EA044362B1 - FUNGICIDES FOR PREVENTION AND CONTROL OF FUNGAL PATHOGENS - Google Patents

Description

Field of technology

The present invention relates to the field of biological fungicides with a broad spectrum of antifungal activity obtained from plant extracts of the Brassica order or molecules having a similar chemical structure. In particular, the authors unexpectedly proposed a new use of a combination of sulfonyl- and sulfinyl-containing aliphatic glucosinolates, their by-products and synthetic analogues as effective antifungal compounds with a broad spectrum of activity.

State of the art

The human population is increasing every year and will reach about 8.6 billion by 2030. To maintain high levels of food production, farmers are forced to use external processing such as

1) chemical pesticides that are highly effective and affordable, but have a negative impact on the environment and human health;

2) biological pesticides that do not have a negative impact on the environment, but demonstrate low efficiency (less than 60% compared to an existing chemical pesticide) and have a high cost.

This makes biological pesticides unsuitable for many countries and opens up opportunities for the development and marketing of new organic treatments that are highly effective, affordable and environmentally friendly.

In recent decades, some biological approaches have been developed to prevent B. cinerea in the field, such as the use of Bacillus subtilis and Trichoderma harzanium, but they are little used by farmers due to their low effectiveness.

In Western European agriculture, copper and sulfur are widely used fungicides for biocontrol. These fungicides are expensive to apply due to the need to reapply after each deposition. In addition, high concentrations of metals in soil have a negative impact on the environment.

Therefore, there is a need to provide an alternative method that is more environmentally friendly as well as a highly effective preventative treatment against fungal pathogens.

Fungal plant pathogens are one of the agronomic pressures that result in large food losses each year. The effectiveness of fungal pathogens is due to their easy dispersal in nature, rapid attachment to host surfaces, and rapid germ tube formation to facilitate entry into plants. On the other hand, plants have developed several defense mechanisms against fungal pathogens, such as necrotrophs:

a) preventing the penetration of the pathogen;

b) increased levels of reactive oxygen species;

c) induction of protective hormones such as jasmonate, ethylene, salicylic and abscisic acids.

In addition, some plants synthesize fungitoxic compounds that prevent the development of fungi on the surface of plants and stop the development of the disease. The identification of plant compounds with potent antifungal activity may lead to the development of new biological fungicides that have the potential to replace currently available chemical treatments.

The order Brassicales consists of economically important plants that are widespread and used as a source of food. This group of plants has been shown to contain a unique set of secondary metabolites - glucosinolates. In recent decades, glucosinolates have been shown to have anticancer, anti-inflammatory, and insecticidal properties.

For example, JP H11139949 A (OGAWA KORYO CO LTD) describes the preparation of an antibacterial antifungal agent without strong irritating odor, high threshold, low volatility and having excellent antibacterial-antifungal activity by compounding a certain compound [omega]-alkenyl isothiocyanate or a certain compound [omega] alkylthioalkylisothiocyanate. [omega]-alkenyl isothiocyanate compound having the formula

CH ₂ CH(CH ₂ ) _m NCS [(m)=2-10] (for example, 3-butenyl isothiocyanate), or an [omega]-alkylthioalkylisothiocyanate compound having the formula

RS(CH2)n2NCS [(n)=1-10], where R represents C ₁ . ₄ αlkyl (e.g. methylthiomethyl isothiocyanate), compounded with food at a concentration of 0.01-100 ppm, preferably 1-50 ppm, or with an oral hygiene agent at a concentration of about 0.01-100 ppm .

Similarly, JP H11246319 A (OGAWA KORYO CO LTD) describes the preparation of an antimicrobial and antifungal agent that can significantly reduce irritating odor and can be used with various types of beverages and foods by using a certain [omega]alkylsulfinylalkylisothiocyanate as an active ingredient. Specified antibacterial

- 1 044362 and the antifungal agent contains [omega]-alkylsulfinylalkylisothiocyanate represented by the formula

RS(O)-(CH2)n-NCS (n is 1-10; R is C _1-4 alkyl).

In one embodiment, the isothiocyanate is 3methylsulfinylpropyl isothiocyanate, 6-methylsulfinylpropyl isothiocyanate, or the like. The compound of formula I, where n is <7, is one of the aromatic components of horseradish, but its content is low. A compound of the indicated formula is prepared by oxidation with [omega]methylthioalkylisothiocyanate peroxide of the formula

CH3S-(CH2)n-NCS

JP 2000086414 A (KINJIRUSHI WASABI KK) describes the preparation of an antimicrobial agent containing horseradish aromatic component as an active ingredient, especially showing a spectrum of antibacterial activity in a wide range from fungi to bacteria and exhibiting strong bacteriostatic and antibacterial effect even at extremely low content of aromatic component. The agent contains, as an active ingredient, horseradish aromatic component n-methylsulfonylalkyl isothiocyanate and one or more horseradish aromatic components selected from the group consisting of p-methylthioalkylisothiocyanate, p-methylsulfinyl alkyl isothiocyanate and allyl isothiocyanate. The agent contains as an active ingredient both the aromatic components of horseradish n-methylthioalkylisothiocyanate and n-methylsulfinylalkylisothiocyanate.

K. GILLIVER The Inhibitory Action of Antibiotics on Plant Pathogenic Bacteria and Fungi (Inhibitory Action of Antibiotics on Plant Pathogenic Bacteria and Fungi) ANNALS OF BOTANY., Vol. 10, No. 3, July 1, 1946 (1946-07-01), p. 271-282, ХР55512767, GB, ISSN: 0305-7364, DOI: 10.1093/oxfordjournals.aob.a083136; reports an example of antagonism between soil microorganisms and plant pathogens that has been studied over many years; and in some cases biological methods of disease control have been developed. Certain antibiotic substances have been isolated from culture filtrates of fungi, bacteria and actinomycetes, and some of them have shown antagonistic effects against organisms that cause plant diseases. In particular, the antifungal effect of cheirolin was discovered.

In T. Sotelo et al., In vitro activity of glucosinolates and their degradation products against Brassica-Pathogenic bacteria and fungi, Applied and Environmental Microbiology , January 1, 2015 (2015-01-01), p. 432-440, XP55512754, DOI: 10.1128/AEM.03142-14, the purpose of the work was to evaluate the biocidal effect of 17 glucosinolates (GZL) and glucosinolate hydrolysis products (PGG), and methanol extracts of leaves of various GZL-enriched cabbage crops on growth suppression in vitro of two bacterial (Xanthomonas campestris pv. Campestris and Pseudomonas syringae pv. maculicola) and two fungal (Alternaria brassicae and Sclerotinia scletoriorum) pathogens of plants of the genus Cabbage. GZL, PGG and methanol extracts of leaves inhibited the development of the studied pathogens compared to the control, and the effect was dose-dependent. In particular, this document describes the antifungal effects of suloraphane.

In L. DROBNICA et al., Antifungal activity of lsothiocyanates and related compounds, APPLIED MICROBIOLOGY, Vol. 15, No. 4, 1967, p. 701-709, the results of a study of the antifungal effect of derivatives of biphenyl isothiocyanates (group A), stilbene (B), azobenzene and benzolazonaphthalene (C), naphthalene (D) and other polycondensed aromatic hydrocarbons (E) are presented. Of all 48 compounds studied, antifungal activity was observed only among compounds of groups A and D. Derivatives of groups B, C and E had extremely low solubility in water and very large molecules; as a result, they were probably unable to penetrate fungal spores or mycelium. It was suggested that the -NCS group was not active. In particular, this document describes the antifungal effects of glucocheirolin, glucoerisoline and glucoberteroin.

DE 1793450 A1 (PHILIPS NV) relates to fungicidal compositions containing certain thiocyanates, i.e. sulfinyl- or sulfonylmethylene rhodanide, and methods for producing and protecting plants from mold infection.

However, there remains a need to provide molecules that are environmentally friendly but exhibit stronger fungitoxic effects and act against a wider range of fungal pathogens.

Brief description of the invention

In the present invention, we have identified a combination of sulfonyl- and sulfinyl-containing aliphatic glucosinolates that exhibit potent fungitoxic effects against a wide range of fungal pathogens. This combination of products can be used as a new line of biological fungicides.

One of the objects of the present invention is to provide the use of a composition containing a combination of at least two compounds selected from the compounds of general formula I

- 2 044362

where Z represents nitrile (-CN), thiocyanate (-SCN) or isothiocyanate (-N=C=S);

X, Y are, independently of each other, a lone pair or O, provided that at least one of the two X or Y is O;

R _b R2 are independently H, saturated linear, cyclic or branched (C ₁ -C ₆ )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1-C ₈ )alkyl, C ₆ aryl;

B represents a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6aryl ;

provided that at least one of A, B or C is different from a covalent bond, and characterized in that at least two compounds in combination contain

1) at least one Z group representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or

3) at least different carbon chain lengths of the fragment -A-B-C- for the prevention or treatment of fungal pathogens in plants.

Another object of the present invention is to provide a composition containing a combination of compounds according to the present invention for use in a method of preventing or treating mycoses in humans or animals.

Another object of the present invention is to provide a fungicidal composition containing a combination of compounds according to the present invention.

The present invention also relates to a food, drink or oral hygiene agent containing the above-mentioned antifungal agent, namely a fungicidal composition.

Other objects and advantages of the present invention will become apparent to one skilled in the art upon consideration of the following detailed description, given with reference to the following illustrative drawings, and the accompanying claims.

Brief description of drawings

The drawing represents a newly identified secondary metabolite on the surface of fad2-3 cotyledons.

(A) Characterization of 7-methylsulfonylheptylglucosinolate. An ion (M-H) with m/z 494.0823 was observed, corresponding to the molecular formula C ₁₅ H ₂₉ NO _n S ₃ (calculated mass 494.0824, error 0.1 mDa). A major fragment ion at m/z 96.9599, typical of a sulfate fragment, was also present. No known compounds with the molecular formula _C15H29NOnS3 have been reported, but the _{data strongly} suggest that this metabolite is 7- _{methylsulfonylheptylglucosinolate} , a new glucosinolate that is a 15-carbon analogue of 8-methylsulfonyloctylglucosinolate (C16H31NO11S3).

(B) Identification of a novel glucosinolate selectively present on the surface of cotyledons and the first true leaves of DT and fad2-3. A relative abundance of 7-methylsulfonylheptylglucosinolate was observed in isopropanol washes from the surface of 7-day-old cotyledons and 14-day-old leaves. Error values represent the mean of four biological replicates (±SD), each from a pool of −980 seedlings. Statistical significance in pairwise comparison was assessed using Student's t test, where **p>0.01; ***p<0.001.

(C) Chemical structure of 7-methylsulfonylheptylglucosinolate.

(D) Structure of 7-methylsulfonylheptyl isothiocyanate.

Detailed Description of the Invention

Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references cited are incorporated by reference in their entirety. The publications and applications discussed are presented solely for the purpose of disclosure prior to the filing date of this application. Nothing should be construed as implying that the present invention does not provide a right of opposition to the source of information with the prior priority of such publication in accordance with the prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the event of a conflict, this description, including definitions, will control.

Unless otherwise indicated, all technical and scientific terms used have the same meanings as commonly understood by one skilled in the art to which the present invention relates. In the present description, the following definitions are provided to facilitate understanding of the present invention.

The term contains is generally used in the sense of inclusion, i.e. it may contain one or more features or components.

In the specification and claims, the singular forms include the plural unless the context clearly dictates otherwise.

The presence in some cases of broadening words and expressions, such as one or more, at least, but not limited to, similar expressions should not mean that a narrower case is assumed or required in those cases where such broadening expressions may not be present.

The terms subject or patient are well known in the art and are used interchangeably to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and most preferably human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject may be a normal subject. The term does not indicate a specific age or gender. Those. the term covers adult and newborn male or female subjects.

The term effective amount refers to the amount necessary to achieve a physiological effect. Physiological effects can be achieved by using a single dose or by repeated doses. Dosages administered may, of course, vary depending on known factors such as the physiological characteristics of the particular composition; age, health status and weight of the subject; the nature and severity of symptoms; type of concomitant treatment; frequency of treatment; and the desired action, and can be determined by one skilled in the art.

The fungus is a eukaryote that digests food externally and absorbs nutrients directly through the cell walls. Most fungi reproduce by spores and have a body (thallus) made up of microscopic tubular cells called hyphae. Fungi are heterotrophic and, like animals, obtain carbon and energy from other organisms. Some fungi obtain nutrients from a living host (plant or animal) and are called biotrophs; others obtain nutrients from dead plants or animals and are called saprotrophs (saprophytes, saprobes). Some fungi infect a living host but kill host cells to obtain nutrients; they are called necrotrophs.

Pathogenic fungi, also called fungal pathogens, are fungi that cause diseases in plants, humans, and other organisms. Approximately 300 species of fungi are known to be human pathogens. The study of fungal pathogens in humans is called medical mycology. Although fungi are eukaryotes, many pathogenic fungi are microorganisms. The study of fungi and other microorganisms that are pathogenic to plants is called plant pathology.

