阅读说明：本技术 抗真菌组合物 (Antifungal composition ) 是由 G·斯坦伯格 S·古尔 M·伍德 于 2020-03-18 设计创作，主要内容包括：本发明提供了一种抗真菌组合物以及所述组合物作为抗真菌剂的用途,所述抗真菌组合物包含式R-S～(+)(R’)-(2)或R-N～(+)(R’)-(3)的抗真菌化合物,其中R为C17-C32直链或支链烷基；并且每个R’独立地为甲基、乙基、丙基、异丙基或丁基。(The present invention provides an antifungal composition comprising the formula R-S and the use of the composition as an antifungal agent + (R’) 2 Or R-N + (R’) 3 Wherein R is a C17-C32 straight or branched chain alkyl group; and each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.)

1. An antifungal composition comprising the formula R-S⁺(R’)₂Or R-N⁺(R’)₃Against fungi

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

2. The antifungal composition of claim 1, wherein R is a C18 alkyl group.

3. The antifungal composition of claim 1 or 2, wherein each R' is methyl.

4. The antifungal composition of any one of claims 1 to 3, wherein the antifungal compound is octadecyl trimethyl ammonium or octadecyl dimethyl sulfonium.

5. The antifungal composition of any one of the preceding claims, wherein the antifungal compound comprises a counterion selected from chloride, bromide, iodide, hydroxide, sulfate, phosphate, bisulfate, or acetate.

6. The antifungal composition of any one of the preceding claims, wherein the antifungal compound is dissolved or suspended in an aqueous carrier.

7. The antifungal composition of any preceding claim, wherein the composition further comprises one or more adjuvants selected from wetting agents, spreading agents, drift control agents, foaming agents, dyes, thickeners, deposition agents (stickers), water conditioning agents, wetting agents, pH buffers, antifoaming agents, uv absorbers, surfactants, oil carriers, Crop Oil Concentrates (COC), vegetable oils, Methylated Seed Oils (MSO), petroleum, silicone derivatives, and nitrogen fertilizers.

8. The antifungal composition of any one of the preceding claims, comprising one or more additional antifungal agents.

9. An antifungal compound having the formula R-S⁺(R’)₂Or R-N⁺(R’)₃

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl;

the antifungal compounds are useful for treating fungal diseases.

10. The antifungal compound of claim 9, wherein the compound is octadecyl trimethyl ammonium or octadecyl dimethyl sulfonium.

11. Formula R-S⁺(R’)₂Or R-N⁺(R’)₃Use of the compounds of (a) as antifungal agents

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

12. By reacting a fungus with a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃By contacting with an antifungal compound

Wherein R is C17-C32 straight chain or branched chain alkyl; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

13. The method of claim 12, wherein the antifungal compound is applied in a composition comprising 0.1-200 μ g/ml of the compound.

14. The method of claim 12 or 13, wherein the method comprises a plant protection method, and wherein the antifungal compound or the composition comprising the antifungal compound is applied to a plant, plant part, or seed of a plant having a fungal infection.

15. The method of claim 12 or 13, wherein the method comprises treating a fungal infection in a human or animal.

16. A method of preventing fungal growth on or in a material, the method comprising applying a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃To said material

Wherein R is C17-C32 straight chain or branched chain alkyl; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

17. The method of claim 16, wherein the material comprises soil, growing medium, wood, wall coverings, or ground coverings.

18. A pharmaceutical composition comprising a compound of formula R-S + (R')₂Or R-N + (R')₃The antifungal agent of

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl;

and a pharmaceutically acceptable carrier or excipient.

19. The use, method or pharmaceutical composition according to any one of claims 11 to 18, wherein the antifungal compound is octadecyl trimethyl ammonium or octadecyl dimethyl sulfonium.

Technical Field

The present invention relates to antifungal compositions comprising mono-alkyl chain cationic antifungal compounds. The invention also relates to the use of monoalkyl chain cationic compounds as antifungal agents. In particular, the present invention relates to antifungal compositions, methods and uses.

Background

Fungi are one of the biggest biological challenges facing crop plant health and, therefore, food safety. This threat is exacerbated by intensive agricultural activities such as single crop planting, where a large population of genetically uniform varieties provides an ideal environment for the generation of new antifungal strains. Indeed, fungicide resistance occurs at a rate that exceeds the rate at which fungicides are found. There are only a few "new" antifungal agents in the "tract", but they are derivatives of commonly used chemicals, such as those targeting ergosterol biosynthesis or specific complexes of the mitochondrial respiratory chain. Clearly, there is an urgent need for new antifungal agents with a new mode of action, low toxicity to mammals and no environmental harm. Furthermore, there is an increasing need to find new antifungal agents to better protect animals, including humans, from fungal diseases. This is done in the context of the emergence of antifungal resistance to currently used clinical drugs (azoles). It is also desirable to replace azoles with a preservative treatment in wall paints and treated wood.

One potential target of the new fungicides is the fungal mitochondria. These organelles are involved in a wide range of cellular processes, but most importantly carry oxidative phosphorylases, which provide chemical energy to fuel the cell. Oxidative phosphorylation is dependent on electron transport through the mitochondrial respiratory chain complex in the inner mitochondrial membrane (Hirst, 2013). Fungal mitochondria differ from mammalian mitochondria in the composition and function of respiratory enzymes, making them attractive targets for new fungicides. In fact, two of the three market leading single-target fungicides, namely succinate dehydrogenase inhibitor (SDHI) and strobilurin, disrupt the fungal mitochondrial respiratory chain, targeting respiratory complexes II and III. Mitochondria also produce reactive oxygen species (mROS) at complexes I and III, which, if deregulated, can damage proteins and lipids in the inner mitochondrial membrane and trigger apoptosis. There is increasing evidence that this programmed cell death pathway exists in fungi, and targeting this pathway is a promising strategy for developing new antifungal agents.

The transport of protons across the inner mitochondrial membrane is triggered by electron transfer through the respiratory chain. This will charge the matrix negatively and thus it becomes the target of the lipophilic cation. These molecules, which bind the cationic head group to the lipophilic moiety, cross the cell membrane and accumulate in the inner membrane of the mitochondria, and expose their cationic moiety to the matrix. This behavior allows the delivery of therapeutic drugs into the mitochondria, but can also inhibit respiratory enzymes. While this effect on mitochondrial function challenges the use of lipophilic cations in medicine, it may be critical to the use of lipophilic cations as plant fungicides/antimycotics.

Lipophilic cations are widely used as "cationic surfactant" disinfectants or as supplements in cosmetic and pharmaceutical preparations. Cationic surfactants, particularly quaternary ammonium compounds, have received acceptance for their antibacterial activity, but have recently received increased attention due to their potential use in controlling human pathogenic fungi. The term cationic surfactant means that these compounds exert their biocidal activity on the surface of pathogens. Indeed, due to their amphiphilic structure, these molecules are expected to intercalate into the plasma membrane, and all antifungal lipophilic n-alkyl chain cations known to date appear to kill fungal cells by altering the permeability or function of the plasma membrane or by interacting with the fungal cell wall. This includes lipophilic monoalkyl chain cation (SACC) dodecylguanidine (hereinafter "C12-G +"), which is the active ingredient of the fungicide dodine. Dodine is a protective fungicide widely used to control fruit scab and foliar diseases in orchards. Although C12-G + is thought to be permeable to fungal cells, it also enters the cell and is reported to inhibit important metabolic enzymes. Whether intracellular activity is the primary mode of action of C12-G + or the result of cell leakage due to increased cell permeability is unclear.

Although C12-G + is useful as a fungicide, it has some toxicity problems. In particular, C12-G + may cause environmental problems if it is used as a fungicide for crops or soil due to its toxicity to Daphnia magna (Daphnia magna), since it may cause water to flow to bodies of water such as lakes and rivers.

Furthermore, although C12-G + is useful as a fungicide, its efficacy is limited when used against certain fungi. For example, when used to inhibit septoria tritici (Zymoseptoria tritici) or Magnaporthe oryzae (Magnaporthe oryzae) on wheat and rice, respectively, C12-G + was nominally effective but failed to completely inhibit the resultant septoria tritici and Magnaporthe oryzae even at nominally high concentrations (75 μ l/ml-100 μ l/ml C12-G +).

Thus, there is a continuing need for more effective treatments for crop pathogens to ensure our future food production. The rapid development of fungicide resistance in market-leading chemicals has made finding new fungicides an urgent necessity. These fungicides should (i) be active against pathogens that destroy crops, (ii) target a fundamental process at multiple sites to reduce resistance development, (iii) have low toxicity to humans and the environment.

It would therefore be advantageous to provide additional cationic surfactant-based antifungal compounds that are improved against a particular fungus or against a wide range of fungi based on the efficacy of known cationic surfactant-based antifungal compounds such as C12-G +. As used herein, the term "antifungal" refers to a broad spectrum of toxic effects against all members of the phylum mycosis and class oomycetes.

It would also be advantageous to provide additional cationic surfactant-based antifungal compounds that have reduced environmental and/or human toxicity as well as paint protection and prevent decay of cut wood products.

