AU2010352387B2 - Diyne compositions - Google Patents

Diyne compositions Download PDF

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AU2010352387B2
AU2010352387B2 AU2010352387A AU2010352387A AU2010352387B2 AU 2010352387 B2 AU2010352387 B2 AU 2010352387B2 AU 2010352387 A AU2010352387 A AU 2010352387A AU 2010352387 A AU2010352387 A AU 2010352387A AU 2010352387 B2 AU2010352387 B2 AU 2010352387B2
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Prior art keywords
diyne
infection
compound
fungus
spp
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AU2010352387A1 (en
Inventor
Arunan V. Gomathi
Katrine Buch Greve
Philipp Knechtle
Jean-Philippe Meyer
Panchapagese Muthuswamy Murali
Alexandra M. P. Santana Sorensen
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Evolva Holding SA
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Evolva AG
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Priority claimed from PCT/US2010/041515 external-priority patent/WO2011006061A1/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D307/00Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/54Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/06Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
    • A01N43/08Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings with oxygen as the ring hetero atom
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10Antimycotics

Abstract

A novel class of diyne compounds and diyne salts provided herein are effective and potent Ole1 protein inhibitors, useful for treating fungal pathogens. Compounds, fungicides and methods are provided as novel, potent and broad spectrum antifungal agents for treatment against a wide variety of fungal pathogens in humans and animals, and in the agricultural setting.

Description

WO 2011/134538 PCT/EP2010/063161 DIYNE COMPOSITIONS FIELD OF THE INVENTION [0001] The present invention relates to diyne salt and in particular potassium 5 diyne salts useful as antifungal agents. The diyne salts are useful as active agent in pharmaceutical compositions for treating infection by a fungus in an individual, such as a mammal, but the diyne salts are also useful for reducing the risk of or treating infection by a fungus in plants. 10 [0002] The present invention also relates to diyne compounds useful for treating fungal infections as well as to methods of treating fungal infections in various organisms, such as plants or mammals. The diyne compounds of the invention are in particular useful for treating fungal infections dependent on the activity of stearoyl CoA desaturase. 15 [0003] This invention further encompasses diynes and diyne salts as Ole protein inhibitors, as well as methods for treating fungal infections in humans, animals and plants. 20 BACKGROUND OF THE INVENTION [0004] The incidence of fungal infections is steadily rising as a consequence of antibiotic treatments, an immunosuppressed or immunocompromised population (mainly caused by cancer treatment, HIV, allergy-treatments, transplantations and general surgery) and an aging population. 25 [0005] Currently, an estimated 15,000 allogenic and 25,000 autologous stem cell transplants are performed worldwide yearly. In addition, from 1998 to 2002, 113,682 solid organ transplants were performed in the United States, which is a 20% increase over the previous 5-year period. Unfortunately, patients undergoing these life-saving 30 procedures are at increased risk for fungal infections for example by Aspergillus funigatus and other Aspergillus spp. due to their immuno-compromised condition. Additionally the populations of immuno-compromised patients due to HIV, cancer therapy, surgical non-transplants and general ageing also continue to increase and with it the number of cases of severe fungal infections for example systemic 1 WO 20111134538 PCT/EP2010/063161 candidiasis. Candida species account for 80% of infections in general medicine, 40% in HIV populations and 90% in both cancer therapy and surgical-non transplant cases. Candida is now the 4t1 largest cause of nosocomial blood stream infections. 5 [0006] Mortality from systemic fungal infections remains high despite the development of new antifungal agents, and Candida bloodstream infections in the United States are associated with a 40% crude mortality rate. Overall, since 1980, the mortality due to Aspergillusfunigatus has increased 357% and is continuing to increase. 10 [0007] Furthermore, during the last decade, there have been changes in the epidemiology of these systemic infections, with five species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) responsible for more than 90% of invasive infections due to Candida Candida spp. are the fourth most common cause 15 of nosocomial bloodstream infections, and while C. albicans was the predominant cause of Candida bloodstream infections in the early 80s, C. glabrata has emerged as the second most common cause in various part of the world, including the United States. 20 [00081 In addition to Aspergillus and Candida infections, other fungal pathogens such as Zygomycetes, Fusarium and Scedosporium spp. are becoming increasingly important. Their susceptibility to existing antifungals is limited and their mortality rate is >70% in patients with hematological malignancies. In other patients the mortality rates vary between 30 and 80%. 25 [0009] Onychomycosis is a fungal infection of the nails which is estimated to affect 2-13% of the general US population and up to 25% of the geriatric and diabetic populations. Common risk factors include age, male gender, diabetes, nail trauma, and chronic Tinea pedis (fungal infection of the foot). Onychomycosis has significant 30 cosmetic, psychological and social implications. In some patient subsets it has serious medical consequences (e.g. foot amputations in diabetics). [0010] Currently the infection is primarily treated with oral drugs, but this is not desirable for what is normally a non life threatening infection, as the currently used 2 WO 20111134538 PCT/EP2010/063161 antifungal drugs have significant toxicities. It also leads to poor compliance - multiple surveys have shown it is counter intuitive to patients to take a pill for c. 6 months to treat a toe nail infection, especially given there is no visible improvement for the first 2-3 months of treatment. Ideally a fast acting topical approach is desired but existing 5 topical drugs have very poor efficacy due to the difficulty of reaching the fungi that are located under the nail. Ciclopirox nail lacquer is the only FDA-approved topical agent available in the US for the treatment of onychomycosis, while amorolfine is a topical agent available in Japan and in Europe. However, these nail lacquer products have very limited efficacy in part because of their inability to penetrate to the nail bed 10 where the infection resides, but also due to the nature of the compounds. As a result of these limitations, only 14% of onychomycosis patients are currently treated with topical drug and just 7% receive systemic drug therapy. In addition, there is a >25% recurrence rate amongst "cures". 15 [0011] At present there are four major compound classes available for the treatment of fungal infections. They are listed in Table A. The use of these drugs may in some cases of fungal infection deliver reasonable results, however, as outlined above mortality caused by fungal infections is still high. Apart from insufficient efficacy there are furthermore several other problems associated with the existing 20 drugs: " Significant toxicity and/or patient sensitivity to the existing drugs e.g. liver toxicity is associated with many of the existing compounds (a significant problem, in particular due to the extended length of treatments) and Lamisil has cardiac toxicity. 25 0 Many pathogenic strains are insensitive or resistant against the anti-fungal drugs and resistance development is also of concern. * High relapse rate [0012] In addition in many cases the efficacy rate is poor. 30 [0013] In relation to treatment of certain fungal infections such as onycomycosis, additional problems are associated with the existing drugs: e Long onset to relief of symptoms 3 WO 20111134538 PCT/EP2010/063161 Long treatment and compliance regimes are necessary, leading to problematic compliance [0014] Additionally, drug interactions are a common problem. In particular, 5 azoles are cytochromes P-450 inhibitors, which may result in that these compounds cannot be administered to a patient receiving medication, the action of which is dependent on cytochrome P-450 activity. [00151 Antifungals currently available for the treatment of systemic infections 10 include Amphotericin B and its less toxic lipid formulations, e.g. AmBisome, and the echinocandins, which include Anidulafungin, Caspofungin, and Micafungin, all of which must be administered intravenously. Along with fluconazole, the newer triazoles such as Posaconazole (oral) and Voriconazole (oral and intravenous) are FDA approved for the treatment and prevention of systemic Candida infections. 15 Despite these new additions to the antifungal armamentarium, treatment failure is still unacceptably high and there is an increase in resistance development to the azole and echinocandin families of drugs. [0016] Caspofungin resistance is still an uncommon occurrence with c. 8% of C. 20 tropicalis and c. 2% of C. glabarata isolates having been defined as resistant (MIC values of > 2mg/L). Nevertheless, taking into consideration the recent introduction of this drug and the observation that 2001-2004 surveillance studies identified > 99.5% of patients as Caspofungin sensitive, it is disconcerting how rapidly echinocandin resistance is spreading. Furthermore, there have recently been reported cases of 25 reduced C. glabarata susceptibility developing during Caspofungin therapy. The target of Caspofungin is the enzyme 1,3-p-D-glucan synthase, encoded by one of several FKS genes, depending on the species. It has been shown that in clinical isolates, mutations in the FKS 1 gene resulting in amino acid changes in the protein were necessary and sufficient to confer reduced susceptibility to Caspofungin. 30 Recently Candida spp. with reduced susceptibility to the newer members of the echinocandin family have also been reported. [0017] In terms of resistance, for the azoles alone, three different resistance mechanisms have been identified: a) alternative pathways for the synthesis of cell 4 WO 20111134538 PCT/EP2010/063161 membrane sterols, b) mutations in the target demethylase site and c) increased efflux of drug from the fungal cell. Table A Summary of existing anti-fungal drug classes and modes of action Drug Class Mode of Action Polyene anti-fungals A molecule with a cyclic part, the (e.g. Amphotericin B) molecule consisting of a hydrophobic and hydrophilic region. The polyene antimycotics bind with sterols in the fungal cell membrane, principally ergosterol. This changes the transition temperature of the cell membrane from a fluid to a more crystalline state. Animal cells contain cholesterol instead of ergosterol and so they are less susceptible. Imidazole and triazole anti-fungals The imidazole and triazole anti-fungal (e.g. Fluconazole or Itraconazole) drugs inhibit the enzyme cytochrome P450 14a-demethylase. This enzyme converts lanosterol to ergosterol, and is required in fungal cell membrane homeostasis. These drugs also block steroid synthesis in humans. Allylamines Allylamines inhibit the enzyme squalene (e.g. Terbinafine) epoxidase, another enzyme required for ergosterol synthesis. Echinocandins Echinocandins inhibit the synthesis of (e.g. Caspofungin) glucan in the cell wall, probably via the enzyme 1,3-P glucan synthase. 5 [00181 Anti-fungals work by exploiting differences between mammalian and fungal cells to selectively inhibit growth or to kill the fungal organism preferably 5 WO 20111134538 PCT/EP2010/063161 without dangerous effects to the host. Unlike bacteria, both fungi and humans are eukaryotes. The basic structure of fungal cells and human cells is similar. This means it is more difficult to find a target for an anti-fungal drug that does not also exist in the infected organism. Consequently, there are often side-effects to some of these drugs. 5 Some of these side-effects can be life-threatening if the drug is not used properly. [0019] Well established examples of the toxicity problems are the nephrotoxicity of Amphotericin B, the liver damage caused by Terbinafine and the generalized intolerance against azoles. For example, up to 20% of females with vaginal 10 candidiosis cannot tolerate Fluconazole. [0020] US patent US6,541,506 describes methods for the synthesis and use of enediynes (compounds with a double bond and two triple bonds, in a given order.) The patent describes that these compounds may inhibit fungal infections and possibly 15 also inhibit growth of fungal cells. [00211 In agriculture, yield losses caused by various fungal pathogens in crops and other plants (for example, ornamental and amenity grasses) are significant, particularly across the major groups of fungal diseases such as rust, rot (root and 20 fruit), leaf spots, mildews and wilts. [0022] To date however, there has been no discovery of an effective antifungal agent for systemic or topical use, lacking the drawbacks of existing antifungal drugs. 6 WO 20111134538 PCT/EP2010/063161 SUMMARY OF THE INVENTION [0023] Accordingly, there is a need for antifungal compounds, which do not have some or all of the above mentioned drawbacks of existing anti-fungal drugs. 5 DIYNE SALTS [0024] It is an objective of the present invention to provide antifungal agents with one or more of the following properties, preferably all of the following properties: 10 [0025] Anti-fungal activity, preferably fungicidal activity [0026] Crystalline [0027] High melting point [0028] High solubility in water [0029] Stability in high humidity 15 [0030] Stability upon storage [00311 The present inventors have found that potassium salts of diynes have some and frequently all of above mentioned properties. 20 [00321 Accordingly, it is one objective of the present invention to provide diyne salts of the formula I: [0033] K Z-[C-C- C-C]-R 3 25 [0034] wherein Z is a carbon chain substituted with -COO- or a bioisostere thereof (preferably -COO-) and optionally also substituted with one or more additional substituents; and [0035] R 3 is a heterocyclic ring, which optionally may be substituted at one or 30 more positions. [0036] The invention also provides pharmaceutical compositions comprising such diyne salts and one or more pharmaceutically acceptable excipients. These 7 WO 20111134538 PCT/EP2010/063161 pharmaceutical compositions are in particular useful for treatment of an infection by a fungus in an individual, such as a mammal in need thereof. [0037] The invention furthermore provides use of the diyne salts for preventing or 5 treating fungal infections of a plant. DIYNES [0038] The present invention provides a new class of compounds with antifungal activity. Thus, diynes compounds disclosed herein generally have a broad spectrum 10 (i.e. activity against a wide range of fungal infections), they generally have potent antifungal activity, including antifungal activity against A. filmigatus and other filamentous fungi and they are generally active via the oral and intravenous routes. Importantly, the diynes disclosed herein have a novel mode of action, hence, their activity is generally unaffected by resistance to existing anti-fungal agents. 15 [0039] Fungi comprise enzymes catalysing fatty acid desaturation, for example conversion of saturated fatty acid to A9-monounsaturated fatty acid. These stearoyl CoA desaturases are found in many fungi. In some fungi such as S. cerevisiae and C. albicans they are referred to as OLE-1. A number of fungi are dependent on stearoyl 20 CoA desaturases (e.g. OLE-1) for viability. The present invention discloses that for these fungi stearoyl-CoA desaturase is an interesting target for novel antifungal compounds. [0040] The present invention discloses diyne compounds that are capable of 25 specifically inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in fungi. [0041] Thus it is one aspect of the present invention to provide pharmaceutical compositions comprising a diyne of formula I': 30 Z-[C=C- C=C]-R 3 8 WO 20111134538 PCT/EP2010/063161 [0042] wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and [0043] R 3 is a heterocyclic ring, which optionally may be substituted at one or 5 more positions [0044] (wherein the diyne may be any of the diynes described herein below in the sections "Diyne" and "Particular diynes") 10 [0045] wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus, [0046] and wherein said pharmaceutical composition is for treatment of an infection by a fungus dependent on activity of stearoyl-CoA desaturase (such as any 15 of the infections described herein below in the section "Fungal infection") in an individual in need thereof (wherein the individual may be any of the individuals described in the section "Individual in need of treatment"). [0047] It is also an objective of the present invention to provide methods of 20 treating infections by a fungus dependent on activity of stearoyl-CoA desaturase in an individual in need thereof, said method comprising administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of a diyne of the formula I': 25 Z-[C-C- C=C]-R 3 [0048] wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and 30 [0049] R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; and 9 WO 20111134538 PCT/EP2010/063161 [0050] wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. [0051] It is furthermore an objective of the present invention to provide methods 5 of treating a fungal infection in an individual in need thereof, said method comprising the steps of a) determining whether an individual is infected by a fungus dependent on activity of stearoyl-CoA desaturase and selecting such individual; b) administering to said selected individual a therapeutically effective 10 amount of a diyne of the formula I': Z-[C=C- C=C]-R 3 [0052] wherein Z is a carbon chain substituted with -COOH or a bioisostere 15 thereof and optionally also substituted with one or more additional substituents; and [0053] R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; and 20 [0054] or a pharmaceutically acceptable salt of said diyne, wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. [0055] It is also an objective of the present invention to provide methods for 25 identifying a fungitoxic compound, said method comprising the steps of, a) providing an indicator composition or cell comprising a gene coding for stearoyl-CoA desaturase and/or a stearoyl-CoA desaturase peptide; b) contacting the indicator composition or cell with a divne compound; c) evaluating the activity of stearoyl-CoA desaturase in the presence and 30 absence of the diyne compound; and d) selecting a diyne compound that down modulates the activity of stearoyl-CoA activity, [0056] thereby identifying a fungitoxic compound. 10 WO 20111134538 PCT/EP2010/063161 [0057] It is furthermore an objective of the invention to provide substantially pure diyne compounds of formula I': 5 Z-[CaC- CaC]-R 3 [0058] wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and 10 [0059] R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; [0060] wherein the diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. These diynes compounds are 15 preferably any of the diyne compounds described in the section "Particular diynes" herein below. [0061] It is also an objective of the present invention to provide diynes of the formula I': 20 Z-[C-C- C-C]-R 3 [0062] wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and 25 [0063] R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; [0064] wherein said diyne is capable of inhibiting conversion of a saturated fatty 30 acid to a A9-monounsaturated fatty acid in a fungus [0065] for inhibiting or treating an infection by a fungus dependent on activity of stearoyl-CoA desaturase in a plant. 11 WO 20111134538 PCT/EP2010/063161 OLE1 PROTEIN INHIBITORS [0066] This invention is also based upon the discovery that a novel class of compounds have been found to inhibit the function of the Olel protein in a wide 5 variety of fungal pathogens, and are thus capable of inhibiting fungal growth of fungi dependent on Ole1 protein function. The inventive thus contemplates antifungal compounds for formulations for treating a subject, and also for use in the agricultural setting. 10 [0067] The invention thus provides a novel fungicide comprising an Olel protein inhibitor. [0068] The invention also provides novel diyne compounds, their salts, derivatives and analogs. 15 [0069] A preferred embodiment of the invention are novel Ole1 protein inhibitors of structure II" a) (Z)-R 1 - C(O) - (C(R2)2)x - C 2
