For the therapy of diseases of this type the Ala-actagardine in particular is suitable. The compatibility of Ala0-actagardine is good in the effective concentration and above it. No cytotoxic effects or other toxicities were observed. Accordingly, the present invention also relates to the use of the compounds according to the invention as medicaments., as well as the use of the compounds in question for the preparation of medicaments for the treatment and / or prophylaxis of bacterial infections. In addition, the present invention relates to medicaments with a content in the compound according to the invention. Said medicament is prepared by mixing at least one compound of the formula I with a coadjuvant and / or physiological support substance and is brought into a suitable administration form. The medicaments according to the invention can be applied enterally (orally), parenterally (intramuscularly or intravenously), rectally or locally (topically). They can be administered in the form of solutions, powders (tablets, capsules, including microcapsules), ointments (creams or gel) or suppositories. Suitable auxiliary substances for formulations of this type are pharmaceutically customary liquid or solid fillers and spreading agents, solvents, emulsifiers, lubricants, flavorings, dyes and / or buffer substances. As a convenient dosage, 0.1-1000, preferably 0.2-100 mg / kg of body weight are administered. The medicaments are conveniently administered in dosage units containing at least the effective daily amount of the compounds according to the invention, for example 30-3000, preferably 50-1000 mg. The present invention has to be explained in more detail by the following exemplary embodiments as well as by the content of the claims.

Example 1: Preparation of a mycelial suspension of the producing strain 100 ml of nutrient solution (10 g of starch, 10 g of glycerol, 10 g of glucose, 2.5 g of maceration liquid of corn, 5 g of peptone and 2 g of yeast extract in 1 1 of tap water , pH value before sterilization: 6.0) in a 500 ml sterile Erlenmeyer flask are inoculated with the strain and incubated for 72 hours at 28 ° C and 140 rpm on a rotary shaker. Then, 120 ml of the culture liquid are uniformly distributed and decanted in a sterile 500 ml Erlenmeyer flask with the nutrient infusion of oatmeal, 2.0 g / 1, to which 15 g of agar were added. / 1 for consolidation. The cultures were incubated for 10 to 14 days at 28 ° C. The resulting mycelium after this time from a flask is extracted with a puncturing element, immediately used again or stored at -22 ° C in 50% glycerol or in 10% dimethyl sulfoxide at -140 ° C.

Example 2: Preparation of a culture or a previous culture of the producing strain in the Erlenmeyer flask A 500 ml sterile rlenmeyer flask with 100 ml of the nutrient solution described in Example 1 is inoculated with a culture grown in an inclined tube or with a piece of agar and incubated in a shaker in the dark a 140 rpm and 28 ° C. The maximum production of the compounds of the formula I has been reached after about 72 hours. For the inoculation of fermenters of 10 and 100 1, a 72-hour immersed culture (amount of inoculation, approximately 5%) of the same nutrient solution is sufficient.

Example 3: Preparation of Ala0-actagardine A 10 1 fermenter is operated under the following conditions: Nourishing medium: 2% soy flour 2% manita Incubation time: 24 or 48 hours Incubation temperature: 28 ° C Agitator speed: 200 rpm Aeration: 5 1 air / min.

By the repeated addition of a few drops of ethanolic polyol solution, foam formation can be suppressed. The maximum production is reached after 48 hours.

Example 4: Isolation of the antibiotic Ala ° -actagardine 27 1 of the culture solution obtained according to Example 3 are separated by centrifugation, and the clear culture filtrate is incorporated in a 3 1 capacity column, filled with the MCI Gel® CHP20P adsorption resin. Dimensions of the column: width x height: 11.3 cm x 30 cm. It is eluted with a solvent gradient of 5% isopropanol in water to 50% isopropanol, and the amount effluent from the column is collected in fractions of 2 1 each. The fractions containing Ala0-actagardine, which is examined by HPLC analysis, collected and concentrated in vacuo as well as lyophilized (4 g).

Example 5: High pressure liquid chromatography (HPLC) of Ala0-actagardine Column: Nucleosil® 100 - 5 C18AB, 250/4. Mobile phase: 32% acetonitrile in 10 mM potassium phosphate buffer, pH 7. Flow: 1 ml per minute. Detection by UV absorption at 210 nm.

For Ala ° -actagardine the retention time of 16 min 50 seconds was found, for the actagardine itself, 11 min and 20 seconds.

