WO1997023486A1

WO1997023486A1 - Active bio-compounds

Info

Publication number: WO1997023486A1
Application number: PCT/DK1996/000543
Authority: WO
Inventors: Helle Demuth; Jens Breinholt; Salka E. Nielsen; Hanne Gürtler
Original assignee: Novo Nordisk A/S
Priority date: 1995-12-21
Filing date: 1996-12-18
Publication date: 1997-07-03
Also published as: AU1093697A

Abstract

The invention relates to biologically active novel compounds having general formula (I), wherein R?1, R2, R3, and R4¿ independently are hydrogen, straight or branched chain alkyl with 1-6 carbon atoms, straight or branched chain alkenyl with 2-6 carbon atoms, straight or branched chain alkynyl with 2-6 carbon atoms, or R?1 and R3¿ independently are acyl (-COR), where R is defined as R1 to R4 above, and R?2 and R4¿ are as above, X is halogen, preferably Cl or Br, especially Cl, mono- or plurisubstituted in the ring. Also disclosed are methods of preparing said compounds, fungicidal compositions comprising, as an active ingredient, these compounds, use of the compounds, and methods of controlling fungi at loci infested or liable to be infested therewith.

Description

Title: Active bio-compounds

FIELD OF THE INVENTION

The invention relates to biologically active compounds, methods for their production, and microorganisms capable of producing such compounds. The invention further relates to fungicidal compositions comprising said compounds and methods of controlling fungi by the use of such compositions.

Also contemplated is an isolated pure culture of a microorganism capable of producing said compounds.

BACKGROUND OF THE INVENTION

Synthetic chemical fungicides, pesticides, acaricides, preservatives etc. have been used for decades, in various fields, such as medicine, agriculture, forestry, horticulture, food industry etc. However, today it is realised that very often in general such chemicals, have a negative impact on the environ¬ ment. Therefore, especially the search for biological agents such as microbes and microbial metabolites, useful for controlling diseases and pests in valuable crops, has been a growing area of research during the last decade.

It is thus well known that microorganisms are capable of producing metabolites associated with interesting biological activities.

Actinomadura spiralis has recently been reported to produce compounds designated pyralomicins (Kawamura et al . Journal of Antibiotics, 48, 435 (1995), Funabashi et al . , Takeda Research Lab, 51, 73 (1992) ) . A general formula is shown below:

wherein R¹ is H or Cl, R² is CH₃ or Cl, R³ is CH₃ or Cl, and R" is H or CH₃.

Pyralomycins were shown to have some antimicrobial activity, especially towards Micrococcus luteus, whereas the activity towards a fungus like Candida albicans was negligible.

The pyralomycins and the compounds of this invention share structural features, but are distinctively different.

SUMMARY OF THE INVENTION

The present inventors have surprisingly succeeded in isolating and characterizing novel biologically active compounds from a bacterium of the genus Streptomyces .

The novel compounds have the generic formula I:

(I) wherein

R¹, R², R³, and R⁴ independently are hydrogen, straight or branched chain alkyl with 1-6 carbon atoms, straight or branched chain alkenyl with 2-6 carbon atoms, straight or branched chain alkynyl with 2-6 carbon atoms, or

R¹ and R³ independently are acyl (-COR) , where R is defined as R¹ to R⁴ above, and R² and R⁴ are as above.

X is halogen, preferably Cl or Br, especially Cl, mono- or plurisubstituted in the ring.

In a preferred embodiment of a compound with the formula

I, R¹ is hydrogen, R² is propyl, R³ and R⁴ are both hydrogen, and X is Cl, resulting in a compound with the formula Ia.

(la)

In another aspect, the invention relates to a method of preparing said compounds comprising a) cultivating a microorganism capable of producing said compound in or on a suitable nutrient medium and under suitable conditions, and b) recovering the compound from the biomass and/or the culture medium.

In an embodiment of the invention the method further comprises the step of c) chemically modifying the compound obtained in step b) .

In a specific embodiment of the invention said microor¬ ganism is a bacterium of the genus Streptomyces, especially of the strain Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof capable of producing a compound of the invention. A third object of the invention is to provide a fungicidal composition comprising, as an active ingredient, said compound alone or in combination with one or more other fungi¬ cidal or pesticidal agents and/or growth regulators. Also contemplated according to the invention is a method of controlling fungi at a locus infested or liable to be infested therewith, which comprises applying to said locus said compound or said composition of the invention.