H
2 - C 4 - R4 II" 20 [0070] wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R4 is a heterocyclic ring, optionally substituted at one or more positions, preferably with one 25 or more substituents selected from the group consisting of a C 1 5 alkyl, a C 1 5 alkenyl, a C 1 5 alkoxy, a C 1 5 alcohol, a hydroxyl, an amine, a nitrate and a halogen; and x is an integer between 4 and 10, inclusive. In a preferred embodiment, R4 is a pyrrole, furan, or thiophene ring. 30 [0071] In both II" and IX" below, specific substituents are contemplated, as set forth in the Description below. [0072] A preferred fungicide is a compound of structure III", 12 WO 20111134538 PCT/EP2010/063161 H H COOH Ii (Z) and its potassium (IV") or sodium salt (V"). 5 [0073] Other preferred compounds are compounds of structure VI"-VIII". [0074] The invention also contemplates a compound of structure IX", 10 a. R 1 - C(O) - (C(R 2
)
2 )x - C 2
H
4 - C 4 - R 4 IX" [0075] wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, H or a monovalent hydrocarbon moiety containing between I and 4 carbon atoms, inclusive; R4 is a 15 heterocyclic ring, optionally substituted at one or more positions, preferably with one or more substituents selected from the group consisting of a C1_- alkyl, a C 1
_
5 alkenyl, a C 1
_
5 alkoxy, a C 1
_
5 alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive. In a preferred embodiment, R 4 is a pyrrole, furan, or thiophene ring. 20 [0076] Preferred compounds include those having the structure of one of X" XII". 13 WO 20111134538 PCT/EP2010/063161 [0077] The invention also contemplates a fungicide comprising an Olel protein inhibitor. [0078] Preferred fungicides are provided wherein the Olel protein inhibitor is 5 selected from the group consisting of compounds of structures II"-XII". [0079] More specifically, the Olel inhibitor is a compound having the structure of II", 10 a. (Z)-R 1 - C(O) - (C(R2)2)x - C 2
H
2 - C 4 - R 4 II" [0080] or is compound having the structure of IX", a. R 1 - C(O) - (C(R 2
)
2 )x - C 2
H
4 - C 4 - R 4 IX" 15 [0081] One preferred Ole1 inhibitor is compound III", H H O COOH gg (Z) 20 or its potassium salt, IV", 14 WO 20111134538 PCT/EP2010/063161 H H COO-,K* gy,, (Z) 5 or its sodium salt, compound V", H H Z isomer COO-,Na' V'' (Z) 10 [0082] Other preferred fungicides comprise an Ole 1 inhibitor having the structure of one of VI", VII" and VIII". [0083] Another preferred Olel inhibitor is a compound having the structure of one 15 of X"-XII". 15 WO 20111134538 PCT/EP2010/063161 [0084] The invention further contemplates methods of providing a fungicide comprising an Olel protein inhibitor, the inhibitor being a compound having the structure of one of II"- XII". 5 [0085] The invention also contemplates methods of providing a formulation against a fungal pathogen or of enhancing the fungicidal activity of a formulation against a fungal pathogen, comprising adding an Ole protein inhibitor to a formulation, wherein the Olel inhibitor is a compound having the structure of one of II"- XII". 10 [0086] In preferred embodiments of the inventive fungicides and the inventive methods, the subject is animal, preferably mammal, more preferably human. [0087] The compounds may also be used in co-therapy with one or more other 15 therapeutically used classes of antifungal substances. [00881 The present invention contemplates the use of the inventive compounds and fungicides against any one or more fungal pathogen selected from the group consisting of Candida spp. (for example C. albicans, C. krusei, C. glabrata, C. 20 tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr or C. dubliniensis), Aspergillus spp. (for example A. fumigatus, A. flavus, A. niger or A. terreus,) Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp. (for example C. neoformans (for example var. neoformans or var. gattii), C. bidus, C. 25 laurentii, or C. fusarium), Zygomycetes (such as Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle bertholletiae, Saksenaea vasifbrmis, Mucor circinelloides, M. ramosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus or Syncephalastrum racemosum), Malassezia spp. (for example 4. furfur or M. globosa), Hyalohyphomycetes (for example Fusarium solani or Scedosporium 30 spp., such as S. prol-ficans or S. apiospermum), Dermatophytes (for example Trichophyton spp. (for example T. mentagrophytes, T. rubrum or T. tonsurans), Epidermophytonfloccosum, Microsporum spp (for example M. cookei, M. canis, M. vanbreuseghemii, M. gallinae or M. gypseum) or Trichosporon terrestre), Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi (for example 16 WO 20111134538 PCT/EP2010/063161 Fonsecaea pedrosoi, F. compacta, Cladophylophora carrionii or Phialophora verrucosa)and Madurella spp. (for example M. mycetoinatis or M. griseum), Pneumocystis jirovecii, Pneumocystis carinii,Ascomycota Botrytis cinerea; Magnaporthe grisea; Anamorph: Pyricularia oryzae Colletotrichum gleoesporioides 5 Chilli strain; Colletotrichum gleoesporioides- mango strain; Fusarium verticillioides; Fusarium oxysporun; Alternaria solani; Uncinula necator Syn Erysiphe necator; Macrophonina phaseolina; Syn. Sclerotium bataticola and Rizoctonia bataticola; Botryodiplodia theobronae; Basidiomycota Sclerotium rolfsii; Rhizoctonia solani; Puccinia arachidis; Oomycota Pythium aphanidermatum; and 10 Plasmopara viticola Syn. Personopora viticola. [0089] The invention also contemplates methods of providing a fungicidal formulation for use in an agricultural setting or of enhancing the fungicidal activity of a formulation for use in an agricultural setting, comprising adding one or more of the 15 inventive fungicides to a formulation. [00901 In preferred embodiments, the formulation is used to combat a fungal pathogen in a plant, a grass or a field. 20 [00911 The invention also contemplates the use of a fungicide comprising any of compounds II" - VIII" for the preparation of a medicament for treating a mammal suffering from or susceptible to a condition which can be improved or prevented by an Olel inhibitor. 25 [0092] The invention also contemplates a kit for treating a fungal pathogen in a subject comprising one or more of the inventive compounds or fungicides of structure II"-VIII,,. [0093] The invention also contemplates the use of a fungicide comprising any of 30 compounds II" - XII" for the preparation of a treatment for an agricultural condition which can be improved or prevented by treatment of the agricultural condition with an Ole1 protein inhibitor. 17 WO 20111134538 PCT/EP2010/063161 [0094] A kit for an agricultural fungicide is also contemplated in the present invention, comprising one or more of the inventive compounds or fungicides of structure II" -XII". 5 [0095] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims. 18 WO 20111134538 PCT/EP2010/063161 BRIEF DESCRIPTION OF DRAWINGS [0096] Figure 1 shows XRPD diffractograms of salts of (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid prepared using potassium ethoxide or sodium 5 ethoxide as indicated in the figure. [0097] Figure 2 shows a high resolution XRPD diffractogram and a low resolustion XPRD diffractogram as indicated in the figure of the potassium (Z)-14 (furan-2-yl)tetradeca-9-en- 11,1 3-diynoate. 10 [0098] Figure 3 shows TGA and DSC analysis (as indicated in the figure) of potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate. [0099] Figure 4 shows a GVS analysis of the potassium salt of potassium (Z)-14 15 (furan-2-yl)tetradeca-9-en- 11,1 3-diynoate . [001001 Figure 5 shows an assay for synergistic effect between (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt and Amphotericin B. 20 [001011 Figure 6 shows a time course of the fungicidal effect of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid [00102] Figure 7 shows the morphological effect of (Z)-14-(furan-2-yl)tetradeca 9-en-11, 1 3-diynoic acid and Fluconazole on growing C. albicans. The % shown in the 25 upper left corners indicates percent cell viability as assessed by MTT (metabolic dye staining). [001031 Figure 8 shows the dose-dependent inhibition of C. albicans germination by (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13 -diynoic acid (A), dose-dependent 30 inhibition of vegetative growth by (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid (B) and dose dependent inhibition of germinated spores by (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid (C) 19 WO 20111134538 PCT/EP2010/063161 [00104] Figure 9 shows 13 C-Acetate incorporation into Ergosterol in C. albicans in the presence (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, Terbinafine or Fluconazole. 5 [00105] Figure 10 shows liver fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue. [00106] Figure 11 shows kidney fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue. 10 [001071 Figure 12 show kidney fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue. [00108] Figure 13 shows topical fungal burden as determined by culture positive 15 hair removed from the site of infection. [001091 Figure 14 shows vaginal fungal burden as determined by colony forming units (CFU) from vaginal lavage fluid. 20 [001101 Figure 15 shows regulation of OLE1 transcriptional activation. [001111 Figure 16 a., b. and c. showing that oleic acid, but not stearic acid, is an antagonist of compound V", b. and c. together showing that oleic acid, but not stearic acid, had an antagonistic effect on compound IV". 25 [00112] Figure 17a shows time dependent expressions of the OLE1 gene in response to compound IV" in S. cerevisiae, numbers indicate time intervals in minutes. Expressions of OLE1 are given as multiples of the expression at 10 min, condition a., and figure 17b shows time and concentration dependent expressions of 30 the OLEI gene in response to compound IV" in C. albicans. [001131 Figure 18 shows (upper pictures, controls) normal hyphal growth as compared with changes in the plane of hyphal growth and abnormal thickening of the hyphac due to the antifungal effect of the potassium salt, XII", (two lower pictures). 20 WO 20111134538 PCT/EP2010/063161 [00114] Figure 19 shows regulation of OLE1 transcriptional activation. Components of the GET complex putatively mediate the insertion of Mga2/Spt23 into the ER membrane. The ERAD complex proteolytically activates Mga2/Spt23, which 5 shuttles to the nucleus where it activates OLE1 transcription. [001151 Figure 20 shows oleic acid antagonises the sodium salt of (Z)-14-(furan-2 yl) tetradeca-9-en-11,13-diynoic acid. YPD agar plates were prepared containing 0, 0.5, 2 and 8 ptg/ml of (Z)-14-(furan-2-yl) tetradeca-9-en-1 1,13-diynoic acid. The 10 plates were either supplemented with 28 tg/ml oleic acid, 28 ptg/ml stearic acid or 0.2 % Tergitol alone as control required for solubilisation of the fatty acids. Suspensions of MGA2, GET 1 and UBX2 deletion strains and wild-type were spotted and the plates incubated for 2d at 30'C. 15 [001161 Figure 21 shows antagonistic effects between the potassium salt of (Z)-14 (furan-2-yl) tetradeca-9-en-11,13-diynoic acid and fatty acids as sodium salt in C. albicans. Compounds were combined in 96-well plates and inoculated with C. albicans. Plates were grown for 48 h and growth determined through visual inspection of growth wells. 20 [00117] Figure 22 shows antagonistic effects between oleic acid and the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en- 11,13-diynoic acid, Amphotericin B, Caspofungin and Voriconauzole on Candida. Compounds were combined in 96-well plates and inoculated with C. albicans or C. parapsilosis for Voriconazole. Plates 25 were grown for 48 h and growth determined through visual inspection of growth wells. [001181 Figure 23 shows time and concentration dependent expressions of OLE1 in response to the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-1 1,13-diynoic 30 acid in Candida albicans. Numbers indicate time intervals in minutes. Expression values are normalised to TUBI and given as multiples of OLE1 expression at 10 minutes in ethanol. 21 WO 20111134538 PCT/EP2010/063161 [00119] Figure 24 shows the liver tissue fungal load of rats infected with C. albicans blastospores and treated with a single infusion of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid, sodium salt, Fluconazole or untreated. 5 [00120] Figure 25 compares the kidney tissue fungal load of rats infected with C. albicans blastospores and treated with a single oral administration of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid, sodium salt or untreated. 10 DETAILED DESCRIPTION Al. Diyne salt [00121] The present invention relates to diyne compounds, in particular diyne salts 15 as well as to use of the diyne salts - mainly in the treatment of fungal infections. [001221 The diyne salts according to the present invention are diyne salts of formula I: 20 K Z-[C-C- C-C]-R 3 wherein Z is a carbon chain substituted with -COO- or a bioisostere thereof, preferably Z is a carbon chain substituted with -COO- and optionally also substituted with one or more additional substituents, preferably with R 2 (wherein R 2 is as 25 described in this section below); and
R
3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this section below. 30 Z is preferably a carbon chain, which is substituted with -COO- or a bioisostere thereof (preferably with -COO-), preferably said -COO- or bioisostere thereof is positioned at the end of said carbon chain, preferably at the distal end of said carbon chain in relation to the diyne moiety. Thus, Z may preferably be selected from the 22 WO 20111134538 PCT/EP2010/063161 group consisting of alkyl and alkenyl, which is substituted with R 4 , wherein R 4 is COO- or a bioisostere thereof, preferably -COO-. In addition, said carbon chain (such as said alkyl or alkenyl) may also optionally be substituted with one or more additional groups, preferably with one or more R 2 groups, wherein R 2 preferably is as 5 defined herein below in relation to diynes of formula II. [001231 Preferably Z is a C 6
-
2 0 , preferably a C 6
_
15 , more preferably C 6
-
12 , even more preferably a C 9
-
2 0 , yet more preferably a C 9
_
15 , such as a C 9 -1 2 alkyl or alkenyl substituted with -COO- or a bioisostere thereof (preferably with -COO-)and 10 optionally also substituted at one or more positions with R 2 , preferably substituted with one or more selected from the group consisting of -COO- and R 2 . More preferably Z is -COO--(C-2 0 alkyl or alkenyl)-, such as -COO--(C 9
-
2 0 alkyl or alkenyl)-, for example -COO--(C 9
_
1 5 alkyl or alkenyl)-, such as -COO--(C6- 12 alkyl or alkenyl)-. 15 [001241 More preferably, the diyne salt is a diyne salt of the formula II: K+ R 4 -Y C C-C C- R 3 20 wherein R 4 is -COO- or a bioisostere thereof, preferably however R 4 is -COO-, and Y is preferably a carbon chain of 6 to 20 carbon atoms, more preferably 9 to 20 carbon atoms, even more preferably 9 to 15 carbon atoms, yet more preferably 9 to 12 25 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds. Depending on whether a carbon atom of said carbon chain is connected to the other carbon atoms of said carbon chain by single bonds and/or double bonds each carbon atom is linked to none, one or two R 2 groups. Thus, a carbon atom connected to both its neighbouring carbon atoms in the carbon chain by single bonds will be linked to 30 two R 2 groups. A carbon atom connected to both its neighbouring carbon atoms in the carbon chain by double bonds will not be linked to any R 2 groups. A carbon atom connected to one neighbouring carbon atom in the carbon chain by a single bond and 23 WO 20111134538 PCT/EP2010/063161 to the other neighbouring carbon atom in the carbon chain by a double bond will be linked to one R 2 group. Y may furthermore be as defined herein below; and
R
3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this 5 section below. [001251 More preferably the diyne salt is a diyne salt of the formula III: C-Y C=C-C=C-R 3 K- -o 10 wherein Y is a carbon chain of 6 to 20 carbon atoms and up to three double bonds, wherein each carbon of said alkyl or alkenyl is linked to none, one or two R 2 groups, wherein each R 2 independently is -H, -OH or a hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R 3 is a heterocyclic ring, which optionally 15 may be substituted at one or more positions; or a pharmaceutically acceptable salt of said diyne, wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. Y is preferably a carbon chain of 6 to 20 carbon atoms, more preferably 9 to 20 20 carbon atoms, even more preferably 9 to 15 carbon atoms, yet more preferably 9 to 12 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds. Depending on whether a carbon atom of said carbon chain is connected to the other carbon atoms of said carbon chain by single bonds and/or double bonds each carbon atom is linked to none, one or two R 2 groups. Thus, a carbon atom connected to both 25 its neighbouring carbon atoms in the carbon chain by single bonds will be linked to two R 2 groups. A carbon atom connected to both its neighbouring carbon atoms in the carbon chain by double bonds will not be linked to any R 2 groups. A carbon atom connected to one neighbouring carbon atom in the carbon chain by a single bond and to the other neighbouring carbon atom in the carbon chain by a double bond will be 30 linked to one R 2 group. 24 WO 20111134538 PCT/EP2010/063161 [00126] Accordingly, Y may be a linear C 6
-
2 0 , preferably a C6_ 1 5 , more preferably C6-12, even more preferably a C 9
-
20 , yet more preferably a C 9
_
15 , such as a C 9
-
12 alkyl, preferably a linear C- 1 1 alkyl, yet more preferably a linear C 8
_
1 0 alkyl, even more preferably a linear C 9 -alkyl. 5 [00127] Y may also be a linear C6- 20 , preferably a C 6
_
1 5 , more preferably C6- 12 , even more preferably a C 9 -20, yet more preferably a C 9
_
1 5 , such as a C 9 -1 2 alkenyl, preferably a linear C 7
_
1 1 alkenyl, yet more preferably a linear C 8 1 0 alkenyl, even more preferably a linear C 9 - alkenyl. The alkenyl may comprise 1, 2 or 3 double bonds, preferably 1 or 10 2 double bonds, even more preferably only 1 double bond. The double bonds may be in the cis or the trans conformation, preferably at least one double bond is in the cis conformation, even more preferably all double bonds are in the cis conformation. Accordingly, Y may be a linear C 6
-
12 , preferably a linear C 7 11 alkenyl, yet more preferably a linear C 8
_