In addition, it would also be advantageous to provide more effective cationic surfactant-based antifungal compounds, compositions and treatments that target fungal metabolism at multiple sites in one or more metabolic pathways. Ideally, such fungicides would employ a novel multi-site mode of action that targets essential processes in pathogenic cells.

Furthermore, it would be advantageous to provide effective cationic surfactant-based biocidal compounds that are effective against human and/or animal pathogens, such as pathogenic fungi, and are effective against plant pathogens.

It is therefore an object of embodiments of the present invention to overcome or mitigate at least one problem of the prior art, whether described herein or not.

Disclosure of Invention

According to a first aspect of the present invention there is provided a composition comprising a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃Antifungal compound (A) of (1)

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

Compound (a) may comprise any suitable counter anion, such as bromide, chloride, iodide, hydroxide, sulfate, bisulfate, and phosphate.

The composition is preferably an antifungal composition, but can also be an antibacterial composition or an antibacterial composition, or any combination thereof (e.g., an antifungal and antibacterial composition, or an antifungal, antibacterial, and antibacterial composition).

The invention also provides pharmaceutical compositions comprising compound (a) and a pharmaceutically acceptable carrier or excipient, the use of compound (a) as an antifungal agent and methods of destroying, controlling or inhibiting fungi by contacting compound (a) with said fungi.

In a preferred embodiment, R is C18-C24, preferably straight chain alkyl, C18-C22, preferably straight chain alkyl, or C18-C20, preferably straight chain alkyl. In a particularly preferred embodiment, R is a C18 straight chain alkyl group.

In some embodiments, at least one R 'is methyl or ethyl, and in preferred embodiments, all R' are methyl.

Particularly useful as compound (a) in the compositions, uses and methods of the present invention are C18-dimethylsulfonium (octadecyldimethylsulfonium or "C18-DMS +") and C18-trimethylammonium (octadecyltrimethylammonium or "C18-TMA +") having the corresponding formulae:

for example, the composition may comprise a solution, suspension, emulsion, powder, paste, granule, gel, mousse, or powder.

The composition may comprise a carrier. The carrier may include a solvent, which may be water or an aqueous solvent. Thus, the composition may be an aqueous composition of compound (a) in water or an aqueous solvent. The aqueous solvent may comprise water and a co-solvent which may be selected from, for example, methanol and ethanol.

The antifungal composition may comprise compound (A) at a concentration of at least 0.1. mu.g/ml, 0.2. mu.g/ml, 0.3. mu.g/ml, 0.4. mu.g/ml, 0.5. mu.g/ml, 0.75. mu.g/ml, 1. mu.g/ml, 1.5. mu.g/ml, 2. mu.g/ml, 2.5. mu.g/ml, 5. mu.g/ml, 7.5. mu.g/ml, 10. mu.g/ml, 15. mu.g/ml, 20. mu.g/ml, 30. mu.g/ml, 40. mu.g/ml, 50. mu.g/ml, 60. mu.g/ml, 70. mu.g/ml, 75. mu.g/ml, at least 100. mu.g/ml, at least 200. mu.g/ml, at least 250. mu.g/ml, at least 500. mu.g/ml or at least 1000. mu.g/ml of the composition.

In some embodiments, the composition comprises compound (A) at a concentration of between 0.1-1000 μ g/ml, such as between 0.1-250 μ g/ml or between 0.1-200 μ g/ml.

For some applications, such as applying the composition to crop plants (e.g. wheat, barley, oats, rice, sorghum, plantain, maize, potatoes, vegetables or fruits), horticultural plants and trees, the composition may comprise compound (a) in a concentration of between 10-1000 μ g/ml, such as between 20-500 μ g/ml, between 25-250 μ g/ml or between 10-100 μ g/ml.

In some embodiments, the composition may comprise an adjuvant. Adjuvants may enhance the efficacy of antifungal compound (a). Suitable adjuvants include mechanisms of action that buffer the water to the pH at which the particular antifungal compound is most active, condition the water (e.g., they may reduce the potential inhibitory effect of minerals such as calcium and magnesium when hard water is used to solvate or dilute the antifungal compound), reduce the surface tension of the water ("wetting agents"), and thereby provide the antifungal composition with greater surface coverage when applied. Adjuvants may include solvents that render surfaces (such as the cuticle of the foliage of the plant to which the composition is applied) more permeable to the antifungal compound; or they may contain nutrients which may contribute to the antifungal compound when applied to crops or other plants, such as to promote plant growth, or to make compound (a) more effective for actively growing plants. Other suitable adjuvants are antifoams, adjuvants which reduce drift during spraying of the antifungal compound, and adjuvants which reduce evaporation and volatility of the antifungal compound.

The adjuvant may be a compound selected from: a pH buffering agent; a water conditioner; a wetting agent; leaf cuticle and/or cell membrane penetration aids; a plant growth promoter; defoaming agents; a spray drift reducing agent; and/or evaporation reducing agents; or any combination thereof.

Adjuvants may be in the form of crop protection spray additives and/or surfactants. Adjuvants can increase the permeability of the plant's stratum corneum and/or cell membrane. The adjuvant may be a non-ionic spreading and penetration aid; and/or act to reduce the surface tension of the composition.

Adjuvants may enhance the fungicidal activity of the compound (a), for example by increasing the permeability of the stratum corneum and/or the cell membrane. Adjuvants may enhance the fungicidal activity of compound (a), for example by increasing the permeability of the plant's cuticle and/or cell membrane.

For example, the adjuvant may be an additive to a crop protection spray, such as a surfactant; a non-ionic spreading and penetration aid; and/or act to reduce the surface tension of the composition.

The adjuvant may include an activating adjuvant or a multi-use adjuvant.

An activating adjuvant is a compound that enhances its antifungal activity when added to a composition comprising compound (a). Activated adjuvants include, for example, surfactants, oil carriers such as phytobland oil (harmless to plants), crop oils, Crop Oil Concentrates (COCs), vegetable oils, Methylated Seed Oils (MSOs), petroleum and silicone derivatives, and nitrogen fertilizers.

Multiple-use adjuvants (also sometimes referred to as spray modifiers) can alter the physical or chemical properties of the composition, making it easier to apply, such as by increasing its adhesion to plant surfaces making it less likely to roll off, or increasing its persistence in the environment.

One or more oils may be used as adjuvant carriers or diluents for compound (a).

The salts may also be used as an activating adjuvant, such as to increase the absorption and action of the antifungal compound in the target surface, material or plant over time.

One or more surfactant adjuvants may be present in the composition to facilitate or enhance the emulsifying, dispersing, spreading, adhering, or wetting properties of the composition.

When the composition is applied to a material (such as a surface, plant foliage, etc.), the surfactant reduces the surface tension in the spray droplets of the composition, which helps the composition spread out and cover the target material with a thin film, resulting in more efficient or faster absorption of the composition into the material. The surfactants may also affect the absorption of the composition by altering the viscosity and crystal structure of the waxes on the surface of the leaves and stems when sprayed onto the stems or leaves of the plant, making them more easily penetrated by compound (a) of the composition.

The surfactant may be selected to enhance the antifungal properties of the composition by any one or more of: a) spreading the composition more evenly over the material to which it is applied; b) improved retention (or 'tack') of the composition on the material; c) for plant or crop protection applications, increasing penetration of the composition through the hair, scale or other leaf surface structure of the plant; d) preventing crystallization of the composition; and/or e) slow the drying of the composition.

Each surfactant may be selected from a nonionic surfactant, an ionic surfactant, an amphoteric surfactant, or a zwitterionic surfactant, or any combination thereof.

Nonionic surfactants are generally biodegradable and compatible with many fertilizers and therefore may be preferred in the compositions of the present invention when used in crop or plant protection applications. Some nonionic surfactants are waxy solids and require the addition of a co-solvent (such as an alcohol or glycol) to dissolve into a liquid. Glycol co-solvents are generally preferred over alcohols because the latter are flammable, evaporate quickly, and can increase the number of fine spray droplets (so that the formulation can drift when sprayed).

The nonionic surfactant may comprise an organosilicone or a silicone surfactant (including siloxanes and organosiloxanes). The organosilicone surfactant significantly reduces the surface tension of the composition, enabling the composition to form a thin layer on the surface of the plant leaf or stem in use. The silicone surfactant also reduces surface tension and allows the composition to penetrate the stomata of the plant leaf. Silicone surfactants also provide protection to the compositions of the present invention by making the compositions very difficult to wash off after application. The silicone surfactant may also affect the amount/rate of uptake of the antifungal compound (a) through the cuticle of the leaf.

In other embodiments, the nonionic surfactant can include a carbamide surfactant (also referred to as a urea surfactant). The carboxamide surfactant may comprise, for example, dihydromono urea sulfate.

Suitable ionic surfactants include cationic and anionic surfactants. Suitable cationic surfactants include tallow amine ethoxylates. Suitable anionic surfactants include sulfates, carboxylates and phosphates linked to lipophilic hydrocarbons, including for example linear alkylbenzene sulfonates.

Amphoteric surfactants contain both positive and negative charges and generally function similarly to nonionic surfactants. Suitable amphoteric surfactants include, for example, lecithin (phosphatidylcholine) and amidopropylamine.

