GB2396349A - Compounds obtained from a fungal microorganism and derivatives thereof - Google Patents

Abstract

The present invention relates to novel fungicidal compounds of the general formula I based on the recently isolated natural fungicidal products and chemically generated derivatives thereof and to the microorganisms producing said fungicidal products and compounds. The inventon encompasses the compound of formula I <EMI ID=1.1 HE=34 WI=120 LX=474 LY=809 TI=CF> <PC>and to the tautomers, optical isomers and enantiomers and to the mixtures thereof, wherein X and Z independently of each other stand for a ring member selected from -CH2- ; -CO-, and R1 to R8 are standard substituents. It further encompasses the microorganisms capable of producing the above natural fungicidal products. The compounds of formula I possess useful plant protecting properties and may advantageously be employed in agricultural practice for controlling or preventing the infestation of plants by phytopathogenic microorganisms, especially fungi.

Description

GB 2396349 A continuation (74) Agent and/or Address for Service: Syngenta

Limited PO Box 3538, Intellectual Property Department, Jealotts Hill Research Centre, BRACKNELL, Berks, RG42 6YA, United Kingdom

Natural Funaicidal Compounds Obtained from a Funnal Microornanism and Derivatives thereof The present invention relates to novel fungicidal compounds of the general formula I based on the recently isolated natural fungicidal products and chemically generated derivatives thereof, to the microorganisms producing said fungicidal products and compounds, to the method of producing the novel compounds by cultivating the microorganisms and isolating the active fungicidal products from the fermentation broth and isolating the active chemical compounds, and to agrichemical formulations containing the fungicidal products and compounds and their use for protecting cultivated plants from damages caused by phytopathogenic microorganisms, especially phytopathogenic fungi.

The chemical molecules according to present invention have been isolated by the inventors as the fungicidally active products from the fermentation broth obtained after a cultivation process of the fungal strain microorganism Panus spec. (AgricalesJ SIN-F-6961 (deposi-

ted in accordance to the Budapest Treaty on the international recognition of the deposit of microorganisms for the purpose of patent procedure on March 18th, 2002 at the "DSMZ -

Deutsche Sammiung fur Mikroorganismen und Zelikulturen", Mascheroder Weg 1 b, D-38124 Braunschweig, Germany, under number DSM 14917) and Panus spec. (Agricales) SIN-F-7459 (deposited in accordance to the Budapest Treaty on the international recognition of the deposit of microorganisms for the purpose of patent procedure on March 18th, 2002 at the "DSMZ- Deutsche Sammiung fur Mikroorganismen und Zelikulturen", Mascheroder Weg 1b, D-38124 Braunschweig, Germany, under number DSM 14918). According to morphological criteria both strains are members of the family Tricholomataceae, order Agaricales, class Basidiomycetes, phylum Basidiomycota (Ainsworth & Bisby's Dictionary of the fungi, eighth edition, 1995). Both strains of fungal microorganisms have been isolated from the fruiting body o the mushroom Panus spec. collected at the Kent Ridge Park, Singapore, by members of the Center for Natural Product Research (CNPR).

The novel chemical molecules have been designated by the inventors as "Aurisins with the annexed designations A to J. These designations shall be used in this document for chemi-

cal compound species which are isolated from the cultivation broth of the above identified strains DSM 14917 and 14918 and shall be referred to in planned future publications. Simple chemical derivatives of the isolated Aurisins have been found to have biological properties which are very similar to the fungicidal properties of the basic Aurisins. Both groups of novel

compounds are integral members of the general concept of present invention.

The novel compounds according to this invention correspond to the general formula I X \ 4 8 R4 ( 1)

YllOb o7' 8A H3C/ 11b CH 1 1H3 CH3 and to the tautomers, optical isomers and enantiomers and to the mixtures thereof, wherein X and Z independently of each other stand for a ring member selected from -CH2-; - CO-, -CHOHand-CHOR5-, Y is a ring member-CO- or-O-, R. and R2 independently of each other are hydrogen, -OH, -OR6, -COOH or -COR7, R3 and R4 independently of each other designate hydrogen, C,-C4alkyl, C2- C4alkenyl, C2-C4alkynyl, C,-C4alkoxy-C,-C4alkyl, benzyl, halobenzyl, C,- C4alkylbenzyl or-CO-R8, R5 and R6 independently of each other designate C, -C4alkyl, C3-C4alkenyl, C3-C4alkynyl, C,-C4alkoxy-C,-C4alkyl or C,- C4alkanoyl, R7 stands for C,-C4alkoxy, C3-C4alkenyloxy, C3-C4alkynyloxy, C,-C4alkylamino, all-C, C4alkylamino, C,-C4alkylthio or C,-C4alkoxy-C,- C4alkoxy; and R8 stands for optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenylamino, optionally substituted benzyl or optionally substituted benzyloxy, or stands for C,- C4alkyl, C2-C4alkenyl, C2-C4-alkynyl, C3-C6cycloalkyl, C,-C4alkoxy, C3- C6cycloalkoxy, C,-C4alkylamino, di-C,.C4alkylamino, N-pyrrolidine, N- piperidine, N-morpholine or C3-C6cycloalkylamino.

In the above definitions "halon or "halogen" includes fluorine, chlorine, bromine and iodine.

The alkyl, alkenyl and alkynyl radicals may be straight-chain or branched. This applies also to the alkyl, alkenyl or alkynyl parts of other alkyl-, alkenyl- or alkynyl-containing groups, such as alkoxy, alkenyloxy, alkynyloxy, alkoxyalkyl, alkylthio, alkanoyl, alkenylcarbonyl, alky-

nylcarbonyl, alkylamino and dialkylamino. Depending upon the number of carbon atoms mentioned, alkyl on its own or as part of another substituent is to be understood as being, for example, methyl, ethyl, propyl, butyl and the isomers thereof, for example isopropyl, isobutyl, tert-butyl or sec-butyl. Depending upon the number of carbon atoms mentioned, alkenyl as a group or as a structural element of other groups is to be understood as being, for example, ethenyl, allyl, 1-propenyl, buten-2-yl or buten-3-yl. Alkynyl as a group or as a structural ele-

ment of other groups is, for example, ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl or butyn-2

- 3 yl, depending on the number of carbon atoms present. Alkoxy thus includes methoxy, eth-

oxy, propoxy, isopropoxy, n-butyloxy, s-butyloxy, i-butyloxy or tertbutyloxy. Examples for cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[3.1.1.]hexyl and bicyclo[3.2.0]hexyl. Alkoxyalkyl represents for example methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethoxy, methoxypropyl, ethoxypropyl, etc., substituted forms like fluo-

romethoxy-methyl, trifluoromethoxy-methyl, phenoxymethyl, benzyloxymethyl, etc.. The sub-

stituents listed under R7 and R8 including the optionally substituted phenyl groups incorpo-

rated in the definitions of RB for phenyl, phenoxy, phenylamino, benzyl and benzyloxy will together with the carbonyl function to which they are attached typically form the radicals like alkylcarbonyl, sometimes addressed as acyl or alkanoyl; alkenylcarbonyl, alkynylcarbonyl, cycloalkylcarbonyl, alkoxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylaminocar-

bonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy-alkoxycarbonyl, cycloalkoxycarbonyl, cycloalkylaminocarbonyl, and optionally substituted phenylcarbonyl, mostly designated as optionally substituted benzoyl; optionally substituted phenoxy, optionally substituted phe-

nylamino, optionally substituted benzyl or optionally substituted benzyloxy. The optional sub-

stituents are predominantly located at the phenyl rings forming parts of the above radicals, and are typically selected from halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, alkoxyalkyl, alkylcarbonyl, alkoxycarbonyl, cyano, nitro and hydroxyl. It is understood that at the same time more than one optional substituent may be present in these groups. For example the groups-CO-R8 encompass benzoyl, benzoyl, halobenzoyl, alkylbenzoyl, C'-C4alkanoyl, C2-C4alkenylcarbonyl, C2-C4alkynylcarbonyl, C,C4alkoxycarbonyl, C,-C4alkylaminocarbonyl, anilinocarbonyl, benzylcarbonyl, ben-zyloxycarbonyl, di-C,-C4alkylaminocarbonyl, C3-C6cyc-

loalkylaminocarbonyl, phenoxycarbonyl, halophenoxycarbonyl or C'C4alkylphenoxycar-

bonyl. Typical combinations of optional substituents in the radicals R7 and R8 are halo and alkyl, e.g. as in 3-chloro-4-methyl-benzyl, 4-chloro3-methyl-benzyl, 4-chloro-2-methyl-ben-

zyl, 2-fluoro-4-methyl-benzyl, 4chloro-3-fluoro-benzyl, 4-fluoro-2-methylbenzyl, 3-chloro4-

ethyl-benzyl, 4-chloro-2-ethyl-benzyl, 4-ethyl-2-fluoro-benzyl, 3-chloro4-methyl-benzoyl, 4-chloro-3-methyl-benzoyl, 4-chloro-2-methyl-benzoyl, 2fluoro-4-methyl-benzoyl, 4-chloro-3-

fluoro-benzoyl, 4-fluoro-2-methyl-benzoyl, 3-chloro-4-ethyl-benzoyl, 4chloro-2-ethyl-benzoyl, 4-ethyl-2-fluoro-benzoyl, 3chloro-4-methyl-phenyl, 4-chloro-3-methyl-phenyl, 4-chloro-2-

methyl-phenyl, 2-fluoro-4-methyl-phenyl, 4-chloro-3-fluoro-phenyl, 4fluoro-2-methyl-phenyl, 3-chloro-4-ethyl-phenyl, 4-chloro-2-ethyl-phenyl, 4-ethyl-2-fluoro-phenyl, 3-chloro-4-methyl-

anilinyl, 4-chloro-3-methyl-anilinyl, 4-chloro-2-methyl-anilinyl, 2fluoro-4-methyl-anilinyl, 4-chloro-3-fluoro-anilinyl, 4-fluoro-2-methylanilinyl, 3-chloro-4-ethyl-anilinyl, 4-chloro-2-ethyl-

anilinyl, 4-ethyl-2-fluoro-anilinyl, 3-chloro-4-methyl-phenoxy, 4-chloro3-methyl-phenoxy,

- 4 4-chloro-2-methyl-phenoxy, 2-fluoro-4-methyl-phenoxy, 4-chloro-3fluoro-phenoxy, 4-fluoro-

2-methyl-phenoxy, 3-chloro-4-ethyl-phenoxy, 4-chloro-2-ethyl-phenoxy, 4ethyl-2-fluoro-phe-

noxy, etc. Alkanoyl includes acetyl, propanoyl, butyroyl and isomers thereof. Alkenylcar-

bonyl and alkynylcarbonyl encompass vinylcarbonyl, methacryloyl, ethynylcarbonyl, etc..