There are thousands of species of plant pathogenic fungi, which together are responsible for 70% of known plant diseases. Plant-pathogenic fungi are parasites, but not all plant-parasitic fungi are pathogenic. Plant parasitic fungi obtain nutrients from the living host plant, but the host plant does not necessarily develop any symptoms. Plant pathogenic fungi are parasites and cause disease characterized by symptoms.

Fungicides are biocidal chemical compounds or biological organisms used to kill parasitic fungi or their spores (referred to herein as fungitoxic). Fungistatic compounds inhibit the growth of fungi. Fungi can cause severe damage to crops, resulting in critical losses in yield, quality and profit. Fungicides are used both in agriculture and medicine to combat fungal infections in animals or humans. Chemicals used to control oomycetes, which are not fungi, are also known as fungicides because oomycetes use the same mechanisms as fungi to infect plants. Fungicides can be contact, translaminar or systemic. Contact fungicides are not absorbed into plant tissue and protect the plant only at the site of application of the spray. Translaminar fungicides are redistributed from the upper, sprayed surface of the leaf to the lower, unsprayed surface. Systemic fungicides are absorbed and distributed throughout the xylem vessels. Some fungicides spread to all parts of the plant. Some are local-systemic, and some move from bottom to top.

Fungistatics are antifungal agents that inhibit the growth of a fungus (without killing the fungus). The term fungistatic can be used both as a noun and an adjective. Fungistatics are used in agriculture, the food industry, the paint and varnish industry and medicine.

Use of a composition containing a combination of at least two compounds selected from compounds of general formula I

- 4 044362 where Z represents nitrile (-CN), thiocyanate (-SCN) or isothiocyanate (-N=C=S);

X, Y are independently of each other a lone pair or O, provided that at least one of the two X or Y is O;

R _b R2 are independently H, saturated linear, cyclic or branched (C ₁ -C ₆ )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6 -aryl;

B represents a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6- aryl;

provided that at least one of A, B or C is different from a covalent bond, and characterized in that at least two compounds in combination contain

1) at least one Z group representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or

3) at least different carbon chain lengths of the fragment -A-B-C-, for the prevention or treatment of fungal pathogens in plants.

Preferably, the aromatic rings may be substituted at the ortho, meta and para positions;

cycloalkyl rings may be substituted in various positions and in cis or trans orientation with respect to the ring; and aliphatic and aromatic rings may be substituted with H, halogen or OMe groups.

Common oxidation states or oxidation states of sulfur range from -2 to +6. If sulfur is neither oxidized nor reduced, it has 6 valence electrons. Certainly in sulfates, SO2 ^-4 , and sulfites, SO2 ^-3 , the sulfur atom has the formal oxidation states VI+ and IV+; in sulfides - II.

In a combination or mixture of at least two compounds, it is believed that the length of the carbon chain of the fragment -A-B-C- is important. The combination may include one short and one long chain, for example, one containing a maximum of 6 linear carbon atoms and another containing a minimum of 7 linear carbon atoms. However, in some cases, the combination may contain at least two compounds with fragment carbon chains -A-B-C- of the same length.

The composition according to the present invention, in order to be effective for the prevention or treatment of fungal plant pathogens, must satisfy at least one of the above criteria according to points 1)-3). Combinations or mixtures of at least two compounds selected from compounds of general formula I, satisfying criteria 1) to 3) showed unexpected synergistic effects compared to the individual components tested separately.

Synergy is generally considered to occur when the combined action of two or more agents is greater than the sum of their individual actions. In other words, synergy is said to occur when the combined effect of two or more agents is greater than would be predicted from the characteristics of the agents when used individually.

In particular, the composition according to the present invention is a mixture of at least one compound in which Z is a nitrile and one compound in which Z is an isothiocyanate.

Alternatively, the mixture corresponds to at least one compound in which Z is an isothiocyanate and one compound in which Z is an isothiocyanate.

Alternatively, the mixture corresponds to at least one compound in which Z is an isothiocyanate and one compound in which Z is a thiocyanate.

Alternatively, the mixture corresponds to at least one compound in which Z is a thiocyanate and one compound in which Z is an isothiocyanate.

Alternatively, the mixture corresponds to at least one compound in which X is a lone pair and Y is oxygen, and at least one compound in which X and Y are oxygen. Alternatively, the mixture corresponds to X and Y representing oxygen. Alternatively, the mixture corresponds to at least one compound in which X represents a lone pair and Y represents oxygen. Alternatively, the mixture according to the present invention contains at least two compounds selected with the same formula A/B/C, where B is a covalent bond and A and C are independently saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1-C ₈ )alkyl. Preferably, the mixture according to the present invention contains at least two compounds selected with different formulas A/B/C, where B represents a covalent bond and A and C represent independently

- 5 044362 from each other saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl.

The term lone pair refers to a pair of valence electrons not shared with another atom, sometimes called an unbonded pair. Lone pairs are found in the outermost electron shell of atoms. They can be identified using the Lewis structure. Therefore, electron pairs are considered lone pairs when two electrons are paired and are not used in the formation of chemical bonds. Thus, the number of electrons in lone pairs plus the number of electrons forming bonds equals the total number of valence electrons around the atom. One lone pair can be found in nitrogen group atoms such as nitrogen in ammonia, two lone pairs can be found in chalcogen group atoms such as oxygen in water, and halogens can carry three lone pairs as in hydrogen chloride.

A covalent bond, also called a molecular bond, is a chemical bond involving the sharing of electron pairs between atoms. Such electron pairs are known as shared pairs or bonding pairs, and the stable equilibrium of attractive and repulsive forces between atoms sharing electrons is known as covalent bonding.

An isothiocyanate is a chemical group -N=C=S formed by replacing the oxygen in the isocyanate group with sulfur. Many naturally occurring isothiocyanates from plants are formed through the enzymatic conversion of metabolites called glucosinolates. These natural isothiocyanates, such as allyl isothiocyanate, are also known as mustard oils. A synthetic isothiocyanate, phenylisothiocyanate, is used for Edman sequencing of amino acids.

The term alkyl refers to saturated and unsaturated aliphatic groups, including linear alkyl groups and branched alkyl groups, optionally substituted with one or more halogens. In some embodiments, the linear or branched alkyl contains about 30 or fewer backbone carbon atoms (e.g., C1-C30 for linear, _C3 - _C30 for branched) and alternatively about 20 or fewer, such as 1 to 6, carbon atoms (defined as lower alkyl). Preferably, the alkyls of the present invention contain from one to thirty, more preferably from one to twenty, even more preferably from one to twelve, more preferably from one to eight, more preferably from one to six, and most preferably from one to four carbon atoms, and are linear or branched. The term C1- _C6 alkyl represents a straight or branched alkyl chain containing from one to six carbon atoms. Examples of C1- _C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, neopentyl, hexyl, isohexyl and the like. Preferably the alkyl is a lower alkyl such as n-butyl, sec-butyl, isobutyl, t-butyl, n-propyl or isopropyl, ethyl or methyl.

The term lower in lower alkyl, lower alkoxyl, lower alkenyl and lower alkynyl refers to a chain containing up to 6 carbon atoms connected in a linear or branched manner.

The term cycloalkyl represents a saturated or partially saturated mono- or polycarbocyclic ring, preferably containing 5-14 carbon atoms in the ring. Examples of cycloalkyls include monocyclic rings containing 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. An example of a cycloalkyl is C ₅ -C ₇ cycloalkyl, which is a saturated hydrocarbon ring structure containing five to seven carbon atoms.

The term alkoxy represents -O-alkyl. An example of an alkoxyl is a C1- _C6 alkoxyl, which is a straight or branched alkyl chain containing from one to six carbon atoms attached to an oxygen atom. Examples of C1- _C6 alkoxy groups include methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, sec-butoxyl, t-butoxyl, pentoxyl, hexoxyl and the like. C ₁ -C ₆ alkoxyl includes in its definition C1 -C ₄ alkoxyl.

The term aryl refers to a carbocyclic or heterocyclic aromatic 5-14 membered monocyclic or polycyclic ring. Examples of aryls include phenyl, naphthyl, anthryl, phenanthryl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, benzo[b]thienyl, naphtho[2,3 -b]thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthenyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxyalinyl, quinzolinyl, benzothiazolyl, benzimidazolyl, tetrahydroquinolinyl, cinnolinyl , pteridinyl, carbazolyl, beta-carbolinyl , phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl and phenoxazinyl.

The term halogen represents chlorine, fluorine, bromine or iodine. The term halogen represents chloro, fluoro, bromo or iodo.

The term carbocycle represents an aromatic or saturated or partially saturated 5-14 membered monocyclic or polycyclic ring, such as a 5-7 membered monocyclic or 7-10 membered bicyclic ring, all of the atoms in the ring being carbon atoms.

The term polycyclic refers to a polycyclic ring system in which at least two rings are connected to each other. Polycyclic refers to any ring system, which may have any degree of saturation, be aromatic or aliphatic, and optionally contain a heteroatom. Systems can be further classified by the number of rings (for example, 2=bicyclic, 3=tricyclic, 4=tetracyclic, etc.) and by the way the rings are connected to each other.

(1) Substituted ring systems. These ring systems do not share any common atoms. In these polycyclic systems, the smaller ring can be considered a substituent on the larger ring. By way of example only, biphenyl is a substituted polycyclic ring system.

(2) Spirocyclic systems. Spirocyclic systems share a single common atom. Therefore, the rings are connected at one point. The connecting atom is also called a spiroatom, most often a quaternary carbon (spirocarbon). For example, a spiro compound consisting of a cyclohexane ring and a cyclopentane ring is called spiro[4.5]decane.

(3) Condensed ring systems. Fused ring systems share two common atoms on one common bond, i.e. the rings separate one side. Examples of fused polycyclic systems include, but are not limited to, naphthalene, benzofuran, indole, benzothiophene, quinoline and anthracene.

(4) Bridge ring systems. Bridging ring systems share more than two common atoms, i.e. Bridged ring systems contain overlapping rings. For example, adamantane, amantadine, biperiden, memantine, methenamine, rimantadine and norbornane are bridged ring systems.

The term sulfonyl represents -SO ₂ -L5, where L5 is preferably an alkyl, aryl, cycloalkyl, heterocycle or amino group. Alkyl, aryl, cycloalkyl and heterocycle may optionally be substituted. An example of a sulfonyl is C1- _C4 alkylsulfonyl, which is a straight or branched alkyl chain containing from one to four carbon atoms attached to a sulfonyl moiety. Examples of C1- _C4 alkylsulfonyl groups include methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl, tert-butylsulfonyl and the like.

As stated above, many groups are optionally substituted. Examples of substituents for alkyl and aryl include mercapto, thioether, nitro (NO2), amino, aryloxyl, halogen, hydroxyl, alkoxyl and acyl, as well as aryl, cycloalkyl and saturated and partially unsaturated heterocycles. Examples of substituents for heterocycle and cycloalkyl include those listed above for alkyl and aryl, and aryl and alkyl.

Examples of substituted aryls include a phenyl or naphthyl ring substituted with one or more substituents, preferably one to three substituents, independently selected from halogen, hydroxy, morpholino(C1- _C4 )alkoxycarbonyl, pyridyl(C1- _C4 )alkoxycarbonyl, halogen(C1 -C ₄ )alkyl, C1-C ₄ alkyl, C1-C ₄ alkoxyl, carboxy, C1-C ₄ alkoxycarbonyl, carbamoyl, N-(C ₁ -C ₄ )αalkylcarbamoyl, amino, C1-C ₄ alkylamino, di(C1 -C ₄ )alkylamine or a group of the formula -(CH ₂ ) _a -R<7>, where a is 1, 2, 3 or 4 and R<7> represents hydroxy, C1- _C4 alkoxy, carboxy, C1-C ₄ alkoxycarbonyl, amino, carbamoyl, C1- _C4 alkylamino or di(C1- _C4 )alkylamino.

Another substituted alkyl is halogen(C1- _C4 )alkyl, which is a straight or branched alkyl chain containing from one to four carbon atoms to which 1-3 halogens are attached. Examples of halogen(C1- _C4 )alkyl groups include chloromethyl, 2-bromoethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl, 3-chloroisobutyl, iodo-tert-butyl, trifluoromethyl and the like groups.

Another substituted alkyl is hydroxy(C1- _C4 )alkyl, which is a straight or branched alkyl chain containing from one to four carbon atoms to which a hydroxy group is attached. Examples of hydroxy(C1- _C4 )alkyl groups include hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxyisopropyl, 4-hydroxybutyl and the like.

Another substituted alkyl is C1- _C4 alkylthio(C1- _C4 )alkyl, which is a straight or branched _C1 - _C4 alkyl group to which is attached a _C1 - _C4 alkylthio group. Examples of C ₁ -C ₄ alkylthio(C ₁ -C ₄ )alkyl groups include methylthiomethyl, ethylthiomethyl, propylthiopropyl, sec-butylthiomethyl and the like.

