WO1998025931A1

WO1998025931A1 - Microorganism capable of producing compounds where pyrrole is fused with 4-oxo-1,3-benzoxazine and method of use as antibacterial and antifungal

Info

Publication number: WO1998025931A1
Application number: PCT/US1997/021694
Authority: WO
Inventors: Eric Olson; Sally J. Trew; Stephen Keith Wrigley; Lydie Pairet; Martin A. Hayes; Steve Martin; David Andrew Kau
Original assignee: Warner Lambert Company
Priority date: 1996-12-09
Filing date: 1997-11-20
Publication date: 1998-06-18
Also published as: AU5459498A

Abstract

Described is a biologically pure culture of a microorganism deposited at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, United Kindgom, under the accession number 40808, which produces any one of the compounds of formula (I), wherein R1 is halogen; R2 is hydrogen or halogen; R3 is an alkyl group of from 1 to 4 carbon atoms; R4 is an hydroxyl or alkoxy group of from 1 to 4 carbon atoms; in a recoverable quantity upon aerobic fermentation in an aqueous nutrient media containing assimilable sources of carbon nitrogen, phosphorus and other essential elements, and processes for the purification of such compounds in a recoverable quantity upon aerobic fermentation in suitable aqueous. The culture and the compounds obtained therefrom are useful as antibacterial or antifungal agents.

Description

MICROORGANISM CAPABLE OF PRODUCING

COMPOUNDS WHERE PYRROLE IS FUSED

WITH 4-OXO- -BENZOXAZINE AND

METHOD OF USE AS ANTIBACTERIAL

AND ANTIFUNGAL

TECHNICAL FIELD

The invention pertains to antibacterial and antifungal materials which contain a 4-oxo-l,3-benzoxazine ring structure and their producing microorganism.

BACKGROUND OF THE INVENTION

Due to ever increasing antibiotic resistance, new anti-bacterials of novel structure have become very important to the treatment of bacterial infections (J. Med. Chem 39, 3853, 1996).

Kinoshita et al (J. Antibiotics 48, 435, 1995; 49, 706, 1996; 49, 651, 1996) has revealed various pyralomicins.

These compounds were isolated from Microtelraspora spiralis and display good activity versus Micrococcus luteiis, but relatively poor activity versus Staphylococcus auretis. The compounds all contain the sugar moiety and are thus higher molecular weight.

Soliveri reveals pyrazoloisoquinolinones from Streptoverticillium griseocarneum (J. Antibiotics 49, 700, 1996).

These compounds show poor activity versus Staphylococci and contain a ring system substantially different from that shown in Formula I, both with regard to the heteroatom placement and location of the substituents.

Saturated fused ring systems have been reported (Tet. Lett 30, 7321, 1989; and 31, 6765, 1990) where the pyrrolidine and piperidine rings are fully saturated rather than aromatic. No biological activities were reported.

A method of preparation has been disclosed where a sulfur replaces the oxygen of the benzoxazine ring structure of Formula I (J. Org. Chem. 57, 3676, 1992). The phenyl ring is not substituted and no biological activity is disclosed.

Sckrob et al. disclose ring systems of Formula I which lack the hydroxyl; also nitrogen has replaced the ring oxygen in the middle ring (J. Gen. Chem. USSR 38, 1970, 1968); no biological activities were revealed.

X = NCH , , 0

SUMMARY OF THE INVENTION

Described is a biologically pure culture of a microorganism deposited at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, the United Kingdom under the accession number 40808, which produces any one of the compounds of Formula I:

FORMULA I

wherein R, is a halogen;

R₂ may be a hydrogen or halogen;

R₃ is an alkyl group of from 1 to 4 carbon atoms;

R₄ is a hydroxyl or alkoxy group of from 1 to 4 carbon atoms; where halogen is any one of fluoro, chloro, bromo or iodo; in a recoverable quantity upon fermentation in an aqueous nutrient medium. The invention also encompasses mutants of the organism. The invention is also concerned with the compounds of Formula I.

