DE4315884C2 - Moenomycin C¶1¶ and derivatives, process for the isolation and derivatization of Moenomycin C¶1¶ and use of Moenomycin C¶1¶ and its derivatives as antibiotics - Google Patents

Description

The invention relates to Moenomycin C₁, a process for the isolation of Moenomycin C₁ from a moenomycin C mixture, moenomycin C₁ derivatives Process for the preparation of moenomycin C₁ derivatives and the use of these compounds as antibiotics.

That from the fermentation broth of Streptomyces ghanaenis and others Moenomycin A obtained from related microorganisms is structurally related with moenomycin C₁. The structure of moenomycin A was published in 1981 (P. Welzel et al. Angew. Chem. Int. Ed. Engl. 20, 121-123 (1981)) and revised in 1990 (H.-W.-Fehlhaber, M. Girg, G. Seibert, K. Hobert, P. Welzel, Y. van Heÿenoort and J. van Heÿenoort, Tetrahedron 46, 1557-1568, (1990)). It is a complicated one built phosphoglycolipid antibiotic. So far, Moenomycin A has mainly used in veterinary medicine as an additive to animal feed.

In DE-OS 37 04 659 is also a Isolation process and the structure elucidation of Moenomycin C₃ from one Moenomycin C mixture described. Moenomycin C₃ is antibiotic and also structurally related to moenomycin C₁.  

In Tetrahedron 49 (15) 1993, pages 3091-3100, Moenomycin-A and its derivatives disclosed that show antibiotic activity and the Moenomycin C₁ are structurally very similar. From the same publication (page 3097) also shows that for the inhibitory effect of this class of substances certain structural requirements are necessary, namely that Presence of those generally designated with the letters E, F, G, H and I. Structural units.

As can be seen from Tetrahedron 49 (8), pp. 1635-1648 (1993) Compounds in this class of substances have different antibiotic activities, some of which can even be classified as antibiotic inactive. One from the same Publication known antibiotic inactive compound differs for example of the antibiotic active moenomycin A derivatives by the spatial arrangement of the OH group at C₄ of the structural unit F, a derivative of moenuronic acid amide. While the OH group on structural unit F des antibiotically inactive derivatives takes an axial position, the antibiotic active moenomycin A derivatives at the appropriate place an equatorial arranged OH group and also an axial methyl group.

The object of this invention is to create a further component from a Isolate and examine moenomycin C mixture to find a substance find that can be used as a drug with good efficacy.  

The object is achieved by a chromatographic Isolation process for obtaining moenomycin C₁ from a moenomycin C mixture, which is a component of a moenomycin A / C complex, and if necessary. The derivatization of Moenomycin C₁.

With the insulation method according to the invention and the following Structure elucidation of the compound Moenomycin C₁ could find a substance which is a new active ingredient in the group of antibiotics and has antibiotic activity.

Moenomycin C₁ and the Moenomycin C₁ derivatives all have C₄ the Structural unit F has an axial OH group. According to those in Tetrahedron 49 (8), S. 1635-1643 (1993), the structure-effect designations presented should therefore no expected antibiotic effects from Moenomycin C₁ and its derivatives his.

Accordingly, it is surprising that moenomycin C₁ and through Derivatization prepared compounds according to the present invention are antibiotic.  

Accordingly, the present invention relates to Moenomycin C₁ of formula I.

a process for the isolation of moenomycin C₁ from a moenomycin C mixture by chromatographic purification, characterized in that moenomycin C₁ with an aqueous eluent mixture of 2-4 components from a moenomycin C mixture is obtained by HPLC chromatography on reversed-phase silica gel,
a compound of formula II

a compound of formula III

and a compound of formula IV

The present invention further relates to a method for producing the Compounds II, III and IV, which is characterized in that the compound of formula I catalytically hydrogenated to compound II, optionally compound II with a Oxidizing agent to compound III and possibly the compound III with a Oxidizing agent for compound IV is implemented.

The compounds of formulas I-IV are preferably for use as Suitable antibiotics.

The invention is described in detail below, in particular in its preferred embodiments. Furthermore, the invention is characterized by the content of the Claims determined.

A moenomycin A / C complex is obtained, for example, from Flavomycin® Extraction using alcohol and purification using filters and Chromatography or from the fermentation mixture of Streptomyces ghanaensis.  

