WO1996026199A1

WO1996026199A1 - Active bio-compounds

Info

Publication number: WO1996026199A1
Application number: PCT/DK1996/000081
Authority: WO
Inventors: Jens Breinholt
Original assignee: Novo Nordisk A/S
Priority date: 1995-02-24
Filing date: 1996-02-23
Publication date: 1996-08-29
Also published as: AU4783896A

Abstract

The invention relates to biologically active novel compounds having general formula (I), wherein R2 and R13 are hydrogen, straight or branched chain alkyl, straight or branched chain alkenyl, straight or branched chain alkynyl, benzyl or aryl, optionally mono- or plurisubstituted with alkyl, atoms, hydroxy, alkoxy, halogen, amino or a nitro group; and R8 is hydrogen, -COR, straight or branched chain alkyl, straight or branched chain alkenyl, straight or branched chain alkynyl, benzyl or aryl; R1, R2, R3, R4, R5, R6, R7, R9, R10, R11, and R12 are hydrogen or together with an adjacent R-group represent an additional bond; and R is a straight or branched chain alkyl, straight or branched chain alkenyl, straight or branched chain alkynyl, aryl. A method of preparing said compounds, fungicidal compositions comprising, as an active ingredient, these compounds, use of the compounds and an isolated pure culture of the microorganism Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof.

Description

Title: Active bio-compounds

FIELD OF THE INVENTION

The invention relates to biologically active compounds, methods for their production, and microorganisms capable of synthesizing such compounds.

The invention further relates to fungicidal compositions com- prising said compounds and methods of controlling fungi by the use of such compositions.

Also contemplated is an isolated pure culture of a microorga¬ nism capable of producing said compounds.

BACKGROUND OF THE INVENTION

Synthetic chemical fungicides, pesticides, acaricides, pre- servatives etc. have been used for decades, in various fields, such as medicine, agriculture, forestry, horticultu¬ re, food industry etc. However, today it is realised that in general such chemicals, have a negative impact on the envi¬ ronment. 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 well known that microorganisms are capable of producing metabolites associated with interesting biological activiti¬ es.

Strains of the fungus Hamigera avellanea are known to produce the compounds Avellanin A and Avellanin B (Yamazaki et al. Chem. Phar , Bull, 1987, 35, 2122; Nakao et al., Chem. Pharm. Bull, 1989, 37, 930) . Ave llanin A

CH2CH3

Avellanin B

R = CH3

From JP-0-2300125-A (Meij i Seika Kaisha) it is known that Avellanin A and Avellanin B can be used for increasing the action of specific anticancer agents .

SUMMARY OF THE INVENTION

The present inventors have surprisingly succeeded in isolat¬ ing and characterizing novel biologically active compounds from a fungus of the genus Hamigera .

The novel compounds have the generic formula I:

Formula I

wherein

RI is hydrogen or together with R2 represents an additional bond,

R2 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

R3 is hydrogen, or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi¬ tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi¬ tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi¬ tional bond,

R7 is hydrogen or together with R6 represents an additional bond, R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

R9 is hydrogen or together with RIO represents an additional bond,

R10 is hydrogen, or together with R9 represents an additional bond,

Rll is hydrogen or together with R12 represents an additional bond,

R12 is hydrogen or together with Rll represents an additional bond,

R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, and when R13 is hydrogen salts thereof,

R is a straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, aryl, optionally mono- or plurisubstituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

In a preferred embodiment of compound with the formula I, RI and R2 together represents an additional bond, R9 and R10 together represents an additional bond, Rll and R12 together represents an additional bond resulting in compounds with the formula la.

Formula la wherein

R3 is hydrogen or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi¬ tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi- tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi¬ tional bond,

R7 is hydrogen or together with R6 represents an additional bond,

R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 10 carbon atoms, straight of branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, and R is a straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisubstituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

In two specific embodiments of the invention the compound I have the below indicated formulas lb or Ic.

Formula Ic

Formula lb

In another aspect, the invention relates to a method of pre¬ paring said compounds comprising a) cultivating a microorganism capable of producing said compound in or on a suitable nutrient medium and under suit¬ able conditions, and b) recovering the compound from the biomass and/or the cul^¬ ture medium.