The cycloaclyl may optionally be substituted with 1, 2 or 3 substituents independently selected from halogen, halogen(C1- _C4 )alkyl, C1- _C4alkyl , C1- _C4alkoxyl , carboxy, C1- _{C4alkoxycarbonyl} , carbamoyl, N- (C ₁ -C ₄ )alkylcarbamoyl, amino, C1-C ₄ alkylamino, di(C1-C ₄ )alkylamino or a group having the structure -(CH ₂ ) _a -R<7>, where a is 1, 2, 3 or 4 and R<7> is hydroxy, C1-C4alkoxy, carboxy, C1-C4alkoxycarbonyl, amino, carbamoyl, C1-C4alkylamino or di(C1-C4)alkylamino. Examples of substituted cycloalkyl groups include 3-methylcyclopentyl, 4-ethoxycyclohexyl, 5-carboxycycloheptyl, 6-chlorocyclohexylyl groups and the like.

Double bonds can, in principle, have an E- or Z-configuration. The compounds of the present invention may therefore exist as isomeric mixtures or individual isomers. If not specified, both isomeric forms are meant. If a compound of the present invention contains a single chiral center, the compound may be present as a single isomer (R or S) or as a mixture of isomers, for example a racemic mixture. If a compound of the present invention contains more than one chiral center, the compound may be present as an enantiomerically pure diastereomer or as a mixture of diastereomers.

A specific embodiment of the present invention provides the use of a composition comprising a combination of at least two isothiocyanate compounds, preferably said at least two isothiocyanate compounds having at least two different oxidation states of the sulfur atom and/or at least different carbon chain lengths of the -A moiety -B-C-, preferably contained between C ₃ and C ₉ .

Preferably, the present invention provides the use of a composition containing a combination of at least two compounds selected from compounds of general formula I (as defined above) wherein Z is isothiocyanate (-N=C=S);

X, Y represent independently of each other O;

R1, R2 are, independently of each other, H, saturated linear or cyclic (C1- _C6 )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear or cyclic (C1-C ₈ )alkyl, provided that at least one of A or C is different from the covalent bond;

B is a covalent bond where the carbon chain length of the fragment -A-B-C- is from C3 to C9, for the prevention or treatment of fungal plant pathogens.

More preferably, the present invention provides the use of a composition containing a combination of at least two compounds selected from a list including

^N ^s y: 0 0 Yfz o _h p O O o o O......-...... o ^zx o —< II ⁰ 11^» 0 o -A ¹ o \ / pp r- ^s W 'S' II o C'' ^S V qS^^, _N 0.0 _r < ^{s N} C. _c > ^s 4F ,c ^s ^N'' ^{C 4} iGG o ^/4 o II 0

- 8 044362

II

O where at least two compounds in combination contain

1) at least one Z group representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or

3) at least a different length of the carbon chain of the fragment -A-B-C-, where the length of the carbon chain of the fragment -A-B-C- is from C ₃ to C ₉ , for the prevention or treatment of fungal plant pathogens.

Preferably, the at least two compounds in combination for preventing or treating fungal plant pathogens are selected from the group consisting of

8MSOH/8MSOOH; 8ASOH/8ASOOH; 8ESOH/8ESOOH; 8CSOH/3MSOOH;

8CSOH/7MSOH; 8CSOH/3MSOH; 8MSOOH/6MSOH; 8MSOH/3MSOOH;

8MSOH/7MSOH; 8MSOH/6MSOH; 8CSOH/8MSOH; 8CSOH/3MSOOH;

8MSOH/9MSOOH; 8CSOH/7MSOH; 8ESOH/7MSOH; 8ESOH/3MSOH;

8MSOOH/7MSOH; 8MSOOH/9MSOH; 8MSOOH/6MSOH; 8CSOH/7MSOH;

8MSOOH/3MSOH; 8MSOOH/8MSOON or 8MSOOH/3MSOOH.

Even more preferably, the at least two compounds in combination for preventing or treating fungal plant pathogens are a mixture of 7-methylsulfonylheptyl isothiocyanate and (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene or a mixture of 8-methylsulfonyl heptyl isothiocyanate and 8- methylsulfinyl locthioisothiocyanate.

The identified compounds of the present invention provide several advantages, being fungitoxic and/or fungistatic against environmental, plant, storage, and medical fungal pathogens.

Moreover, the identified compounds according to the present invention do not exhibit phytotoxicity, are stable relative to exposure to light and can easily be applied to plants.

- 9 044362

The composition used according to the present invention has been shown to extend shelf life by at least one week for fruits, vegetables and cut flowers infected with fungal pathogens in storage areas. The compounds used (i.e. the mixture) were shown to be non-toxic to insects and humans. The composition according to the present invention is easily applicable with a special effect on the ripening of perishable food products and does not require additional installation costs. The composition according to the present invention may be of interest to companies involved in storage (ie, reducing packaging costs), forestry industries, horticulturists and farmers.

Thus, the composition according to the present invention is useful as a fungitoxic and/or fungistatic agent for plants. The composition according to the present invention, used as a fungicide, has shown high effectiveness when treating various plants or plant families (hosts). In fact, the composition according to the present invention can be used to treat more than 1400 species of agronomically important crops or plants, including Solanaceae, Rosaceae, Vinaceae, Grassaceae, etc.

The composition of the present invention can be applied to any part of a plant during any part of its life cycle, including, without limitation, seeds, seedlings, plant cells, plants or flowers.

According to the present invention, all plants and plant parts can be treated. Plant refers to all plants and plant populations, such as desirable and undesirable wild plants, cultivars and plant varieties (whether or not protected by plant variety rights or plant breeder rights). Cultivars and plant varieties may be plants obtained by traditional methods of propagation and breeding, which may be supplemented or accompanied by one or more biotechnological methods, such as the use of double haploids, protoplast fusion, random and targeted mutagenesis, molecular or genetic markers, or bioengineering methods and genetic engineering. Parts are understood as all above-ground and underground parts and organs of plants, such as shoot, leaf, flower and root, examples include leaves, needles, stems, branches, flowers, fruiting bodies, fruits and seeds, as well as roots, corms and rhizomes. Crops and vegetative and generative propagation material, such as cuttings, corms, rhizomes, tendrils and seeds, are also plant parts.

Among the plants that can be protected using the composition according to the present invention are the main field crops such as corn, soybeans, cotton, oilseeds of the genus brassica such as rapeseed (for example, canola), turnips, mustard and Abyssinian cabbage, rice, wheat, sugar beets, sugar cane, oats, rye, barley, millet, triticale, flax, grapes, and various fruits and vegetables of various botanical taxa, such as various rose species (e.g., pome fruits such as apples and pears, and stone fruits fruits such as apricots, cherries, almonds and peaches, berries such as strawberries), currants, nuts, birch, sumac, beech, mulberries, olives, actinides, bays, bananas (e.g. banana trees and plantings), madder (e.g. , coffee), Teaaceae, Sterculiaceae, Rutaceae (e.g. lemons, oranges and grapefruit), Solanaceae (e.g. tomatoes, potatoes, peppers, eggplant), Liliaceae, Asteraceae (e.g. lettuce, artichoke and chicory, including chicory root, endive or common chicory), umbelliferous (for example, carrots, parsley, celery and root celery), cucurbits (for example, cucumber, including gherkins, squash, watermelon, pumpkins and melons), alliums (for example, onions and leeks) , cruciferous vegetables (e.g. cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choy, kohlrabi, radish, horseradish, watercress, bok choy), legumes (e.g. peanuts, peas and beans - such as curly beans and broad beans), chenoraceae (e.g. chard, chard, spinach, beets), malvaceae (e.g. okra), asparagus (e.g. asparagus), horticultural and forest crops, ornamental plants and flowers, including cut flowers, herbs, including golf courses, lawn, and genetically modified homologues of these crops.

For example, the composition of the present invention can be used to control common fungal diseases, such as powdery mildew, rust, downy mildew and anthracnose, of field crops, fruit trees and vegetables.

In addition, the composition of the present invention can be used for the treatment of persistent diseases, mainly for the control of wheat powdery mildew, rice blast, rice smut, melon powdery mildew, tomato powdery mildew, apple rust, watermelon anthracnose and flower powdery mildew. In addition, the composition showed very good control action against cucumber downy mildew, grape downy mildew, scab, anthrax and spotted leaf drop.

In a specific embodiment of the present invention, the composition of the present invention is used to treat or prevent tree diseases caused by fungal pathogens, for example Panama disease of bananas, ash blight.

In addition, the composition according to the present invention can be applied directly to the field on

- 10 044362 plant cultures, but also in vitro, for example, for introduction into plant crops.

Surprisingly, the composition according to the present invention, used as a fungicide, showed extremely high activity against at least 43 fungal pathogens (see list below) from 4 different types, 8 different classes and 14 different orders._______________

View Type Class Order Rhizoctonia solani Basidiomycetes Agaricomycetes Cantarellaceae Thamnidium elegans Zygomycetes Zygomycetes Mucoraceae Phytophthora ca ctor urn Oomycetes Oomycetes Peronosporaceae Phytophthora syringae Oomycetes Oomycetes Peronosporaceae Phytophthora infestans Oomycetes Oomycetes Peronosporaceae Phytophthora agathidicida strain 18407 Oomycetes Oomycetes Peronosporaceae Phythium sp. Oomycetes Oomycetes Peronosporaceae PI as mo para viticola Oomycetes Oomycetes Peronosporaceae Aspergillus versicolor Ascomycetes Eurocyomycetes Eurociaceae Aspergillus pseudoglaucus Ascomycetes Eurocyomycetes Eurociaceae Aspergillus sp. Ascomycetes Eurocyomycetes Eurociaceae Penicillium vulpinum Ascomycetes Eurocyomycetes Eurociaceae Penicillium wortmannii Ascomycetes Eurocyomycetes Eurociaceae Trichophyton rubrum Ascomycetes Eurocyomycetes Onigenaceae Lasiodiplodia theobromae Ascomycetes Dothideomycetes Bothryosphere Guignardia bidwellii Ascomycetes Dothideomycetes Botryospherial

- 11 044362

Alternaria ra di ci na Ascomycetes Dothideomycetes Pleosporans Phom a exigua Ascomycetes Dothideomycetes Pleosporans Phoma betae Ascomycetes Dothideomycetes Pleosporans Helminthosporium solani Ascomycetes Dothideomycetes Pleosporans Cladosporium langeronii Ascomycetes Dothideomycetes Capnodial Cladosporium haloterans Ascomycota Dothideomycetes Capnodial Cladosporium sp. Ascomycetes Dothideomycetes Capnodial Fusarium verticillioides Ascomycetes Sordariomycetes Hypocrete Fusarium gramineum Ascomycetes Sordariomycetes Hypocrete Fusarium equiseti Ascomycetes Sordariomycetes Hypocrete Fusarium culmorum Ascomycetes Sordariomycetes Hypocrete Fusarium avenaceum Ascomycetes Sordariomycetes Hypocrete Acremonium sp. Ascomycetes Sordariomycetes Hypocrete Pyricularia oryzae Ascomycetes Sordariomycetes Magnaportovye Scopulariopsis brevicaulis Ascomycetes Sordariomycetes Microasceae Scopulariopsis fusca Ascomycetes Sordariomycetes Microasceae Colletotrichum acutatum Ascomycetes Sordariomycetes Glomerella Colletotrichum coccodes Ascomycetes Sordariomycetes Glomerella Colletotrich urn gloeosporioides Ascomycetes Sordariomycetes Glomerella Plectosphaerella cucumerina Ascomycetes Sordariomycetes Glomerella Monilinia laxa Ascomycetes Leociomycetes Helociaceae Botrytis cinerea Ascomycetes Leociomycetes Helociaceae Sclerotinia sclerotiorum Ascomycetes Leociomycetes Helociaceae Gloeosporium album Ascomycetes Leociomycetes Helociaceae Hymenoscyphus fraxineus Ascomycetes Leociomycetes Helociaceae Geotrichum candidum Ascomycetes Saccharomyces Saccharomycetes Pichia fermentans Ascomycetes Saccharomyces Saccharomycetes

In particular, the composition according to the present invention has shown effectiveness against fungal plant pathogens selected from the phyla including basidiomycetes, zygomycetes, oomycetes or ascomycetes.

For example, the composition of the present invention has shown particular effectiveness against the fungal plant pathogens Botrytis cinerea, Colletotrichum graminicola, Fusarium oxysporum, Sclerotiana sclerotiorum, Verticillium dahlia, Mycospharella gramincola and Sphacelotheca reliana.

In another embodiment, the composition of the present invention (according to General Formula I, as defined above) can be used as an antimycotic agent in a food or beverage. The present invention also relates to a food, beverage and oral hygiene agent containing the above-mentioned antifungal agent.