10 alkenyl preferably a linear C 9 alkenyl, wherein all double 15 bonds are cis double bonds. The double bonds may be at any suitable position, however in a preferred embodiment at least one double bond is situated at the C 8 . C 9 o, CiO position, preferably at the C 9 position (the C in the carbonyl group being C 1 ), even more preferably at least one double bond in the cis conformation is situated at the C 8 ,
C
9 0 , CIO position, preferably at the C 9 position (the C in the carbonyl group being C 1 ). 20 [00128] Thus, in a preferred embodiment of the invention the diyne salt is a compound of the formula IV: KI R4-(C(R 2
)
2 )n-X-(C(R 2
)
2 )m-[C-C- C--C]-R 3 25 wherein R 4 is -COO- or a bioisostere thereof, preferably however R4 is -COO-, and n is an integer, preferably an integer in the range of 4 to 10, inclusive, preferably in the range of 5 to 9, even more preferably in the range of 6 to 8, yet more preferably n 30 is 7; and 25 WO 20111134538 PCT/EP2010/063161 m is an integer, preferably an integer in the range of 0 to 10, such as in the range of 0 to 8, for example in the range of 0 to 6, such as in the range of 0 to 4, for example in the range of 0 to 2, such as 0; and 5 each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; and X is -CH 2
-CH
2 - or -CH=CH- or phenyl, preferably X is -CH 2
-CH
2 - or -CH=CH-; and 10 R 3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this section below. [001291 In a preferred embodiment of the invention the diyne salt is a compound of formula V: 15 O
C-(C(R
2 )2)n--X- C C- C C- R 3 K* -O wherein each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; n is an integer between 4 and 10, inclusive; X is -CH 2
-CH
2 - or -CH=CH- or phenyl, preferably X is -CH 2
-CH
2 - or -CH=CH-; and 20 R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions. n is an integer in the range of 4 to 10, preferably in the range of 5 to 9, even more preferably in the range of 6 to 8, yet more preferably n is 7. 25 [001301 In one preferred embodiment of the invention, the diyne salt is a compound of formula V, wherein X is -CH=CH-, wherein the double bond is in the trans or cis conformation, preferably in the cis conformation. Also in this embodiment it is preferred that n is as outlined above and R 2 is as outlined below. 30 26 WO 20111134538 PCT/EP2010/063161 [00131] In another preferred embodiment of the invention, the diyne is a compound of formula V, wherein X is -CH 2
-CH
2 -. Also in this embodiment it is preferred that n is as outlined above and R 2 is as outlined below. 5 [00132] R 3 of the diyne salt of formula I, II, III, IV or V is a heterocyclic ring, which optionally may be substituted at one or more positions. If substituted, the heterocyclic ring is preferably substituted with one or more, preferably one or two selected from the group consisting of lower alkyl, lower alkenyl, lower alkoxy, lower alcohol, hydroxyl, amine, -NO 2 and halogen. Lower alkyl is preferably C1_s, more 10 preferably C 1
-
3 , even more preferably C 1 atkyl. Lower alkenyl is preferably C 1
_
5 , more preferably C 1
-
3 , even more preferably C1- 2 alkenyl. Lower alkoxy is preferably C 1
_
5 , more preferably C 1
_
3 , even more preferably C 1 alkoxy. Lower alcohol is preferably C1_ 5, more preferably C 1
-
3 , even more preferably C 1 alcohol comprising one or more -OH groups, preferably only one -OH group. Halogen may be any halogen, but is 15 preferably -F. It is however preferred that R 3 is a heterocyclic ring, which is not substituted or that R 3 is a heterocyclic ring substituted with a small substituent, preferably a small substituent selected from the group consisting of methyl, methoxy, hydroxyl, -CH 2 -OH, amine and halogen, preferably methyl. 20 [001331 R 3 of the diyne salt of formula 1, 11, III, IV or V is preferably an aromatic heterocyclic ring. [00134] R 3 of the diyne salt of formula I, II, III, IV or V is preferably a 3 to 7 membered heterocyclic ring, more preferably a 5 to 6 membered heterocyclic ring, 25 even more preferably a 5 membered heterocyclic ring. The heterocyclic ring may be aromatic or non-aromatic. In one embodiment the heterocyclic ring is a 3 to 7 membered aromatic heterocyclic ring, more preferably a 5 to 6 membered aromatic heterocyclic ring, even more preferably a 5 membered aromatic heterocyclic ring. 30 [00135] The heterocyclic ring may comprise one or more heteroatoms, preferably in the range of 1 to 3 heteroatoms, more preferably in the range of 1 to 2 heteroatoms, yet more preferably I heteroatom. Said heteroatom(s) are preferably selected from the group consisting of S, N and 0. 27 WO 20111134538 PCT/EP2010/063161 [00136] In a very preferred embodiment of the invention, R 3 is selected from the group consisting of pyrrole, furan and thiophene, which may optionally be substituted as outlined above at one or more positions. R 3 may also be selected from the group consisting of imidazole, oxazole, cyclopentadiene and triazole. Preferably, R 3 is furan, 5 which is not substituted except for being linked to the diyne chain or is substituted at one or more positions as outlined above, even more preferably R 3 is furan, which is not substituted except for being linked to the diyne chain or is substituted at one or more positions with a small substituent, preferably a small substituent selected from the group consisting of methyl, methoxy, hydroxyl, amine and halogen, preferably 10 methyl. [00137] In a very preferred embodiment of the invention, R, is furan. [001381 It is preferred that the heterocyclic ring is 2-substituted with the -[CaC 15 C-C]-Z chain. In particular in embodiments wherein the heterocyclic ring contains only one heteroatom it is preferred that the heterocyclic ring is 2-substituted with the [C=C- C=C]-Z chain. Thus, in embodiments of the invention wherein R 3 is pyrrole, furan or thiophene, in particular when R 3 is furan, then it is preferred that the heterocyclic ring is 2-substituted with the -[CaC- CaC]-Z chain. 20 [001391 Each R 2 of a diyne salt of formula IV or V as well as each R2 when contained in diynes of formula 1, 11 or Il is preferably, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6, preferably 1 to 4 carbon atoms, inclusive. The hydrocarbon moiety may be an alkyl, alkenyl or alkynyl, such as C 14 25 alkyl, C 14 alkenyl or C 1
_
4 alkynyl. It is however also possible that two neighboring
R
2 groups are connected to form a hydrocarbon ring system. Said ring system may for example be a 3 to 10 membered ring, preferably a 3 to 7 membered ring, more preferably a 5 to 6 membered ring system (numbers include carbon in carbon chain plus R2 carbons). The ring system may be aromatic or none aromatic, for example the 30 ring system may be an 6 membered aromatic ring. However, preferably neighbouring
R
2 groups are not connected. 28 WO 20111134538 PCT/EP2010/063161 [00140] Thus, any particular diyne may contain a plurality of different R 2 groups. It is preferred that the majority of the R 2 groups of a diyne compound of formula 1, 11, III, IV or V is -H. It is even more preferred that all R 2 groups of a diyne compound of formula I, II, III, IV or V except for in the range of 0 to 5 R 2 groups, preferably all R 2 5 groups except for in the range of 0 to 3 R 2 groups, more preferably all R 2 groups except for in the range of 0 to 1 R 2 groups are -H. It is even more preferred that each
R
2 group of a diyne compound of formula I, II, III, IV or V is -H. [001411 In embodiments of the invention where two R 2 groups are connected to 10 form a ring system, and in particular if said ring system is aromatic, then it is preferred that said R 2 groups are positioned on C 7 and Cs; or on Cs and C 9 ; or on C 9 and C; or on C 10 and CII, preferably on C 9 and C 10 position (the C in the carbonyl group being C 1 ). 15 [001421 A preferred diyne compound according to the invention is potassium (Z) 12-(furan-2-yl)dodeca-7-en-9, 11-diynoate. [001431 Another preferred diyne compound according to the invention is potassium (Z)-1 3-(furan-2-yl)trideca-8-en- 10,12-diynoate. 20 [00144] Yet another preferred diyne compound according to the invention is potassium (E)- 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate. [001451 Yet another preferred diyne compound according to the invention is the 25 diyne compound potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoate. [00146] Another preferred diyne compound according to the invention is the diyne compound potassium 14-(furan-2-yl)tetradeca-11,13-diynoate. 30 [00147] In yet another embodiment of the invention the diyne compound may be selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-1l,13-diynoic acid, potassium salt; (Z)-14-(5-methylfuran-2-yl)tctradeca-9-en-1 1,13-diynoic acid, potassium salt; 8-(2-(4-(furan-2-yl)buta-1,3-diynyl)phenyl)octanoic acid, potassium 29 WO 20111134538 PCT/EP2010/063161 salt; (Z)-14-(4,5-dimethylfuran-2-yl)tetradeca-9-en-1 1,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca- 11,13-diynoic acid, potassium salt. 5 A2. Preparation of diyne salts [001481 The diyne salts according to the present invention may be prepared using the corresponding carboxylic acids as starting material. Said corresponding diyne carboxylic acids may be prepared essentially as described in US patent US6,541,506, 10 which is hereby incorporated by reference. [00149] The diyne carboxylic acid is then mixed with a base in a useful solvent. In some embodiments, the diyne carboxylic acid is dissolved in solvent and the base is dissolved in solvent prior to mixing. For preparation of potassium diyne salts, then the 15 base is a base comprising a potassium cation, such as potassium ethoxide. [001501 The solvent may be any useful solvent. In preferred embodiments of the invention the solvent is a water miscible organic solvent, such as a solvent selected from the group consisting of IPA, Dioxane, EtOH and acetone. 20 [00151] The method may also comprise a heating step, for example the step of dissolving the diyne carboxylic acid may comprise a heating step, such as heating to in the range of 40 to 80'C, such as to 50'C. If a heating step is employed, then usually also a cooling step is employed - typically, the heating solution comprising diyne 25 carboxylic acid and base is allowed to cool slowly, for example to a temperature in the range of 0 to 30 0 C, such as to 15-25'C, for example to 18 0 C. [001521 Diyne salt may then precipitate as solids. The solvent or part thereof may also be allowed to evaporate. 30 [00153] A useful method for preparing potassium (Z)-14-(furan-2-yl)tetradeca-9 en-11,13-diynoate is described in Example 2 herein below. The skilled person will be able to prepare other potassium diyne salts starting from the corresponding carboxylic acids using a similar approach. 30 WO 20111134538 PCT/EP2010/063161 A3. Properties of diyne salts 5 [00154] The present invention relates to diyne salts per se as well as to use of the diyne compounds in treatment of fungal infections. The structural properties of the diynes according to the invention are described herein above in the section "Diyne salts". In addition to these structural properties it is preferred that the diyne salts according to the invention also have the functional properties described in detail in 10 this section. a. Inhibition of conversion of a saturated fatty acid to a A9 monounsaturated fatty acid 15 [001551 It is very preferred that the diyne salts according to the invention is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. In particular it is preferred that the diyne compound is capable of inhibiting conversion of stearic acid to oleic acid in a fungus. It is also preferred that the diyne compound is capable of inhibiting conversion of palmitic acid to palmitoleic acid. 20 [00156] Whether a diyne compound is capable of inhibiting conversion of stearic acid to oleic acid in a fungus may be determined in any useful method known to the skilled person. According to the present invention one method for determining whether a diyne compound is capable of inhibiting conversion of stearic acid to oleic 25 acid in a fungus comprises the steps of a) providing a fungus or a fungal extract or an in vitro assembled complex comprising stearoyl-CoA 9 desaturase (such as OLE-1) b) optionally providing co-factors for stearoyl-CoA 9 desaturase (such as OLE-1) c) providing a substrate selected from the group consisting of stearic acid and 30 activated forms of stearic acid d) providing a diyne compound c) incubating said fungus or fungal extract or in vitro assembled complex with said substrate, optionally co-factors and with a predetermined amount of said diyne compound for a predetermined amount of time 31 WO 20111134538 PCT/EP2010/063161 f) detecting the presence of product formed, wherein the product is selected from the group consisting of oleic acid and activated forms of oleic acid. [00157] Said activated form of stearic acid is preferably stearyl-CoA and said 5 activated from of oleic acid is preferably oleoyl-CoA. If stearyl-CoA is provided in step b), then preferably step d) comprises detecting formation of oleoyl-CoA. [00158] Simiarly, whether a diyne compound is capable of inhibiting conversion of palmitic acid to palmitoleic acid in a fungus may be determined in any useful method 10 known to the skilled person. According to the present invention one method for determining whether a diyne compound is capable of inhibiting conversion of palmitic acid to palmitoleic acid in a fungus comprises the steps of a) providing a fungus or a fungal extract or an in vitro assembled complex comprising stearoyl-CoA 9 desaturase (such as OLE-1) 15 b) optionally providing co-factors for stearoyl-CoA 9 desaturase (such as OLE-1) c) providing a substrate selected from the group consisting of palmitic acid and activated forms of palmitic acid d) providing a diyne compound e) incubating said fungus or fungal extract or in vitro assembled complex with 20 said substrate, optionally cofactors and a predetermined amount of said diyne compound for a predetermined amount of time f) detecting the presence of product formed, wherein the product is selected from the group consisting of palmitoleic acid and activated forms of palmitoleic acid. 25 [00159] Said activated form of palmitic acid is preferably palmitoyl-CoA and said activated from of palmitoleic acid is preferably palmitoleoyl-CoA. If palmitoyl-CoA is provided in step b), then preferably step d) comprises detecting formation of palmitoleoyl-CoA. 30 [00160] Said fungus may be provided as an intact viable fungus or as intact fungal cells. The fungal extract may be any extract comprising stcaroyl-CoA 9 desaturase (such as OLE-1). In Sacchoromyces cerevisiae and other fungi the stearoyl-CoA desaturase is mainly located in the membranes of the endoplasmatic reticulum and 32 WO 20111134538 PCT/EP2010/063161 accordingly it is preferred that said fungal extract comprises endoplasmatic reticulum or parts thereof For example, the extract may be prepared by lysis of fungal cells, followed by separation of fractions for example by centrifugation. In particular, differential centrifugation may be used to enrich for endoplasmatic reticulum. 5 However, fractions or extracts comprising endoplasmatic reticulum may also be obtained by other useful methods known to the skilled person. Alternatively, stearoyl CoA desaturase may be obtained by in vitro assembly by methods known to the skilled person. 10 [00161] Preferably, the substrate provided in step c) is labelled allowing easy detection of the product in step f). The substrate may be labelled with any suitable label, such as a radioactive label, a dye, a heavy metal, a fluorescent label or a bioilluminescent label. Preferably, the substrate is radioactively labelled. Detecting product formed may then be performed by detecting labelled product. The detection 15 method will depedent on the particular label used. [001621 One non-limiting example of determining inhibition of conversion of stearic acid to oleic acid is described herein below in Example 6. The skilled person will understand that a similar method may be performed for determining inhibition of 20 conversion of palmitic acid to palmitoleic acid, by exchanging the substrate provided. [001631 Preferably, the diyne compounds according to the invention are capable of inhibiting at least 50%, more preferably at least 60%, even more preferably at least 70%, yet more preferably at least 80%, yet more preferably at least 90%, even more 25 preferably at least 95%, yet more preferably essentially 100% of the formation of oleic acid, wherein "essentially 100%" means that no detectable product is formed. Inhibition is determined in relation to a control, wherein said fungus or fungal extract is incubated with said substrate in the absence of said diyne compound for the same predetermined amount of time. 30 b. Inhibition of stearoyl-CoA 9 desaturase 33 WO 20111134538 PCT/EP2010/063161 [00164] It is preferred that the diyne salt of the invention is capable of inhibiting the activity of a fungal steraroyl-CoA 9 desaturase, preferably the diyne compound is capable of inhibiting the activity of a fungal OLE-1. 5 [00165] The activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be inhibited by different means by said diyne compound. Thus, the diyne may directly inhibit the enzymatic activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1). Thus, the diyne compound may preferably be an inhibitor of the fatty acid desaturase activity of OLE-i polypeptide. 10 [001661 Whether said diyne salt is capable of directly inhibiting the activity of fungal stearoyl-CoA 9 desaturase (such as OLE-1) may for example be determined using an in vitro assay for fungal stearoyl-CoA 9 desaturase (such as OLE-1) activity. Thus, for example fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be 15 incubated with either stearic acid and/or palmitic acid and/or activated forms thereof (such as stearyl-CoA or palmitoyl-CoA) optionally together with co-factors to form a reaction mixture and the formation of oleic acid and/or palmitoleic acid and/or activated forms thereof may then be determined. Addition of a diyne salt to said reaction mixture preferably significantly inhibits the formation of oleic acid and/or 20 palmitoleic acid or activated forms thereof. Thus, preferably addition of a diyne salt to said reaction mixture reduces the formation of oleic acid and/or palmitoleic acid to less than 30%, preferably less than 20%, more preferably to less than 10%, for example to less than 5%, for example to less than 3%, such as to less than 1%. Said fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be provided to said reaction 25 mixture in a purified form or as part of a crude extract, for example a fungal extract or as prepared in vitro. . [001671 Mammalian desaturases are significantly different to fungal stearoyl-CoA 9 desaturases (such as OLE-1), for example mammalian desaturases lacks an integral 30 cytochrome b 5 domain (Krishnamurthy et al., 2004, Microbiology, 150, 1991-2003. Accordingly, inhibitors of fungal stearoyl-CoA 9 desaturase (such as OLE-1), may be specific for the fungal enzymes in the sense that they do not inhibit mammalian desaturases to any significant extent. 