Further the invention relates to the use of the novel compounds as a fungicide, preservative and/or additive for com¬ bating plant diseases, especially fungal attack or control fungi in timber, wood, cosmetics, paints, growth media, feeds and foods.

Lastly the invention relates to the use of an isolated pure culture of the microorganism Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof, for the production a compound of the invention of formula Ia.

DETAILED DESCRIPTION OF THE INVENTION

As indicated above the invention relates, in its first aspect, to novel compounds having the generic formula I:

tf

( I ) wherein

R¹ and R³ independently are acyl (-C0R), where R is defined as R¹ to R⁴ above, and R² and R⁴ are as above.

X is halogen, preferably Cl or Br, especially Cl, mono- or plurisubstituted in the ring. In connection with the compounds of the present invention having the formula I, the term "alkyl with 1-6 carbon atoms" is intended to include methyl, ethyl, propyl, butyl, pentyl, hexyl etc. straight, branched or cyclic where appro¬ priate.

The term "alkenyl with 2-6 carbon atoms" is intended to include ethenyl, propenyl, butenyl, pentenyl, hexenyl etc. straight, branched or cyclic where appropriate. Also polyenyl (dienyl, trienyl etc.) is intended to be included in the term.

The term "alkynyl with 2-6 carbon atoms" is intended to include ethynyl, propynyl, butynyl pentynyl, hexynyl etc. straight, branched or cyclic where appropriate. Also polyynyl (diynyl, triynyl etc.) is intended to be included in the term. The term "plurisubstituted" covers di-, or tri- substitution.

In a preferred embodiment of compound with the formula I, R¹ is hydrogen, R² is propyl, R³ and R⁴ are both hydrogen, and X is Cl, resulting in a compound with the formula Ia.

(la)

Another object of the invention is to provide a method 5 of preparing compounds with the formula I, comprising a) cultivating a microorganism capable of producing said com¬ pound in or on a suitable nutrient medium and under suitable conditions, and b) recovering the compound from the biomass and/or the culture o medium.

Also contemplated according to the invention are methods which further comprise a step c) of chemically modifying the com¬ pound obtained in step b) . In a specific embodiment said microorganism is a bacterium, preferably of the genus Strep tomyces, especially of the species Streptomyces armeniacus, in particular the strain thereof identified by the deposition number ATCC 15676, or a mutant thereof capable of producing a compound of the invention. An isolate of the bacterium Streptomyces armeniacus NN029813 (ATCC 15676) is available from ATCC.

A suitable nutrient medium is one which comprises the micro- and macronutrients required to obtain a satisfactory growth of the microorganism in question and at the same time give rise to a production of the compound of the invention when subjected to suitable cultivation conditions.

Normally, a suitable nutrient medium contain sources of carbon and nitrogen assimilable by the microorganism and normally a low level of inorganic salts. In addition, the nutrient medium may contain traces of metals and other components necessary for the growth of the microorganisms and the production of the desired compound. Such other components may be in sufficient concentrations in the complex sources of carbon and nitrogen, typically used as nutrient sources, but can, of course, be added separately to the medium if desired.

The conditions under which the microorganism is cultivated may be chosen so as to optimize the production of secondary metabolites therefrom. The optimization of the production of secondary metabolites may be performed by methods known in the art, such as methods based on batch fermentation, fed-batch fermentation or continuous fermentation.

The compound produced may be contained in the biomass or may alternatively be excreted into the culture medium, fully or partially, depending on the microorganism in question.

The recovery of the compound of the invention from the bio-mass and/or culture medium produced in accordance with step a) above may be performed by any suitable technique useful for the microorganism in question. The recovery of the compound may comprise harvesting the biomass, e.g. by filtration and/or cen¬ trifugation, and subsequently isolating the compound from either the biomass and/or the centrifugate. Suitable methods for isolating the compound includes extraction of the biomass or filtrate/centrifugate or both simultaneously using a suitable solvent such as polar solvents like methanol, ethanol, ethyl acetate, or acetone, and solid phase extraction using a hydrophobic resin, an example of which is XAD-8 (Rohm and Haas Co.) . Further purification may be accomplished by chromatography and/or crystallisation.