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

In a specific embodiment of the invention said microorganism is a fungus of the genus Hamigera , preferably of the species Hamigera avellanea , especially of the strain Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof ca¬ pable 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 com¬ pound 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 infes¬ ted 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 co - pounds 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 an isolated pure culture of the microorganism Hamigera avelleanea NN0054922 (CBS No. 501.94) or a mutant thereof capable of producing a compound of the invention. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the infra red spectrum (IR-spectrum) of the compound with the formula lb, and

Figure 2 shows the UV spectrum of the compound with the for¬ mula lb.

Figure 3 shows the UV spectrum of the compound with the for- mula Ic.

DETAILED DESCRIPTION OF THE INVENTION

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

Formula I

wherein

RI is hydrogen or together with R2 represents an additional bond,

R3 is hydrogen, or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi¬ tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi¬ tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi¬ tional bond,

R7 is hydrogen or together with R6 represents an additional bond,

R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

R9 is hydrogen or together with R10 represents an additional bond,

R10 is hydrogen, or together with R9 represents an additional bond,

Rll is hydrogen or together with R12 represents an additional bond,

R12 is hydrogen or together with Rll represents an additional bond, R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, and when R13 is hydrogen salts thereof,

In connection with the compounds of the present invention having the formula I, the term "alkyl with 1-10 carbon atoms" is intended to include methyl, ethyl, propyl, butyl, pentyl, hexyl etc. straight, branched or cyclic where appropriate.

The term "alkenyl with 2-10 carbon atoms" is intended to in¬ clude 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 2-10 carbon atoms" is intended to in¬ clude 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 "aryl" is intended to include aromatic radicals like phenyl, naphtyl, phenantryl etc. and hetero aromatic radicals like furanyl, thiophenyl, pyridinyl, imidazolyl, oxazolyl etc. The term "plurisubstituted" covers di-, tri-, tetra- or higher substitution.

The term "halogen" covers fluorine, chlorine, bromine and iodine.

Further, it is to be understood that "-COR" specifies either a carbonyl or an ether group.

In a preferred embodiment of compound with the formula I, RI and R2 together represents an additional bond, R9 and RIO to¬ gether represents an additional bond, Rll and R12 together represents an additional bond resulting in compounds with the formula la.

Formula la wherein

R3 is hydrogen or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi¬ tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi¬ tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi¬ tional bond, R7 is hydrogen or together with R6 represents an additional bond,

R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, and

R is a straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisubstituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

In another embodiment compounds of the invention have the formula lb, :

Formula Ibl wherein

R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

Further the compounds of the invention may have the formula Ic,:

Formula Icl wherein

R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, when R13 is hydrogen salts thereof, and

Formula Ic

Formula lb

Another object of the invention is to provide a method of pre¬ paring compounds with the formula I, comprising a) cultivating a microorganism capable of producing said compound in or on a suitable nutrient medium and under suit¬ able conditions, and b) recovering the compound from the biomass and/or the cul¬ ture medium.

Also contemplated according to the invention are methods which further comprise a step c) of chemically modifying the compound obtained in step b) .

In a specific embodiment said microorganism is a fungus, pre- ferably of the genus Hamigera, especially of the species Hamigara avellanea , in particular the strain thereof identi- fied by the deposition number CBS No. 501.94, or a mutant thereof capable of producing a compound of the invention.

An isolate of the fungus Hamigera avellanea NN005492 (CBS No. 5501.94) has been deposited with the Centraalbureau voor Schim elcultures, P.O. Box 273, 3740 AG Baarn, The Nether^¬ lands, for the purposes of patent procedure on the date indi¬ cated below. CBS being an international depository under the Budapest Treaty affords permanence of the deposit in accor- o dance with rule 9 of said treaty.