34 WO 20111134538 PCT/EP2010/063161 [00168] In a preferred embodiment of the invention the diyne salt according to the invention is a selective inhibitor of a fungal stearoyl-CoA 9-desaturase (such as OLE 1). Thus it is preferred that the diyne compound is capable of inhibiting the activity of at least one fungal stearoyl-CoA 9 desaturase, preferably of more than one fungal 5 stearoyl-CoA 9 desaturase. It is furthermore preferred that the diyne compound of the invention does substantially not inhibit at least one mammalian stearoyl-CoA 9 desaturase, preferably human stearoyl-CoA 9 desaturase. Accordingly, it is preferred that if using the above-described in vitro assay, then the diyne compounds according to the invention are capable of reducing the formation of oleic acid and/or palmitoleic 10 acid to less than 30%, preferably less than 20%, more preferably to less than 10%, for example to less than 5%, for example to less than 3%, such as to less than 1% in the presence of one or more fungal stearoyl-CoA 9 desaturase (such as OLE-1), but in the absence of any mammalian stearoyl-CoA 9 desaturase. In addition it is preferred that in a similar in vitro assay said diyne compound is substantially not capable of 15 reducing the formation of oleic acid and/or palmitoleic acid and thus in the presence of said diyne compound at least 80%, preferably at least 90%, yet more preferably at least 95% oleic acid and/or palmitoleic acid is formed compared to in the absence of said diyne compound, when incubating either stearic acid and/or palmitic acid with one or more mammalian stearoyl-CoA 9 desaturases, preferably in the presence of 20 human stearoyl-CoA 9 desaturase, but in the absence of any fungal stearoyl-CoA 9 desaturase (such as OLE-1). [00169] The diyne salt may also indirectly inhibit the activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1) by down modulating the level of said 25 fungal stearoyl-CoA 9 desaturase (such as OLE-1) in a fungus. Thus, the diyne compound may decrease the stability or the half life of said fungal stearoyl-CoA 9 desaturase (such as OLE-1), thereby down modulating the level. The diyne compound may also inhibit the expression of said fungal stearoyl-CoA 9 desaturase (such as OLE-1), for example by inhibiting transcription or translation of fungal stearoyl-CoA 30 9 desaturase (such as OLE-1). [001701 In one embodiment of the invention, the diyne compound may down modulate the expression of the OLE-i polypeptide by modulating the activity of a transcriptional regulator of the gene encoding the OLE-I polypeptide. The activity of 35 WO 20111134538 PCT/EP2010/063161 the transcriptional regulator may for example be down modulated by inhibition of the binding of the transcriptional regulator to an OLE-I promoter or enhancer region. [00171] Spt23p/Mga2p is a fungal transcriptional regulator that amongst others 5 controls the expression of fungal stearoyl-CoA 9 desaturase (such as OLE-1). Thus, the diyne salt may be capable of inhibiting the activity of Spt23p/Mga2p. c. Antifungal activity 10 [00172] The MIC (minimal Inhibitory Concentration) is the minimal concentration of diyne salt required for inhibiting essentially 100%, such as 100% growth of a fungi. Preferably, the MIC of the diyne salts according to the invention is at the most 500 ng/ml, preferably at the most 250 ng/ml, yet more preferably at the most 100 ng/ml, for example at the most 60 ng/ml, such as at the most 40 ng/ml, for example at the 15 most 20 ng/ml, such as at the most 10 ng/ml in relation to at least 3 different fungi. [001731 It is also preferred that the diyne salts according to the invention are capable of killing one or more fungi, preferably capable of killing at least 2, more preferably at least 5, even more preferably at least 10 different fungi. Preferably, the 20 diyne compound has a minimum fungicidal concentration (MFC) of at the most 100 gg/ml, preferably at the most 50 pg/ml, yet more preferably at the most 10 gg/ml, even more preferably at the most 2 gg/ml, against one or more fungi, preferably one or more pathogenic fungi, even more preferably against at least 2, yet more preferably at least 5, even more preferably at least 10 different fungi. Preferably, said MFC for a 25 given fungus is determined in a method comprising the steps of a) cultivating said fungus in vitro b) contacting said fungus with various concentrations of test diyne compound c) incubating said fungus with said diyne test compound for a predetermined 30 amount of time d) transferring said fungus to another in vitro culture medium e) determining growth of said fungus in said another in vitro culture medium 36 WO 20111134538 PCT/EP2010/063161 [00174] The lowest concentration of diyne test compound resulting in essentially no growth in step e), preferably in no detectable growth in step e) is the MFC. [00175] In addition it is preferred that contacting a fungus with said diyne 5 compound leads to a rapid loss of viability. This may for example be determined by a method comprising the steps of: a) cultivating one or more fungi in vitro, thereby obtaining a fungal culture 10 b) determining the CFU/ml in said fungal culture c) contacting the fungal culture with a test diyne compound d) incubating said fungal culture with said test diyne compound for a predetermined amount of time e) determining the CFU/ml in said fungal culture 15 [001761 If the CFU/ml determined in step e) is at the most 20%, preferably at the most 15%, yet more preferably at the most 10% of the CFU/ml determined in step b), wherein said predetermined amount of time is in the range of 0.5 to 24 hours, preferably in the range of 0.5 to 12 hours, even more preferably in the range of 0.5 to 20 6 hours, yet more preferably in the range of 0.5 to 2 hours, such as in the range of 1 to 24 hours, even more preferably in the range of 1 to 12 hours, yet more preferably in the range of 1 to 6 hours, even more preferably in the range of 1 to 2 hours then said diyne compound is said to be capable of leading to rapid loss of viability of said fungus. Thus, it is preferred that at least a 1000 fold reduction in CFU/ml is 25 determined in step e) compared to step b), when said predetermined amount of time is at least 3 hours, such as 3 hours. [001771 As outlined above it is preferred that the diyne compound has fungicidal activity against one or more fungi and in addition it is preferred that said diyne 30 compound is capable of leading to rapid loss of viability of one or more fungi. [001781 It is also preferred that the diyne compounds according to the invention are capable of inhibiting growth of at least 1, preferably of at least 2, such as of at least 3 different fungi, at a concentration of at the most 100 pg/ml, preferably at the most 50 37 WO 20111134538 PCT/EP2010/063161 pig/ml, yet more preferably at the most 10 pg/mi, even more preferably at the most 2 ptg/ml. Inhibition of growth may for exampe be determined as described herein below in Example 7. 5 d. Solubility [001791 In order to obtain a desired antifungal activity in a clinical setting, it is important that the antifungal compound is soluble in water. Thus, it may be challenging to prepare pharmaceutical compositions comprising antifungal 10 compounds, which are not water soluble, and which still may reach the site of disease and be functional there. This is in particular the case with infections by a fungus which involve infection of inner organs or disseminated infections. Also in relation to treatment or reduction of risk of infection by a fungus in a plant, it is preferable that the antifungal compound is soluble in water. 15 [001801 Accordingly, it is one objective of the present invention to provide antifungal compounds, which are not soluble in water. Interestingly, in contrast to for example diyne carboxylic acids, the diyne salts disclosed herein are readily soluble in water. 20 [00181] Thus, the diyne salt of the invention preferably has a solubility in water of at least 50 mg/ml, preferably at least 60 mg/ml, more preferably at least 70 mg/ml, yet more preferably at least 80 mg/ml, even more preferably at least 90 mg/ml. 25 [001821 The solubility in water is preferably determined as described in Example 1 herein below. e. Crystal 30 [00183] For pharmaceutical applications it is furthermore generally preferred that the active compound is provided in a crystalline form. [001841 Accordingly, it is also an objective to provide antifungal compounds, which are crystalline. 38 WO 20111134538 PCT/EP2010/063161 [00185] Interestingly, and in contrast to many other diyne salts, the diyne potassium salts provided herein are available in a crystalline form. 5 [00186] Whether a compound is crystalline may be determined by any suitable method known to the skilled person, however, preferably it is determined using X Ray Powder Diffraction (XRPD). One non-limiting method for performing XRPD is described in Example 1. Crystalline compounds gives rise to a XRPD pattern with distinctive peaks, whereas an amorphous compound gives rise to an XPRD pattern 10 without distinctive peaks. An example of an XPRD pattern for a crystalline compound (potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoate) with distinctive peaks and an amorphous compound (sodium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoate) without distinctive peaks is shown in figure 1. 15 [001871 In a preferred embodiment of the invention, the diyne salt is potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate. In this particular embodiment it is preferred that said potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoate is in the crystalline form. 20 [001881 More preferably, said potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13 diynoate crystals have a high resolution XPRD pattern comprising peaks at the following 20 angles. Said 20 angles are wavelength dependent and calculated at Cu-K a, X =1.54173 A. Preferably only peaks with an intensity of >3 % are included in the pattern. The high resolution XPRD pattern may be obtained using any suitable method 25 known to the skilled person, but in one preferred embodiment it is prepared as described herein below in Example 2. [001891 Preferably, the potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13 diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the 30 following 20 angles: 2.42' and 4.78'. [001901 It is also preferred that the potassium (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 20 angles: 7.14', 9.52' and 11.89'. 39 WO 20111134538 PCT/EP2010/063161 [00191] Thus, in a preferred embodiment the potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 20 angles: 2.42' and 4,780, 7.14', 9.52 and 5 11.890. [001921 In a very preferred embodiment the potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 20 angles: 2.420, 4,780, 7.140, 9.520, 9.520, 10 16.450, 17.270, 18.410, 19.110, 19.680, 21.270, 21.950, 23.060, 23.860, 24.90, 26.980, 27.820, 28.680, 28.860 and 38.770. In this embodiment it is preferred that the high resolution XPRD pattern does not comprises any other peaks with an intensity of >5%. 15 [001931 In a very preferred embodiment the potassium (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoate crystals have a high resolution XPRD pattern as shown herein in figure 2B 20 f. Moisture uptake [001941 It is also preferred that the diyne salt according to the invention does not take up to much moisture from the surroundings. 25 [001951 Accordingly, it is preferred that the weight change of the compound is less than 5%, preferably less than 4%, more preferably less than 3%, even more preferably less than 2 %, for example even less than 1% at a humidity of 60% RH compared to a humidity of 10% RH. 30 [00196] It is also preferred that the weight change of the compound is less than 5%, preferably less than 4% at a humidity of 70% RH compared to a humidity of 10% RH. 40 WO 20111134538 PCT/EP2010/063161 [00197] In addition, it is preferred that if the diyne salt is exposed to humidity higher than 80%, then upon return to a lower humidity, then moisture is lost and the crystalline form of the diyne salt is kept/regained. 5 [00198] The weight change at various humidity conditions may be determined by any useful method known to the skilled person, however in a preferred embodiment it is determined using GVS, for example as described in Example 2 herein below. . g. Melting temperature 10 [001991 It is also preferred that the diyne salt has a sufficiently high melting temperature in order to allow handling during manufacture of pharmaceutical compositions and storage at ambient temperature. 15 [002001 Thus, it is preferred that the melting point of said compound is at least 100 C, preferably at least 1 10 C, more preferably at least 120'C, yet more preferably at least 130'C, even more preferably at least 140'C. [00201] The melting point of a compound may be determined using any suitable 20 method known to the skilled person, however in a preferred embodiment the melting temperature is determined by an DSC analysis, for example as described herein below in Example 1. h. Stability 25 [00202] It is also preferred that the diyne salt according to the present invention is stable upon storage. [002031 Thus it is preferred that when stored as a solid, the diyne salt is stable for 30 at least 3 months, preferably at least 4 months, more preferably at least 5 months, even more preferably at least 6 months, such as for in the range of 3 to 12 months, for example in the range of 4 to 12 months, such as for in the range of 5 to 12 months, for example in the range of 6 to 12 months, such as for in the range of 3 to 6 months, for example in the range of 4 to 6 months, such as for in the range of 5 to 6 months. 41 WO 20111134538 PCT/EP2010/063161 [00204] In particular, it is preferred that the content of diyne salt in a diyne salt solid has not decreased significantly as determined by HPLC after storage for at least 3 months, preferably at least 4 months, more preferably at least 5 months, even more 5 preferably at least 6 months, such as for in the range of 3 to 12 months, for example in the range of 4 to 12 months, such as for in the range of 5 to 12 months, for example in the range of 6 to 12 months, such as for in the range of 3 to 6 months, for example in the range of 4 to 6 months, such as for in the range of 5 to 6 months at a temperature in the range of 2 to 25'C, such as at a temperature of 2 to 8'C, for example at a 10 temperature of 25'C and a humidity of in the range of 10 to 6 0%, such as 30 to 60%, for example 60%. In this context the term "content of diyne salt in a diyne salt solid has not decreased significantly" preferably means that the content has not decreased to less than 95%, preferably not to less than 96%, more preferably not to less than 97%, yet more preferably not to less than 98%. 15 [002051 It is also preferred that the content of diyne salt in a diyne salt solid has only decreased slightly as determined by HPLC after storage for at least 3 months, preferably at least 4 months, more preferably at least 5 months, even more preferably at least 6 months, such as for in the range of 3 to 12 months, for example in the range 20 of 4 to 12 months, such as for in the range of 5 to 12 months, for example in the range of 6 to 12 months, such as for in the range of 3 to 6 months, for example in the range of 4 to 6 months, such as for in the range of 5 to 6 months at a temperature in the range of 35 to 50'C, such as at a temperature of 35 to 45 0 C, for example at a temperature of 40 0 C and a humidity of in the range of 60 to 7 5%, such as 70 to 75%, 25 for example 75%. In this context the term "content of diyne salt in a diyne salt solid has only decreased slightly" preferably means that the content has not decreased to less than 90%, preferably not to less than 92%, more preferably not to less than 94%, yet more preferably not to less than 96%. 30 A4. Pharmaceutical composition [002061 The pharmaceutical compositions comprising dine salts according to the present invention are useful for treating fungal infections in an individual in need 42 WO 20111134538 PCT/EP2010/063161 thereof The pharmaceutical compositions may be in any suitable form depending on the fungal infection to be treated. [00207] Thus, the pharmaceutical composition may be formulated for topical 5 administration or for systemic administration. Typically, if the fungal infection is a local infection on a body surface, then the pharmaceutical composition is formulated for topical administration. If the fungal infection is a disseminated infection and/or an infection of one or more inner organs, tissues or cells then the pharmaceutical composition is typically formulated for systemic administration, such as parenteral 10 administration or oral administration. [00208] In addition, to said diyne salts the pharmaceutical compositions according to the invention will frequently comprise one or more pharmaceutically acceptable excipients. 15 [002091 For therapeutic uses, the pharmaceutical compositions comprising diyne salts may be administered systemically, for example, formulated in a pharmaceutically acceptable buffer such as physiological saline. Treatment may be accomplished directly, e.g., by treating the individual (such as a human being) with a 20 diyne salt or pharmaceutical composition comprising a diyne salt of the invention. Preferable routes of administration include, for example, inhalation or subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the diyne compounds in the patient. Treatment of human beings or other animals is carried out using a therapeutically effective amount 25 of a diyne compound of the invention in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro A R., 1995, Mack Publishing Company, Easton, Pa. 30 [00210] In one preferred method, one or more diyne salts of the invention are formulated in combination with a solid or a liquid dermatologically acceptable carrier. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, alcohols, or glycols (or water-alcohol/glycol blends), in which the present compounds can be 43 WO 20111134538 PCT/EP2010/063161 dissolved or dispersed at effective levels. Adjuvants (such as flavourings and/or fragrances), surfactants, and additional antimicrobial agents can be added to optimize the properties for a given use. The compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area 5 using pump-type or aerosol sprayers. The liquid compositions can also be employed as eye drops, mouth washes, douches, etc. [00211] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be 10 employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user, which is mainly relevant for treating fungal infections of the skin. [00212] In other medical applications, diyne salts can be added to materials used to 15 make medical devices such as catheters, including but not limited to intravenous, urinary, intraperitoneal, ventricular, spinal, and surgical drainage catheters, in order to prevent colonization and systemic seeding by potential fungal pathogens. Similarly, an antifungal compound may be added to the materials that constitute various surgical prostheses and to dentures to prevent colonization by fungal pathogens and thereby 20 prevent more serious invasive infection or systemic seeding by these pathogens. [002131 The amount of the diyne salt to be administered may vary depending upon the manner of administration, the age and body weight of the individual, and the type of fungal infection and extensiveness of the infection. 25 [00214] However, preferably the pharmaceutical compositions comprising diyne salts according to the invention are formulated for administration of in the range of 0.001mg/Kg to 100mg/kg, preferably in the range of 0.001mg/Kg to 100mg/kg daily, in particular when the individual to be treated is a mammal, such as a human being. 