The invention is also concerned with producing the compounds of Formula I from the aforementioned culture or mutants thereof. The invention is also concerned with pharmaceutical compositions containing aforementioned culture as well as pharmaceutical compositions containing the compounds of Formula I. The invention is also concerned with utilizing the compounds and pharmaceutical compositions as antibacterial agents.

BRIEF DESCRIPTION OF THE DRAWING

FIGURE 1 shows changes in pH, dissolved oxygen tension (DOT) and titre of compound 1 during 75L fermentation of Streptomyces rimosus X10/78/978.

DESCRIPTION OF PREFERRED EMBODIMENT

The compounds of Formula I have been isolated from a microorganism which has been designated X10/78/978. This microorganism has been deposited, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, UK, under the accession number 40808. Organism X10/78/978 has been identified according to the methods of Williams et al

(Williams, S. T., M. Goodfellow, E. M. H. Wellington, J. C. Vickers, G. Alderson, P. H. A. Sneath, M. J. Sackin & A. M. Mortimer: A Probability Matrix for Identification of Streptomycetes. J. Gen. Microbiol. 129, pl815-1830, (1983)) as a strain of Streptomyces rimosus (cluster 42) on the basis of the following observations and data contained in Table 1.

On oatmeal agar the organism grew as flat, circular colonies with a sparse pasty to powdery aerial mycelium. The substrate mycelium was vellum coloured (BSI 365) and the aerial mycelium white. On glucose yeast extract agar the colonies were slightly raised and the substrate mycelium light brown (BSI 320). On Emmersons agar the colonies were again slightly raised and the substrate mycelium beige (BSI 388). No diffusible pigments were produced on any of these media. Microscopic examination of the organism revealed a highly branched substrate mycelium of lμm diameter hyphae. The aerial mycelium bore Rectiflexibiles, sympodial chains of more than 20 spores. The LL- isomer of diaminopimellic acid was present in the cell wall.

Organism XI 0/78/978 exhibits the following characteristics which are atypical for the species Streptomyces rimosus. It is able to utilise rhamnose and DL- aminobutyric acid, hydrolyses pectin, produces a red pigment in the substrate mycelium, has a grey rather than white or yellow spore mass, and the spore chains are Rectiflexibiles.

TABLE 1

The compounds of Formula I can be obtained from cultures of the aforementioned organism upon aerobic fermentation in suitable aqueous nutrient media. Such nutrient media contain assimilable sources of carbon, nitrogen, and phosphorus, together with any additional elements necessary for the growth of the organism and production of the desired compounds. The compounds of the invention are associated primarily with the biomass on termination of the fermentation and may be recovered and purified. The separation and purification of these compounds from the biomass and their recovery can be achieved using solvent extraction followed by application of conventional chromatographic fractionations with various chromatographic techniques and solvent systems.

The invention is also concerned with pharmaceutical compositions. Contemplated are salts of amino acids such as arginate and the like and gluconate, galacturonate (see, for example, Berge S.M., et al., "Pharmaceutical Salts," J. Pharma. Sci., 1977;66: 1).

Pharmaceutically acceptable base addition salts can be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of such metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see Berge, Supra, 1977).

The base addition salts of said acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.

Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms and are intended to be encompassed within the scope of the present invention.

Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R(D) or S(L) configuration. The present invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof. The compounds of the present invention can be prepared and administered in a wide variety of routes of administration such as parenteral, oral, topical, rectal, inhalation and the like. Formulations will vary according to the route of administration selected. Examples are oral and parenteral dosage forms. Thus, the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intra-cutaneously, subcutaneously, intra-duodenally, or intra-peritoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. The following dosage forms may comprise as the active component, a compound of Formula I or a corresponding pharmaceutically acceptable salt of a compound of Formula I.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier can be a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component can be mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% or 10% to about 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component can be dispersed homogeneously therein, as by stirring. The molten homogenous mixture can be then poured into convenient sized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The quantity of active component in a unit dose preparation may be varied or adjusted for example from about 0.1 mg to 200 mg, preferably about 0.5 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.

