HK1155762B - Highly bridged peptides from actinomadura namibiensis - Google Patents

Description

Highly bridged peptides from Actinomadura namibiensis

Several highly bridged peptides have been described in the literature, such as conopeptide (conopeptide) isolated from conus (cone snail) (see, e.g., the reviews by Terlau & Olivera, Physiol. Rev.2004, 84, 41-68) or the so-called lantibiotic (lantibiotic) from gram-positive bacterial sources (Chatterjee et al, chem. Rev.2005, 105, 633-) 683). The peptides have different uses. For example, nisin, a lantibiotic, has been used for many years as a food preservative.

The conopeptide is useful for treating pain, diabetes, multiple sclerosis and cardiovascular diseases, and is now under preclinical or clinical development. Examples of conopeptides include alpha-GI (sequence: ECCNPACGRHYSC)^*，^*Amidation, linkage: 1-3, 2-4), and α -GID (sequence: IR γ CCSNPACRVNNOHVC, connection: 1-3, 2-4) where O/Hyp is hydroxyproline and the crosslinks indicate the position of the cysteines involved in each particular disulfide bond, e.g., the first cysteine with the third cysteine, the second with the fourth cysteine in the α -GID:

a new type of highly bridged peptide, called Labyrinthopeptin (european patent application EP06020980.6), has recently been discovered. The so-called labyrinth peptides exhibit a unique bridging motif (motif) throughout their peptide chain, as shown by the compounds of the following formula:

it has now been found that more highly bridged peptides of the labyrinth peptin class can be isolated from the microbial strain actinomaduranaminoensis (DSM 6313). Said compounds differ significantly from the labyrinth peptide derivatives described in patent application EP 06020980.6.

One embodiment of the present invention is a compound of formula (I)

Wherein

{ A } is a group selected from:

{ B } is a group selected from:

{ C } is a group selected from:

R₁is R₁' group or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O);

R₂is OH, NH₂，(C₁-C₆) alkyl-NH, phenyl- (C)₁-C₄) alkylene-NH or pyridyl- (C)₁-C₄) alkylene-NH;

R₃and R₄Independently of one another, are H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene group, (C)₁-C₄) Alkyl NHC (O) - (C)₁-C₆) Alkylene radicalOr [ (C)₁-C₄) Alkyl radical]₂NC(O)-(C₁-C₆) An alkylene group or a substituted alkylene group,

or R₃And R₄Together with the S atom to which they are attached form a disulfide group S-S;

R₅and R₆Independently of one another, is H or OH, or R₅And R₆Together are ═ O;

m and n are each independently of the other 0, 1 or 2;

provided that if

{ A } is

{ B } is

{ C } is

Then R is₃And R₄May not form together with the S atom to which they are attached a disulfide group S-S.

The above-mentioned compounds can be in any stereochemical form or a mixture of any stereochemical forms in any ratio, or a physiologically acceptable salt thereof.

Preferably, the first and second electrodes are formed of a metal,

{ A } is

{ B } is

{ C } is

More preferably still, the first and second liquid crystal compositions are,

{ A } is

{ B } is

{ C } is

R₁Preferably a radical R₁’，

R₁' is preferably H.

R₂OH is preferred.

R₃And R₄Preferably independently of one another, are H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene groups or together with the S atom to which they are attached form a disulfide group S-S. More preferably, R₃And R₄Is H, or together with the S atom to which they are attached forms a disulfide group S-S. Most preferably, R₃And R₄Together with the S atom to which they are attached form a disulfide group S-S.

R₅And R₆Preferably H or OH, wherein if R₅Is OH, then R₆Is H, and if R is₅Is H, then R₆Is OH, or R₅And R₆Together are ═ O. More preferably, R₅Is OH and R₆Is H, or R₅Is H and R₆Is OH.

Preferably, compound (I) is characterized by a compound of formula (II):

wherein R is₁Is R₁' or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O), preferably H.

More preferably, compound (I) is characterized by a compound of formula (III):

wherein

R₁Is R₁' or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O), preferably H;

R₂is OH, NH₂，(C₁-C₆) -alkyl-NH [ (C)₁-C₆) -alkyl radical]₂N, phenyl- (C)₁-C₄) alkylene-NH or pyridyl- (C)₁-C₄) alkylene-NH, R₂Preferably H; and is

R₃And R₄Independently of one another, are H, (C)₁-C₆) Alkyl or NH₂C(O)-(C₁-C₄) An alkylene group.

Compounds of the formulae (II) and (III), in which R₁Is R₁', hereinafter designated as labyrinth peptide A1.

Compounds of the formulae (II) and (III), in which R₁Is a group

Hereinafter designated as labyrinth peptide A3.

More preferably, compound (I) is characterized by formula (IV):

wherein

R₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O), and

R₂is OH, NH₂，(C₁-C₆) -alkyl-NH, N [ (C)₁-C₆) -alkyl radical]₂Benzene(s)Radical- (C)₁-C₄) alkylene-NH or pyridyl- (C)₁-C₄) alkylene-NH, to

R₃And R₄Independently of one another, are H, (C)₁-C₆) Alkyl or NH₂C(O)-(C₁-C₄) An alkylene group.

The compound of formula (IV) is named labyrinth peptide A2.

Preferably, in the compounds of formula (I), m and n are both 0, or m and n are both 2, or m is 0 and n is 2, or m is 2 and n is 0. Most preferably, m and n are both 0.

The invention also relates to all the obvious chemical equivalents of the compounds of formula (I) according to the invention. These equivalents are compounds which show only slight chemical differences and have the same pharmacological effect, or which are converted under mild conditions into the compounds of the invention. Such equivalents also include, for example, salts, reduced products, oxidized products, partially hydrolytically treated (partially hydrolyzed) esters, ethers, acetals, or amides of the compounds of formula 1, and equivalents which may be prepared using standard methods by those skilled in the art, in addition to all optical antipodes (optical antipodes) and diastereomers (diastereomers) and all stereoisomeric forms (stereoisomeric forms).

Unless otherwise indicated, the chiral centers of the compounds of formula (I) may be present in the R configuration or in the S configuration. The present invention relates to optically pure compounds and stereoisomeric mixtures, such as meso mixtures and diastereomeric mixtures.

Physiologically acceptable salts of the compounds of formula (I) are understood to be organic and inorganic salts thereof, as described, for example, in Remington's Pharmaceutical Sciences (17 th edition, page 1418 (1985)). For the acid group, sodium, potassium, calcium, and ammonium salts, etc. are preferable because of their physical and chemical stability and solubility; as the basic group, a salt of hydrochloric acid, sulfuric acid or phosphoric acid, or a salt of a carboxylic acid or sulfonic acid (e.g., acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid), and the like are preferable.

More preferably, the compounds of formulae (I) to (IV) are characterized by the stereochemistry shown by the compounds of formula (V), which are compounds of formula (I), wherein

{ A } is

{ B } is

{ C } is

R1 is

Wherein R is₁' is H;

R₂is H;

R₃and R₄Together with the sulfur atom to which they are attached form a disulfide group S-S;

R₅is H;

R₆is OH; while

m and n are 0:

most preferably, as described in formula (VI):

a further embodiment of the invention is a compound of formula (I), characterized by formula (VII):

preferred are compounds characterized by formula (VIII)

Wherein formulae (V) and (VI) relate to labyrinthin A3 and formulae (VII) and (VIII) relate to labyrinthin A1.