Deposit date : 18. October 1994 Depositor's ref.: NN005492

CBS designation : CBS No. 501.94 5

Further the isolate of the fungus Hamigera avelleane a NN005492 (CBS No. 501.94) has been deposited under conditions that assure that access to the isolated fungus will be avail¬ able during the pendency of this patent application to one 0 determined by the commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C § 122. The deposit represents a substantially pure culture of the isolated fungus. The deposit is available as required by foreign patent laws in countries wherein counterparts of the 5 subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

0 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. 5

Normally, a suitable nutrient medium contain sources of car¬ bon and nitrogen assimilable by the microorganism and normal- ly a low level of inorganic salts. In addition, the nutrient medium may contain traces of metals and other components ne¬ cessary for the growth of the microorganisms and the produc¬ tion 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.

In the case of a fungus, such as a fungus of the species Hamigera , especially the Hapmigera avellanea NN005492 ident¬ ified by the deposition number (CBS No. 501.94), the compound is normally contained in the biomass, such as in or on the mycelium, the spores, and in the culture filtrate.

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 com- pound comprises harvesting the mycelium, e.g. by filtration and/or centrifugation, and subsequently isolating the com¬ pound from either the biomass and/or the centrifugate. Suit¬ able methods for isolating the compound includes extraction of the biomass or filtrate/centrifugate using a suitable sol- vent 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 chro^¬ matography 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 spec¬ trum, potency, UV or heat resistance or the sensitivity of the compound to pH variations, etc. as well as membrane per¬ meability 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. Al¬ ternatively, modification may be achieved by feeding suitable precursors to the medium in which the microorganism producing the compound is cultured to obtain production of the deriva- tive. 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 formula I is given below. The example describes the production of the specific compounds with the formula lb and Ic, from a strain of the deposited microorganism of the invention.

While it is contemplated that compounds of the invention hav¬ ing formula I may be prepared by the general method outlined above, i.e. from a microorganism capable of producing such compounds, some compounds of the invention may advantageously be prepared from the compounds with the formula lb or Ic using a synthetic process.

Ester derivatives of the compound with the formula I (wherein R2 is acyl) may be prepared by treating the compound I (where- in R2=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 cata- lyst (pyridine, other amines etc.). Ester derivatives of com¬ pound I wherein R13 is defined as R above (except H) may be prepared by treating compounds of formula I (wherein R13=H) with the appropriate alcohol (ROH) under acid catalysis, with a suitable diazo compound, or by preparing activated esters and subsequent reaction with the appropriate alcohol (ROH) . Ether derivatives of compound I may be prepared by treating compound I (wherein R2=H) , dissolved in a suitable solvent, with an appropriate alkylating reagents, such as diazo com- pounds 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 fungi- cidal composition comprising, as an active ingredient, said novel compound with the formula I.

In an embodiment of the invention said fungicidal composition 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 fungus of a species belonging to the genus Hamigera capable of producing said novel compound, preferably of the species Hamigera avellanea , especially a strain of the Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof capable of producing said compound of the invention.

In this context it was specifically found that organic ex¬ tracts of the fungus Hamigera avellanea NN005492 (CBS No. 501.94) fermented in submerged culture inhibited the growth of various plant pathogenic fungi. The principle responsible for the observed activity was isolated and characterized spec- troscopically, chemically and biologically as described below. Fungicidal compositions according to the invention, exhibit¬ ing 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 com^¬ patible 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 pur¬ pose. 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 carboxy- late, 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; ethoxy- lated fatty alcohol sulphates; ethoxylated alkylphenol sul¬ phates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzene sulphonates or lower alkyl- naphthalene 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 sul- phonate of dioctyl succinate. Non-ionic agents include con¬ densation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- of alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alco¬ hol ethers, e.g. sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g. polyoxye- thylene 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 acety¬ lenic glycols.

Examples of a cationic surface-active agent include, for in- stance, 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 formu¬ lation, 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 ingre^¬ dient 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 ingredi¬ ent absorbed or adsorbed on a pre-granular diluent for exam¬ ple, Fuller's earth, attapulgite or limestone grit. Wettable powders, granules or grains usually comprise the active in- gredient 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 con¬ tain 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 bio¬ synthesis inhibitors ("EBIs") . These are generally imidazole or triazole derivatives and examples include those known by the common names prochloraz, triadimefon, propiconazole, di- clobutrazol, triadiminol, flusilazole, flutriafol, myclobuta- nil, penconazole, quinconazole, imazalil and diniconazole. Examples of non azole EBis include nuarimol, fenarimol, fen- propimorph, tridemorph, fenpropidine and dimethomorph.