In therapeutic use as antibacterial agents, the compounds utilized in the pharmaceutical methods of this invention can be administered at an initial dosage of about 0.01 mg to about 200 mg/kg daily (dosage amount/kg of mammal to be treated). A daily dose range of about 0.01 mg to about 50 mg/kg is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired. Having described the invention herein, listed below are preferred embodiment or working examples wherein all temperatures are degrees Centigrade and all parts are parts by weight unless otherwise indicated.

EXAMPLES

Example 1: 75L Batch Fermentation of Streptomyces rimosus XI 0/78/978

A 1.5ml cryovial containing 1ml of macerated vegetative mycelium suspended in a 10% (V/V) glycerol solution was retrieved from storage at -135°C. A preculture was produced by aseptically placing 1ml of starting material in a 250ml baffled Erlenmeyer flask containing 40ml of nutrient solution S (1% D-glucose, 1.5% glycerol,

1.5% soya bean peptone, 0.3% NaCl , 0.1% CaCO₃, 0.5% malt extract, 0.5%) yeast extract, 0.1% Tween 80, 0.1% Antifoam A [suppliers: Sigma], 0.1 % Junlon PW1 10 [suppliers: Honeywill and Stein Ltd., Times House, Throwley Way, Sutton, Surrey, SMI 4AF] adjusted to pH 7.0) shaken at 240 rpm for 2 days at 28°C.

An intermediate culture was generated by aseptically transferring the preculture to 2L of nutrient solution S in a 3L fermenter. The fermenter was agitated at 500 rpm, aerated at 0.5vvm, and the temperature controlled at 28°C for 3 days. A production culture was generated by aseptically transferring the intermediate culture to a 75L fermenter containing 50L of nutrient solution P (1.77% soya bean flour, 2.0% mannitol, 0.1% Antifoam A [suppliers: Sigma], adjusted to pH 7.0). The production fermenter was stirred at 350 rpm, aerated at 0.5vvm, and temperature controlled at 28°C. After 5 days incubation the fermentation was stopped and the culture was harvested. Example 2: 20L Batch Fermentation of Streptomyces rimosus in the Presence of Sodium Bromide or Sodium Iodide

A preculture was produced following the procedure of Example 1.

An intermediate culture was generated by transferring the 40ml preculture to two litres of nutrient solution S in a 3L fermenter. The fermenter was agitated at 500rpm, aerated at 0.5vvm, and the temperature controlled at 28°C for three days. A production culture was generated by aseptically transferring 560ml of the intermediate culture to a 20L fermenter containing 14L of nutrient solution PBr (1.77% soya bean flour, 2.0% mannitol, 0.05%o sodium bromide, 0.1% Antifoam A [suppliers: Sigma], adjusted to pH 7.0) or nutrient solution PI (as for PBr except for 0.05% sodium iodide in place of sodium bromide). The production fermenter was stirred at 350rpm, aerated at 0.5vvm, and temperature controlled at 28°C. After five days incubation, the fermentation was stopped and the culture was harvested.

Example 3: Purification of compound 1 from a 75L fermentation

A 75L fermentation obtained as described in Example 1 was harvested by filtration using a Schenk Niro 430 filter press, the clarified filtrate was discarded and the retained biomass was extracted with 30L of recirculating methanol for 24 hours. The methanolic extract was harvested via filtration through the filter press and evaporated to an aqueous concentrate using a thin film evaporator.