In order to improve certain properties of the metabolite such as its solubility in aqueous media, its hydrophobicity, hy- drophilicity, stability, specificity, toxicity, target spectrum, potency, UV or heat resistance or the sensitivity of the compound to pH variations, etc. as well as membrane permeability and translocation of the compound in the host plant to which it is applied, it may be advantageous to subject the isolated natural metabolite to a chemical modification. Alternatively, modifica¬ tion may be achieved by feeding suitable precursors to the medium in which the microorganism producing the compound is cultured to obtain production of the derivative. Furthermore, derivatives may be produced by chemical synthesis using the natural metabolite as a lead structure. The compounds produced by such modifications may either belong to the group of compounds having the general formula I or may be different from these compounds.

An example of the production of compounds with the for¬ mula I is given below. The example describes the production of the specific compound with the formula Ia, from a culture of the deposited microorganism (ATCC 15676) of the invention.

While it is contemplated that compounds of the invention having formula I may be prepared by the general method outlined above, i.e. from a microorganism capable of producing such compounds, compounds of the invention may advantageously be prepared from the compound with the formula Ia using a synthetic process.

Ester derivatives of the compound with the formula I (wherein R¹ or R³ is acyl) may be prepared by treating compounds of the formula I (wherein R¹ or R³ is H) , dissolved in a suitable solvent, with appropriate acylating reagents such as acid halides, acid anhydrides or activated esters, optionally under influence of a basic catalyst (pyridine, other amines etc.) . Ether derivatives of compounds with the formula I may be prepared by treating compounds of formula I (wherein R¹ or R³ is H) , dissolved in a suitable solvent, with an appropriate alkylating reagents, such as diazo compounds or with appropriate alkyl halides and alike, under influence of a catalyst (potassium carbonate, silver oxide etc.) .

It is also contemplated that compounds, according to the invention, may be produced entirely by well known chemical synthetic processes using available starting materials. Still another object of the invention is to provide a fungicidal composition comprising, as an active ingredient, said novel compound with the formula I .

In an embodiment of the invention said fungicidal compo¬ sition comprises one of said compounds in an amount of from 0.001 μg/ml to 100 mg/ml. Alternatively, the active ingredient may be a bacterium of a species belonging to the genus Streptomyces capable of producing said novel compound, especially a strain of the Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof capable of producing said compound of the invention. In this context it was specifically found that organic extracts of the bacterium, especially of the strain Strepto/nyces armeniacus N 029813 (ATCC 15676) fermented in submerged culture inhibited the growth of various plant pathogenic fungi a d bacteria. The principle responsible for the observed activity was isolated and characterized spectroscopically, chemically a d biologically as described below.

Fungicidal compositions according to the invention, exhibiting fungicidal and optionally antibacterial activity, having compounds of the invention as an active ingredient, may for agronomical and/or horticultural applications be formulated by mixing the active principle with suitable inert and compatible carriers or diluents to obtain a composition of the type generally used in agricultural compositions, examples of which are further discussed below. The diluent or carrier in the composition of the invention may be a solid or a liquid conventionally used for the purpose. As solid carriers bentonite, diatomaceous earth, apati- te, gypsum, talc, pyrophyllite, vermiculite, ground shells, and clay may be mentioned.

In order to obtain a homogeneous and/or stable formulation, a surface-active agent may be associated with the diluent or carrier. The surface-active agent may, for instance, be a dispersing agent, an emulsifying agent or a wetting agent, examples of which are anionic compounds such as a carboxylate, for example a metal carboxylate of a long chain fatty acid; an N- acylsarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate or sodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylated alkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl- benzene sulphonates or lower alkylnaphthalene sulphonates, e.g. butyl-naphthalene sulphonate; salts of sulphonated naphthalene- formaldehyde condensates; salts of sulphonated phenol- formaldehyde condensates; or more complex sulphonates such as the amide sulphonates, e.g. the sulphonated condensation product of oleic acid and N-methyl taurine or the dialkyl sulphosuccinates, e.g. the sodium sulphonate of dioctyl succinate. Non-ionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- of alkenyl- substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol ethers, e.g. sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g. polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7, 9-tetraethyl-5-decyn-4, 7-diol, or ethoxylated acetylenic glycols.

Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate; an oxygen-containing amine such as an amine oxide or polyoxyethylene alkylamine; an amide-linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt.