To further characterize the compounds of the invention, the peptide residues were converted back to their possible precursors from ribosomal peptide synthesis. The alpha, alpha-disubstituted amino acids of residues 4 and 13 are not mentioned in the literature. The amino acid can be described as a hydroxyl-substituted serine at the beta-position, as shown below for compounds of formula (II) and (III):

in the prior european patent application (EP06020980.6) relating to maze pep a2, in the case where the biosynthetic pathway is unknown, the ribosome precursors are presumed to be as follows:

based on the new insight into the biosynthesis of labyrinth peptin (new insight) (see below), the precursors for the biosynthesis of the compounds of formula (IV) are as follows:

the invention also relates to a process for the preparation of a compound of formula (I) according to claim 1, comprising

a) Fermenting the strain Actinomadura namibiensis (DSM6313), or one of its variants and/or mutants, in a medium under suitable conditions until one or more compounds of formula (I) are produced in said medium,

b) isolating the compound of formula (I) from the culture medium, and

c) derivatizing the compound isolated in step b), if appropriate, and/or converting the compound isolated in step b) or a derivative of the compound isolated in step b), if appropriate, into a physiologically acceptable salt.

Preferably, the compound isolated in step b) is characterized by formula (II), wherein m and n are both 0,

R₁is R₁' or group

Wherein R is₁Is H, and

R₂is OH.

More preferably, the compound isolated in step b) is labyrinthin A2, which is subsequently derivatized in step c) to a compound of formula (IV), wherein

m and n are both 0, and n is,

R₁is a compound of formula (I) wherein the compound is H,

R₂is OH, and

R₃and R₄Independently of one another, are H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene group, (C)₁-C₄) Alkyl NHC (O) - (C)₁-C₆) Alkylene or [ (C)₁-C₄) Alkyl radical]₂NC(O)-(C₁-C₆) An alkylene group.

The medium is a nutrient solution or a solid medium which contains at least one conventional (custom) carbon source and at least one conventional nitrogen source, and one or more conventional inorganic salts.

The process of the invention is useful for fermentation on a laboratory scale (microliter to liter scale) and fermentation on an industrial scale (cubic meter scale).

Suitable carbon sources for the fermentation are assimilable sugars and sugar alcohols, such as glucose, lactose, sucrose or D-mannose, and sugar-containing natural products, such as malt extract and yeast extract. Examples of nitrogen-containing nutrients are amino acids, peptides and proteins and their breakdown products (e.g. casein, peptone or tryptone), meat extracts, yeast extracts, gluten (gluten), ground seeds (e.g. seeds from corn, wheat, beans, soy or cotton trees), distillation residues from ethanol production, meat meal (meat metals), yeast extracts, ammonium salts, nitrates. Preferred nitrogen sources are one or more peptides, which are obtained synthetically or biosynthetically. Examples of inorganic salts are the hydrochlorides, carbonates, sulfates or phosphates of alkali metals, alkaline earth metals, iron, zinc, cobalt and manganese. Examples of trace elements are cobalt and manganese.

Conditions particularly suitable for forming the labyrinth peptide of the present invention are as follows: yeast extract, from 0.05 to 5%, preferably from 0.1 to 2.5%; tyrose peptone, from 0.2 to 5.0%, preferably from 0.1 to 2%; CaCl₂×2H₂O, from 0.02 to 1.0%, preferably from 0.05 to 0.5%; MgSO (MgSO)₄×7H₂O, from 0.02 to 1.5%, preferably from 0.05 to 0.7%, and 0.00001% to 0.001% of vitamin B₁₂(cyanocobalamin). Each percentage value given is by weight of the total nutrient solution.

The microorganisms are subjected to aerobic cultivation, i.e.for example by submerging them in shake flasks or fermenters with shaking or stirring, or submerging them on solid medium, if appropriate with simultaneous aeration with air or oxygen. The microorganisms are cultured at a temperature in the range from about 18 to 35 ℃, preferably from about 20 to 32 ℃, in particular from 27 to 30 ℃. The pH should be in the range of 4 to 10, preferably 6.5 to 7.5. The microorganisms are usually cultured under these conditions for a period of2 to 10 days, preferably 72 to 168 hours. It is advantageous to carry out the multistage cultivation of the microorganisms in question, i.e.initially to prepare one or more preliminary cultures (precultures) in a liquid nutrient medium and then to inoculate these preliminary cultures, for example, in a volume ratio of from 1: 10 to 1: 100, into the actual production medium (i.e.main culture). The preliminary culture is obtained, for example, by inoculating a strain in the form of vegetative cells (vegetative cells) or spores into a nutrient solution and allowing it to grow for about 20 to 120 hours, preferably 48 to 96 hours. Vegetative cells and/or spores can be obtained, for example, by growing the strain on a solid or liquid nutrient substrate (e.g., yeast agar) for about 1 to 15 days, preferably 4 to 10 days.

The labyrinthin derivative can be isolated and purified from the culture medium using known methods, taking into account the chemical, physical and biological properties of natural substances. The concentration of the various labyrinthin derivatives in the medium or in the various separation steps is tested using HPLC, and the amount of material produced can be conveniently compared to a calibration solution.

For separation, optionally, the culture broth or the culture together with the solid medium is lyophilized, and the labyrinthin derivative is extracted from the lyophilizate using an organic solvent or a mixture of water and an organic solvent (preferably containing 50-90% of an organic solvent). Examples of organic solvents are methanol and 2-propanol. The organic solvent phase contains the natural substance; when appropriate, it was concentrated in vacuo and subjected to further purification.

Further purification of one or more compounds of the invention is carried out by chromatography on suitable materials, for example on molecular sieves, silica gel, alumina, ion exchangers or on absorbent resins or Reversed Phase (RP). Chromatography is used to isolate the maze peptin derivatives. The labyrinth peptide derivative is chromatographed using a buffered, basic or acidified aqueous solution or a mixture of an aqueous solution and an organic solution.

By water or mixture of organic solutions is understood an organic solvent which is miscible with water, preferably methanol, 2-propanol or propionitrile, an organic solvent in a concentration of 5 to 99%, preferably 5 to 50%, or an aqueous buffer solution which is all miscible with organic solvents. The buffers to be used are the same as those indicated above.

The labyrinth peptide derivatives are separated on the basis of their different polarity by means of reverse phase chromatography, for example on MCI (absorbent resin, Mitsubishi, Japan) or Amberlite XAD (TOSOHAAS), or other hydrophobic materials (for example on RP-8 or RP-18 phases). Furthermore, the separation can be carried out by means of normal phase chromatography (e.g. on silica gel, alumina and the like).

Buffered, basic or acidified aqueous solutions are to be understood as meaning, for example, water, phosphate buffers, ammonium acetate and citric acid buffers, the concentration of which is up to 0.5M, and also formic acid, acetic acid, trifluoroacetic acid, aqueous ammonia and triethylamine, or all commercially available acids or bases known to the person skilled in the art, preferably the concentration of which is up to 1%. In the case of an aqueous buffer solution, 0.1% ammonium acetate is particularly preferred.

Chromatography can be performed using a gradient starting with 100% water and ending with 100% organic solvent, the chromatography preferably being run with a linear gradient of 5 to 95% propionitrile.

Alternatively, gel chromatography or chromatography in a hydrophobic phase may be performed. Gel chromatography may, for example, be performed on polyacrylamide gel or copolymer gel. The order of the above chromatograms can be reversed.