In particular, the antifungal or antimycotic agent according to the present invention can be introduced into foods or beverages to prevent food spoilage by fungus or contamination by pathogens.

In a specific embodiment, the composition of the present invention may be advantageously used by food retailers to extend the shelf life of fruits and vegetables by preventing the development of fungal plant pathogens. For example, the compositions of the present invention can extend the shelf life of vegetables, berries, fruits or cut flowers by at least one week.

Preferably, the amount administered is in the range of 0.01 to 100 ppm. by weight of food, more preferably from 1 to 50 ppm.

Examples of foods and drinks with which the antifungal agent can be mixed according to the infusion

- 12 044362 of this invention include fish products, fish sausages, ham, fish sausages and hams, surimi products, dried fish and shellfish products, smoked products, salted fish, shrimp, semi-solid seafood such as pickled cod roe and similar products; livestock products such as ham, sausages, bacon and minced meat products; prepared foods such as salads, hamburgers, dumplings, boiled beans, bok choy, cucumbers, pickled vegetables such as kimchi, sweet potatoes, Japanese omelette and rosemary lotus roots; liquid dressings such as sweet sauce and soup; seasonings such as miso and soy sauce; noodles such as raw or cooked wheat noodles, udon, pasta, various drinks such as fruit juices, carbonated drinks, tea, milk drinks, lactic acid drinks, coffee, cocoa, soy milk and the like; fruits and vegetables, milk powder, fermented milk, butter, cheese, ice cream, dairy products such as cream, caramel, candy, chewing gum, jams, margarine and similar products.

In a preferred embodiment, the antimycotic composition according to the present invention contains a combination of at least two compounds selected from the group consisting of

8MSOH/8MSOOH; 8ASOH/8ASOOH;

8ESOH/8ESOOH; 8CSOH/3MSOOH; 8CSOH/7MSOH; 8CSOH/3MSOH;

8MSOOH/6MSOH; 8MSOH/3MSOOH; 8MSOH/7MSOH; 8MSOH/6MSOH;

8CSOH/8MSOH; 8CSOH/3MSOOH; 8MSOH/9MSOOH; 8CSOH/7MSOH;

8ESOH/7MSOH; 8ESOH/3MSOH; 8MSOOH/7MSOH; 8MSOOH/9MSOH;

8MSOOH/6MSOH; 8CSOH/7MSOH; 8MSOOH/3MSOH; 8MSOOH/8MSOON or 8MSOOH/3MSOOH.

Even more preferably, the antimycotic composition according to the present invention contains at least two compounds in combination, which are a mixture of 7-methylsulfonylheptyl isothiocyanate and (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene or a mixture of 8-methylsulfonyloctyl isothiocyanate and 8 -methylsulfinyl locthioisothiocyanate.

In another embodiment, the composition of the present invention (according to General Formula I, as defined above) can be used as a disinfectant for oral hygiene or in sanitary products.

Preferably, the oral hygiene product is selected from a dentifrice, a lozenge, a liquid or powder mouthwash, a coating solution, a halitosis preventing agent, and a chewing gum.

The dosage form of the oral hygiene agent with which the antimycotic agent of the present invention is mixed may be any of the following: a liquid preparation, a solid preparation and a semi-solid agent; and may be a dentifrice, a lozenge, a liquid or powdered mouthwash, a coating solution, a halitosis preventing agent, chewing gum, and the like.

In addition, the disinfectant according to the present invention can be used in various products, so-called anti-fungal product(s), such as kitchen utensils such as cutting boards, washing utensils such as toothbrushes, office supplies such as writing instruments, erasers and note paper, clothing such as linen, automotive interior items such as handles and seats, home applications such as typewriters and refrigerators, interior decorative materials such as tatami mats and wallpaper. Those. the antimycotic agent is mixed into the raw material at the production stage of the above utensils and mixed or, with the addition of an auxiliary substance such as a surfactant, if necessary with a solvent, applied to the surface of the product by application or spray. The liquid or spray formulation can similarly be applied to sanitary compartments, toilet seats, bathroom disinfectants and anti-mold agents. Additionally, they can be used as sanitary products, such as disinfectant cleaner or wet wipes, by impregnating the agent in liquid form onto an absorbent surface such as paper or fabric.

In a preferred embodiment, the disinfectant composition according to the present invention contains a combination of at least two compounds selected from the group consisting of

8MSOH/8MSOOH; 8ASOH/8ASOOH;

8ESOH/8ESOOH; 8CSOH/3MSOOH; 8CSOH/7MSOH; 8CSOH/3MSOH;

- 13 044362

8MSOOH/6MSOH; 8MSOH/3MSOOH; 8MSOH/7MSOH; 8MSOH/6MSOH;

8CSOH/8MSOH; 8CSOH/3MSOOH; 8MSOH/9MSOOH; 8CSOH/7MSOH;

8ESOH/7MSOH; 8ESOH/3MSOH; 8MSOOH/7MSOH; 8MSOOH/9MSOH;

8MSOOH/6MSOH; 8CSOH/7MSOH; 8MSOOH/3MSOH; 8MSOOH/8MSOON or 8MSOOH/3MSOOH.

Even more preferably, the disinfectant composition of the present invention contains at least two compounds in combination, which are a mixture of 7-methylsulfonylheptyl and (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2 or a mixture of 8-methylsulfonyloctyl isothiocyanate and 8-methylsulfinyl octioisothiocyanate .

Advantageously, the composition of the present invention can also be used to treat or prevent a microbial infection selected from the list consisting of bacterial, fungal, yeast and/or viral infection.

According to one embodiment of the present invention, the bacterial infection includes a gram-positive or gram-negative infection.

Specifically, the bacterial or yeast infection is an infection of Pseudomonas aeruginosa, Escherichia coli, S. aureus, S. epidermis, Klebsiellae pneumoniae, Acinetobacter baumannii, B. subtilis, E. aerogenes, C. freundii, Staphylococcus spp, Streptococcus spp, Enterococcus spp , Proteus spp, Candida spp, Apophysomyces spp, Aspergillus, Mucor spp., Porphymonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythensis or Aggregatibacter actinomycetemcomitans.

Another object of the present invention is to provide a composition containing a combination of at least two compounds selected from the compounds of general formula I

X U s //

where Z represents nitrile (-CN), thiocyanate (-SCN) or isothiocyanate (-N=C=S);

X, Y are independently of each other a lone pair or O, provided that at least one of the two X or Y is O;

R _b R ₂ independently represent H, saturated linear, cyclic or branched (C ₁ -C ₆ )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6 -aryl;

B represents a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6- aryl;

provided that at least one of A, B or C is different from a covalent bond, and characterized in that at least two compounds in combination contain

1) at least one Z group representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or,

3) at least different lengths of the carbon chain of the fragment -A-B-C-, for use in a method for preventing or treating mycoses in humans or animals.

Preferably the composition is a pharmaceutical composition.

The pharmaceutical composition according to the present invention can be used for the preparation of a medicinal product suitable for the therapeutic or prophylactic treatment of fungal diseases of humans or animals, such as, for example, mycoses, chytrid infections (Bd; Bs), dermatoses, trichophytic diseases and candidiasis, or diseases caused by Aspergillus spp., for example Aspergillus fumigatus.

In one embodiment of the present invention, mycosis is caused by infections with Candida albicans or Trichophyton rubrum.

In particular, the antifungal agent of the present invention has an excellent antimicrobial effect against Candida albicans, which is considered one of the yeasts that causes denture-associated stomatitis, so when mixed with an oral hygiene agent in a traditional way, the infection can be easily prevented and treated caused by Candida.

Moreover, the antifungal agent of the present invention can be used in combination with other antimicrobial agents such as alcohol, spice ingredients such as sage and rosemary, organic acids such as citric acid, lactic acid, acetic acid and the like.

In a preferred embodiment, the antifungal agent of the present invention for preventing or treating mycosis in a human or animal contains at least two

- 14 044362 units selected from the group consisting of

8MSOH/8MSOOH; 8ASOH/8ASOOH; 8ESOH/8ESOOH; 8CSOH/3MSOOH;

8CSOH/7MSOH; 8CSOH/3MSOH; 8MSOOH/6MSOH; 8MSOH/3MSOOH;

8MSOH/7MSOH; 8MSOH/6MSOH; 8CSOH/8MSOH; 8CSOH/3MSOOH;

8MSOH/9MSOOH; 8CSOH/7MSOH; 8ESOH/7MSOH; 8ESOH/3MSOH;

8MSOOH/7MSOH; 8MSOOH/9MSOH; 8MSOOH/6MSOH; 8CSOH/7MSOH;

8MSOOH/3MSOH; 8MSOOH/8MSOON or 8MSOOH/3MSOOH.

More preferably, the at least two compounds in combination for use in a method of preventing or treating mycosis in a human or animal are a mixture of 7-methylsulfonylheptyl isothiocyanate and (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene or a mixture of 8- methylsulfonyloctylisothiocyanate and 8-methylsulfonyloctioisothiocyanate.

Another object of the present invention is to provide a fungicidal composition containing a combination of at least two compounds selected from the compounds of general formula I

where Z represents nitrile (-CN), thiocyanate (-SCN) or isothiocyanate (-N=C=S);

X, Y are independently of each other a lone pair or O, provided that at least one of the two X or Y is O;

R _b R2 are independently H, saturated linear, cyclic or branched (C ₁ -C ₆ )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1-C ₈ )alkyl, C ₆ aryl;

B is a covalent bond, saturated or unsaturated linear, cyclic, spirocyclic, bicyclic, polycyclic or branched (C1- _C8 )alkyl, _C6 aryl, provided that at least one of A, B or C is different from the covalent bond , and characterized in that at least two compounds in combination contain

1) at least one Z group representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or

3) at least different lengths of the carbon chain of the fragment -A-B-C-.

Preferably, the fungicidal composition contains a combination of at least two compounds selected from compounds of general formula I, where

Z is isothiocyanate (-N=C=S);

X, Y represent independently of each other O;

R1, R2 are, independently of each other, H, saturated linear or cyclic (C1- _C6 )alkyl;

A, C represent, independently of each other, a covalent bond, saturated or unsaturated linear or cyclic (C1-C ₈ )alkyl, provided that at least one of A or C is different from the covalent bond;

B represents a covalent bond, where the length of the carbon chain of the fragment -A-B-C- is from C ₃ to C ₉ .

In a specific embodiment, the fungicidal composition of the present invention contains a combination of at least two isothiocyanate compounds, preferably said at least two isothiocyanate compounds have at least two different oxidation states of the sulfur atom and/or at least different carbon chain lengths of the -A- moiety. B-C-, preferably the component from C3 to C9.

More preferably, the fungicidal composition of the present invention contains a combination of at least two compounds selected from a list including

- 15 044362

- 16 044362

wherein at least two compounds in combination contain

1) at least one group Z representing an isothiocyanate; and/or

2) at least two different oxidation states of the sulfur atom; and/or

3) at least different lengths of the carbon chain of the fragment -A-B-C-, where the length of the carbon chain of the fragment -A-B-C- ranges from C ₃ to C ₉ .

Preferably, at least two compounds in the combination of the fungicidal composition according to the present invention are selected from the group consisting of

8MSOH/8MSOOH; 8ASOH/8ASOOH; 8ESOH/8ESOOH; 8CSOH/3MSOOH;

8CSOH/7MSOH; 8CSOH/3MSOH; 8MSOOH/6MSOH; 8MSOH/3MSOOH;

8MSOH/7MSOH; 8MSOH/6MSOH; 8CSOH/8MSOH; 8CSOH/3MSOOH;

8MSOH/9MSOOH; 8CSOH/7MSOH; 8ESOH/7MSOH; 8ESOH/3MSOH;

8MSOOH/7MSOH; 8MSOOH/9MSOH; 8MSOOH/6MSOH; 8CSOH/7MSOH;

8MSOOH/3MSOH; 8MSOOH/8MSOON or 8MSOOH/3MSOOH.

Even more preferably, the at least two compounds in the combination of the fungicidal composition are a mixture of 7-methylsulfonylheptyl isothiocyanate and (E)-1-isothiocyanato8-(methylsulfinyl)oct-2-ene or a mixture of 8-methylsulfonyloctylisothiocyanate and 8-methylsulfinylocthioisothiocyanate.

In particular, the fungicidal composition of the present invention has shown effectiveness against fungal plant pathogens selected from the phyla including basidiomycetes, zygomycetes, oomycetes or ascomycetes.

For example, the fungicidal composition of the present invention has shown particular effectiveness against the fungal plant pathogens Botrytis cinerea, Colletotrichum graminicola, Fusarium oxysporum, Sclerotiana sclerotiorum, Verticillium dahlia, Mycospharella gramincola and Sphacelotheca reliana.

In one embodiment of the present invention, the fungicidal composition of the present invention is used in combination with suitable carriers or diluents, for example, in a spray.