The multiple-use adjuvant (sometimes referred to as a spray modifier) can alter the physical or chemical properties of the composition of the invention, making the composition easier to apply, which can increase its adherence to the plant surface, resulting in a composition with a reduced risk of removal from the surface; or to enhance the persistence of the composition in the environment or treatment area in which the composition is present.

Examples of different functional classes of adjuvants of multiple use suitable for use in the compositions and uses of the present invention include wetting agents, spreading agents, drift control agents, foaming agents, dyes, thickeners, deposition agents (stickers), water conditioning agents, wetting agents, pH buffers, antifoam agents and uv absorbers. Some multiple use adjuvants may function in more than one of the above functional classes. Some activated adjuvants are also versatile adjuvants.

Wetting or spreading agents reduce the surface tension in the composition and cause the composition to form a large, thin layer on the leaves and stems of the target plant. Since these agents are usually nonionic surfactants diluted with water, alcohol or glycol, they can also be used as an activating adjuvant (surfactant). However, some wetting or spreading agents only affect the physical properties of the composition and do not affect the behavior of the composition after contact with the plant.

Drift control agents can be used to reduce spray drift of the composition, for example when spraying the composition onto plants, which is most often caused when fine (<150 μm diameter) spray droplets are carried away from the target area by the gas stream. Drift control agents are capable of altering the viscoelastic properties of the spray solution, producing a coarser spray with a larger average droplet size and weight, and minimizing the number of small droplets that are easily airborne. Suitable drift control agents may include large polymers such as polyacrylamides, polysaccharides, and certain types of gums.

Suitable deposition agents (stickers) include, for example, film-forming vegetable gels, emulsifiable resins, emulsifiable mineral oils, vegetable oils, waxes, and water-soluble polymers. The deposition agent can be used to reduce composition loss from the target plant due to composition volatilization from the target surface or composition beading and falling off. The deposition agents are particularly suitable for use in the compositions of the present invention in dry (wettable) powder and granular formulations.

Defoamers and antifoams are capable of reducing, inhibiting or destroying the formation of foam in the container in which the composition of the present invention may be contained. Suitable defoamers include, for example, oils, polydimethylsiloxanes and other silicones, alcohols, stearates and glycols.

One or more adjuvants may includeS 240、SP 131、SP 133、S 233、OE 446, Aduro (RTM) and/or Transport Ultra (RTM).S240 is a polyether trisiloxane, which imparts super-spreadability and greatly reduces surface tension. The BREAK-THRU SP131 is composed of polyglycerol fatty acid ester and polyethylene glycol, and can improve the performance of antifungal compound.

SP 133 is based on polyglycerol esters and fatty acid esters.S233 is a nonionic trisiloxane surfactant that can increase deposition of agricultural sprays and improve penetration of pesticide actives in plant tissues.OE 446 is a polyether polysiloxane.

The Transport Ultra (RTM) comprises a blend of a nonionic surfactant, an ammoniated ion, a water conditioning agent and an antifoam agent.

Aduro (RTM) contains dihydromono urea sulfate and alkylamine ethoxylates.

In some preferred embodiments, the at least one adjuvant is selected from the group consisting of silicone, siloxane, alkylamine ethoxylate, or carbamide. The adjuvants are particularly useful for enhancing the action of antifungal compounds, or otherwise increasing or accelerating the absorption of antifungal compounds by plants, particularly vascular plants and mosses.

In some embodiments, the adjuvant may comprise: a nonionic surfactant; and/or an antifoam agent; and/or ammonium ions; and/or a water conditioning agent; and/or polyether-polymethylsiloxane-copolymers; and/or a polyether polysiloxane; and/or polyglyceryl fatty acid esters and polyethylene glycols; and/or polyglycerol esters and fatty acid esters; and/or a nonionic trisiloxane.

In a preferred embodiment, the composition comprises at least one surfactant, which may be a nonionic surfactant. In some embodiments, the composition comprises at least one silicone or siloxane that can act as a surfactant and/or an antifoaming agent and/or a wetting agent.

In addition to compound (a), the antifungal composition may further comprise one or more additional antifungal agents. The additional antifungal agent may be selected from azoles; an amino derivative; strobilurin; specific anti-powdery spore compounds; an anilinopyrimidine; benzimidazoles and the like; a dicarboximide; polyhalogenated fungicides; systemic Acquired Resistance (SAR) inducers; a phenyl pyrrole; an acylalanine; an anti-efflorescence compound; a dithiocarbamate; (ii) an arylamidine; phosphorous acid and its derivatives; a fungicidal copper compound; vegetable based oils (botanical preparations); chitosan; a sulfur-based fungicide; a fungicidal amide; and a nitrogen heterocycle; or any combination thereof.

According to a second aspect of the present invention there is provided a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃As an antifungal agent, antibacterial agent or antibacterial agent against archaea

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

Compound (a) may be as described and defined above for the first aspect of the invention, and is preferably octadecyldimethyl sulfonium or octadecyltrimethyl ammonium. Compound (a) may be present in the antifungal composition of the first aspect of the present invention.

According to a third aspect of the present invention there is provided a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃Biocidal compounds of (A)

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl;

the biocidal compounds (A) are useful for the treatment of diseases, preferably fungal diseases.

Compound (a) may be as described and defined above, and is preferably octadecyldimethyl sulfonium or octadecyltrimethyl ammonium. Compound (a) may be present in the antifungal composition of the first aspect of the present invention.

The disease may be a phytopathogenic disease. The phytopathogenic disease may be a fungal disease of a plant or its seeds such as cereals (wheat, barley, rye, oats, rice, corn, sorghum, etc.), fruit trees (apples, pears, plums, peaches, apricots, cherries, bananas, grapes, strawberries, raspberries, blackberries, etc.), citrus trees (oranges, lemons, oranges, grapefruit, etc.), beans (soybeans, peas, lentils, soybeans, etc.), vegetables (spinach, lettuce, asparagus, cabbage, carrots, onions, tomatoes, potatoes, eggplants, peppers, etc.), cucurbits (squash, zucchini, cucumbers, melons, etc.), oil-producing plants (sunflower, oilseed rape, peanuts, castor beans, coconut, etc.), tobacco, coffee, tea, cocoa, beets, sugar cane, cotton and/or horticultural plants.

The plant pathogenic fungi and oomycete species in which the compounds (A) can be used include basidiomycetes, ascomycetes, deuteromycetes or incomplete fungi, chytrida, zygomycetes, microsporidia and oomycetes. Including, but not limited to, Puccinia spp, Ustilago spp, Tilletia spp, Monomyces spp, Phellinus spp, Rhizoctonia spp, Erysiphe spp, Sphaerotheca spp, Podosphaea spp, Rhizoctonia spp, Helminula spp, Helminthosporium spp, Rhinocladium spp, Pyrenophora spp, Sphaerotheca spp, Spirosporium spp, Sphaerotheca spp, Spirosporium spp, Spirillus spp, Spirosporium spp, Spirillum spp, Spirillus spp, Spirosporium spp, Spirillum spp, Spirillus spp, Spirillum, and Spirillum, and Spirillum, and Spirillum, etc. Spirillum, etc, Wheat-based Pythium species (Cercospora herpotrichoides), anthrax species (Colletotrichum spp.), Pyricularia oryzae (Pyricularia oryzae), Sclerotinia species (Sclerotium spp.), Phytophthora species (Phytophthora spp.), Pythium species (Pythium spp.), Plasmopara viticola (Plasmopara viticola), Peronospora species (Peronospora spp.), Pseudoperonospora cubensis (Pseudoperonospora cubensis), and Bremia lactucae (Bremia lactucae).

Specific fungal species infections that may be combated with compound (a) include: wheat powdery mildew in cereals (Erysiphe graminis), Septoria tritici in cereals (especially wheat), pyricularia grisea in cereals (especially rice), asteraceous powdery mildew (Erysiphe cichororarum) and Sphaerotheca fuliginea in cucurbitaceae, sphacelosia leucotricha (podospora leucotricha) in apples, devilpox vitis vinifera (Uncinula necator) in vines, Venturia inaequalis (Venturia inaequalis) in apples, Helminthosporium (Helminthosporium) species in cereals, Septoria nodorum (Septoria nodorum) in wheat, Botrytis cinerea (Botrytis cinerea) in strawberries and grapes, Cercospora arachidicola (Cercospora), verticillium and verticillium (Fusarium), and fusarium graminearum in peanuts, and various species of rice, rice and fusarium, and various species of fruits and vegetables.

Examples of plant fungal diseases that can be combated with compound (a) include, but are not limited to: spot disease (in particular septoria tritici), rot disease, fusarium wilt, stem rot, black root rot, root moniliforme root rot, blast disease (in particular rice blast), cotton rot, smut, soybean rust, cereal rust, potato blight, mildew, rhizomatosis, anthracnose, damping off, rhizoctonia rot, bottom rot, hollow leaf spot, target spot, leaf blight, ring spot, black shank, stem blight, black knot disease, ergot, leaf blister disease, scab, snow mold, smut and verticillium wilt.

In some embodiments, the disease may be an animal pathogenic mycosis, which in some embodiments may be independently selected from the diseases of the following table:

thus, compound (a) is useful in the treatment of many common human and animal fungal infections, including candidiasis, tinea nigra, and dermatophytosis.