In the case where the labyrinth peptides exist in the form of stereoisomers, they can be separated using known methods, for example, by means of separation using a chiral column (chiral column).

Derivatization of the OH groups into ester or ether derivatives using methods known per se (J.March, Advanced Organic Chemistry, John Wiley&Sons, 4 th edition, 1992), for example by means of reaction with an acid anhydride or by reaction with a carbonic acid diester or a sulfuric acid diester. Derivatization of COOH to an ester or amide derivative Using methods known per se (J.March, advanced organic Chemistry, John Wiley&Sons, 4 th edition, 1992), e.g. by reaction with ammonia to form the corresponding CONH₂By means of radicals or by means of reaction with optionally activated alkyl compounds to give the corresponding alkyl esters. Will be-CH₂-S-CH₂Oxidation of the-group to-CH₂-S(O)-CH₂-or-CH₂-S(O)₂-CH₂This can be achieved when the corresponding labyrinthin derivative is exposed to oxygen or air. The reduction of the disulfide bonds, and optionally also the subsequent alkylation of the free SH groups, is carried out using Methods known per se (A. Henschen, Analysis of cycle (e) amine derivatives, disulphides, and sulfhydryl groups in proteins, in: B.Wittmann-Liebold, J.Salnikov, V.A.Erdman (eds.), Advanced Methods in Protein Analysis, Springer, Berlin, 1986, pp. 244-255), for example reduction by dithiothreitol and alkylation with alkyl iodides. Reduction of thioether (sulfide) to a compound of formula (I) (wherein R is₃And R₄Is H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene group, (C)₁-C₄) Alkyl NHC (O) - (C)₁-C₆) Alkylene or [ (C)₁-C₄) Alkyl radical]₂NC(O)-(C₁-C₆) Alkylene) groups, obtainable as follows: in the presence of a disulfideIn the presence of threitol, wherein R is₃And R₄Compounds of formula (I) which form a disulfide bond with the S atom to which they are attached and C₁-C₆) Haloalkyl or halo- (C)₁-C₆) alkylene-C (O) NH₂Halo- (C)₁-C₆) alkylene-C (O) NH (C)₁-C₄) Alkyl or halo- (C)₁-C₆) alkylene-C (O) N [ (C)₁-C₄) Alkyl radical]₂The reaction is carried out (general literature). Halogen is F, Cl, Br or I.

Isolates of the microorganism strain Actinomardra namibiensis were deposited by Hoechst AG, Frankfurt, Germany under the identification reference FH-A1198 at 23.1.1991 under the Budapest treaty (Budapest treaty) under the accession number DSM6313 in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, (DSMZ), Mascheroder Weg 1B (as of 2008: Inhofftress. 7B), 38124Braunschweig, Germany. The microbial strain Actinomadura namibiensis is further described by Wink et al in International Journal of systematic and evolution Microbiology 2003, 53, 721-.

In addition to the strain Actinomadura namibiensis (DSM6313), mutants and/or variants thereof which synthesize one or more compounds of the invention may be used.

Mutants are microorganisms in which one or more genes in the genome have been modified with one or more genes which enable the organism to produce the compounds of the invention, remain functional and can be inherited.

Such mutants may be generated in a manner known per se, using physical means (e.g.irradiation, such as with UV or X-rays), or chemical mutagens (e.g.ethyl methanesulfonate (EMS), 2-hydroxy-4-Methoxybenzophenone (MOB) or N-methyl-N' -nitro-N-nitrosoguanidine (MNNG)), or as described by Brock et al in "Biology of Microorganisms", Prentice Hall, pp.238-247 (1984)).

The variant is a phenotype of said microorganism. Microorganisms have the ability to adapt to their environment and thus exhibit highly developed physiological plasticity. All cells of the microorganism are involved in phenotypic adaptation, which is not gene-based in nature and is reversible upon a change in conditions (H.Stolp, microbiology: organissm, hashites, activities. Cambridge University Press, Cambridge, GB, page 180, 1988).

Screening for mutants and/or variants of one or more compounds of the invention is performed by: optionally, the fermentation medium is lyophilized and the lyophilizate or fermentation broth is extracted with an organic solvent or a mixture of water and organic solvent as described above and analyzed by HPLC or TLC means or by testing for its biological activity.

The fermentation conditions may be applied to Actinomydra namibiensis (DSM6313) and mutants and/or variants thereof.

A further embodiment of the invention is the use of a compound of formula (I) as defined above for the treatment of bacterial infections, in particular bacterial infections caused by gram-positive bacteria, for the treatment of fungal infections, and/or for the treatment of pain, in particular neuropathic pain or pain caused by inflammation.

The medicament (also called pharmaceutical formulation or pharmaceutical composition) as described above comprises an effective amount of at least one compound of formula (I), which may be in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a pharmaceutically acceptable salt or a chemical equivalent thereof, as described above, and at least one pharmaceutically acceptable carrier, preferably one or more pharmaceutically acceptable carrier substances (or vehicles) (vehicle), and/or additives (or excipients).

The medicaments can be administered orally, for example in the form of pills, tablets, lacquered tablets (lacquered tablets), coated tablets (coated tablets), granules (granules), hard and soft gelatin capsules, solutions, syrups, emulsions, suspensions or aerosol mixtures. However, administration may also be effected rectally (e.g. in the form of suppositories), parenterally, e.g. intravenously, intramuscularly or subcutaneously in the form of injections or infusions, microcapsules, implants or sticks (rod), or transdermally, or topically, e.g. in the form of ointments, solutions or tinctures, or in other ways, e.g. in the form of aerosols or nasal sprays.

The medicaments according to the invention are prepared in a manner known per se and familiar to the person skilled in the art, using, in addition to the compounds of formula (I), which, as described above, can be in any stereochemical form, or a mixture of any stereochemical forms in any proportions, or a physiologically acceptable salt or a chemical equivalent thereof, a pharmaceutically acceptable inert inorganic and/or organic carrier material and/or additives. For the production of pills, tablets, coated tablets and hard gelatine capsules, use may be made of, for example, lactose, maize starch or derivatives thereof, talc, stearic acid or its salts and the like. Carrier substances for soft gelatine capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils (hardenoil) and the like. Suitable carrier materials for the preparation of solutions (e.g. injection solutions), or emulsions or syrups are, for example, water, saline, alcohols, glycerol, polyols, sucrose, invert sugar, glucose, vegetable oils and the like. Suitable carrier materials for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid. The pharmaceutical preparations generally contain from about 0.5 to about 90% by weight of a compound of formula (I) and/or a physiologically acceptable salt thereof and/or a prodrug (produgs) thereof. As mentioned above, the amount of active ingredient of formula (I) in a medicament, in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically acceptable salt or a chemical equivalent thereof, is usually from about 0.5 to about 1000mg, preferably from about 1 to about 500 mg.