The aqueous concentrate (5L) was then back extracted with 2 x 7L of ethyl acetate. The solvent extracts were pooled and evaporated to a gum under reduced pressure and redissoved in 50L of ethyl acetate: hexane (2:8). Purification was achieved by normal phase chromatography using a Biotage Flash 75 chromatography system and a Flash 75 KP-Sil silica (32-62_μm 60 A°) column (ID 7.5 x 30cm length) with an isocratic mobile phase (ethyl acetate: hexane 2:8 mix, 200ml/min flow rate). 1L fractions were collected and analysed by thin layer chromatography using the same mobile phase as the developing solvent.

Fractions rich in compound 1 (Rf 0.50) were pooled, evaporated to dryness under reduced pressure and subjected to further purification by preparative reversed phase HPLC using a Beckman 350 HPLC with a Shandon Hyper prep HS BOS C₁₈ (100 A⁰ 12_μm) column

(ID 10 x 30cm length) and an isocratic mobile phase (80% acetonitrile: 20%) water, flow rate 170ml/min). Wavelength monitoring was at 242nm. The peak collected between 22-32 minutes was evaporated to dryness to yield compound 1 (4.5g).

Example 4: Purification of compounds 2, 3, 4, 7, 8 and 9

Flash chromatography fractions rich in compound 1 obtained by purification of the 75L fermentation of S. rimosus as described in Example 3 were also found to contain a number of minor components related to compound 1 and with similar chromatographic properties. After the preparative reversed phase HPLC purification step for compound 1 described in Example 3, the HPLC fractions eluting immediately before and after those containing compound 1 were combined and concentrated to dryness. The material thus obtained was further purified by preparative reversed phase HPLC using a Waters NovaPak C_I8 (100A⁰ 5μM) column (ID 2.5 x 20cm length) and a linear acetonitrile: water gradient starting at 70% acetonitrile: 30% water increasing to 100% acetonitrile over 30 minutes, flow rate 55ml/minute. Wavelength monitoring was at 252nm. The peaks collected at 5 minutes, 9.5 minutes, 10 minutes, 15 minutes, 20 minutes and 23 minutes were evaporated to dryness to yield compound 2 (50mg), compound 3 (4mg), compound 7 (200mg), compound 8 (1.5mg), compound 4 (lOmg) and compound 9 (lmg) respectively.

Example 5: Purification of Compounds 5 and 6 from a 20L

Fermentation

The biomass of a 20L fermentation obtained in the presence of sodium bromide as described in Example 2 was collected by centrifugation, lyophilised and then extracted with 4L of methanol. The methanolic extract was then concentrated in vacuo and resuspended in 10ml of methanol.

This extract was purified by reversed phase HPLC on a Waters Novapak C_I8 (6μM, 6θA) column (2 x (ID 40 x 100mm length)) eluted isocratically (70% acetonitrile: water, 50ml/min, monitoring at

250nm). The fractions eluting after 12 minutes were combined and concentrated in vacuo to yield a brown solid (20mg) which was further purified by preparative thin layer chromatography (Merck, silica gel 60 F₂₅₄, thickness 2mm, 20 x 20cm plates, 50% ethyl acetate: hexane). The main fraction (Rf = 0.60) was finally purified by reversed phase HPLC on a Waters Novapak C₁₈ (6μM, 6θA) column (ID 8 x 100mm length) using an isocratic mobile phase (80%) methanol: water, 2ml/min). The peak eluting after 19 minutes was concentrated in vacuo to yield compound 5 (1.4mg). The biomass of a 20L fermentation obtained in the presence of sodium iodide as described in Example 2 was collected by centrifugation and extracted with 1 OL of methanol. The fermentation did not produce any detectable iodinated analogues of compound 1. The methanol extract was evaporated to an aqueous concentrate using a thin film evaporator. The concentrate (3 L) was back extracted with 2 x 3.5L of ethyl acetate. The solvent extracts were pooled, evaporated in vacuo to a gum and redissolved in 30ml of 60% hexane: ethyl acetate:

This extract was purified by normal phase chromatography using a Biotage flash 75 system with a 75 KP-Sil silica (32-62μM, 6θA) column (ID 7.5 x 30cm length) eluted isocratically

(60%o hexane: ethyl acetate, 200ml/min). 1 L fractions were collected and analysed by thin layer chromatography using the same mobile phase as the developing solvent. Compound 6 (Rf = 0.77-0.96) rich fractions were pooled, evaporated to dryness in vacuo and resuspended in 30ml of methanol.