According to a fourth aspect of the invention there is provided a method of treating a fungus by contacting the fungus with a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃Method for destroying, controlling or inhibiting fungi by contacting compound (A) with

Wherein R is C17-C32 straight chain or branched chain alkyl; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl.

Compound (a) may be as described and defined above, and is preferably octadecyldimethyl sulfonium or octadecyltrimethyl ammonium. Compound (a) may be present in the antifungal composition of the first aspect of the present invention.

The method may comprise contacting the fungus with an antifungal composition of the first aspect of the present invention.

Controlling or inhibiting fungi may include controlling the growth and/or longevity of the fungi.

The method may include destroying, controlling or inhibiting fungal infection of plants (including cut or grown plants or plant parts), seeds, animals (including humans, non-human mammals and other non-human animals), soil or ecosystems.

When compound (a) or a composition thereof is applied to soil or an ecosystem floor, the application rate can be 0.02kg to 3kg or more of active ingredient per hectare, depending on the type of effect desired.

The plant may be a vascular plant, and in some embodiments is selected from a crop plant or a tree. The compound (a) or a composition comprising the compound (a) can be applied by spraying or sprinkling the plant with the active ingredient or treating the seed of the plant with the active ingredient. They may be applied before or after infection of the plant or seed by one or more fungi.

In other embodiments, the method may include destroying, controlling, or inhibiting fungi in the building material or construction material. The building or construction material may comprise wood (raw or treated), wall coverings, bricks, blocks, plasterboards, floor coverings or paints.

Suitable wall coverings include, for example, cladding, wallpaper, ceramic wall coverings (such as tile), rubber, polymeric paint, varnish, and lacquer. Floor coverings include, for example, carpets, linoleum, wood boards or blocks, tiles, varnishes and lacquers. The compound (a) or the composition containing the compound (a) may be applied to a wall covering or a floor covering by spraying or sprinkling the covering, or by impregnating the covering with the compound (a) or the composition containing the compound (a).

Compositions such as solutions, emulsions, suspensions, powders, dusts, pastes and granules comprising compound (a) can be applied to construction or construction materials by, for example, spraying, atomising, sprinkling, dusting, dressing or watering.

According to a fifth aspect of the present invention there is provided a building or construction material or composition comprising a compound of formula R-S⁺(R’)₂Or R-N⁺(R’)₃Compound (A)

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl or ethyl.

For example, compound (a) may be impregnated in a building or construction material or composition, or may comprise a coating thereon. The building or construction material may be selected from wood (raw or treated), wall coverings, bricks, plasterboard, floor coverings or paints.

The building or construction material or composition may be one which has been treated (such as sprayed, coated or impregnated) by the method of the fourth aspect of the invention.

According to a sixth aspect of the present invention there is provided a pharmaceutical composition comprising a compound of formula R-S + (R')₂Or R-N + (R')₃Antifungal compound (A) of (1)

Wherein R is a C17-C32 straight or branched chain alkyl group; and is

Each R' is independently methyl, ethyl, propyl, isopropyl, or butyl;

and a pharmaceutically acceptable carrier or excipient.

Compound (a) may be as described and defined above for the first aspect of the invention, and in a preferred embodiment is octadecyldimethyl sulfonium ("C18-DMS +") or octadecyltrimethylammonium (C18-TPP +).

The pharmaceutical composition of the second aspect of the invention may be used for the treatment of a human or non-human animal, in particular a non-human mammal. Suitable non-human mammals include, for example, sheep, goats, cattle, pigs, dogs, cats, and horses. Other animals may include, for example, chickens, geese, ducks, turkeys, or other birds.

The pharmaceutically acceptable carrier or excipient may be a solvent, preferably an aqueous solvent. In some embodiments, the aqueous solvent may be water. In other embodiments, the aqueous solvent may comprise a mixture of water and a co-solvent. The co-solvent may be an alcohol and may be selected from the group consisting of methanol, ethanol, and combinations thereof.

The pharmaceutical composition may be in a form for topical administration. Thus, the pharmaceutical composition may be a topically administrable pharmaceutical composition.

The term "topical application" relates to the application of a substance to a body surface, such as skin. In a preferred embodiment, topical application is applied to the skin (directly on the skin surface), also referred to as "dermal application".

The pharmaceutical composition may be provided in any form suitable for topical administration, including, but not limited to, ointments, gels, creams, lotions, foams, sprays, mousses, patches, powders, pastes, hydrogels, emulsions (including oil-in-water, water-in-oil, oil-in-water-in-oil, water-in-oil-in-water emulsions), or any combination thereof. In some preferred embodiments, the pharmaceutical composition is provided as a cream, ointment, lotion, or gel, most preferably as a cream or ointment.

In some embodiments, the carrier or diluent may be an aqueous carrier or diluent, which may itself comprise water, such as deionized water, or a mixture of water and another solvent. Suitable mixtures include, for example, water and polar protic solvents (such as methanol, ethanol, propanol, isopropanol and butanol).

The carrier or diluent may alternatively comprise a hydrophobic carrier or diluent, which may be selected from oils or fats, natural waxes, petroleum waxes, hydrocarbons or any suitable mixture thereof. Alternatively, the carrier or diluent may comprise an organic solvent.

Suitable natural waxes include beeswax (including white or yellow beeswax), carnauba wax, wool wax, lanolin (such as purified lanolin or anhydrous lanolin), or any suitable combination thereof.

Suitable petroleum waxes include hard paraffin waxes and microcrystalline waxes.

Suitable hydrocarbons include liquid paraffin, soft paraffin (including white or yellow soft paraffin), white petrolatum, yellow petrolatum or any suitable combination thereof.

The pharmaceutical composition may comprise any other suitable carrier or diluent, such as those described in British Pharmacopoeia, 2017 edition or European Pharmacopoeia, 9 th edition.

Suitable organic solvents include, but are not limited to, nonpolar solvents, polar aprotic solvents, and polar protic solvents.

Suitable non-polar solvents include alkanes (such as hexane and pentane), cycloalkanes (such as cyclopentane and cyclohexane), benzene, toluene, chloroform, diethyl ether and dichloromethane.

Suitable polar aprotic solvents include tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, and propylene carbonate.

Polar protic solvents include, but are not limited to, alcohols (such as methanol, ethanol, propanol, isopropanol, and butanol), formic acid, and acetic acid.

The use of an organic solvent as defined above is particularly useful for pharmaceutical compositions, for example in the form of a spray. Sprays can be used to ensure that hard to reach areas of the skin are coated with the pharmaceutical composition, for example between hooves or under skin folds.

In some embodiments, compound (a) may be encapsulated. In some embodiments, compound (a) may be incorporated into liposomes. For encapsulated compound (a) and liposome-entrapped compound (a), the pharmaceutical compositions of the invention may comprise an aqueous carrier, including water itself. The compound (a) may be microencapsulated. Suitable encapsulating agents for encapsulation or microencapsulation include, for example, yeast cells, exine shell materials (such as from pollen grains), and the like.

The pharmaceutical compositions of the present invention may also comprise one or more pharmaceutical excipients. Suitable pharmaceutical excipients include, but are not limited to, emulsifiers, surfactants, solvents, co-solvents, preservatives, stabilizers, buffers, solubilizers, dispersants, antioxidants, thickeners, emollients, lubricants, emollients, and one or more additional skin healing or conditioning agents.

The pharmaceutical compositions of the present invention may also comprise one or more pharmaceutical excipients. Suitable pharmaceutical excipients include, but are not limited to, emulsifiers, surfactants, solvents, co-solvents, preservatives, stabilizers, buffers, solubilizers, dispersants, antioxidants, thickeners, emollients, lubricants, emollients, and one or more additional skin healing or conditioning agents.

Suitable preservatives include one or more selected from the list comprising: quaternary ammonium compounds such as benzalkonium chloride (N-benzyl-N- (C8-C18-alkyl) -N, N-dimethyl ammonium chloride), benzozylammonium chloride, benzethonium chloride, cetrimide (cetyl-trimethyl ammonium bromide), chlorospasazole, cetylpyridinium chloride, domiphen bromide, and the like; quaternary ammonium cyclodextrin compounds (such as QACD compounds described, for example, in US3,453,257 or US 5,241,059); alkyl mercury salts of thiosalicylic acid, such as thimerosal, phenylmercuric nitrate, phenylmercuric acetate, or phenylmercuric borate; parabens, such as methyl paraben or propyl paraben; alcohols such as chlorobutanol, benzyl alcohol or phenethyl alcohol; biguanide derivatives such as chlorhexidine or polyhexamethylene biguanide; sodium perborate; imidazolidinyl urea; sorbic acid; a stable oxy-chloro complex; polyethylene glycol-polyamine condensation resins; stabilized hydrogen peroxide generated from a hydrogen peroxide source for providing effective trace amounts of the resulting hydrogen peroxide, such as sodium perborate tetrahydrate; and/or any suitable combination thereof.

Preferred preservatives are quaternary ammonium compounds, in particular benzalkonium chloride, cetrimide and phenylethyl alcohol. Where appropriate, sufficient preservatives are added to the pharmaceutical compositions to ensure prevention of secondary contamination during use.

Suitable surfactants or emulsifiers include, but are not limited to, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and zwitterionic surfactants.