In addition to the active ingredient of formula (I) in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically acceptable salt or a chemical equivalent thereof, as described above, and a carrier, the pharmaceutical preparation may contain one or more additives, such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavoring agents (flavoring agents), fragrances (aromatizers), thickeners, diluents, buffering substances, solvents, solubilizers, agents for achieving a depot effect (agents for improving a depot effect), salts for varying the osmotic pressure, coating materials or antioxidants. It may also contain two or more compounds of formula (I) in any stereochemical form, a mixture of any stereochemical forms in any ratio, or a physiologically acceptable salt or a chemical equivalent thereof. If a pharmaceutical formulation contains two or more compounds of formula (I), the selection of each compound may be targeted to a particular overall pharmacological profile of the pharmaceutical formulation. For example, a compound with high potency but short duration of action may be combined with a compound with lower potency but long duration of action. The plasticity allowed for the choice of substituents on the compounds of formula (I) enables a large number of modulations in the biological and physicochemical properties of the compounds, and thus the selection of such desired compounds. Furthermore, the pharmaceutical preparations may comprise, in addition to at least one compound of formula (I), one or more therapeutically or prophylactically active ingredients.

When using the compounds of formula (I), the dosage may vary within a wide range to suit the individual condition in each individual case, as is conventional and known to the physician. For example, it will depend on the particular compound used, the nature and severity of the disease to be treated, the mode and schedule of administration, or whether the condition being treated is acute or chronic, or whether prophylaxis is being effected. Suitable dosages may be established using clinical methods well known in the medical arts. In general, the daily dose to achieve the desired result in an adult human weighing about 75kg is from about 0.01 to about 100mg/kg, preferably from about 0.1 to about 50mg/kg, in particular from about 0.1 to about 10mg/kg, (in each case mg per kg body weight). The daily dose may be divided into several portions, for example 2, 3 or 4 portions, especially when relatively large amounts are administered. Typically, depending on the individual's condition, it may be desirable to adjust the indicated daily dose up or down.

Example 1: preparation of frozen culture of Actinomadura namibiensis (DSM6313)

For 100ml of medium (10g of starch, 2g of yeast extract, 10g of glucose, 10g of glycerol, 2.5g of corn steep powder, 2g of peptone, 1g of NaCl, 3g of CaCO)₃pH 7.2 in 1 liter of tap water, then sterilized) was inoculated with the strain Actinomadura namibiensis (DSM6313) in a 500ml sterile Erlenmeyer flask and incubated at 27 ℃ for 72 hours at 120rpm on a shaker. Then, 1ml of the culture was mixed with 1ml of sterilized preservation solution (20g of glycerol, 10g of sucrose, 70ml of deionized water) and stored at-80 ℃. Alternatively, small pieces of well-grown culture on agar were transferred to Cryotubes with 1.5ml of 50% sterilized glycerol solution(Vangard International) and stored in liquid nitrogen at-196 ℃.

Example 2: preparation of labyrinth peptide

A500 ml sterilized Erlenmeyer flask containing 100ml of the medium described in example 1 was inoculated with a culture of Actinomadura namibiensis (DSM6313), which was grown on agar plates and incubated at 27 ℃ on a shaker at 120 rpm. After 72 hours, other Erlenmeyer flasks each containing the same medium in the same amount were inoculated with 2ml of the preculture and incubated under the same conditions for 168 hours. Alternatively, a 300ml Erlenmeyer flask containing 100ml of the medium described in example 1 was inoculated with a culture of Actinomadura namibiensis (DSM6313) and incubated at 25 ℃ and 180 rpm. After 72 hours, other Erlenmeyer flasks containing the same medium in the same amount were inoculated with 5ml of the preculture and incubated under the same conditions for 168 hours.

Example 3: solid extraction of labyrinth peptide

After completion of the 40L fermentation of Actinomardura namibiensis (DSM6313), the fermentation broth was filtered. Tissue permeate (about 30L) was loaded onto a column (size: 160X 200mm) packed with about 3L CHP-20P material. The compound was eluted at a flow rate of 250 ml/min using an isopropanol/water gradient from 5% to 95%. Fractions were collected every 4 minutes over a 45 minute period. Fractions containing maze peptin were pooled and lyophilized (fraction 8: MW 2190 Da; fraction 9: MW 2190 and 2074 Da; fraction 10-12: MW 2074Da)

Example 4: prepurification of maze Peptidin A1 Using RP-18 chromatography

Fractions 10-12 from example 3 (670mg) were dissolved in 500ml methanol and purified with Phenomenex Luna10 μ C18(2) pre-column (size: 21.2mm x 60mm) loaded on Phenomenex Luna10 μ C18(2) column (size: 50 mm. times.250 mm). The compounds were eluted with a 5% to 75% propionitrile/water gradient over a period of 40 minutes at a flow rate of 190 ml/min (buffer: 0.1% ammonium acetate, pH9.0, adjusted with 30% aqueous ammonia). Fractions per minute were collected. Fractions 21-22 contained the desired maze peptin (MW ═ 2074 Da). After lyophilization, 332mg of crude product was obtained.

Example 5: final purification of maze Peptidin A1

Fractions 21-22 from example 4 (60mg) were dissolved in 50ml methanol and washed with WatersXTerraPrep MS C1810 μ Pre-column (size: 19X 10mm) Loading to Phenomenex Luna5 μ C18(2) Axia column (size: 30 mm. times.100 mm). The compound was eluted with a gradient of 5% to 75% propionitrile/water over a period of 40 minutes at a flow rate of 70 ml/min (buffer: 0.1% ammonium acetate, pH4.6, adjusted with aqueous acetic acid). The eluate was collected as 10 ml-fractions using UV-triggering (triggering). Fractions containing maze pep-min (fractions 9-12) were pooled. After lyophilization, 17mg of labyrinth peptide A1 was obtained.

Example 6: prepurification of maze Peptidin A3 Using RP-18 chromatography

Fraction 8 (. about.850 mg) from example 3 was dissolved in 500ml methanol and purified by Phenomenex Luna10 μ C18(2) pre-column (size: 21.2mm x 60mm) loaded on Phenomenex Luna10 μ C18(2) column (size: 50 mm. times.250 mm). The compounds were eluted with a gradient of 5% to 75% propionitrile/water over a period of 40 minutes (buffer: 0.1% ammonium acetate, pH 7.0) at a flow rate of 190 ml/min. Fractions per minute were collected. Fraction 19 contained the desired labyrinth peptide (MW 2190 Da). After lyophilization, 48mg of crude product was obtained.

Example 7: final purification of maze Peptidin A3

Fraction 19 from example 6 (48mg) was dissolved in 50ml methanol and washed with WatersXTerraPrep MS C1810 mu pre-column (size: 19 mm. times.10 mm) loaded on PhenomenexLuna5 μ C18(2) Axia column (size: 30 mm. times.100 mm). The compound was eluted with a gradient of 5% to 75% propionitrile/water over a period of 40 minutes at a flow rate of 70 ml/min (buffer: 0.1% ammonium acetate, pH9.0, adjusted using 30% aqueous ammonia). The eluate was collected as fractions using UV-trigger. Fractions containing the maze pep-min were pooled (F9-12). After lyophilization, 12mg of labyrinth peptide A3 was obtained.

Example 8: identification of maze Peptidin A1 and A3 by high Performance liquid chromatography (HPLC-DAD-MS) with diode array and Mass Spectrometry detection

The maze peptides A1 and A3 were analyzed on a Waters acquisition UPLC System with Sample Manager, Binary Solvent Manager and PDA (Photodiode Array Detector). A Waters Acquity UPLC BEH C18(1.7 μ; 2.1X100mm) was used as the UPLC column and eluted at a flow rate of 0.6 ml/min with the following gradient: from water propionitrile (9: 1) to 100% propionitrile in 15 minutes, all the solvent was buffered to pH4.6 with 6.5mM ammonium acetate. The UV spectrum is recorded by a PDA detector at a wavelength between 200 and 600 nm. Mass spectra were recorded using a Bruker μ TOFLC MS using orthogonal electrospray ionization (orthogonal electrospray ionization) at a sampling rate of 0.5Hz and a detection limit of 150-.