30 [00215] The total concentration of one or more diyne salts of the invention in the present compositions can be varied widely, and will depend on factors such as the compatibility of the active ingredient(s) with the vehicle, the potency of the active ingredient(s) and the condition to be treated. Generally, the concentration of the diyne 44 WO 20111134538 PCT/EP2010/063161 compound(s) in a t composition for topical administration, such as a lotion, will be from about 0.01 to 25% by weight, such as from 0.1-25% by weight, preferably from about 0.5-10% by weight, and more preferably from about 0.5% to 5% by weight. In liquid formulations the concentration may be from 0.01 to 90%, preferably from 0.01 5 to 50%, such as from 0.01 to 25% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.01-99% by weight, such as from 0.01 to 50%, for example from 0.01 to 25% and preferably about 0.5-2.5% by weight. 10 [00216] The pharmaceutical compositions according to the invention may in addition to the diyne salts also comprise one or more additional active agents. [002171 Said additional active agents may for example be antifungal agents. 15 A5. Individual in need of treatment [002181 The present invention relates to pharmaceutical compositions comprising diyne salts for treatment of fungal infections in an individual in need thereof. 20 [002191 Said individual may be any individual suffering from a fungal infection, preferably an animal, more preferably a mammal, even more preferably a human being suffering from a fungal infection. In some preferred embodiments of the invention, the individual is however a non-human mammal suffereing from a fungal infection, preferably a mammal selected from the group consisting of horses, cattle, 25 dogs and cats. Said fungal infection may be any of the fungal infections described herein below in the section "Fungal Infections". [002201 In one embodiment, the individual is an individual susceptible to fungal infections, for example an immunocompromised individual, such as an 30 immunocompromised human being. Said immunocompromised individual may be immunocompromised for various reasons, for example the individual may receive immune suppressing medication (for example in connection with transplantation) or the individual may be suffering from an immunocompromising condition, such as HIV infection. 45 WO 20111134538 PCT/EP2010/063161 [00221] Interestingly, the present invention discloses that the diyne salts of the invention does reduce the activity of cytochromes P450 significantly. Whether a diyne salt is capable of reducing the activity of of cytochromes P450 may be determined by 5 any suitable assay known to the skilled person, preferably by an assay comprising the steps of: a) providing a cytochrome P450 b) providing a substrate for said cytochrome P450 10 c) incubating said cytochrome P450 with said substrate under conditions allowing for cytochrome P450 activity in the presence and absence of the diyne salt d) determining a reduction in product formed in the presence of said diyne salt compared to in the absence of said diyne salt 15 [002221 A diyne salt is said to not "reduce the activity of cytochromes P450 significantly" if at least 60%, preferably at least 70% product is formed in the presence of 10 pM of the diyne salt compared to in the absence of said diyne salt. Preferably, said diyne salt does not significantly reduce the activity of at least 3, 20 preferably at least 4, more preferably at least 5, even more preferably at least 6, such as in the range of 3 to 10, for example in the range of 5 to 10, such as 7 different cytochromes P450. Said substrate may be labelled, and the product formed may then be detected by detecting labelled product. However, frequently the detection may also be direct using for example chromatographic methods, for example HPLC-UV/VIS or 25 HPLC-MS/MS. A preferred method for determining whether a compound reduces the activity of cytochromes P450 is described in Example 9 herein below, [002231 The substrate used is dependent on the particular cytochrome P450. Substrates useful for individual cytochromes P450 are well known to the skilled 30 person. For example the catalogue of Cerep, France (as of 10 July 2009) describes suitable substrates for various cytochromes P450. Useful substrates for some cytochrome P450s are also described in Example 9 herein below. 46 WO 20111134538 PCT/EP2010/063161 [00224] Accordingly, the pharmaceutical compositions according to the invention are particularly useful for treating individuals receiving one or more other active agents, in particular such active agents wherein the activity of said active agents is increased or even depending on the activity of cytochromes P450. Typically, 5 immunocompromised individuals will receive such active agents, and the pharmaceutical compositions of invention are accordingly in particular useful for treatment of immuno compromised individuals. [002251 Thus, in a preferred embodiment, the pharmaceutical compositions 10 comprising diynes are in particular useful for treating fungal infections in individuals to whom one or more active agents selected from the following group is being administered, has been or is foreseen to be administered, preferably is being administered, has been administered within the past 48 hours or is foreseen to be administered within the next 48 hours. 15 [002261 Said active agents, wherein the activity is increased or even depending on the activity of cytochromes P450 is preferably one or more selected from the group consisting of acetaminophen, alfentanil, alprazolam, alprenolol, aminophyllin, amiodarone, amitriptyline, amlodipine, amphetamine, amprenavir, aniline, 20 artemisinin, astemizole, atorvastatin, azelastine, azithromycin, barnidepine, benzene,bufuralol, bupropion, buspirone, bezafibrate, caffeine, carbamazepine, carisoprodol, carvedilol, celecoxib, cerivastatin, chlorpheniramine, chlorpromazine, chlorzoxazone, cimetidine, ciprofloxacin, cisapride, citalopram, clarithromycin, clemastine, clomipramine, clopidogrel, clozapine, cocaine, codeine, cyclobenzaprine, 25 cyclophosphamide, cyclosporine, dapsone, debrisoquine, delavirdine, desipramine, dexamethasone, dexfenfluramine, dextromethorphan, dextropropoxyphene, diclofenac, diazepam, diltiazem, N,N-dimethyl formamide, diphenhydramine, disulfiram, docetaxel, dofetilide, dolasetron, econazole, efavirenz, encainide, enflurane, enoxacin, ergotamine, estradiol, erythromycin, ethanol, ethinylestradiol, 30 etomidate, etoposide, felbamate, felodipine, fenofibrate, fentanyl, finasteride, flecainide, fluconazole, fluorouracil, fluoxetine, flurbiprofen, fluvastatin, fluvoxamine, gemfibrozil, glibenclamide, glipizide, glyburide,granisetron, growth hormone, halofantrine, haloperidol, halothane, hexobarbital, hydrocortisone, hydroxyzine, ibuprofen, ifosfamide, imipramine, indinavir, indoramine, insulin, 47 WO 20111134538 PCT/EP2010/063161 indomethacin, irbesartan, irinotecan, isoflurane, isoniazid, isradipine, itraconazole, ketoconazole, lansoprazole, lercanidipine, levomepromazine, lidocaine, lignocaine, loratadine, lomoxicam, losartan, lovastatin, meloxicam, mephenytoin, mephobarbital, mequitazine, mestranol, methadone, methoxsalen, methoxyamphetamine, 5 methoxyflurane, metoclopramide, metoprolol, metronidazole, mianserin, mibefradil, miconazole, midazolam, mifepristone, mirtazapine, mepyramine, methoxyamphetamine, metoclopramide,metyrapone, mexiletine, midazolam, minaprine, moclobemide, montelukast, naproxen, nefazodone, nelfinavir, nicardipine, nifedipine, nilutamide, nisoldipine, nitrendipine, norethindrone, norfloxacin, 10 nortriptyline, omeprazole, ondansetron, orphenadrine, oxcarbazepine, pantoprazole, paracetamol, paroxetine, pefloxacin, perhexiline, perphenazine, pethidine, pentobarbitone, phenacetin, phenformin, phenobarbitone, phenytoin, pimozide, piroxicam, prednisone, primidone, procainamide, progesterone, proguanil, promethazine, propafenone, propofol, propranolol, quanoxan, quinidine, quinine, 15 ranitidine, rifabutin, rifampicin, riluzole, risperidone, ritonavir, ropinirole, ropivacaine, rosiglitazone, salmeterol, saquinavir, secobarbital, selegiline, sildenafil, simvastatin, sertraline, sevoflurane, Snaproxen, sparteine, sufentanil, suprofen, sulphamethoxazole, sulphonamides (sulfonamides), tamoxifen, tacrine, tacrolimus, taxol,teniposide, terbinafine, terfenadine, terfenidine, testosterone, theophylline, 20 thiopental, thioridazine, ticlopidine, timolol, tirilazad, tobacco, tolbutamide, tolterodine, topiramate, torsemide, tramadol, tranylcypromine, trazodone, triazolam, trofosfamide, troglitazone, troleandromycin, tropisetron, valsartan, venlafaxine, verapamil, vesnarinone, vigabatrin, vinblastine, vincristine, warfarin, zafirlukast, zaleplon, zanamivir, zileuton, zolmitriptan, zolpidem, zonisamide, zotepine and 25 zuclopenthixol. A6. Fungal infection [002271 The present invention in one aspect relates to pharmaceutical compositions 30 comprising diyne salts (such as any of the diyne salts described herein above in the section "Diyne salt") for treatment of infections by a fungus. The invention also relates to methods of treating an infection by a fungus by administering to an individual in need thereof a therapeutically effective amount of a diyne salt (such as any of the diyne salts described herein above in the section "Diyne salts"). 48 WO 20111134538 PCT/EP2010/063161 [00228] In general said fungus is a fungus dependent on the activity of stearoyl CoA desaturase (such as OLE-1). The fungus may also be a fungus dependent on the activity of Spt23p/Mga2p, preferably a fungus dependent on both the activity of 5 stearoyl-CoA desaturase (such as OLE-1) and Spt23p/Mga2p. [002291 In general, the diyne salts according to the present invention are capable of killing fungi, i.e. they have fungicidal activity. Accordingly, the pharmaceutical compositions comprising diyne compounds according to the invention are in 10 particular suitable for treating infections by fungus, wherein it is desirable to kill the fungus, rather than just to inhibit growth of the fungus. Thus, the pharmaceutical compositions comprising diyne salts according to the invention are particularly useful for treating recurrent infections by fungus, such as an infection by a fungus, which is expected to be recurrent or an infection by fungus, which has re-occurred at least 15 once, for example at least twice, such as at least 3 times. [002301 Preferably, the pharmaceutical compositions comprising diyne compounds according to the invention are prepared for killing at least 50%, preferably at least 80%, more preferably at least 95% of the infecting fungus. 20 [00231] Another very interesting aspect of the present invention is that the pharmaceutical compositions comprising diyne salts according to the invention are particularly useful for treating infection by a fungus under hypoxic conditions. Without being bound by theory it is believed that this is based on OLE-1 being 25 particularly important for fungal growth under hypoxia. Thus, OLE-I transcript levels are upregulated in fungi under hypoxia (for example in C. albicans). [002321 Accordingly, the infection by a fungus may preferably be an infection involving at least partly infection of tissue, organs or cells with hypoxic conditions, 30 preferably the infection may be infection of tissues, organs or cells with hypoxic conditions. Thus, said infection may at least partly involve infection of one or more inner organs, tissues or cells of a mammal, preferably a human being. More preferably, said infection may be infection of one or more inner organs, tissues or cells of a mammal, preferably a human being. 49 WO 20111134538 PCT/EP2010/063161 [00233] Said hypoxic condition is preferably an oxygen partial pressure (p02) of at the most 140 mmHg, preferably at the most 110 mmHg, such as at the most 80 mmHg. Such conditions may in general be found in inner organs, for example in the 5 liver, pancreas, gut, duodenum, skeletal muscles, brain, kidney or peritoneal cavity. [002341 It is also comprised within the present invention that the pharmaceutical compositions comprising diyne salts according to the invention may be for treatment of a disseminated infection or a local infection. 10 [002351 The infection by said fungus may also involve at least partly infection of a body surface, for example infection of skin, nails or mucosal membranes of body surfaces. Thus, said infection may be infection of a body surface, for example infection of skin, nails or mucosal membranes of body surfaces. Body surfaces may 15 include the oral cavity, the genital organs, nose or eyes. [002361 Accordingly, the fungal infection may be one or more selected from the group consisting of oropharyngeal fungal infections (such as thrush, glossitis, stomatitis or angular cheilitis), cutaneous fungal infections (such as intertrigo, diaper 20 candidiasis, paronychia or onychomycosis), paronychia, onychomycosis, vulvovaginal fungal infection, balanitis, mucocutaneous fungal infection, neonatal fungal infection, congenital fungal infection, oesophageal fungal infection, gastrointestinal fungal infection, pulmonary fungal infection, peritonitis, urinary tract fungal infections, renal fungal infection, meningitis associated with fungi, hepatic 25 fungal infection, hepatosplenic fungal infection, endocarditis, myocarditis, pericarditis, ocular fungal infection, endophthalmitis and osteoarticular fungal infection. [002371 Interestingly, the diyne compounds according to the present invention are 30 even useful for treating onychomycosis, i.e. fungal infection of the nails. [002381 The infection by a fungus may be infection by one species of fungus or infection by more than one fungal species, such as two, for example 3, such as 4, for example 5, such as more than 5 different fungal species. 50 WO 20111134538 PCT/EP2010/063161 [00239] The fungus may be any fungus, but usually it is a pathogenic fungus, such as a fungus pathogenic in the individual to be treated. In one preferred embodiment of the invention, the individual to be treated is a mammal, preferably a human being, and 5 then the fungus is a fungus pathogenic in mammals, preferably in human beings. [002401 The fungus may preferably be selected from the group consisting of wherein one or more fungus is selected from the group consisting of Candida spp., Aspergillus spp., Histoplasina capsulatun, Coccidioides immitis, Coccidioides 10 posadasii, Cryptococcus spp., Zygomycetes, Malassezia spp., Hyalohyphomycetes, Dermatophytes, Epiderinophyton floccosum, Microsporum spp, Blastonyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi and Madurella spp. [00241] Thus the fungus may be selected from the group consisting of Candida 15 spp., preferably from the group consisting of C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guillierniondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis. [00242] The fungus may also be selected from the group consisting of Aspergillus 20 spp., preferably from the group consisting of A. fumigatus, A. flavus, A. niger and A. terreus. [00243] The fungus may also be selected from the group consisting of Cryptococcus spp., Preferably from the group consisting of C. neoforinans, C. bidus, 25 C. laurentii, and C. fusarium. Said C. neoformans is preferably selected from the group consisting of var. neoformans and var. gattii. [002441 The fungus may also be selected from the group consisting of zygomycetes, preferably from the group consisting of Rhizopus oryzae, R. 30 nicropsorus, R. pusillus, Cunninghainelle bertholletiae, Saksenaea vasiformis, Mucor circinelloides, M. rainosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeronyces recurvatus and Syncephalastrun racenosun. 51 WO 20111134538 PCT/EP2010/063161 [00245] The fungus may also be selected from the group consisting of Malassezia spp., preferably from the group consisting of M. furfur and M. globosa. [00246] The fungus may also be selected from the group consisting of 5 Hyalohyphomycetes, preferably from the group consisting of Fusarium solani and Scedosporium spp., wherein said Scedosporium spp. preferably is selected from the group consisting of S. prolificans and S. apiospermumn. [002471 The fungus may also be selected from the group consisting of 10 Dermatophytes. This is in particular the case when the infection is partly or entirely an infection of the skin. Said Dermatophyte may preferably be selected from the group consisting of Trichophyton spp., Epidermophyton floccosum, Microsporum spp and Trichosporon terrestre. Said Trichophyton spp. may preferably be selected from the group consisting of T. mentagrophytes, T. rubrun and T. tonsurans. Said 15 Microsporum spp may preferably be selected from the group consisting of M. cookei, M. canis, M. vanbreuseghemini, M. gallinae and A. gypseum. [002481 The fungus may also be selected from the group consisting of Chromomycotic fungi, preferably from the group consisting of Fonsecaea pedrosoi, 20 F. comnpacta, Cladophylophora carrionii and Phialophora verrucosa. [002491 The fungus may also be selected from the group consisting of Madurella spp., preferably from the group consisting of M. mycetomnatis and M. griseun. 25 [002501 In embodiments of the invention wherein the individual is a non-human animal, preferably a mammal, more preferably a mammal selected from the group consisting of horses, cattle, dogs and cats then the fungus may for example be selected from the group consisting of Aspergillus spp., Batrachochytrium dendrobatidis, Blastomyces spp., Branchionyces spp., Candida spp., Cladosporiumn spp., 30 Coccidioides spp., Cryptococcus neoformans, Entomnophthora spp., Epidermnophyton spp., Fonsecaea spp., Geotrichuni spp., Histoplasma spp., Ichthyophonus hoferi, Lacazia loboi, Malassezia spp., Metarhiziun spp., Microsporun spp., Mucor spp., Ochroconis spp., Paecilomyces spp., Penicillium spp., Phialophora spp., Saprolegnia spp., Sporothrix schenckii, Trichophyton spp. and Wangiella spp. 52 WO 20111134538 PCT/EP2010/063161 A7. Resistant fungus [00251] Another very interesting aspect of the present invention is that the diyne 5 salts according to the present invention are capable of treating infections by fungi which are resistant to one or more conventional antifungal agents, in particular antifungal agents, which are not capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. 10 [00252] Said fungi may be resistant for any reason. Thus, for example that particular species of fungus may be resistant to treatment with that particular antifungal agent. Alternatively, the fungus may have acquired resistance, i.e. in general said fungal species is not resistant to treatment with the particular antifungal agent, but this particular fungus has become resistant. In a preferred embodiment of 15 the invention, the fungus has acquired resistance to one or more conventional antifungal agents. [002531 Thus, the pharmaceutical compositions comprising diyne salts according to the invention are useful for treating infection by a fungus, which is resistant to one or 20 more antifungal agents, which are not of formula I. In particular, the pharmaceutical compositions comprising diyne salts according to the invention are useful for treating infection by a fungus, which is resistant to one or more antifungal agents capable of at least one of a) inhibiting ergosterol biosynthesis; 25 b) binding to ergosterol; c) inhibiting 1,3--glucan synthase; d) inhibiting epoxidase; e) inhibiting Leucyl-tRNA synthetase; and/or f) inhibition of elongation factor 2. 