The fungicidal composition of the present invention will often be concentrated formulations that can be diluted with water or other liquid for application. In some embodiments, the fungicidal composition may also be formulated as particulate or granular formulations that are sprayed or applied without further processing.

Preferably, carriers or diluents for use in the present invention are phytologically acceptable.

- 17 044362

As used herein, the term phytologically acceptable composition refers to compositions, diluents, excipients and/or carriers that are generally suitable for use on any part of a plant during any part of its life cycle, including without limitation seeds, seedlings, plant cells, plants or flowers . The compositions can be prepared according to techniques, methods and formulas conventional in the field of agriculture. By following the teachings of the present invention, one skilled in the field of agriculture and/or chemistry will easily be able to prepare the desired compositions. Most commonly, the fungicidal composition of the present invention may be formulated to be stored and/or applied as aqueous or non-aqueous suspensions or emulsions, prepared in pure form or from concentrated formulations. Water-soluble, water-suspendable or emulsifiable compositions can also be converted or formulated into solids (eg, wettable powders), which can then be diluted to form a finished composition. In some formulations, the fungicidal compositions of the present invention may also be provided in a growth medium, such as in vitro growth media for plants or other cell types, in laboratory plant growth media, in soil, or for spraying onto seeds, seedlings, roots, stems , petioles, leaves, flowers or the whole plant.

These phytologically acceptable compositions are prepared in a known manner, for example by mixing the fungicidal composition according to the present invention with excipients, i.e. liquid solvents, liquefied gases under pressure and/or solid carriers, optionally using surfactants, i.e. emulsifiers and/or dispersants and/or foaming agents. If water is used as filler, it is also possible, for example, to use organic solvents as auxiliary solvents. Substantially suitable liquid solvents include aromatics such as xylene, toluene or alkyl naphthalenes, chlorinated aromatic or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins such as petroleum fractions, alcohols such as butanol or glycol, or their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, highly polar solvents such as dimethylformamide and dimethyl sulfoxide, and water. Liquefied gaseous excipients or carriers are understood as liquids that are gaseous at room temperature and atmospheric pressure, such as aerosol propellants such as butane, propane, nitrogen and carbon dioxide. Suitable solid carriers are, for example, ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals such as finely divided silica, alumina and silicates. Suitable solid carriers for the granules are, for example, crushed and fractionated natural minerals such as calcite, marble, pumice, sepiolite and dolomite, or synthetic granules of inorganic and organic powders such as sawdust, coconut husks, corn cobs and tobacco stalks. Suitable emulsifiers and/or foaming agents are, for example, nonionic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkyl sulfonates, alkyl sulfates, arylsulfonates, as well as protein hydrolysates. Suitable dispersants are: for example lignin sulphite wastewater or methylcellulose.

The fungicidal composition of the present invention can be used in various forms, such as aerosol dispenser, capsule suspension, cold aerosol concentrate, spray powder, emulsifiable concentrate, oil-in-water emulsion, encapsulated granules, fine granules, bulk treatment concentrate seeds, gas (pressurized), gas-releasing product, granules, hot spray concentrate, macrogranules, microgranules, oil-dispersible powder, oil-miscible bulk concentrate, oil-miscible liquid, paste, plant stick, dry seed treatment powder , pesticide coated seed, soluble concentrate, soluble powder, seed treatment solution, suspension concentrate (bulk concentrate), ultra low volume liquid (ULV), ultra low volume suspension (ULV), water dispersible granules or tablets, water dispersible powder for suspension treatments, water soluble granules or tablets, water soluble seed treatment powder and wettable powder. Such compositions include not only those ready to be applied to the plant or seed being treated using a suitable device, such as a spray or duster, but also concentrated commercial compositions that must be diluted before application to the crop.

In a preferred embodiment of the present invention, the composition can be specifically applied to fruits and vegetables in storage using an ultrasonic aerosol sprayer. An ultrasonic aerosol atomizer is a device that uses ultrasonic waves to break water into small droplets (<10 microns) and spray them into the air as a dense, cool mist (i.e. not produced by boiling water). Examples of ultrasonic aerosol nebulizers and systems are described, among others, in the following US patents: US Patent No. 4,042,016; US Patent No. 4058253; US Patent No. 4118945; US Patent No. 4564375; US Patent No. 4667465;

- 18 044362 US patent No. 4702074; US Patent No. 4,731,990; US Patent No. 4731998; US Patent No. 4773846; US Patent No. 5454518; US Pat. No. 6,854,661 Typically, an ultrasonic aerosol atomizer includes a generally cylindrical body having an axial channel exiting at a front surface of the body; gas supply and liquid supply connected to the channel; at least a portion of the front surface has a curved convex contour, the front surface has a flat central annular portion surrounding the exit of the channel; and a resonator spaced and located opposite the channel exit. Such devices are widely used to control humidity levels in greenhouses, to deliver nutrients to the plant in aeroponic crops, or to create optimal humidity levels in homes.

The authors showed that this technique can be used to apply products used to prolong the freshness of fruits and vegetables in storage, such as natural fungicides. This technique can effectively treat fruits and vegetables that are not accessible to spray or other application treatments due to packaging (i.e. they are difficult to spray directly because the fruit and vegetables are stored, for example, in a container, or because spraying may damage fruits and vegetables).

Interestingly, the authors conducted an effectiveness test on fruits and vegetables in a 30 m ³ storage facility using industrial ultrasonic humidifiers MHS15 (http://www.mainlandmart.com/humidify.html), generating 15 kg of mist per hour consisting of droplets size between 1-10 microns, average 5 microns. The composition according to the present invention, presented in powder form, was added to a water container in an aerosol spray at a dosage of 400-600 μM depending on the plant species being tested. The powder was dissolved in water and applied to the fruit in the form of cold steam. The authors confirmed equal distribution of the product across all treated crops and easy access to previously unattainable parts of the package.

The fungicidal composition of the present invention may also be mixed with one or more insecticides, pesticides, attractants, sterilants, bactericides, acaricides, nematicides, other fungicides, growth regulators, herbicides, fertilizers, antidotes, chemical signal agents, odor control agents or other compounds in biological activity. The resulting mixtures usually have an extended spectrum of action. Mixtures with other fungicidal compounds are especially beneficial.

The fungicidal composition of the present invention containing a mixture with a bactericidal compound may also be particularly advantageous. Examples of suitable bactericidal mixture components may be selected from the following: bronopol, dichlorophen, nitrapyrin, nickel dimethyldithiocarbamate, kasugamycin, octylinone, furancarboxylic acid, oxytetracycline, probenazone, streptomycin, teclophthalam, copper sulfate and other copper preparations.

The dose of the fungicidal composition typically applied in the treatment of the present invention is typically and advantageously from 10 to 800 g/ha, preferably from 50 to 300 g/ha for field applications. The dose of the fungicidal composition applied is usually and advantageously from 2 to 200 g per 100 kg of seed, preferably from 3 to 150 g per 100 kg of seed, in the case of seed treatment.

It is understood that these dosages are given as illustrative examples of the treatment method according to the present invention. One skilled in the art will understand how to adapt the doses used, particularly according to the nature of the plant or crop being treated.

Glucosinolates are sulfur- and nitrogen-containing secondary metabolites found in the Caperaceae order, which includes the agronomically important Brassica family. Approximately 200 glucosinolates have been described. All of them can be divided into three main groups based on their amino acid precursors: aliphatic, indole and aromatic glucosinolates. The role of these metabolites in plants is not fully understood, but they have been shown to be involved in defense responses against herbivores, some fungi (Schreiner and Koide, 1993) and bacterial pathogens (Dufour et al., 2015). Glucosinolates are not active in their intact form, but when tissue is damaged during, for example, a herbivore attack, they are broken down by the enzyme myrosinase to form three main bioactive compounds: nitriles, thiocyanates and isothiocyanates. All three active compounds have been shown to be toxic to a large group of plant pathogens (Lambrix et al., 2001). In this study, the authors unexpectedly discovered a new role for aliphatic glucosinolates, specifically methylsulfonyl and methylsulfinyl linear alkyl compounds ( _CH38 (O) ₂ ( _CH2 ) _n ), in toxicity to fungal pathogens.

For this purpose, the authors selected Arabidopsis thaliana mutants with changes in cuticle composition and performed fungitoxicity tests using Botrytis cinerea, the second most agronomically important fungal pathogen for which there is currently no effective organic treatment. As the results showed, the following mutants showed strong resistance to B. cinerea: with mutations in fatty acid desaturase (fad2; fad3-2 fad7-2 fad8), cytochromes P450 belonging to the CYP77 family/protective cuticle ridges (sur77 dcr), glycerol acyltransferase -3phosphate/permeable cuticle 1 (gpat6 pec1) and glycerol 3phosphate acyltransferase double knockout (gpat4 gpat8). All selected mutants showed strong resistance to B. cinerea or only

- 19 044362 at the cotyledon stage (fad mutants), or at both stages - seedlings and adults (double mutants sur77 dcr; gpat6 pec1; gpat4 gpat8). Various approaches, such as molecular biology, microscopy and biochemistry, have been used to understand what causes fungal resistance in these plants. Test for currently known mechanisms involved in resistance to B. cinerea, i.e. hormonal changes, higher ROS concentrations and surface modification cannot fully explain resistance to B. cinerea. This led the authors to use a surface metabolism approach to identify potential antifungal compounds on the plant surface. Using this technique, the authors were able to identify 2 compounds that are very abundant on the surface of various cuticle mutations: 7-methylsulfonylheptylglucosinolate (on the cotyledons of fad2 mutants), as far as the authors know, not previously identified on Arabidopsis plants, and 8-methylsulfonyloctylglucosinolate (on the cotyledons of fad3- mutants). 2 fad7-2 fad8 and gpat6 pec1 mutant leaves). Careful consideration of the overall structure of the first identified compound led the authors to the identification of 2 major groups of aliphatic glucosinolates and their mixtures strongly implicated in fungal resistance.

One skilled in the art will appreciate that the described invention is subject to variations and modifications beyond those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics of the invention. The invention also includes all of the steps, features, compositions and compounds specified or referred to herein, separately or together, and any and all combinations of any two or more of these steps or features. This description is intended to be illustrative and not restrictive in all respects, the scope of the present invention is determined by the appended claims, and all changes made within the meanings and ranges of equivalence are considered to be within the scope stated.

The above description will be more clear with reference to the following examples. Such examples are examples of methods for practicing the present invention and are not intended to limit the scope of the invention.

Examples

Materials and methods.

Arabidopsis thaliana accession Columbia-0 (Col-0) was used as the wild type (WT). The following alleles in the Col-0 background were obtained from the Arabidopsis Biological Resource Center (ABRC): fad2-1 (CS8041), fad2-3 (SK18137), fad2-1 fad6 and fad2-3 fad6 were obtained by crosses of individual mutants in this work. The gpat4 gpat8 double mutant was kindly provided by Frederic Beisson, the fad3-2 fad7-2 fad8 seeds were obtained from John Browse, the sur77 dcr and gpat6pec1 double mutants were kindly provided by Dr. Christiane Nawrath. Before planting, the seeds were stratified for 2 days at 40°C and then grown at 21°C under 100 μE ^m'2 -s'1 light with photoperiods depending on the application (seedling experiments: 14 h light, 10 h dark (long day); on soil for seed germination: 24 hours of light, continuous day).

Seedlings for experiments were grown on half-strength Murashige-Skoog solid medium (0.5xMS, 2.15 g/L, pH 5.7; Duchefa Biochemie, Haarlem, The Nederlands) supplemented with 0.5 g/L MES hydrate (Sigma, Buchs, Switzerland) and 0.7% plant agar (Sigma, Buchs, Switzerland). After stratification, the seeds were placed on a nylon mesh (mesh size 200 μm; product number AH03444, Lanz-Anliker AG, Rohrbach, Switzerland) to provide firm support for uniform seed germination and growth. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich.

In vivo fungal biological test with B. cinerea.

In this work, two strains of B. cinerea were used: BMM and B05.10, provided by Dr. Christiane Nawrath and Prof. Michael Hahn respectively. The fungus was grown on potato dextrose agar (PDA) for 10 days. Conidia were filtered and diluted to 5x105 spores/µl in 1/2 strength potato dextrose broth (PDB). 2 μl of the resulting suspension was applied to 7-day-old Arabidopsis cotyledons grown in Petri dishes. Infected seedlings were incubated for 48 h under long-day conditions.

In vitro fungal biological test.

Methylsulfonyl and methylsulfinyl nitrile and thiocyanates were obtained from SpiroChem (Basel, Switzerland), methylsulfonyl and methylsulfinyl isothiocyanates from SpiroChem and LKT LABS (Minnesota, United States of America). All obtained compounds were dissolved in dimethyl sulfoxide (DMSO). The fungal strains used in the in vitro fungal biological test were obtained from the Agroscope mycotheca (www.mycoscope.bcis.ch).