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

Dithiocarbamates, e.g. thiram, 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. fosetylaluminiu ;

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;

Pyrimidines, 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 inven- tion 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 invention also relates to the use of these active ingre- dients 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 con- trolling 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 composi- tion 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 Graminea genera, such as Poa cea ; Leguminosea ; vegetables; fruits; etc.

Specifically the compounds of the invention can be used for controlling plant pathogenic fungi on vegetables, such as po- tatoes, turnips, beets, etc.

The compounds of the invention can be also be used for con¬ trolling plant pathogenic fungi on fruits, such as apples and pears, and citrus fruits, such as oranges and lemons.

Specifically the compounds of the invention are especially useful for controlling plant pathogenic fungi on cereals, such as wheat, barley, oats, corn, and rice.

Compounds of the invention have been found to be particularly potent towards fungi belonging to the classes Ascomycota , Ba - sidiomyceta and imperfect fungi, especially plant pathogenic 24

fungi belonging to the classes Ascomycota , Basidiomyceta and imperfect fungi.

Examples of fungi which has been found to be sensitive to compounds of the invention are fungi of the genera Aspergil - lus, especially Aspergill us niger, or Pyricularia , especially Pyricularia oryzae, or Cochliobol us, especially Cochliobol us sa tivus, or Septoria, especially Septoria nodorum, or Ventu- ria, especially Venturia inaeqalis, or Ga umannomyces, especi- ally Ga umannomyces graminis, or Rhizoctonia , especially Rhi ¬ zoctonia solani .

In connection with the method of the invention for control¬ ling fungi, the composition may for agronomical or horticul- tural 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 trea¬ tment. 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 practi¬ cable to treat the roots of a plant before or during plant¬ ing, for example by dipping the roots in a suitable liquid or solid 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 alco¬ hols as described in the pending US patent application no. 4017.000 (Entotech, Inc.) .

Examples of suitable lignins comprise lignin sulfulfonate 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 composition. 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 en- hance 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 al¬ cohol 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 contai¬ ning 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 sup¬ plemented with other components.

Therefore the composition according to the invention may fur- ther 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 deposi¬ ted. 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 pro¬ tein which can be conveniently and economically added to the formulation are soy protein, potato protein, soy flour, pota¬ to 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 alco¬ hols 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 moisture 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, hexy- lene glycols; the sugar alcohol component may also contribute to this function, but it is desirable to add a second poly¬ hydric alcohol to the composition. Overall, the second poly- hydric alcohol component of the composition, if present, should comprise from about 1 to about 20% w/w of the total composition.

The active preparation or the compositions of the invention can be applied directly to the plant by, for example, spray¬ ing 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 g 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 pre¬ ferably 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 impreg¬ nation 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 cer- tain bacteria or yeast.

A final aspect of the invention is to provide an isolated pure culture of the microorganism Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof capable of producing a compound of the invention.

MATERIALS AND METHODS

Materials:

PD agar slants: P_otato Dextrose agar (Difco) slant 812 ml/slant) autoclaved for 121°C for 40 minutes. Tween® 80: Merck Art. 822187

SKP X medium: 1 litre was prepared by mixing Potato flour (75 g), Ban 800 MG (0.075 g) , and tap water (800 ml), heating 5 at 60-70°C for 10 minutes and at 70°C for 20 minutes and was then quickly brought to the boiling point. To this mixture was added soyabean meal (40 g) , Na,HP0₄, 12H,0 (9 g) , KH₂P0₄ (1.5 g) , Pluronic 100% (0.1 ml), and tap water to a total vo¬ lume of 1 litre. The medium was homogenized and sterilized at 10121°C for 40 minutes.