30 [00254] In particular, the pharmaceutical compositions comprising diyne salts according to the invention are useful for treating infection by a fungus, which is resistant to one or more antifungal agents selected from the group consisting of 53 WO 20111134538 PCT/EP2010/063161 polyene antifungal agents, azole antifungal agents, allylamine antifungal agents and echinocandins. [00255] Polyene antifungal agents are antifungal agents with multiple conjugated 5 double bonds. Typically, polyene antifungal agents also comprise a heavily hydroxylated region. Non-limiting examples of polyenes include Natamycin, Rimocidin, Filipin, Nystatin, Amphotericin B or Candicin. [002561 Azole antifungal agents may for example be imidazole or triazole or 10 thiazole antifungal agents. Non-limiting examples of imidazole antifungal agents include miconazole, ketoconazole, clotromazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, seraconazole, sulconazole or tioconazole. Non-limiting examples of triazole antifungal agents include fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole or terconazole. A non 15 limiting example of a thiazole antifungal is abafungin. [002571 Non-limiting examples of allylamine antifungals include Terbinafine, Amorolfine, Naftifine or Butenafine. 20 [002581 Non-limiting examples of echinocandins include Anidulafungin, Caspofungin or Micafungin. [00259] The pharmaceutical compositions comprising diynes according to the invention may also be useful for treating infection by a fungus, which is resistant to 25 one or more antifungal agents selected from the group consisting of benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate. [002601 By the term "resistant to an antifungal agent", it is meant that said 30 infection by fungus in said individual cannot be treated in a curable manner with said antifungal agent. 54 WO 20111134538 PCT/EP2010/063161 [00261] The pharmaceutical compositions for treating fungal infections according to the invention may in addition to one or more diyne salts also comprise additional active agents, preferably one or more antifungal agents. 5 [00262] Thus said pharmaceutical compositions may in addition to one or more diynes also comprise one or more antifungal agents capable of at least one of a) inhibiting ergosterol biosynthesis; b) binding to ergosterol; c) inhibiting 1,3-p-glucan synthase; 10 d) inhibiting epoxidase; e) inhibiting Leucyl-tRNA synthetase; and/or f) inhibition of elongation factor 2. [00263] Thus, the additional antifungal agent may be for example be selected from 15 the group consisting of polyene antifungal agents (such as any of the polyene antifungal agents described herein above in the section), azole antifungal agents (such as any of the azole antifungal agents described herein above in the section), allylamine antifungal agents (such as any of the allylamine antifungal agents described herein above in the section) and echinocandins (such as any of the 20 echinocandins described herein above in the section). [002641 In particular, due to the synergistic effect the pharmaceutical compositions of the invention may preferably comprise a diyne compound as described herein above and a polyene antifungal agent. 25 [00265] Said polyene antifungal agent may preferably be Amphotericin B. A8. Fungal infection of plants 30 [00266] The present invention also relates to methods of reducing the risk of an infection by a fungus or to methods of treating an infection by a fungus in a plant by contacting said plant with a diyne salt. 55 WO 20111134538 PCT/EP2010/063161 [00267] Thus, in one aspect the invention relates to use of a diyne salt of the formula I: K Z-[C=C- C=C]-R 3 5 wherein Z and R 3 are as defined herein above in the section "Diyne salts" such as any of the diyne salts described herein above in the section "Diyne salts", for inhibiting or treating an infection by a fungus in a plant, preferably by a plant 10 pathogenic fungus. Said infection by a fungus is preferably an infection by a fungus dependent on activity of stearoyl-CoA desaturase, more preferably an infection by a plant pathogenic fungus dependent on the activity of stearoyl-CoA desaturase. [002681 Said diyne salt may in particular be a diyne salt of formula II, 15 K+ R 4 - Y C C--C C--R 3 wherein R 4 , Y and R 3 are as described herein above in the section "Diyne salt". 20 [002691 The diyne compound for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may also be a diyne of formula III: C- Y C-C--C C- R 3 25 K* -0 wherein Y and R 3 is as described herein above in the section "Diyne salts", [002701 The diyne compound for treating or reducing the risk of infection by a 30 fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may also be a diyne salt of formula IV, 56 WO 20111134538 PCT/EP2010/063161 K R4-(C(R2)2)n-X-(C(R2)2)m-[C--C- C--C]-R3 wherein R4, R 2 , n, m and R 3 are as described herein above in the section "Diyne salts" 5 in relation to formula IV. [002711 The diyne salt for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may also be a diyne salt of formula V, 10 C -(C(R 2 ))-X -C C C C-R 3 K* -O wherein R 1 , R 2 , n, X and R 3 are as described herein above in the section "Diyne salt" in relation to formula V. 15 [00272] The diyne salt for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may preferably be selected from the group consisting of potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate and potassium 14-(furan-2-yl)tetradeca- 11,13-diyne diynoate. 20 [002731 Thus, for example the plant pathogenic fungus may be selected from the group consisting of Albugo spp., Alternaria spp., Anisogranna anonala, Apiosporina niorbosa, Ascochyta spp., Aureobasidiuin zeae, Bipolaris spp., Bluneria spp., Blumeriella jaapii, Botritis spp., Botryosphaeria dothidea, Ceratocystic paradoxa, 25 Cercospora spp., Cercosporidium spp., Cladosporiuin spp., Cochiliobolus spp., Colletotrichum spp., Corynespora cassiicola, Cristulariella inoricola, Diaporthe phaseolorumn, Didymella bryoniae, Drechslera tritici, Entylona oryzai, Erysiphe spp., Fluvia fluva, Fusarium spp., Gaeunannomyces graminis, Gnomonia spp., Gremmeniella abietina, Helininthosporium spp., Leptosphaerulina crassiasca, 30 Leveillula taurica, Lophoderniun hypophyllun, Macrophonina phaseoli, Magnaporthe spp., Microdochiuni spp., Microsphaera spp., Monilinia spp., 57 WO 20111134538 PCT/EP2010/063161 Mycosphaerella spp., Myrothedium roridum, Oidiopsis sicula, Passalora puncta, Penicillium spp., Peronospora spp., Phaeociyptopus gaeumannii, Phakopsora spp., Phona arachidicola, Phraginidium potentillae, Phytophtora spp., Plasmopara spp., Plectosporiun tabacinum, Pleospora herbaruin, Podosphaera spp., 5 Pseudocercosporella spp., Pseudoperonospora cubensis, Puccinia spp., Pucciniastruin vaccinii, Pyrenophora spp., Pythiun spp., Ramularia cynarae, Rhizoctonia spp., Rhizosphaera spp., Rhynchosporium secalis, Sclerotinia spp., Sclerotium spp., Selenophoina spp., Septoria spp., Setosphaeria turcica, Sirococcus conigenus, Sphaerotheca spp., Stagonospora nordorum, Stemphyllium botryosum, 10 Taphrina deformans, Thielaviopsis spp., Tilletia barclayana, Tranzschelia discolor, Uncinula necator, Uronyces appendiculatus, Ustilaginoidea virens, Ustilago spp., Venturia spp., Verticillium spp. and Wilsonomyces carpophilus. [00274] Treatment of infections by a fungus in a plant may be done by any suitable 15 means, for example the diyne salts may be applied as sprays or dusts on the foliage of plants, or in irrigation systems. Typically, the diyne salts according to the invention are administered on the surface of the plant in advance of the pathogen in order to prevent or reduced the risk of infection. Seeds, bulbs, roots, tubers, and/or corms may also be treated to prevent pathogenic attack after planting or reducing the risk of 20 infection and for example thereby controlling pathogens carried on them or existing in the soil at the planting site. However, plants, may also be treated once an infection is already present in order to eliminate or reduce the infection, preferably eliminate the infection. Similarly, seeds, bulbs, roots, tubers and/or corms may also be treated once an infection is already present in order to eliminate or reduce the infection, preferably 25 eliminate the infection. [00275] Soil to be planted with vegetables, ormementals, shrubs, or trees can also be treated with the diyne salts of the invention for control of a variety of fungal pathogens. Treatment is preferably done several days or weeks before planting. The 30 diyne salts can be applied by either a mechanized route, e.g., a tractor, or with hand applications. [002761 In most applications said diyne salts are used with an agronomically acceptable carrier. An "agronomically acceptable carrier" is a solid or liquid which is 58 WO 20111134538 PCT/EP2010/063161 biologically, chemically and physically compatible with the diyne salts of the present invention, and which may be used in agricultural applications. Agronomically acceptable carriers suitable for use in the method of the present invention include organic solvents, and finely divided solids, and aqueous solutions or suspensions. For 5 example, the diyne salts for use in treatment or prevention of an infection by a fungus in a plant can be formulated as wettable powders, emulsifiable concentrates, dusts, granular formulations, aerosols, or flowable emulsion concentrates. In such formulations, the diyne compounds may be extended with a liquid or solid carrier and, when desired, suitable surfactants may be incorporated. 10 [002771 Optionally added components or additives, not required for fungicidal activity but useful or required for other properties, include, but are not limited to, adjuvants such as wetting agents, spreading agents, dispersing agents, stickers, adhesive and the like. Such adjuvants are well known in the art. 15 [002781 In general, the diyne salts of this invention may be dissolved in solvents such as water or other aqueous solutions, acetone, methanol, ethanol, dimethylformamide, pyridine or dimethyl sulfoxide and such solutions can further be diluted with water. The concentrations of the solution after dilution may vary from 20 1% to 90% by weight, with a preferred range being from 5% to 50%. [002791 For the preparation of emulsifiable formulations and concentrates of the diyne compounds of the present invention, the diyne compound can be dissolved in suitable organic solvents, or a mixture of solvents, together with an emulsifying agent 25 to enhance dispersion of the diyne compound in water. The concentration of the diyne compound in emulsifiable concentrates is usually from 10% to 90%, and in flowable emulsion concentrates, can be as high as 75%. [002801 Wettable, powdered formulations suitable for spraying can be prepared by 30 admixing the diyne salt with a finely divided solid, such as clays, inorganic silicates and carbonates, and silicas and incorporating wetting agents, sticking agents, and/or dispersing agents in such mixtures. The concentration of total active ingredients in such formulations is usually in the range of from 20% to 99% by weight, preferably from 40% to 75%. A typical wettable powder is made by blending 50 parts of a diyne 59 WO 20111134538 PCT/EP2010/063161 salt, 45 parts of a synthetic precipitated hydrated silicon dioxide, such as that sold under the trademark Hi-SilR, and 5 parts of sodium lignosulfonate. In another preparation a kaolin type (Barden) clay is used in place of the Hi-Sil in the above wettable powder, and in another such preparation part of the Hi-Sil is replaced with a 5 synthetic sodium silicoaluminate sold under the trademark Zeolex. RTM. 7 (J. M. Huber Corporation). [00281] Dusting formulations may be prepared by mixing the diyne salts with finely divided inert solids which can be organic or inorganic in nature. 10 [002821 Materials useful for this purpose include botanical flours, silicas, silicates, carbonates and clays. One convenient method of preparing a dust is to dilute a wettable powder with a finely divided carrier. Dust formulations or concentrates containing from 20% to 80% of the active ingredient are commonly made and are 15 subsequently diluted to from 1% to 10% use concentration. [002831 The diyne salt and formulations may be applied as fungicidal sprays by methods commonly employed, such as conventional high-gallonage hydraulic sprays, low-gallonage sprays, air-blast spray, aerial sprays and dusts. 20 [00284] The dilution and rate of application will depend upon the type of equipment employed, the method of application, plants to be treated and diseases to be controlled. Generally, the diyne compounds of this invention will be applied in an amount of from 0.06 to 60 kilograms (kg) per hectare and preferably from I to 28 kg 25 per hectare of the active ingredient. [00285] As a seed protectant, the diyne salt formulation may be coated on the seed. The dosage rate may for example be from 3 g of diyne salt per hundred kg of seed, to 1000 g per hundred kg of seed. As a soil fungicide the fungicidal formulation may be 30 incorporated in the soil or applied to the surface for example at a rate of from 0.02 to 20 kg per hectare. As a foliar fungicide, the diyne compounds may be applied to growing plants for example at a rate of from 0.01 to 10 kg per hectare. 60 WO 20111134538 PCT/EP2010/063161 [00286] The diyne salts of the present invention may be combined with other known fungicides. B1. Diyne 5 [00287] The present invention relates to diyne compounds per se as well as to the use of the diyne compounds. [002881 Thus, the present invention relates to pharmaceutical compositions 10 comprising a diyne, to methods of treatment of fungal infections with diynes in an individual in need thereof as well as to uses of diynes for inhibiting or treating an infection of a plant by a fungus. The diyne may be any of the diynes described in this section. Fungal infections which may be treated with the diynes described in this section are disclosed in more detail in the section "Fungal Infections" herein below. 15 [002891 The diynes according to the present invention are diynes of formula I': Z-[C=C- C=C]-R 3 20 wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus, and Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents, preferably with R 2 ; and 25
R
3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this section below. Z is preferably a carbon chain, which is substituted with -COOH or a bioisostere 30 thereof, preferably said -COOH or bioisostere thereof is positioned at the end of said carbon chain, preferably at the distal end of said carbon chain in relation to the diyne moiety. Thus, Z may preferably be selected from the group consisting of alkyl and alkenyl, which is substituted with R 4 , wherein R4 is -COOH or a bioisostere thereof 61 WO 20111134538 PCT/EP2010/063161 In addition, said carbon chain (such as said alkyl or alkenyl) may also optionally be substituted with one or more additional groups, preferably with one or more R2 groups, wherein R 2 preferably is as defined herein below in relation to diynes of formula II'. 5 [00290] Said bioisostere of -COOH may for example be -CO-R 1 , wherein R 1 preferably is as defined herein below in relation to diynes of formula II'. [002911 The bioisostere of -COOH may also preferably be selected from the group 10 consisting of tetrazoles, preferably from the group consisting of tetrazoles, tetrazolates and salts thereof [002921 It is also comprised in the present invention that the bioisostere of -COOH may be selected from the group of azoles, preferably the bioisostere may be a 1 ,2,4 15 oxadiazole heterocycle. [002931 Preferably Z is a C 6
-
2 0 , preferably a C 6
_
1 5 , more preferably C6- 12 , even more preferably a C 9
-
2 0 , yet more preferably a C 9
_
15 , such as a C 9
-
12 alkyl or alkenyl substituted with -COOH or a bioisostere thereof and optionally also substituted at one 20 or more positions with R 2 , preferably substituted with one or more selected from the group consisting of -CO-R 1 and R 2 . More preferably Z is R 1 -CO-(C6-20 alkyl or alkenyl)-, such as R 1
-CO-(C
9
-
2 0 alkyl or alkenyl)-, for example R 1
-CO-(C
9
_
15 alkyl or alkenyl)-, such as R 1
-CO-(C
6
-
12 alkyl or alkenyl)-. 25 [002941 More preferably, the diyne is a diyne of the formula II':
R
4 -Y C C-C C-R 3 wherein R 4 is -COOH or a bioistere thereof, wherein said bioisostere of -COOH may 30 be any bioisostere of -COOH, preferably any of the bioisosteres of -COOH mentioned herein above in relation to compounds of formula I', and Y is preferably a carbon chain of 6 to 20 carbon atoms, more preferably 9 to 20 carbon atoms, even more preferably 9 to 15 carbon atoms, yet more preferably 9 to 12 62 WO 20111134538 PCT/EP2010/063161 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds. Depending on whether a carbon atom of said carbon chain is connected to the other carbon atoms of said carbon chain by single bonds and/or double bonds each carbon atom is linked to none, one or two R 2 groups. Thus, a carbon atom connected to both 5 its neighbouring carbon atoms in the carbon chain by single bonds will be linked to two R 2 groups. A carbon atom connected to both its neighbouring carbon atoms in the carbon chain by double bonds will not be linked to any R 2 groups. A carbon atom connected to one neighbouring carbon atom in the carbon chain by a single bond and to the other neighbouring carbon atom in the carbon chain by a double bond will be 10 linked to one R 2 group. Y may furthermore be as defined herein below; AND
R
3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this section below. 15 [002951 More preferably the diyne is a diyne of the formula III': C-Y C=C- C=C- R3 R1 wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group 20 in a hydrolysis reaction; Y is a carbon chain of 6 to 20 carbon atoms and up to three double bonds, wherein each carbon of said alkyl or alkenyl is linked to none, one or two R 2 groups, wherein each R 2 independently is -H, -OH or a hydrocarbon moiety containing between I and 4 carbon atoms, inclusive; R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; or a pharmaceutically 25 acceptable salt of said diyne, wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. [002961 Y is preferably a carbon chain of 6 to 20 carbon atoms, more preferably 9 to 20 carbon atoms, even more preferably 9 to 15 carbon atoms, yet more preferably 9 30 to 12 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds. Depending on whether a carbon atom of said carbon chain is connected to the 63 WO 20111134538 PCT/EP2010/063161 other carbon atoms of said carbon chain by single bonds and/or double bonds each carbon atom is linked to none, one or two R 2 groups. Thus, a carbon atom connected to both its neighbouring carbon atoms in the carbon chain by single bonds will be linked to two R 2 groups. A carbon atom connected to both its neighbouring carbon 5 atoms in the carbon chain by double bonds will not be linked to any R 2 groups. A carbon atom connected to one neighbouring carbon atom in the carbon chain by a single bond and to the other neighbouring carbon atom in the carbon chain by a double bond will be linked to one R 2 group. 10 [00297] Accordingly, Y may be a linear C 6
-
2 0 , preferably a C6_ 1 5 , more preferably C6- 12 , even more preferably a C 9
-
20 , yet more preferably a C 9
_
15 , such as a C 9
-
12 alkyl, preferably a linear C> 11 alkyl, yet more preferably a linear C 8
_
1 0 alkyl, even more preferably a linear C 9 -alkyl. 15 [002981 Y may also be a linear C 6
-
20 , preferably a C 6
_
1 5 , more preferably C6- 12 , even more preferably a C 9
-
20 , yet more preferably a C 9
_
1 5 , such as a C 9 -1 2 alkenyl, preferably a linear C7_ 1 1 alkenyl, yet more preferably a linear C 8 10 alkenyl, even more preferably a linear C 9 - alkenyl. The alkenyl may comprise 1, 2 or 3 double bonds, preferably 1 or 2 double bonds, even more preferably only 1 double bond. The double bonds may be 20 in the cis or the trans conformation, preferably at least one double bond is in the cis conformation, even more preferably all double bonds are in the cis conformation. Accordingly, Y may be a linear C 6
