DE19617283A1 - Process for the degradation of quinolones and naphthyridones - Google Patents

Abstract

The invention concerns two methods of treating quinolones and naphthyridones having an antimicrobial, in particular antibacterial, action, as well as derivatives of such compounds with wood-rotting fungi, optionally in the presence of water and/or inert solid or liquid substrates and other auxiliaries.

Description

The present invention relates to two methods for the degradation of antimicrobial, especially antibacterial quinolonic and naphthyridonecarboxylic acids and their derivatives with the help of wood rot fungi.

Antimicrobial substances are widely used in medicine and Veterinary medicine. There are problems with the manufacture of these active ingredients in the removal of active ingredient-containing production residues, waste water and Packagings. Waste water from Großver also contains problems users such as clinics, animal breeders and fatteners. There may be other problems with the decontamination of water or soil when in accidents Active ingredient is released undesirably.

In such cases, the antimicrobial activity of the active ingredients can be the usual Prevent degradation by bacteria in soils, water, sewage treatment plants. It can U. even to an undesirable disturbance, e.g. B. biological wastewater treatment plants, come when larger amounts of wastewater containing active substances are processed must, d. H. in all of these cases there is a complex pretreatment of the waste water, Residues etc. required.

For these cases, simple treatment should be found by which Active substances are broken down to such an extent that they lose their antibiotic effect and their further degradation take place using the usual biological degradation methods can.

From a) DE-OS 41 04 624 and b) EP 0 192 237 A1 were processes for the degradation of organic pollutants with the help of lignin-degrading fungi.

However, the degradation in a) is only based on the example of aromatic hydrocarbons shown. Halogen-containing hydrocarbons cannot according to DE-OS 41 04 624 be broken down in this way.  

EP 0 192 237 A1 contains some examples of chlorinated hydrocarbons called, which is broken down by the white rot fungus Phanerochaete chrysosporium will.

The fluorine-containing heteroaromatic quinolones and Naphthyridones from white rot and especially brown rot or even Wood-degrading Ascomycetes are attacked. On the contrary, there is public belief that there are no microorganisms that can break down such connections (see e.g. in Swedish Journal "Land" June 1995, page 4). There is in scientific literature no indication of a significant degradation of such connections Microorganisms. Likewise, the brown rot fungi are generally only very limited ability to split aromatic rings with awarded significantly lower capacities (compared to white rot fungi) (J. Buswell, 1991). In the abstracts of the "Division of Agrochemicals of the American Chemical Society 1993 issue No. 44 "is published in an abstract (No. 92) on the extremely low bioavailability of ¹⁴C-labeled sarafloxacin in Earth samples reported. Sarafloxacin was found to be affected by microorganism not significantly degraded in the soil.

The present invention relates to

• 1. Method for the treatment of antimicrobial, in particular antibacterial acting quinolonic and naphthyridonecarboxylic acids and their derivatives with wood, especially white rot or brown rot fungi, if necessary in the presence of water and / or inert solid or liquid substrates as well as other auxiliary substances.
• 2. Process for the degradation of antibacterial and antimicrobial Quinolonic and naphthyridonecarboxylic acids and their derivatives, thereby characterized in that the active ingredients with wood, especially white or Brown rot fungi optionally in the presence of water and / or inert treated solid or liquid substrates and other auxiliary substances will.  
• 3. Process for treating wastewater which is antibacterial and anti microbial quinolonic and naphthyridonecarboxylic acids and their Derivatives with wood, especially white or brown rot fungi.
• 4. Process for the treatment of manure from intensive animal husbandry, the antibacterial rial and antimicrobial quinolonic and naphthyridonecarboxylic acids and their derivatives contains with wood, especially white or Brown rot fungi.
• 5. Method of treating floors that are antibacterial and antimicrobial acting quinolonic and naphthyridonecarboxylic acids and their derivatives contain with wood, especially white or brown rot fungi.