Suitable cationic surfactants include, for example, quaternary ammonium salts. Suitable anionic surfactants include carboxylates, such as the sodium or potassium salts of fatty acids; and sulfates of fatty acid salts, such as sodium laureth sulfate and sodium lauryl sulfate.

Suitable nonionic surfactants include, but are not limited to, fatty alcohol ethers, polyol esters, polyoxyethylene esters, poloxamers, and the like. Suitable polyoxyethylene esters include, but are not limited to, polyethylene glycol (PEG). Suitable polyol esters include, but are not limited to, glycols and glycerides, and sorbitan derivatives.

It will be appreciated that the pharmaceutical compositions of the invention for topical application should not contain ingredients that may cause irritation to the skin, even when used for extended periods. The use of compounds which may cause allergy should be avoided. Therefore, balanced amphoteric surfactants may be preferred as the surfactant.

The term "amphoteric surfactants" is well known to those skilled in the art. Such surfactants (which may also be referred to as amphoteric surfactants) have at least one anionic group and at least one cationic group and thus may have anionic, nonionic or cationic properties depending on the pH. A molecule is said to be in equilibrium if its isoelectric point occurs at pH 7. Amphoteric surfactants may have detergent and disinfectant properties. Balanced amphoteric surfactants may be particularly non-irritating to the skin and are therefore preferred for use in the topical pharmaceutical compositions of the present invention.

Suitable amphoteric surfactants include aminocarboxylic acids, aminopropionic acid derivatives, imidazoline derivatives, doxine, penethamine or long chain betaines, or cocamidopropyl betaine.

Suitable complexing agents include, but are not limited to, those selected from: disodium ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid (EDTA); chelating agents having a phosphonic acid or phosphonate group, preferably organic phosphonates, specifically aminotri (lower alkylene phosphonic acid) and the like; cyclodextrins, such as α -, β -or γ -cyclodextrins, for example alkylated, hydroxyalkylated, carboxyalkylated or carboalkoxyalkylated derivatives, or mono-or di-glycosyl- α -, β -or γ -cyclodextrins, mono-or di-maltosyl- α -, β -or γ -cyclodextrins or panosyl-cyclodextrins, and any suitable mixtures thereof.

The pharmaceutical compositions of the present invention may also comprise antioxidants such as ascorbic acid, acetylcysteine, cysteine, sodium bisulfite, butylated hydroxyanisole, butylated hydroxytoluene, or natural or synthetic vitamin E derivatives such as alpha-tocopherol or alpha-tocopherol acetate.

In yet another embodiment, the pharmaceutical compositions described herein comprising compound (a) may comprise, in addition to the antifungal compound (a), a second agent (second active ingredient, second active agent) having the desired therapeutic or prophylactic activity. Such second active agents may include, but are not limited to, additional antifungal agents, antibiotics, antibodies, antiviral agents, anticancer agents, analgesics (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, COX-2 inhibitors), immunostimulants (e.g., cytokines), hormones (natural or synthetic), Central Nervous System (CNS) stimulants, antiemetics, antihistamines, erythropoietin, complement stimulators, sedatives, muscle relaxants, anesthetics, anticonvulsants, antidepressants, antipsychotics, and combinations thereof.

According to a seventh aspect of the present invention there is provided compound (a) for use in the treatment of a fungal condition in a human or mammal. Compound (a) may be in a pharmaceutical composition as described above for the sixth aspect of the invention. The fungal condition in humans or mammals may be as described above.

Detailed Description

For a more clear understanding of the present invention, embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, in which:

FIG. 1 is a table (Table 1) showing the relative efficacy of C12-G +, C18-TMA +, and C18-DMS + in antifungal activity and toxicity against two plant fungal infections (Septoria tritici on wheat and Pyricularia oryzae on rice);

FIG. 2 comprises 4 panels showing the inhibition of mitochondrial potential in two phytopathogenic fungi and two human pathogenic fungi by SACC C18-TMA + and C18-DMS +.

FIG. 3 comprises 3 panels summarizing the results of LIVE/DEAD staining experiments for assessing the mortality of C18-TMA + and C18-DMS + in a phytopathogenic fungus causing wheat leaf blight and in two Candida species causing infectious diseases in humans.

FIG. 4 is a graph showing the additional mode of action (mitochondrial reactive oxygen species generation) of C18-DMS + in three phytopathogenic fungi causing rice blast (Pyricularia oryzae), Septoria oryzae (Septoria tritici) and maize smut (Ustilago maydis) in wheat and in two Candida species causing infectious diseases in humans;

FIG. 5 is a graph showing the additional mode of action of C18-DMS + (induction of programmed cell death, shown by two staining methods in Septoria tritici);

FIG. 6 is a graph showing the increased potential of C18-DMS + to protect crops from septoria tritici and rice blast;

FIG. 7 is a diagram showing the additional mode of action (induction of the plant defense system) of C18-DMS +;

FIG. 8 is a table comparing the relative efficacy and toxicity of C12-G + and C18-DMS + using the data in the table of FIG. 1 (Table 2); and is

Figure 9 shows a model of the effect of SACC on fungal oxidative phosphorylation.

Determination of the Effect of dodecyl guanidine (Doudodin) on the integrity of the fungal plasma Membrane

The physiological effects and mode of action of the known antifungal compound dodine (hereinafter referred to as "C12-G +") on the basidiomycetous corn smut fungus, Ustilago zeae and ascomycetous septoria tritici, Septoria tritici (hereinafter referred to as "MoA") were studied. Both fungi are economically important crop pathogens for which cell biology techniques and tools have recently been developed, including fluorescent markers for live cell imaging of all organelles.

Fungal growth was inhibited in a concentration-dependent manner on agar plates supplemented with C12-G +, in which septoria tritici was paired with C₁₂Sensitivity of-G + is about 4 times (50) that of Ustilago zeae% growth inhibitory concentration: EC (EC)₅₀Septoria tritici: 0.6 mu g/ml; EC (EC)₅₀Maize smut: 2.3. mu.g/ml. Next, toxicity of C12-G + was tested in liquid cultures of cells expressing the fluorescent plasma membrane marker GFP-Sso1 using live/dead cell staining. In these assays, live cells are green, but change from yellow to red upon death. Likewise, C12-G + was more effective in Septoria tritici than in Ustilago zeae and killed all cells after 1 hour of 100. mu.g/ml hold>80% (in contrast, maize smut killed about 15% of the cells under the same conditions). After incubation with C12-G + at concentrations as high as 50. mu.g/ml (Ustilago zeae) or 100. mu.g/ml (Septoria tritici) for 30-45 minutes, most of the cells remained viable, indicating that studying cells under these conditions would be able to understand the major cellular response to the drug.

C12-G + is thought to act on the fungal plasma membrane. Exposed cells expressing the fluorescent marker GFP-Sso1 were monitored to determine changes in plasma membrane appearance. High concentrations of C12-G + induced the formation of GFP-Sso1 "plaques" around the cells of both fungi, but C12-G + was approximately 2-fold less sensitive to Septoria tritici (at 50. mu.g/ml). Electron microscopy studies showed that C12-G + -treated cells formed plasma membrane invaginations. These structures were recognized by antibodies directed against GFP, indicating that they represent GFP-Sso1 plaques. It is believed that plasma membrane invagination may be the result of excessive insertion of C12-G + into the fungal plasma membrane, thus monitoring lateral diffusion of GFP-Sso1 in C12-G + treated cells. The reporter was photobleached in the plasma membrane and the recovery of fluorescence was monitored, indicating lateral movement of unbleached GFP-Sso 1. The addition of ≥ 10 μ G/ml C12-G + significantly reduced the movement of GFP-Sso1 in Ustilago zeae, however, this effect was not observed in C12-G + -treated Septoria tritici cells, indicating a difference in the effect of SACC on plasma membranes of both fungi.

Then, the membrane potential changes in the C12-G + treated cells were visualized by using the voltage sensitive fluorescent probe bis- (1, 3-dibutylbarbituric acid) trimethyoxonol, DiBAC4(3), which stains only depolarized cells, to determine drug-induced ion leakage from the cells. The high concentration of C12-G + increased the number of depolarized cells in Ustilago zeae, but had little effect on Septoria tritici. This result confirms that C12-G + has a stronger effect on plasma membranes in Ustilago zeae than on Septoria tritici. However, the low effect of C12-G + on the plasma membrane of septoria tritici is not compatible with its toxicity in this fungus (see above). Thus, the effect on plasma membranes is likely not the major MoA of C12-G + in fungi.

