FIELD OF THE INVENTION

The invention relates to biologically active compounds of te- tracyclic structures, related to the tetracyclines, methods for their production, and microorganisms capable of synthe¬ sizing such compounds. The compounds of this invention are tetracyclic structures, related to the tetracyclines.

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, horticul¬ ture, food industry etc. However, today it is realised that in general such chemicals, have a negative impact on the en¬ vironment. Therefore especially, the search for biological agents such as microbes and icrobial 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.

One group of compounds exhibiting biological activities are the tetracyclines. The tetracyclines are important drugs used extensively in treating bacterial infections in humans and animals.

O96/16014 PC-7DK95.00461

The linear arrangement of four six-membered rings is a struc¬ tural feature common to the tetracyclines isolated from Strep- tomyces spp..

Tetracyclin

Further, the fungal metabolite viridicatumtoxin of tetracy- clinic structure has been reported as a metabolic product of Penicillium expansum, earlier P. viridicatum (Kabuto, C. et al., J. Chem. Soc. Chem. Commun. , p. 728, 1976).

Viridicatumtoxin exhibits acute toxicity when administered to laboratory animals (Hutchinson et al., Toxicol. Appl. Pharm. ,

24, p. 507, 1973)

Viridicatumtoxin

The genus Hypomyces is known to produce metabolites of indus- trial interest. However these are not of tetracyclic struc¬ ture.

Examples are alkaloids produced from the strain Hypomyces aurantius IFO 7773, as described in JP-A-5-9140892 (TAKADA CHEMICALS) .

Further, JP-A-5-9102396 (ENDO A) concerns a metabolite ML- 236B prepared by culturing Hypomyces chrysospermuε, which exhibits inhibitory effect on cholesterol synthesis.

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 Hypomyces .

The novel compounds have the general formula I

(I) wherein

R 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, in which

Rι is 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,

R ₂ 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,

R ₃ 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,

R ₄ 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 ₅ 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,

In a preferred embodiment of compound I, R ₂ is hydrogen and R ₄ is methyl, R ₃ is 3-methyl-2-butenyl, R ₅ is hydrogen, resulting in compounds with the formula:

(la)

A specific embodiment of the invention is the compound with the specific formula lb

(lb)

In another aspect, the invention relates to a method of pre-

paring said compound 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 Hypomyces , preferably of the species Hypomyces aurantius , especially of the strain Hypomyces au- rantius (CBS No. 654.93) or a mutant thereof capable of pro¬ ducing 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 in- fested 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 com¬ pounds as a fungicide, preservative and/or additive for co - 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 Hypomyces aurantius A03892 (CBS No. 654.93) or a mutant thereof capable of producing a compound of the invention.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the Infrared spectrum (IR spectrum) of com¬ pound with the formula lb.

DETAILED DESCRIPTION OF THE INVENTION

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

(i) wherein R 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, in which

R, is 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,

R ₂ 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 ₃ 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,

R ₄ 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 ₅ 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.

In connection with the compounds of the present invention having the formula I, respectively, 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

O 96/16014 PC--7DK95/00461

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.

In an embodiment of the invention R ₂ and R ₅ is hydrogen or hydroxy.

In another embodiment R, is hydrogen or a straight or branch¬ ed chain alkyl with 1-10 carbon atoms.

Further, in a preferred embodiment R ₂ is hydrogen, R ₃ is 3- methyl-2-butenyl, R, is methyl, and R _s is hydrogen, resulting in compounds of the invention with the general formula la:

(la)

In a specific embodiment of the invention is the compound with the formula lb.

(lb)

Another object of the invention is to provide for a method of preparing compound having 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 Hypomyces , especially of the species Hypomyces aurantius, in particular the strain thereof ident¬ ified by the deposition number CBS No. 654.93, or a mutant thereof capable of producing a compound of the invention.

An isolate of the fungus Hypomyces aurantius A03892 (CBS No. 654.93) has been deposited with the Centraalbureau voor Schimmelcultures, 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¬ dance with rule 9 of said treaty.

Deposit date : December 8, 1993 Depositor's ref.: A03892

CBS designation : CBS No. 654.93

Further the isolate of the fungus Hypomyces aurantius A03892 (CBS No. 654.93) has been deposited under conditions that assure that access to the isolated fungus will be available during the pendency of this patent application to one deter¬ mined by the commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. S 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 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.

A suitable nutrient medium is one which comprises the micro- and macronutrients required to obtain a satisfactory growth

of the microorganism in question and at the same time give rise to a production of the compound of the invention when subjected to suitable cultivation conditions.