The best-known wood-destroying Basidiomycetes are divided into 2 large groups subdivided: on the one hand into the white rot decomposing lignin, on the other hand into the Cellulose and hemicellulose decomposing brown rot fungi, which lignin in the do not degrade essentially, but only by hydroxylation and Modify demethylations. White rot fungi are characterized by extracellular Enzymes such as lignin peroxidases, manganese peroxidases and laccases, which the Brown rot fungi are usually absent. Both groups can be identified by chemical Distinguish detection methods (see e.g. Stalpers, 1978, Studies in Mycology, No. 16, Centralbureau voor Schimmelcultures, Baarn, Holland). At Brown rot fungi is of crucial importance to the hydroxyl radical the mining activity too. The big differences between white and Brown rot fungi, especially with regard to the molecular mechanisms of the Timber degradation are documented in the specialist literature (see e.g. Evans et al., FEMS Microbiological Reviews 13 (1994) 235-240).

As examples of species of wood rot fungi, which in the invention Methods that can be used include:

White rot fungi:
Bjerkandera sp.
Cladoderris sp.
Coriolopsis sp.
Coriolus sp.
Ganoderma sp.
Irpex sp.
Phanerochaete sp.
Phellinus sp.
Phlebia sp.
Pleurotus sp.
Trametes sp.
Trichaptum sp.

Brown rot fungi:
Fomitopsis sp.
Gloeophyllum sp.
Leatiporus sp.
Pholiota sp.
Serpula sp.

Litter remover:
Agrocybe sp.
Hypholoma sp.
Stropharia sp.

Other:
Xylaria sp. (Ascomycet)
The following species are preferred:

White rot fungi:
Bjerkandera adusta DSM 4708
Cladoderris dendritica DSM 9611
Coriolopsis rigida DSM 9596
Coriolus sp.
Ganoderma lucidum DSM 9621
Irpex lacteus DSM 9595
Phanerochaete chrysosporium DSM 9620
Phanerochaete chrysosporium DSM 1556
Phanerochaete chrysosporium ATCC 24725
Phellinus gilvus DSM 9602
Phlebia radiata DSM 5111
Pleurotus sp. DSM 9618
Trametes versicolor DSM 3086
Trametes villosa DSM 9589
Trichaptum bysogenum DSM 9597

Brown rot fungi:
Fomitopsis spraguei DSM 9600
Gloeophyllum sepiarium DSM 6420
Gloeophyllum striatum DSM 9594
Gloeophyllum striatum DSM 9592
Gloeophyllum striatum DSM 10335
Gloeophyllum trabeum DSM 3087
Leatiporus sulphureus DSM 2785
Pholiota aurivella DSM 5070
Serpuia lacrymans DSM 8728

Litter remover:
Agrocybe sp.
Hypholoma sp.
Stropharia rugosoannulata DSM 9616
Stropharia sp.

Other:
Xylaria digitata DSM 914 (Ascomycet)
The following are particularly preferred:

White rot fungi:
Irpex lacteus DSM 9595
Phanerochaete chrysosporium DSM 1556
Phanerochaete chrysosporium DSM 9620

Brown rot fungi:
Gloeophyllum striatum DSM 9592
Gloeophyllum striatum DSM 9594
Gloeophyllum striatum DSM 10335

Litter remover:
Stropharia rugosoannulata DSM 9616

Wood rot fungi are known. The above species of wood rot fungi are contained in public collections of microorganisms and freely accessible, e.g. B. those marked with DSM at the German Collection for Microorganisms (DSMZ), Mascheroder Weg 1b, 38116 Braunschweig.

It is easily possible to change one of the species mentioned to another to replace the same group.

As an antibacterial and antimicrobial quinolone and naphthyri Doncarboxylic acids are the compounds of the following formulas (I) and (II) called:

in which
X¹ and X² are independently hydrogen, halogen, C _1-4 alkyl, C _1-4 alkoxy, NH₂,
Y for structural remains