Obtaining a Moenomycin C Mixture from a Moenomycin A / C DE-OS 37 04 659, for example, is complex described. This method is a chromatographic one Cleaning. The Moenomycin A / C complex is used for this Column chromatography e.g. B. on silica gel using a polar organic solvent and an organic or inorganic base as Moenomycin A is removed from the solvent. For this purpose it is useful to use (C₁-C₃) - Alcohols, low molecular weight ketones and esters with max. 8 carbon atoms, alkali eyes, Ammonia solution and secondary or tertiary amines such as Methanol, ethanol, n-propanol, isopropanol, acetone, sodium hydroxide solution, triethylamine and Pyridine. However, isopropanol and aqueous ammonia solution are preferred. The polar organic solvents and the organic or inorganic base are used in a ratio of 5: 5 to 9: 1, preferably in a ratio of 8: 3.

The moenomycin C mixture thus obtained is subsequently in the Process according to the invention for the isolation of moenomycin C₁ by chromatographic cleaning used. Chromatographic purification done by HPLC chromatography on reverse phase silica gel Moenomycin C₁ with an aqueous eluent mixture (eluent) from 2-4 Components, preferably 3 components, from a moenomycin C mixture wins. The ratio of the components to each other is chosen so that each Component at least a 5% share - based on the total amount of Elution mixture - has, however, all components of the mixture together 100% result. The total amount of the elution mixture is 0.5-10 l, preferably 1 l, used.

Appropriately used as eluents: halogenated (C₁-C₂) alkanes, (C₁- C₃) alcohols, low molecular weight ketones, aliphatic esters and nitriles with a maximum 8 carbon atoms and aliphatic and heterocyclic ethers such. B. methylene chloride, Chloroform, methanol, ethanol, n-propanol, isopropanol, acetonitrile, ethyl acetate, Diethyl ester, tetrahydrofuran and dioxane. Methanol, acetonitrile and water are preferred.  

Based on the spectroscopic investigations for Moenomycin C₁ a structure of formula I:

By successively applying the degradation processes already used for Moenomycin A had led to fission products and those in the DE-OS 33 01 430 are described, is by catalytic hydrogenation of moenomycin C₁ on a platinum dioxide catalyst, a hydrogenation product of formula II producible. If you put the compound of formula II in a subsequent Reaction step with an oxidizing agent, preferably with Potassium hexacyanoferrate (III) or with ozone, can be a compound of Isolate formula III, in which the rest called chromophore the Compound of formula II is split off. Implementation of the connection of the Formula III again with an oxidizing agent, preferably with 2 mol Sodium metaperiodate, leads to the splitting off of the terminal sugar unit Obtaining a compound of formula IV.  

The following sequence explains the reaction sequence:

The isolation method according to the invention makes it possible to find the Compound moenomycin C₁, which as a medicament, in particular as an antibiotic, can be used. The Moenomycin C₁ derivatives are antibiotic effective.

The antibiotic activity of Moenomycin C₁ and its derivatives is based on an inhibition of transglycosylase, a key enzyme in biosynthesis the bacterial cell wall. As an exact evaluation criterion for these substances consequently use an in vitro test in which an inhibition of the Transglycosylase can be detected. Such a test model was made as early as 1982 by Höltje et al. presented (K. Schaller, J. V. Höltje, V. Braun; J. Bacteriology 152/3, 994-1000 (1982)).

The test is based on the following principle:
C-UDP-N-acetylglucosamine and N-acetylmuramic acid polymerize in the presence of a transglycosylase obtained from membrane preparations of E. coli. The degree of polymerization can be concluded from the radioactive incorporation rate of the ¹⁴C atom in the macromolecular substance. The result is given as a percentage inhibition of transglycosylase at a certain concentration.

Moenomycin C₁, Moenomycin C₁ derivatives and the corresponding physiologically compatible salts are suitable due to their valuable pharmacological Properties very good for use as a remedy. Subject of the invention hence medicinal products containing at least one compound of Formula IV. The medicaments are preferably suitable for prophylaxis and / or Therapy of bacterial diseases.

The pharmaceuticals according to the invention are generally orally or parenterally administered, but also rectal use is possible in principle. Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, Powders, tablets, coated tablets, (micro) capsules. Suppositories, syrups, emulsions, Suspensions, aerosols, drops or injectable solutions in ampoule form as well Preparations with protracted active ingredient release, usually in their manufacture Carriers and additives and / or auxiliaries such as explosives, binders, coatings, Swelling, lubricating or lubricating agents, flavorings, sweeteners or Find solution brokers. As a commonly used carrier or Auxiliaries are z. B. magnesium carbonate, titanium dioxide, lactose, mannitol and others Sugar, talc, milk protein, gelatin, starch, vitamins, cellulose and their derivatives, animal and vegetable oils, polyethylene glycols and solvents such as sterile Called water, alcohols, glycerin and polyhydric alcohols.

The pharmaceutical preparations are preferably in dosage units manufactured and administered, each unit as an active ingredient contains a certain dose of at least one compound of the formula I-V. With fixed Dosage units such as tablets, capsules and suppositories can reduce this dose up to about 500 mg, but preferably about 50 to 300 mg, and at  Injection solutions in ampoule form up to 150 mg, but preferably about 10 to 100 mg.