The composition of the invention can be in any form known in the art for the formulation of agrochemicals, for example, an emulsifiable concentrate, a concentrated emulsion, a multiple emulsion, an aqueous emulsion, a solution, a dispersion, a suspension concentrate, a release formulation (including a slow release formulation) , a seed dressing, a granular formulation, a water soluble powder, a wettable powder, a dusting powder, a dispersible powder, an alginate, a xanthan gum and/or an aerosol. Moreover, it can be in a suitable form for direct application or as a concentrate or primary composition which requires dilution with a suitable quantity of water or other diluent before application.

An emulsifiable concentrate comprises the active ingredient dissolved in a water-immiscible solvent which is formed into an emulsion with water in the presence of an emulsifying agent. Another suitable concentrate is a flowable suspension concentrate which is formed by grinding the active ingredient with water or other liquid, a wetting agent and a suspending agent .

A dusting powder comprises the active ingredient intimately mixed and ground with a solid pulverulent diluent, for example, kaolin. A granular solid comprises the active ingredient associated with similar diluents to those which may be employed in dusting powders, but the mixture is granulated by known methods. Alternatively it comprises the active ingredient absorbed or adsorbed on a pre-granular diluent for example, Fuller's earth, attapulgite or limestone grit. Wettable powders, granules or grains usually comprise the active ingredient in admixture with a suitable surfactant and an inert powder diluent such as china clay.

Depending on the circumstances such as the crop wherein fungi are to be controlled, the environmental conditions or other factors, a composition of the invention in addition to said fungicidally active compounds of the invention may also contain other active ingredients, e.g. fungicides, pesticides, herbicides, insecticides, nematocides or acaricides, or plant nutrients or fertilizers.

Examples of fungicides which can be combined with the active compounds of the invention include especially ergosterol biosynthesis inhibitors ("EBIs") . These are generally imidazole or triazole derivatives and examples include those known by the common names prochloraz, triadimefon, propiconazole, diclobu- trazol, triadiminol, flusilazole, flutriafol, myclobutanil, penconazole, quinconazole, imazalil and diniconazole. Examples of non azole EBIs include nuarimol, fenarimol, fenpropimorph, tridemorph, fenpropidine and dimethomorph.

Further fungicides which can be combined with compounds of the invention include:

Dithiocarbamates, e.g. thira , maneb, zineb and mancozeb; Phatalimides, e.g. captan, folpet and captafol;

Carboxines, e.g. carboxin and oxycarboxin;

Benzimidazoles, e.g. benomyl, carbendazim and fuberidazole;

Carbamates, e.g. prothiocarb and propamocarb;

Isoxazoles, e.g. hymexazol; Cyanoacetamides, e.g. cymoxanil;

Ethylphosphonates, e.g. fosetylaluminium;

Phenylamides, e.g. furalaxyl, metalaxyl, benalaxyl, ofurace, cyprofuram and oxandixyl;

Dicarboximides, e.g. procymidone, iprodione and vinclozolin; Organophosphorous fungicides, e.g. pyrazophos, triamiphos, ditalimfos and tolcofosmethyl;

Aromatic hydrocarbon fungicides, e.g. quintozene, dichloren, and diphenyl;

Pyri idines, e.g. pyrimethanil, and Dinitroanilies, e.g. fluazinam.

The concentration of the active compounds of the invention described herein in the compositions of the invention may vary within a wide range depending on the type of formulation and the field of application.

In order to provide the antifungal composition of the invention with a satisfactory activity, the active compound should normally be present in an amount from 0.001 μg/ml to 100 mg/ml, such as 0.1 μg/ml to 5 mg/ml. The concentration of the biologically active compounds of the invention in the compositions of the present invention when used alone or in combination with a conventional fungicide, as applied to plants is preferably within the range from about 0.001 to about 30 per cent by weight, especially 0.01 to 3.0 per cent by weight, although it may vary more widely and be, for instance, within the range from about 5 to about 95 per cent by weight of the composition.

The concentration of the other fungicidally active ingredient in the mixed composition of the present invention, as applied to plants is preferably within the range of 0.001 to 50 per cent by weight, especially 0.01 to 10 per cent by weight, al- though it can vary widely and can be, for example, from 5 to 80 per cent by weight of the composition.

The composition according to the invention may also comprise compounds which contributes to various functions, such as protection of the fungicidal properties of the active compo- nents from sun, or UV damage.

Examples of preferred UV protectants are lignins or lignin derivatives, which are readily available by-product of the pulp and paper industry, alone or combined with sugar alcohols as described in the pending international patent application no. PCT/US95/01760 (Entotech, Inc.) .