This fraction was further purified by reversed phase HPLC on a Beckman 350 system with a Shandon Hyper prep HS BOS Cis (12μM, lOOA) column (ID 10 x 300cm length) under isocratic conditions (75% acetonitrile; water, 200ml/min, monitoring at 290nm). The peak collected between 24-25 minutes was evaporated to dryness to yield compound 6 (7.9mg).

Example 6: Determination of Structure of Compound 1

The structure of compound 1 was established by interpretation of data obtained using a variety of spectroscopic techniques including mass spectrometry (MS), infra-red spectroscopy (IR), UV-visible spectrophotometry (UV-vis) and an extensive range of one- and two-dimensional nuclear magnetic resonance (NMR) experiments. Final confirmation of the structure was obtained by x-ray crystallography.

The structures of compounds 2-9 were determined by comparison of their MS, IR, UV-vis, Η and ¹³C NMR data with those of compound 1. The physico-chemical properties and characteristic spectroscopic data for compounds 1-9 are summarised in Tables 2, 3 and

4.

Table 2: Physico-chemical Properties of Compounds 1-9

Compound 1

Appearance White powder While powder White powder EI- S (m/z) 293/295 (M^*) 279/281 (M^*) 327/329/331 (M^*) 264/266 ([M- H,]^*) 264/266 ([M -CH,]') 298/300/302 ([M-C₂

Molecular formula C, H,₂N0₄CI C,₃H,„N0₄CI C, II, N0₄CI₂

HRE1-MS

Found 2930478 2790301 3270067

Calculated 2930454 2790298 3270065

UV λ„,„ (HjO-acetonitπleJπm 254, 293, 336 245, 293, 336 247, 295, 342

IR v ( Br)cm' 3343,3149,2965, 2930, 2868 1643 , 3267, 2966, 2933, 2874, 1641, 1594, 3430, 3215, 3140, 2962

,1118.1101,785,757,704,624 580

Compound 5 6 1

Appearance White powder White powder White powder White po

EI-MS (m/z) 337/339 (M^*) 313/315/317 (M^*) 307/309 (M^*) 293/295 (

308/310 ([M-C_jl .]') 264/266 ([M C,M,]') 278/2

248/2

Molecular formula C,₄H,₂N0₄Br C.jH.NO.CI; C_lsH„Nθ₄Cl C₁₄H„CI

HREI-MS

Found 3369957 - 3070600 2930448

Calculated 3369950 - 3070611 2930454

UV λ,„_4< (Hjθ-acetonιtπle)nm 243, 295, 333 245, 293,336 240, 295, 340 248,291,

IRv..,„(KBr)cm' - - 3260, 2956, 2929, 2864, -

Table 3: Η NMR Assignments for Compounds 1 to 9

a) referenced to acetone signal at 2.05 ppm referenced to external TMS b) measured in CDCI,; referenced to CHCI, signal at 7.26 ppm referenced to external TMS n.o. = not observed

Table 4: ³C NMR Assignments of Compounds 1-5

a) referenced to acetone signal at 29.8 ppm referenced to external TMS b) measured in CDC1₃; referenced to CHC1₃ signal at 76.9 ppm referenced to external TMS

*, ≠ these assignments are interchangable n.o. = not observed

Example 7: Histidine Kinase Assay

Histidine kinases have an important role in bacteria; they control the switching on and off of genes to enable the bacterium to adapt to stressful or changing conditions. The target rationale is that inhibitors of these systems could severely limit the ability of the bacteria to colonise and cause disease in the host organism, so they would have a role in the treatment of infections. The purpose of the assay is to determine inhibition of bacterial signal transduction.