Determination of the Effect of C12-G + on fungal mitochondrial organization and respiration

The lipophilic cation C12-G + was tested to determine if it targets negatively charged fungal mitochondria. Accumulation of lipophilic cations in the inner membrane causes a change in mitochondrial ultrastructure. The effect of C12-G + was tested using a fungal strain containing a mitochondrial fluorescent marker (fluorescent reporter protein). The low concentration of C12-G + severely affected the shape of mitochondria and induced disruption of organelles. Electron microscopy showed alterations in the organization of the inner mitochondrial membrane, disorganized and swollen ridges. This result supports the notion that C12-G + targets mitochondria and inserts into the inner membrane. The membrane contains protein complexes of the respiratory chain, which are capable of generating the membrane potential required for ATP synthesis. Whether C12-G + interferes with this oxidative phosphorylation was also determined by staining C12-G + treated cells with the mitochondrial potential dye tetramethylrhodamine methyl ester (TMRM), and it was found that even small amounts of C12-G + depolarize the mitochondria in Ustilago zeae. Measurement of oxygen consumption by cells in cell suspension confirmed that C12-G + inhibited cellular respiration. A similar effect on membrane potential was observed in Septoria tritici and the sensitivity of Septoria tritici to C12-G + treatment was about 4-fold higher than that of Ustilago zeae. This corresponds well to an increased mortality of about 4 times for septoria tritici in the presence of C12-G + (see above). Thus, inhibition of ATP production is likely to be the major MoA of C12-G + in fungi.

The effect on mitochondria may explain the fungal specificity of C12-G +. To test this, mammalian skin C109 fibroblasts were treated with C12-G + and their effects on mitochondrial shape and membrane potential were monitored.

C12-G + had little effect on mitochondrial tissue at concentrations as high as 20. mu.g/ml, but exceeding this concentration resulted in organelle fragmentation and ridge swelling. This indicates that C12-G + is inserted into the human mitochondrial inner membrane, but at a concentration about 5-fold higher than that of Septoria tritici (as shown in Table 1 of FIG. 1; compare EC50 values for mitochondrial fragmentation). We also found that C12-G + had little effect on mitochondrial membrane potential and that human cells were approximately 50-fold less sensitive to lipid cations (as shown in Table 1 of FIG. 1; compare EC50 values for depolarization in Table 1). Thus, inhibition of mitochondrial respiration most likely supports the specificity of C12-G + for fungi.

Determination of the Effect of longer alkyl chains on antifungal Activity

After establishing a quantitative monitoring of the effect of C12-G + on mitochondria, synthesis and identification of novel monoalkyl chain cations (hereinafter referred to as "SACC") were performed. It is reported that the length of the cationic head group and alkyl chain determines the activity of the SACC. Thus, SACCs combining different cationic (and one anionic) moieties containing sulfonium head groups with alkyl chains of different lengths were synthesized and tested for their effect on septoria tritici mitochondria. As an indicator of mitochondrial membrane insertion, the degree of fragmentation was monitored and mitochondrial depolarization was measured using TMRM staining.

The following compounds were synthesized:

(i) c12 alkyl cations with different moieties:

(dodecyltriphenylphosphonium-hereinafter referred to as "C12-TPP +";

dodecyltrimethylammonium-hereinafter referred to as "C12-TMA +";

dodecyl triethyl ammonium ═ C12-TEA +;

dodecyl dimethyl sulfonium-hereinafter referred to as "C12-DMS +"),

(ii) lipophilic cation with short C6 alkyl chain:

(hexyltrimethylammonium-hereinafter referred to as "C6-TMA",

hexyldimethylsulfonium-hereinafter referred to as "C6-DMS +") and

(iii) lipophilic cation with longer C18 alkyl chain:

(octadecyl trimethyl ammonium-hereinafter referred to as "C18-TMA +",

octadecyldimethylsulfonium-hereinafter referred to as "C18-DMS +",

(iv) lipophilic alkyl anion:

dodecyl phosphate-hereinafter referred to as "C12-PO 4")

(v) A symmetric lipophilic cation with a dimethylsulfonium moiety at each end of the C12 alkyl chain:

(dodecane-1, 12-diylbis (dimethylsulfonium-hereinafter referred to as "+ (DMS) 2-C12")

At low concentrations (2.5 μ g/ml), only the molecules in groups (i) and (iii) induced significant mitochondrial fragmentation.

The EC50 for septoria tritici was tested for each compound in groups (i) and (iii) with the following results:

EC50[C12-G+]：6.63μg/ml；EC50[C12-TPP+]：6.63μg/ml；EC50[C12-TMA+]：3.85μg/ml；EC50[C12-TEA+]：2.38μg/ml；EC50[C12-DMS+]：7.39μg/ml；EC50[C18-TMA+]：1.55μg/ml；EC50[C18-DMS+]：1.72μg/ml)。

the results show that only cationic amphiphilic molecules with long alkyl chains are inserted into the mitochondrial membrane. However, when mitochondria were tested for depolarization, only C12-TPP + and two C18 alkyl chain cations (C18-TMA +, C18-DMS +) showed significant mitochondrial respiration inhibition in septoria tritici. All three SACCs were tested for their ability to depolarize mitochondria in the plant pathogenic fungi septoria tritici (causing septoria tritici) and magnaporthe oryzae (causing magnaporthe oryzae). In addition, C18-TMA +, C18-DMS + were tested for similar effects on mitochondrial potential in Candida albicans and Candida glabrata, both of which cause various infectious candidiasis. All compounds reduced the mitochondrial potential required for ATP synthesis (the major function of mitochondria) (figure 2). This supports the following conclusions: inhibition of respiration is a major physiological role of SACC. Consistent with interfering with this basic cellular pathway, C18-TMA +, C18-DMS + effectively killed Septoria tritici (12-14 hour exposure, 10. mu.g/ml) and two Candida species (FIG. 3; results from LIVE/DEAD staining experiments), with C18-DMS + being the most toxic in Candida albicans (5 hour exposure, 100. mu.g/ml). The toxicity of C18-TMA + and C18-DMS + to Septoria tritici is 2-7 times that of C12-G +.

As a result of these experiments, only some C12 and C18 SACCs were used for subsequent testing, since it is clear that C6 SACCs and some C12 SACCs are not suitable as effective antifungal agents.

Determining the toxicity of C18-TMA + and C18-DMS + in human cells and daphnia magna

Low toxicity to humans and the environment is an important requirement for fungicides. Thus, C12-TPP +, C18-TMA +, and C18-DMS + were tested for toxicity on C109 human skin fibroblasts. Mitochondrial tissue and mitochondrial membrane potential were monitored after incubation with various concentrations of the compounds. These experiments show that C12-TPP + has little effect on human mitochondrial tissue (EC [ C12-TPP + ]: 5.62. mu.g/ml), but at low concentrations (EC 50C 12-TPP +: 0.355. mu.g/ml) it affects human mitochondrial respiration and is therefore considered too toxic as an effective antifungal agent for most applications.

In contrast, the C18-alkyl chain cations C18-TMA + and C18-DMS + induced changes in mitochondrial morphology in fungi at 1.5-1.8 μ G/ml, whereas human mitochondria were approximately 29-fold less sensitive to both SACCs than C12-G + (as shown in Table 1 of FIG. 1). Furthermore, the two compounds inhibited septoria tritici at much lower concentrations than human cells, indicating that the two SACCs are about 75-fold more specific for fungi than humans (C18-TMA +) and about 53-fold more specific for fungi (C18-DMS +) (as shown in table 1 of fig. 1). Similar mitochondrial respiratory depression was found in the human pathogens candida albicans and candida glabrata (fig. 2).

In addition, the toxicity of C12-G +, C18-TMA + and C18-DMS + to daphnia magna was also studied. Such freshwater crustaceans have been recognized as reporter organisms in toxicity tests. First, daphnia magna was treated with 1. mu.g/ml of C12-G +, C18-TMA +, and C18-DMS + for 30 minutes, and then mitochondrial potential was observed by TMRM. This treatment does not affect the crustacean's locomotion. Daphnia incubated with solvent control (no SACC) showed red fluorescence indicating that its mitochondria were healthy. The C12-G + treated organisms lose most of the signal, indicating that even small amounts of C12-G + rapidly affect mitochondrial respiration in daphnia magna. In contrast, the two C18 alkyl chain cations had little effect on mitochondrial potential.

Next, the daphnia was treated with all three SACCs at different concentrations for 24 hours and its locomotor activity and "escape response" were monitored as indicators of mortality. In these experiments, C12-G + killed all daphnia magna at about 1. mu.g/ml, whereas C18-TMA + and C18-DMS + had little effect at this concentration. The higher amount of the two C18 SACCs eventually killed daphnia magna, where C18-TMA + was 1.8 times more toxic than C18-DMS +. However, both C18 alkyl cations were about 5-8 times less toxic than C12-G + (as shown in Table 1 of FIG. 1).

In conclusion, toxicity experiments showed that C18-TMA + and C18-DMS + are less toxic to human cultured cells and freshwater plankton, especially compared to C12-G + and C12-TPP +.

Determining whether C18 SACC inhibits the mitochondrial respiratory chain in various ways

The results of the above experiments show that C12-G +, C18-TMA +, and C18-DMS + depolarize the inner mitochondrial membrane of plant and human pathogenic fungi. In mammalian cells, the lipophilic cation C12-TPP + plays this role by acting as a proton carrier. We began to test whether C12-G +, C18-TMA +, and C18-DMS + have a similar mechanism of action in Septoria tritici. Proton carriers increase the formation of mitochondrial reactive oxygen species (mROS), which can be detected in living cells using the fluorescent dye dihydrorhodamine-123 (DHR-123). The increase in the level of mitochondrial reactive oxygen species (hereinafter mROS) was confirmed by treating septoria tritici cells with the proton carrier carbonyl cyanide metachlorophenylhydrazone (CCCP) for 30 minutes. The mROS levels in cells treated with C12-G + and C18-TMA + were then measured. Both compounds reduced the mROS concentration compared to CCCP, indicating that their effect is different from CCCP (fig. 4, septoria tritici). Thus, the proton carrier activity may be unique among C12-TPP +.