Examples of suitable lignins comprise lignin sulfonate and salts thereof (e.g. Na, K, Ca, and Mg salts) , oxylignins and salts thereof, lignin liquors, Kraft lignins and derivatives thereof and low and high lignins. An "effective amount" of lignin refers to an amount which when combined with an effective amount of sugar alcohol, under normal sun conditions, increases the UV protection of the composition at least 25%, and preferably at least 50%, relative to the protection provided by lignin alone in the same composi- tion. The amount of lignin in the composition is at least 2% w/w, up to about 95% w/w, and preferably at least about 5% w/w, most preferably at least about 15% w/w, up to about 50% w/w.

The sugar alcohols have the formula CH₂OH (CHOH)_nCH₂OH, wherein n is an integer from 2 to 5. Among the sugar alcohols useful for this purpose are sorbitol, mannitol and xylitol .

An "effective amount" of a sugar alcohol is that amount, which in combination with a given amount of lignin, will enhance the UV protective properties of lignin at least 25%, and preferably at least 50%, relative to lignin alone in the same composition. A preferred concentration of the sugar alcohol in the composition is at least 4% w/w, up to about 95% w/w, and preferably at least about 10% w/w, up to about 35% w/w. In order to achieve maximum efficacy, the formulation containing a fungicide or a pesticide must first be deposited di¬ rectly on the plants to be treated, and then must adhere to and remain active on the surface to which it is applied. To achieve this goal, the UV protecting components may be supplemented with other components.

Therefore the composition according to the invention may further advantageously comprise at least one agent which is capable of enhancing deposition (hereinafter, "deposition agent") of the composition, i.e., a component which will assist in keeping it from drifting from the target area as it is being applied (e.g. as it is sprayed from a plane), or from being blown away from the plant once it has been deposited. The deposition agent thus should be a component which has sufficient density to prevent random dispersal. Any animal or vegetable protein is suitable for this purpose, in dry or in liquid form. Examples of useful sources of protein which can be conveniently and economically added to the formulation are soy protein, potato protein, soy flour, potato flour, fish meal, bone meal, yeast extract, blood meal and the like.

However, protein is not the only material which can be used in this manner. Alternative deposition agents include modi¬ fied cellulose (e.g. carboxymethylcellulose), botanicals (e.g. grain flours, ground plant parts) , natural earths (talc, vermiticulite, diatomaceous earth), natural clays (e.g. attapulgite, bentonite, kaolinite, montmorillonite) , or synthetic (e.g. Laponite) .

When utilized, the deposition agent preferably constitutes at least about 1% w/w up to about 50% w/w, more preferably at least 2% w/w up to about 20% w/w.

Retention of the composition can be aided by inclusion of an adherent component. To this end, one or more polyhydric alcohols are added to the composition. This component can serve a number of functions. First, it functions as an adherent which permits the composition to stick to the plant surface. In ad¬ dition, these components serve as a humectant, to attract moi¬ sture to the composition in si tu . Such components also can be useful in preventing freezing of the composition, thereby protecting the activity of the fungicidal component .

The alcohol component can be chosen from one or more of the following: ethylene glycol, propylene glycol, dipropylene glycol, glycerol, butylene glycols, pentylene glycols, hexylene glycols; the sugar alcohol component may also contribute to this function, but it is desirable to add a second polyhydric alcohol to the composition. Overall, the second polyhydric alcohol component of the composition, if present, should comprise from about 1 to about 20% w/w of the total composition. The invention also relates to the use of these active ingredients alone or in combination with one or more other fungi¬ cidal or pesticidal agents and/or growth regulators for con¬ trolling fungi in agriculture, forestry, horticulture etc.

In a further aspect the invention relates to a method of controlling fungi at a locus infested or liable to be infested therewith, which comprises applying to said locus a compound or a composition of the invention as defined above.

In an embodiment of the invention said compound or composition is applied to a locus selected from the group consisting of plants, timber, wood, cosmetics, feeds and foods.

In relation to plants the compounds of the invention can be used on both monocotyledons and dicotyledons. Specific examples are members of the Gra inea genera, such as Poacea ; Leguminosae ; vegetables; fruits; etc. Specifically the compounds of the invention can be used for controlling plant pathogenic fungi on vegetables.