The histidine kinase used in this assay was a fusion of the NRIIc histidine kinase and the carboxy terminal end of the maltose binding protein, both from E.coli. Construction of the expression plasmid encoding the gene for this protein, and the methods of production and isolation of the purified protein is described in Kamberov, E.S. et al., Effect of mutations in Escherichia coli glnL (ntrB), encoding nitrogen regulator II (NRII or NtrB), on the phosphatase activity involved in bacterial nitrogen regulation. J. Biol. Chem. 269: 28294-9

(1994). Assays were performed in 96 well polypropylene microtitre plates.

In each experimental well, 20μl of a 3μM solution of enzyme made up in assay buffer (3mM AEBSF, 50mM Tris, 50mM KC1, lOmM MgCl₂, pH8.0) was mixed with 5μl of a known concentration solution of test compound in methanol. A 25μl aliquot of ATP solution (0.46pM [³³P]-ATP, 70μM ATP in assay buffer) was then added and the reaction left to proceed at room temperature for 15 minutes before being terminated by the addition of lOOμl EDTA solution (50mM, pH8.0).

The reaction mixture in each well was then transferred to the corresponding well in a 96-well high protein binding 0.45 μm filter plate

(Millipore) and the liquid removed by filtration under vacuum. The enzyme retained on the filter was washed with 6 x 1.5ml wash buffer (lOmM Tris, lOmM EDTA, pH8) per well. After drying of the filters at 65°C for 1 hour, 20μl of Microsint 0 (Packard) was dispensed into each well and the radioactivity measured using a Canberra Packard Top Count liquid scintillation counter.

Total binding was determined by replacing the sample with assay buffer (50mM Tris, 50mM KCl, lOmM MgCl₂, pH8.0). Non-specific binding was determined by replacement of the enzyme with assay buffer. The inhibitory activities of representative compounds of the current invention in the histidine kinase assay are shown in Table 5.

Table 5: Activity in Histidine Kinase Assay

Example 8: Minimum Inhibitory Concentration (MIC) Determination

The minimum inhibitory concentration (MIC) of compounds against a series of micro-organisms was tested by the broth dilution method performed in microtitre plates.

Pre-cultures were prepared for all organisms. Cells from a suitable agar slope (see Table 6) were suspended in 5ml sterile saline solution and 1ml of this suspension used to inoculated 30ml of the appropriate preculture medium (see Table 6) in a 250ml conical flask. The pre-cultures were then incubated under the conditions given in Table 1 and the cell count determined using a bacterial cell counting chamber (depth 0.02mm) for bacterial cultures or a haemocytometer (depth 0.1mm) for the strains of Cryptococcus or Candidia. The pre-cultures were then used to inoculate the assay medium to the appropriate cell density, as shown in Table 6. The inoculated assay medium was dispensed in 190μl aliquots into the microtitre plate wells.

A serial 1 in 2 dilution in methanol was made from a 1 mg/ml stock solution of the test compound and lOμl per well of the resultant solutions added to the inoculated assay medium in the microtitre plate. For control wells, lOμl of methanol was added in place of the test compound solution. The plates were then sealed and incubated under the conditions shown in Table 6. The MIC was determined by calculating the lowest final concentration of test compound in a well where the medium remained clear at the end of the incubation period, indicating complete inhibition of growth. MIC data for representative compounds of the current invention are shown in Table 7.