Normally, a suitable nutrient medium contain sources of 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 bio ass 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 Hypomyces , especially the Hypomyces aurantius A03892 ident¬ ified by the deposition number (CBS No. 654.93), the compound is normally contained in the biomass, such as in or on the mycelium, the spores, and the like.

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. When the compound is con¬ tained in the biomass, e.g. a fungal mycelium, the recovery comprises harvesting the mycelium, e.g. by filtration and/or centrifugation, and subsequently isolating the compound the-

refrom. Suitable methods for isolating the compound includes extraction using a suitable solvent such as polar solvents like methanol, ethanol, ethyl acetate, or acetone, and solid phase extraction using a hydrophobic resin, an example of which is XAD-8 (Rohm and Haas Co.). Further purification may be accomplished by chromatography and/or crystallisation.

In order to improve certain properties of the metabolite such as its solubility in aqueous media, its hydrophobicity, hy- drophilicity, stability, specificity, toxicity, target 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.

A specific example of the production of a compound with the formula I and la is given in the examples below. This example describes the production of the specific compounds lb, 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 compound ,-some compounds of the invention may advantageously be prepared from the compound I using a synthetic process.

Ester derivatives of the compound with the formula la yield¬ ing compounds of formula I (wherein R is acyl) may be pre¬ pared by treating the compound la (wherein R=H) , dissolved in

a suitable solvent, with appropriate acylating reagents such as acid halides, acid anhydrides or activated esters, optio¬ nally under influence of a basic catalyst (pyridine, other amines etc.). Ether derivatives of compound la yielding com- pounds of formula I may be prepared by treating la (wherein R-H) , dissolved in a suitable solvent, with an appropriate alkylating reagents, such as diazo compounds or with appro¬ priate alkyl halides and alike, under influence of a catalyst (potassium carbonate, silver oxide etc.).

The oxygen substitution pattern of lb indicates that the tetracyclic core is constructed by folding and cyclization of a linear decaketide (10 acetate units) , with C13-C14 being the starting unit. Thus, derivatives where R ₄ varies may be obtained by directed biosynthesis, i.e. forcing the producing microorganism to build in other starter units than acetate by feeding to the growth media appropriate substances acceptable for the enzyme system involved i the biosynthesis.

Stereogenic centers of lb may independently be of R- or S configuration.

Compounds of formula I carrying substitutents at C7 and C9 may be prepared from compounds of formula la by nucleophilic substitution using standard methods.

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

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 said compound in an amount of from 0.001 μg/ml to 100 mg/ml. Alternatively, the active ingredient may be a fun-

gus of a species belonging to the genus Hypomyces capable of producing said novel compound, preferably of the species Hy¬ pomyces aurantius, especially a strain of the Hypomyces au¬ rantius (CBS No. 654.93) or a mutant thereof capable of pro- ducing said compound of the invention.

In this context it was specifically found that organic ex¬ tracts of the fungus Hypomyces aurantius A03892 (CBS No. 654.93) fermented in submerged culture inhibited the growth of various plant pathogenic fungi. The principle responsible for the observed activity was isolated and characterized spectroscopically, chemically and biologically as described below.

Fungicidal compositions according to the invention, exhibit¬ ing fungicidal and antibacterial active, having compounds of the invention as its active ingredient, may for agronomical and/or horticultural applications be formulated by mixing the active principle with suitable inert and compatible carriers or diluents to obtain a composition of the type generally used in agricultural compositions, examples of which are fur¬ ther 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 conden¬ sation 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 prod¬ ucts 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 acetyle- nic 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 ingredient in admixture with a suitable surfactant and an inert powder diluent such as china clay.

Depending on the circumstances such as the crop wherein fungi are to be controlled, the environmental conditions or other factors, a composition of the invention in addition to said fungicidally active compounds of the invention may also con¬ tain other active ingredients such as biocides, e.g. fungi¬ cides, 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 nuari ol, fenari ol, fen- propimorph, tridemorph, fenpropidine and dimethomorph.

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

Dithiocarbamates, e.g. thira , maneb, zineb and mancozeb;

Phatalimides, e.g. captan, folpet and captafol;

Carboxines, e.g. carboxin and oxycarboxin;

Benzimidazoles, e.g. benomyl, carbendazim and fuberidazole; Carbamates, e.g. prothiocarb and propamocarb;

Isoxazoles, e.g. hymexazol;

Cyanoacetamides, e.g. cymoxanil;

Ethylphosphonates, e.g. fosetylaluminium;

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

Dicarboximides, e.g. procymidone, iprodione and vinclozolin;

Organophosphorous fungicides, e.g. pyrazophos, tria iphos, 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/ l, 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 and bacteria 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.

Compounds of the invention have been found to be particularly potent towards plant pathogenic fungi, preferably fungi be¬ longing to the classes of Oomycetes, Basidiomycetes and Asco- mycetes .

