WO1993022444A1

WO1993022444A1 - Fungicidally active compounds

Info

Publication number: WO1993022444A1
Application number: PCT/DK1993/000145
Authority: WO
Inventors: Jens Breinholt; Connie Ninna Rosendahl; Helle Demuth
Original assignee: Novo Nordisk A/S
Priority date: 1992-05-01
Filing date: 1993-04-30
Publication date: 1993-11-11
Also published as: JPH07505781A; EP0652970A1; AU4260693A; DK58392D0

Abstract

Compounds of formula (I), wherein R1 is hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH¿2?OH, straight or branched alkylethers with 1 to 6 carbon atoms or optionally substituted aromatic ethers; each R?2¿ independently are hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH¿2?OH, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and each R?3¿ independently are hydrogen, hydroxy, methoxy, acetoxy, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and wherein the chiral centres a, b, c, d, e, f, and g each independently may have the R or S configuration; except for the compound, wherein R1 is -CH¿2?-OH, all of R?2¿ are -CH¿3?, and all of R?3¿ are -OH (formula Ib); or agronomically acceptable salts or esters thereof have been found to have fungicidal activity. Furthermore processes for producing such compounds by cultivation of a species belonging to the genus Fusarium on suitable nutrient media are given. The invention also relates to fungicidal compositions comprising these compounds, either alone or in combination with known biocides or growth regulators, and the use of the compounds or compositions of the invention for controlling fungi.

Description

Title: FUNGICIDALLY ACTIVE COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to fungicidally active compounds and derivatives thereof, and processes and microorganisms for producing such compounds. Furthermore the invention relates to fungicidal compositions comprising these compounds, either alone or in combination with other biocides or growth regulators, and to the use of the compounds or compositions of the invention for controlling fungi.

BACKGROUND OF THE INVENTION

For a number of years it has been known that various microorganisms produce secondary metabolites exhibiting biological activity and that these can be useful as biocides for controlling diseases and pests.

Although progress has been made in the field of identifying and developing biological biocides for controlling various diseases and pests in plants and animals of agronomical importance, most of the pesticides in use are still synthetic compounds that are difficult to decompose in nature and have a broad spectrum of activity.

In the last decade there has been an increasing concern for the impact of such pesticides on the environment and the ecosystems surrounding farmlands, and consequently there exists an outspoken need for biocides that are more specific in their activity, and are readily degradable in the natural environment.

Natural products can often provide an alternative to synthetic fungicides in that they may have another mode of action, may be more biodegradable and safer in the environment. It is consequently an object of the invention to identify novel fungicidally active compounds such as microbial secondary metabolites which originates from natural sources or are direct derivatives of such compounds.

US Patent No. 5,008, 187 and Chaiet et al (The Journal of Antibiotics 42 1718-1721, 1989) describe a secondary metabolite of formula lb below as an antifungal agent obtainable by fermentation of a microorganisms belonging to the genus Fusaήum designated MF 5000 (ATCC No. 20883)

The invention concerns compounds closely related to the compound described in the above US Patent No. 5,008,187 and their use in agriculture for controlling fungi.

SUMMARY OF THE INVENTION

Briefly stated the invention relates to compounds of the formula I below

wherein R 1¹ is hydrogen, straight or branch ^ωed alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms or optionally substituted aromatic ethers; each R² independently are hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and each R3 independently are hydrogen, hydroxy, methoxy, acetoxy, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and wherein the chiral centres a, b, c, d, e, f, and g each independently may have the R or S configuration; except for the compound, wherein RMs -CH2-OH , all of R² are -CH3, and all of R^ are -OH (formula lb); or agronomically acceptable salts or esters thereof.

Compounds of formula I wherein all of R² are -CH3, all of R^ are -OH, and R* is CH₃ (formula la) or -CH2-OH (formula lb), are obtainable as secondary metabolites from microorganisms belonging to the genus Fusarium, specifically microorganisms belonging to the species Fusarium pallidoroseum, and more specifically the strain designated CL534-1 (IMI CC No. 351439)

Said compounds exhibit antifungal activity and can be used as an agricultural, horticultural antifungal compound for protection of plants against fungi.

According to a second aspect of the invention the compounds of formulae la and lb are obtainable by cultivation of a microorganisms belonging to the genus Fusarium, specifically microorganisms belonging to the species Fusarium pallidoroseum, and more specifically the strain designated Fusarium pallidoroseum (IMI CC No. 351439) and subsequent recovery of the active compounds from the biomass. These isolated compounds can thereafter be treated chemically to produce the derivatives indicated above.