Example 9: identification of labyrinth Peptidin A1

Maze pep 1 eluted at 5.46 minutes (PDA). UV spectra from 218nm (sh) and lambda of 279nm_maxAnd (5) characterizing.

In the anionic mode (negative mode), a two-charge molecular ion is observed at m/z (I): 1035.87(4539), 1036.37(5566), 1036.87(4086), 1037.37(2296), 1037.87(1034) and 1038.37 (280). Two-charge molecular ions having the following m/z (i) were observed in the positive ion mode (positive mode): 1037.88(2925), 1038.38(3252), 1038.88(2492), 1039.38(1396) and 1039.88 (623).

Labyrinth peptin a1 was identified by high resolution ESI-FTICR-mass spectrometry: maze pep 1/methanol solution (c ═ 0.2mg/ml) was introduced into Bruker Apex III FTICR MS (7T magnet) equipped with an electrospray source by a syringe pump at a flow rate of2 μ l/min. Spectra were recorded in cationic mode using external calibration (external calibration).

Observed M/z, Da (z ═ 2, M +2 Na)+Ion)	1059.8693
		Exact monoisotopic molecular weight of neutral species [ M ]]	2073.7592
C92H119N23O25S4Theoretical molecular weight of [ M ]]	2073.7630
		Molecular formula	C92H119N23O25S4

Example 10: identification of maze Peptidin A3

LabyrinthPeptidin A3 eluted at 4.79min (PDA). UV spectra from λ of 218nm (sh) and 274nm (sh)_maxAnd (5) characterizing.

In the anionic mode, two charge-charged molecular ions are observed at m/z (I): 1093.38(1262), 1093.88(1587), 1094.39(1201), 1094.89(686) and 1095.38 (195). Two-charge molecular ions having the following m/z (I) were observed in the cationic mode: 1095.40(365), 1095.91(433) and 1096.41 (294).

Maze pep 3 was identified by high resolution ESI-FTICR-mass spectrometry (method described in example 9):

observed M/z, Da (z ═ 2, M +2 Na)+ion)	1117.3847
		Exact monoisotopic molecular weight of neutral species [ M ]]	2188.7900
C96H124N24O28S4Theoretical molecular weight of [ M ]]	2188.7900
		Molecular formula	C96H124N24O28S4

Example 11: amino acid analysis of maze Peptidin A1

Hydrolysis: labyrinth peptide A1(0.05mg) was hydrolyzed under nitrogen atmosphere with 6N HCl, 5% phenol at 110 ℃ for 24 hours. The hydrolysate was dried in a nitrogen stream.

Achiral (achiral) GC-MS: the hydrolysate was heated with bis- (trimethylsilyl) trifluoroacetamide (BSTFA)/propionitrile (1: 1) at 150 ℃ for 4 hours. For the GC-MS experiments, DB 5-fused-silica capillary (fused-silica-capillary) (l ═ 15m × 0.25 μm, fused silica coated with dimethyl- (5% -phenylmethyl) -polysiloxane, d_f0.10 μm; temperature program: t is 65 °/3'/6/280 deg.C.

Chiral GC-MS: the hydrolysate was esterified with 200. mu.l 2N HCl/ethanol for 30min at 110 ℃ and dried. The mixture was then acylated with 25. mu.l trifluoroacetic anhydride (TFAA) in 100. mu.l dichloromethane at 110 ℃ for 10 minutes and dried. For the GC-MS experiments, fused silica was spread using fused silica capillary tubes (l ═ 22m × 0.25 μm with Chirasil-S-Val (Machery-Nagel), d_f0.13 μm; temperature program: t55 °/3'/3, 2/180 ℃).

Example 12: identification of structural genes of labyrinth peptide A1 and A3

The cosmid library of the microorganism Actinomydra namibiensis (DSM6313) was generated from Agowa GmbH, Berlin on the basis of pWEB-cosmid vectors (Epicentre Biotechnologies, Madison, USA). The filter paper is manufactured by RZPD GmbH, Berlin usage described in Zehetner& Methods mol. biol.2001, 175, 169-188.

Based on the known structure of pepstatin A2, extended degenerate primers were deduced from the N-and C-termini (Fw: 5 '-CAGGAAACAGCTATGACCGAYTGGWSNYTNTGGG-3' (SEQ ID NO: 4); Rev: 5 '-TGTAAAACGACGGCCAGTRCANGANGCRAANARRC-3' (SEQ ID NO: 5); Dabard et al, appl.environ.Microbiol.2001, 4111-. The 5' extension of the primers was to increase the size of the expected PCR product for better detection and processing (PCR conditions: 3min 95 ℃, 30X (60 sec 95 ℃, 30 sec 50 ℃, 60 sec 72 ℃), 7 min 72 ℃, Taq polymerase). The PCR product was gel purified and cloned into the vector pDrive (Qiagen). Sequencing gave a 18 nucleotide long sequence (AGTGCTGTAGCACGGGAA, SEQ ID NO: 6) from the middle of the A2 gene. Based on this known fragment of 18 nucleotides in length, more sequence information was obtained by two-step PCR. In the first step, monospecific primer PCR (PCR conditions: 3min 95 ℃; 10X (45 sec 95 ℃; 45 sec 38 ℃; 3.5min 72 ℃); 30X (45 sec 95; 45 sec 52;. 3.5min 72 ℃) 5min 72;. Taq polymerase) was performed with degenerate reverse (rev) primers (5 '-RCARCANGCRAANARRCTTCC-3', SEQ ID NO: 7) and a non-specific forward (fw) primer (5 '-CACGGTACCTAGACTAGTGACCAAGTGCGCCGGTC-3', SEQ ID NO: 8) from the C-terminus of A2. After exonuclease I digestion to digest the primers (5. mu.l PCR sample + 0.5. mu.l exonuclease I (20U/. mu.l); 15 min 37 ℃; 15 min 80 ℃ heat inactivation), a second PCR was performed using the PCR sample as a template (PCR conditions: 3min 95 ℃; 30X (45 sec 95 ℃; 45 sec 56 ℃; 3.5min 72 ℃); 5min 72 ℃; Taq polymerase). The second PCR was performed as a nested (nested) PCR with a primer pair consisting of fw primer, which was not specific in the first PCR, and specific rev primer, which contained 18 nucleotides (5 '-CTTCCCGTGCTACAGCACTCCC-3', SEQ ID NO: 9), as known. The resulting 0.4kbp product was gel purified and cloned into pDrive. Sequencing revealed the expected amino acid sequence of the C-terminus of a 2. From this 0.4kbp sequence, a probe labeled with Dig was constructed by PCR (Fw: 5 '-ATGGACCTCGCCACGGGCTC-3', SEQ ID NO: 10; 5 '-CTTCCCGTGCTACAGCACTCCC-3', SEQ ID NO: 11). The Dig-labeled probe was used to screen filter paper by hybridization and detection by an anti-Dig antibody labeled with alkaline phosphatase. In this way, a positive cosmid was obtained and sequenced.