Examples of fungal genera and species which has been found to be sensitive to compounds of the invention are fungi of the genera Pyt iuπr, especially Pythium ultimum, or Aphanomyces , especially Aphanomyces euteiches , or Phytophthora , especially Phytophthora cryptogea , or Colletotrichum, especially Colle- totrichum graminicola , or Gaeu annomyces , especially Gaeuman- nomyces graminis , or Pyricularia , especially Pyricularia ory-

zae, or Sclerotium, especially Sclerotium rolfsii , or Alter- naria, especially Alternaria alternata .

In connection with the method of the invention for control- 5 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 treatment. Depending on the crop and circumstances the treat- ιo ment may be postponed until seeds or fruits appear on the plants, wherein fungi are to be controlled. Sometimes, it is practicable to treat the roots of a plant before or during planting, for example by dipping the roots in a suitable liquid or solid composition.

15

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.

20

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. 254017.000 (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 30 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 35 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 CH ₂ OH(CHOH) _n CH ₂ OH, 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. carboxymethyIcellulose) , 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 situ . 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 Hypomyces aurantius A03892 (CBS No. 654.93) or a mutant thereof capable of producing a compound of the invention.

MATERIALS AND METHODS

Materials: PD agar: P_otato βextrose agar (Difco) Tween® 80: Merck Art. 822187 PCS medium: 1 % Pharmamedia (Traders Protein, Southern Cotton Oil Company, Tennessee, USA), 0.5 % spray dried corn steep liquet (Solulys® A ST, Roquette Freres, France) and 3 % glucose. The pH was adjusted to 7 before the substrate was autoclaved at 121°C for 40 minutes.

Compound lb: The compound was dissolved in EtOH (96%) - and thereafter to final concentrations (0 and 125 ppm ai) containing 70% EtOH and 0.025% Tween®

20.

TFA: Triflouroacetic acid (Merck Art. 8178)

Ecpύ ment:

Reversed phase MPLC-column:

2.6cm ID, 23 cm of length packed with LiChroprep RP18 15-25 μm material (Merck, 5 Darmstadt, Germany) .

Methods:

Cultivation of the strain 10 A culture of the strain can be grown on Potato Dextrose agar (Difco) slants (12 ml/slant), autoclaved at 121°C for 40 min¬ utes. Hereafter referred to as PD agar.

Fungicide production (submerged)

15 A slant culture grown on PD agar for 16 days ^' at 26 ^β C was mixed with 10 ml of sterile water containing 0.1 % Tween® 80 and inoculated into 500 ml Erlenmeyer flasks containing 100 ml PCS medium using tap water. After inoculation, the flasks were incubated at 26°C and shaken at 150 rpm for 12 days.

20

Extraction

The whole broth (1 litre, 10 shake flasks) was extracted (pH 7) with EtOAc (1 1) . The organic extract was dried, evapor¬ ated to dryness under reduced pressure and residue redissol-

25 ved in MeOH-EtOAc 1:1 (8 ml) for further purification. The extract was (in portions of 2 ml) applied to a reversed phase MPLC-column eluted at a flow rate of 15 ml/min using a water- MeOH gradient (starting at 60% MeOH (0.05% TFA) rising lin¬ early to 100% MeOH (0.05% TFA) in 60 min) and UV detection at

30225 nm.

The activity started eluting after 50 min. The active frac¬ tions were concentrated to dryness, and compound with the formula lb was obtained as yellow crystals by crystallization 35 from EtOAc or acetone.

EXAMPLES

EXAMPLE 1

Characterization of compound lb A culture of the strain Hypomyces aurantius A03892 (CBS No. 654.93) was cultivated, incubated, extracted and purified as described above in the section "METHODS AND MATERIALS".

The obtained compound was found to have the following physical and spectroscopic properties:

Melting point: 195-210°C Optical rotation [α] _D : +116°C (C=0.16,MeOH) UV _^ nm loge) : 236(4.43), 277(4.67), 323(3.77),

340(sh), 415(4.09)

Elementary analysis: Found: C: 62.36; H: 5.51

Calc. for C ₂₆ H ₂₆ O, ₀ : C: 62.65; H: 5.26

HR-EIMS: Found: 498.1560

Calc. for 498.1526

IR spectra: Figure 1 ^! H-NMR spectra: Table 1 ¹³ C-NMR spectra: Table 1

TABLE 1

No. Ή ^» c

1 189.8

2 110.4

3 194.3

4 2.76(lH,d,-7«19 Hz) 40.5 2.88(lH,d,-7=19 Hz)