For the treatment of an adult patient - depending on the effectiveness of the Compounds according to formula I-V in humans - daily doses of about 20 to 500 mg of active ingredient, preferably about 50 to 300 mg, when administered orally and from about 5 to 300 mg, preferably about 10 to 100 mg, with intravenous administration indicated. However, higher or lower may also be possible Daily doses may be appropriate. The administration of the daily dose can be both by single administration in the form of a single dosage unit or several smaller dosage units as well as doses divided into multiple doses at certain intervals.

The medicaments according to the invention are produced by at least one compound of formula IV with conventional carriers - and optionally additives and / or auxiliary substances in the or a suitable Dosage form brings.

The methods for obtaining compounds mentioned in the description of the formulas I to V are explained in more detail by the following examples.

Example 1 Preparation of the Moenomycin A / C complex

75 kg of spray-dried Flavomycin® in 300 l of 80% methanol Stirred for 2 hours. The insoluble residue is separated off and the filtrate is after concentration to approx. 45 l, adjusted to pH 7.0 with 35% sulfuric acid. The combined organic phases are extracted twice with 10 l each of n-butanol are washed with 5 l of water and the combined aqueous phases finally concentrated, centrifuged and filtered clear. This degreased, aqueous Concentrate is washed dialysis (Amicon dialysis station) chloride-free. The  resulting retenate is mixed with 16 l ®Dowex 1 × 2 (chloride form) and stirred. After filtration, dialysis is carried out again. This process is done five times carried out. Then the ion exchanger with the following solutions washed: 60 l water, 60 l 10% formic acid, 80 l one Methanol / formic acid / water mixture of the composition 8/1/1 and 60 l 80% methanol. The product is then with a 1% Potassium chloride solution eluted in 80% methanol. After concentrating the 180 l resulting eluate is concentrated to an aqueous solution, dialysis-free chloride washed and concentrated to 3.5 l. At -5 ° C is slowly in 130 l acetone added dropwise, stirred for a further 2 hours and left to stand overnight. Of the Precipitate is then separated off, it is again with 10 l of acetone offset and separated again. After drying in vacuo at room temperature an amount of 1.2 kg of moenomycin A / C complex.

Example 2 Purification of the Moenomycin A / C complex, extraction of the Moenomycin C links

12 g moenomycin A / C complex containing 7 g antibiotic active Components are added to 300 g of silica gel (manufacturer: Grace, 60-200 pm) chromatographed. The substance is added to 24 g of the same Silica gel material and grown on with isopropanol / 2 N aqueous Ammonia solution in 8/2 equilibrated column. It comes with 1 l elutes this system, then isopropanol / 2 N aqueous ammonia solution in Ratio 8/3 used and after 600 ml is in fractions of 100 ml caught. The fractions are thin layer chromatographed in the system Isopropanol / concentrated aqueous ammonia solution in a ratio of 65/35 as well via HPLC on Spherisorb ODS 5 µm with the system methanol / acetonitrile / 0.02 % phosphate buffer (pH 7.8) in the ratio 4/1/5 and UV detection at 258 nm analyzed. After the corresponding fractions have been combined, it is neutralized concentrated and precipitated with 20 times the volume of acetone. In fractions 2 to 5  1.05 g of moenomycin C mixture are obtained in this way.

Example 3 Preparation of Moenomycin C₁ (Compound of Formula I)

1.35 g of moenomycin C mixture are dissolved in the eluent (methanol: acetonirtile: water = 52: 8:40) and on 500 g of Merck LiChroprep RP-18 (25-40 µm) in a cartridge system with a flow of 25 ml / min and one Fractionation of 15 ml / fr. chromatographed. The corresponding fractions are combined, concentrated to the aqueous phase and then lyophilized. There will be 128 mg of pure Moenomycin C₁, which is characterized by NMR and mass spectrum.
FAB mass spectrum:
m / z = 1450.6 ([M + Na + KH] ⁺), 1434.5 ([M + 2Na-H] ⁺), 1412.6 ([M + Na] ⁺), 1006.2, 886.3, 668.2, 459.2

Example 4 Preparation of Decahydromoenomycin C1 (Compound of Formula II)

A mixture of moenomycin C1 (123.0 mg, 89 mmol) methanol (12.4 ml), PtO2.H₂O (37.2 mg) and acetic acid (0.46 ml) is stirred at 20 ° C under normal pressure under H₂ for 3 hours. After filtration and evaporation of the filtrate, the residue is chromatographed on RP-18 silica gel with the eluent methanol: water: acetonotrile = 6: 3: 1. Fractions containing compound II are combined, the organic solvent is removed on a rotary evaporator and the aqueous phase is lyophilized (yield 84.6 mg of compound II).
FAB mass spectrum:
m / z = 1460.6 ([M + Na + KH] ⁺), 1422.7 ([M + Na] ⁺), 1006.3, 886.3, 668.3, 559.5, 459.2