Example 6: Enrichment of the Ala ° -actagardine 3 g of the product obtained according to Example 4 are incorporated into a 3 liter capacity column loaded with Fractogel® TSK HW-40 s (width x height = 10 cm x 50 cm). The eluent: 50% methanol in water is pumped on the column at a flow rate of 50 ml per minute and the amount effluent from the column is collected fractionally (65 ml). In fractions 24 to 28 is mainly the antibiotic Ala ° -acta-gardina, 140 mg.

Example 7: Final purification of Ala ° -actagardine The enriched antibiotic Ala-actagardine (280 mg), obtained according to Example 6, is separated on a Nucleosil® 12C18AB-HPLC column (width x height = 3.2 cm x 25 cm) in a gradient procedure with 5% a 30% acetonitrile of 0.05% trifluoroacetic acid. The fractions investigated by analytical HPLC (see Example 5) are combined in a corresponding manner, concentrated in vacuo and lyophilized. They provide 185 mg of Ala ° -actagardine with a purity of 98%. Molecular weight calculated by ESI + mass spectroscopy of Ala0-actagardine: M + H "= 1962.6.

Example 8: Obtaining the Lys0-actagardine In 10 ml of anhydrous dimethylformamide (DMF), 94.5 mg (0.05 mmol) of actagardine are dissolved and 22 mg (0.05 mmol) of di-Boc-lisin-ON-hydroxysuccinimide are added, as well as 100 μl of Triethylamine (TEA) and allowed to stand at room temperature. The course of the reaction is monitored analytically by HPLC (see Example 5). After 96 hours, the reaction is stopped by removing the DMF and TEA in high vacuum, and the reaction product is purified by preparative HPLC in the gradient procedure with 25 to 50% acetonitrile in trifluoroacetic acid. (TFA) at 0.05%. The dimensions of the column are of height x width: 25 mm x 250 mm; Support: Select B®. After lyophilization of the fractions with reaction product content, 33 mg (0.015 mmol) of di-Boc-Lys ° -actagardine are obtained. The Boc protecting groups are completely separated with 60% TFA. For this, 25 mg (0.011 mmol) of the protected derivative are dissolved in 5 ml of 60% TFA at room temperature. After 90 minutes the separation has ended. The free lysyl-actagardine is purified with the same gradient, as described above, on a preparative HPLC column (10 mm x 250 mm, LiChrospher®). Lyophilization of the purified material provides 14 mg (0.007 mmol) of Lys0-actagardine. The molecular weight of the final product is examined by mass spectrometry. This amounts to (M + H) +: 2019, corresponding to the empirical formula C87H138025N:, 2S4.

Example 9: The preparation of Ile ° -actagardine 189 mg (0.1 mmol) of actagardine are reacted, as described in Example 8, with 33 mg (0.1 mmol) of Boc-Ile-O-N-hydroxysuccinimide ester. 210 mg of Boc-Ile ° -actagardine are obtained. Separation of the Boc protecting group and final purification provide 84 mg (0.042 mmol) of Ile-actagardi-na.

The molecular weight calculated by mass spectrometry is (M + H) +: 2004, corresponding to the empirical formula C87H135025N21S4.

Example 10: The preparation of N-a-aminobutyryl-actagardine [Abu0-ac agardine] 94.5 mg (0.05 mmol) of actagardine are reacted, as described in Example 8, with 16.3 mg (0.05 mmol) of para-nitrophenyl ester of N-Boc-Gi- amino-butyric. After 9 days, 54 mg of N-Boc-Abu ° -acctagardine and, after separation of the protecting group, 19 mg (0.01 mmol) of N-a-aminobutyryl-octagardine. Molecular peak (M + H) +: 1976, corresponding to the empirical formula Cß5H131025N21S4.

Example 11: The preparation of Gln ° -actagardine 94.5 mg (0.05 mmol) of actagardine are reacted, as described in Example 8, with 16.4 mg (0.05 mmol) of Boc-glutamin-paranitrophenyl ester. After removing the protecting group, 38 mg (0.019 mmol) of Gln ° -actagardina. Molecular peak (M + H) +: 2019, corresponding to the empirical formula C86H13202SN22S4.

Example 12: The preparation of Phe0-actagardine 94.5 mg (0.05 mmol) of actagardine are reacted, such as described in Example 8, with 15.6 mg (0.05 mmol) of Boc-Phe-O-N-hydroxysuccinimide ester. After removing the protecting group, 26 mg (0.013 mmol) of Phe0-actagardine. Molecular peak (M + H) +: 2038, corresponding to the empirical formula C90H133? 2sN21S4.