-
12 , preferably a linear C7_1 alkenyl, yet more preferably a linear Cs_ 10 alkenyl preferably a linear C 9 alkenyl, wherein all double bonds are cis double bonds. The double bonds may be at any suitable position, 25 however in a preferred embodiment at least one double bond is situated at the CS, C 9 or
C
1 0 position, preferably at the C 9 position (the C in the carbonyl group being C 1 ), even more preferably at least one double bond in the cis conformation is situated at the CS,
C
9 or C1o position, preferably at the C 9 position (the C in the carbonyl group being C 1 ). 30 [00299] Thus, in a preferred embodiment of the invention the diyne compound is a compound of the formula IV': R4-(C(R2)2)n-X-(C(R2)2)m-[C-C- C-4C]-R3 64 WO 20111134538 PCT/EP2010/063161 wherein R4 is -COOH or a bioistere thereof, wherein said bioisostere of -COOH may be any bioisostere of -COOH, preferably any of the bioisosteres of -COOH mentioned herein above in relation to compounds of formula I', and 5 n is an integer, preferably an integer in the range of 4 to 10, inclusive, preferably in the range of 5 to 9, even more preferably in the range of 6 to 8, yet more preferably n is 7; and 10 m is an integer, preferably an integer in the range of 0 to 10, such as in the range of 0 to 8, for example in the range of 0 to 6, such as in the range of 0 to 4, for example in the range of 0 to 2, such as 0; and each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 15 6 carbon atoms, inclusive; and X is -CH 2
-CH
2 - or -CH=CH- or phenyl, preferably X is -CH 2
-CH
2 - or -CH=CH-; and
R
3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this 20 section below. [003001 In a preferred embodiment of the invention the diyne is a compound of formula V': C -- (C(R2
)
2)n- X- C C -C C - R 3 25 R 1 wherein, R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; n is an integer between 4 and 10, 30 inclusive; X is -CH 2
-CH
2 - or -CH=CH- or phenyl, preferably X is -CH 2
-CH
2 - or 65 WO 20111134538 PCT/EP2010/063161 CH=CH-; and R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions. n is an integer in the range of 4 to 10, preferably in the range of 5 to 9, even more 5 preferably in the range of 6 to 8, yet more preferably n is 7. [003011 In one preferred embodiment of the invention, the diyne is a compound of formula III', wherein X is -CH=CH-, wherein the double bond is in the trans or cis conformation, preferably in the cis conformation. Also in this embodiment it is 10 preferred that n is as outlined above and R 2 is as outlined below. [00302] In another preferred embodiment of the invention, the diyne is a compound of formula III', wherein X is -CH 2
-CH
2 -. Also in this embodiment it is preferred that n is as outlined above and R 2 is as outlined below. 15 [003031 R 1 of the diyne either of formula II', III' or V' or of formula I', or IV' when comprised therein is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction. Preferred moieties that can be replaced by a hydroxyl group in a hydrolysis reaction may for example be selected from the group 20 consisting of amines and lower linear or branched alkoxy groups. Lower alkoxy are preferably Ci-, more preferably C 1 3 , even more preferably C1- 2 alkoxy, preferably linear alkoxy. Thus, preferably R 1 of the diyne either of formula II' or formula III' may be selected from the group consisting of -OH, -NH 2 , -OCH 3 and -OC 2
H
5 , preferably R 1 is -OH. 25 [00304] R 3 of the diyne of formula I', II', III', IV' or V' is a heterocyclic ring, which optionally may be substituted at one or more positions. If substituted, the heterocyclic ring is preferably substituted with one or more, preferably one or two selected from the group consisting of lower alkyl, lower alkenyl, lower alkoxy, lower 30 alcohol, hydroxyl, amine, -NO 2 and halogen. Lower alkyl is preferably C 1 5 , more preferably Ci , even more preferably C1 alkyl. Lower alkenyl is preferably C 1 5 , more preferably Ci 3 , even more preferably C 1 2 alkenyl. Lower alkoxy is preferably C 1 5 , more preferably C 1 , even more preferably C 1 alkoxy. Lower alcohol is preferably Cp 5, more preferably C 1 3 , even more preferably C 1 alcohol comprising one or more -OH 66 WO 20111134538 PCT/EP2010/063161 groups, preferably only one -OH group. Halogen may be any halogen, but is preferably -F. It is however preferred that R 3 is a heterocyclic ring, which is not substituted or that R 3 is a heterocyclic ring substituted with a small substituent, preferably a small substituent selected from the group consisting of methyl, methoxy, 5 hydroxyl, -CH 2 -OH, amine and halogen, preferably methyl. [003051 R 3 of the diyne of formula I', II', III', IV' or V' is preferably an aromatic heterocyclic ring. 10 [00306] R 3 of the diyne of formula I', II', III', IV' or V' is preferably a 3 to 7 membered heterocyclic ring, more preferably a 5 to 6 membered heterocyclic ring, even more preferably a 5 membered heterocyclic ring. The heterocyclic ring may be aromatic or non-aromatic. In one embodiment the heterocyclic ring is a 3 to 7 membered aromatic heterocyclic ring, more preferably a 5 to 6 membered aromatic 15 heterocyclic ring, even more preferably a 5 membered aromatic heterocyclic ring. [003071 The heterocyclic ring may comprise one or more heteroatoms, preferably in the range of 1 to 3 heteroatoms, more preferably in the range of 1 to 2 heteroatoms, yet more preferably 1 heteroatom. Said heteroatom(s) are preferably selected from the 20 group consisting of S, N and 0. [003081 In a very preferred embodiment of the invention, R 3 is selected from the group consisting of pyrrole, furan and thiophene, which may optionally be substituted as outlined above at one or more positions. R3 may also be selected from the group 25 consisting of imidazole, oxazole, cyclopentadiene and triazole. Preferably, R 3 is furan, which is not substituted except for being linked to the diyne chain or is substituted at one or more positions as outlined above, even more preferably R 3 is furan, which is not substituted except for being linked to the diyne chain or is substituted at one or more positions with a small substituent, preferably a small substituent selected from 30 the group consisting of methyl, methoxy, hydroxyl, amine and halogen, preferably methyl. [003091 In a very preferred embodiment of the invention, R 3 is furan. 67 WO 20111134538 PCT/EP2010/063161 [00310] It is preferred that heterocyclic ring is 2-substituted with the -[C-C- C-C] Z chain. In particular in embodiments wherein the heterocyclic ring contains only one heteroatom it is preferred that the heterocyclic ring is 2-substituted with the -[CaC C-C]-Z chain. Thus, in embodiments of the invention wherein R 3 is pyrrole, furan or 5 thiophene, in particular when R 3 is furan, then it is preferred that the heterocyclic ring is 2-substituted with the -[CaC- CaC]-Z chain. [003111 Each R 2 of a diyne of formula IV' or V' as well as each R 2 when contained in diynes of formula I', II' or III' is preferably, independently, -H, -OH or a 10 hydrocarbon moiety containing between 1 and 6, preferably 1 to 4 carbon atoms, inclusive. The hydrocarbon moiety may be an alkyl, alkenyl or alkynyl, such as C 14 alkyl, C 1 4 alkenyl or C 1 4 alkynyl. It is however also possible that two neighbouring R2 groups are connected to form a hydrocarbon ring system. Said ring system may for example be a 3 to 10 membered ring, preferably a 3 to 7 membered ring, more 15 preferably a 5 to 6 membered ring system (numbers include carbon in carbon chain plus R 2 carbons). The ring system may be aromatic or none aromatic, for example the ring system may be an 6 membered aromatic ring. Thus, any particular diyne may contain a plurality of different R 2 groups. It is preferred that the majority of the R 2 groups of a diyne compound of formula I', IF1, Ill', IV' or V' is -H. It is even more 20 preferred that all R 2 groups of a diyne compound of formula I', 11', III', IV' or V' except for in the range of 0 to 5 R 2 groups, preferably all R2 groups except for in the range of 0 to 3 R2 groups, more preferably all R2 groups except for in the range of 0 to 1 R2 groups are -H. It is even more preferred that each R2 group of a diyne compound of formula I', II', III', IV' or V' is -H. 25 [00312] In embodiments of the invention where two R2 groups are connected to form a ring system, and in particular if said ring system is aromatic, then it is preferred that said R2 groups are positioned on C 7 and C 8 ; or on Cs and C 9 ; or on C 9 and C. or on C 10 and C 11 , preferably on C 9 and C 10 position (the C in the carbonyl 30 group being C 1 ). 68 WO 20111134538 PCT/EP2010/063161 [00313] A preferred diyne compound according to the invention is (Z)-12-(furan-2 yl)dodeca-7-en-9,11-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof 5 [00314] Another preferred diyne compound according to the invention is (Z)-13 (furan-2-yl)trideca-8-en-10,12-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof [003151 Yet another preferred diyne compound according to the invention is (E) 10 14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof [003161 Yet another preferred diyne compound according to the invention is the diyne compound (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid or a salt 15 thereof, preferably a pharmaceutically acceptable salt thereof [003171 Another preferred diyne compound according to the invention is the diyne compound (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof 20 [00318] Yet another preferred diyne compound according to the invention is (Z) methyl 14-(furan-2-yl)tetradeca-9-en-11,13-diynoate or a salt thereof, preferably a pharmaceutically acceptable salt thereof 25 [003191 Another preferred diyne compound according to the (Z)-ethyl 14-(furan-2 yl)tetradeca-9-en-11,13-diynoate or a salt thereof, preferably a pharmaceutically acceptable salt thereof [003201 In yet another embodiment of the invention the diyne compound may be 30 selected from the group consisting of (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, (Z)- 14-(5-methylfuran-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, 8-(2-(4-(furan-2 yl)buta-1,3-diynyl)phenyl)octanoic acid, (Z)-14-(4,5-dimethylfuran-2-yl)tctradeca-9 en- 11,13-diynoic acid and 14-(furan-2-yl)tctradeca-11,13-diynoic acid. 69 WO 20111134538 PCT/EP2010/063161 [00321] Pharmaceutically acceptable salts of the diyne compounds of the invention and in particular diyne compounds selected from the group consisting of (Z)-12 (furan-2-yl)dodeca-7-en-9,11-d iyno i c acid, (Z)-13-(furan-2-yl)trideca-8-en-10,12 diynoic acid, (E)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, (Z)- 14-(furan-2 5 yl)tetradeca-9-en- 11,13-diynoic acid, (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, (Z)-14-(5-methylfuran-2 yl)tetradeca-9-en-11,13-d i y n o i c a c i d , 8-(2-(4-(furan-2-yl)buta-1,3 diynyl)phenyl)octanoic acid, (Z)-14-(4,5-dimethylfuran-2-yl)tetradeca-9-en-11,13 diynoic acid and 14-(furan-2-yl)tetradeca-11,13-diynoic acid may be any 10 pharmaceutically acceptable salt. Preferably, said pharmaceutically acceptable salt is an alkali metal salt, such as a potassium salt or a sodium salt, preferably a potassium salt. [00322] The diyne compounds according to the invention may be prepared 15 essentially as described in US patent US6,541,506, which is hereby incorporated by reference. B2. Particular Diynes 20 [003231 The following section relates to diynes according to the invention which are suitable for being contained within the pharmaceutical compositions according to the invention, as well as for use in methods of treatment of fungal infections according to the invention. However, in some embodiments the invention also relates to substantially pure diyne compounds, such as pure diyne compounds, and the diyne 25 compounds described in this section are in particular useful in these embodiments. [00324] Thus, in certain embodiments of the invention, and in particular in such embodiments of the invention relating to the diyne compounds per se it is preferred that the diyne compound is a diyne compound of formula I': 30 Z-[C=C- C=C]-R 3 70 WO 20111134538 PCT/EP2010/063161 wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and R 3 is a heterocyclic ring, which optionally is substituted and wherein the diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a 5 fungus, with the proviso that the diyne is not a compound selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, (E)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid, (Z)-13-(furan-2-yl)trideca-8-en-10,12-diynoic acid, (Z)- 12-(furan-2-yl)dodeca-7-en-9, 11 -diynoic acid, (9Z, 16Z)-octadeca-9,16-dien 12,14-diynoic acid, (Z)-methyl 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate and 10 (1 OE, 1 6Z)-9-hydroxyoctadeca- 10,1 6-dien- 12,14-diynoic acid [00325] In these embodiments it is also preferred that the diyne compound is not a compound selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid, (Z)-13-(furan-2-yl)trideca-8-en-10,12-diynoic acid and (Z)-12 15 (furan-2-yl)dodeca-7-en-9,11 -diynoic acid. [003261 In these embodiments it is even more preferred that the diyne compound is not a compound according to formula VII', 0 ||
R
1 -C- (C(R 2
)
2 )x- C 2
H
2
-C
4
-R
3 20 wherein R 1 is a hydroxyl group or a moiety that can be replaced by e hydroxyl group in a hydrolysis reaction, each R 2 is independently H or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive, R 3 is a pyrrole, furan or 25 thiophene ring and X is an integer between 4 and 10, inclusive. [00327] In these embodiments it may also be preferred that R 3 of said diyne compound is a heterocyclic ring, which optionally may be substituted at one or more positions with the proviso that R 3 is not pyrrole, furan or thiophene. 30 71 WO 20111134538 PCT/EP2010/063161 [00328] The diyne compound of these embodiments may also be a diyne of formula VIII',
R
4 -Y C C-C C-R 3 5 wherein R 4 , Y and R 3 are as described herein above in the section "Diyne", with the proviso that the diyne compound is not any of the above mentioned compounds, which are preferably excluded. 10 [00329] The diyne compound of these embodiments may be a diyne of formula IX': C-Y C--C--C C--R 3 15 R 1 wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; Y is a carbon chain of 6 to 12 carbon atoms and up to three double bonds, wherein each carbon of said alkyl or alkenyl is linked to one or two R 2 20 groups, wherein each R 2 independently is -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions, with the proviso that the diyne compound is not any of the above mentioned compounds, which are preferably excluded. 25 [00330] The diyne compound of these embodiments may also be a diyne of formula IV', R4-(C(R2)2)n-X-(C(R2)2)m-[C-C- C-C]-R3 30 72 WO 20111134538 PCT/EP2010/063161 wherein R 4 , R 2 , n, m and R 3 are as described herein above in the section "Diyne" in relation to formula IV', with the proviso that the diyne compound is not any of the above mentioned 5 compounds, which are preferably excluded. [003311 The diyne compound of these embodiments may also be a diyne of formula V', C- ((R 2
)
2 )n-X C--C- C- C R 3 10 R1 wherein R 1 , R2, n, X and R 3 are as described herein above in the section "Diyne" in relation to formula V', 15 with the proviso that the diyne compound is not any of the above mentioned compounds, which are preferably excluded. [003321 In one of these particular embodiments it is preferred that the diyne is a diyne of the formula ', wherein Z is alkyl, which optionally may be substituted. Z 20 may for example be substituted with one or more selected from the group consisting of -CO-R 1 and R2, wherein R1 and R2 preferably are as defined herein above in relation to dynes of formula II'. [003331 Thus, preferably Z may be a C6- 20 alkyl, preferably a C 6 _1 5 , more preferably 25 C 6 -1 2 , even more preferably a C9-20, yet more preferably a C 9 _1 5 , such as a C 9 -1 2 alkyl, optionally substituted, preferably substituted with one or more selected from the group consisting of_-COOH, bioisosters of -COOH and R2, more preferably from the group consisting of -CO-R 1 and R 2 , yet more preferably substituted with one group selected from the group consisting of -COOH and bioisoters of -COOH and with one 30 or more selected from the group consisting of R 2 . In this context bioisosters of COOH may be any of the bioisosters of-COOH described herein above in the section 73 WO 20111134538 PCT/EP2010/063161 "Diynes" and R 2 is as described herein above in the section "Diynes" in relation to formula IV' and V'. More preferably Z is R 4
-(C
6
-
2 0 alkyl)-, for example R 4
-(C
6
_
15 alkyl), such as R4-(C 6 -1 2 -alkyl), for example R 4
-(C
9 -20-alkyl), such as R4-(C 9
_
15 -alkyl), for example R 4
-(C
9
-