Pure cultures of various species of fungal plant pathogens were inoculated into KDA medium supplemented with aliphatic glucosinolate by-products pre-dissolved at various concentrations (e.g., 10, 100, 250, 400, 550, 700, 850, 1100, 1400 μM) in KDA and placed into 24-well plates (VWR International, Dietikon, Switzerland). Control samples were treated similarly with DMSO alone. 2 mm agar plug (Dufour et al., 2015) with pre-grown

- 20 044362 fungal cultures were placed in each well, and 3 biological replicates were performed for each concentration. The plates were incubated for one week in a phytotron (80% relative humidity, constant temperature 23°C, with alternating cycles of 16 hours day and 8 hours night). Mycelial growth was measured after 7 days using ImageJ (http://imagej.net/Welcome), _EC50 was assessed as described in

Schnee et al., 2013.

Metabolomics analyses.

To conduct this experiment, we used ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS) on an Acquity ultra-high performance liquid chromatograph (UPLC) and a Synapt G2 quadrupole time-of-flight mass spectrometer (QTOF; Waters). The Acquity UPLC was equipped with a C ₁₈ ethylene bridged hybrid (EBH) column (50 x 2.1 mm, 1.7 μm; Waters) with a flow rate of 400 μL/min and maintained at 25°C. Gradient elution was used with mobile phase A 0.05% formic acid in water and mobile phase B 0.05% formic acid in acetonitrile: 0.0-7.0 min 2-100% B, 7.0-9.0 min 100% B, 9.0-11.0 min 2% B. The sample injection volume was 2.5 μl. The KVP operated in negative electrospray mode using MS ^E mode (simultaneous recording of the entire spectrum and all fragment ions). The mass spectrometer parameters were as follows: mass range 85–1200 Da, scan time 0.2 s, source temperature 120°C, capillary voltage -2.0 kV, cone voltage -25 V, extraction cone -4.5 V, flow and desolvation gas temperature 800 l/h and 400°C, respectively, gas flow through the cone 20 l/h, collision energy 4 eV (low-energy detection function) and 10-35 eV (high-energy detection function), collision gas (argon) 7x10 -3 mbar. A solution of 400 ng/mL synthetic leucine-enkephalin peptide in water:acetonitrile:formic acid (50:50:0.1) was fed into the mass spectrometer continuously as an internal standard to ensure accurate mass measurements. Data were recorded using Masslynx v.4.1 (Waters). Marker detection was carried out using Markerlynx XS (Waters) with the following parameters: initial and final retention time 0.0 and 9.0 min, mass range 85-1200 Da, mass window 0.02 Da, retention time window 0.06 min, intensity threshold of 500 events, automatic calculation of peak width and noise of the zero line between peaks, isotope elimination function applied. Data were mean centered and Pareto scaled before applying principal component analysis. Markers of interest were tentatively identified based on determination of their molecular formula and fragments obtained by collision-induced dissociation.

Example 1.

7-Methylsulfonylheptylglucosinolate was identified on the surface of the fatty acid desaturase 2 (fad2-3) mutant of Arabidopsis thaliana, which showed strong resistance to the necrotrophic fungal pathogen Botrytis cinerea at the cotyledon stage (see drawing). Confirmation of the antifungal activity of 7-methylsulfonylheptyl isothiocyanate (7MSOOH, the only commercially available by-product of 7-methylsulfonylheptylglucosinolate) against B. cinerea and other fungal pathogens was carried out in vitro by direct application of the isothiocyanate to potato dextrose agar at various dosages. The results obtained did not reveal the indicated compound to be fungitoxic to Botrytis cinerea in the dose range from 10 to 1500 μM. Higher doses were not used due to the possible toxic effects of this compound. However, biological test data on other fungal pathogens have determined that 7-methylsulfonylheptyl isothiocyanate is fungitoxic to some of the fungal species shown in Table 1. 1.

Table 1

Antifungal properties of 7-methylsulfonylheptyl isothiocyanate revealed in an in vitro antifungal test

# Types of mushrooms Host organism ED50 (µM) 1 Septoria tritici Wheat 350 2 Colletotrichum dematium Various types of plants 650 3 Serpula lacrymans Wood 150 4 Hemenoscyphus fraxineus Ash 770

Conclusion. 7-Methylsulfonylheptyl isothiocyanate showed a narrow spectrum of fungitoxic activity against several fungal plant pathogens and was inactive against a wide range of fungal plant pathogens such as Botrytis cinerea (gray mold) or Fusarium oxysporum. However, the authors unexpectedly found that the combination of glucosinolate byproducts resulted in potent fungitoxic effects on a wide range of agronomically important fungal pathogens (43 species, see table above for list of species).

Example 2.

Study of other natural glucosinolate byproducts and their combinations led the authors to identify mixtures of compounds with high antifungal effectiveness. The authors used

- 21 044362 an effective dose that had a quantitative effect on 95% of the population (ED ₉₅ ) at a maximum concentration of 1500 μM to study the fungitoxic effect of the studied compounds. The authors selected two of the most widespread fungal pathogens for toxicity testing: Botrytis cinerea (a pathogen of 1,400 plant species) and Fusarium oxysporum (a pathogen of a wide range of plant species, e.g., tomatoes, bananas). The selection of such fungal pathogens was driven by the lack of any effective organic treatments (less than 60% effective) on the market, creating a need to identify new effective products that farmers can use without risking their health and the environment.

The authors initially tested individual molecules of 8-methylsulfonyloctyl (8MSOOH) and 8-methylsulfonyloctyl (8MSOH) isothiocyanates, which did not show fungitoxic effects on Botrytis cinerea and Fusarium oxysporum. However, when 8-methylsulfonyloctyl (8MSOOH) and 8-methylsulfonyloctyl (8MSOH) isothiocyanates were combined, they became highly toxic to B. cinerea (1400 µM) and F. oxysporum (923.64 µM).

Surprisingly, toxicity tests of 8-methiosulfonyloctylnitrile (8MSOON) and 8-methylsulfonyloctylisothiocyanate (8MSOOH) showed different effects on the two fungal species. The combination of nitrile and isothiocyanates was toxic to B. cinerea at a dose of 850 µM (lower than the combination of two isothiocyanates). However, this combination had a moderate effect on the development of F. oxysporum. For this fungal pathogen, a fungitoxic effect was obtained for the combination 8MSOOH+8MSOH, with an EC _{of 95,923.64} μM.

The combination of three molecules (isothiocyanates and nitriles) did not increase the fungitoxic effect of these molecules and was at the same level as for a single compound, with the most effective combination of 2 molecules. The results for individual molecules and their combinations are presented in table. 2.

table 2

Fungitoxic effects of various glucosinolate by-products (combinations and individual molecules) on Botrytis cinerea and Fusarium oxysporum

ED95 Botrytis cinerea F. oxysporum 8MSOH <1500 <1500 8MSOON <1500 <1500 8MSOOH <1500 <1500 8MSOH+8MSOON <1500 <1500 8MSOOH+8MSOON 850 <1500 8MSOOH+8MSOH 1400 923.64 8MSOOH+8MSOON+8MSOH 850 1125.08

Conclusion. Methylsulfonyl and methylsulfinyl glucosinolate by-products, when used alone, did not show fungitoxic effects on the most common fungal plant pathogens at tested concentrations. However, combinations of methylsulfonylisothiocyanates with methylsulfonylnitriles, -thiocyanates or methylsulfonylisothiocyanates can lead to fungitoxic effects.

Example 3.

Screening of other aliphatic isothiocyanates led the authors to test combinations of 6-methylsulfonylhexyl isothiocyanates (6MSOOH) with 6-methylsulfinylhexyl (6MSOH), 4-methylsulfinylbutyl (4MSOH), 8-methylsulfinyloctyl (8MSOH) and 8-methylsulfonyloctyl (8MSOOH) isothiocyanates for their fungitoxic effects on Fusarium gramineum (a pathogen of barley and wheat) and Phytophthora cactorum (a pathogen of 200 plant species).

The authors found that creating a combination with weakly active compounds could unexpectedly lead to increased fungitoxicity against a large number of fungal pathogens. For example, both 6MSOOH and 6MSOH are not fungistatic for Fusarium gramineum at 1500 µM. However, when used in a mixture (combination of 6MSOOH and 6MSOH) they become highly fungitoxic with an effective dose of 116.8 µM having a quantitative effect on 50% of the population ( _ED50 ). Similar effects were observed for other combinations for both mushroom species.

- 22 044362

Table 3

Fungitoxic effect of isothiocyanates in various combinations on P. cactorum and F. gramineum

Conclusion. A combination of methylsulfonyl and methylsulfinyl isothiocyanates with varying carbon chain lengths has shown potent fungitoxic properties against a wide range of fungal pathogens. The results have led to the development of new natural organic products that can be used in agriculture, for example to prevent pathogen contamination. The development of new product combinations is particularly useful in reducing the use of chemical pesticides as well as their dosages. Moreover, due to their high efficiency, these new combinations are very useful for promoting the development of biofarming.

Example 4. Chemical synthesis.

NaSMe about O

VN ₃

RuCI ₃ , NalO ₄

ACN, AcOEt

Methyl(oct-7-en-1-yl)sulfane (2). To a suspension of sodium thiomethoxide (3.23 g, 46.0 mmol) in dimethylformamide (85 ml) was added a solution of 8-bromoct-1-ene (8 g, 41.9 mmol) in dimethylformamide (195 ml) at 0°C and Stir the reaction mixture at room temperature for 14 hours. Dilute the reaction mixture with saturated ammonium chloride solution and then extract with ether (3 times), dry the combined organic layers over sodium sulfate, filter and concentrate under reduced pressure to give methyl (oct-7- en-1-yl)sulfan (7 g, 44 mmol), which was used in the next step without further purification.

^1H NMR (300 MHz, chloroform-d) δ 1.26-1.45 (m, 6H), 1.50-1.67 (m, 2H), 2.00-2.08 (m, 2H) , 2.09 (s, 3H), 2.44-2.54 (m, 2H), 4.84-5.06 (m, 2H), 5.68-5.90 (m, 1H).

7-(methylsulfonyl)heptanoic acid (3). To an ice-cold solution of methyl(oct-7-en-1-yl)sulfane (2) in acetonitrile (139 ml) and ethyl acetate (139 ml) was added ruthenium trichloride trihydrate (197 mg, 0.834 mmol), followed by water (139 ml) and stirred the resulting mixture using a mechanical stirrer. Sodium periodate (71.3 g, 334 mmol) was added to this solution with stirring in portions over 10 minutes (caution may cause explosion). After addition was complete, the mixture was stirred overnight at room temperature. The layers were separated and the organic layer was washed with sat. NaHCO ₃ for now

- 23 044362 wash solutions will not show an alkaline pH. All collected aqueous layers were washed with ethyl acetate and then acidified using 6 N HCl, followed by extraction with ethyl acetate (3 times). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give 7-(methylsulfonyl)heptanoic acid (3) (7.25 g, 34.8 mmol) in 83% yield. The analytical sample was prepared by trituration of the crude product with ether, filtering and air drying, and the remaining material was used in the next step without further purification.

1H NMR (300 MHz, chloroform-d) δ 1.34-1.56 (m, 4H), 1.66 (m, 2H), 1.79-1.93 (m, 2H), 2.37 ( t, J=7.3 Hz, 2H), 2.90 (s, 3H), 2.95-3.07 (m, 2H).

OO but ^x s ^z

7-(methylsulfonyl)heptan-1-ol (4). To a solution of 7-(methylsulfonyl)heptanoic acid (3) (1 g, 4.80 mmol) in tetrahydrofuran (60 ml) was added a 1 M solution of borane-tetrahydrofuran complex (14.40 ml, 14.40 mmol) in THF dropwise at 0°C in an inert atmosphere and stirred the reaction mixture at room temperature for 3 hours. The reaction mixture was filled with a saturated ammonium chloride solution, then the layers were separated and the aqueous layer was extracted with ethyl acetate (3 times). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified using PC (Biotage, KP Sil-25g, DCM, ethyl acetate, 0-100%), yielding 7-(methylsulfonyl)heptan-1-ol (4) (0.72 g, 3.71 mmol, yield 77%) as a white solid.

1H NMR (300 MHz, chloroform-d) δ 1.35-1.51 (m, 6H), 1.57 (m, 2H), 1.86 (m, 2H), 2.90 (s, 3H) , 2.96-3.07 (m, 2H), 3.65 (td, J=6.5, 1.0 Hz, 2H).

OO ^MsO \z^A\ZX/ ^s \

7-(methylsulfonyl)heptylmethanesulfonate (5). To a solution of 7-(methylsulfonyl)heptan-1-ol (4) (0.7 g, 3.60 mmol) in dichloromethane (36.0 mL) was added triethylamine (1.023 mL, 7.21 mmol) and the mixture was cooled to 0 °C. Methanesulfonyl chloride (0.349 mL, 4.50 mmol) was added to this mixture, and the reaction mixture was then stirred at room temperature for 14 hours. The reaction mixture was washed with 1 M HCl and the aqueous layer was extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting crude product was purified using PC (Biotage, KP Sil-25g, DCM - ethyl acetate, 0-40%), yielding 7-(methylsulfonyl)heptylmethanesulfonate (5) (940 mg, 3.45 mmol, 96% yield) as white solid.