Halobenzyl includes chlorobenzyl, fluorobenzyl, chlorofluorobenzyl, dichlorobenzyl or difluorobenzyl, while alkylbenzyl is exemplified by methylbenzyl, dimethylbenzyl, ethylbenzyl, and similar homologues. Alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl, tert-

butyloxycarbonyl, etc. while alkyl-aminocarbonyl, dialkylaminocarbonyl, anilinocarbonyl, cycloalkylaminocarbonyl for example stand for methylaminocarbonyl, dimethylaminocar-

bonyl, ethylaminocarbonyl, diethylaminocarbonyl, ethylmethylaminocarbonyl, propylami-

nocarbonyl, isopropylaminocarbonyl, bromophenylaminocarbonyl, chlorophenylaminocar-

bonyl, fluorophenylaminocarbonyl, methylphenylaminocarbonyl, methoxyphenylaminocar-

bonyl, trHluoromethylphenylaminocarbonyl, trifluoromethoxyphenylaminocarbonyl, meth-

oxycarbonylphenylaminocarbonyl, ethoxycarbonylphenylaminocarbonyl, cyclopropylami-

nocarbonyl, cyclopentylaminocarbonyl or cyclohexylaminocarbonyl.

In formula I also the various alkyl, alkenyl, alkynyl groups defined for the radicals R. to R8 may optionally be substituted with further common organic substituents like halogen, hydro-

xyl, alkyl, haloalkyl, alkoxy, haloalkoxy, cyano, nitro, alkylcarbonyl, alkoxycarbonyl, benzyl, phenyl, and the like. However, preferred are the low-molecular weight substituents like methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, fluoro, chloro, hydroxyl, etc..

Related to the 1,3-positioning of the various carbonyl ring member groups in formula I in positions 2, 4, 6 and 8 very often the molecules of formula I are present in their natural balance of tautomeric forms. For example, where R3 and R4 are hydrogen the enol-groups in positions 3 and 7 may be displayed in their tautomeric form as carbonyl groups, which will in most cases be in their natural balance of tautomers. When only one of R3 and R4 is hydro-

gen and the other has a different meaning, only some of the tautomeric forms are suppres-

sed. Where the groups X, Y or Z are carbonyl functions depending on the strength of the respective hydrogen-bridge built up with the 1,3positioned carbonyl function some tauto-

meric enol forms are indicated. The tautomeric forms are understood to be likewise encom-

passed by formula 1. When R3 and R4 are different from hydrogen most of the tautomers are suppressed. For illustration purposes some of the possible tautomeric forms for some impor-

tant subgroups are displayed as subformulae: for example subformula la (X, Y and Z are C=O, and R3 and R4 are hydrogen) which includes the Aurisin A,

t - 5 O,49,O,fO 000 H mr < H3C,H3 CH3 11 CH 3 CH3 H3C;H3 lH3 11 lH3 t-CH3 o o - O,W:O H3CCH3 H3C CH3

00,,f,t,fo 000 H3CCH3 H3C CH3

or subformula lb (X and Z are CHOH, Y is C=0 and R3 and R4 are hydrogen) which includes the Aurisin B. HOOH HO {:OH

H C CH 3 CH3 H C CH3 CH3 CH3

3 3 11 CH3 3 CH3 11 3 CH3

Ho:l,o,:' ( lb),W,OH Y H I H. _ <

3CH 1 '- '; CH3 1 1 CH3

H3C,H3 3 11;H3 H3C 11 H3CH3

HO-T',o.,'oH HO_: O,:roH _, H3C' CH3 1 1 CH3 -CH3 H3C' CH3 1 1 CH3 H3 CH3

or subformula Ic (X and Z are CHOH, Z is C=O, R. is hydrogen and R3 and R4 are hydrogen) which encompasses Aurisin C

I. - 6

HO, OH HO OH

H. C CH3 CH3 CH3 CH3 CH3 CH

3 CH3 CH3 11 CH3 CH3 H3C CH CH3 11 CH3 CH3

HO,M,O,L,OH HO jOH >1<, Yl: CH3 I CH3 CH3 I I CH3

H3C'1:H3 CH3 11 1H3; CH3 H3C,H3 CH3 11 CH3;H3 CH3

HO,,O OH HOOH

<, r< H3C' CH31 1 CH3 H3 CH3 H3C CH3 1 1 CH3 H3 CH3

or subformula Id (X and Y are C=0, Z is CHOH and R3 and R4 are hydrogen) which encompasses Aurisin D W\'OH O W,O,,:1,OH

3 CH3 CH3 11 CH3 CH3 H3CKH 3CH3 11 CH 3 -CH3

OH O R. R2 0 0 ( Id) OH O R. R2 OH O OOOH O,T,O: FOH

1 1-b' > H3C H 3CH 11 CH CH3 H3 CH3 H3C H CH3 || CH 3 -CH3

OW O WOH O?,GO,W,OH

' > - <

H3C' CH3| 1 CH3 H3CH3 H3C,H 1H3 1 CH3; H3CH3

or subformula le (X and Y are C=0, Z is CHOH, R., R3 and R4 are hydrogen) which encompasses Aurisin E

1, - 7

O OtOH o OOH H3C - CH3

O'OW,T,OH O - O,,,LOH

cH3,r H3C CH 3CH3 11 CH3 C H3 CH3 H3C 1H CH3 li CH3 ( H3 CH3 O5[,O L OH O1,;,O,F,!FOH

I r H3C' CH3I CH3 CH3 H3C' CH3| | CH3 H3 CH3

or subformula If (X and Y are C=0, Z is CHOH, R2, R3 and R4 are hydrogen) which encompasses Aurisin F 0 0 0 0

ogoOH o,( <OH CH3 J:<1

H3C CH CH3 CH3 CH3 H3C CH CH3 11 CH3 C 3

O TOOH O/<O,,,R,OH

,R<. - - - >W<:

H3C ( H CH3 11 CH3 (-CH3 H3C' 'H. CH3 11 CH3 (-CH3

O1O OH O-O,,F,!LOH

H3C' CH3l CH3 CH3 H3C' CH3I I CH3; H3CH3 or the subformula 19 (X, Y and Z are C=O, R2 is methoxy and R3 and R4 are hydrogen) which encompasses Aurisin G

- 8 OCH3 OCH3

0;0 00

H. C CH3 CH3 CH3 CH3 CH3 CH3

3 CH3 CH3 CH3 CH3 H3C CH CH3 CH3 CH

1 OCH3 3 11 OCH3 3

000 I9 Or,Ot4irO < R <

:H CH3 I CH3 / CH3CH | CH3

H3C 11 OCH3 -CH3 H3C 3 11 OCH3 -CH3

O:OtO 0!,00 YP-<. H3C' CH3 1 1 CH3 H3 CH3 H3C' CH3 j CH3 CH3 or the subformula Ih (X and Z are C=O, Y is -O-, R. is COOH and R3 and R4 are hydrogen) which encompasses Aurisin H o H OH CH3 _ -

H3C H3 CH3 11 CH3 -CH3 H3C H3 CH3 11 CH3 -CH3

( Ih) OH COOH R2 O O OH,COOH R2 O OH

0000 000

> 1 r< CH31 3 CH3 CH31 1 CH3 -CH

H3C H3 CH3 11 CH3 CH3 H3C,H3 CH3 11 CH3 C H3 3

OH CooH R2 O O OH COOH R2 OH O 0 = 0 00

H3C CH CH3 CH3 CH CH3 H3C CH CH3 CH3 CH CH3

or subformula li (X and Z are C=O, Y is'-, R. is COOH3 and R3 and R4 are hydrogen) which encompasses Aurisin I

- 9 - OOCH COOCH3

o:o oo 'OOo W _ H3C 'H. CH3 11 CH3 CH3 H3C H3 CH3 11 CH3 H3 CH3

( 1i) COOCH 0070 00400

' _ CH3 CH3 CH3 3 CH3

H3C,H3 1H3 11 1H3 H3 CH3 H3C I H3 CH3 11 CH3 CH3

COOCH3

:'CH, OH

CH CH3 3 CH3 H C CH3 CH3 CH3

H3C CH3 CH3 CH3 3 CH3 CH3 CH3 CH3

or subformula Ij (X, Y and Z are C=O, R2 is ethoxy and R3 and R4 are hydrogen) which encompasses Aurisin J OC2H5 C2Hs oo:o oo 'Wo wK - CH3 CH3 CH CH3 CH

H3C H3 CH3 11 CH3 ( 1j),H3 CH3 11 CH3 H3 3 oo?o o o;o >1 1 ><

CH3 j 3 CH3 CH3 1 1 CH3 H3C: H3 CH3 11 CH3 CH H3C/ 5H3 CH3 11 CH3 H3 CH3

C2H5 C2H6

00,0 00,:0

- H3C' CH31 CH3 CH3 H3C' CH31 1 CH3 'H3 CH3

t, -10 The compounds of formula I have now been found to provide in particular, anti-fungal, anti-

viral and anti-tumor activity, but are of special interest for use in agriculture and horticulture.

However, due to their excellent microbiocidal activity, especially their efficacy against phyto-

pathogenic fungi allowing to control a wide variety of crop damaging fungi by treating the plants that are infested or likely to be infested by a phytopathogenic microorganism, the compounds of formula I and the method or protecting crop plants by employing them as plant protecting agent are subject to present invention.

The protection of cultivated plants is preferably achieved by applying the compounds of formula I to the locus where the crop plants are growing or where they will be planted.

The harvested Aurisin compounds of formula I may also be of use as an intermediates in the preparation of further active compounds of formula 1, which in turn may again serve as intermediates for further derivatisized active compounds within the scope of this invention.

The Aurisins "A" to "J", characterized as individual species among the compounds of formu-

la 1, may be obtained either by cultivating of the mentioned microorganism strains producing fungicidal compounds by common methods of cultivating the microorganism strains and of harvesting the active products from the fermentation broth being produced by such cultiva-

tion, and finally by isolation of the fungicidally active group or individual chemical compound.

It has been found that the individual chemical compounds isolated from the fermentation and having all a very similar biological activity profile belong to the chemicals of formula I which characterizes the similarities of the common molecule skeleton. The microbiocidally active compounds isolated from the fermentation product consisting of the microorganism and the fermentation broth have been designated "Aurisin A" through to "Aurisin Jn. They are all members of the group defined by the general formula 1. The displayed individual formulae for the Aurisins "A" to "J" within the scope of formula I are believed to be the true molecular structures of the products isolated from the fermentation process, but naturally the Aurisins "A" to "J" as claimed likewise are characterized by the method of preparation, i.e. by being produced by cultivating the indicated fungal microorganisms and isolating the fungicidally active compounds from the fermentation product.

The other compounds of formula I which are not directly obtained from cultivating the two microorganisms DSM 14917 and DSM 14918, may be obtained from the harvested molecu-

les by simple chemical derivatization reactions, such as oxidation, reduction, acylation, ethe-

rification or esterification. Where more than one reactive radical is present being susceptible to a planned derivatization reaction the technique of employing protection groups in order to preserve a comparatively reactive radical is recommended, and also in fact employed for

- 1 1 -

preparing chemically synthesized derivatives of the harvested Aurisins "At to "J".