Sequence data were analyzed by local BLAST and FramePlut. This analysis yielded the following open reading frames (orf) comprising the structural gene of labyrinth peptide a 2:

TGACGCCCGCACACCGTTCCACCGATGAGAGGTGACAGTCCCATGGCGTCGATCCTGGAACTCCAGAACCTGGACGTCGAGCACGCCCGCGGCGAGAACCGCTCCGACTGGAGCCTGTGGGAGTGCTGTAGCACGGGAAGCCTGTTCGCCTGCTGCTGA (SEQ ID NO：12)

within this orf, the following sequence represents the structural gene of prepro-labyrinthin a2 (leader sequence followed by propeptide coding sequence followed by stop codon TGA):

ATGGCGTCGATCCTGGAACTCCAGAACCTGGACGTCGAGCACGCCCGCGGCGAGAACCGCTCCGACTGGAGCCTGTGGGAGTGCTGTAGCACGGGAAGCCTGTTCGCCTGCTGCTGA (SEQ ID NO：13)

shown in SEQ ID NO: 13 to obtain the following amino acid sequence of prepro-labyrinth peptide A2(SEQ ID NO: 14) and of proparalabyrinth peptide A2(SEQ ID NO: 15):

MASILELQNLDVEHARGENR SDWSLWECCSTGSLFACC (SEQID NO：14)

SDWSLWECCSTGSLFACC (SEQ ID NO：15)

the propeptide sequence is converted to the maze peptoid a2 by post-translational modification by the enzyme of the microorganism Actinomadura namibiensis (DSM 6313).

Example 13: determination of the Structure of labyrinth Peptidin A1 and A3

The upstream region of the a2 gene shows another small orf with high homology to the structural gene of maze peptide a 2. The open reading frame (orf) contains the structural genes of the labyrinth peptide elements a1 and A3. Orf of maze pep 1 has the following gene sequence:

TGAACATCCACCATGGCATCCATCCTTGAGCTCCAGGACCTGGAGGTCGAGCGCGCCAGCTCGGCCGCCGACAGCAACGCCAGCGTCTGGGAGTGCTGCAGCACGGGCAGCTGGGTTCCCTTCACCTGCTGCTGA

(SEQ ID NO：16)

within this orf, the following sequences represent the structural genes of prepro-labyrinth peptides a1 and A3 (leader sequence followed by propeptide coding sequence followed by stop codon TGA):

ATGGCATCCATCCTTGAGCTCCAGGACCTGGAGGTCGAGCGCGCCAGCTCGGCCGCCGACAGCAACGCCAGCGTCTGGGAGTGCTGCAGCACGGGCAGCTGGGTTCCCTTCACCTGCTGCTGA (SEQ IDNO：17)

shown in SEQ ID NO: 17 to obtain the following amino acid sequence of prepro-labyrinth peptide A1(SEQ ID NO: 18) and of prepro-labyrinth peptide A1(SEQ ID NO: 19):

MASILELQDLEVERASSAADSNASVWECCSTGSWVPFTCC(SEQ IDNO：18)

SNASVWECCSTGSWVPFTCC (SEQ ID NO：19)

this amino acid sequence is consistent with the expected amino acid composition of maze peptin a1 based on the amino acids performed on maze peptin a1 and the results of MS analysis (see above). The post-translational modifications to the side chains were deduced to be similar to the corresponding modifications of maze pep 2. The stereochemistry of amino acids was taken from the amino acid analysis (example 11). Finally, it was deduced that the threonine (Thr) residue was dehydrated to dehydrobutyric acid to match the empirical formula calculated by high resolution MS. Based on the similar stereochemistry of the post-translationally modified amino acids of labyrin a2, labyrin a1 was deduced to be of formula (VIII).

Previous molecular weight analysis indicated that the difference between maze peptins A1 and A3 was one Asp. This is confirmed by the encoded sequence, which contains an Asp at position-1 before the proteolytic cleavage site of maze pep A1. Based on the hypothesis that maze pep a1 and A3 are encoded by the same gene, differing only in the proteolytic cleavage of the leader sequence, the additional Asp is at the N-terminus of maze pep A3. Thus, maze pep 3 was deduced to be formula (V). Based on the similar stereochemistry of the post-translationally modified amino acids of labyrin a2, labyrin A3 was deduced to be of formula (VI).

Example 14: cleavage of labyrinthin A1 disulfide bridge and subsequent alkylation with methyl iodide

Labyrinth peptide A1(50mg, 0.024mmol) was dissolved in methanol (3ml) and dithiothreitol solution (1ml, 75mg dithiothreitol dissolved in 40mg NaHCO) was added at room temperature₃Freshly prepared in 1ml aqueous solution). The mixture was stirred at 60 ℃ for 1 hour. After that, it was cooled to room temperature, and methyl iodide (50. mu.l, 0.80mmol) was added. After 4 hours at room temperature, the mixture was filtered and subjected to reverse phase HPLC using a filter equipped with Waters XTerraPhenomenex Luna of Prep MS C1810 μm pre-column (size: 19 mm. times.10 mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm). The gradient was run from 5% to 95% propionitrile/water over 30min (buffer: pH 2.0, adjusted with formic acid). The flow rate was 60 ml/min and the peaks were fractionated by UV. Fractions 12 and 13 were combined to yield 23.1mg (45.5%) of the desired compound after lyophilization. The product was characterized by UV spectroscopy and mass spectrometry (bruker daltonics microtorf).

RT_min5.46min (PDA; LC-method, as in example 8)

UV(λ_max)：217nm(sh)，279nm

ESI-MS(neg)：[M-2H]₂ ^-＝1050.894

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2103.802

From C₉₄H₁₂₅N₂₃O₂₅S₄Calculated neutral monoisotopic molecular weight: 2103.810

The molecular formula is as follows: c₉₄H₁₂₅N₂₃O₂₅S₄

Formula weight 2105.44.

Example 15: cleavage of labyrinthin A1 disulfide bridge and subsequent alkylation with iodoacetamide

Labyrinth peptide A1(50mg, 0.024mmol) was dissolved in methanol (3ml) and dithiothreitol solution (1ml, 75mg dithiothreitol dissolved in 40mg NaHCO) was added at room temperature₃Freshly prepared in 1ml aqueous solution). The mixture was stirred at 60 ℃ for 1 hour. After that, it was cooled to room temperature, and methyl iodide (40mg, 0.216mmol) was added. The mixture was stirred at room temperature overnight. The compounds were filtered and subjected to reverse phase HPLC using a equipped Waters XTerraPhenomenex Luna of Prep MS C1810 μm pre-column (size: 19 mm. times.10 mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm). The gradient was run from 5% to 95% propionitrile/water over 30min (buffer: 0.1% ammonium acetate, pH adjusted to 4.6 with acetic acid). The flow rate was 60 ml/min and the peaks were fractionated by UV. The following compounds were obtained:

diethylaminoated labyrinth peptide a 1:

fractions 7 and 8 were combined to yield 13.3mg (25.2%) of the desired compound after lyophilization. The product was characterized by UV spectroscopy and mass spectrometry (Bruker Daltonics MicroTof).

RT_min5.09min (PDA; LC-method, as in example 8)

UV(λ_max)：218nm(sh)，280nm

ESI-MS(neg)：[M-2H]₂ ^-＝1093.9022

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2189.819

From C₉₆H₁₂₇N₂₅O₂₇S₄Calculated neutral monoisotopic molecular weight: 2189.822

The molecular formula is as follows: c₉₆H₁₂₇N₂₅O₂₇S₄

Formula weight 2191.49.