1H NMR (300 MHz, chloroform-d) δ 1.34-1.53 (m, 6H), 1.68-1.94 (m, 4H), 2.90 (s, 3H), 2.96- 3.06 (m, 5H), 4.18-4.27 (m, 2H).

OO

1-(methylsulfonyl)-7-thiocyanatoheptane (6). To a solution of 7-(methylsulfonyl)heptylmethanesulfonate (5) (0.2 g, 0.734 mmol) in acetone (5 ml) was added potassium thiocyanate (0.714 g, 7.34 mmol) and the resulting mixture was stirred at 60 °C in a sealed test tube in for 18 hours. Then the cooled mixture was filtered through a porous glass filter and the resulting residue was washed several times with ether. The combined filtrates were evaporated to dryness and then purified by PC (Biotage, KP Sil-25g, DCM, 0-40% ethyl acetate), yielding 1-(methylsulfonyl)-7-thiocyanatoheptane (6) (156 mg, 0.663 mmol, 90% yield %) in the form of a colorless oil.

1H NMR (300 MHz, chloroform-d) δ 1.35-1.55 (m, 6H), 1.84 (m, 4H), 2.91 (s, 3H), 2.94 (t, J= 7.2 Hz, 2H), 2.98-3.05 (m, 2H).

OO

NC

8-(methylsulfonyl)octanenitrile (7). To a solution of 7-(methylsulfonyl)heptylmethanesulfonate (5) (0.2 g, 0.734 mmol) in dimethyl sulfoxide (5 ml) was added sodium cyanide (0.180 g, 3.67 mmol) and the mixture was stirred at 60 °C in a sealed tube for 18 hours. The cooled reaction mixture was diluted with ethyl acetate and washed with saturated ammonium chloride solution. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting crude product was purified using PC (Biotage, KP Sil-25g, DCM, ethyl acetate, 0-40%), yielding 8-(methylsulfonyl)octanenitrile (7) (0.136 g, 0.669 mmol, 91% yield) as a colorless oil .

1H NMR (300 MHz, chloroform-d) δ 1.33-1.55 (m, 6H), 1.60-1.74 (m, 2H), 1.79-1.94 (m, 2H), 2.35 (td, J=7.0, 0.6 Hz, 2H), 2.90 (s, 3H), 2.96-3.05 (m, 2H).

- 24 044362

Synthesis of 5MSOOH.

With

ABOUT

Conditions:

A. phthalimide, N-bsnzyl-MN-diethylethanamine chloride. K2CO3, acetone, 24 °C, 2 days, 38%;

b. NaSMe, DMF, 0-RT °C, 16 h; quantitative yield;

With. mCPBA, DCM, 0 °C, 14 h, quantitative yield;

d. NH ₂ NH ₂ .H ₂ O, MeOH, 24 °C, 16 h; 92%;

e. thiophosgene, NaOH, СНС1 ₃ , 0 °C, 2 h, 59% 2-(5-bromopentyl)isoindoline-1,3-dione (SVB-18-025).

To a solution of phthalimide (3 g, 20.39 mmol) in acetone (102 ml) was added N-benzyl-N,Ndiethylethanamine chloride (0.511 g, 2.243 mmol), K ₂ CO ₃ (8.45 g, 61.2 mmol) and 1,5-dibromopentane (14.07 g, 61.2 mmol) and stirred the reaction mixture at RT for 14 hours. The reaction mixture was evaporated to dryness and the resulting residue was resuspended in water, extracted with DCM (3 times), and the combined organic layers were dried. over sodium sulfate, filtered and concentrated under reduced pressure to give the rough product. It was purified using PC (SiO ₂ , hexane, ethyl acetate, 0-40%) to give 2-(5-bromopentyl)isoindoline-1,3-dione (2.3 g, 7.77 mmol, 38.1% yield ) in the form of a colorless oil.

1H NMR (300 MHz, chloroform-d) δ 7.89-7.80 (m, 2H), 7.77-7.66 (m, 2H), 3.75-3.63 (m, 2H), 3.44-3.31 (m, 2H), 1.99-1.84 (m, 2H), 1.79-1.63 (m, 2H), 1.60-1.42 (m, 2H ).

2-(5-(methylthio)pentyl)isoindoline-1,3-dione (SVB-18-033).

To a suspension of NaSMe (0.833 g, 11.89 mmol) in DMF (21.83 ml) was added a solution of 2-(5-bromopentyl)isoindoline-1,3-dione (3.2 g, 10.80 mmol) in DMF ( 50.2 ml) at 0°C and stirred the reaction mixture at RT for 14 hours. The reaction mixture was diluted with a solution of NH ₄ Cl and then extracted with ether (3 times), the combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure, obtaining a rough product. It was used in the next step without further purification.

^1H NMR (300 MHz, chloroform-d) δ 7.90-7.80 (m, 2H), 7.78-7.66 (m, 2H), 3.74-3.63 (m, 2H) , 2.54-2.42 (m, 2H), 2.08 (s, 3H), 1.80-1.57 (m, 5H), 1.52-1.37 (m, 2H).

2-(5-(methylsulfonyl)pentyl)isoindoline-1,3-dione (SVB-18-038).

To a solution of 2-(5-(methylthio)pentyl)isoindoline-1,3-dione (2.8 g, 10.63 mmol) in DCM (53.2 ml) at 0°C, m-CPBA (6, 55 g, 26.6 mmol) and stirred the reaction mixture at RT for 14 hours. 100 ml of a 1:1 diluted saturated solution of NaHCO ₃ and a 10% solution of sodium thiosulfate were added to the reaction mixture and stirred for 1 hour, the layers were separated and extracted aqueous layer of DCM (3 times), the combined organic layers were dried over sodium sulfate, filtered and concentrated to give 2-(5-(methylsulfonyl)pentyl)isoindoline-1,3-dione (3.1 g, 10.50 mmol, yield 99%) as a white solid.

¹ H NMR (300 MHz, chloroform-d) δ 7.89-7.82 (m, 2H), 7.77-7.68 (m, 2H), 3.71 (t, J=7.0 Hz , 2H), 3.07-2.95 (m, 3H), 2.93-2.87 (m, 4H), 1.99-1.86 (m, 2H), 1.82-1.67 (m, 2H), 1.61-1.43 (m, 2H).

5-(methylsulfonyl)pentan-1-amine (SVB-18-042).

- 25 044362

To a solution of 2-(5-(methylsulfonyl)pentyl)isoindoline-1,3-dione (3.1 g, 10.50 mmol) in methanol (52.5 mL) was added hydrazine hydrate (1.020 mL, 20.99 mmol) and stirred the reaction mixture at RT for 2 hours. The reaction mixture was concentrated in vacuo, then the resulting residue was suspended in 1 M KOH solution, extracted with ethyl acetate (3 times), the combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure , yielding 5-(methylsulfonyl)pentan-1-amine (1.6 g, 9.68 mmol, 92% yield) as a white solid. The resulting material was pure enough to be used in the next reaction.

1-isothiocyanato-5-(methylsulfonyl)pentane (SVB-18-048).

To a solution of 5-(methylsulfonyl)pentan-1-amine (0.25 g, 1.513 mmol) in chloroform (7.56 ml) at 0°C was added thiophosgene (0.150 ml, 1.967 mmol) followed by the addition of 5% aqueous NaOH and the resulting mixture was stirred at 0°C for 2 hours, the reaction mixture was diluted with water, then the layers were separated and the aqueous layer was extracted with DCM (3 times). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. It was purified using PC (SiO ₂ , hexane, ethyl acetate, 0-40%) to give 1-isothiocyanato-5-(methylsulfonyl)pentane (0.185 g, 0.892 mmol, 59.0% yield).

1H NMR (300 MHz, chloroform-d) δ 3.56 (t, J=6.3 Hz, 2H), 3.10-3.00 (m, 2H), 2.93 (d, J=0, 7 Hz, 3H), 1.98-1.86 (m, 2H), 1.83-1.70 (m, 2H), 1.68-1.53 (m, 2H).

Example 5.

Studying natural combinations of isothiocyanates with different carbon chain lengths and sulfinyl or sulfonyl groups led the authors to the identification of highly effective antifungal mixtures of compounds. The authors used an effective dose producing a quantitative effect on 95% of the population (ED ₉₅ ), at a maximum concentration of 1500 μM to study the fungitoxic effect of the analyzed compounds. The authors selected the second most common fungal pathogen for toxicity testing, Botrytis cinerea (a pathogen of 1,400 plant species), due to its worldwide occurrence and the paucity of effective organic products available to control this fungal pathogen.

The authors initially selected 4 compounds that showed fungitoxicity to a wide range of fungal pathogens, such as Phytophthora infestarts, Fusarium gramineum: 5-methylsulfonylpentyl- (5MSOOH), 5-methylsulfinylpentyl- (5MSOH), 8-methylsulfonyloctyl- (8MSOOH) and 8-methylsulfonyloctyl (8MSOH ) isothiocyanates. However, none of these compounds alone showed fungitoxic effects on Botrytis cinerea. To improve the properties against Botrytis, the authors carried out combinations of compounds 5MSOOH, 5MSOH, 8MSOOH and 8MSOH. Surprisingly, only two combinations showed antifungal properties: the first, 8MSOOH with 8MSOH (ED ₉₅ 1400 μM), and the second, 8MSOH with 5MSOOH, which became highly toxic to B. cinerea at a dosage of 550 μM. The results for individual molecules and their combinations are presented in table. 4.

Conclusion. Glucosinolate by-products such as methylsulfonyl and methylsulfinyl isothiocyanates, when used separately, did not show strong fungitoxic effects on the most widespread fungal plant pathogen, Botrytis cinerea, at concentrations tested. However, when combining compounds with different carbon chain lengths and containing sulfonyl or sulfonyl groups, it resulted in a strong fungitoxic effect at low dosages, thereby increasing the fungitoxic effect compared to combinations of molecules with the same carbon chain lengths and differences only in the functional groups ( i.e. sulfinyl and sulfonyl groups).

Example 6.

The study of the fungitoxic properties of individual and aliphatic isothiocyanates in combination led the authors to an in-depth study of the effects of 8-methylsulfonyloctyl (8MSOOH) and 8-methylsulfonyloctyl (8MSOH) isothiocyanates. The authors conducted fungitoxicity studies using: Fusarium gramineum (barley and wheat pathogen), Phytophthora infestans (nightshade pathogen), and Guignardia bidwellii (grape pathogen).

The authors measured the fungitoxic effects caused by single and 8MSOOH molecules and 8MSOH in combination on 50% of the fungal population (ED50). The test results are included in the table. 5.

Table 5

Fungitoxic effect of isothiocyanates in various combinations on P. infestans, F. gramineum and G. bidwellii

ED50 R. infestans F. gramineum G. bidwellii 8MSOH 26.36 292.03 30.38 8MSOOH 350 22.18 463.88 8MSOOH+8MSOH 10 61.73 40.2

Conclusion. The combination of molecules with the same carbon chain lengths of methylsulfonyl and methylsulfin isothiocyanates does not enhance the fungitoxic properties against F. gramineum and G. bidwellii, but was effective against P. infestans.

Example 7. Fungitoxic effects of isothiocyanates in combination on three necrotrophic species of fungi.

The authors tested the fungitoxic effect of isothiocyanates in combination on three necrotrophic fungi, i.e. Alternaria radicina (class Dothideomycetes; order Pleosporaceae; host carrot), Fusarium gramineum (class Sordariomycetes; order Hypocrete; host Cereals) and Plectosphaerella cucumerina (class Sordariomycetes; order Glomerellaceae; host Tomato and Cucurbitaceae). A total of 133 combinations were tested on three types of mushrooms.

Isothiocyanates were obtained from SpiroChem (Basel, Switzerland) and LKT LABS (Minnesota, United States of America). All obtained compounds were dissolved in dimethyl sulfoxide (DMSO). The fungal strains used for the in vitro fungal biological test were obtained from the Agroscope mycotheca (www.mycoscope.bcis.ch).