Another method for producing the compounds of the invention is by traditional chemical synthesis from appropriate starting materials.

In a third aspect of the invention it relates to fungicidal compositions comprising the novel compounds of the invention in combination with suitable excipients, such as diluents, car¬ riers, etc.

The invention also relates to the use of microorganisms producing the compounds of the invention as fungicidal compositions. Within the context of the invention it is also contemplated to combine the novel compounds of the invention with known biocides or plant growth regulators for making up novel combination compositions.

Furthermore the invention in a fourth aspect relates to methods of controlling plant diseases by application of compositions comprising the novel compounds of the invention to infested areas.

In a fifth aspect the invention relates to the use of the novel compounds or compositions of the invention for combating or controlling plant diseases, especially fungal attack.

Lastly the invention relates to an isolated pure culture of the microorganism Fusarium pallidorseum (IMI CC No. 351439).

DEPOSITION OF MICROORGANISMS

For the purpose of describing this invention in detail a strain of the fungus Fusarium pallidoroseum (IMI CC No. 351439) has been deposited with the International Mycological Institute Culture Collection (IMI CC), Ferry Lane, Kew, Surrey TW9 3AF, England, for the purposes of patent procedure on the date indicated below. IMI CC being an interna¬ tional depository under the Budapest Treaty affords permanence of the deposit in ac¬ cordance with rule 9 of said treaty.

Deposit date: 18 February, 1992

Depositor's ref.: CL534-1

UVU CC designation: IMI CC No. 351439

IMI CC No. 351439 belongs to the class Deuteromycetes, and the order Moniliales. The fungus was isolated from plant material collected in the tropical area. DETAILED DESCRIPTION OF THE INVENTION

As indicated above the invention relates in its first aspect to novel compounds of the general formula I below

wherein R 1¹ is hydrogen, straight or branch ^roed alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms or optionally substituted aromatic ethers; each R² independently are hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and each R-^> independently are hydrogen, hydroxy, methoxy, acetoxy, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and wherein the chiral centres a, b, c, d, e, f, and g each independenfly may have the R or S configuration; except for the compound, wherein R s -CH2-OH , all of R² are -CH3, and all of R-> are -OH (formula lb); or agronomically acceptable salts or esters thereof.

Compounds of formula I, wherein all of R² are -CH3, all of R-^> are -OH, and R* is -CH3 (formula la), or -CH2-OH (formula lb), have been isolated as metabolites from a strain of the genus Fusarium, Fusarium pallidoroseum (IMI CC No. 351439) According to its second aspect the invention relates to processes for the production of the novel compounds of the composition I.

The compounds of formula I may be prepared by aerobic cultivation on suitable nutrient media under conditions hereinafter described with a strain of a fungus belonging to the genus Fusarium especially the fungus Fusarium pallidoroseum (UvU CC No. 351439) and subsequent recovery of the active components from the biomass and/or fermentation medium.

The natural metabolite may subsequently be modified chemically or by biotransformation in order to obtain various derivatives thereof. The derivatives may be produced in order to improve certain properties of the metabolite such as its solubility in aqueous media, its hydrophobicity, hydrophilicity, stability, specificity, etc.

It is also contemplated that compounds according to the invention may be produced by well known chemical synthetic processes using available starting materials.

The compounds of the invention have been found to exhibit antifungal activity, and they are consequently useful as active ingredients in fungicidal compositions.

The present invention therefore also embraces fungicidal compositions containing the compounds of formula I as active components. The invention contemplates the use of any of the compounds of the invention used alone or in combination with any other of the compounds of the invention or any other biocidally active agent or plant growth regulator as active components in any fungicidal composition.

A fungicidal composition according to the invention having a fungicidally active compound of the invention as its active ingredient may for agronomical and/or horticultural applica¬ tions 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 such as a wettable powder, an emulsifiable concentrate, a concentrated emulsion, a granular formulation, a water soluble powder, an alginate, a xanthan gum and/or an aerosol. As solid carriers bentonite diatomaceous earth, apatite, gypsum, talc, pyrophyllite, vermiculite, ground shells, and clay may be mentioned. A surface active agent may also be added with the purpose of producing a homogeneous and stable formulation.