Equipment:

10 L tank fermentor (Chemap) XAD-1180 resin column (1.3 litre) 15 Column of silica gel (Merck Si60, 63-200 μm, 160x40 mm Normal Phase Column (Merck Lobar B) Reversed Phase Column (Dupont ODS, 10 μm, 150 x 20 column

Methods:

20

Cultivation of the strain

A culture of the strain can be grown on JPotato Dextrose agar slants.

25 Metabolite production (submerged)

Hamigera avellanea NN005492 was progagated on Potato Dextrose ager slants for 16 days at 26°C. The slants were washed with sterile, distilled water containing 0.1% Tween®80 (10 ml/slant) , and the resulting spore/mycelium suspension was

30 used to inoculate the shake flasks (3 ml/flask) . The fermen¬ tation was conducted in 500 ml Erlenmeyer flasks with 2 baf¬ fles, each containing 100 ml SKP-X growth media. The pH was after sterilization 6.4. The flasks were shaken (200 rpm) for 12 days at 26°C.

35 Tank Fermentation

Hemigera avellanea (CBS No. 501.94) was fermented in 10 litre Chemap-fermentors containing SKP-X medium. The tanks were inoculated with 3-5% (v/v) of 48 hours old precultures (SKP-X shake flasks prepared as described above) . The tanks were continuously stirred (500 rp ) and aerated (1.0 vvm) and the temperature was kept at 26°C. The fermentations were har¬ vested after 163 hours.

Isolation of compound having formula lb and Ic Hamigera avellanea (CBS No 501.94) was fermented in two 10 litre tank fermentors. The mycelia was harvested by vacuum filtration to yield 3 kg of filter cake and 12.5 litre of filtrate. The culture filtrate was passed through a column of XAD-1180 resin (1.3 litre) at a flow rate of approximately 3 litre/hour. After washing with water (1.3 litre) the column was eluted step-wise with increasing concentrations of EtOH and the following fractions collected: (#1) 25% (1.3 litre), (#2) 50% (1.3 litre), (#3) 75% (1.3 litre), (#4) 100% (400 ml), (#5) 100 % (400 ml), (#6) 100% (400 ml), (#7) 100% (400 ml), (#8) 100% (400 ml), (#9) 100% (400 ml), (#10) 100% (2.4 litre) . Fractions #5-9 were pooled and evaporated to yield 6.6 g syrup. Fraction #4 contained more coloured impurities and was evaporated separately (0.95 g) .

Silica Gel Column Chromatography

The crude extract from the XAD-column (fraction #5-9, 6.6 g) was in two portions applied to a short columns of silica gel (Merck Si60, 63-200 μm, 160 x 40 mm) equilibrated with hep- tane-EtOAc 9+1. The columns were eluted with step-gradients of heptane-EtOAc-MeOH and 200 ml fractions collected: heptane-EtOAc 9+1 (400 ml,#l and #2), heptane-EtOAc 1+1 (400 ml,#3 and #4), EtOAc (400 ml,#5 and #6), EtOAc-MeOH 3+1 (400 ml, #7 and #8), EtOAc-MeOH 1+1 (800 ml, #9, 10, 11, and 12). Fractions #9 and #10 and fractions #11 and #12 from both columns were pooled and evaporated to yield 853 mg (Fraction A) and 194 mg (Fraction B) of oily residues, respectively.

Silica Gel Column Chromatography or TLC Further purification was achieved using normal phase column chromatography (Merck Lobar B eluted at a flow rate of 7.5 ml/min with linear gradient from CH₂C1₂ to CH₂Cl₂-MeOH (1:1) over 60 minutes, Retention time (R_t) was approximately 24 minutes or preparative TLC (Merck Si60 (2 mm layer) , mobile phase CH₂Cl₂-MeOH 6+1, R_f (the relationship between solvent front and the centre of the band) approximately 0.5. 400 mg of Fraction A yielded about 250 mg of semi-pure material, comprising a 10:1 mixture of compound lb and Ic.