Table 6:

NA = Nutrient Agar SDA = Sabouraud Dextrose Agar SDB = Sabouraud Dextrose Broth MHB = Mueller Hinton Broth D B = Defined medium

DMB (Modified after Fang and Demain, 1989)

Component per litre

Glucose 20g

Glycerol 2.0g Trace Elements Solution per litre

L-Aspartic acid 2.0g H₂S0₄ (IM) 1ml

L-Arginine 0.85g ZnS0₄.7H₂0 1.722g

L-Methionine 0.4g FeS0₄.7H₂O 1.112g

Glycine 0.4g MnS0₄.4H₂0 0.223g

Na₂S0₄ 0-2g CuS0₄.5H₂0 0.125g

KH₂P0₄ 3.5g Na₂MoO₄.2H₂O 0.048g

K₂HP0₄ l.Og CoCl₂.6H₂0 0.048g

MnS0₄ lOmg KI 0.082g

CaCl₂ 2mg

MgSO₄ 0.04g

Trace Elements Solution 5ml

_PII 6.0

Reference: Fang, A.Q. and Demain, A.L.. A New Chemically-Defined Medium for RAC-Certified and Other Strains of Bacillus subtilis. Appl. Microbiol Biotechnol., 30, 144-147 (1989).

Table 7: Antimicrobial Activity Data

Table 8: Antimicrobial Activity Data for Compound #5

While the forms of the invention herein disclosed constitute presently preferred embodiments, many others are possible. It is not intended herein to mention all of the possible equivalent forms or ramifications of the invention. It is understood that the terms used herein are merely descriptive rather than limiting and that various changes may be made without departing from the spirit or scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A biologically pure culture of a microorganism deposited at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, the United Kingdom under the accession number 40808, which produces any one of the compounds of Formula I

FORMULA I

wherein R, is halogen;

R₂ is hydrogen or halogen;

R₃ is an alkyl group of from 1 to 4 carbon atoms;

R₄ is an hydroxyl or alkoxy group of from 1 to 4 carbon atoms; in a recoverable quantity upon fermentation in an aqueous nutrient medium.

2. The culture of claim 1 in freeze-dried form.

3. The culture of claim 1 wherein R₂ may be chloro or bromo.

4. A biologically pure culture of a microorganism deposited at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, the United Kingdom under the accession number 40808 or mutants thereof.

5. A process for producing the compound of Formula I or a pharmaceutically acceptable salt thereof, comprising fermenting a culture of a microorganism deposited at the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland, the United Kingdom under accession number 40808 or mutants thereof, which produces any one of the compounds of Formula I

FORMULA I

wherein R, is a halogen; R₂ is a hydrogen or halogen;

R₃ is an alkyl group of from 1 to 4 carbon atoms;

R₄ is a hydroxyl or alkoxy group of from 1 to 4 carbon atoms; under aerobic conditions in an aqueous nutrient medium and recovering any one of the compounds of Formula I by separating from the fermentation medium.

6. The process of claim 5, wherein the separating step comprises freeze-drying or spray-drying the fermentation medium and recovering the compound.

7. A pharmaceutical composition comprising an effective amount of the culture of Claim 1, together with a pharmaceutically acceptable carrier.

8. A method of treating a mammal in need thereof comprising administering to said mammal an effective antibacterial or antifungal amount of the culture of Claim 1.

9. A compound of Formula I

wherein K_} is halogen; R₂ is hydrogen or halogen; R₃ is an alkyl group of from 1 to 4 carbon atoms; R₄ is an hydroxyl or alkoxy group of from 1 to 4 carbon atoms or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition comprising an effective amount of the compound of Claim 9, together with a pharmaceutically acceptable carrier.

11. A method of treating a mammal in need thereof comprising administering to said mammal an effective antibacterial or antifungal amount of

\ the compound of Claim 9.

12. A method of inhibiting bacterial signal transduction in a mammal in need thereof comprising administering to such mammal an effective inhibiting amount of a compound of Formula I

FORMULA I

wherein R, is halogen; R₂ is hydrogen or halogen; R₃ is an alkyl group of from 1 to 4 carbon atoms; R₄ is an hydroxyl or alkoxy group of from 1 to 4 carbon atoms or a pharmaceutically acceptable salt thereof.