The compounds of the invention can be also be used for controlling plant pathogenic fungi on fruits, such as citrus fruits, e.g. oranges and lemons. Specifically the compounds of the invention are especially useful for controlling plant pathogenic fungi on ce¬ reals, such as wheat, barley, oats, corn, and rice. Compounds of the formula Ia has been found to exhibit activity towards fungi of the Class Ascomycota , such as Botryfcis cinerea, Colletotrichum graminicola, and Septoria nodorum, of the

Imperfect fungi, such as Pyricularia oryzae (sexual stage Magnaporthe grisea) , and Al ternaria al ternata .

Compounds of the formula Ia has also been found to exhibit activity towards Yeast, such as Saccharomyces cerevisiae, and Bacteria, such as Bacillus subtilis .

In connection with the method of the invention for controlling fungi, the composition may for agronomical or horti¬ cultural uses be applied to a region to be treated either direct¬ ly to the soil as a pre-emergence treatment or to the foliage or fruits of the plants as a pre- and/or post-emergence treatment. Depending on the crop and circumstances the treatment may be postponed until seeds or fruits appear on the plants, wherein fungi are to be controlled. Sometimes, it is practicable to treat the roots of a plant before or during planting, for example by dipping the roots in a suitable liquid or solid composition.

The active preparation or the compositions of the invention can be applied directly to the plant by, for example, spraying or dusting either at the time when the fungus has begun to appear on the plant or before the appearance of fungus as a protective measure. In both such cases the preferred mode of application is by foliar spraying. It is generally important to obtain good control of fungi in the early stages of plant growth as this is the time when the plant can be most severely damaged. The spray or dust can conveniently contain a pre-or post-emergence herbicide if this is thought necessary. When the active preparation of the invention is applied directly to the plant a suitable rate of application is from 0.001 to 50 kg per hectare of active com¬ pound, preferably from 0.05 to 5 kg per hectare of active compound.

In the method of the invention the active preparation of the invention alone or in combination with a conventional biocide can also be applied to seeds or other habitats. Thus the preparation can be applied directly to the soil before, at or after drilling so that the presence of active ingredient in the soil can control the growth of fungi which may attack seeds.

The compositions may be applied in amounts corresponding to from about 1 to about 50 kg biologically active compound per hectare.

When the soil is treated directly the active preparation alone or in a mixture with the conventional biocide can be applied in any manner which allows it to be intimately mixed with the soil such as by spraying, by broadcasting a solid form of granules, or by applying the active ingredient at the same time as drilling by inserting it in the same drill as the seeds. A suitable application rate is within the range of from 0.01 to 50 kg of active compound per hectare, more preferably from 0.05 to 5 kg of active compound per hectare. Although the present invention has been described in detail in connection with controlling fungi on plants, it is also anticipated that the active compounds of the invention may be used for controlling fungi in mammals, including humans; for the preservation of cosmetics; for the preservation for wood by adding said compounds to wood preservation and/or impregnation compositions; for the preservation of food or feed by adding the compounds directly to the food or feed or to the containers in which it is present; and for the preservation of cosmetics. Also, the active compounds of the invention may be useful as a fungicide and preservant in paints - both to prevent growth in the paint during storage, and growth on the painted object such as the plastered surface of a house - and as an additive to growth media, e.g. for cultivation of certain bacteria or yeast.

A final aspect of the invention relates to the use of an isolated pure culture of the microorganism Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof for the pro¬ duction of compounds of the invention. EXAMPLES

Example 1 Fermentation

Cultivation of the Strain

Streptomyces armeniacus NN029813 (ATCC 15676) was propagated on oat meal agar (OMA) slants (12 ml/slant) for 6 days at 30°C. OMA was prepared by mixing oat meal (20 g) , yeast extract (2.5 g) , and water (to 1000 ml total volume) . This mixture was brought to the boiling point and then stirred at 60°C for 4 hours. Merck agar (20 g) was added and the mixture again brought to the boiling point. Finally the medium was autoclaved at 121°C for 20 min.

Submerged Fermentation

The submerged fermentation was conducted in 250 ml baffled shake flask each containing 100 ml of Emerson medium. Fermentation was carried out for 7 days at 30°C and 250 rpm. The growth medium was prepared by mixing yeast extract (4 g) , soluble starch (15 g) , K₂HP0₄ (1 g) , and MgS0₄,7 H₂0 (0.5 g) , and adding distilled water to a final volume of 1000 ml. The medium was autoclaved for 20 min at 121°C. Inoculum for the submerged fermentation was prepared by washing one OMA-slant with sterile water (10 ml) containing 0.1% tween. 5 ml of the resulting suspension was used to inoculate one shake flask.