Preparative Reversed Phase HPLC

Final purification was achieved by reversed phase HPLC

(Dupont ODΞ (10 μm) , 150 x 20 mm column eluted isocratically with 55 % acetonitrile (0.05% H₃P0₄) at a flow rate of 10 ml/min, Rt_lc=8.4 minutes and Rt_Ifc=9.5 minutes). Fractions con- taining pure compound lb and compound Ic were combined and extracted twice with one volume portions of CH₂C1₂. The or¬ ganic phases were washed with water (one volume) and dried over anhydrous Na₂S0₄ to yield pure compound lb and Ic as clear, colourless glasses.

EXAMPLES

Example 1 Characterization of compound Ic

A culture of the strain Hamigera avelleanea NN005492 (CBS no.

501.94) was cultivated, incubated, fermented, isolated and purified as described above in the section "METHODS AND

MATERIALS" .

The materials obtained were found to have the following physical and spectroscopic properties: Compound ex Compound Ic

Appearance: Colourless glass Colourless glass

Optical rotation [o]_D: -345° (c=0.5 MeOH) -7° (c=0.4 MeOH)

HR-LSIMS ([M-H]-) :

Found: 411.2160 413.2325

Calc: 411.2171 (C₂₅H₃₁0₅) 413.2328 (C₂₅H₃₃0₅)

10 IR spectra: Figure 1

UV-spectra Figure 2 Figure 3

^XH-NMR spectra: Table 1 Table 2 15 ¹³C-NMR spectra: Table 1 Table 2

Table 1 H and ¹³C NMR data for compound or in chloroform-d] and Benzene-d₆

20

CDCl, C*D₆ CDClj C,D₆

2.15 1.75 29.48 28.97

25 206.37 204.37

2.73 2.60 46.55 46.52 3.43 3.12

122.29 122.96

127.55 127.26

30 1.82 55 18.24 18.11 5.72 40 122.67 122.45

137.51 138.21

02 .66 42.46 42.44 65 .23

35 10 39 .90 48.54 48.51 11 09 1.06 14.83 15.21 12 212.31 210.03 13 39 2.26 49.01 49.21 14 07 26 10.81 11.28

40 15 1.74 64 49.79 49.93 16 3.13 ,19 38.04 38.22 17 5.31 52 129.25 129.21 18 127.94 128.54 19 71 ,50 14.18 14.17

45 20 24 ,00 67.73^a 67.73 21 60.36 60.32 22 24 .02 13.91 13.75 23 85 ,95 151.09 151.49 24 01 .14 121.90 121.87

50 25 168.79 170.73 ^{a 1}J_CH=176 Hz Table 2

^:H and ¹ C NMR data for compound Ic in chloroform-d_!

*H ¹³C

1 1.33 24.07

2 4.60 64.35

3 5.62 131.2

4 - 136.00

5 - 132.83

6 1.91 19.95

7 5.65 131.3

8 2.77 32.39

9 1.60 40.84

2.10 -

10 2.49 41.03

11 1.00 14.43

12 - 214.04

13 2.32 43.81

14 1.08 11.43

15 1.74 50.28

16 3.79 36.91

17 5.49 128.73

18 - 128.73

19 1.78 14.02

20 3.30 67.64^a

21 - 60.66

22 1.29 13.92

23 6.90 151.53

24 6.05 120.47

25 - 168.66

' ^:J_CH=174 Hz

These obtained data provides evidence for the structure of compound and compound Ic.

Example 2

Preparation of the methylester of compound 2 mg of compound was dissolved in chloroform (500 μl and diazomethane in ether added in excess. The solvent was removed in a stream of nitrogen and the product purified by reversed phase HPLC (Dupont 250 x 10 mm, linear gradient from 25 to 100 % acetonitrile over 45 minutes, flow 7.5 ml/minutes) to yield the methylester of compound .