The diluent or carrier in the composition of the invention can as indicated be a solid or a liquid optionally in association with a surface-active agent, for example a dispersing agent, emulsifying agent or wetting agent. Suitable surface-active agents include 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 compositions of the invention can take any form known in the art for the formulation of agrochemicals, for example, a solution, a dispersion, an aqueous emulsion, a dusting powder, a seed dressing, a dispersible powder, an emulsifiable concentrate or granules. 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.

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.

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 suspending agent.

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 such as other biocides, such as fungicides, pesticides, herbicides, insecticides, nematocides, acaricides or plant nutrients or fertilizers.

Examples of other 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, diclobutrazol, triadiminol, flusilazole, flutriafol, myclobutanil, penconazole, quinconazole, imazalil and diniconazole. Examples of non azole EBis include nuarimol, fenarimol, fenpropimorph, tridemorph and fenpropidine. Other fungicides which can be combined with the fungicide preparation of the invention include anilides, e.g. carboxin, matalaxyl, furalaxyl, ofurace, benalaxyl, mepronil, flutolanil, pencycuron and oxadixyl; benzimidazoles, e.g. benomyland carbendazim; carbamates, e.g. maneb, mancozeb and propamocarb; dicarboximides, e.g. iprodione, vinclozolin and procymidone; phosphorus derivatives, e.g. pyrazophos, tolclofos-methyl and fosetyl aluminum; and miscellaneous compounds, including dithiocarbamates, bisdithiocarbamates, imidazoles, triazoles, phathalonitriles, polyhalogenalkylthio compounds, iminoctadine, guazatine, dicloran, chlorothalonil, pyrifexox, ethirimol, cymoxanil and anilazine.

In its fourth aspect the invention relates to methods of combating fungi ON plants, wherein an effective amount of a fungicidally active compound of the invention is applied to a region to be treated.

In connection with this aspect the compositions of the invention may for agronomical or horticultural uses be applied to a region to be treated either directly 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.

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. 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.

The concentration of the fungicidally 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.

In a primary composition or concentrate the amount of active compound can also vary widely and can be, for example, in 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. In a primary composition the amount of other active ingredient can vary widely and can be, for example, from 5 to 80 per cent by weight of the composition.

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. 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, preferably from 0.05 to 5 kg per hectare.

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 fungicidally 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 per hectare, more preferably from 0.05 to 5 kg per hectare.

Although the present invention has been described in detail in connection with controlling fungi in plants, it is also anticipated that the fungicidally active compounds of the invention may be used for combating or controlling fungi in mammals, including humans; and for the preservation of wood by adding said compounds to wood preservation and/or impreg¬ nation compositions. Also, the active compounds of the invention may be useful as a fungi¬ cide 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.

Description of the strain

Taxonomy and morphology of the organism:

The fungus is identified as Fusarium pallidoroseum (Cooke) Sacc. (syn. F. semitectum Berk. & Rav) using: Burgess, W. & Liddell CM. (1983), Laboratory manual for Fusarium research: Incorporation a key and descriptions of common species found in Australia, The University of Sydney, Sydney, (ISBN 0-909798-39-7), Booth, C. (1971). The genus Fusarium. Commonwealth Mycological Institute, Kew Surrey, England, and Booth, C & B.C. Sutton, 1984. Fusarium pallidoroseum, the correct name for F. semitectum Auct. Trans. Br. Mycol. Soc. 83 (4): 702-704.

The fungus belongs to the class Deuteromycetes, order Moniliales.

The aerial mycelium on Potato Sucrose Agar is floccose white gradually changing through peach, avellaneous to buff brown. Conidia of two types, primary and secondary. The primary conidia are somewhat shorter than the secondary. They have a wedge-shaped foot cell, are hyaline, 0-5 septate and measure 7.5-35 x 2.5-4 μm. These primary conidia are formed as holoblastic conidia from polyblastic sympodially proliferating cells with up to six separate spores formed from each cell. The secondary conidia are formed from simple phialides. These conidia have a typical heeled foot cell (for further details see Booth and Sutton, 1984).

Cultivation of the strain

A culture of the strain can be grown on potato dextrose agar (Difco) slants (12 ml/slant), autoclaved at 121 °C for 40 minutes and will hereafter be referred to as PD agar. After inoculation it can be incubated at 20 - 25° C for 2 days or longer.