Example 5 Preparation of a compound of formula III

To a solution of compound II (84.5 mg, 0.06 mmol) in water (2.7 ml), solutions of K₂CO₃ (136 mg, 0.986 mmol) in water (0.21 ml) and K₃ [Fe (CN) ₆] ( 409.9 mg, 1.245 mmol) in water (0.5 ml) added. The mixture is stirred for 30 min, then warmed to RT, stirred for a further 2.5 hours and adsorbed on HP-20 resin. It is washed with 600 ml of water. Compound III is eluted with 1 liter of methanol. After evaporating the methanol and lyophilizing the aqueous phase, 80.6 mg of the pure compound III, which was characterized as follows:
FAB mass spectrum:
m / z = 1305 ([M + H] ⁺), 768, 550, 537, 363, 176

Example 6 Preparation of the compound of formula IV

To perform the cleavage reactions with sodium metaperiodate, two Reagent solutions are made:

The oxidation solution is composed of 1.07 g (5 mmol) Sodium metaperiodate and 1.38 g (10 mmol) sodium acetate trihydrate in 12 ml 50% acetic acid. In order to obtain a clear solution, the mixture must be added 80 ° C are stirred. The solution must be freshly made for every attempt. For Preparation of the dimethylhydrazine solution is 0.94 ml of N, N-dimethylhydrazine 0 ° C in 2.80 ml isopropanol and with 6.4 ml of 2 N sulfuric acid pH 4.5 adjusted. For each aldehyde group there are 2 equivalents of the freshly prepared one Add solution.

1.4 ml of the oxidizing solution are added to a solution of compound III (288.7 mg, 0.221 mmol) in the smallest possible volume of water and the mixture is stirred at 40 ° C. for 3 hours with the exclusion of light. After the reaction has ended, the reaction solution is chromatographed on 60 g of HP-20 with initially 600 ml of water and then 1 l of methanol, the pH of the eluate rising from pH 3.5 to 6.5. Aqueous eluates from pH 5.5 and the methanol eluate are combined and the solvents are removed by vacuum distillation and subsequent lyophilization. The residue (221.1 mg) is taken up in as little water as possible, mixed with 0.62 ml of the dimethylhydrazine solution and stirred at 80-85 ° C for 3.5 hours. After cooling, it is adsorbed on 60 g of HP-20, washed with 600 ml of water and eluted with 1 l of methanol. After vacuum distillation and lyophilization, the methanol eluate gives 142.7 mg of crude mixture with compound IV. The purification is carried out on silica gel (three times) using the chloroform: methanol: water system = 10: 6: 1 and gave 97 mg of pure compound IV, which is characterized as follows has been:
FAB mass spectrum:
m / z = 1168 ([M + K] ⁺), 1152 ([M + Na] ⁺), 1130 ([M + H] ⁺), 615, 593, 559, 537, 397, 375.188

Example 7 Transglycosylase test

The following table gives inhibition values for transglycosylase in three different types Concentrations of the substances tested in each case.

Claims (11)

1. Moenomycin C₁ of formula I. 2. Process for the isolation of Moenomycin C₁ from a Moenomycin C- Mixture by chromatographic purification, characterized in that by HPLC chromatography on reverse phase silica gel Moenomycin C₁ with an aqueous eluent mixture of 2-4 Components obtained from a moenomycin C mixture. 3. A method for isolation according to claim 2, characterized in that the aqueous elution mixture from one or more of the following Components consist: halogenated (C₁-C₂) alkanes, (C₁-C₃) alcohols, low molecular weight ketones, aliphatic esters and nitriles with max. 8 carbon atoms as well as aliphatic and heterocyclic ethers. 4. A method for isolation according to one or more of claims 2 and 3, characterized in that the aqueous eluent mixture from 3 Components. 5. A method for isolation according to one or more of claims 2 to 4, characterized in that as an aqueous eluent mixture Methanol, acetonitrile and water can be used.   6. Compound of formula II 7. Compound of formula III 8. Compound of formula IV 9. A process for the preparation of the compounds II, III and IV, thereby characterized in that the compound of formula I catalytically Compound II hydrogenated, possibly compound II with an oxidizing agent Reacts compound III and possibly the compound III with an oxidizing agent to compound IV. 10. Use of at least one of the compounds of the formulas I-V as Antibiotic. 11. Use of at least one of the compounds of the formulas I-V for Manufacture of a drug with an antibiotic effect.