ANTIBACTERIAL AND ANTIFUNGAL SUBSTANCES BIPHENYLYL COMPOUNDS

The invention relates to the use of a substance of the general form (I) to produce an antibacterial and/or antifungal drug, wherein X is a methylene group or a carbonyl group; R1, R2, and R3 are each selected from the group comprising hydrogen, an alkyl group having a chain length of 1-4 carbon atoms, an alkoxy group having a chain length of 1-3 carbon atoms, and a halogen; R4 and R5 are each selected from the group comprising hydrogen and an alkyl group having a chain length of 1-4 carbon atoms; and n=3 to 6.

25.7 mmol (6 g) 4-brombiphenyl, 25.7 mmol (3.6 g) 4-chloro butyryl chloride and 32.2 mmol (4.2 g) aluminiumtrichloride are weighed out in a 250 mL piston before 50 mL dryed dichloromethane are added. After stirring overnight at room temperature, the mixture is placed on ice and the precipitated aluminum hydroxide is brought into solution with concentrated hydrochloric acid. Subsequently, the organic phase is separated and extracted with water as long as the water phase shows a neutral pH value. The organic phase is than dried over Na2SO4, and the solvent is filtered and removed under vacuum. The residuum is recrystallized from a mixture of cyclohexane/ethyl acetate.

1.2 mmol (500 mg) NaI are added to 1.2 mmol (184 mg) of the resulting pure product in 5 mL ethylmethylketone and heated for 1½ h at 90° C. in an oil bath. Subsequently, the solution is removed in a vacuum, 1 mL of dimethylamine as well as 30 mL absolute ethanol are added and heated for further 8 h at reflux.

After removing the solvent in the vacuum, ice is added to the residue and the precipitated raw product is aspirated. Recrystallization from cyclohexane/ethyl acetate results 1 in a yield of 82% (377 mg); mp=224° C.

0.02 mol (6.96 g), 1, 5 mL hydrazine monohydrate and 60 mL absolute ethanol are added in a 100 mL piston. Subsequently, it is heated at the reflux until a complete solution occurs (12 h). Afterwards, 40 mL ethanol are removed under vacuum and 40 mL triglycol as well as 10 g of potassium hydroxide and 3 ml hydrazine monohydrate are added. The mixture is heated a further hour at 80° C. (development of gas!) before the temperature is up-regulated until the thermometer indicates 200 to 220° C. The mixture is cooled down and the resulting precipitation is aspirated and recrystallized from ethanol. Yield 85% (5.6 g); mp=245° C.

The melting points of the synthesized substances were measured with the melting point apparatus Büchi 510 device and microhotplate Thermovar (Company Reichert). The NMR spectra were measured with the nuclearmagneticresonancespectrometer Bruker ARX 300 and the IR spectra (as KBr-Presslinge) with a Perkin-Elmer FT-IR 16 PC spectrometer. The mass spectra were measured with a device of type Hewlett-Packard 5989. Elementary analysis was perfomed in the Institute of Inorganic Chemistry, of the CAU Kiel using a CHNS-analyzator of the company Hekatech GmbH. Unless otherwise stated, the chemicals including vancomycin-HCl and tetracycline-HCl at the highest purity were purchased at the company Sigma-Aldrich GmbH.

The minimum inhibitory concentrations of the substances of the present inventions against various infectious germs, i.a. against methicillin-resistantStaphylococcus aureuswere determined with the Bouillon-microdilutionmethod in accordance with the procedure M07-A8 of the Clinical and Laboratory Standards Institute (Pennsylvania, USA).

Test Organisms

Test Vessels

Sterile 96-well microtiterplates of plastic with rounded bottoms of the test wells (wells; BRANDplates™, Ref. 781960).

Cultivation of Test Organisms

The long-term cultures of the test organisms are incubated at titled agar at 22° C. and each inoculated after 4 weeks on fresh medium. After every fifth culture passage or ascertainment of impurities to the correspondent culture is discarded and newly grown from the lyophilized.

Production of Inocula

Suspensions are produced on titled agar by floating with 0.9% sterile NaCl solution. The turbidity is adjusted photometrically by dilution according to the turbidity of the Mc Farland-standard 0.5. Subsequently, the bacterial suspensions are diluted in the ratio of 1:10, the Pi-suspension of the fungi remains undiluted.

The number of the germs in the inocula is adjusted so that there are approximately 5×105colony forming units per milliliter after inoculation of the test wells.

Preparing Stock Solutions of the Antibiotics

The amount of a substance to be tested in a trial is weighed out on a microbalance with an Eppendorf reaction vessel and dissolved in an appropiate volume of dimethyl sulfoxide so that the concentration of the resulting solution corresponds to the 21 times highest final test concentration. The stock solution is subsequently diluted in a serial in a ratio respectively 1 ad 2 with DMSO resulting in seven different concentrations in the wells of a predilutionplate. In the eighth well only DMSO is pipetted.

Loading the Plates

In each of the 96 wells of a test plate 95 μl sterile Mueller Hinton II medium (Cation adjusted, BBL™ Ref 212322) for bacteria and sterile Sabouraud 2% glucose medium (Difco™ Ref 238230) for fungis are added with an eight-channel pipette (Socorex 50-200 μL). 5 μL of the solution to be tested are pipetted in each of the test wells of the plate (8-channel pipette, Eppendorf-Research, 5-10 μL). They are transferred in this way from the pre-dilutions that the horizontal row A of the highest concentration and to row G which shows a half reduced concentration of the substance to be tested and in row H where only DMSO is pipetted. Subsequently, 5 μL of the inocula are pipetted in the test wells using an eight-channel pipette. In one test at least two, maximum four rows are inoculated with the same substance and the same germs. The concentrations of the test substance are each 128 μg/mL to 2 μg/mL.

Evaluation and Incubation of the Microtiter Plates

The prepared plates are converted to a microtiter plate reader (Anthos htlll) which is connected to a printer. In this device, the plates are shaken for 60 seconds with high frequency before each measurement and the absorption of the probes is measured at the wavelength of light of 590 nm.

After determination of the initial values the plates are incubated at 34° C. for 16 to 20 h for bacteria andCandida albicans, and 68 to 72 h at 34° C. for mould fungis.

Following this the microtiterplates are measured again as described above.

Determination of the Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration is the lowest concentration of the tested substance which is able to inhibit the complete growth of the particular test organism, i.e. where after incubation no turbidity is measurable. The particular determination is valid whether in the growth control (DMSO and medium) a distinct turbidity is observed.