EI-MS: mlz (rel.int.%) 426 (7, M⁺) , 408 (5), 394 (3), 376 (4), 365 (5), 351 (6), 333 (4), 323 (6), 298 (12), 281 (8), 265 (9), 255 (21), 253 (13), 241 (28), 223 (12), 225 (12), 240 (40), 213 (13), 201 (15), 197 (20), 185 (13), 183 (14), 171 (22), 169 (15), 157 (16), 149 (14), 112 (100), 97 (17). ^XH NMR (CDC1₃) : 6.82 (lH,d,15.6), 6.04 (lH,d,15.6), 5.72 (lH,s), 5.32(lH,d,10) , 3.76 (3H,s), 3.44 (lH,d,15), 3.26 (lH,s), 3.15 (lH,d,10), 2.75 (lH,d,15), 2.67 (1H,dd, 6/12) , 2.40 (2H,m), 2.16 (3H,s), 2.04 (1H,dd, 10/12) , 1.83 (3H,s), 1.75 (lH,d), 1.71 (3H,s), 1.26 (3H,s), 1.11 (3H,d,6), 1.10 (3H,d,6) .

Example 3

Fungicidal activity Agar diffusion assay The compound has been found to have an in vi tro inhibitory effect on the growth of fungi belonging to the classes Basidiomycota , Ascomycota and imperfect fungi.

It was found to be particularly potent towards

Class Ascomycota : Cochliobol us sa tivus Septoria nodorum Venturia inaequalis Gaumannomyces graminis

Class Basidiomycota Rhizoctonia solani

Imperfect fungi

Aspergillus niger

Pyricularia oryzae (sexual stage Magnaporthe grisea) .

In the inhibition assays the test organisms were embedded in agar media. Small wells were punched in the agar and 15 μl sample was applied to the wells. Inhibition zones were scored after incubation in dark for 2 days at 26°C.

Table 3 shows the activity observed when applying a 1000 ppm (1 mg/ml) solution of compound m in EtOH. Table 3

Test organism mm inhibition zone

VeΛturia inaequalis 52

Gaumannomyces graminis 26

Cochliobolus sa tivus 24

Septoria nodorum 20

Rhizoctonia solani 17

Aspergill us niger 17

Pyricularia oryzae 21

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 inven¬ tion in addition to those shown and described herein will from the foregoing description become apparent to those skil¬ led in the art. Such modifications are intended to fall with¬ in the scope of the appended claims.

Claims

PATENT CLAIMS

A compound having the generic formula I

Formula I wherein RI is hydrogen or together with R2 represents an additional bond,

R2 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

R3 is hydrogen, or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi- tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi¬ tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi^¬ tional bond,

R9 is hydrogen or together with RIO represents an additional bond,

R10 is hydrogen, or together with R9 represents an additional bond,

Rll is hydrogen or together with R12 represents an additional bond,

R12 is hydrogen or together with Rll represents an additional bond,

R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

2. The compound according to claim 1, in which R2 is hydro- gen or together with RI represents an additional bond.

3. The compound according any of claims 1 and 2, in which R13 is hydrogen.

4. The compound according to any of claims 1 to 3, in which RI and R2 represents an additional bond, R9 and RIO repre¬ sents an additional bond, and Rll and R12 represents an addi¬ tional bond, having the formula la:

Formula la wherein

R3 is hydrogen or together with R4 represents an additional bond,

R4 is hydrogen or together with R3 or R5 represents an addi- tional bond,

R5 is hydrogen or together with R4 or R6 represents an addi¬ tional bond,

R6 is hydrogen or together with R5 or R7 represents an addi¬ tional bond,

R7 is hydrogen or together with R6 represents an additional bond,

R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub- stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group, and

5. The compound according to any of claims 1-4, with the formula ₁ :

Formula Ibl wherein R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

6. The compound according to any of claims 1-4, with the formula Ic_x:

Formula Icl wherein

R8 is hydrogen, -COR, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2- 510 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group,

10 R13 is hydrogen, straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon atoms, benzyl or aryl, optionally mono- or plurisub¬ stituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy,

15 halogen, amino or a nitro group, and

R is a straight or branched chain alkyl with 1-10 carbon atoms, straight or branched chain alkenyl with 2-10 carbon atoms, straight or branched chain alkynyl with 2-10 carbon 20 atoms, benzyl or aryl, optionally mono- or plurisubstituted with alkyl with 1-10 carbon atoms, hydroxy, alkoxy, halogen, amino or a nitro group.