A preferred group of compounds including some of the harvested Aurisins and derivatives obtainable from the harvested Aurisins by reacting the enol-forms of the carbonyl functions in positions 3 and 7 is characterized by the formula 11 onto H3C CH CH3 CH3 CH CH3

wherein R' and R2 independently of each other are hydrogen or hydroxyl, and R3 and R4 have the same meaning and are selected from hydrogen, C'C4alkyl, allyl, benzyl, benzoyl, acetyl, methoxymethyl, benzyloxymethyl, cyclohexylcarbamoyl and phenylcarbamoyl.

Most preferred are compounds of formula I wherein R. and R2 is OH and R3 and R4 are hydrogen, acetyl or C,-C4alkyl.

The fungal strains Panus spec. (Agaricales) SIF-6961 (DSM 14917) and Panus spec.

(Agaricales) SIN-F-7459 (DSM 14918) have been isolated at the Center for Natural Products Research (CNPR) in Singapore. According to morphological criteria both strains are members of the order Agaricales, class Basidiomycetes, phylum Basidiomycota (Ainsworth & Bisby's Dictionary of the fungi, eighth edition, 1995).

The invention encompasses the two deposited strains of microorganisms, but also extends to further new fungal strains which possess similar cultural characteristics as SIN-F-6961 (DSM-14917) or SIN-F-7459 (DSM 14918) and which are capable of producing a compound of formula 1. The invention extends also to mutant strains of SIN-F-6961 (DSM-14917) or SIN- F-7459 (DSM 14918) capable of producing a compound of formula 1. Mutants may be naturally occurring or may have developed through natural selection and cultivation, or may be produced by dedicated recombinant gentechnological DNA alternations or mutagenesis of the parent strain using conventional mutagenesis techniques such as ionizing radiation; chemical methods, e.g. treatment with N-methyl-N-nitrosoguanidine; heat; genetic techniques such as recombination, transduction, transformation, Iysoge-nization, and selective techniques for spontaneous mutants.

The production of a compound of formula I by cultivation of a suitable strain of SIN-F-6961 (DSM 14917) or SIN-F-7459 (DSM 14918) may be effected by conventional means; i.e. by culturing the fungal strains or mutants thereof in the presence of assimilable sources of

- 1 2 carbon, nitrogen and mineral salts.

Assimilable sources of nitrogen, carbon, and minerals may be provided by either simple or complex nutrients. Sources of carbon will generally include glucose, maltose, starch, glyce rol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise form 0.5% to 10% by weight of the fermentation medium.

Sources of nitrogen will generally include cereal meals from e.g. soya beans, rice, oat, wheat, German wheat (spelt), corn or others, corn steep liquors, distillers solubles, yeast extracts, cottonseed meal, peptones, proteins, malt extract, molasses, casein, amino acid mixture, ammonia, ammonium salts or nitrates, urea and other amides. Sources of nitrogen will generally comprise from 0.1% to 10% by weight of the fermentation medium.

Nutrient mineral salts which may be incorporated into the culture medium include for example, salts capable of generating sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions. The microorganism of the invention will generally be cultivated at a temperature of from +1 8 C to +40 C, preferably from +22 C to +35 C, in particular from +25 C to +30 C, for example at +25 C, +27 C, +28 C or +30 C.

Cultivation will desirably take place with aeration and agitation. The cultivation will generally be carried out in the pH range 2 to 8, preferably 3 to 7, most preferably pH 4 to 6.

The fermentation may be carried out for a period of 2 to 20 days, e.g. 8 to 10 days.

Adsorption resins similar or identical to XAD-16 may be added to the medium prior to cultivation or added to the culture during incubation to stimulate or improve production and/or stabilize the product by binding to the resin granules.

Where it is desired to separate the compounds of the invention from the entire fermentation broth this may be achieved using conventional isolation and separation techniques. The compounds of the invention are found in the mycelia of the cells as well as in the mycelia free parts of the fermentation broth. In case of femmentation with addition of XAD resin to the medium or culture the compounds are found exclusively in the XAD resin containing fraction of the fermentation broth.

The compounds of the invention may be isolated and separated by a variety of fractionation techniques, for example, adsorption-elusion, precipitation, fractional crystallization and solvent extraction which may be combined in various ways.

Centrifugation, filtration, extraction and chromatography have been found to be most suitable for isolating and separating the compounds of the invention.

Following the fermentation, the mycelia may be harvested using conventional techniques

- 1 3 such as filtration or centrifugation. The compounds of the invention may be extracted from the mycelia or adsorption resin containing mycelia fraction with an appropriate organic solvent such as a lower ketone e.g. acetone; a lower alcohol e.g. methanol or ethanol; a lower dial like ethanediol or glycerine; or an ester e.g. methyl acetate or ethyl acetate.

Generally more than one extraction is desirable to achieve optimum recovery. Preferably the first extraction is performed using a water soluble solvent, preferably methanol. The com-

pounds of the invention may then be recovered as a crude extract by removal of the solvent e.g. by evaporation. The concentrate may then be re-suspended in an aqueous media e.g. water and further purified using a water immiscible solvent such as ethyl acetate, sufficient water being added to achieve satisfactory partition of the compounds of the invention.

Removal of the water miscible phase yields the compounds of formula 1.

Where the compounds of the invention are to be recovered from the supernatant of the fermentation broth, the entire fermentation broth may be centrifuged and the solid material discarded. The compounds of the invention are then further purified from the supernatant material. Purification of an individual compound of the invention may be effected by conventional techniques such as for example chromatography, preferably gel permeation chromatography followed by high performance liquid chromatography, on a suitable support such as silica. The support may be in the form of a bed or preferably packed in a column.

A solution of such compounds in a suitable solvent will generally be loaded on to the silica, if desired after reducing the volume of the solvent. The column may optionally be washed and eluted with a solvent of suitable polarity. In the case of silica, alcohols such as methanol or acetonitrile in combination with water and acidic modifiers like trifluoroacetic acid may be used. Elution and separation of such compounds may be monitored by conventional techniques such as chromatography preferably high performance liquid chromatography.

Further purification may be achieved by crystallization. The invention extends also to the crystalline form of the iolated and purified compounds of formula 1.

The invention further includes a process for controlling phytopathogenic fungi, viruses or tumor cells which process comprises exposing said fungi, viruses or tumor cells in crop plants to at least one compound of formula 1.

The invention extends also to the use of compounds of formula I with a biological system which has modified or enhanced resistance to fungi and/or viruses. In the case of a plant system the enhanced resistance of the phytopathogenic microorganisms may have been introduced into the plant using conventional breeding techniques where plants showing enhanced resistance are selected over many generations. Altematively or as well as, the

- 1 4 enhanced resistance may due to the introduction into said plant by genetic modification

recombinant DNA techniques of one or more DNA sequences, the expression of which enhances the resistance of the host plant. This approach is often referred to as integrated crop management.

In a further aspect therefore the invention provides a method of controlling fungal infection of a plant or part thereof said plant having been genetically modified to enhance resistance to fungi either by selective breeding and/or preferably by genetic modification where one or more DNA sequences, the expression of which enhances the resistance of the plant to fungi have been introduced into said plant using recombinant DNA techniques, said method comprising exposing said plant or part thereof to a compound of formula 1.

In a further aspect therefore the invention provides a method of controlling viral infection of a plant or part thereof said plant having been genetically modified to enhance resistance to viruses either by selective breeding and/or preferably by genetic modification where one or more DNA sequences, the expression of which enhances the resistance of the plant to viruses have been introduced into said plant using recombinant DNA techniques, said method comprising exposing said plant or part thereof to a compound of formula 1.

In a yet further aspect the invention provides the use of the compounds of formula I as a biocidal agent.

In a preferred embodiment of this aspect the invention provides the use of a compound of formula I as an anti-fungal agent.

In a further embodiment of this aspect the invention provides the use of a compound of formula I as an anti-viral agent. In a further embodiment of this aspect the invention provides the use of a

compound of formula I as an anti-tumor agent.

The general methods of preparing chemical derivatives of the Aurisins are known per se in the art and are all very common in the literature, as the reactions of the following Schemes dealing with methods of preparing chemical derivatives of the harvested Aurisin "A" to "J".

This means that, where novel derivatives of an Aurisin are prepared, the respective process of manufacture is also deemed novel. The few procedures for the preparation of the preferred compounds of formula 11 may be outlined in the following Schemes 1 to 3, but the other compounds of formula I may be obtained by suitably adapted methods.

Scheme 1

- 15 z R3 X; j: Z H3C;<:CH ( 111) (:CH3

According to Scheme 1 compounds of formula 11 wherein R3 and R4 are optionally substituted C'-C.alkyl, C3-C4alkenyl, C3-C4alkynyl, or optionally substituted phenyl and X and Z indepen-

dently of each other stand for a ring member selected from -CH2-, -CO- and Y is a ring member -CO- or -O-, in its shown form or in form of the possible tautomers, may be prepa-

red, by treating the natural product, an Aurisin from the group of the Aurisins "A" to "J" as encompassed by the formula I, wherein R', R2, X, Y and Z are as defined for formula 1, with an alkylating agent of formula lil wherein R3 and R4 are as defined for fommula I and W is a leaving group like a halide such as chloride or bromide or a sulfonic ester group such as a tosylate, mesylate or triflate in a polar solvent like DMF, preferably in the presence of an inorganic base, like a metal hydroxide or a metal carbonate, preferably potassium carbonate or an organic base like tertiary amine such as triethylamine, N,N-diisopropyl-ethyamine, pyridine, N-methyl-piperidine or N-methyl-morpholine at temperatures ranging from 0 C to +200 C, preferentially at temperatures ranging from 0 C to +120 C.

Alternatively the reaction as shown in Scheme 1 can be performed by treating the natural Aurisin product of formula I selected from "A" to aJ" with an alkyl oxonium reagent like the "Meerwein salts". This reaction is preferably performed in a solvent like aromatic, non-aro-

matic or halogenated hydrocarbons, such as benzene, toluene, xylene, hexane, pentane, cyclohexane, etc. or chlorohydrocarbons, preferably in the presence of an organic base like a tertiary amine, such as triethylamine, N,N-diisopropyl-ethyamine, pyridine, N-methyl-piperi-

dine or N-methyl-morpholine at temperatures ranging from - 0 C to +200 C, preferentially at temperatures ranging from 0 C to +1 20 C.

Scheme 2 R,R,, H C CH3 ( I) CH3 CH3 ( IV) H C CH3 ( 11a) CH 3 CH3 CH3 CH3 CH3 3 CH3 CH3 CH3 CH3

Alternatively as shown in Scheme 2. compounds of the subformula lla wherein R8 is optio nally substituted phenyl, optionally substituted phenoxy, optionally substituted phenylamino,

- 16 optionally substituted benzyl or optionally substituted benzyloxy, or stands for C,-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C3-C6cycloalkyl, C, -C4alkoxy, C3-C6cycloalkoxy, C' -C4alkylami-

no, all-C, C4alkylamino or C3-C6cycloalkylamino and X and Z independently of each other stand for a ring member selected from -CH2-, -CO- and Y is a ring member -CO- or -O-, in its shown form or the possible tautomers, may be prepared, by treating the natural Aurisin products of formula I with a carboxyl-activated derivative of the acid of formula IV, optionally in the presence of a base and optionally in the presence of a diluting agent.