stands in what
R¹⁶ = H, -CH₃, -CH = CH-CO₂R¹⁶ ', -CH₂-CH₂-CO₂R¹⁶', -CH₂-CO-CH₃, -CH₂-CH₂-CN
R¹⁶ ′ = Me, Et
D = O, -CH₂-
R⁴ represents hydrogen, optionally substituted by hydroxyl or methoxy, straight-chain or branched C₁-C₄-alkyl, cyclopropyl, acyl having 1 to 3 C atoms,
R⁵ represents hydrogen, methyl, phenyl, thienyl or pyridyl,
R⁶ represents hydrogen or C _1-4 alkyl,
R⁷ represents hydrogen or C _1-4 alkyl,
R⁸ represents hydrogen or C _1-4 alkyl,
R⁹ represents hydrogen, methyl or -CH₂-NR⁷R⁸,
R¹⁰ for hydrogen, C _1-4 alkyl, amino, optionally substituted by hydroxy substituted alkyl or dialkylamino with 1 or 2 carbon atoms in the alkyl part, amino-C _1-4 alkyl, C _1-4 alkylamino-C _{1- 4} -alkyl or 1-imidazolyl,
R¹¹ represents hydrogen, hydroxy, methoxy, methylthio, halogen, methyl, hydroxymethyl,
R¹² represents hydrogen or methyl,
R¹³ represents hydrogen or C _1-4 alkyl,
R¹⁴ represents hydrogen or C _1-4 alkyl and
R¹⁵ represents hydrogen or C _1-4 alkyl,
R 1 represents hydrogen, an optionally halogen-substituted alkyl radical having 1 to 3 carbon atoms, cyclopropyl, methoxy, 4-fluorophenyl, 2,4-difluorophenyl or methylamino,
R² represents hydrogen or alkyl which has 1 to 6 carbon atoms and is optionally substituted by methoxy or 2-meth oxyethoxy, and also cyclohexyl, benzyl, 2-oxopropyl, phenacyl, ethoxycarbonylmethyl, pivaloyloxymethyl,
R³ represents hydrogen, methyl or ethyl and
X¹ and Y together with the adjacent carbon atoms represent a dioxolanyl radical or dioxoamyl radical.

A stands for nitrogen or = CH-, = C (halogen) -, = C (OCH₃) - = C (CH₃) -, = C (CN) -,
B represents oxygen, optionally substituted by methyl or phenyl = N- and = CH₂,
Z stands for = CH- or = N-,
and their pharmaceutically usable hydrates, acid addition salts and salts with bases. The compounds of the formulas I and II can be present in the form of their racemates or in enantiomeric forms.

Compounds of the formulas (1) and (II) are preferred
in which
R¹ represents C₁-C₃-alkyl optionally substituted by fluorine or cyclopropyl,
R¹ represents an alkyl radical with 1 to 3 carbon atoms, cyclopropyl, 2-fluoroethyl, methoxy, 4-fluorophenyl, 2,4-difluorophenyl or methyl amino,
R² represents hydrogen or alkyl which has 1 to 6 carbon atoms and is optionally substituted by methoxy or 2-methoxyethoxy and cyclohexyl, benzyl, 2-oxopropyl, phenacyl, ethoxycarbonyl methyl, pivaloyloxymethyl,
R³ represents hydrogen, methyl or ethyl and
A stands for nitrogen or = CH-, = C (halogen) -, = C (OCH₃) -, = C (CH₃) -, = C (CN) -,
B represents oxygen, optionally substituted by methyl or phenyl = N- and = CH₂,
Z stands for = CH- or = N-,
X¹ and X² independently of one another represent hydrogen, halogen or NH₂,
Y for structural remains

stands in what
R¹⁶ = H, -CH₃,
D = O, -CH₂-
R⁴ represents hydrogen, optionally substituted by hydroxy straight-chain or branched C₁-C₃-alkyl, oxalkyl with 1 to 4 C-Ato men,
R⁵ represents hydrogen, methyl or phenyl,
R⁶ represents hydrogen or methyl,
R⁷ represents hydrogen or methyl,
R⁹ represents hydrogen, methyl or -CH₂NH₂,
R¹⁰ represents hydrogen, methyl, amino, methylamino, dimethylamino, aminomethyl, methylaminomethyl or ethylaminomethyl,
R¹¹ represents hydrogen, hydroxy, methyl, fluorine, methyl or hydroxymethyl,
R¹³ represents hydrogen or methyl,
R¹⁴ represents hydrogen or methyl,
R¹⁵ represents hydrogen or methyl,
and their pharmaceutically usable hydrates and acid addition salts and their alkali, alkaline earth, silver and guanidinium salts.