The fungitoxic effect was tested in 48-well tissue culture plates. In more detail, fungal plant pathogens were inoculated onto KDA supplemented with isothiocyanates pre-dissolved at various concentrations (10, 150, 250, 375, 500, 700, 850 μM) in KDA. A 2 mm agar plug (Dufour et al., 2015) with pre-grown fungal cultures was placed in each well, and 3 biological replicates were used for each concentration. The plates were incubated for one week in a phytotron (80% relative humidity, constant temperature 23°C, with alternating cycles of 16 hours day and 8 hours night). Mycelial growth was measured after 7 days using ImageJ (http://imagej.net/Welcome), ED ₅₀ was assessed as described in Schnee et al., 2013. Synergetic fungitoxic effects were assessed as described in Chou (2006), and used CompuSyn software (www.combosyn.com) to determine the additivity index (AD) for combinations of two molecules and the presence of synergism (AD<1). The results obtained for the best combinations for each type of mushroom are shown in Table. 6, 7 and 8. Shown are the ED50 (in µM) for two molecules alone, for a combination of molecules (always less), and the lowest additivity index for at least one given concentration ranging from 10 to 850 µM. All PAs shown in table. below were less than 0.26, indicating that the molecules in combination were highly synergistic and effective against the fungal species tested.

Additional molecules tested in these combinations are 1-(isothiocyanatomethyl)-3-(4-(methylsulfinyl)butyl)benzene (8ASOH), 1-(isothiocyanatomethyl)-3-(4(methylsulfonyl)butyl)benzene (8ASOOH), (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene (8CSOH), 1-(ethylsulfinyl)-8-isothiocyanatooctane (8ESOH), 1-(ethylsulfonyl)isothiocyanatooctane (8ESOOH), 1-isothiocyanato-3 -(methylsulfinyl)propane (3MSOH), 1-isothiocyanato-3-(methylsulfonyl)propane (3MSOOH), 1-isothiocyanato-9-(methylsulfinyl)nonane (9MSOH) and 1-isothiocyanato-9(methylsulfonyl)nonane (9MSOOH).

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Table 6

Best combinations of molecules against Alternaria Radicina (molecules with an EC ₅₀ of 850 were not fungitoxic in that range)

Combination EU50 molecules 1 EC50 molecules 2 EC50 combinations PA combinations 8ASOH/8ASOOH 723.3 850 156.08 0.02872 8CSOH/8ESOH 704.24 472.4 390 0.08735 8CSOH/8ESOOH 472.4 850 474.46 0.115 8CSOH/3MSOOH 704.24 468.89 479.2 0.02991 8CSOH/7MSOH 704.24 536.96 534.04 0.08249 8CSOH/3MSOH 704.24 786.17 625.78 0.25268

Table 7

Best combinations of molecules against Fusarium gramineum (molecules with an EC ₅₀ of 850 were not fungitoxic in that range)

Combination EU50 molecules 1 EC50 molecules 2 EC50 combinations PA combinations 8MSOH/3MSOOH 316.78 386.88 252.41 0.24857 8MSOH/7MSOH 316.78 586.15 258.16 0.16426 8MSOH/9MSOH 316.78 552.9 263.27 0.15388 8MSOH/6MSOH 316.78 850 266.17 0.25732 8CSOH/8MSOH 850 316.78 274.62 0.13763 8CSOH/3MSOOH 850 386.88 282.16 0.10929 8MSOH/9MSOOH 316.78 850 283.52 0.13736 8CSOH/3MSOH 850 525.24 383.96 0.04368 8CSOH/7MSOH 850 586.15 389.42 0.03555 8ESOH/7MSOH 533.3 586.15 414.39 0.12419 8ESOH/3MSOH 533.3 525.24 455.21 0.15703 8MSOOH/7MSOH 850 586.15 540.78 0.06049

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Table 8

Best combinations of molecules against Plectosphaerella cucumerina (molecules with an EC ₅₀ of 850 were not fungitoxic in that range)

Combination EU50 molecules 1 EC50 molecules 2 EC50 combinations PA combinations 8CSOH/7MSOH 272.83 337.32 145.68 0.18832 8MSOOH/3MSOH 850 626.83 483.44 0.09631 8MSOOH/3MSOOH 850 536.05 521.17 0.03061

Example 8. In vivo fungal biological test with Plasmopara viticola (class oomycetes; order Peronosporaceae; biotrophic pathogen of grapevine).

1-isothiocyanato-8-(methylsulfonyl)octane and 1-isothiocyanato-8-(methylsulfinyl)octane were obtained from SpiroChem (Basel, Switzerland). The compounds were dissolved in dimethyl sulfoxide (DMSO).

Leaf discs were collected from the 4th and 5th leaves of Vitis vinifera cv. Cabernet Sauvignon, pre-washed with water. For each concentration (0, 100, 250, 500, 750, and 1000 μM), 10 dry leaf disks were placed in Petri dishes on wet filter paper. Leaf discs were sprayed with various concentrations of active molecules and dried under a sterile hood. After drying, they were sprayed with a solution of Plasmopara viticola (10 ⁵ spores/ml), Petri dishes were covered and incubated in the dark. After 1 h, the Petri dishes were randomly placed on a shelf under normal day/night conditions. The percentage of infection was calculated after 7 days as described by Schnee et al. (2013).

Plasmopara viticola.

Table 9

Fungitoxic activity of isothiocyanates in combination against Plasmopara viticola

Combination EU50 8MSOH/8MSOOH 94.2

The tested combination showed high effectiveness against this biotrophic fungal pathogen (EC50 94.2).

Example 9. Tricophyton rubrum (class Eurocyomycetes; order Onigenes; pathogen of human skin and nails).

1-isothiocyanato-8-(methylsulfonyl)octane and 1-isothiocyanato-8-(methylsulfinyl)octane were obtained from SpiroChem (Basel, Switzerland). The compounds were dissolved in dimethyl sulfoxide (DMSO).

The fungitoxic effect was tested in 48-well tissue culture plates. Tricophyton rubrum was inoculated into KDA supplemented with isothiocyanates pre-dissolved at various concentrations (10, 150, 250, 375, 500, 700, 850 μM) in KDA. 2 mm agar plugs (Dufour et al., 2015) with pre-grown fungal cultures were placed in each well, and 3 biological replicates were used for each concentration. The plates were incubated for one week in a phytotron (80% relative humidity, constant temperature 23°C, with alternating cycles of 16 hours day and 8 hours night). Mycelial growth was measured after 7 days using ImageJ (http://imagej.net/Welcome), and EC ₅₀ was assessed as described in Schnee et al., 2013.

Tricophyton rubrum.

Table 10

Fungitoxic effect of isothiocyanates in combination against Tricophyton rubrum

Combination EU50 8MSOH/8MSOOH 275.2

The tested combination showed high effectiveness against a human fungal pathogen (EC50 275.2).

Example 10. Thamnidium elegans (class Zygomycetes; order Mucoraceae; stored meat).

1-isothiocyanato-8-(methylsulfonyl)octane and 1-isothiocyanato-8-(methylsulfinyl)octane were obtained from SpiroChem (Basel, Switzerland). The compounds were dissolved in dimethyl sulfoxide (DMSO).

The fungitoxic effect was tested in 48-well tissue culture plates. Thamnidium elegans was inoculated into KDA supplemented with isothiocyanates pre-dissolved at various concentrations (10, 150, 250, 375, 500, 700, 850 μM) in KDA. 2 mm agar plugs (Dufour et al., 2015) with pre-grown fungal cultures were placed in each well, and 3 biological replicates were used for each concentration. The plates were incubated for one week in a phytotron (80% relative humidity, constant temperature 23°C, with alternating cycles of 16 hours day and 8 hours night). Mycelial growth was measured after 7 days using ImageJ

- 29 044362 (http://imagej.net/Welcome), EC50 was assessed as described in Schneeetal., 2013.

Table 11

Fungitoxic effect of isothiocyanates in combination against Thamnidium elegans

Combination EU50 8MSOH/8MSOOH 395.0

The tested combination showed effectiveness against this type of fungus growing on stored meat (EC5 ₀ 395).

List of molecules used in examples

Abbreviation for molecule IUPAC name 7MSOH 1-isothiocyanato-7-(methylsulfinyl)heptane 8MSOOH 1-isothiocyanato-8-(methylsulfonyl)octane 8MSOH 1-isothiocyanato-8-(methylsulfinyl)octane 8MSOON 8-(methylsulfonyl)octylnitrile 6MSOH 1-isothiocyanato-6-(methylsulfinyl)hexane 4MSOH 1-isothiocyanato-4-(methylsulfinyl)butane 6MSOOH 1-isothiocyanato-6-(methylsulfonyl)hexane 5MSOOH 1-isothiocyanato-5-(methylsulfonyl)pentane 5MSOH 1-isothiocyanato-5-(methylsulfinyl)pentane 8AS0H 1-(isothiocyanatomethyl)-3-(4- (methylsulfinyl)butyl)benzene 8AS00H 1-(isothiocyanatomethyl)-3-(4- (methylsulfonyl)butyl)benzene 8CS0H (E)-1-isothiocyanato-8-(methylsulfinyl)oct- 2-en 8ES0H 1-(ethylsulfinyl)-8-isothiocyanatooctane 8ES00H 1-(ethylsulfonyl)-8-isothiocyanatooctane 3MSOOH 1-isothiocyanato-3-(methylsulfonyl)propane 3MSOH 1-isothiocyanato-3-(methylsulfinyl)propane 9MSOH 1-isothiocyanato-9-(methylsulfinyl)nonane 9MSOOH 1-isothiocyanato-9-(methylsulfonyl) nonane

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Claims (8)

Links 1. Schreiner RP, Koide RT. 1993a. Antifungal compounds from the roots of mycotrophic and non-mycotrophic plant species. New Phytol. 123:99-105. 2. Dufour V, Stahl M, Baysse C. 2015. The antibacterial properties of isothiocyanates. Mycrobiol. 161(Pt 2):229-43. 3. Lambrix V, Reichelt M, Mitchell-Olds T, Kliebenstein DJ, Gershenzon J. 2001. The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. Plant Cell. 13(12):2793-807. 4. Schnee S, Queiroz EF, Voinesco F, Marcourt L, Dubuis P-H, Wolfender J-L, Gindro K. 2013. Vitis vinifera canes, a new source of antifungal compounds against Plasmopora viticola, Erysiphe necator, and Botrytis cinerea. J. Agric. Food Chern. 61(23):5459-67. 5. Chou TC. 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58:621681,2006. CLAIM 1 . Use of a composition containing a combination of at least two compounds selected from the group consisting of 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-8-(methylsulfonyl)octane; 1-(isothiocyanatomethyl)-3-(4-(methylsulfinyl)butyl)benzene/1-(isothiocyanatomethyl)-3-(4(methylsulfonyl)butyl)benzene; (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-isothiocyanato-3-(methylsulfonyl)propane; (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-isothiocyanato-7-(methylsulfinyl)heptane; (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-isothiocyanato-3-(methylsulfinyl)propane; 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-3-(methylsulfonyl)propane; 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-7-(methylsulfinyl)heptane; 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-6-(methylsulfinyl)hexane; (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-isothiocyanato-8-(methylsulfinyl)octane; 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-9-(methylsulfonyl)nonane; 1-(ethylsulfinyl)-8-isothiocyanatooctane/1-isothiocyanato-7-(methylsulfinyl)heptane; 1 -(ethylsulfinyl)-8-isothiocyanatooctane/1-isothiocyanato-3-(methylsulfinyl)propane; 1-isothiocyanato-8-(methylsulfonyl)octane/1-isothiocyanato-7-(methylsulfinyl)heptane; 1-isothiocyanato-8-(methylsulfonyl)octane/1-isothiocyanato-9-(methylsulfinyl)nonane; 1-isothiocyanato-8-(methylsulfonyl)octane/1-isothiocyanato-3-(methylsulfinyl)propane; 1-isothiocyanato-8-(methylsulfonyl)octane/1-isothiocyanato-3-(methylsulfonyl)propane; (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-(ethylsulfinyl)-8-isothiocyanatooctane; or (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene/1-(ethylsulfonyl)-8-isothiocyanatooctane, for the prevention or treatment of fungal pathogens in plants. 2. Use of a composition according to claim 1, characterized in that said combination of at least two compounds is a mixture of 1-isothiocyanato-7-(methylsulfinyl)heptane and (E)-1-isothiocyanato-8-(methylsulfinyl)oct-2-ene or a mixture of 1-isothiocyanato-8-(methylsulfonyl)octane and 1-isothiocyanato-8-(methylsulfinyl)octane. 3. Use of a composition according to any one of claims 1, 2, characterized in that said composition is fungitoxic and/or fungistatic for plants. 4. Use of the composition according to any of claims 1-3 for plant crops in the field or for their practical implementation in vitro. 5. Use of a composition containing a combination of two compounds consisting of 1-isothiocyanato-8-(methylsulfinyl)octane/1-isothiocyanato-8-(methylsulfonyl)octane as an antimycotic agent in food or drink. 6. A method for preventing or treating mycosis in a human or animal, comprising administering a composition containing a combination of two compounds consisting of 1-isothiocyanato-8(methylsulfinyl)octane/1-isothiocyanato-8-(methylsulfonyl)octane. 7. The method according to claim 6, characterized in that the mycosis is caused by infections with Candida albicans or Trichophyton rubrum. 8. A fungicidal composition containing a combination of at least two compounds selected from the group consisting of -