Carboxyl-activated derivatives of the acid of formula IV are all compounds carrying an acti-

vated carboxyl group like an isonitrile like alkyl- or aryl isonitriles such as cyclohexyl isonitrile or like acid halide such as an acid chloride, like symmetrical or mixed anhydrides with O-al-

kylcarbonates, like activated esters such as p-nitrophenylesters or Nhydroxysuccinimid-

esters, as well as in-situ-formed activated forms of the acid of formula IV with condensing agents, such as dicyclohexylcarbodiimide, carbonyidiimidazole, benzotriazol-1-yloxy-tris-

(dimethylamino)phosphonium hexafluorophosphate, O-benzotriazol-1-yl N,N, N',N'-bis(penta-

methylene)uronium hexafluorophosphate, O-benzotriazol-1-yl N,N,N'N'bis(tetramethylene)-

uronium hexafluorophosphate or benzotriazol-1-yloxytripyrrolidiniophosphonium hexafluo-

rophosphate. The present reaction is preferably performed in a solvent like aromatic, non-

aromatic or halogenated hydrocarbons such as chlorohydrocarbons or toluene, ketones such as acetone, esters such as ethyl acetate, amides such as N,N-dimethylformamide, nitri-

les such as acetonitrile or ethers such as diethyl ether. The reaction is performed optionally in the presence of an organic or inorganic base like tertiary amine such as pyridine or trie-

thylamine, like a metal hydroxide or a metal carbonate such as potassium carbonate at tem-

peratures ranging from - 0 C to +150 C, preferentially at temperatures ranging from -40 C to +40 C. It is also possible to use an organic base such as pyridine as the solvent per se.

Scheme 3 Reduction O fOH Oxidation Y \^ H3C H3;H31 H3 CH3 H3 CH3 H. C/ H3, H3CH3 CH3 H3 H3CH3

(la) (lb) Compounds of the subformula la, in its shown form or the possible tautomers, may be prepared by oxidizing a compound of the subformula lb, in its shown form or the possible tautomers, with an organic oxidizing agent like an alkyl hydroperoxide, a DMSO-based reagent (T.T. Tidwell, Org. React., 1990, 39, 297-572), an hypervalent iodine reagent, a

- 17 dioxirane, a nitroxyl radical; or an inorganic oxidizing agent, like peroxides, hypochlorites, transition metal oxide, (like Cr. On, Ru, Re, Os), sodium percarbonate, sodium perborate, silver carbonate, or air in the presence of transition metal catalysts.

The reaction of the compound of formula lb with the oxidizing agent advantageously takes place in an inert solvent such as THE, dichloromethane, water or a ketone such as acetone, or a mixture thereof, in absence or in the presence of an acid or in the absence or in the presence of a base, at temperatures between -80 C to +150 C.

Compounds of the subformula lb, in its shown fomm or the possible tautomers, may be prepared by reducing a compound of the subformula la, in its shown form or the possible tautomers, by reacting it with an reducing agent like a metal hydride such as aluminum or borane based hydrides, molecular hydrogen or a source producing molecular hydrogen (like cyclohexadiene, ammonium formats) in the-presence of a transition metal based catalyst, (like Pd. Pt. Rh, Ir), alkali metal dissolved in liquid ammonia, or single electron transfer reagents based on cerium or samarium.

The reaction of the compound of formula la with the reducing agent advantageously takes place in an inert solvent, like an ether such as diethylether or THE, a hydrocarbon or haloge-

nated hydrocarbon such as dichloromethane, or alcohols such as methanol, ethyl acetate, water or a mixture thereof, in absence or in the presence of an acid or in the absence or in the presence of a base, at temperatures between -80 C to +150 C. The acid or the base can be used as the solvent per se.

The compounds I are used in unchanged form or preferably together with customary exci-

pients in formulation techniques. To this end, they are conveniently processed into agriche-

mical compositions in known manner, e.g. into emulsion concentrates, coatable pastes, directly sprayable or diluable solutions, diluted emulsions, wettable powders, soluble powders, dusts or granules, e.g. by encapsulation into for example polymeric materials. As with the type of medium, the application processes, such as spraying, atomizing, dusting, scattering, coating or pouring are similarly chosen according to the desired aims and the prevailing conditions.

The invention also relates to compositions which comprise the compounds of the formula I as an active component, in particular plant-protecting compositions, and also to their use in the agricultural sector or related areas.

- 18 Active compounds of the formula I are customarily used in the form of compositions and may be added, simultaneously or successively, to the surface or plant to be treated together with additional active compounds and further auxiliaries. These additional active compounds may be either fertilizers, trace element-supplying agents or other preparations which influence plant growth or other active ingredients for obtaining special desirable biological effects. It is also possible, in this context, to use selective herbicides, such as insecticides, fungicides, bactericides, nematicides or molluscicides, or mixtures of several of these preparations, additionally, where appropriate, together with excipients, surfactants or other administration-

promoting additives which are customary in formulation technology (designated collectively as carrier materials herein).

Suitable excipients and additives may be solid or liquid substances which are appropriate in formulation technology, for example natural or regenerated minerals, solvents, dispersants, wetting agents, adhesives, thickening agents, stabilizers, defoamers, preservatives, viscosity regulators,, tackifiers or binding agents or other active substances for achieving specific effects. A preferred method for applying a compound of formula 1, or an agrochemical composition which comprises at least one of these compounds, is administration to the leaves (foliar application). The frequency and rate of administration depend upon the risk of infestation by the corresponding pathogen. The compounds of formula I can, however, also penetrate the plant through the roots via the soil (systemic action). If the locus of the plant is impregnated with a liquid formulation or if the substances are introduced in solid form into the soil, e.g. in the form of granules (soil application). In paddy rice crops, such granules can be applied in metered amounts to the flooded rice fields. In order to treat seeds, the compounds of formu-

la I can, however, also be applied to the seeds (coating), either by impregnating the grains or tubers with a liquid formulation of the active ingredient, or by coating them with a solid formulation. Advantageous rates of application are in normally from 59 to 2kg of active ingredient (a.i.) per hectare (ha), preferably from 1Og to 1 kg of a.i./ha, especially from 2Og to 60Og a.i./ha.

When the compound are used as seed dressings, dosages of from 10mg to 19 of active in-

gredient per kg seed are advantageous employed. The agrochemical compositions generally comprise 0.1 to 99% by weight, preferably 0.1 to 95% by weight, of a compound of formula 1, 99.9 to 1% by weight, preferably 99.8 to 5% by weight, of a solid or liquid adjuvant and O to

- 19 25% by weight, preferably 0.1 to 25 % by weight, of a surfactant. Whereas commercial pro-

ducts will preferably be formulated as concentrates, the end user will normally employ dilute formulations. The compounds of formula I may be used preventatively and/or curatively in the sector of agronomics and related technical areas as active ingredients for controlling plant pests. The active ingredients of formula I according to the invention are notable for their good activity even at low concentrations, for their good plant tolerance and for their environmentally friendly nature. They have very advantageous, especially systemic, properties and may be used to protect a plurality of cultivated plants. Using the active ingredients of formula I on plants or plant parts (fruit, flowers, leaves, stems, tubers, roots) of various crops, the pests appearing can be controlled or destroyed, whereby the parts of plants which grow later also remain protected, e.g. from phytopathogenic microorganisms.

The compounds I may additionally be used as a dressing to treat seeds (fruits, tubers, corms) and plant cuttings to protect against fungal infections and against phytopathogenic fungi occurring in the soil.

The compounds I are effective for example against the following classes of related phyto-

pathogenic fungi: Fungi imperfect) (e.g. Botrytis, Pyricularia, Helminthosporium, Fusarium, Septoria, Cercospora and Altemaria); Basidiomycetes (e.g. Rhizoctonia, Hemileia, Puccinia); Ascomycetes (e.g. Venturia and Erysiphe - now often called Blumeria, Podosphaera, Monilinia, Uncinula) and Oomycetes (e.g. Phytophthora, Pythium, Plasmopara).

In particular, the compounds of formula I demonstrate a high level of fungicidal activity against phytopathogenic fungi, particularly Phytophthora infestans, Plasmopara viticola, Botrytis cinema, Sphaerotheca fuliginea, Erysiphe (Blumeria) graminis, Pyrenophora teres, Pythium ultimum, Leptosphaeria nodorum, Fusarium culmorum, Rhizoctonia solani, Puccinia triticina, Pyricularia oryzee, Rhizoctonia solani, Phytophthora cinnamomi, Ustilago maydis and Monilinia fructigena. The method of controlling plant pathogens by applying a fungicidally effective amount of a compound of formula I to the plant pathogens or their habitat is another aspect of present invention.

Target crops for the plant-protecting usage in terms of the invention are for example the following crop plant species: cereals (wheat, barley, rye, oats, rice, maize, sorghum and related species); beet (sugar beet and fodder beet); pome, stone and berry fruit (apple, pear,

- 20 plum, peach, almond, cherry, strawberry, raspberry and blackberry); legumes (bean, lentil, pea, soya); oil crops (rape, mustard, poppy, olive, sunflower, coconut, castor oil, cocoa, peanut); cucumber plants (squash, cucumber, melon); citrus fruits (orange, lemon, grape-

fruit, mandarins); vegetables (spinach, lettuce, asparagus, cabbage varieties, carrot, onion, tomato, potato, paprika); laurels (avocado, cinnamonium, camphor) and plants such as tobacco, nuts, coffee, aubergine, sugar cane, tea, pepper, vine, hops, banana and natural rubber plants, as well as ornamental plants.

Further areas of application for the active ingredients according to the invention are the protection of stored food, cattle feed and other organic material, where the stored matter is protected against putrescence and mould.