C12-G + and C18-TMA + significantly reduced mROS in Septoria tritici (FIG. 4). A similar reduction in mROS was found in C18-TMA + -treated Candida glabrata, the human pathogenic fungus (FIG. 4). The major site of mROS formation in mitochondria is respiratory complex I, and to a lesser extent respiratory complex III. Specific complex I inhibitors rotenone and antimycin a were used to block the activity of complexes I and III, respectively. Blocking complex I reduced mROS levels and was 3-4 times more effective than C12-G + and C18-TMA +. Thus, both SACCs inhibit complex I, but not as tightly as rotenone. The inhibitor antimycin a blocks the binding of reduced coenzyme Q at complex III, thereby increasing mROS in mammalian cells. Increased mROS in septoria tritici treated with antimycin a. This is significantly reduced in the presence of C12-G + and C18-TMA +. Thus, in septoria tritici, both SACCs appear to reduce electron delivery via coenzyme Q. This supports the notion that SACC can interfere with early steps of electron transfer in the inner mitochondrial membrane.

Determining whether C18-DMS + induces mROS production and programmed fungal cell death

mROS production in C18-DMS + treated cells was also determined in the plant pathogenic fungi Septoria tritici, Ustilago zeae and Pyricularia oryzae, and the human pathogens Candida albicans and Candida glabrata. Surprisingly, the effect of C18-DMS + differs from that of C12-G + and C18-TMA +; since this compound induced significantly mROS levels in all fungi tested, as shown in figure 4. In septoria tritici, this effect mimics CCCP, and incubation with CCCP for 24 hours reduced mROS, while treatment with C18-DMS + for 24 hours further induced the production of mROS. C18-DMS + did not increase mROS levels when Septoria tritici cells were incubated with the complex I inhibitor rotenone. Thus, C18-DMS + may induce mROS production at respiratory complex I. Next, the importance of alkyl chain length in the ability of C18-DMS + to trigger mROS formation was investigated. For this purpose, C16 alkyl chain dimethylsulfonium cation (C16-DMS +) was also synthesized. This compound and C12-DMS + (see above) were both tested for their ability to induce mROS production in Septoria tritici. Incubation with both SACCs for 30 minutes did not increase mROS levels, either at 5. mu.g/ml or 20. mu.g/ml. Thus, it appears that a chain length greater than C16 alkyl is required for the alkyl-DMS + compound to induce mROS.

There is increasing evidence that fungi can undergo programmed cell death, and mROS are thought to be able to induce this pathway. Thus, wheat husk needles treated with C12-G +, C18-TMA + and C18-DMS + were testedProgrammed cell death in spores. Using fluorescent caspase Activity marker CaspACE^TMFITC-VAD-FMK, which allows the detection of apoptosis in fungi. Co-staining was performed with propidium iodide, a proton membrane integrity reporter, to distinguish early apoptotic cells from dead post-apoptotic cells. A significant increase in early apoptotic cells was found 24 hours after treatment with C18-DMS + (CaspACE)^TMFITC-VAD-FMK positive, but propidium iodide negative; as shown in fig. 5). Only a few apoptotic cells were found after incubation with C12-G + or C18-TMA +. This result was confirmed using annexin-V-fluorescein staining for phosphatidylserine exposed at the plasma membrane in early apoptotic cells. Furthermore, membrane-associated fluorescence in apoptotic cells was only found in cells treated with C18-DMS +, as shown in fig. 5. Thus, C18-DMS + induced mROS production leads to activation of the programmed cell death pathway in septoria tritici and is likely to be present in other plant and human pathogens as well.

The protective effect of C18-DMS + and C18-TMA + on plants against the infection of septoria tritici and rice blast is determined.

As described above, C12-G +, C18-TMA +, and C18-DMS + depolarize the mitochondrial membrane and inhibit ATP synthesis, thereby cutting off the pathogen's "energy supply". In addition, C18-DMS + induces mROS production, thereby causing mitochondrial oxidative damage and triggering programmed cell death. This multiple mode of action was investigated how to enhance the defense against plant pathogens in leaf infection assays. Wheat plants whose leaves were pretreated with all SACC were spray inoculated with Septoria tritici IPO 323. In addition, the ability of C12-G +, C18-TMA +, and C18-DMS + to protect rice leaves from the rice blast fungus Magnaporthe grisea was also tested. It was found that all SACCs tested inhibited the germination of rice blast fungus and disrupted the formation of their adherent cells-these are very critical development steps for subsequent rice infection. Within 3 hours on the slides, conidia treated with the solvent control germinated and formed adherent cells. In fact, all three SACCs suppressed this, with C18-TMA + being slightly more potent than C18-DMS +. This is accompanied by mitochondrial fragmentation, changes in the intimal tissue, and depolarization of the mitochondria. mROS levels were also determined in the presence of all SACCs, and only C18-DMS + was found to induce mROS production. These results confirm the findings in septoria tritici and indicate that C18-DMS + may have greater potential to protect against rice blast. As a further preparation of the plant infection experiments, it was investigated whether wheat and rice leaves are sensitive to C12-G +, C18-TMA + or C18-DMS +. The whole plant was sprayed to "leaf drip" with water containing a small amount of methanol solvent in solvent/water (negative control), 10% Tween 20 (positive control) and 1000 μ G/ml C12-G +, C18-TMA + and C18-DMS +, respectively. Despite these high concentrations, none of the SACCs induced sallowness or necrosis of wheat or rice leaves after 7 days. This indicates that neither C12-G +, C18-TMA + nor C18-DMS + is phytotoxic.

Next, quantitative wheat and rice leaf infection assays were performed. All 3 SACCs of different concentrations were sprayed onto wheat and rice, the plants were left for 24 hours, and then septoria tritici (strain IPO323) and magnaporthe oryzae (strain Guy11) were applied and the occurrence of disease lesions/symptoms quantified. In the control experiment, after 21 days, septoria tritici formed black spots on the chlorosis leaves, which represent the melanized conidiophores, i.e. the symptoms of septoria tritici. Infection with Pyricularia oryzae resulted in the formation of brown lesions after 4 days of incubation. When leaves of both crops were treated with C12-G +, C18-TMA +, or C18-DMS +, the development of symptoms was inhibited. This protection is concentration dependent. Even at high concentrations, C12-G + did not completely inhibit the infection by Septoria tritici or Pyricularia oryzae, and C18-TMA + did not completely protect against Septoria tritici and Pyricularia oryzae, as shown in FIG. 6. In contrast, C18-DMS + almost abolishes symptom development in wheat and rice at 75. mu.g/ml and 100. mu.g/ml, and is therefore particularly useful as an antifungal agent for crop protection. Indeed, a direct comparison of disease symptom development at high concentrations indicates that C18-DMS + is significantly more defensive against septoria tritici and magnaporthe oryzae (as shown in table 1 of fig. 1 and table 2 of fig. 6).

The observed increase in plant protection by C18-DMS + may be due to the multiple moas in fungal pathogens. However, C18-DMS + may also induce innate defenses in plants, thereby alerting the plants to potential plant concernsPathogen attack. This initiation results in an oxidative burst, including the production of hydrogen peroxide, thereby protecting the plant from pathogen invasion. The three SACCs were tested to determine whether they induced this early plant defense response by treating rice leaves with 150. mu.g/ml C12-G +, C18-TMA +, and C18-DMS + followed by staining with Diaminobenzidine (DAB). The dye and topical H₂O₂The reaction occurred, producing a reddish-brown precipitate, indicating the presence of a plant defense response. The leaves treated with the solvent showed almost no precipitation of DAB, whereas treatment with 15mM salicylic acid resulted in brown color. The C12-G + and C18-TMA + treatments induced slight peroxide production after 6 hours, but a much stronger DAB reaction was found when the leaves were sprayed with C18-DMS + (as shown in FIG. 7 and Table 1 of FIG. 1). This indicates that C18-DMS + elicits plants against pathogen attack, thereby increasing the protective activity of the SACC.

Uses and advantages of the invention

New fungicides are needed to protect our thermal crops from fungal diseases, to ensure food safety and to protect the health of humans/animals and the ecosystem. Thus, the challenge is to find antifungal agents that combine low human toxicity and low environmental impact. Furthermore, such chemicals must be elastic, i.e. have a multi-site mode of action, and not fail to function because of the development of resistance. Mitochondria are important targets for fungicide development because these organelles provide cellular ATP, but also control lipid homeostasis and programmed fungal cell death. A group of novel SACCs with alkyl chain lengths greater than 16 have been synthesized and used as antifungal agents. In particular, both SACCs (i.e., C18-TMA + and C18-DMS +) combine high antifungal activity with relatively low toxicity to humans, daphnia magna and plants. C18-DMS + in particular exhibits low toxicity and is active against fungal pathogens in a number of ways, namely: (i) inhibition of oxidative phosphorylation, (ii) induction of destruction of mROS, (iii) triggering of fungal apoptosis, and (iv) triggering of plant defense.