Fungicide production

A slant culture grown on PD agar for 7 days at room temperature, consisting of mycelia and spores, was mixed with 10 ml of sterile water containing 0.1% Tween^® 80 and inoculated into 500 ml Erlenmeyer flasks containing 100 ml Yeast-Extract-Sucrose medium using tap water (2% yeast extract (Difco); 15% sucrose; 10 ppm ZnSO₄-7H₂O; 5 ppm CuSO₄ ^«5H₂O and 0.3 % agar (Difco). The Ph was adjusted to 6.4 before the substrate was autoclaved at 121 °C for 40 minutes. After inoculation, the flasks were placed at 26°C for 13 days.

The fungicide can also be produced in surface cultures in accordance with well known procedures.

The fermentation can also be carried out in media containing other sources of carbon and nitrogen assimilable by the microorganism and generally low levels of inorganic salts. In addition, the media may be supplemented with trace metals, although if complex sources of carbon and nitrogen are employed, they are usually present in the complex sources.

Extraction of the compound

100 ml methanol was added to each flask and hereafter the flasks were shaken (200 rpm) at 18 °C for 3 hours. The methanol supernatant containing the fungicide was separated from the mycelium by centrifugation for 10 min. at 20.000 rpm. Hereafter the pellet containing the mycelium was discarded and the supernatant was analyzed for fungicidal activity. The compounds of the invention can also be extracted from the biomass and/or growth medium by any other conventional extraction method known in the art, such as with a lower chain alcohol, acetone, DMSO, ethylacetate etc. The concentrated extract can be purified by adsorption to a hydrophobic resin. The resin used are non-ionic, especially cross-linked styrene(divinyl-benzene-copolymers and halogenated styrene/divinyl-benzene- copolymers. A generally used adsorption resin is XAD-8 (Rohm and Haas Co.). A mixture of the two compounds can be obtained more than 90% pure by elution of this resin with increasing concentrations of a lower alcohol/acetone in water, preferably methanol or ethanol. A crude mixture of the two compounds can be obtained by combination and concentration of these fractions. If required the compounds can be further purified by known reversed phase chromatography using an acidic mobile phase, yielding two pure compounds.

For simplicity the compound (formula la) isolated hereinafter will be designated BK219.

Characterization of the isolated compound

Fermentation and Extraction:

A slant culture of the strain grown on PD agar was inoculated into yeast extract-sucrose medium, fermented, and extracted as disclosed above.

Isolation/purification :

1115 ml supernatant was concentrated under reduced pressure to 250 ml. The concentrated extract was applied to a 200 ml column of Amberlite XAD-8 (Rohm and Haas) which was eluted with a step gradient (0%, 30%, 50%, 70% and 100% each 200 ml) of aqueous ethanol. The activity of all fractions were monitored by a disk diffusion assay with Botrytis cinerea. The biological activity was found in the pale yellow fractions eluting with 30 to 50 % ethanol, which upon evaporation of the solvent yielded 780 mg of a yellow oil. Further purification and separation of compound la and compound lb was carried out by reversed phase HPLC [Bϋchi column 26x230 mm, LiChroprep^® C,₈, 15-25 μm; linear gradient of CH₃CN/H₂O buffered with 0.1 % formic acid starting at 60% CH₃CN ending after 60 min with 100% CH₃CN, flow 10 ml/min, detection λ = 225 nm] yielding 449 mg of compound lb (R. = 33 min) and 251 mg of compound la (R. = 50 min), both as a colourless glass.

Anal tical HPLC method:

The novel compound BK 219 assigned formula la, is a colourless solid characterized by the following physico-chemical properties.

Molecular weight: 590 (negative FAB-MS)

Molecular composition C₃₄H₅ (calcd. mass 589.3740 found 589.3792) by negative FAB-MS.

UV spectral data HV^ 216 nm (32,000) 239 nm (26,000) 281 nm (7,900).

Mf = -23.6 (c = 0.4, MeOH).

The active compound assigned formula lb, is a colourless solid characterized by the following physico-chemical properties.

Molecular weight: 606 (negative FAB-MS)

Molecular composition

(calcd. 605.36896 found 605.3645 by negative FAB-MS.

UV spectral data UV-_^ 216 nm (28,000) 239 nm (24,000) 281 nm (6,500). [α]? = -21.2 (c = 0.3 MeOH).

Η-NMR data are given in table 1 and "C-NMR data are given in table 2. The spectra were obtained at ambient temperature in CD₃OD and the signals at δ=3.3 (*H) and δ=49 ppm (¹³C) were used as internal references.

Table. 1. Η NMR data of compound la and lb.