Where the compounds of formula I are mixed with further active ingredients used in the plant protection science, e.g. especially further fungicides, in some cases synergistic enhancement of the biological effects may occur. Preferred active ingredients advantageous as additives to the compositions comprising the active ingredient of formula I are: Azoles, such as azaconazole, bitertanol, bromuconazole, cyproconazole, dHenoconazole, diniconazole, epoxiconazole, fenbuconazole, fluquinconazole, flusilazole, flutriafol, hexacon-

azole, imazalil, S-imazalil, imibenconazole, ipconazole, metconazole, myclobutanil, pefura-

zoate, penconazole, pyrifenox, prochloraz, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol, triflumizole, triticonazole; pyrimidinyl carbinole, such as ancymidol, fenarimol, nuarimol; 2-amino-pyrimidines, such as bupirimate, dimethirimol, ethirimol; morpholines, such as dodemorph, fenpropidine, fenpropimorph, spir-

oxamine, tridemorph; an ilinopyrimidines, such as cyp rodinil, mepanipyrim, pyrimethan il; pyrroles, such as fenpiclonil, fludioxonil; phenylamides, such as benalaxyl, furalaxyl, meta-

laxyl, R-metalaxyl, of urace, oxadixyl; benzimidazoles, such as benomyl, carbendazim, deba-

carb, fuberidazole, thiabendazole; dicarboximides, such as chlozolinate, dichlozoline, iprodi-

one, myclozoline, procymidone, vinclozoline; carboxamides, such as carboxin, fenfuram, flutolanil, mepronil, oxycarboxin, thifluzamide; guanidines, such as guazatine, dodine, imin-

octadine; strobi lu rin es, such as azoxystrobin, dimoxystrobin, fluoxystrobin, kresoxi m-methyl, metominostrobin, SSF-129, trifloxystrobin, orysastrobin, picoxystrobin, pyraclostrobin; dithiocarbamates, such as ferbam, mancozeb, maneb, metiram, propineb, thiram, zineb, ziram; N-halomethylthiotetrahydrophthalimides, such as captafol, captan, dichlofluanid, fluoromides, folpet, tolyfluanid; Cucompounds, such as Bordeaux mixture, copper hydro-

xide, copper oxychloride, copper suHate, cuprous oxide, mancopper, oxinecopper; nitrophe

-21 nol-derivatives, such as dinocap, nitrothal-isopropyl; organo-pderivatives, such as edifen-

phos, iprobenphos, isoprothiolane, phosdiphen, pyrazophos, tolclofosmethyl; various others, such as acibenzolar-S-methyl, anilazine, benthiavalicarb, blasticidin-S, chinomethio-

nate, chloroneb, chlorothalonil, IKF-916 (proposed name cyazofamid), cyflufenamid, cym-

oxanil, dichlone, diclomezine, dicloran, diethofencarb, dimethomorph, ethaboxam, feoxanil, SYP-LI90 (proposed name: flumorph), dithianon, etridiazole, famoxadone, fenamidone, fen-

tin, ferimzone, fluazinam, flusuHamide, fenhexamid, fosetyl-aluminium, hymexazol, iprova-

licarb, kasugamycin, methasulfocarb, metrafenone, nicobHen, pencycuron, phthalide, polyo-

* xins, probenazole, propamocarb, pyroquilon, proquinazid, quinoxyfen, quintozene, silthi-

of am, sulfur, triazoxide, tricyclazole, triforine, validamycin, orzoxamide.

One preferred method of application of an active ingredient of formula I or of an agrochemi-

cal composition containing at least one of these active ingredients is foliar application. The frequency and amount of application depend on the severity of the attack by the pathogen in question. However, the active ingredients I may also reach the plants through the root sys-

tem via the soil (systemic action) by drenching the locus of the plant with a liquid preparation or by incorporating the substances into the soil in solid form, e.g. in the form of granules (soil application). In rice cultivations, these granules may be dispensed over the flooded paddy field. The compounds I may however also be applied to seed grain to treat seed material

(coating), whereby the grains or tubers are either drenched in a liquid preparation of the active ingredient or coated with a solid preparation.

The compositions are produced in a per-se known manner, e.g. by intimately mixing and/or grinding the active ingredient with extenders such as solvents, solid carriers and optionally surfactants. Favorable application rates are in general 1 g to 2kg of active substance (AS) per hectare (ha), preferably 1 Og to 1 kg AS/ha, especially 2Og to 60Og AS/ha. For usage as a seed dres sing, it is advantageous to use dosages of 10mg to 19 active substance per kg of seed grain.

The compound of the formula I are employed in unaltered form, i. e., as a pure crystalline substance or as the biomass obtained from the femmentation, dried and ground, or preferably together with the auxiliaries conventionally used in the art of formulation, and they can therefore be processed in a known manner for example to emulsion concentrates, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules and also encapsulations in polymeric substances. The application methods, such

- 22 as spraying, misting, atomizing, scattering or pouring, as well as the compositions, are selected to suit the intended aims and the prevailing circumstances.

While concentrated compositions are preferred for commercial usage, the end user normally uses diluted compositions.

The formulations, i. e. the compositions, preparations or combinations, containing the active substance of the formula I or combinations of this active substance with other fungicides, insecticides or acaricides, and if desired a solid or liquid additive, are prepared in a known manner, for example by intimately mixing and/or grinding the active substances with diluents, for example with solvents, solid carriers, and, if desired, surface-active compounds (surfactants). Formulations may be prepared analogously to those described for example in WO 97/33890.

As a rule, the plant-protective preparations contain 0.1 to 99%, in particular 0.1 to 95%, to the active substance of the formula I or combinations of this active substance with other fungicides, insecticides or acaricides, 5 to 99.9% of a solid or liquid diluent(s) and O to 25%, in particular 0.1 to 20%, of a surfactant. Most preferred plant- protective compositions according to this invention contain 5 to 40% of a compounds of formula 1. While concen-

trated compositions are often preferred as commercially available goods, the end user generally uses dilute preparations containing considerably lower concentrations of active substance. Typical application concentrations are between 0.1 and 1,000 ppm, preferably between 0.1 and 500 ppm. The application rates per hectare are generally 1 to 1'000g of active substance per hectare, preferably 1 to 1 OOg/ha.

More appropriate application rates can be determined by routine experiments by those skilled in the art, or by comparing the activity of the compounds of formula I with standards for which the application rate is known. In general, satisfactory control of phytopathogens is obtained when employing the compounds of formula I at a rate of from about 2g active ingredient (a.i.)/ha to 500g/ha of phytopathogen-infested habitat, preferably from about 1 OOg/ha to 40Og/ha. Satisfactory control of plantfungi is obtained when employing an Aurisin or an Aurisin derivative at a rate from about 2g active ingredient (a.i.)/ha to 100 g/ha of plant fungiinfested habitat.

The following examples are intended to illustrate the invention, but do not restrict it.

Examples:

- 23 Example 1: Production of Aurisins A-F Culture of strain SIN-F-7459 (DSM 14918) a) Start culture The strain SIN-F-7459 (DSM 14918) is grown on agar plates. It is transferred to TSM-40 liquid medium and cultivated at +25 C for 5 days for strain preservation. The strain is stored at-80 C and in liquid nitrogen in the presence of 15% (vol) glycerol.

composition of liquid medium TSM-40: Glucose (Merck) 49 Malt Extract (Difco) Fog Yeast Extract (Difco) 49 Demineralized water to 1000 ml) The medium is adjusted to pH 7 and then sterilized for 20 minutes at +121 C.

b) Seed culture 1 The strain from -80 C stock (start culture) is inoculated into in TSM-40 liquid medium for the first seed culture and incubated in 500ml shaker flasks with one baffle containing 100ml medium at +25 C and 120 rpm on the rotary shaker for 3 days.

c) Seed culture 2 From seed culture 1 a second seed culture is inoculated. 5% inoculum (2 x 20 ml) is added to 2 x 400 ml TSM-40 medium respectively (2 liter shake flasks with one baffle) and incubated at +25 C and 120 rpm on the rotary shaker for 3 days.

d) Main culture: A fermentation vessel (Infors ISF 200 stirred tank bioreactor) containing 15 liter of the main culture medium is inoculated with 0.75 liter (= 5%) of seed culture 2.

The composition of the main culture medium is as follows: Malt Extract ( Difco) 2Og Spelt meal Grain 2Og CaCO3 ( Fluka) 0.59 pH (adjusted with NaOH) to pH 7 Sterilization for 20 minutes at +121 C.

The main culture is incubated for 5 days at +25 C. The culture is constantly aerated with 7.5 liter air / min. (0.5 wm) and agitated at 400 rpm by stirrer devices. Head presure is kept constant at 250mbar. Foam prevention is done by addition of 2ml antifoam agent (PPG 2025) prior to sterilization.

e) Harvest Separation of supematant and biomass is performed by centrifugation (Beckman, model J2 21 M induction drive centrifuge) with rotor JA -10,6000 rpm,10 min. +4 C.

Workup procedure The biomass is extracted three times with 2 liter of acetone, the supernatant is decanted off and the organic solvent is evaporated under reduced pressure at +40 C to yield 1 liter of the

- 24 aqueous layer. The water phase is then extracted three times with 1 liter of ethyl acetate and the combined extracts are dried with sodium sulphate, filtrated and evaporated to dryness to yield 15.79 of the crude extract.

Isolation of Aurisins from the crude extract with LH20 and RP18 chromatoaraphv The crude extract is subjected to chromatography on Sephadex LH20 (eluent: methanol, column height 92cm, column width 7cm, flow rate 2mVmin). The fraction which eluates between 11.5 and 16.5 hours is evaporated to dryness (9.59) and then 300ml of methanol containing 15 ml of water are added and then extracted three times with 300 ml of heptane to remove the non-polar components. The methanol layer is evaporated to dryness to yield 8.3g of an enriched fraction containing the Aurisins. Further purification is accomplished by preparative RP18- HPLC (column length: 250mm; column width: 20mm; Kromasil 100 C18 10,um; gradient: 0-5min water/ methanol 90:10 v/v, 5-25min linear gradient to water/ metha-

nol 25:75 v/v, 25-45min water / methanol 25:75 vhf 45-50 min linear gradient to 100% methanol; flow rate 10 ml/mini detection wavelength 300 nm; 6 runs) to yield 77mg of Aurisin A (Rt= 34.5 min), 52mg of Aurisin B (Rt= 41.5 min), 31 mg of Aurisin C (Rt= 45.5 min), and 105 mg of a fraction containing an Aurisin mixture (Rt= 37 min). This fraction is separated by an additional preparative HPLC on the same column (gradient: 0-5min water / acetonitrile 90:10 v/v, 5-20min linear gradient to water/ acetonitrile 40:60 vh, flow rate 10 ml/mini detection wavelength 300 nm; 3 runs) to yield 40mg of Aurisin D (Rt= 32.5 min), 3mg of Aurisin E (Rt= 36.0 min), and 22mg of Aurisin F (Rt= 38.5 min).

Snectroscopic characterizations Aurisin A yellow solid, molecular formula: C30H36Og; molecular weight: 540.62 MS- ESI(+): 541.4 [M+H]+, 523. 4 [M+H-H2O]+

HR-TOF-MS-ESI(+): 523.2339 [M+H]+, calculated 523.2332 [a]D = +278 (c= 0. 53, CHC13) IR- and UV-spectra confirm the associated molecular structure.

The molecule is symmetrical. Therefore, the number of observed signals is only haH of the number of carbon atoms comprised in the molecular formula.

-25 OH 0 0 OH

8a if 1a 3C-NMR (CD30D) 10b 54.6 C-# ppm 11 38.6 1 29.5 11 a 38.7 1 a 38. 3 11 b 39.2 2 193.5 1- CH3 endo 15.9 3 160.7 1- CH3 exo 29.9 3a 121.1 11 CH3 14.3 4 201.7 11 a-CH3 25.7 4a 102 Aurisin B Molecular formula: C30H40Og; molecular weight: 544.64; yellow solid MS-ESI(-): 543 [M-H]

HR-TOF-MS-ESI(-): 543.2613 [M-H], calculated 543.2594 IR- and UV-spectra confirm the associated molecular structure.