Compounds of the formula (I) in which
R¹ represents an alkyl radical with 1 to 3 carbon atoms, cyclopropyl, 2-fluoroethyl, methoxy, 2,4-difluorophenyl or methylamino,
R² represents hydrogen or alkyl which has 1 to 6 carbon atoms and is optionally substituted by methoxy or 2-meth oxyethoxy, and also cyclohexyl, benzyl, 2-oxopropyl, phenacyl, ethoxycarbonylmethyl, pivaloyloxymethyl,
A represents nitrogen or = CH-, = C (halogen) -, = C (CH₃) -, = C (OCH₃) -, = C (CN) -,
X¹ and X² are independently hydrogen, fluorine, NH₂,
Y for structural remains

stands in what
R¹⁶ = H, -CH₃
D = O, -CH₂-
R⁴ represents hydrogen, optionally substituted by hydroxy straight-chain or branched C₁-C₃-alkyl, hydroxyalkyl having 1 to 4 carbon atoms,
R⁵ represents hydrogen, methyl or phenyl,
R⁶ represents hydrogen or methyl,
R⁷ represents hydrogen or methyl,
R⁹ represents hydrogen, methyl or -CH₂NH₂,
R¹⁰ represents hydrogen, methyl, amino, methylamino, dimethylamino, aminomethyl, methylaminomethyl or ethylaminomethyl,
R¹¹ represents hydrogen, hydroxy, methyl, fluorine, methyl or hydroxymethyl,
R¹³ represents hydrogen or methyl,
R¹⁴ represents hydrogen or methyl,
R¹⁵ represents hydrogen or methyl,
and their pharmaceutically usable hydrates and acid addition salts and their alkali, alkaline earth, silver and guanidinium salts.

The active ingredients with the common names are particularly preferred Enrofloxacin, ciprofloxacin, danofloxacin, ofloxacin, norfloxacin, benofloxacin, Sarafloxacin, Difloxacin, Orbifloxacin, Marbofloxacin and Oxolinic Acid.

The active substances enrofloxacin, ciprofloxacin and marbo may be mentioned in particular floxacin, ofloxacin, norfloxacin and oxolinic acid.

The process according to the invention is preferably in liquid, solid or semi-solid homogeneous or heterogeneous media performed.

In addition to the active ingredient to be broken down and the wood rot fungus Material containing lignin, lignocellulose and / or cellulose. These include straw, Chopped wood, wood chips, wood flour and grain residue.

The procedures can be described as follows:  

1. Process for the degradation of quinolone dissolved in water

The preculture of one of the listed wood rot fungi that

• - in malt bouillon, which malt extract or an equivalent Carbon and energy source in the concentration of 1-50 g / l, preferably contains 5-30 g / l, in particular 5-8 g / l,
• at 5-40 ° C, preferably 15-30 ° C, especially at 18-22 ° C,
• - standing or shaken, preferably shaken at 80-120 rpm,
• for a period of 3-28 days, preferably 5-14 days, especially 5-7 days,
• - The lighting in the dark, in daylight or at all intermediate levels cultivated

a rinse water, e.g. B. from the manufacturing process for a quinolone containing drug, containing from 0.01 to 500 ppm, preferred 0.1 to 50 ppm, especially 1 to 20 ppm of the quinolone in the ratio from 0.01 to 100 g dry weight TG / l, preferably 0.1 to 10 g TG / l, in particular 1 to 3 g TG / l added.

The pH of the suspension can be 3 to 8, preferably 4 to 7, in particular 5 to 6; Phosphate is not added. Organic acids, such as B. Acetic acid or oxalic acid can be used to adjust the pH will.

The iron ²⁺ or iron ³⁺ content should be 0.1 to 100 μM (10 ^-6 mol per liter), preferably 1 to 30 μM, in particular 2 to 20 μM,
the content of NH₄⁺ can be 0.001 to 20 mM (10 ^-3 mol per liter), preferably 0.1 and 10 mM, in particular between 0.1 to 1 mM.

The content of divalent cations should be between 0.1 and 10 mM, preferably be 1 mM (e.g. 0.2 mM CaCl₂ + 0.8 mM MgSO₄).  

This suspension is incubated under the same conditions as the previous one Culture.