Example 13: The preparation of Phe-Ala ° -actagardine 94.5 mg (0.05 mmol) of actagardine are reacted for 3 hours, as described in Example 8, with 21.7 mg (0.05 mmol) of Boc-Phe-Ala-ON ester -hydroxysuc-cinimide. After removing the protecting group, 37 mg (0.018 mmol) of Phe-Ala ° -actagardine are obtained. Molecular peak (M + H) +: 2109, corresponding to the empirical formula C93H138026N22S4.

Example 14: The preparation of D-Ala ° -actagardine 94.5 mg (0.05 mmol) of actagardine are reacted, during 24 hours, as described in Example 8, with 14.5 mg (0.05 mmol) of Boc-DAla-0- ester N-hydroxysuc-cinimide. After separating the protective group, they are obtained 47 mg (0.024 mmol) of D-Ala ° -actagardine. Molecular peak (M + H) +: 1961, corresponding to the empirical formula Ca4H?:! ,, 02SN21S4.

Tables 4 to 6 show the in vitro antibacterial activity (MIC values [μg / ml]) of the actagardine (acta) and of the compounds according to the invention.

Table 4: Table 5 Table 6

Claims (16)

1. - Compound of general formula I - Ala1 - Se2 - Gly3 - Trp4 - Val5 - Ala6 - Abu7 - Leu8 - Abu9 - lie10 - Glu1 - Ala12 HOOC - Ala19 - Ala18 - Ala17 - Lie16 - Val15 - Abu14 s = o - wherein R means the remainder of an amino acid, as well as its physiologically compatible salts.

2. Compound of the formula I according to claim 1, wherein R means the rest of a natural amino acid.

3. Compound of formula I according to claim 2, wherein the amino acid means Ala lie, Lys, Phe or Val.

4. Compound of the formula I according to claim 1, 2 or 3, wherein the amino acid means Ala.

5. Compound of formula I according to one or more of claims 1 to 4, wherein the amino nitrogen of the amino acid carries a separable protecting group.

6.- Compound of the empirical formula: C84H129N21025S4 (Ala ° --actagardine), obtainable by fermentation of Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis, DSM 11796 or one of its variants and / or mutants in a culture medium until the Ala0-actagardine compound accumulates in the culture broth and by subsequent isolation of the compound, as well as its pharmacologically compatible salts.

7. - Chemical derivatives that are derived from a compound of the empirical formula C84H123N2102SS4 (Ala ° -actagardine), obtainable by fermentation of Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796 or one of its variants and / or mutants, in a medium of culture until the Ala-actagardine compound accumulates in the culture broth and by subsequent isolation of the compound and transformation into chemical derivatives, as well as its pharmacologically compatible salts.

8. Process for the preparation of a compound according to one or more of claims 1 to 5, characterized in that actagardine is reacted with an amino acid and optionally transformed into its pharmacologically compatible salts.

9. - Process for the preparation of a compound according to one or more of claims 1 to 5, characterized in that the microorganism Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796 or one of its variants or mutants is fermented in a culture medium, a compound of the formula I is isolated and optionally transformed into its pharmacologically compatible salts.

10. Process according to claim 9, characterized in that Actinoplanes liguriae, DSM 11797 or Actinoplanes garbadiensis DSM 11796 or their mutants and / or variants are fermented, under aerobic conditions, in culture media containing a carbon and nitrogen source as well as the usual inorganic salts and trace elements.

11. Process according to claim 9 or 10, characterized in that the fermentation is carried out in a nutrient medium that as a carbon source contains 0.5 to 5% of mannitol and 0.5 to 5% of soybean meal.

12. Method according to one or more of claims 9 to 11, characterized in that the fermentation is carried out under aerobic conditions at a temperature between 20 and 35 ° C and at a pH between 4 and 10.

13. - Compound according to one or various of claims 1 to 7 for use as a medicament.

14. Use of a compound according to one or more of claims 1 to 7 for the preparation of a medicament for the treatment and prophylaxis of bacterial infectious diseases.

15. A medicament having a content in at least one compound according to one or more of claims 1 to 7 and one or more physiologically acceptable carriers and optionally suitable excipients.

16. Process for the preparation of a medicament according to claim 15, characterized in that at least one compound according to one or more of claims 1 to 7 is brought into a suitable administration form with a coadjuvant and / or physiological support substance.