257. The compound according to any of claims 1 to 4, with the formula :

30

Formula Ic

8. The compound according to any of claims 1 to 4, with the formula Ic

Formula lb

7. A method of preparing a compound according to claim 1 to 6, comprising a) cultivating a microorganism capable of producing said compound in or on a suitable nutrient medium and under suit¬ able conditions, and b) recovering the compound from the biomass and/or the cul¬ ture medium.

8. The method according to claim 7, which further comprises c) chemically modifying the compound obtained in step b) .

9. The method according to any of the claims 7 and 8, where¬ in the microorganism is a fungus, preferably of the genus Hamigera , preferably of the species Hamigera avellanea .

510. The method according to claim 9, wherein the fungus is a strain of the Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof capable of producing a compound as defined in any of the claims 1 to 6.

o

11. The method according to claim 10, in which the compound prepared is the compound with the formula .

12. The method according to claim 10, in which the compound prepared is the compound with the formula Ic. 5

13. A fungicidal composition comprising, as an active ingre¬ dient, a compound according to claim 1 to 6.

14. The fungicidal composition according to claim 13, wherein 0 said compound is present in an amount of from 0.001 μg/ml to

100 mg/ml.

15. The fungicidal composition according to claim 13 and 14, comprising as an active ingredient, a fungus of a species be- 5 longing to the genus Hamigera capable of producing a compound as defined in any of the claims 1 to 6.

16. The fungicidal composition according to claim 15, wherein the Hamigera is Hamigera avellanea, and preferably a strain 0 of the Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof capable of producing a compound as defined in any of the claims 1 to 6.

17. The fungicidal composition according to claim 13 to 16, 5 comprising as an active ingredient a compound of any of the claims 1 to 6 in combination with one or more further fungi¬ cidal or pesticidal agents and/or growth regulators.

18. The fungicidal composition according to claim 17, wherein said other active agent (s) is/are present in an amount of 0.001 % (w/w) to 50 % (w/w) by weight, preferably 0.01 % (w/w) to 10 % (w/w) .

5

19. The fungicidal composition according to claim 13 to 18, further comprising adjuvants, such as UV protective com¬ pounds, retention compounds, surface active compounds.

ιo 20. A method of controlling fungi 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 6 or a composition according to any of the claims 13 to 19.

1521. The method according to claim 20, in which the locus is selected from the group consisting of plants, timber, wood, cosmetics, feeds and foods.

22. The method according to claim 21, in which the locus is 20 a cereal.

23. The method according to claim 22, in which the locus is a rice plant, especially the rice plant Oryzae sa tiva .

2524. The method according to any of claims 20 to 23, in which the fungus to be controlled is a plant pathogenic fungus, preferably one which belongs to the classes Ascomycota , Basidiomycota and imperfect fungi.

3025. The method according to any of the claims 20 to 24, in which the fungus to be controlled belongs to the genera Pyricularia , especially Pyri cularia oryzae, or Cochliobol us, especially Cochliobolus sa tivus, or Septoria , especially Septoria nodorum, or Ven turia , especially Venturia inaeqalis,

35 or Ga umannomyces, especially Ga umannomyces graminis, or Rhizoctonia , especially Rhizoctonia solani , or Aspergill us, especially Aspergillus niger.

26. Use of a compound according to any of the claims 1 to 6 for the preservation of cosmetics.

27. Use of a compound according to any of the claims 1 to 6 5 for the preservation of foods and feeds.

28. Use of a compound according to any of the claims 1 to 6 as an additive to growth media.

1029. Use of a compound according to any of the claims 1 to 6 for the preservation of paints.

30. Use of a compound according to any of the claims 1 to 6 for the preservation of wood or timber.

15

31. Use of the compound according to any of claims 1 to 6 for combating fungi on cereals.

32. Use of the compound according to claims 31 for combating 20 fungi on rice plants, especially on the rice plant Oryzae sa tiva .

33. An isolated pure culture of the microorganism Hamigera avellanea NN005492 (CBS No. 501.94) or a mutant thereof

25 capable of producing a compound as defined in any of the claims 1 to 6.