^•in Methanol-d,,, 600 MHz-

40 Table. 2. ¹³C NMR data of compound I and II.

Ib^a Ia^a δ m δ m [ppm] [ppm]

14.4

23.6

32.9

28.3

30.6

38.2

32.2

20.0 q

40.9 t

133.9 s

23.8 q

131.8 d

32.9 d

22.1 q

139.4 d

131.4 s

12.9 q

139.1 d

125.9 d

78.1 d

45.4 s

22.9 q

20.3 q

172.4 s

100.8 d

170.4 s

100.2 s

166.9 s

75.6 d

73.5 d

74.0 d

32.9 t

78.4 d

65.6 t

in Methanol-d₄, 150 Mhz; " in MeOH, 75 Mhz.

From the foregoing spectral data the compound BK 219 is believed to have the formula la.

da)

The compound is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulphoxide, ethylacetate and the like.

From the spectral data the other compound is believed to have the formula lb.

(lb)

The compound is soluble in various organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulphoxide, ethylacetate and the like.

Compound lb is identical to the compound described in US Patent No. 5,008,187. The compounds la and lb have been found to be active against a broad spectrum of fungi, rendering them adaptable for agricultural/horticultural applications. Activity against a bacteria and a yeast was also showed.

Although the invention discussed herein below, principally deals with the specific strain Fusarium pallidoroseum, it is well known in the art that the properties of the microorganisms may be varied naturally and artificially. Thus all strains belonging to the genus Fusarium including variants, recombinants and mutants, whether obtained by natural selection, produced by the action of mutating agents such as ionizing radiation or ultraviolet irradiation, or be the action of chemical mutagens such as nitrosoguanidine, are included in this invention.

Fungicidal activity

A mixture of compound la and lb (1:4) has been found to have an in vitro inhibition on the growth of fungi belonging to the Oomycetes, Ascomycetes and Deuteromycetes species. It was found to be particularly potent towards:

Class Oomycetes Pythium sp. Type F. Phytophthora i festans.

Class Ascomycetes Venturia inaqualis Phoma betae Phoma lingam Ascochyta pisi Sclerotinia sclerotiorum Pyrenophora teres Saccharomyces cerevisiae

Class Deuteromycetes Fusarium oxysporum Botrytis cinerea Rhizoctonia solani AG-3. Monilinia fructigena Alternaria alternata Aspergillus niger

The same mixture of compound la and lb has been found to have in vitro inhibition effect 5 on the growth of a bacteria belonging to the order Eubacteriales. It was found to be particular potent towards:

Order Eubacteriales Bacillus subtilis.

10

EXAMPLES

Example I

15 Fermentation and Isolation/purification

500 ml Erlenmeyer flasks containing 100 ml Yeast-Extract-Sucrose medium (2% yeast extract (Difco), 15 % sucrose, 10 ppm ZnSQ, 7H₂O, 5 ppm CuSO₄ 5H₂oO 0.3% agar), were inoculated with a suspension of spores and mycelϊa from strain CL 534-1. The flasks were placed at 26°C for 13 days. Each flask was extracted with 100 ml MeOH, shaken at 20 18°C for 3 hours (200 rpm). Hereafter the mixture was centrifuged, and the supernatant containing the metabolites thereby separated from the mycelia.

Isolation and purification was performed as described above under "Characterization of the isolated compound". 25

Example II

Activity against Botrytis cinerea

Residual protective activity. 30 Host: Lycopersicon esculentum (Tomato, var First in Field)

Tomato plants (5 weeks old) were sprayed to run off with a liquid suspension using a handhold sprayer (Bink Bullows 900). The suspension contained different concentrations of either compound la and lb in combination or only compound la. The plants were kept 24 hours in a glasshouse to dry before they were inoculated with a spore suspension containing lxlO⁵ spores per ml and 25 % grape juice. The inoculation was carried out with a handhold atomizer (Wagner, Pico Bel). The plants were then incubated (16 hours light (1000 lux) and 8 hours dark) at 15-20°C in clear polythene bags to raise the relative humidity to 95-100%

After 6 days the assessments were done giving the results indicated below:

Example III

Preparation of Methyl ether of lb

To a solution of lb (24 mg) in 1 ml of dichloromethane excess diazomethane in ether solution was added at 0 C After concentration to dryness in vacuo the reaction mixture was purified by reversed phase chromatography yielding 16 mg of Ic

Ic

Activity of Ic

Compound Ic has been found to have an in vitro inhibition on the growth of fungi belong to Ascomycetes and Deuteromycetes. It was found particular potent towards:

Class Ascomycetes Saccharomyces cereviviae

Class Deuteromycetes Botrytis cineres Rhizoctonia solani AG-3 Aspergillus niger

The compound also showed in vitro inhibition effect on the growth of a bacteria belonging to the order Eubacteriales.