Monoacetaylated labyrinth peptide a 1:

fractions 10 and 11 were combined to yield 5.3mg (10.3%) of the desired compound after lyophilization. The product was characterized by UV spectroscopy and mass spectrometry (Bruker Daltonics MicroTof).

RT_min5.31min (PDA; LC-method, as in example 8)

UV(λ_max)：217nm(sh)，280nm

ESI-MS(neg)：[M-2H]₂ ^-＝1065.390

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2132.794

From C₉₄H₁₂₄N₂₄O₂₆S₄Calculated neutral monoisotopic molecular weight: 2132.800

The molecular formula is as follows: c₉₄H₁₂₄N₂₄O₂₆S₄

Formula weight 2134.44.

Example 16: synthesis of Boc-protected labyrinth peptide A1

To labyrinth peptide A1(50mg, 0.024mmol) in dimethylformamide (3ml) was added di-tert-butyl dicarbonate (11mg, 0.048mmol) and N-ethyldiisopropylamine (6mg, 0.048mmol) at room temperature. The mixture was stirred at room temperature for 2 hours. Thereafter, it was subjected to reverse phase HPLC using a HPLC equipped with Waters XTerraPhenomenex Luna of Prep MS C1810 μm pre-column (size: 19 mm. times.10 mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm). The gradient was run from 5% to 95% propionitrile/water over 30min (buffer: 0.1% ammonium acetate, pH 7.0). The flow rate was 60 ml/min and the peaks were fractionated by UV. Fractions 4-7 were combined and, after lyophilization, 21.4mg (40.8%) of the desired compound was obtained. The product was characterized by UV spectroscopy and mass spectrometry (bruker daltonics microtorf).

RT_min5.30min (PDA; LC-method, as in example 8)

UV(λ_max)：219nm(sh)，278nm

ESI-MS(neg)：[M-2H]₂ ^-＝1085.895

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2173.805

From C₉₇H₁₂₇N₂₃O₂₇S₄Calculated neutral monoisotopic molecular weight: 2173.815

The molecular formula is as follows: c₉₇H₁₂₇N₂₃O₂₇S₄

Formula weight 2174.49.

Example 17; benzyl derivatives of labyrinthin A1

To labyrinth peptide A1(50mg, 0.024mmol) in dimethylformamide (2ml) was added di-tert-butyl dicarbonate (10mg, 0.046mmol) and N-ethyldiisopropylamine (7mg, 0.054mmol) at room temperature. After 1 hour at room temperature, the maze pep 1 completely disappeared. Benzylamine (6.8mg, 0.063mmol) and n-propylphosphonic anhydride (T3P) were added50 μ l, 0.072mmol in DMF) and the mixture stirred at room temperature for 2 h. Thereafter, it was subjected to reverse phase HPLC using a HPLC equipped with Waters XTerraPhenomenex Luna of PrepMS C1810 μm pre-column (size: 19 mm. times.10 mm)Axia 5. mu. mC18(2) column (size: 100 mm. times.30 mm). The gradient was run from 5% to 95% propionitrile/water over 30min (buffer: 0.1% ammonium acetate, pH 7.0). The flow rate was 60 ml/min and the peaks were fractionated by UV (220 nm). The following two compounds were obtained:

monobenzyl derivative of labyrinthin a 1:

fractions 13 and 14 were combined and 10.4mg (19.1%) of the desired compound was obtained after lyophilization. The product was characterized by UV spectroscopy and mass spectrometry (Bruker Daltonics MicroTof).

RT_min7.03min (PDA; LC-method, as in example 8)

UV(λ_max)：217nm(sh)，275nm

ESI-MS(neg)：[M-2H]₂ ^-＝1130.427

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2262.868

From C₁₀₄H₁₃₄N₂₄O₂₆S₄Calculated neutral monoisotopic molecular weight: 2262.878

The molecular formula is as follows: c₁₀₄H₁₃₄N₂₄O₂₆S₄

Formula weight 2264.63.

Dibenzyl derivatives of labyrinthin a 1:

fractions 7 and 8 were combined and 9.9mg (17.5%) of the desired compound was obtained after lyophilization. The product was characterized by UV spectroscopy and mass spectrometry (Bruker Daltonics MicroTof).

RT_min8.31min (PDA; LC-method, as in example 8)

UV(λ_max)：214nm(sh)，276nm

ESI-MS(pos)：[M+2(NH₄)]₂ ⁺＝1194.002

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2351.937

From C₁₁₁H₁₄₁N₂₅O₂₅S₄Calculated neutral monoisotopic molecular weight: 2351.941

The molecular formula is as follows: c₁₁₁H₁₄₁N₂₅O₂₅S₄

Formula weight 2353.77.

Example 18: acylation reaction at N-terminus of labyrinth peptide A1

To 2-chloro-4, 6-dimethoxy-1, 3, 5-triazine (CDMT, 5mg, 0.028 mmol)/dimethylformamide (2ml) was added N-methylmorpholine (8.6mg, 0.085mmol) at room temperature. After 1 hour at room temperature, n-hexanoic acid (3.3mg, 0.028mmol) was added. After stirring the mixture for 30 minutes, labyrinth peptide A1(50mg, 0.024mmol) was added, followed by stirring at room temperature for 2 h. The mixture was subjected to reverse phase HPLC using a Waters XTerraPhenomenex Luna of Prep MS C1810 μm pre-column (size: 19 mm. times.10 mm)Axia 5. mu. m C18(2) column (size: 100 mm. times.30 mm). The gradient was run from 5% to 95% propionitrile/water over 30min (buffer: 0.1% ammonium acetate, pH 7.0). The flow rate was 60 ml/min and the peaks were fractionated by UV (220 nm). Fraction 39 after lyophilization gave 2.0mg (3.8%) of the desired compound. The product was characterized by UV spectroscopy and mass spectrometry (bruker daltonics microtorf).

RT_min5.41min (PDA; LC-method, as in example 8)

UV(λ_max)：216nm(sh)，266nm

ESI-MS(neg)：[M-2H]₂ ^-＝1084.906

Experimentally determined neutral monoisotopic molecular weight, [ M ] ═ 2171.827

From C₉₈H₁₂₉N₂₃O₂₆S₄Calculated neutral monoisotopic molecular weight: 2171.836

The molecular formula is as follows: c₉₈H₁₂₉N₂₃O₂₆S₄

Formula weight 2173.52.

Example 19: antibacterial activity of labyrinth peptide and derivatives thereof

The compound was dissolved in 10% MeOH in water to a final concentration of 1 mg/ml. For the bioassay, sterilized Nunc plates of 24 x 24cm size were used. For one plate 200ml agar was used. The agar was cooled to 55 ℃ after autoclaving and 2-4ml of tissue suspension of the test organism was added before plating. To each plate, 64 filter plates of 6mm diameter were added. To each filter paper sheet (filter plate) 20. mu.l of the test solution was added and incubated at 28 ℃ or 37 ℃ for 1 to 3 days. The inhibition zone is recorded in mm. For a detailed description of the method, see Bauer et al, amer.j.clin.pathol.1966, 45, 493-; muller & Melchinger, Methoden in der Mikrobiologie, Franckhsche Verlagshandlung, Stuttgart (1964); mueller & Hinton, Proc. Soc. Expt. biol. Med.1941, 48, 330-.