4a 70.9

5a 130.3

6 130.7

6a 140.6

7 6-86(lH,d,J=2-3 Hz) 99.8

8 164.5

9 6.62(lH,d,J~2.3 Hz) 102.0

10 160.7

10a 108.5

11 166.7

11a 104.1

12 195.8

12a 79.8

13 202.3

15 3.87(lH,dd,J-«7/16 Hz) 27.2 3.60(lH,dd,J»5/16 Hz)

16 5.04(lH,m) 122.1

17 133.2

18 1.74(3H,d,-r=l Hz) 18.3

OH'β 18.1(lH,s), 15.7(lH.β),

9.94(lH,β), 5.46(lH,s)

4.09 (lH,β(br)), 3.29 (lH,β(br)

Η and ^l3 C-NMR data (chloroform-d,, 297 K) for lb

These obtained data provides evidence for a tetracyclic structure with the formula lb.

EXAMPLE 2

Fungicidal activity Agar diffusion assay The compound has been found to have an in vitro inhibitory effect on the growth of fungi belonging to the classes Oomycetes , Basidiomycetes and As corny ce t eε.

It was found to be particularly potent towards:

Class Oomycetes: Pythium ultimum

Aphanomyces euteiches Phytophthora cryptogea

Class Ascomycetes : Colletotrichum graminicola

Gaeumannomyces graminis Pyricularia oryzae

Class Basidiomycetes .' Sclerotium rolfsii

Anamorph fungus: Alternaria alternata

In the gross inhibition assays the test organisms were em¬ bedded 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 at 26°C. Table 2 show the activ¬ ity observed when applying a 1000 ppm (1 mg/ml) solution of the compound lb in EtOH.

Table 2

Test organism mm inhibition zone (clear)

Pythium ultimum 21

Aphanomyces euteiches 25

Phytophthora cryptogea 20

Colletotrichum graminicola 25

Gaeumannomyces graminis 18

Pyricularia oryzae 21

Sclerotium rolfsii 16

Alternaria alternata 14

EXAMPLE 3

Fungicidal activity (Greenhouse test)

Activity of compound lb

Host: Vitis vinifera (Vine, var. Ugni Blanc) .

Target: Plasmopara viticoa (downy mildew)

Vine plants (5 weeks old) were sprayed to run off (6.5 ml/plant) with the liquid solution containing the active com¬ pound with the formula la (see the section "MATERIALS AND METHODS") - 5 repetitions per treatment - using a robot atom- izer. The plants were kept for ventilation 24 hours (16 hours light, approximately 20°C) in the cabinet of the robot. They were then inoculated with a spore suspension (seven days old) containing 1*10 ⁵ spores/ml. The inoculation was carried out with a handhold atomizer (Wagner, Pico Bel) using approxima- tely 3.5 ml/plant. The plants were then incubated in a plexi glass box to keep a high humidity (>95%RH) . The box was plac¬ ed in a climate chamber at the following conditions: 16 hours light, approximately 10.000 lux, 20°C and 8 hours darkness.

The assessment was done 9 days after starting incubation.

The compound lb (125 ppm ai) gave 90-95% control to downy mildew (Plasmopara viticola) on vine.

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.

REFERENCES

1) R.C. Evans, Ed.: The Technology of the Tetracyclines, Quadrangle Press, New York, 1968

5

2) Kabuto, C. ; Silverton, J.V. ; Akiarna, T, ; Sankawa, U. ; Hutchinson, R.D. Steyn, P.S. & Vleggaar, R. : J. Chem. Soc. Chem. Commun. p. 728, 1976

103) Hutchinson, R.D. Steyn, P.S. & van Rensburg, S.J.. Toxicol. Appl. Pharm. , 24, p. 507, 1973

4) JP-A-5-9140892 (TAKADA CHEMICALS)

155) JP-A-5-9102396 (ENDO A)

6) US patent application no. 4017.000 (Entotech, Inc.)

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 11 , line 19-21

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [ |

Name of depositary institution

CENTRAALBUREAU VOOR SCHIMMELCULTURES

Address of depositary institution (including postal code and country)

Oosterstraat 1, Postbus 273, NL-3740 AG Barn, Nether- land

Date of deposit Accession Number

8 December, 1993 CBS No . 654.93

C. AD DrπONA INDI CATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

In respect of those designations in which a European and/or Australian patent is sought, during the pendency of the patent application a sample of the deposited microorganism is only to be provided to an independent expert nominated by the person requesting the sample (Rule 28(4) EPC / Regulation 3.25 of Australia Statutory Rules 1991 No 71) .

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will besubmitted to the International Bureau later (specify the general nature of the in£cationse.g., "Accession Number of Deposit")

For International Bureau use only

I j This sheet was received by the International Bureau on:

Authorized officer