Class Eubacteriales Bacillus subtilis. International Application No: PCT/

Claims

1. An antifungal compound producible from a strain belonging to the class Deuteromycetes, preferably belonging to the order Moniliales, more preferably to the genus Fusarium, specifically belonging to the species Fusarium pallidoroseum, and especially the strain Fusarium pallidoroseum (HvH CC No. 351439) .

2. An antifungal antibiotic compound having the formula I.

(I) wherein R is hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms or optionally substituted aromatic ethers; each R^ independently are hydrogen, straight or branched alkyl with 1 to 6 carbon atoms, such as methyl, straight or branched hydroxyalkyl with 1 to 6 carbon atoms, such as CH₂OH, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and each R-* independently are hydrogen, hydroxy, methoxy, acetoxy, straight or branched alkylethers with 1 to 6 carbon atoms, or optionally substituted aromatic ethers; and wherein the chiral centres a, b, c, d, e, f, and g each independently may have the R or S configuration; except for the compound, wherein R is -CH2-OH , all of R^ are -CH3, and all of R³ are -OH (formula lb); or agronomically acceptable salts or esters thereof.

3. The antifungal antibiotic compound of claim 2 having the formula la.

(la) or agronomically acceptable salts or esters thereof.

4. A process for producing a compound of claim 2 or 3 or formula lb comprising cul¬ tivation of a species belonging to the class Deuteromycetes, preferably belonging to the order Moniliales, more preferably to the genus Fusarium, specifically belonging to the species Fusarium pallidoroseum, and especially the strain Fusarium pallidoroseum (IMI CC No. 351439) on a suitable nutrient media under suitable conditions, subsequently isolating an active compound from the biomass and the fermentation media of formula (la) or (lb)

(lb) and optionally chemically modifying any one of said compounds to obtain a compound of formula (J).

5. The process of any of the claims 3 or 4, wherein the microorganism is Fusarium 5 pallidoroseum (IMI CC No. 351439).

6. A fungicidal composition which comprises at least one compound of one of the claims

I, 2 or 3.

10 7. The fungicidal composition of claim 6, which comprises a mixture of compound (la) and (lb).

8. A fungicidal composition which comprises as active ingredients at least one compound of one of the claims 1, 2, or 3 in combination with other biocidal agents and/or

15 plant growth regulators.

9. The fungicidal composition of claim 8, which comprises as an active ingredient a mixture of compounds of formula la and lb.

20 10. A method of producing a fungicidally active composition, comprising mixing of at least one active compound according to claim 1, 2, or 3 with an inert carrier.

II. The method of claim 10, wherein said at least one active compound is further combined with one or more biocidally active compounds and/or plant growth regulators.

25

12. A method of combating fungi at a locus infested or liable to be infested therewith, which comprises applying to said locus at least one compound of formula (I) according to any of claims 1 or 2, or a composition according to any of the claims 6 to 9.

30 13. The method of claim 12, wherein said compound(s) or composition is applied to plants.

14. The method of claim 12, wherein the active ingredient of formula (I) is applied in a concentration ranging from 0.001 to 50 kg, preferably 0.05 to 5 kg.

15. The method of claim 12 for combating fungi on timber, in cosmetics, feeds or foods. 5

16. The method of any of claims 12 to 15 to control attack from fungal species belonging to the classes Ascomycetes, Oomycetes or Deuteromycetes.

17. The method of claim 16 to control attacks from fungal species belonging to the genus 10 Botrytis, especially Botrytis cinerea.

18. Use of a compound of one of the claims 1, 2, or 3, or a composition of any of the claims 6 to 9 for controlling fungi.

15 19. The use of claim 18 to control attack from fungal species belonging to the classes Ascomycetes, Oomycetes or Deuteromycetes.

20. The use of claim 19 to control attacks from fungal species belonging to the genus Botrytis, especially Botrytis cinerea.

20

21. A substantially pure culture of a microorganism belonging to the class Deuteromycetes, preferably belonging to the order Moniliales, more preferably to the genus Fusarium, specifically belonging to the species Fusarium pallidoroseum, and especially the sttain Fusarium pallidoroseum (IMI CC No. 351439) useful for the production of

25 fungicidally active compounds according to any of claims 1 to 3.