Example 2

Extraction and Purification

Extraction : Whole culture broth (2.0 L) from 25 shake flasks was extracted with EtOAc (2.0 L) under stirring for 2 h. After phase separation, achieved by centrifugation, the organic phase was dried by freezing followed by removal of the separated ice by filtration. The solvent was evaporated under reduced pressure to a brownish, oily residue (453 mg) . Purification : The compound was purified by reversed phase HPLC, using a Dupont ODS, lOμm, 250 x 20 mm column eluted at a flow rate of 15 ml/min with a linear gradient from 50 % aqueous acetonitrile to 90 % acetonitrile over 35 min; UV detection at 210 and 225 nm. The cut between 19 and 21 min afforded pure Ia. The yield of Ia from 25 shake flasks was 15 mg. Final purification was achieved by crystallization from methanol.

Chemical and Spectroscopic Data Ia purified as described above appear homogenous (NMR,

HPLC) and exhibit the following physical and spectroscopic properties:

Appearance: colourless needles Melting point: 181-183°C (dec)

Molecular formula/weight: C₁₄H₁₂C1N0₄ (mw=293) UV^nmdogε) : 241 (4.51) , 294 (4.30), 335 (3.65) IR cm^"1 (KBr) : 2950, 2928, 1640, 1584, 1514, 1467, 1297, 1193, 1120, 1100 EIMS (m/z (% rel.int.) : 295 (13) , 293 (37), 266 (33), 264 (100) , 149 (6) , 138 (8) , 94 (6) ,

40 ( 10 )

HR-EIMS : Formula Calculated Found Error (ppm)

C₁₄H₁₂ ³⁵C1N0₄ 293 . 0455 293 . 0457 + 0 . 7

C₁₄H₁₂ ³⁷C1N0₄ 295 . 0425 295 . 0436 + 3 . 7

NMR : ³C and ^lH nmr data in table 1

Table 1 ^XH and ¹³C NMR data for Ia (500 μl CDC1₃ + 10 drops CD₃0D) . δ-values relative to solvent peak at 77.0 and 7.27 ppm for ¹³C and ^XH, respectively.

Position* ^lU

2 103.9 7.10

3 118.1

4 90.1 5.73

5 140.8

7 153.2

8 93.3 6.23

9 163.5

10 112.4

11 159.7

12 92.8

13 158.4

14 24.1 2.58

15 21.8 1.55

16 13.9 0.90

* According to formula Ia below

la

These data provide evidence for the structure of compound Ia. Fungicidal Activity

Agar diffusion assay

The compound has been found to have an in vi tro inhibitory effect on the growth of fungi, yeast, and bacteria: It was found to have activity towards:

Class Ascomvcota :

Botrytis cinerea, Colletotrichu graminicola, and Septoria nodorum

Imperfect fungi :

Pyricularia oryzae (sexual stage Magnaporthe grisea) . , and

Altemaria alteraaCa

Yeast:

Saccharomyces cerevisiae

Bacteria: Bacillus subtilis

In the inhibition assays the test organisms were em¬ bedded in agar media. Small wells were punched in the agar and 15 μl of sample was applied to the wells. Inhibition zones were scored after incubation in dark for 1-2 days at 26°C for fungi and 30°C for bacteria.

Table 2 shows the activity observed when applying a 1000 ppm (1 mg/ml) and 100 ppm (0.1 mg/ml) solution of compound Ia in EtOH. Table 2 .

Antimicrobial activity of Ia

Organism Ia Ia

1000 ppm 100 ppm

Activity Activity

Botrytis cinerea Colletotrichum graminicola ++ ++ Septoria nodorum Pyricularia oryzae Al ternaria al ternata Saccharomyces cerevisiae ++ ++ Bacillus subtilis (G+) ++ ++

+ : < 10 mm

++ : 11-20 mm

- : not active

The present invention is not to be limited in scope by the above examples since they are only intended as illustrations of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will from the foregoing description become apparent to those skilled in the art. Such modifications are intended to fall within the scope of the appended claims.

Claims

PATENT CLAIMS

1. A compound having the generic formula I

wherein

R¹ and R³ independently are acyl (-COR) , where R is defined as R¹ to R⁴ above, and R² and R⁴ are as above,

X is halogen, preferably Cl or Br, especially Cl, mono- cr plurisubstituted in the ring.