Example 20: neuropathic pain assay

Maze peptide a1 was studied in a mouse model of reserve nerve injury (SNI) for neuropathic pain to confirm its activity on tactile allodynia. Under general anesthesia, adult male C57B6 mice (weight: 22.8+/-0.35SEM) had both major branches of the sciatic nerve ligated and transected, while the sural nerve (sural nerve) was left intact. Tactile allodynia was determined using an automated von Frey test (automated von Frey test): the plantar dermis of its hind paw (plantar skin) was exposed to a pressure stimulus using stack needle stick (dump needle) which increased in intensity up to 5 g. The force (in grams) in response to hindpaw withdrawal was taken as a readout of tactile allodynia. The study was performed 7 days after nerve injury for 6 hours and was again assayed 24 hours later. Within 2 days of nerve transection, tactile allodynia developed completely and remained stable for at least two weeks. The compound was administered intravenously as a single application (3 mg/kg). As the solvent vehicle (vehicle) for the intravenous administration, a 1: 18 solvent vehicle (ethanol: Solutol: phosphate buffered saline) was selected.

The determination of Paw Withdrawal Threshold (PWT) was used to calculate significant therapeutic effect, as well as AUC calculation over the reference period (6 hours) and subsequent% benefit calculation. For statistical analysis, PWT values for the ipsilateral hind paw were used in two ways: the first was done with two-way ANOVA (two-way ANOVA) based on PWT values at a specific time (over a 24 hour period), and the second was done with one-way ANOVA (one-way ANOVA) for the unsealed (non-transformed) AUC difference (delta AUC value) | AUC1-6 hours |.

The use of a two-way Analysis of variance (two-way Analysis of variance) with repeated measures (repeat factor: "TIME", analytical variable: PWT), followed by a Complementary Analysis (comparative Analysis) (effect of factor for each "TIME" factor level "GROUP (GROUP)" factor (wine Analysis), analytical variable: PWT) and subsequently Dunnett's test (two-sided comparison of vs levels VEHICLE) for TREATMENT factors on each level of the TIME factor revealed highly significant differences between each compound and the solvent VEHICLE GROUP over 1 to 6 hours of intravenous administration. This effect disappeared after 24 hours of application. A one-way ANOVA using the difference | AUC1-6 hr | showed a p value of p < 0.0001. Dunnett's analysis gave significant therapeutic effect on maze pep a 1. The percent benefit of the treatment was evaluated using the | AUC1-6 hr | values for the ipsilateral solvent vehicle group (0% benefit) and all | AUC1-6 hr | values contralateral to all three groups (100% benefit-the greatest possible effect). Compared to these limits, the peptide maze A1 achieved 95% benefit.

In conclusion, in the neuropathic pain SNI mouse model, the compound of formula (I) significantly reduced tactile allodynia.

Claims (22)

1. A compound of formula (I)

Wherein

{ A } is a group selected from:

{ B } is a group selected from:

{ C } is a group selected from:

R₁is a group R₁' or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O);

R₂is OH, NH₂，(C₁-C₆) alkyl-NH, phenyl- (C)₁-C₄) alkylene-NH or pyridyl- (C)₁-C₄) alkylene-NH;

R₃and R₄Independently of one another, are H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene group, (C)₁-C₄) Alkyl NHC (O) - (C)₁-C₆) Alkylene or [ (C)₁-C₄) Alkyl radical]₂NC(O)-(C₁-C₆) Alkylene, or R₃And R₄Together with the sulfur atom to which they are attached form a disulfide group S-S;

R₅and R₆Independently of one another, is H or OH, or R₅And R₆Together are ═ O;

m and n are each independently of the other 0, 1 or 2;

wherein the above compounds may be in any stereochemical form, or a mixture of any stereochemical forms in any ratio, or a physiologically acceptable salt thereof.

2. A compound of formula (I) as claimed in claim 1 wherein R₁' is H.

3. A compound of formula (I) as claimed in claim 1 wherein R₂Is OH.

4. A compound of formula (I) as claimed in claim 2 wherein R₂Is OH.

5. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein R₃And R₄Independently of one another, are H, (C)₁-C₆) Alkyl radical, NH₂C(O)-(C₁-C₆) Alkylene, or R₃And R4 together with the S atom to which they are attached form a disulfide group S-S.

6. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein R₃And R₄Is H, or R₃And R₄Together with the S atom to which they are attached form a disulfide group S-S.

7. A compound of formula (I) according to any one of claims 1 to 4, wherein,

R₅and R₆Is H or OH, wherein if R is₅Is OH, then R₆Is H, and if R is₅Is H, then R₆Is OH; or R₅And R₆Together are ═ O.

8. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein R₅Is OH and R₆Is H, or R₅Is H and R₆Is OH.

9. A compound of formula (I) according to any one of claims 1 to 4, characterized by having the formula (II):

wherein R is₁Is R₁' or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (O) or (C)₁-C₆) alkyl-O-C (O), preferably H.

10. A compound of formula (I) according to any one of claims 1 to 4, characterized by having the formula (III):

wherein R is₁Is R₁' or group

Wherein R is₁Is H, (C)₁-C₆) alkyl-C (0) or (C)₁-C₆) alkyl-O-C (O), preferably H;

R₂is OH, NH₂，(C₁-C₆) -alkyl-NH [ (C)₁-C₆) -alkyl radical]₂N, phenyl- (C)₁-C₄) alkylene-NH or pyridyl- (C)₁-C₄) alkylene-NH, preferably R₂Is OH; and

r3 and R4 independently of one another are H, (C)₁-C₆) Alkyl or NH₂C(O)-(C₁-C₄) An alkylene group.

11. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein

m and n are 0; or m and n are 2; or m is 0 and n is 2; or m is 2 and_nis 0.

12. A compound of formula (I) as claimed in any one of claims 1 to 4 wherein m and n are 0.

13. A process for the preparation of a compound of formula (I) according to claim 1, comprising

a) Fermenting the strain Actinomadura namibiensis (DSM6313), or one of its variants and/or mutants, in a medium under suitable conditions until one or more compounds of formula (I) are produced in said medium,

b) isolating the compound of formula (I) from the culture medium, and

c) derivatizing the compound isolated in step b), if appropriate, and/or converting the compound isolated in step b) or a derivative of the compound isolated in step b), if appropriate, into a physiologically acceptable salt.

14. The method of claim 13, wherein the compound isolated in step b) is of formula (II):

wherein m and n are 0, and wherein,

R₁is R₁' or group

Wherein R is₁' is H, and

R₂is OH.

15. Use of a compound according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment of bacterial infections, fungal infections and/or pain.

16. A pharmaceutical composition comprising at least one compound of any one of claims 1 to 12 and at least one pharmaceutically acceptable carrier.

17. DNA encoding prepro-maze pep 2, consisting of the amino acid sequence shown in SEQ ID NO: 13, or a nucleic acid sequence of seq id no.

18. Prepro-maze pep 2, encoded by the amino acid sequence shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.

19. Pro-maze pep 2, consisting of the amino acid sequence shown in SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

20. DNA encoding prepro-maze pep 1, consisting of the amino acid sequence shown in SEQ ID NO: 17.

21. Prepro-maze pep 1, encoded by the amino acid sequence shown in SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof.

22. Pro-maze pep 1, consisting of the amino acid sequence shown in SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof.