RELATED APPLICATION

The present application gains priority from U.S. Provisional Patent Application No. 61/193,278 filed 13 November 2008, which is included by reference as if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of antimicrobial compounds and compositions and especially anti-fungal and anti-oomycete compounds and compositions.

Various microbes cause infections that exact a high toll in terms of pathogenesis in humans, non-human animals and plants as well as in terms of direct economic damage to animals and animal products, plants and plant products and industrial products. There are many different types of microbes that cause such infection including fungi (including yeasts and molds) and oomycetes.

Fungi is a kingdom of eukaryotic organisms. Many species of fungi are known, about 400 of which are pathogenic to man, animals or plants. Some fungi cause damage to surfaces, e.g. mildew on walls.

Most fungi are multicellular. Such fungi germinate from spores and grow as filaments termed hyphae. As the hyphae grow, they branch repeatedly. Hyphae from individual fungal cells interconnect with hyphae from other cells, forming one larger structure termed the mycelium. There is some disagreement as how to classify fungi, althouh one approach is to divide fungi (including molds) into phyla based on characteristics of the reproductive organs. Yeasts are unicellular, with individual cells being spheroid or elongated, and reproduce by dividing in a fashion similar to animal cells. However, some yeasts, such as Candida, can grow in hyphal form and also produce strucutres known as chlamydospores.

Oomycetes are a class of eukaryotic organisms of the phylum heterokontophyta popularly known as water molds, which superficially resemble Fungi. Some species of Oomycetes are plant or animal pathogens, the main pathogenic species being Phytophthora, Pythium, and downy mildew.

Species of Phytophthora include Phytophthora infestans which causes potato blight; Phytophthora sojae which causes stem and root rot of soybean; Phytophthora alni which causes alder root rot; Phytophthora cactorum which causes rhodendron root rot affecting rhodendrons and azaleas; Phytophthora capsi which affects Cucurbitacea fruits, such as cucumbers and squash; Phytophthora cinnamomi which causes cinnamon root rot affecting woody ornamentals including arborvitae, azalea, Chamaecyparis, dogwood, forsythia, Fraser fir, hemlock, Japanese holly, juniper, Pieris, rhodendron, Taxus, white pine, American chestnut and Australian Jarrah; Phytophthora fragariae which causes red root rot affecting strawberries; Phytophthora kernoviae, a pathogen of beech and rhodendron, also occurring in other trees and shrubs, including oak; Phytophthor palmivora which causes fruit rot in coconuts and betel nuts; Phytophthora ramorum which causes Sudden Oak Death; and Phytophthora quercina which causes oak death.

A large number of Pythium species are known, many of which are plant pathogens. Some species of Pythium kill seedlings in greenhouses by damping off. For example, Pythium aphanidermatum is an aggressive pathogen that causes damping off, root and stem rots, and blights, especially in greenhouses. Economic damage is primarily in greenhouses, beets, peppers, cucurbits, beans, broccoli, chrysanthemums, begonia, pansy, cotton and grasses. Pythium aphanidermatum infection is usually treated by soil drenches of fungicide compositions both prophylactically and curatively, by irrigation or by foliar spray (both air and ground). Pythium aphanidermatum is considered a re-emerging disease due to strains developing resistance to previously known treatments (e.g., mefenoxam). Pythium insidiosum is known to infect mammals.

Downy mildews cause the appearance of white "mildew" on leaf surfaces. There are very few effective treatments for Oomycete infections and many Oomycete are able to survive dormant in soil for decades.

It is known to use antimicrobial compounds for treatment of fungal infections and oomycete infections. Examples of anti-fungal compounds include polyene antifungals, imidazole, triazole and thiazole antifungals, allylamines, echinocandines, and others. In many agricultural practices, fungicides are extensively used to prevent and curb the progression of plant diseases that have severe adverse effects on crop yields and quality. In some cases, the prolonged and repeated use of many fungicides has resulted in the proliferation of plant pathogen isolates that are resistant to the fungicides employed. Therefore, identifying new classes of antifungal compounds based on structures not previously used in combating plant diseases may assist in the control of plant pathogens that are tolerant or resistant to known compounds.

Further, many prior art antimicrobial compounds exhibit serious side effects in mammals. For example, exposure to fungicides commonly used with food stuffs such as vinclozolin, prochloraz and procymidone during pregnancy is suspected to cause feminization of male fetuses, by blocking testosterone or mimicking estrogen.

It would be useful to have new antimicrobial compounds and compositions for treatment of microbial infections.

SUMMARY OF THE INVENTION

Some embodiments of the present invention provide for compounds, antimicrobial compositions, methods and uses that relate to treatment of infections by microbes, for example fungi and oomycetes. According to an aspect of some embodiments of the invention, there is provided, an antimicrobial composition comprising at least one compound selected from the group consisting of a compound of Formula I:

and a compound of Formula II:

esters thereof, and salts thereof, wherein:

Z is chosen from the group consisting of O and S; n is an integer from 0 to 20; each X is independently selected from the group consisting of H and F; each of R ¹ , R ² R ³ and R ⁴ is independently selected from the group consisting of hydrogen, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, amino and -NR ⁵ R ⁶ , or, alternatively, two of R ² , R ³ and R ⁴ form a five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and

R ⁵ and R ⁶ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl and sulfonyl, or, alternatively R ⁵ and R ⁶ form a five- or six- member aromatic, heteroaromatic, alicyclic or heteroalicyclic ring.

According to an aspect of some embodiments of the invention, there is also provided a method of treating an infection by microbes, comprising administering an effective amount of the compositions described above.

According to an aspect of some embodiments of the invention, there is also provided the use of the composition or method described above for the treatment of an infection by microbes. In some embodiments, the compound of Formula I is selected from the group consisting of compounds having the Formulae NP-25, NP- 18a, NP-5, NP- 17, NP-6, NP-IO, NP- 19a, AG-5, AG-2 and AG-4.

In some embodiments, the compound of Formula II comprises NP- 18b.

In some embodiments, the microbes are selected from the group consisting of fungi and oomycetes.

In some embodiments, the fungi are members of a phylum selected from the group consisting of Chytridiomycota; Blastocladiomycota; Neocallimastigomycota; Zygomycota; Glomeromycota; Ascomycota; Basidiomycota; and Deuteromycota.

In some embodiments, the fungi are selected from the group consisting of yeast and mold. In some embodiments, the mold is a member of a phylum selected from the group consisting of Zygomycota, Deuteromycota, and Ascomycota.

In some embodiments, the Ascomycota is selected from the group consisting of Aspergillus, Sclerotinia, Botrytis, Penicillum, Neurospora, Alternaria; Verticillium and Candida. In some embodiments, the Aspergillus is selected from the group consisting of

Aspergillus fumigatus and Aspergillus niger.

In some embodiments, the Sclerotinia is selected from the group consisting of Sclerotinia sclerotiorum, Sclerotinia trifoliorum and Sclerotinia minor. In some embodiments, the Sclerotinia sclerotiorum comprises sclerotiorum and the compound of Formula I comprises AG-5.

In some embodiments, the Botrytis is selected from the group consisting of Botrytis cinerea, Botrytis convolute, Botrytis polyblastis, Botrytis allii and Botrytis fabae. In some embodiments, the Botrytis comprises Botrytis cinerea, and the compound of

Formula I is selected from the group consisting of NP- 19a, NP- 18a and AG-5.

In some embodiments, the Penicillum is selected from the group consisting of Penicillium chrysogenum, Penicillium citrinum, Penicillium janthinellum, Penicillium marneffei, Penicillium purpurogenum, Penicillium expansum, Penicillium funiculosum and Penicillium digitatum. In some embodiments, the Penicillium comprises Penicillium digitatum, and the compound of Formula I or Formula II is selected from the group consisting of NP- 19a, NP-18a and NP-IO.

In some embodiments, the Neurospora comprises Neurospora crassa, and the compound of Formula I or Formula II is selected from the group consisting of NP-6, NP- 19a, NP- 18b, NP- 18a, AG-5 and AG-4.

In some embodiments, the Alternaria comprises Alternaria alternaria, and the compound of Formula I comprises NP- 18a.

In some embodiments, the Verticillium comprises Verticillium dahlia.

In some embodiments, the Candida comprises Candida albicans. In some embodiments, the Basidiomycota comprises Rhizoctonia solani and the compound of Formula I is selected from the group consisting of NP-6 and NP- 18a.

In some embodiments, the oomycete is selected from the group consisting of

Phytophthora, Pythium, and downy mildew. In some embodiments, the Phytophthora is selected from the group consisting of Phytophthora infestans; Phytophthora sojae; Phytophthora alni; Phytophthora cactorum; Phytophthora capsi; Phytophthora cinnamomi;

Phytophthora fragariae; Phytophthora kernoviae; Phytophthor palmivora; Phytophthora ramorum; and Phytophthora quercina. In some embodiments, the Pythium is selected from the group consisting of Pythium aphanidermatum and Pythium insidiosum. In some embodiments, the Pythium comprises Pythium aphanidermatum and the composition comprises at least one selected from the group consisting of a compound of Formula AG-5,

NP-18a, NP-10 and NP- 18b.

In some embodiments, the composition comprises a sprayable composition.

In some embodiments, the composition comprises a liquid carrier. According to an aspect of some embodiments of the invention, there is also provided a use of a composition described above for prevention or delay of development of an infection by microbes in a food stuff. In some embodiments, the foodstuff is selected from the group consisting of a fruit, vegetables and a grain. In some embodiments, the composition is applied to the foodstuff post-harvest.

In some embodiments, a composition described above is a medicament for human use. In some such embodiments, the compositions is for the treatment of a condition selected from the group consisting of diaper rash, an infection of the mouth, a genital infection, a nail infection, tinea pedis, tinea crutis; tinea capitis; finger tinea unguium; tinea corporis; tinea barbae and Aspergilliosis.

In some embodiments, a composition described above is a medicament for veterinary use. In some embodiments, the composition is used as a medicament for veterinary medicine.

In some embodiments, a composition described above is configured for use in agriculture. In some embodiments, the composition is used in agriculture. In some embodiments, a composition described above is configured for use in animal husbandry. In some embodiments, the composition is used in animal husbandry.

In some embodiments, a composition described above is configured for use in treating plants or plant matter. In some embodiments, the composition is used for treating plant or plant matter. In some embodiments, the plants or plant matter comprises living plants, seeds, harvested fruit and grain.

In some embodiments, a composition described above is configured for use in treating soil. In some embodiments, the composition is used for treating soil. In some embodiments, the composition is applied to soil during soil irrigation

In some embodiments, the composition described above is a topical composition. In some embodiments, the composition is topically applied. In some embodiments, the composition is configured for application to a surface selected from the group consisting of a surface of an animal, a surface of a plant, a soil surface and a surface of an inanimate object.

In some such embodiments, the surface of an animal is selected from the group consisting of skin, keratinous surface and mucous membrane. In some such embodiments the surface of a plant is selected from the group consisting of a surface of a leaf, a surface of a stalk, a surface of a fruit and a surface of a seed. In some such embodiments, the composition comprises a form selected from the group consisting of a cream, a spray, an ointment, a lotion, a solution, a suspension, a spray, a wash, a paint and a lacquer. In some embodiments, the composition is configured for use with edible products. In some embodiments, the composition is used by applying to an edible product. In some embodiments, the composition is applied to a wrapping for an edible product, such as a food wrapping, a food container, or a food additive. According to an aspect of some embodiments of the invention, there is also provided a use of the composition described above, for the prevention or delay of onset of a microbial infection.

According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising a compound selected from the group consisting of NP- 18b and NP-100 for the treatment of an infection by Penicillium digitatum.

According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising a compound selected from the group consisting of NP- 19a, NP- 18a, and AG-5 for the treatment of an infection by Botrytis cinerea.

According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising a compound selected from the group consisting of NP- 18b, NP-19a, NP-IO and AG-5, for the treatment of an infection by Pythium aphanidermatum.

According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising a compound selected from the group consisting of NP-6, NP- 19a, NP-18b, NP- 18a, NP-10, AG-5 and AG-4, for the treatment of an infection by Neurospora crassa.

According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising NP- 18a for the treatment of an infection by Alternaria alternate. According to an aspect of some embodiments of the invention, there is also provided a use of a composition comprising a compound selected from the group consisting of NP-6 and NP- 18a for the treatment of an infection by Rhizoctonia solani.

In some embodiments, a composition described above, further comprises an additional agent for the treatment of infection by microbes. In some such embodiments, an amount of the compound selected from the group consisting of a compound of Formula I and a compound of Formula II, esters thereof, and salts thereof alone, and the amount of the additional antifungal agent alone is insufficient to achieve the antifungal effect achieved by the administration of the combination of the two or more ingredients. According to an aspect of some embodiments of the invention, there is also provided a compound of Formula I:

esters thereof, and salts thereof, wherein Z, n, X, and R ¹ , R ² R ³ and R ⁴ are defined as above.

In some embodiments, the compound of Formula I is (Z)-3-amino-l-(4- chlorophenyl)-4,4,5,5,5-pentafluoropent-2-en-l-one (NP-25) wherein Z=O, n=2, all X=F, R ⁱ =H, R ² =p-Cl, R ³ =H and R ⁴ =H.

In some embodiments, the compound of Formula I is (Z)-3-amino-4,4,4-trifluoro-l- (4-hydroxyphenyl)but-2-en-l-one (NP- 18a) wherein Z=O, n=l, all X=F, R^H, R ² =p-OH, R ³ =H and R ⁴ =H.

In some embodiments, the compound of Formula I is (Z)-3-amino-4,4,4-trifluoro-l- (3-(trifluoromethyl)phenyl)but-2-en-l-one (NP-5) wherein Z=O, n=l, all X=F, R ¹ =!!, R ² =m- CF ₃ , R ³ =H and R ⁴ =H. In some embodiments, the compound of Formula I is (Z)-3-amino-4,4,4-trifluoro-l-

(naphthalen-2-yl)but-2-en-l-one (NP- 17) wherein Z=O, n=l, all X=F, R^H, R ² R ³ =naphtalen-2-yl and R ⁴ =H.

In some embodiments, the compound of Formula I is(Z)-3-amino-l-(3-bromophenyl)- 4,4,4-trifluorobut-2-en-l-one (NP-6) wherein Z=O, n=l, all X=F, R^H, R ² =m-Br, R ³ =H and R ⁴ =H.

In some embodiments, the compound of Formula I is (Z)-3-amino-l-(4- chlorophenyl)-2-ethyl-4,4,4-trifluorobut-2-en-l-one (NP-IO) wherein Z=O, n=l, all X=F, R^C ₂ H ₅ , R ² =p-Cl, R ³ =H and R ⁴ =H.

In some embodiments, the compound of Formula I is (Z)-tert-butyl-4-(3-amino-4,4,4- trifluorobut-2-enoyl)phenyl carbamate (NP- 19a) wherein Z=O, n=l, all X=F, R^H, R ² =p- NHBoc, R ³ =H and R ⁴ =H. In some embodiments, the compound of Formula I is (Z)-3-amino-4,4,4-trifluoro-l- (4-chlorophenyl)but-2-en-l-one (AG-5) wherein Z=O, n=l, all X=F, R^=H, R ² =p-Cl, R ³ =H and R ⁴ =H (AG-5).

In some embodiments, the compound of Formula I is (Z)-4-(3-amino-4,4,4- trifluorobut-2-enoyl)benzonitrile (AG-2) wherein Z=O, n=l, all X=F, R^H, R ² =p-CN, R ³ =H and R ⁴ =H (AG-2).

In some embodiments, the compound of Formula I is (Z)-3-amino-4,4,4-trifluoro-l- (4-fluorophenyl)but-2-en-l-one (AG-4) wherein Z=O, n=l, all X=F, R^H, R ² =p-F, R ³ =H and R ⁴ =H (AG-4). According to an aspect of some embodiments of the invention, there is also provided a use of a compound selected from the group consisting of a compound of Formula I:

and a compound of Formula II:

II

esters thereof, and salts thereof, wherein Z, n, X, and R ¹ , R ² R ³ and R ⁴ are defined as above in the manufacture of a medicament for the treatment of a microbial infection.

Some embodiments of the present invention have at least one beneficial effect in treating microbial infections on plant and animal (including human) species. Beneficial effects include effects such as curing a microbial infection, preventing a microbial infection (i.e., prophylaxis), treating symptoms of a microbial infection, curing symptoms of a microbial infection, ameliorating symptoms of a microbial infection, treating effects of a microbial infection, ameliorating effects of a microbial infection, and preventing results of a microbial infection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the patent specification, including definitions, will control.

As used herein, the terms "comprising", "including", "having" and grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms "consisting of" and

"consisting essentially of".

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying figures. The description, together with the figures, makes apparent how embodiments of the invention may be practiced to those skilled in the art. It is stressed that the particulars shown in the figures are by way of example and for purposes of illustrative discussion of embodiments of the invention.

In the figures:

FIG. IA shows the chemical structure of compounds of Formula I;

FIG. IB shows the chemical structure of compound NP-25;

FIG. 1C shows the chemical structure of compounds of Formula II; FIG. ID shows the chemical structure of compound NP- 18b;

FIG. 2 shows the synthetic route taken to prepare compounds of the invention;

FIG. 3A and 3B are bar charts showing the plant toxicity of tested compounds on Arabidopsis plant;

FIGS. 4 and 5 are bar charts showing the in vitro antimicrobial efficacy of tested compounds;

FIGS. 6A-6D show the in vitro antimicrobial efficacy of compounds NP- 18a, NP- 18b and AG-5 against Botrytis cinerea and Sclerotinia sclerotiorum in Petri dishes;

FIG. 6E is a bar chart summarizing the results shown in FIGS. 6A-6D; FIG. 7 is a bar chart showing the effect of compounds NP- 18a, NP- 18b, AG-5, NP-6 and NP-25 on radial growth of Sclerotina sclerotiorum and Botrytis cinerea; and

FIG. 8 is a table showing the in vitro antimicrobial efficacy of tested compounds on Candida albicans, Aspergillus fumigates, and Aspergillus niger.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

Some embodiments of the present invention provide for compounds, antimicrobial compositions, methods and uses that relate to treatment of infections by microbes, such as fungi and oomycetes. Specifically, some embodiments relate to (Z) β-trifluroalkyl aminovinyl ketones and thioketones, that in some instances have antimicrobial activity. In some embodiments, the compounds of the present invention are compounds of Formula I (see Figure IA and below), Formula II (see Figure 1C and below), esters thereof or salts thereof, or combinations thereof.

According to some aspects of the present invention there is provided an antimicrobial composition comprising at least one of the compounds from the group of compounds consisting of a compound of Formula I, esters thereof, salts thereof, a compound of Formula II, esters thereof, and salts thereof.

According to some aspects of the present invention, there is provided a method of treating an infection by microbes, comprising applying or administering an effective amount of a composition as described above.

According to some aspects of the present invention, there is provided a use of at least one compound selected from the group consisting of a compound of Formula I and a compound of Formula II, esters thereof, and salts thereof for the treatment of an infection by microbes. According to some aspects of the present invention there is provided a use of at least one compound selected from the group consisting of a compound of Formula I and a compound of Formula II, esters thereof or salts thereof, in the manufacture of an antimicrobial composition for the treatment of an infection by microbes.

In some embodiments, by microbe is meant a fungus (including a yeast or a mold) and by antimicrobial is meant antifungal. In some embodiments, by microbe is meant an oomycete and by antimicrobial is meant anti-oomycete.

The principles, uses and implementations of the teachings of the invention may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art is able to implement the teachings of the invention without undue effort or experimentation.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth herein. The invention can be implemented with other embodiments and can be practiced or carried out in various ways. It is also understood that the phraseology and terminology employed herein is for descriptive purpose and should not be regarded as limiting.

Compounds In some embodiments, the present invention relates to compounds having Formula I:

ϊ wherein:

Z is chosen from the group consisting of O and S; n is an integer from 0 to 20; each X is independently selected from the group consisting of H and F (each of the between 1 and 42 Xs is independently selected from the group consisting of H and F); each of R ¹ , R ² R ³ and R ⁴ is independently selected from the group consisting of hydrogen, alkyl, hydroxyalkyl, haloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, amino and -NR ⁵ R ⁶ , or, alternatively, two of R ² , R ³ and R ⁴ form a five- or six-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and

R ⁵ and R ⁶ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl and sulfonyl, or, alternatively R ⁵ and R ⁶ form a five- or six- member aromatic, heteroaromatic, alicyclic or heteroalicyclic ring; and salts and esters thereof.

In some embodiments, at least one of R ² , R ³ and R ⁴ is in an ortho or para position relative to the functional group bearing Z. In some embodiments, at least one of R ² , R ³ and R ⁴ is an electron donating group, for example is selected from the group consisting of alkyl, hydroxyalkyl, cycloalkyl, halo, hydroxy, alkoxy, aryloxy, amino and -NR ⁷ R ⁸ , where R ⁷ and R ⁸ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl and sulfonyl, or, alternatively R ⁷ and R ⁸ form a five- or six-member aromatic, heteroaromatic, alicyclic or heteroalicyclic ring. Exemplary compounds of Formula I prepared and tested are shown in Figure IB.

In some embodiments, the present invention relates to compounds having Formula II: z

11

In Formula II, Z, n, X, R ² , R ³ and R ⁴ are as defined above with reference to Formula I. An exemplary compound of Formula II tested was NP- 18b depicted in Figure ID

Synthesis

Synthesis of the compounds of the invention is within the ability of a person having ordinary skill in the art of synthetic chemistry upon reading the disclosure herein, including the examples section hereinbelow and Figure 2. Additionally, synthesis of compounds having Formula I has been described in "Reactions of aromatic and heteroaromatic β-amino- β- (polyfluoroalkyl)vinyl ketones with ethylenediamine. A new synthesis of N,N'-unsubstituted imidazolidines" by Sosnovskikh VY and Kutsenko VA in Russian Chemical Bulletin (Izvestiya Akademii Nauk, Seriya Khimicheskaya) 1999, 48(3), 540-551.

Compositions

According to some embodiments, compositions comprising compounds of Formula I and/or Formula II are used for treating an infection by microbes, especially microbes such as fungi and oomycetes. The present invention provides various different types of compositions. As noted, some embodiments of the present invention provide for compounds, antimicrobial compositions, methods and uses that relate to treatment of infections by microbes.

In some embodiments, the infections are infections by fungi, such as yeasts or molds. The fungi may comprise one or more members of the phyla Chytridiomycota; Blastocladiomycota; Neocallimastigomycota; Zygomycota; Glomeromycota; Ascomycota; Basidiomycota or Deuteromycota.

The Ascomycota may comprise, for example, Aspergillus, including Aspergillus fumigatus and Aspergillus niger; Sclerotinia, such as Sclerotinia sclerotiorum, Sclerotinia sclerotiorum, Sclerotinia trifoliorum and Sclerotinia minor; Botrytis, such as Botrytis cinerea, Botrytis convolute, Botrytis polyblastis, Botrytis allii and Botrytis fabae; Penicillum, including Penicillium chrysogenum, Penicillium citrinum, Penicillum italicum, Penicillium janthinellum, Penicillium marneffei, Penicillium purpurogenum, Penicillium expansum, Penicillium funiculosum and Penicillium digitatum; Neurospora, including Neurospora crassa; Alternaria, including Altemaria altemata, Altemaria arborescens, Alternaria arbusή, Alternaria blumeae, Alternaria brassicae, Alternaria brassicicola, Alternaria brunsii, Alternaria carotiincultae, Alternaria conjuncta, Alternaria dauci, Alternaria euphorbiicola, Alternaria gaisen, Alternaria infectoria, Alternaria japonica, Alternaria molesta, Alternaria panax, Alternaria petroselini, Alternaria selini, Alternaria solani, and Alternaria smyrnii; Verticillium, including Verticillium dahlia, Verticillium albo-atrum, and Verticillium lecanii; and Candida, such as Candida albicans.

The Basidiomycota may comprise, for example, Agaricomycotina (including Rhizoctonia, such as Rhizoctonia solani), Ustilaginomycotina, Pucciniomycotina, Wallemiomycetes and Entorrhizomycetes. In some embodiments, the infections are infections by oomycetes. The oomycetes may comprise one or more members of the group consisting of Phytophthora (such as, for example, Phytophthora infestans; Phytophthora sojae; Phytophthora alni; Phytophthora cactorum; Phytophthora capsi; Phytophthora cinnamomi; Phytophthora fragariae; Phytophthora kernoviae; Phytophthor palmivora; Phytophthora ramorum; or Phytophthora quercind), Pythium (such as, for example, Pythium aphanidermatum or Pythium insidiosum), and downy mildew.

In some embodiments, the present invention is applied to the field of pharmaceuticals, both human and non-human (veterinary medicine) to treat microbial infections. In some embodiments, the composition of the present invention is applied to the treatment of an infection of humans involving fungi or yeast. The infection may occur, for example, at the site of an injury, in mucous membranes, the sinuses or the lungs. In some embodiments, the infection may be caused by a species of the yeast Candidia, such as Candida albicans, and may comprise diaper rash, infection of the mouth, genital infection in women, or nail infection.

In some embodiments, the composition of the present invention is applied to the treatment of fungal infection of humans such as Athlete's foot (tinea pedis); jock itch (tinea crutis); scalp and hair infections (tinea capitis); finger or toenail infection (tinea unguium); ringworm of the body (tinea corporis); and barber's itch (tinea barbae).

In some embodiments, the composition of the present invention is applied to the treatment of a systemic fungal infection of mammals, including humans, such as

Aspergillosis, caused by Aspergillus fumigatus or other Aspergillus species; an infection caused by Alternaria alternate, such as upper respiratory infections, particularly in AIDS patients; or an infection caused by Pythium insidiosum in humans, dogs, cattle and horses.

In some embodiments, the present invention is applied to the field of agriculture, both of animal husbandry and of plants. In some such embodiments, the present invention is applied prophylactically, for example to prevent or delay the onset of mold.

In some embodiments, the present invention is applied for use in treating plants or plant matter. Such embodiments include, but are not limited to, treating living plants, treating seeds, treating harvested fruit, grain and other plant products.

In some embodiments wherein plants or plant matter are treated, the microbe is a fungus of the phylum Ascomycota, such as Aspergillus, including Aspergillus niger;

Sclerotinia, such as Sclerotinia sclerotiorum, Sclerotinia trifoliorum and Sclerotinia minor; Botrytis, such as Botrytis cinerea, Botrytis convolute, Botrytis polyblastis, Botrytis allii and

Botrytis fabae; Penicillum, such as Penicillium chrysogenum, Penicillium citrinum,

Penicillum italicum, Penicillium janthinellum, Penicillium marneffei, Penicillium purpurogenum, Penicillium expansum, Penicillium funiculosum and Penicillium digitatum;

Verticillium, including Verticillium dahlia, Verticillium albo-atrum, and Verticillium lecanii. A plant in which an Aspergillus niger infection occurs may comprise, for example, a grape, an onion or a peanut.

A plant in which a Sclerotinia sclerotiorum infection occurs may comprise, for example, cabbage, common bean, citrus, celery, coriander, melon, squash, soybean, tomato, lettuce, and cucumber. A plant in which a Botrytis cinerea infection occurs may comprise, for example, asparagus, bean, beet, carrot, celery, chicory, crucifers, cucurbits, eggplant, endive, grape, lettuce, onion, pepper, potato, raspberry, rhubarb, rutabaga, shallot, strawberry, tomato, turnip; or an ornamental plant such as ornamental plants including: anemone, begonia, calendula, chrysanthemum, dahlia, dogwood, fuchsia, geranium, hawthorn, heather, hydrangea, marigold, pansy, periwinkle, petunia, rose, snapdragon, sunflower, sweet pea, violet, and zinnia.

A plant in which a Penicillium digitatum infection occurs may comprise, for example, a citrus fruit. In some embodiments, the present invention is applied for preventing formation of mold on baked goods, such as bread, for example formation of molds similar to the mold such as Neurospora, such as Neurospora crassa.

In some embodiments, the present invention is applied for use in treating soil. Such embodiments include but are not limited to treating agricultural land during soil irrigation, prior to sowing, prior to sprouting or when the plants have already emerged.

In some embodiments, the present invention is applied for use in treating edible products. Such embodiments include but are not limited to wrappings for food products such as cheese and fruit, a bread additive, a jam additive or a tomato product (e.g., tomato juice and paste) additive. In some embodiments, the present invention is applied to other fields, for example in the field of surface treatments for the formulation of paint compositions (such as for prevention of growth of Alternaria alternata on paint surfaces), lacquers or anti-mold and mildew compositions.

Compositions of the present invention may comprise, in addition to the antimicrobial agents, an approved carrier or a diluent that does not cause significant damage to an organism and does not abrogate the biological activity and properties of the administered antimicrobial agent.

The compositions of the present invention may comprise, in addition to the compounds of Formula I and/or Formula II, an additional antifungal agent. The additional antifungal agent may comprise any antifungal agent known in the art, such as polyene antifungals; imidazole, triazole and thiazole antifungals; allylamines; echinocandins; and others.

Examples of polyene antifungals include natamycin, rimocidin, filipin, nystatin, amphotericin B, and candicin. Examples of imidazoles include miconazole, ketocanazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole,

Examples of triazoles include fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, and terconazole.

Examples of thiazoles include abafungin.

Examples of allylamines include terbinafine, amorolfine, natifine, and butenafine.

Examples of echinocandins include anidulafungin, caspofungin and micafungin.

Examples of other suitable antifungal agents include ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofluvin, haloprogin and sodium bicarbonate.

The additional antifungal agent may comprise a natural product having antifungal activity, such as, for example, allicin, tea tree oil, citronella, iodine, lemon grass, olive leaf, orange oil, palmarosa oil, patchouli, lemon myrtle, neem seed oil, coconut oil, zinc or selenium. According to some embodiments, the compound of Formula I and/or the compound of

Formula II, esters thereof or salts thereof, and the additional antifungal agent, are present in a single composition. Alternatively, the compound of Formula I and/or the compound of Formula II, esters thereof or salts thereof, and the additional antifungal agent, may be administered sequentially or simultaneously in separate dosage forms. A combination of the compound of Formula I and/or the compound of Formula II, esters thereof or salts thereof, and the additional antifungal agent may produce a synergistic effect, such that the effect of the combination is greater than the effect of the compound of Formula I and/or the compound of Formula II, esters thereof or salts thereof alone, and of the additional antifungal agent alone. According to some embodiments, there is provided a composition comprising a combination of a compound of Formula I and/or a compound of Formula II, esters thereof or salts thereof, and an additional antifungal agent, wherein the amount of the compound of Formula I and/or the compound of Formula II, esters thereof or salts thereof, and the amount of the additional antifungal agent alone is insufficient to achieve the therapeutic effect achieved by the administration of the combination of the two or more ingredients.

AG-5 and NP-18b compositions for treating Oomycete infections

In a preferred embodiment, a composition of the invention, especially a composition comprising a compound of Formula AG-5 and/or NP- 18b is used to treat a microbial infection of plants, especially to treat an Oomycete infection, especially of Pythium, especially Pythium aphanidermatum.

In an embodiment, a sprayable composition, comprising a liquid carrier and an effective concentration of AG-5 and/or NP- 18b, is applied as a foliar spray on affected plants. In an embodiment, a composition comprising a liquid carrier and an effective concentration of AG-5 and/or NP- 18b is applied to soil as a soil wash or during plant irrigation.

NP -18a compositions for treating Penicillium infections Penicillium is a genus of molds that attacks stored foodstuffs, including cooled food stuffs. Exceptionally vulnerable are fruit such as citrus fruit, bread and cereal products and meats. Stored grains, including feed grains, are susceptible to Penicillium infection which leads to the production of mycotoxins that may damage mammals consuming the grain.

The genus Penicillium has several species. Examples include Penicillium chrysogenum, Penicillium citrinum, Penicillum italicum, Penicillium janthinellum,

Penicillium marnejfei, Penicillium purpurogenum, Penicillium expansum, Penicillium funiculosum and Penicillium digitatum.

Penicillium digitatum is a common mold found on post-harvest citrus and grain, and recognized by the appearance of green brush-like fruiting structures. In a preferred embodiment, a composition of the invention, especially a composition comprising a compound of Formula NP- 18a is used to treat a microbial infection of food stuffs, especially to prevent and/or delay development of a Penicillium infection, especially of Penicillium digitatum, especially for harvested fruit such as citrus and other fruits or grain for animal feed. In an embodiment, a sprayable composition, comprising a liquid carrier (and in some embodiments additional inert materials) and an effective concentration of NP- 18a, is sprayed on the harvested fruit.

In an embodiment, the harvested fruit is washed, for example in a bath, with a composition comprising a liquid carrier and an effective amount of NP- 18a.

NP-18b and AG-5 compositions for treating mold on baked goods

In a preferred embodiment, a composition of the invention, especially a composition comprising a compound of Formula AG-5 and/or NP- 18b is used to treat or prevent growth of mold on baked goods, such as bread, for example formation of molds similar to Neurospora such as Neurospora crassa.

Preparation Generally, a composition comprises at least one compound having Formula I or

Formula II and a carrier. A carrier is formulated in accordance with the desired use of the composition. One skilled in the relevant art for which a given composition is to be used is able to formulate a desired composition.

Compositions of the present invention are generally formulated to be topical compositions, whether to be applied to a surface of an animal (e.g., skin, mucous membranes), a surface of a plant (e.g., leaves, stalks, fruit, seeds), soil or surfaces such as walls and other inanimate surfaces. Typical topical compositions include creams, sprays, ointments, lotions, solutions, suspensions, sprays, foams, washes, paints and lacquers.

In the field of pharmaceuticals (both human and non-human), one skilled in the art may consult standard references such as the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, which is herein incorporated by reference as if fully set-forth herein for guidance and techniques for Formulation and administration of compositions of the invention.

In other fields, one skilled in the art may consult standard references used in a particular field for guidance and techniques for formulation and administration of compositions of the invention.

For example, one skilled in the art of polymer technology is able to integrate the compounds described herein into a food wrapping, a paint, a lacquer, generally by mixing an effective amount of the composition into a known non-polymerized precursor. For example, one skilled in the art of food wrapping and papers is able to integrate the compounds described herein into a food wrapping, by application of a compound in a suitable carrier to a food wrapping or container (e.g. of paper, cardboard or plastic). In some embodiments, the carrier includes an adhesive or polymerizing component that assists in maintaining the applied compound associated with the wrapping or container. In some embodiments, the carrier includes volatile components that, subsequent to application and evaporation of the volatile components, leave the compounds associated with the wrapping or container. In some embodiments, at least some of an applied compound is absorbed into a wrapper or container. For example, one skilled in the art of food technology is able to integrate the compounds described herin into food stuffs, for example adding a compound to dough prior to baking in bread and baked goods; adding a compound to a meat mixture during the preparation of a processed meat product (sausage); adding a compound to a fluid edible product such as jam, a syrup or a tomato product.

For example, one skilled in the art of plant care and agriculture is able to integrate the compounds described herein into compositions useful in agriculture, useful for administering to plants and useful for treating plant products such as adding compounds to growth media for tissue culture and hydroponics; soil washes and treatments; as additives to irrigation water; as aerial and terrestrial sprays suitable for sprouts, trunks, branches, stems, leaves and fruit and as baths, washes and sprays for harvested fruit, vegetables and grains.

EXPERIMENTAL

Reference is now made to the following examples which, together with the above description, illustrate the invention in a non-limiting fashion.

Synthesis of Compounds

Analytical HPLC was performed on a 250x4.2 mm Lichroprep RP- 18 column from

Merck, with a 1 niL/min flow and detection at 214 nm. The eluents were triply distilled water and HPLC-grade CH ₃ CN (containing 0.1% TFA) or MeOH. Mass spectra were measured in the positive and negative modes using a quadruple mass spectrometer equipped with an electrospray ionization source and cross-flow inlet.

¹ H and ¹³ C NMR spectra were recorded at 300 and 75 MHz, respectively in CDCl ₃ , unless otherwise indicated. Assignments in the final products were supported by 2D COSY, TOCSY, NOESY, ROESY, HMBC and/or HMQC spectroscopy. AU chemical shifts are reported with respect to TMS. Chromatography was carried out by standard flash chromatography and TLC on silica-gel (Merck 7735).

Mass spectra were measured in the positive mode using a quadrupole mass spectrometer equipped with an electrospray ionization source and cross-flow inlet. Unless otherwise stated, chemicals and materials were obtained from Sigma-Aldrich

(St. Louis, MO, USA). NP- 18b was commercially available and obtained from Sigma- Aldrich (St. Louis, MO, USA).

NP- 18a, NP-5, NP-17, NP-6, NP-IO, NP- 19a, AG-5, AG-2, AG-4, NP-25 were synthesized using standard methods and commercially available materials substantially as depicted in Figure 2 and detailed hereinbelow where compound 2 was trifluoroacetamide (prepared from ammonia and trifluoroethyl ester) and compound 3 was trifluoroacetonitrile, excepting NP-25 where compound 2 was pentafluoropropionamide (prepared from ammonia and pentafluoropropyl ester) and compound 3 was pentafluoropropiononitrile.

Preparation of 3, trifluoroacetonitrile and pentafluoropropiononitrile

Sulfuric acid (2 ml) was slowly added to 100 ml methanol while stirring in an ice bath. Trifluoroacetic acid (118 gr, 78 ml) or pentafluoropropionic acid (170 gr) was added to the cooling methanol solution. The mixture was stirred while heating (40-45 ⁰ C) for 6 h. After cooling to r.t, water was added and the resulting trifluoromethyl ester or pentafluoroethyl ester organic phase was separated and dried over anhydrous sodium sulfate and filtered.

Ammonia in methanol (IL, 2.0 M) was added to the filtrate including the fluorinated ester while cooling with ice. The mixture was stirred overnight at room temperature. Solvent and unreacted ammonia were evaporated and the remaining white solid was dried under vacuum. As depicted in Figure 2, the obtained trifluoroacetamide or pentafluoropropionamide 2 was rapidly mixed with phosphorus pentoxide in 3 times excess in a 500 ml flask so that the condensation reaction started immediately. The reaction mixture was heated at 15O ⁰ C and the thus-obtained gaseous trifluoroacetonitrile or pentafluoropropiononitrile 3 was directly bubbled into the reaction mixture described in the next step.

First general procedure for preparation of β-trifluroalkyl aminovinyl ketones

As depicted in Figure 2, to a mixture of 10 mmol sodium tert-butoxide in 80 ml of dry

THF, 4 mmol of an acetophenone 4 was added drop wise and then trifluoroacetonitrile 3

(produced from 40 mmol trifluoroacetamide 2 and 120 mmol phosphorus pentoxide as described above) was bubbled during 2 hour through the reaction mixture using a dry ice- acetone condenser. After ending of the bubbling of 3, the mixture was stirred for 5 h at RT.

A solution of HCl- water IN was added drop wise and after 10 min ethyl acetate was added and the organic layer was separated and washed with brine. The organic layer was separated, dried with anhydrous Na ₂ SO ₄ , filtered, and concentrated under vacuum. AG-2: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19 (s, IH), 7.68 (d, J=10.7 Hz, 2H), 8(d, J=10.7 Hz, d). 1 ³ CNMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 142.2, 132.7, 131.5, 130.6, 118.4, 115.7, 95.5.

5 AG-4: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19(s, IH), 7.06 (t, J=5.35 Hz, 2H), 7.68 (m, 2H). ¹³ CNMR (δ, ppm, CDCl ₃ ): 189.7, 168.7, 160.7, 133.5, 131.5, 116, 95.5.

AG-5: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19 (s, IH), 7.41 (d, J=10.7 Hz, 2H), 7.82 (d, J=10.7 Hz, 2H). ¹³ CNMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 140.1, 136, 131.5, 131.3, 129.3, 95.5. 10

NP-18a: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19 (s, IH), 6.89 (d, J=10.7 Hz, 2H), 7.82 (d, J=10.7 Hz, 2H). ¹³ CNMR (δ, ppm, CDCl ₃ ): 189.7, 164.3, 160.7, 131.3, 130.5, 125.5, 116.4, 95.5.

NP-19a: ¹ HNMR (δ, ppm, CDCl ₃ ): 1.5 (s, 9H), 6.19 ( s, IH), 7.45 (d, J=10.7 Hz, 2H), 7.85 15 (d, J=10.7 Hz, 2H). ¹³ CNMR (δ, ppm, CDCl ₃ ): 189.7, 164.3, 160.7, 131.5, 131.3, 130.5, 116.4, 95.5.

NP-17: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19 (s, IH), 7.58 (m, 3H), 7.99 (m, 4H), 8.40 (s, IH). 1 ³ CNMR (δ, ppm, CDC13): 189.7, 160.7, 135.6, 134.7, 132.5, 131.5, 129.5, 128.6, 128.5, 20 128.3, 127.7, 126.9, 124.2, 95.5.

HRMS (DI, m/z) calculated for C _I4 HI ₀ F ₃ NO (MH ⁺ ) 266 found 266.077.

NP-5: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.21 (s, H), 7.6 (t, J=8.82 Hz, IH), 7.8 (d, J=8.82 Hz, 2H), 8.09 (d, J=8.82 Hz, 2H), 8.19 (s, IH). ¹³ CNMR (δ, ppm, CDC13): 189.7, 160.7, 138.2, 133.2, 25 131.5, 130.9, 129.6, 126.2, 124.2, 95.5.

HRMS (DI, m/z) calculated for CnH ₇ F ₆ NO (MH+) 284.04 found 284.051.

NP-6: ¹ HNMR (δ, ppm, CDCl ₃ ): 6.19 (s, IH), 7.38 (t, J=6.25, IH), 7.69 (d J=12.5 Hz, IH), 7.81 (d, J=12.5 Hz, IH), 8.09 (s, IH). ¹³ CNMR (δ, ppm, CDC13): 189.7, 160.7, 140.1, 137.4, 30 133.4, 131.5, 128.9, 123.6, 95.5 .

HRMS (DI, m/z) calculated for Ci ₀ H ₇ BrF ₃ NO (MH ⁺ ) 293.295 found 293.97.

NP-10: ¹ HNMR (δ, ppm, CDCl ₃ ): 0.98 (t, J=9.31Hz, 3H), 2.00 (q, J=9.31 Hz, 2H), 7.51 (d, J=10.34, 2H), 7.86 (d, J=10.34 Hz). ¹³ CNMR (δ, ppm, CDC13): 190.5, 152.5, 140.1, 136,

35 131.3, 129.3, 125.3, 112.9, 12.6, 11.6. Second general procedure for preparation of β-trifluroalkyl aminovinyl ketones

As depicted in Figure 2, 4 mmol of acetophenone were added drop wise to a mixture of 10 mmol of sodium tert-butoxide in 100 ml of dry THF and then trifluoroacetonitrile or, when preparing NP-25, pentafluoropropiononitrile 3 (produced from 15 g trifluoroacetamide 5 or equivalent amount of pentafluoropropionamide 2 and 45 g of P ₂ Os as described above) was bubbled during 2 hour through the reaction mixture using a dry ice-acetone condenser. After ending of the bubbling of 3, the mixture was stirred for 5 h at RT.

10 mmol commercial 4N HCl-dioxane solution was added drop- wise and after 10 min the mixture was filtered and the dioxane was evaporated under vacuum. The crude product 10 was recrystallized from hexane.

AG-2 was prepared from 3 and 4-cyanoacetophenone (1 g, 6.9 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent, yielded 1.44 g AG-2 (87 % yield) as a colorless oil. iw 15 (KBr): 2880, 1670, 1635, 1270 cm ¹ ; HRMS (DI, m/z) calculated for CnH ₇ F ₃ N ₂ O (MH ⁺ ) 241.05 found 241.04; ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, CH=C), 7.68 (d, 2, /=10.7 Hz, H-2 ), 8.14 (d, 2, /=10.7 Hz, H-3). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 142.2, 132.7, 131.5, 130.6, 118.4, 115.7, 95.5.

20 AG-4 was prepared from 3 and 4-fluoroacetophenone (1 g, 7.3 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent yielded 1.28 g of AG-4 (85 % yield) as a colorless oil. iw (KBr): 1670, 1650, 1190 cm ¹ ; HRMS (DI, m/z) calculated for Ci ₀ H ₇ F ₄ NO (MH ⁺ ) 234.046 found 234.052; ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, CH=C), 7.06 (t, 2, /=5.35 Hz, H-3), 7.66 (m,

25 2, H-2). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 168.7, 160.7, 133.5, 131.5, 116, 95.5.

AG-5 was prepared from 3 and 4-chloroacetophenone (1 g, 6.5 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent yielded 1.38 AG-5 (81 % yield) as a colorless oil.iw (KBr): 30 1670, 1590, 1380, 1060 cm ¹ ; HRMS (DI, m/z) calculated for CI ₀ H ₇ CIF ₃ NO (MH ⁺ ) 250.017 found 250.020: 252.018 (3:1); ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, CH=C), 7.41 (d, 2, /=10.7 Hz, H-3), 7.82 (d, 2, /=10.7 Hz, H2). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 140.1, 136, 131.5, 131.3, 129.3, 95.0. NP-18a was prepared from 3 and 4-hydroxyacetophenone (1 g, 7.4 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (6:4) as eluent yielded 1.19 g NP-18a (76 % yield) as a white powder: iw (KBr): 3500-3180(bs), 1660, 1520, 1450, 1230 cm ¹ ; HRMS (DI, m/z) calculated for 5 Ci ₀ H ₈ F ₃ NO ₂ (MH ⁺ ) 232.05 found 232.04; ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, GH=C), 6.89 (d, 2, /=10.7 Hz, H-3), 7.82 (d, 2, /=10.7 Hz, H-2). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 164.3, 160.7, 131.3, 130.5, 125.5, 116.4, 94.5.

NP-19a was prepared from 3 and 4-BOC-amino acetophenone (1 g, 4.3 mmol) following the

10 second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (6:4) as eluent yielded 1.01 g NP-19a (72 % yield) as a colorless oil. iw

(KBr): 1660, 1500, 1350, 1120 cm ¹ ; HRMS (DI, m/z) calculated for Ci ₅ Hi ₇ F ₃ N ₂ O ₃ (MH ⁺ )

331.119 found 331.126; ¹ H NMR (CDCl ₃ ): δ 1.5 (s, 9, ¹ Bu), 6.19 ( s, 1, CH=C), 7.45 (d, 2,

/=10.7 Hz, H-3), 7.85 (d, 2, /=10.7 Hz, H-2). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 164.3, 160.7,

15 131.5, 131.3, 130.5, 116.4, 90.0.

NP-17 was prepared from 3 and acetonapthone (1 g, 5.9 mmol) following the second general procedure. Purification by column chromatography on silica gel using w-hexane-EtOAc (7:3) as eluent yielded 1.1 Ig NP-17 (74 % yield) as a colorless oil; υ _max (KBr): 1655, 1570, 1360, 20 1240 cm ¹ ; HRMS (DI, m/z) calculated for CI ₄ HI ₀ F ₃ NO (MH ⁺ ) 266.071 found 266.077J ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, CH=C), 7.58 (m, 3, H-3,7,8), 7.99 (m, 3, H-4,5,6), 8.40 (s, 1, H- 2). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 135.6, 134.7, 132.5, 131.5, 129.5, 128.6, 128.5, 128.3, 127.7, 126.9, 124.2, 93.0.

25 NP-5 was prepared from 3 and 3-trifluoromethyl acetonapthone (I g, 4.29 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent yielded 1.02 NP-5 (84 % yield) as a colorless oil. iw (KBr): 1665, 1570, 1360, 1240 cm ¹ ; HRMS (DI, m/z) calculated for CnH ₇ F ₆ NO (MH+) 284.048 found 284.051; ¹ H NMR (CDCl ₃ ): δ 6.21 (s, 1, CH=C), 7.6 (t, 1, /=8.82 Hz, H-3), 7.8 (d, 2,

30 /=8.82 Hz, H-2), 8.09 (d, 2, /=8.82 Hz, H-4), 8.19 (s, 1, H-6). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 138.2, 133.2, 131.5, 130.9, 129.6, 126.2, 124.2, 94.0.

NP-6 was prepared from 3 and 3-bromoacetophenone (1 g, 5.0 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent yielded 1.32g NP-6 (89 % yield) as a colorless oil: iw (KBr): 1675, 1640, 1410. 1305, 1060 cm ¹ ; HRMS (DI, m/z) calculated for Ci ₀ H ₇ BrF ₃ NO (MH ⁺ ) 294.295 found 293.972, 295.974 (1:1); ¹ H NMR (CDCl ₃ ): δ 6.19 (s, 1, CH=C), 7.38 (t, 1, /=6.25, IH, H-4), 7.69 (d, 1 /=12.5 Hz, H-3), 7.81 (d, 1, /=12.5 Hz, H-2), 8.09 (s, 1, H-6). ¹³ C NMR (δ, ppm, CDCl ₃ ): 189.7, 160.7, 140.1, 137.4, 133.4, 131.5, 128.9, 123.6, 95.5.

NP-10 was prepared from 3 and 4-chlrobutyrophenone (1 g, 5.5 mmol) following the second general procedure. Purification by column chromatography on silica gel using n- hexane-EtOAc (8:2) as eluent yielded 1.17g NP-10 (68 % yield) as a colorless oil: iw (KBr): 1665, 1630, 1390 cm ¹ ; HRMS (DI, m/z) calculated for Ci ₂ HnClF ₃ NO (MH ⁺ ) 277.048 found 277.043: 277.044 (3:1); ¹ H NMR (CDCl ₃ ): δ 0.98 (t, 3, /=9.3 IHz, CH ₂ CH ₃ ), 2.00 (q, 2, /=9.31 Hz, C=CCH ₂ CH ₃ ), 7.51 (d, 2, /=10.34m H-3), 7.86 (d, 2, /=10.34, H-4). ¹³ C NMR (δ, ppm, CDCl ₃ ): 190.5, 152.5, 140.1, 136, 131.3, 129.3, 125.3, 112.9, 12.6, 11.6.

NP-25 was prepared from gaseous 2,2,3,3, 3-pentafluoropropanenitrile (prepared from commercial CF ₃ CF ₂ CONH ₂ in the same manner as for 3) and 4-chlrobutyrophenone (1 g, 6.9 mmol) following the second general procedure. Purification by column chromatography on silica gel using w-hexane-EtOAc (8:2) as eluent yielded 1.18g NP-25 (57 % yield) as a colorless oil: υ _max (KBr): 1660, 1600, 1470, 1290 cm ¹ ; HRMS (DI, m/z) calculated for C _II H _I7 C1F ₅ NO (MH ⁺ ) 300.014 found 300.016:302.017 (3:1); ¹ H NMR (CDCl ₃ ): δ 6.23 (s, 1, CH=C), 7.40 (d, 2, /=10.7 Hz, H-3), 7.84 (d, 2, /=10.7 Hz, H-4). ¹³ C NMR (δ, ppm, CDCl ₃ ): 191.2, 161.6, 141.7, 1364, 136.9, 133.8, 130.6, 127.1, 97.3.

Phytotoxicity Phytotoxicity of the compounds was tested by placing seeds of Arabidopsis on water agar containing the test compounds (using a stock solution prepared as described above).

Germination rates and radicle length were measured and compared to those of seeds germinating on dishes lacking the test compounds.

Figure 3A shows the effect of the tested compounds on germination of Arabidopsis seeds. Figure 3B shows the effect of the compounds on the growth of Arabidopsis leaves.

Further assessment of phytotoxicity was performed by placing seeds of alfalfa

(Medicago sativa L.) on water agar containing the compounds, as described above (not shown). Additional phytotoxicity tests included the exposure of freshly-detached tomato

{Solatium lycopersicum L.) leaves (maintained on moist filter paper placed in Petri dishes) to the various compounds. The test compounds were administered, by drop- wise topical application (10 or lOOμl per leaflet). The appearance of necrosis in the vicinity of the droplet administration point within 7 days was considered as a phytotoxic response. The compounds were found to have no significant phytotoxic effect.

Anti-microbial activity

Preliminary

Various compounds having Formula I were tested and found to have in vitro antimicrobial activity in concentrations of 5 μM - 100 μM against Botrytis cinerea, Aspergillus niger and Verticillium dahliae. It was seen that the antimicrobial activity varied with different n and different substituents R ¹ , R ² , R ³ and R ⁴ . Preliminary results indicate that thioketone compounds (where Z=S) have a greater antifungal activity than ketone compounds (where Z=O).

In vitro anti microbial activity

The ten compounds synthesized as described above, as well as commercially available compound NP- 18b (4'-hydroxyacetophenone), were tested at a concentration of 10 μM and 100 μM for antimicrobial activity against the fungi Penicillium digitatum (a mold), Botrytis cinerea (a mold), Neurospora crassa (a mold), Altemaria alternata, Rhizoctonia solani and Sclerotinia sclerotiorum (results not shown) and also the oomycete Pythium aphanidermatum.

The fungi Sclerotinia sclerotiorum (isolate 1980; 12), Botrytis cinerea (isolate BcI16; 13), Penicillium digitatum (14) Altemaria alternata and Rhizoctonia solani (both from the fungal collection of the Dept. of Plant Pathology and Microbiology) and Neurospora crassa (strain 74-OR23-1A, The Fungal Genetics Stock Center; 75) were used during this study. In addition, the oomycete Pythium aphanidermatum (from the Dept. of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel) was also used as a test organism. Strains were cultured on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI), unless otherwise stated.

Initial toxicity against the fungi (excluding S. sclerotiorum) and the oomycete was assessed by culturing the test organsims on Petri dishes containing 10ml of PDA amended with either 10 or 100 μM of the ten original test compounds. Stock solutions of the different compounds were prepared in dimethyl sulfoxide (DMSO) and aliquots (not exceeding 0.001% v/v, a concentration that had no observable effect on the test organisms) were added to warm (5O ⁰ C) sterile medium prior to dispensing to the Petri dishes.

The results of these tests are show in Figure 4, and summarized in Figure 5 and Table 1.

Table 1: Inhibitory effect (percent of control) of various compounds on radial growth of five fungi and one Oomycete ^Indicates radial growth was faster than the control)

It is seen that all the test compounds of Formula I and Formula II have antifungal and antioomycete activity. Nonetheless, significant differences in toxicity were observed among these compounds. For example, the highest toxicity levels were evident with AG-5 while NP- 25, NP-10 and NP-19a were significantly less toxic. The fungi least susceptible to most of the compounds tested were A. alternata and P. digitatum. None of the compounds tested inhibited radial growth of A. alternaria more than 50%

(even at a 100 micromolar concentration) and only AG-5 inhibited P. digitatum by more than 50%. In contrast, N. crassa appeared to be the most sensitive to the compounds at the higher concentrations tested, AG-5, NP-6 and AG-4 inhibited radial growth almost entirely.

The only oomycete phytopathogen tested in this study, P. aphanidermatum, was sensitive mainly to higher concentrations of NP- 18a, NP- 18b, AG-5 and NP-6, while the other compounds tested inhibited radial growth only to a limited extent.

Anti-microbial activity against Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are necrotrophic phytopathogenic filamentous ascomycetes known to attack more than 400 and 200 plant species, respectively (1-7). Diseases caused in economically important plants by these fungi occur worldwide, cause considerable damage, have proven difficult to control (culturally or chemically) and host resistance to these fungi is inadequate. Annual losses of crops from diseases caused by

S. sclerotiorum and B. cinerea are in the multimillion dollar range. In addition, their ubiquitous prevalence along with the intensive use of fungicides for their control has resulted in repeated appearance of fungicide-resistant strains (8-11).

Based on the outcome of the initial assessment of toxicity of the compounds to the test organisms, further analysis was conducted on certain of the original compounds tested and their toxicity to B. cinerea and S. sclerotiorum.

The in vitro antimicrobial activity of NP- 18a, NP- 18b and AG-5 was tested at a concentration of 10 μM and 100 μM for antifungal activity towards Botrytis cinerea and Sclerotinia sclerotiorum.

The results are presented in Figures 6A, 6B, 6C, and 6D, and summarized in Figure 6E. As shown in the Figures, each of the compounds tested exhibited antifungal activity against Sclerotinia sclerotiorum and Botrytis cinerea, with the most effective being AG-5, particularly at concentration of 100 μM.

Further analysis was focused on the effects of compound concentrations (0-50) micromolar of five of the original compounds tested (NP- 18a, NP- 18b, AG-5, NP-6 and NP-

25) on S. sclerotiorum and B. cinerea. The two fungi were cultured at 17 ⁰ C and 22 ⁰ C, respectively, for 48 hrs, after which radial growth was measured and compared to that of the fungal strains cultured in medium not amended with the antifungal compounds.

Assessment of antimicrobial activity of the various compounds was performed by culturing mycelial discs of the test phytopathogens on Petri dishes containing 10ml of standard growth media (potato dextrose agar) amended with various concentrations of the test compounds. Stock solutions of the different compounds were prepared in dimethylsulf oxide (DMSO) and aliquots (not exceeding 0.001% v/v, a concentration that had no observable effect on the fungi) added to warm (5O ⁰ C) sterile medium prior to dispensing to the Petri dishes.

Results are shown in Figure 7. In most instances, the fungal response to the tested 5 compounds was dose dependent. Both fungi exhibited very similar sensitivity to NP- 18a and AG-5. Commercially available NP- 18b, that lacks the trifluoroprop-l-en-2-amine part in the backbone, was the least active compound and, interestingly, at the lower concentrations may have even stimulated the radial growth of S. sclerotiorum. This was also evident at the lower concentrations of NP-25. Overall, the most effective compound (in terms of curbing radial

10 growth) was AG-5, even though the sensitivity of B. cinerea to this compound was significantly higher, at all concentrations, than that of S. sclerotiorum.

The in vitro antimicrobial activity of NP-5, NP-6, NP-IO, NP-17, NP-18b, NP- 19a, AG-4 and AG-5 was further tested for antifungal activity against the fungi Candida albicans (a yeast), Aspergillus fumigatus (a mold) and Aspergillus niger (a mold), using the minimum

15 inhibitory concentration test (MIC), and compared to the activity of Fluconazole and Amphotericin B. The results are summarized in Figure 8.

Synergism with fluconazole

A commercially available composition comprising fluconzole is diluted 10, 100 and 20 1000-fold. To each of eleven samples at each dilution are added 5 μM of one of the compounds having a Formula I or II (NP- 18b, AG-2, AG-4, AG-5, NP-18a, NP- 19a, NP-17,

NP-5, NP-6, NP-10 or NP-25).

The antimicrobial activity of each of the thirty-three thus-obtained compositions is compared to that of the commerically- available composition alone at 10, 100 and 1000-fold 25 dilution against Blastomyces dermatitidis, Candida spp., Coccidioides immitis, Cryptococcus neoformans, Epidermophyton spp., Histoplasma capsulatum, Microsporum spp. and

Trichophyton spp.

The antimicrobial activity of each of the thirty-three composition is further compared to that of 10 and 100 μM compositions of the compounds of Formula I or II alone. 30 Observed is that compositions comprising fluconazole together with compounds having Formula I and Formula II have a synergistic antimicrobial effect, such that the effect is achieved at concentrations of each component which would be insufficient alone to produce the effect. Synergism with amphotericin B

A commercially available composition comprising amphotericin B is diluted 10, 100 and 1000-fold. To each of eleven samples at each dilution are added 5 μM of one of the compounds having a Formula I or II (NP- 18b, AG-2, AG-4, AG-5, NP-18a, NP- 19a, NP-17, NP-5, NP-6, NP-IO or NP-25).

The antimicrobial activity of each of the thirty-three thus-obtained compositions is compared to that of the commerically- available composition at 10, 100 and 1000-fold dilution against Candida spp. and Cryptococcus neoformans.

The antimicrobial activity of each of the composition is further compared to that of 10 and 100 μM compositions of the compounds of Formula I or II alone.

Observed is that compositions comprising amphotericin B together with compounds having Formula I and Formula II have a synergistic antimicrobial effect, such that the effect is achieved at concentrations of each component which would be insufficient alone to produce the effect.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the invention. Section headings are used herein to ease understanding of the specification and should not be construed as necessarily limiting.