2. The compound according to claim 1, with the formula Ia

Ia

3. A method of preparing a compound according to claim 1 or 2, comprising a) cultivating a microorganism capable of producing said compound in or on a suitable nutrient medium and under suitable conditions, and 5 b) recovering the compound from the biomass and/or the culture medium.

4. The method according to claim 3, which further comprises

0 c) chemically modifying the compound obtained in step b) .

5. The method according to any of the claims 3 and 4, wherein the microorganism is a bacterium, preferably of the genus Streptomyces, preferably of the species Streptomyces armeniacus . 5

6. The method according to claim 5, wherein the bacterium is a strain of Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof capable of producing a compound as defined in any of the claims 1 to 2. 0

7. The method according to claim 6, in which the compound prepared is the compound with the formula Ia.

8. A Biocidal composition comprising, as an active 25 ingredient, a compound according to claim 1 to 2.

9. The biocidal composition according to claim 8, which is a fungicidal composition.

30 10. The fungicidal composition according to claim 9, wherein said compound is present in an amount of from 0.001 μg/ml to 100 mg/ml.

11. The fungicidal composition according to claim 9 and 10, 35 comprising as an active ingredient, a bacterium of a species belonging to the genus Streptomyces capable of producing a compound as defined in any of the claims 1 to 2.

12. The fungicidal composition according to claim 11, wherein the genus is Streptomyces, and preferably a strain of the species Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant

5 thereof capable of producing a compound as defined in any of the claims 1 to 2.

13. The fungicidal composition according to claim 9 to 12, comprising as an active ingredient a compound of any of the 0 claims 1 to 6 in combination with one or more further fungicidal or pesticidal agents and/or growth regulators.

14. The fungicidal composition according to claim 13, wherein said other active agent (s) is/are present in an amount of 5 0.001 % (w/w) to 50 % (w/w) by weight, preferably 0.01 % (w/w) to 10 % (w/w) .

15. The fungicidal composition according to claim 9 to 14, further comprising adjuvants, such as UV protective compounds, 0 retention compounds, surface active compounds.

16. A method of controlling microbes at a locus infested or liable to be infested therewith, which comprises applying to said locus a compound according to any of the claims 1 to 2 or a

25 composition according to any of the claims 8 to 15.

17. The method according to claim 16, which method is a method of controlling fungi.

30 18. The method according to claims 16 or 17, in which the locus is selected from the group consisting of plants, timber, wood, cosmetics, feeds and foods.

19. The method according to claim 18, in which the locus is 35 a cereal.

20. The method according to claim 18, in which the locus is a vegetable.

21. The method according to claim 18, in which the locus is 5 a fruit.

22. The method according to any of claims 17 to 20, in which the fungus to be controlled is a plant pathogenic fungus, preferably one which belongs to the Class Ascomycota, such as 0 Botrytis cinerea, Colletotrichum graminicola, and Septoria nodorum .

23. The method according to any of claims 17 to 21, in which the fungus to be controlled is a plant pathogenic fungus, 5 preferably one which belongs to the Imperfect fungi, such as Pyricularia oryzae (sexual stage Magnaporthe grisea) , and Al ternaria al ternata .

24. The method according to any of claim 16, in which the 0 microbe to be controlled belongs to the Yeasts, such as

Saccharomyces cerevisiae .

25. The method according to any of claim 16, in which the microbe to be controlled belongs to the Bacteria, preferably gram

25 positive bacteria, such as Bacillus subtilis .

26. Use of a compound according to any of the claims 1 to 2 for the preservation of cosmetics.

30 27. Use of a compound according to any of the claims 1 to 2 for the preservation of foods and feeds.

28. Use of a compound according to any of the claims 1 to 2 as an additive to growth media.

35

29. Use of a compound according to any of the claims 1 to 2 for the preservation of paints.

30. Use of a compound according to any of the claims 1 to 2 for the preservation of wood or timber.

31. Use of the compound according to any of claims 1 to 2 for combating fungi on cereals.

32. Use of the compound according to claims 1 to 2 for com¬ bating fungi on vegetables.

33. Use of the compound according to claims 1 to 2 for combating fungi on fruits.

34. Use of an isolated pure culture of the microorganism Streptomyces armeniacus NN029813 (ATCC 15676) or a mutant thereof capable of producing a compound as defined in any of the claims 1 to 2 for the production of said compounds.