The molecule is symmetrical. Therefore, the number of observed signals is only half of the number of carbon atoms comprised in the molecular formula.

HO, i, O: OH = 3C-MR (CD3OD) 10b 51.7 C-# oom 11 39.1 . 1 20.1 11a 37.9 1 a 26.7 11 b 31.9 2 69.7 1- CH3 endo 15.3 3 201.9 1- CH3 ego 30.5 3a 110. 8 11 -CH3 13.3 171 11 a-CH3 24.7 4a 101.3

- 26 Aurisin C Molecular formula: C30H40O8; molecular weight: 528.64; yellow solid MS- ESI(+): 529.2 [M+H]+, 5211.2 [M+H-H20]+

HR-TOF-MS-ESI(-): 527.2668 [M-H], calculated 527.2645 IR- and UV-spectra confirm the associated molecular structure.

O OH oH O HO,f OOH 3C-NMR (CD3OD) l 9a 31.9 C-# ppm 9b 38.4 1 19.9 10 39. 0

1 a 26.8 1 Oa 52.4 2 69.5 1 Ob 45.9 3 199.4 11 39.9

3a 111.6 11a 379 4 176.8 11b 31.9 4a 73.9 1- CH8 endo 15.2 5a 108.8 1 CH3 exo 30.4 6 170.2 9- CH3 endo 15.5 6a 110.9 9- CH3. exo 30.4 7 201. 3 9b-CH3 25.1 8 69.5 10- CH3 12.7

8a 26.8 11 -CH3 14.3 9 20.2 11 a-CH3 24 2 Aurisin D Molecular formula: C30H38Og,molecular weight: 542.62, yellow solid MS- ESI(-): 541 [M-H]

HR-TOF-MS-ESI(-): 541.2458 [M-H]-, calculated 541.2438 IR- and UV-spectra confirm the associated molecular structure.

OoOH p | 13C-NMR (CD3OD) I | C-# | ppm l

- 27 1 20.1 9b 1 377 1 1 a 26.5 10 36.4 2 69.5 1 Oa 51.1 3 201.8 1 Ob 52. 3 3a 110.9 11 38.6 4 170.8 11a 4a 101.0 11 b 31.7 5a 100.4 1 - CH3, endo 15.0 6 199.1 1 - CH3, exo 30.2 6a 118.6 9- CH3, endo 15.5 7 160.7 9- CH3, exo 29.9 8 191.0 9b-CH3 26.1 8a 37.1 10- CH3 14.0 9 27.9 11 -CH3 13.9

9a 37.4 11 a-CH3 24.4 Aurisin E Molecular formula: C3OH380,,, molecularweight: 526.62, yellow solid MS- ESI(-): 525 [M-H]'

IR- and UV-spectra confirm the associated molecular structure.

qo OH p 3C-NMR (CD3OD) 9a 37.9 C-# 1 ppm 9b 37.1 1 20.3 10 39.7

1 a 26.7 1 Oa 46.4 2 69.6 1 Ob 52.4 3 201.4 11 39.5

3a 111.3 11 a 38.4 4 169.1 11b 31.9 4a 100.6 1 - CH3, endo 15.4 5a 77.1 1 - CH3, exO 30.6 6 204.5 9- CH3, endo 15.5 6a 119.3 9- CH3. exo 29.8 159. 7 9b-CH3 25.2 8 192.0 10- CH3 15.2

8a 37.3 11 -CH3 12.5 9 28.2 11 a-CH3 25.2 Aurisin F

-28 Molecular formula: C30H3808; molecular weight: 526.63; yellow solid HR-TOF-MS-ESI(+): 527.2655 [M+H]+, calculated 527.2645 IR- and UV-spectra confirm the associated molecular structure.

0 'OH 8 |8a 5 4a 4 2 8a i; 1a C-NMR (CD3OD)

C-# ppm 1 20.1

1 a 26.9 2 69.4

3 199.8

3a 11.7 4 _ 176.1

4a 73.6 5a 99.8 6 198.2

6a 119.0 7 160.0 _

8 190.8

_ 8a 37.2 _ 9 28.2 _

Aurisin G Molecular formula: C3,H38Og; molecular weight: 554.632; yellow solid HR-TOF-MS-ESI(+): 555.2576 [M+H]+, calculated 555.2594 IR- and UVspectra confirm the associated molecular structure.

CH q: 5 4:0 Per Aurisin H Molecular formula: C30H36O'o; molecular weight: 556.604; yellow solid HR-TOF-MS-ESI(+): 557.2469 [M+H]+, calculated 557. 2386 IR- and UV-spectra confirm the associated molecular structure.

-29 TH iOOH O TH 0!'06 O- O

B Sa S 4a 2 aa \: 108 1C 1a 13C-NMR (CD3OD)

C-# DDm _.. 1 29.5

1 a 38.2 2 193.3

3 160.7

3a 1 21.0 200.6 4a 1 02.5 5a 107.1 6 1 71.6

6a 153.0 7 134.8

8 192.5

8a 34.5 9 26.6

Aurisin I Molecularformula:C3,H38O,0; molecularweight:570.631; yellow solid HR-TOF-MS-ESI(+): 571.2288 [M+H]+, calculated 571.2543 H-NMR-, IRand UV-spectra confirm the associated molecular structure.

COOCH Oq:OIO:O Aurisin J Molecular formula: C3,H38O'o; molecular weight: 570.631; yellow solid HR-TOF-MS-ESI(+): 569.3122 [M+H]+, calculated 569. 2750 H-NMR-, IR- and UV-spectra confirm the associated molecular structure.

) 5 p; ExamoIe 2: Derivatives of harvested Aurisins obtainable bv preparatory chemical treatment

- 30 The chemical methods for preparing derivatives of the harvested Aurisins are illustrated by the following preparatory methods (the abbreviations have the given meanings): ml = milliliters DMF = dimethylformamide Al = microliters NMR = nuclear magnetic resonance mg = milligrammes ppm = parts permillion Preparation 1: 3,7-Dimethvlation of Aurisin A. compound No. 01 Aurisin A (10 mg), anhydrous potassium carbonate (10 mg) and methyl iodide (50 Ill) in dry DMF (2ml) is mixed at room temperature in a sealed tube. The reaction mixture is stirred for 6 hours at room temperature, poured into brine and extracted with ether. The ether extracts are combined and washed with water, dried over anhydrous magnesium sulphate, and concentrated by removing the ether solvent. The residue is purified by chromatography on silica gel (ethyl acetate-hexane 1:1) to give the 3,7-dimethyl-derivative of Aurisin A (8.6 mg, 81 % yield) as yellow foam.

1H-NMR(CDC13): 8-values: 4.78(s, 1 H), 3.65(s, 3H), 2.85(dq, J = 11.6, 6. 7 Hz, 1 H), 2.21 (d, J = 11.6 Hz,1H), 1.89(d, J = 8.0 Hz, 1H),1.35(d, = 8. 5, 1H), 1.33(s, 3H), 1.21 (s, 3H), 1.17(d, J = 6.7 Hz, 3H), 1.14(s, 3H) ppm.

Preparation 2: 3.7-Di-tert-butvioxvcarbonYlization of Aurisin A. compound No. 10 Aurisin A (10 mg) and tert-butyloxycarbonic acid anhydride (50 pI) in dry pyridine (2ml) are mixed at room temperature in a sealed tube and stirred for 6 hours at room temperature, then poured into brine and extracted with ether. The ether extracts are washed with water, dried over anhydrous magnesium sulphate, the ether solvent is removed and the residue is chromatographed over silica gel (ethyl acetate-hexane 1:1) to give the 3,7-di-tert-butyloxy-

carbonyl derivative of Aurisin A (8.3 mg, 61% yield) in form of white crystals, m.p. 192 C.

H-NMR(CDC13): 6-values: 4.53(s, 1 H), 2.88(dq, J = 1 1.6, 6.7 Hz, 1 H), 2. 23(d, J = 12.2 Hz, 1 H), 1.89(d, J = 8.0 Hz, 1 H), 1.45(s, 9H), 1.35(d, J = 8.0, 1 H), 1.32(s, 3H), 1.22(s, 6H), 1.19(d, J = 6.7 Hz, 3H) ppm.

In analogous methods the following derivatives of Aurisin A are obtained.

-31 Table 1: Selected physico-chemical data for examplatory compounds owl o H3C CH3 CH3 ICY

Comp.-No | R3 and R4 | M+, m.p. or/and 1H-NMR (CDCI3) 01 -CH3 4.78(s,1H), 3.65(s,3H),2.85(dq,J=11.6, 6.7 Hz, 1H), 2.21 (d, J=11.6 Hz, 1H),1.89(d, J=8.0 Hz,1H),1.35(d,J=8.5,1H), 1.33(s,3H),1.21(s,3H),1.17(d,J=6.7 Hz, 3H), 1.14(s,3H).

02 -CzH5 4.75(s,1H),3.88(q,J= 6.7 Hz, 2H),2,85(dq,J=11.6, 6.7 Hz), 2.19(d, J=11.6 Hz,1H),1.87(d,J=8.5 Hz, 1H),1.34(d,J= 8.6 Hz, 1H),1.33(s,3H),1. 21(t,J=6.7 Hz, 3H),1.20(s,3H), 1.16(d,J=6.7 Hz, 3H),1.13(s,3H).

03 -C3H, 624 (m/z) 04 -CH2-CH=CH2 5.87-5.79(m,1H),5.20(d,J=17.1 Hz, 1H),5. 12(d,J=10.4 Hz, 1H),4.76(s,1H),4.45-4.35(m,2H),2.86(m,1H),2.19(d,J= 12.8 Hz, 1H),1.87(d,J= 7.9 Hz, 1H),1.35(d,J=8.0,1H), 1.31(s,3H),1.20(s,3H),1. 17(d,J= 6.7 Hz, 3H),1.13(s,3H).

05 -CH2-C6Hs 7.42-7.29(m,5H),5.32-5.11(m,2H),4.34(s,1H),2.75(dq,J= 11.6,6. 7 Hz,1H),2.15(d,J=11.6 Hz,1H),1.93(d,J=8.0 Hz, 1H),1.36(d,J=8.0,1H),1. 23(s,3H),1.17(s,3H),1.16(d,J= 6.7 Hz, 3H),1.08(s,3H).

06 -CO-CH3 4.6(brs,1H),2.85(dq,J=11.1,6.7 Hz, 1H),2.25(d,J=11.0 Hz, 1H),2. 17(s,3H),1.90(d,J=8.0 Hz, 1H),1.36(d,J= 8.0, 1H),1.33(brs,3H),1.22(s 6H) 1.19(d,J=6.7 Hz, 3H).

07 -CO-C6H5 8.00(d,J-7.3 Hz, 2H),i.i9(t,i=7.i Hz, 1H),7.35(t,J=7.3 Hz, 2H) ,4.55(brs,1H),2.93(dq,J=11.6, 6.7 Hz,1H),2.28(d,3 =12.2 Hz, 1H), 1.95 (d, J=8.0 Hz, 1H),1.41(d,J=8.0,1H), 1.27(s,3H),1.24(s,6H),1.21(d,J=6.7 Hz, 3H) .