The addition of coniferyl or veratryl alcohol in a concentration of 0.01 to 10 mM, preferably 0.1 to 3 mM, in particular 0.5 to 1 mM an increase in the degradation activity for the quinolone.

2. Process for mining straw, manure or soil Quinolone

Starting from stock cultures of wood rot fungi on petri dishes with Malt agar are substances containing lignocellulose, in particular straw, Wood flour or wood shavings, with those cut from the stock culture Inoculated agar blocks, whereby 0.1 to 100 g agar per kg solid substrate, preferably 1 to 10 g of agar are used per kg of substrate.

The solid matrix (straw, manure, quinolone (0.1 to 200 ppm) Soil, mud) is inoculated with water before inoculation a water content of 10 to 90%, preferably 40 to 50% (based on the weight).

When inoculated, the inoculum is distributed in the solid matrix. The subsequent incubation at 5 to 40 ° C, preferably 15 to 30 ° C, ins especially at 18 to 22 ° C.

The following examples illustrate the processes according to the invention:

Examples example 1

One liter of rinsing water from a formulation kettle with a content of 10 ppm Enrofloxacin is used with a preculture of Gloeophyllum striatum DSM 9592 or Inoculates Gloeophyllum striatum DSM 10335, the fungal mycelium from the Pre-culture in 1/4 concentrated malt broth in this way into the rinse water is transferred that a concentration of 2 g dry weight per liter is reached becomes. This suspension is at room temperature with shaking at 100 rpm Incubated in the dark. After 2 weeks, 10 to 20% of the [Carbo nyl-¹⁴C] -Enrofloxacins degraded to ¹⁴CO₂.

Example 2

1 kg of wheat straw, which contained 2 mg of enrofloxacin adsorptively bound, was mixed with 1 kg of water moistened and inoculated with Gloeophyllum striatum DSM 9594. The Inoculum consisted of 20 agar blocks, each weighing approximately 0.5 g. This Blocks were from a preculture on malt agar from the mushroom colony in one Petri dish has been cut out. The approach was at room temperature in Incubate in the dark with humidified air. After 8 weeks 50% of the enrofloxacin is broken down.

Example 3

0.5 kg of dampened wood shavings were made with Phanerochaeta chrysosporium DSM Inoculated 9620 and pre-incubated for 1 week at room temperature. To this approach were 0.5 kg of manure from an animal population treated with enroflaxacin was added. The enrofloxacin content in the manure was 2 ppm. The approach was incubated at room temperature.

Example 4

0.1 kg of wheat straw was moistened with 100 ml of water and with Gloeophyllum inoculated striatum DSM 9594. After 7 days of pre-incubation at room temperature the straw was grown with 0.6 kg of arable soil (with a water content of about 50%)  layered containing 10 ppm enrofloxacin and at room temperature incubated. After 15 weeks, 6% of the enrofloxacin used had been broken down.

Claims (9)

1. Method for the treatment of antimicrobial, in particular antibacterial acting quinolonic and naphthyridonecarboxylic acids and their derivatives with wood rot fungi, optionally in the presence of water and / or inert solid or liquid substrates and other auxiliary substances. 2. Process for the degradation of antimicrobial, in particular antibacterial acting quinolonic and naphthyridonecarboxylic acids and their derivatives, characterized in that the active substances with rot rot fungi if necessary, in the presence of water and / or inert solid or liquid Substrates and other auxiliaries are treated. 3. Method of treating wastewater that is antimicrobial, in particular antibacterial quinolonic and naphthyridonecarboxylic acids and their Derivatives contain with wood rot fungi. 4. Process for the treatment of manure from intensive animal husbandry of the anti microbial, especially antibacterial quinolone and naphthyri Doncarbonic acids and their derivatives contains with wood rot fungi. 5. Method of treating floors which are antimicrobial, in particular antibacterial quinolonic and naphthyridonecarboxylic acids and their Derivatives contain with wood rot fungi. 6. The method according to claims 1 to 5, characterized in that as Active ingredient enrofloxacin or ciprofloxacin is present. 7. The method according to claims 1-6, characterized in that White rot fungi can be used for degradation. 8. The method according to claims 1-6, characterized in that Brown rot fungi can be used for degradation. 9. The method according to claims 1-6, characterized in that wood-degrading ascomycetes are used.