Single alkyl chain cationic targeting of fungal mitochondria

Lipophilic cations have long been known for their antibacterial toxicity and have recently been recognized as antifungal compounds in medical applications. One of the biocidal cations is SACC, which combines a cationic head group and a long n-alkyl chain. This simple amphiphilic organization indicates that SACC can be inserted into membranes. In fact, many studies report that SACCs such as CTAB (cetyltrimethylammonium bromide) or C12-G + (dodecylguanidine, also known as dodine) alter the permeability or function of plasma membranes. Before SACCs reach the plasma membrane, they must pass through the fungal cell wall. In this process, they can alter the surface charge of fungal cells, which is described as supporting the major role of fungal toxicity of these compounds. Thus, antifungal lipid cations, including SACC, are thought to act on fungal cell surfaces, which is reflected in the name "cationic surfactants".

However, many results contradict this postulated MoA in fungi. Early reports on the mode of action of dodine showed that at high concentrations this fungicide penetrated the plasma membrane but the fungal cells had died. Other reports indicate that many fruits are able to inhibit important metabolic enzymes, suggesting that lipophilic cations cross the plasma membrane and exert their major fungitoxic effects within fungal cells. Once beyond the plasma membrane, the positively charged SACC may accumulate in the negatively charged mitochondrial matrix where it is inserted into the inner mitochondrial membrane. The results detailed herein support this notion. Although high concentrations of C12-G + affected fungal plasma membranes (EC50 in septoria tritici was about 20-50 μ G/ml), all SACCs tested induced mitochondrial fragmentation and altered the appearance of the mitochondrial inner membrane (EC50 in septoria tritici <7 μ G/ml). Furthermore, all 7 long-chain SACCs tested showed more potent mitochondrial respiration inhibition (EC50 <2 μ g/ml in Septoria tritici), which was confirmed using the selected SACCs in Ustilago zeae and Pyricularia oryzae. Thus, SACC is believed to target mitochondria and interfere with oxidative phosphorylation in fungi.

Multiple effects of single alkyl chain cations on mitochondrial respiration

The electron transfer through the mitochondrial respiratory chain complex pumps protons into the mitochondrial inner space, leaving the matrix negatively charged. This space charge separation generates proton motive forces for ATP synthesis. SACC depolarizes the inner membrane, disrupting ATP production, effectively "cutting off" the energy supply to the fungal cell. Previous studies using isolated mitochondria have shown that C12-TPP + depolarizes mitochondria by interacting with negatively charged fatty acid residues, which results in a slight uncoupling effect. Thus, the SACCs tested may promote uncontrolled proton access to the mitochondrial matrix. However, monitoring mROS production in septoria tritici revealed that the effect of SACC was different from that of the protic carrier CCCP. Furthermore, the uncoupling activity of C12TPP + requires a delocalized cationic charge, whereas SACC carries a local charge. Thus, C12-TPP + may have a unique impact on the fungal mitochondrial respiratory chain, which may explain its high toxicity. However, the results do not exclude the mild uncoupling effect of the tested SACCs.

Without being bound by any theory, it is believed that lipophilic cations may inhibit respiratory complex I and/or complex III in isolated mitochondria. In fungi, complex I is supplemented by a fungus-specific replacement dehydrogenase. Both are capable of reducing coenzyme Q, which in turn delivers electrons to complex III. As a byproduct, both complexes I and III produce low levels of mROS. C12-G + and C18-TMA + reduced the basal level of mROS in Septoria tritici and also reduced antimycin A-induced mROS formation in Complex III. Thus, SACC may interfere with early steps of the electron transport chain. One possibility is that membrane-inserted SACCs alter the surface charge of these membrane-bound enzymes, thereby interfering with their activity. In addition, SACC can alter membrane fluidity, interfering with complex I assembly and function, as it requires its diffusion through the inner membrane and has been shown to be dependent on the lipid composition of the inner membrane. In addition, lipophilic cations may bind directly to proteins of the respiratory complex. In fact, the specific complex I inhibitor 2-decyl-4-quinazolinamine (DQA) binds directly to purified complex I, and is expected to be a lipophilic cation at physiological pH. Thus, interfering with early steps in the respiratory chain via inhibition of fungal replacement NADH dehydrogenase and/or fungal specific proteins in complex I can explain the specificity of SACC for fungal cells.

C18-DMS + and C18-TMA + act against fungal pathogens through multiple modes of action.

As shown, the most effective antifungal SACC against Septoria tritici is C18-DMS +. This compound is more fungitotoxic and more effective in inhibiting respiration than C12-G + (a known fungicide used to control orchard pathogens) (as shown in table 2 of figure 8). Importantly, the enhanced antifungal properties of C18-DMS + are accompanied by lower toxicity to human cells and daphnia magna. Although C18-DMS + is less effective at inhibiting oxidative phosphorylation than the quaternary ammonium cation C18-TMA + (as shown in table 1 of fig. 1), it is significantly more effective at protecting wheat and rice against fungal pathogens than other C12-G + and C12-TPP +. This improved protective performance may be due to the C18-DMS + and C18-TMA + "attacking" the fungal pathogen in a number of ways. C18-DMS + has been shown to inhibit and kill fungi, inter alia, by a variety of actions, namely: (i) like other SACCs, this compound inhibits oxidative phosphorylation, thereby depriving fungal cells of ATP; (ii) it induces the production of mROS, probably involving complex I, which leads to oxidative damage of mitochondrial lipids and proteins; (iii) C18-DMS + distributes pathogens along irreversible programmed cell death pathways; (iv) finally, C18-DMS + triggers the formation of hydrogen peroxide in plants, which indicates an oxidative burst that triggers an early plant defense response. Thus, C18-DMS + elicits plants against pathogen attack, thereby reducing the chance of successful fungal infection.

C18-TMA + and C18-DMS + differ only in their head group, suggesting that additional physiological effects in fungal cells are due to the dimethylsulfonium moiety. The key to the physiological role of C18-DMS + in fungi is its ability to induce the production of mROS. This is seen in Septoria tritici, Pyricularia oryzae and Ustilago zeae and is therefore a general feature of C18-DMS +. Interestingly, the dimethylsulfonium moiety does not induce mROS when attached to C12 or C16 alkyl chains, even at high concentrations. The results indicate that the length of the alkyl chain is critical to the function of the cationic head group. This finding is surprising because even slightly shorter hydrophobic C16 alkyl chains do not integrate efficiently into the inner mitochondrial membrane and only chain lengths longer than C16 show mROS induction.

Conclusion

Surprisingly, C18-DMS + and C18-TMA + are more effective antifungal agents than any of the C6 or C12 alkyl chain length compounds tested. C18-DMS + and C18-TMA + were generally more effective against the maize smut fungi maize smut, the ascochyta tritici and the pyricularia oryzae, the rice blast fungi, than the SACCs tested elsewhere (in essentially all modes of action in terms of C18-DMS +). Overall, the pathogen tested a crop that provided two-thirds of the calories in a human diet. Table 1 of figure 1 shows that C18-DMS + and C18-TMA + have higher efficacy and lower toxicity compared to the well-known SACC antifungal compound dodine, and table 2 of figure 8 also shows the extent to which C18-DMS + is more effective compared to dodine.

Secondly, both C18-DMS + and C18-TMA + target the fungal respiratory chain in multiple ways, with C18-DMS + causing mROS at respiratory complex I, which is expected to damage mitochondrial proteins and lipids, but also trigger fungal apoptosis, as shown in fig. 9.

Finally, the tested C18 SACC showed relatively low toxicity (compared to known SACC fungicides) in human cultured cells and daphnia magna, where C12-TPP + showed unexpectedly high toxicity to human cultured cells; C12-G + does not protect plants to an adequate extent from pathogens.

The determination of C18-DMS + and C18-TMA + and thus C17-C32 alkyl chain cations with di-or tri-alkyl substituted cation moieties as particularly effective antifungal agents that function through a range of modes of operation and have significantly lower toxicity (human and/or environmental toxicity) compared to C12-G + indicates that the compounds and compositions of the present invention have great potential as fungicides in crop protection.

Other uses

Due to their low toxicity to humans and antifungal efficacy, the C17-C32 SACC compositions of the present invention are useful for treating (preventing or ameliorating) building and construction materials, particularly wood, wall coverings (such as wallpaper), floor coverings (such as carpet or linoleum), and paints. The SACC composition may be incorporated during the manufacture of the building or construction material, or may be impregnated or coated onto the material after manufacture.

The SACC compositions of the invention may also be used in cleaning or disinfecting compositions, both against fungi and against other microorganisms (e.g. oomycetes, archaea), which may for example be used prophylactically to prevent the growth of pathogens on surfaces or to remove infestation of surfaces.

The SACC compositions of the invention can also be used in soil or other plant growth media to remove, limit the spread, or control the growth of fungi. It may be applied in any suitable manner, such as by spraying or sprinkling.

The C18-DMS +, C18-TMA +, and other C17+ SACC compositions of the invention may be incorporated into pharmaceutical compositions, particularly for topical administration to humans, non-human mammals, and other animals.

The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention, which is defined in the appended claims.