08 -CH2-O-CH3 628 (m/z) 09 -CH2-O-CHz-C6Hs 752 (m/z) 10 -CO-O-C(CH3)3 1 92 C;4.53(s,1H),2.88(dq,J=11.6, 6.7 Hz,1H),2.23(d,J= 12.2 Hz, 1H),1.89(d, J=8.0 Hz, 1H),1.45(s,9H),1.35(d,J= 8.0,1H),1.32(s,3H),1.22(s,6H),1.19(d, J=6.7 Hz, 3H).

1 1 -CO-NH-C6H1 1 790 (m/z) 12 -CO-NH-C6Hs 778 (m/z) Biolonical Assav's In the following, examples of test systems in plant protection are provided which can demonstrate the efficiency of the compounds of the formula I (designated as nactive ingredient"or "test compounds"): as a rule formulations of the Aurisins A to J or derivatives thereof are employed in diluted form.

- 32 Example 3: Biological Tests a) Leaf Disk Assav: Leaf disks of various plant species (diameter 14 mm) are cut from plants grown in the greenhouse (1 to 5-leaf stage depending of the size of the leaves). The cut leaf disks are placed in multiwell plates (24-well format) onto water agar. Immediately after cutting the leaf disks are sprayed with a test solution prepared from a standard dispersion of the tested compound and diluted to a standard concentration Compounds to be tested are uniformly confectionated as DMSO solutions (active ingredient contents: 10 mg/ml) and then diluted to the appropriate sprayable concentrations with an aqueous solution of 0.025% Tween20. The tested compounds are applied onto the leaf disks in form of sprayable dispersions (comprising 200, 67 and 22,ug a.i./ml) by spraying 12 Saul of the test dispersion using an airbrush system (Iwata Ltd.). The dispersion of the tested compound is then left on the leaf surface until the liquid has evaporated.

The leaf disks are subsequently inoculated with a spore suspension of the appropriate pathogenic fungus.

After an incubation time of 3 to 7 days after inoculation at defined conditions (standadizing temperature, relative Humidity: rH, exposure to light, etc.) according to optimal setting of the respective test system, the activity of the test compound is assessed as antifungal activity, using the following rating system: 10 = complete inhibition of disease, no growth of the pathogen, 7 = good - but incomplete inhibition of disease, 3 = slightly decreased disease development, and 0 = no control of disease observed.

In this test system the growth of the phytopathogenic fungi Phytophthora infestans (on tomato), Plasmopara viticola (on grape vines), Botrytis cinerea (on beans), Erysiphe graminis fop horder (on barley) and Pyricularia oryzee (on rice) is controlled by compounds of formula I (selected from the Aurisins Flu to A" and from the compounds of Table 1) to an extent that the growth of the phytopathogenic fungi is substantially affected.

Specifically, disease incidence caused by P. infestans on tomato was reduced at 60 ug/ml with Aurisins A and B. and P. viticola was completely inhibited at 200 ppm in the presence of Aurisin B. and at 67 ppm in the presence of Aurisin F. B. cinerea growth on bean leaves was reduced at 200 ppm when treated with Aurisin B. Aurisins A, C, F. and compounds 07 and 1 1 were highly active against E. graminis f.sp. horder on barley with complete inhibition of disease at 200 ppm and good control at 67 ppm with Aurisins A and C. Best control was attained with compound 07 with full protection of the plant at 22 ug/ml.

Compound 06 displayed good activity at 67 ppm against E graminis f.sp. tritici on wheat.

P. oryzee on rice was fully controlled by compound 06 at 20 ppm, also good intrinsic activity

-33 against this pathogen was found for Aurisins C, F. and compounds 06 and 11 at 20 ppm.

b) Aaar Droplet Test: Suspensions containing homogenized mycelium of the test organisms were mixed with nutrient agar (1.5%) at +42 C. Agar droplets of 1001 were placed in Petri dishes. After hardening of the agar, the solution of a compound of formula I (1 O,ul) in ethanol (p.a.) is administered on top of the droplets. Following evaporation of the solvent, the plates are incubated at +24 C (96% rH). The growth of pathogenic organisms is evaluated visually after 7 days using the following rating system: 0: strong growth of pathogen, like untreated; 3: slightly reduced; 7: weak pathogen-growth; 10: complete control of phytopathogen, no disease.

In this test the growth of Phytophthora connamomi, Pythium ultimum, Monilinia fructigena, Rhizoctonia so/ani, Ustilago maydis, Botrytis cinerea, Phytophthora infestans, Plasmopara viticola, Botrytis cinerea, Erysiphe graminis, Pyricularia oryzae, Leptosphaeria nodorum, Fusarium culmorum and Pyricularia oryzee is controlled by compounds of formula I (selected from the Aurisins "A" to RJl. and the compounds of Table 1) to an extent that the growth of the phytopathogenic fungi is substantially affected.

c) Liquid Culture Assav: Mycelial fragments or conidia suspensions of a phytopathogenic fungus, prepared either freshly from liquid cultures of the fungus or from cryogenic storage, are directly mixed into nutrient broth (e.g. PDB = potato dextrose broth).10,ul of the test compound (in concentra-

tions of 200, 67, 22 g a.i./ml = 200, 67, 22 ppm) is pipetted into a microtiter plate (96-well format) and the nutrient broth containing the fungal spores/mycelial fragments is then added in measured amounts to give an end concentration of the liquid of 20, 6.7, and 2.2,ug a.i./ml = 20, 6.7, 2.2 ppm. The test plates are incubated at +24 C and 96% rH in the dark. The inhibition of fungal growth is determined photometrically after 48 to 72 hours and expressed using the following rating system: 10 = complete inhibition of the pathogen, 7 = good but not complete inhibition of the pathogen, 3 = partial inhibition of the pathogen, and 0 = no inhibition of the pathogen measured.

In this test system the growth of the phytopathogenic fungi Phytophthora infestans, Plasmopara viticola, Botrytis cinerea, Erysiphe graminis f.sp hordei, Pyricularia oryzee, Septoria tritici (Mycosphaerella graminicola), Pythium ultimum, Fusarium culmorum, Rhizoctonia solani and Leptosphaeria nodorum is controlled by compounds of formula I (selected from the Aurisins "AN to J and the compounds of Table 1) to an extent that the growth of the phytopathogenic fungi is substantially affected.

- 34 , Specifically, the Aurisins A, B. C, D, F. and compounds 06, 07 and 11 showed good activity against P. ultimum, in particular Aurisin D and compound 11 gave complete control at 2 ppm, where Aurisins B. C, F. and compound 06 controlled the fungus at 6.7 ppm.

S. tritici was controlled at 20 ppm by compounds 06 and 11. Moderate activity against F. culmorum and R. solaniwas found with compound 06.

d) Effect against Puccinia araminis on wheat (brownrust on wheat) i) Residual protective activity 1 week old wheat plants cv. Arina are treated with the formulated test-compound (0.02 % active substance) in a spray chamber. Two days after application wheat plants are inocula ted by spraying a spore suspension (1 x 105 ureidospores/ml) on the test plants. After an incubation period of 1 day at +20 C and 95% relative atmospheric humidity (r. h.) plants are kept for 9 days at +20 C and 60% r.h. in a greenhouse. The disease incidence is assessed 10 days after inoculation.

Compounds of Table 1 and Aurisins A to J show moderate activity in this test.

ii) Svstemicactivitv An aqueous spray liquor prepared from the formulated test compound (0.002 % active substance, based on the volume of soil) is poured onto wheat plants 5 days after sowing.

Care is taken that the spray liquor does not come into contact with the above-ground parts of the plant. 48 hours later, the plants are inoculated with a spore suspension of the fungus.

After an incubation period of 48 hours (95 to 100 % r.h. at +20 C), the plants are placed in a greenhouse at +20 C. 12 days after infection, the disease incidence is evaluated.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

e) Effect against Phvtoabthora infestans on tomatoes (late blight on potato) i) Residual protective activity 3 week old tomato plants cv. Roter Gnom are treated with the formulated test compound (0.02 % active substance) in a spray chamber. Two day after application the plants are inoculated by spraying a sporangia suspension (2 x 104 sporangia/ml) on the test plants.

After an incubation period of 4 days at +18 C and 95% r. h. in a growth chamber the disease incidence is assessed.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

At the indicated concentration Aurisins A and F exhibited over 70% control of the fungal infection in this test, while Aurisin A had the same level of activity at 60 ppm.

ii) Svstemicactivitv An aqueous suspension prepared from the formulated test compound (0.002 % active

- 35 substance, based on the volume of soil) is poured onto tomato plants which have been cultivated for three weeks. Care is taken that the spray liquor does not come into contact with the above-ground parts of the plant. 48 hours later, the plants are inoculated with a sporangia suspension of the fungus. Evaluation of the disease incidence takes place 5 days after infection, during which period conditions of 90 to 100 % r.h. and +20 C are maintained.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

f) Effect against PhYtonhthora infestans / potato (late blight on potato) 5 week old potato plants cv. Bintie are treated with the formulated test compound (0.02 % active substance) in a spray chamber. Two days after application the plants are inoculated by spraying a sporangia suspension (1.4 x 105 sporangia/ml) on the test plants. After an incubation period of 4 days at +1 8 C and 95% r. h. in a growth chamber the disease incidence is assessed.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

q) Effect against Plasmonara viticola on grapevine (grape downy mildew) 5 week old grape seedlings cv. Gutedel are treated with the formulated test compound (0.02 % active substance) in a spray chamber. One day after application grape plants are inoculated by spraying a sporangia suspension (4 x 104 sporangia/ml) on the lower leaf side of the test plants. After an incubation period of 6 days at +22 C and 95% r. h. in a green-

house the disease incidence is assessed.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

h) Residual protective activity against Ventuna inaeoualis on apples (scab on angle) 4 week old apple seedlings cv. McIntosh are treated with the formulated test compound (0.02 % active substance) in a spray chamber. One day after application apple plants are inoculated by spraying a spore suspension (4 x 105 conidia/ml) on the test plants. After an incubation period of 4 days at +21 C and 95% r. h. the plants are placed for 4 days at +21 C and 60% r. h. in a greenhouse. After another 4 day incubation period at +21 C and 95% r. h. the disease incidence is assessed.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

k) Effect against ErvsiPhe araminis on barley (powdery mildew on barley) i) Residual protective activity Barley plants of approximately 8 cm height are sprayed to drip point with an aqueous spray liquor prepared from wettable powder of the active ingredient (0.02 % active substance), and

- 36 dusted 3 to 4 hours later with conidia of the fungus. The infected plants are placed in a greenhouse at +22 C. 12 days after infection, the fungal attack is evaluated.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

ii) Systemic activity An aqueous spray liquor prepared from the formulated test compound (0.002 % active substance, based on the volume of soil) is poured onto barley plants of approximately 8 cm height. Care is taken that the spray liquor does not come into contact with the above- ground parts of the plant. 48 hours later, the plants are dusted with conidia of the fungus. The infected plants are placed in a greenhouse at + 22 C. 12 days after infection, the disease incidence is evaluated.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

m) Botrvtis cinerea / crane (botrytis on grapes) 5 week old grape seedlings cv. Gutedel are treated with the formulated test compound (0. 02% active substance) in a spray chamber. Two days after application grape plants are inoculated by spraying a spore suspension (1 x 1 o6 conidia/ml) on the test plants. After an incubation period of 4 days at + 21 C and 95% r. h. in a greenhouse the disease incidence is assessed. Compounds of Table 1 and Aurisins A to J show good activity in this test.

n) Effect against Botrvtis cinerea / tomato (botrytis on tomatoes) 4 week old tomato plants cv. Roter Gnom are treated with the formulated test compound 0.02 % active substance) in a spray chamber. Two days after application tomato plants are inoculated by spraying a spore suspension (1 x 105 conidia/ml) on the test plants. After an incubation period of 4 days at +20 C and 95% r. h. in a greenhouse the disease incidence is assessed. Compounds of Table 1 and Aurisins A to J show good activity in this test.

o) Effect against Pvricularia orvzae / rice (rice blast) 3 week old rice plants cv. Sasanishiki are treated with the formulated test compound (0. 02 % active substance) in a spray chamber. Two days after application rice plants are inoculated by spraying a spore suspension (1 x 105 conidia/ml) on the test plants. After an incubation period of 6 days at + 25 C and 95% r. h. the disease incidence is assessed.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

At the indicated concentration Aurisin C exhibited over 80% control of the fungal infection in this test.

- 37 p) Effect against PYrenophora teres (Helminthosporium) / barley (net blotch on barley) 1 week old barley plants cv. Regina are treated with a formulated test compound (0.02 % active substance) in a spray chamber. Two days after application barley plants are inoculated by spraying a spore suspension (3 x 104 conidia/ml) on the test plants. After an incubation period of 2 days at +20 C and 95% r.h. plants are kept for 2 days at +20 C and 60% r.h. in a greenhouse. The disease incidence is assessed 4 days after inoculation.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

q) Effect against Fusarium culmorum / wheat (fusarium head blight on wheat) A conidia suspension of F. culmorum (7 x 105 conidia/ml) is mixed with the formulated test compound (0.002 % active substance)The mixture is applied into a pouch which has been equipped before with a filter paper. After the application wheat seeds (cv. Orestis) are sown into the upper fault of the filter paper. The prepared pouches are then incubated for 11 days at approx. +1 0 C to +1 8 C and a relative humidity of 100% with a light period of 14 hours.

The evaluation is made by assessing the degree of disease occurrence in the form of brown lesions on the roots.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

r) Effect againsteDtoria nodorum /wheat (septoria leaf soot on wheat) 1 week old wheat plants cv. Arina are treated with a formulated test compound (0.02 % active substance) in a spray chamber. One day after application wheat plants are inoculated by spraying a spore suspension (5 x 105 conidia/ml) on the test plants. After an incubation period of 1 day at +20 C and 95% r.h. plants are kept for 10 days at +20 C and 60% r.h. in a greenhouse. The disease incidence is assessed 11 days after inoculation.

Compounds of Table 1 and Aurisins A to J show good activity in this test.

Claims (43)

- 38 CLAIMS

1. A compound of the general formula I HaCCH, and the tautomers, optical isomers and enantiomers and to the mixtures thereof, wherein X and Z independently of each other stand for a ring member selected from -CH2-; CO-, -CHOH- and -CHOR5-, Y is a ring member-CO- or-O-, R. and R2 independently of each other are hydrogen, -OH, -OR6, -COON or -COR7, R3 and R4 independently of each other are hydrogen, C,-C4alkyl, C2-C4alkenyl, C2-C4alkynyl, C,-C4alkoxy-C,-C4alkyl, benzyl, halobenzyl, C,C4alkylbenzyl or-CO-R8, R5 and R6 independently of each other are C,C4alkyl, C3-C4alkenyl, C3-C4alkynyl, C, -C4alkoxy-C,-C4alkyl or C, C4alkanoyl, R7 is for C,-C4alkoxy, C3-C4alkenyloxy, C3-C4alkynyloxy, C,C4alkylamino, all-C, C4alkylamino, C,-C4alkylthio or C,-C4alkoxy-C,C4alkoxy; and R8 is for optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenylamino, optionally substituted benzyl or optionally substituted benzyloxy, or stands for C,C4alkyl, C2-C4alkenyl, C2 C4-alkynyl, C3-C6cycloalkyl, C,-C4alkoxy, C3C6cycloalkoxy, C'-C4alkylamino, all-C, C4alkylamino, N-pyrrolidine, Npiperidine, N-morpholine or C3-C6cycloalkylamino..

2. A compound according to claim 1 of the general formula 11 oo H3C CH CH3 CH3 CH CH3

wherein R. and R2 independently of each other are hydrogen or hydroxyl, and R3 and R4 have the same meaning and are selected from hydrogen, C,C4alkyl, allyl, benzyl, benzoyl, acetyl, methoxymethyl, benzyloxymethyl, cyclohexylcarbamoyl and phenylcarbamoyl.

3. A compound according to claim 2, wherein R. and R2 are OH and R3 and R4 are

- 39 . hydrogen, acetyl or C,-C4alkyl.

4. A compound according to claim 1 of the subformula la (la) H3C CH CH3 CH3 OH CH3

and of the tautomers, optical isomers and enantiomers thereof, wherein R. and R2 are as defined for formula I in claim 1.

5. The compound according to claim 4 wherein R. and R2 are OH.

6. The compound Aurisin A according to claim 5 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SINF-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F-7459 (DSM 14918) and isolating the compound from the fermentation broth.

7. A compound according to claim 1 of the subformula lb HO - Out, OH :1,<] ( lb) H3CCH CH3 CH3 C|-CH3

and of the tautomers, optical isomers and enantiomers thereof, wherein R. and R2 are as defined for formula I in claim 1.

8. The compound according to claim 7 wherein R' and R2 are OH.

9. The compound Aurisin B according to claim 8 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SINF-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F-7459 (DSM 14918) and isolating the compound from the fermentation broth.

- 40

10. A compound according to claim 1 of the subformula Ic HO'OW - POOH

(Ic) H3C: CH3 CH3 H3 CH3

and of the tautomers, optical isomers and enantiomers thereof, wherein R. is as defined for formula I in claim 1.

11. The compound according to claim 10 wherein R. is OH.

12. The compound Aurisin C according to claim 11 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

13. A compound according to claim 1 of the subformula Id OO POOH

H3C CH CH3 CH3 CH CH3

and of the tautomers, optical isomers and enantiomers thereof, wherein R. and R2 are as defined for formula I in claim 1.

14. The compound according to claim 13 wherein R. and R2 are OH.

15. The compound Aurisin D according to claim 14 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

-41

16. A compound according to claim 1 of the subformula le O'O,,OH

If ( le) H3C'lCH CH3 CH3 CH3 and of the tautomers, optical isomers and enantiomers thereof, wherein R2 is as defined for formula I in claim 1.

17. The compound according to claim 16 wherein R2 is OH.

18. The compound Aurisin E according to claim 17 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

19. A compound according to claim 1 of the subformula If 0 0 0 0

0,OH ( If) H3C CH CH3 CH3 CH CH3

and of the tautomers, optical isomers and enantiomers thereof, wherein R. is as defined for formula I in claim 1.

20. The compound according to claim 19 wherein R. is OH.

21. The compound Aurisin F according to claim 20 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

- 42

22. A compound according to claim 1 of the subformula 19 H ( 19)

and of the tautomers, optical isomers and enantiomers thereof, wherein R. is as defined for formula I in claim 1.

23. The compound according to claim 22 wherein R. is OH.

24. The compound Aurisin G according to claim 23 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

25. A compound according to claim 1 of the subformula In H CH3 ( Ih) and of the tautomers, optical isomers and enantiomers thereof, wherein R2 is as defined for formula I in claim 1.

26. The compound according to claim 25 wherein R2 is OH.

27. The compound Aurisin H according to claim 26 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

- 43

28. A compound according to claim 1 of the subformula li H3 ACHE ( hi) and of the tautomers, optical isomers and enantiomers thereof, wherein R2 is as defined for formula I in claim 1.

29. The compound according to claim 28 wherein R2 is OH.

30. The compound Aurisin I according to claim 29 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

31. A compound according to claim 1 of the subformula Ij 3 ( 1j) and of the tautomers, optical isomers and enantiomers thereof, wherein R. is as defined for formula I in claim 1.

32. The compound according to claim 31 wherein R. is OH.

33. The compound Aurisin J according to claim 32 obtained from cultivating a fungal microorganism strains selected from Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F- 7459 (DSM 14918) and isolating the compound from the fermentation broth.

34. A compound according to any one of claims 6,9,12,15,18,21,24, 27, 30 and 33 wherein the employed microorganism is the Panus spec. (Agancales) SIN-F-7459 (DSM-

14918).

35. The fungal microorganism strains selected from the order of the Agaricales strains or

- 44 Trogia spec. strains capable of producing the Aurisins A to J as defined in claims 8,11,14, 17,20,23,26,29 and 32.

36. The fungal microorganism strains according to claim 35, selected from the Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agaricales) SIN-F-7459 (DSM 14918) or mutants or modified forms thereof.

37. A method of preparing the compounds of formula I comprising a first step of cultivating a fungal microorganism strain Panus spec. (Agaricales) SIN-F-6961 and Panus spec. (Aga-

ricales) SIN-F-7459 and harvesting and isolating the Aurisins A to J. and an optional second step of derivatizing the harvested fermentation product by well-known oxidation-, reduction-, hydrolysis-, acylation-, esterification-, etherification- or amidation- methods, if desired.

38. A method according to claims 36 or 37 wherein the fungal microorganism strain Panus spec. (Agaricales) SIN-F-6961 (DSM 14917) and Panus spec. (Agancales) SIN-F-7459 (DSM 14918), or mutants or modified forms thereof are employed. i

39. A method according to any one of claims 36 to 38, which comprises cultivating the microorganisms or mutants or modified forms thereof under aerobic or immersion conditions in a culture medium containing assimilable sources of carbon, nitrogen and mineral salts to produce and accumulate the Aurisins A to J in said culture medium.

40. A method of controlling or preventing infestation with phytopathogenic fungi on crop plants which comprises applying to the fungus, its locus or the host plant or part of the host plant on which the fungus is living or the locus of the host plant a fungicidally effective amount of a compound of formula I according to claim 1.

41. An agricultural composition comprising fungicidally effective amount of at least one compound of formula I according to claim 1 and an agriculturally acceptable carrier.

42. The use of a compound of formula I according to claim 1 in protecting plants against infestation by phytopathogenic microorganisms by treating the host plant, or parts thereof with a fungicidally effective amount of a compound of fommula I according to claim 1.

43. The use according to claim 42, wherein the phytopathogenic microorganisms are fungal organisms.