12 -alkyl), wherein R 4 is as described herein above in the section 5 "Diynes" in relation to formula II'. B3. Properties of diyne compounds [003341 The present invention relates to diyne compounds per se as well as to use 10 of the diyne compounds in treatment of fungal infections. The structural properties of the diynes according to the invention are described herein above particularly in the section "Diynes", but also in the section "Particular diynes". In addition to these structural properties it is preferred that the diynes according to the invention, in particular the diyne compounds contained in the pharmaceutical compositions of the 15 invention also have the functional properties described in detail in this section. [003351 It is very preferred that the diyne compound according to the invention is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. In particular it is preferred that the diyne compound is capable 20 of inhibiting conversion of stearic acid to oleic acid in a fungus. It is also preferred that the diyne compound is capable of inhibiting conversion of palmitic acid to palmitoleic acid. [003361 Whether a diyne compound is capable of inhibiting conversion of stearic 25 acid to oleic acid in a fungus may be determined in any useful method known to the skilled person. According to the present invention one method for determining whether a diyne compound is capable of inhibiting conversion of stearic acid to oleic acid in a fungus comprises the steps of a) providing a fungus, a fungal extract or an in vitro assembled complex 30 comprising stearoyl-CoA 9 desaturase (such as OLE-1) b) optionally providing co-factors for stearoyl-CoA 9 desaturase (such as OLE-1) c) providing a substrate selected from the group consisting of stearic acid and activated forms of stearic acid d) providing a diyne compound 74 WO 20111134538 PCT/EP2010/063161 e) incubating said fungus, fungal extract or in vitro assembled complex with said substrate, co-factors and with a predetermined amount of said diyne compound for a predetermined amount of time f) detecting the presence of product formed, wherein the product is selected from 5 the group consisting of oleic acid and activated forms of oleic acid. [003371 Said activated form of stearic acid is preferably stearyl-CoA and said activated from of oleic acid is preferably oleoyl-CoA. If stearyl-CoA is provided in step b), then preferably step d) comprises detecting formation of oleoyl-CoA. 10 [003381 Simiarly, whether a diyne compound is capable of inhibiting conversion of palmitic acid to palmitoleic acid in a fungus may be determined in any useful method known to the skilled person. According to the present invention one method for determining whether a diyne compound is capable of inhibiting conversion of palmitic 15 acid to palmitoleic acid in a fungus comprises the steps of a) providing a fungus, a fungal extract or an in vitro assembled complex comprising stearoyl-CoA 9 desaturase (such as OLE-1) b) optionally providing co-factors for stearoyl-CoA 9 desaturase (such as OLE-1) c) providing a substrate selected from the group consisting of palmitic acid and 20 activated forms of palmitic acid d) providing a diyne compound e) incubating said fungus, fungal extract or in vitro assembled complex with said substrate, co-factors and a predetermined amount of said diyne compound for a predetermined amount of time 25 f) detecting the presence of product formed, wherein the product is selected from the group consisting of palmitoleic acid and activated forms of palmitoleic acid. [003391 Said activated form of palmitic acid is preferably palmitoyl-CoA and said 30 activated from of palmitoleic acid is preferably palmitoleoyl-CoA. If palmitoyl-CoA is provided in step b), then preferably step d) comprises detecting formation of palmitoleoyl-CoA. 75 WO 20111134538 PCT/EP2010/063161 [00340] Said fungus may be provided as an intact viable fungus or as intact fungal cells. The fungal extract may be any extract comprising stearoyl-CoA 9 desaturase (such as OLE-1). In Sacchoronyces cerevisiae and other fungi the stearoyl-CoA desaturase is mainly located in the membranes of the endoplasmatic reticulum and 5 accordingly it is preferred that said fungal extract comprises endoplasmatic reticulum or parts thereof. For example, the extract may be prepared by lysis of fungal cells, followed by separation of fractions for example by centrifugation. In particular, differential centrifugation may be used to enrich for endoplasmatic reticulum. However, fractions or extracts comprising endoplasmatic reticulum may also be 10 obtained by other useful methods known to the skilled person. Alternatively, stearoyl CoA desaturase may be obtained by in vitro assembly by methods known to the skilled person. [00341] Preferably, the substrate provided in step c) is labelled allowing easy 15 detection of the product in step f). The substrate may be labelled with any suitable label, such as a radioactive label, a dye, a heavy metal, a fluorescent label or a bioilluminescent label. Preferably, the substrate is radioactively labelled. Detecting product formed may then be performed by detecting labelled product. The detection method will depedent on the particular label used. 20 [00342] One non-limiting example of determining inhibition of conversion of stearic acid to oleic acid is described herein below in Example 12B. The skilled person will understand that a similar method may be performed for determining inhibition of conversion of palmitic acid to palmitoleic acid, by exchanging the 25 substrate provided. [00343] Preferably, the diyne compounds according to the invention are capable of inhibiting at least 50%, more preferably at least 60%, even more preferably at least 70%, yet more preferably at least 80%, yet more preferably at least 90%, even more 30 preferably at least 95%, yet more preferably essentially 100% of the formation of oleic acid, wherein "essentially 100%" means that no detectable product is formed. Inhibition is determined in relation to a control, wherein said fungus or fungal extract is incubated with said substrate in the absence of said diyne compound for the same predetermined amount of time. 76 WO 20111134538 PCT/EP2010/063161 [00344] It is preferred that the diyne compound of the invention is capable of inhibiting the activity of a fungal steraroyl-CoA 9 desaturase, preferably the diyne compound is capable of inhibiting the activity of a fungal OLE-1. 5 [00345] The activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be inhibited by different means by said diyne compound. Thus, the diyne may directly inhibit the enzymatic activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1). Thus, the diyne compound may preferably be an inhibitor of the fatty acid 10 desaturase activity of OLE-i polypeptide. [00346] Whether said diyne is capable of directly inhibiting the activity of fungal stearoyl-CoA 9 desaturase (such as OLE-1) may for example be determined using an in vitro assay for fungal stearoyl-CoA 9 desaturase (such as OLE-1) activity. Thus, for 15 example fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be incubated with either stearic acid and/or palmitic acid and/or activated forms thereof (such as stearyl CoA or palmitoyl-CoA) optionally together with co-factors to form a reaction mixture and the formation of oleic acid and/or palmitoleic acid and/or activated forms thereof may then be determined. Addition of a diyne compound to said reaction mixture 20 preferably significantly inhibits the formation of oleic acid and/or palmitoleic acid or activated forms thereof. Thus, preferably addition of a diyne compound to said reaction mixture reduces the formation of oleic acid and/or palmitoleic acid to less than 30%, preferably less than 20%, more preferably to less than 10%, for example to less than 5%, for example to less than 3%, such as to less than 1%. Said fungal 25 stearoyl-CoA 9 desaturase (such as OLE-1) may be provided to said reaction mixture in a purified form or as part of a crude extract, for example a fungal extract or as prepared in vitro. [003471 Mammalian desaturases are significantly different to fungal stearoyl-CoA 30 9 desaturases (such as OLE-1), for example mammalian desaturases lacks an integral cytochrome b 5 domain (Krishnamurthy et al., 2004, Microbiology, 150, 1991-2003. Accordingly, inhibitors of fungal stearoyl-CoA 9 desaturase (such as OLE-1), may be specific for the fungal enzymes in the sense that they do not inhibit mammalian desaturases to any significant extent. 77 WO 20111134538 PCT/EP2010/063161 [00348] In a preferred embodiment of the invention the diyne compound according to the invention is a selective inhibitor of a fungal stearoyl-CoA 9-desaturase (such as OLE-1). Thus it is preferred that the diyne compound is capable of inhibiting the 5 activity of at least one fungal stearoyl-CoA 9 desaturase, preferably of more than one fungal stearoyl-CoA 9 desaturase. It is furthermore preferred that the diyne compound of the invention does substantially not inhibit at least one mammalian stearoyl-CoA 9 desaturase, preferably human stearoyl-CoA 9 desaturase. Accordingly, it is preferred that if using the above-described in vitro assay, then the diyne compounds according 10 to the invention are capable of reducing the formation of oleic acid and/or palmitoleic acid to less than 30%, preferably less than 2 0%, more preferably to less than 10%, for example to less than 5%, for example to less than 3%, such as to less than 1% in the presence of one or more fungal stearoyl-CoA 9 desaturase (such as OLE-1), but in the absence of any mammalian stearoyl-CoA 9 desaturase. In addition it is preferred that 15 in a similar in vitro assay said diyne compound is substantially not capable of reducing the formation of oleic acid and/or palmitoleic acid and thus in the presence of said diyne compound at least 80%, preferably at least 90%, yet more preferably at least 95% oleic acid and/or palmitoleic acid is formed compared to in the absence of said diyne compound, when incubating either stearic acid and/or palmitic acid with 20 one or more mammalian stearoyl-CoA 9 desaturases, preferably in the presence of human stearoyl-CoA 9 desaturase, but in the absence of any fungal stearoyl-CoA 9 desaturase (such as OLE-1). [003491 The diyne may also indirectly inhibit the activity of said fungal stearoyl 25 CoA 9 desaturase (such as OLE-1) by down modulating the level of said fungal stearoyl-CoA 9 desaturase (such as OLE-1) in a fungus. Thus, the diyne compound may decrease the stability or the half life of said fungal stearoyl-CoA 9 desaturase (such as OLE-1), thereby down modulating the level. The diyne compound may also inhibit the expression of said fungal stearoyl-CoA 9 desaturase (such as OLE-1), for 30 example by inhibiting transcription or translation of fungal stearoyl-CoA 9 desaturase (such as OLE-1). [003501 In one embodiment of the invention, the diyne compound may down modulate the expression of the OLE-i polypeptide by modulating the activity of a 78 WO 20111134538 PCT/EP2010/063161 transcriptional regulator of the gene encoding the OLE-I polypeptide. The activity of the transcriptional regulator may for example be down modulated by inhibition of the binding of the transcriptional regulator to an OLE-I promoter or enhancer region. 5 [00351] Spt23p/Mga2p is a fungal transcriptional regulator that amongst others controls the expression of fungal stearoyl-CoA 9 desaturase (such as OLE-1). Thus, the diyne compound may be capable of inhibiting the activity of Spt23p/Mga2p. [003521 It is very preferred that the diyne compounds according to the present 10 invention are capable of reducing or preferably inhibiting formation and/or growth of hyphal filaments and/or formation of germ tubes from blastospores. Formation and/or growth of hyphal filaments may be determined by a method comprising the steps of: a) cultivating one or more fungi in vitro under conditions allowing hyphal filament formation and/or growth 15 b) contacting said fungi with a test diyne compound c) visually inspecting whether hyphal filaments form and/or elongate. [003531 Induction of germ tube formation from blastospores may be determined by a method comprising the steps of 20 a) providing one or more fungi in the form of blastospores b) cultivating said blastospores in vitro under conditions allowing induction of germ tube formation c) contacting said blastospores with a test diyne compound d) visually inspecting whether germ tubes are formed 25 [00354] Preferably, these methods involve also cultivating one or more fungi under conditions allowing hyphal filament growth or germ tube formation but in the absence of the test diyne compound as control. 30 [00355] If the visual inspection reveals significantly reduced hyphal filament or germ tube formation of said one or more fungi cultivated in the presence of said diyne compared to in the absence of said diyne compound, then the diyne compound is said to be capable of reducing formation and/or growth of hyphal filaments or formation of germ tubes. 79 WO 20111134538 PCT/EP2010/063161 [00356] If the visual inspection reveals essentially no hyphal tube formation, preferably no hyphal tube formation in the presence of said diyne compound, then the diyne compound is said to be capable of inhibiting formation of hyphal filaments or to 5 be capable of inhibiting hyphal growth. [003571 It is also very preferred that the diyne compounds according to the present invention are capable of reducing or preferably inhibiting clamydospore formation. This may be determined by a method comprising the steps of: 10 a) cultivating one or more fungi in vitro under conditions allowing clamydospore formation b) contacting said fungi with a test diyne compound c) visually inspecting whether clamydospores are formed 15 [003581 Preferably, the method involves also cultivating one or more fungi under conditions allowing clamydospore formation but in the absence of the test diyne compound as control. [00359] If the visual inspection reveals significantly reduced clamydospore 20 formation of said one or more fungi cultivated in the presence of said diyne compared to in the absence of said diyne compound, then the diyne compound is said to be capable of reducing formation of clamydospores. [003601 If the visual inspection reveals essentially no clamydospore formation, 25 preferably no clamydospore formation in the presence of said diyne compound, then the diyne compound is said to be capable of inhibiting formation of clamydospores. [003611 The MIC (minimal Inhibitory Concentration) is the minimal concentration of diyne compound required for inhibiting essentially 100%, such as 100% growth of 30 a fungi. Preferably, the MIC of the diyne compounds according to the invention is at the most 500 ng/ml, preferably at the most 250 ng/ml, yet more preferably at the most 100 ng/ml, for example at the most 60 ng/ml, such as at the most 40 ng/ml, for example at the most 20 ng/ml, such as at the most 10 ng/ml in relation to at least 3 different fungi. 80 WO 20111134538 PCT/EP2010/063161 [00362] It is also preferred that the diyne compounds according to the invention are capable of killing one or more fungi, preferably capable of killing at least 2, more preferably at least 5, even more preferably at least 10 different fungi. Preferably, the 5 diyne compound has a minimum fungicidal concentration (MFC) of at the most 100 pg/ml, preferably at the most 50 ag/ml, yet more preferably at the most 10 gg/ml, even more preferably at the most 1 tg/ml, against one or more fungi, preferably one or more pathogenic fungi, even more preferably against at least 2, yet more preferably at least 5, even more preferably at least 10 different fungi. Preferably, said MFC for a 10 given fungus is determined in a method comprising the steps of a) cultivating said fungus in vitro b) contacting said fungus with various concentrations of test diyne compound c) incubating said fungus with said diyne test compound for a predetermined 15 amount of time d) transferring said fungus to another in vitro culture medium e) determining growth of said fungus in said another in vitro culture medium [00363] The lowest concentration of diyne test compound resulting in essentially 20 no growth in step e), preferably in no detectable growth in step e) is the MFC. The MFC may preferably be determined using the assay described herein below in Example 10. [003641 In addition it is preferred that contacting a fungus with said diyne 25 compound leads to a rapid loss of viability. This may for example be determined by a method comprising the steps of: a) cultivating one or more fungi in vitro, thereby obtaining a fungal culture 30 b) determining the CFU/ml in said fungal culture c) contacting the fungal culture with a test diyne compound d) incubating said fungal culture with said test diyne compound for a predetermined amount of time e) determining the CFU/ml in said fungal culture 81 WO 20111134538 PCT/EP2010/063161 [00365] If the CFU/ml determined in step e) is at the most 20%, preferably at the most 15%, yet more preferably at the most 10% of the CFU/ml determined in step b), wherein said predetermined amount of time is in the range of 0.5 to 24 hours, 5 preferably in the range of 0.5 to 12 hours, even more preferably in the range of 0.5 to 6 hours, yet more preferably in the range of 0.5 to 2 hours, such as in the range of 1 to 24 hours, even more preferably in the range of 1 to 12 hours, yet more preferably in the range of 1 to 6 hours, even more preferably in the range of 1 to 2 hours then said diyne compound is said to be capable of leading to rapid loss of viability of said 10 fungus. Thus, it is preferred that at least a 1000 fold reduction in CFU/ml is determined in step e) compared to step b), when said predetermined amount of time is at least 3 hours, such as 3 hours. One example of how rapid loss of viability may be determined is described in Example 11. 15 [003661 As outlined above it is preferred that the diyne compound has fungicidal activity against one or more fungi and in addition it is preferred that said diyne compound is capable of leading to rapid loss of viability of one or more fungi. [00367] It is also preferred that the diyne compounds according to the invention are 20 capable of inhibiting growth of fungi. The IC50 indicates the concentration where 50% growth inhibition is obtained. Preferably, the IC50 of the diyne compounds according to the present invention is at the most 100 ng/ml, preferably at the most 50 ng/ml, even more preferably at the most 25 ng/ml, yet more preferably at the most 10 ng/ml, for example at the most 5 ng/ml, such as at the most 1 ng/ml in respect of at 25 least 1, preferably at least 3 different fungi. [00368] Said one or more fungi are preferably one or more fungi selected from the group consisting of Ascomycete, Basidiomycete, Deuteromycete, Oomycete, and combinations thereof. More preferably said one or more fungi are fungal pathogens of 30 mammals. [003691 Thus said one or more fungi may be selected from the group consisting of Candida spp. (for example C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guillierinondii, C. haenulonii, C. lusitaniae, C. lipolytica, C. 82 WO 20111134538 PCT/EP2010/063161 norvegensis, C. viswanathii, C. kefyr or C. dubliniensis), Aspergillus spp. (for example A. funigatus, A. flavus, A. niger or A. terreus), Histoplasma capsulatun, Coccidioides immitisCoccidioides posadasii, Cryptococcus spp. (for example C. neoformans (for example var. neoformans or var. gattii), C. bidus, C. laurentii, or C. 5 fusarium), Zygomycetes (such as Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle bertholletiae, Saksenaea vasiformis, Mucor circinelloides, M. ramosissim us, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus or Syncephalastruni racemosum), Malassezia spp. (for example M. furfur or M. globosa), Hyalohyphomycetes (for example Fusarium solani or Scedosporium 10 spp., such as S. prolificans or S. apiospermum), Dermatophytes (for example Trichophyton spp. (for example T. mentagrophytes, T. rubrum or T. tonsurans), Epidermophyton floccosum, Microsporum spp (fbr example M. cookei, M. canis, M. vanbreuseghemii, M. gallinae or M. gypseum) or Trichosporon terrestre), Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi (for example 15 Fonsecaea pedrosoi, F. compacta, Cladophylophora carrionii or Phialophora verrucosa)and Madurella spp. (for example M. mycetomatis or M. griseum). [003701 In some embodiments of the invention the diyne compound substantially does not significantly inhibit ergosterol synthesis; however in other embodiments of 20 the invention it is possible that the diyne compounds in addition to above mentioned functions also are capable of inhibiting ergosterol synthesis. In these embodiments, then upon addition of said diyne compound to a fungus cultivated in vitro, then ergosterol is produced at substantially the same level as in the absence of said diyne, i.e. that at least 80%, preferably at least 85%, more preferably at least 90% of the 25 ergosterol produced in the absence of said diyne compound is produced in the presence of said diyne compound. Within the present context "does not significantly inhibit" means that at concentrations similar to the MFC, such as at a concentration of at the most 2 times MFC, more preferably at a concentration of at the most 1.5 time MFC, such as at a concentration of at the most MFC of said particular diyne against a 30 given fungus, then said diyne does not inhibit ergosterol synthesis in said fungus (i.e. ergosterol is produced at substantially the same level as in the absence of said diyne as described above). Ergosterol synthesis may be determined by any suitable method known to the skilled person, for example by incubating fungal cells with 13 C labelled 83 WO 20111134538 PCT/EP2010/063161 acetate and determining the ratio of 13 C labelled to unlabelled ergosterol. Preferably ergosterol synthesis may be determined as described herein in Example 16. [00371] In some embodiments of the invention the diyne compound does not 5 significantly inhibit chitin synthase and/or B-glucan synthase; however in other embodiments of the invention it is possible that the diyne compounds in addition to above mentioned functions also are capable of inhibiting chitin synthase and/or B glucan synthase. Within the present context "does not significantly inhibit" means that at concentrations similar to the MFC, such as at a concentration of at the most 2 times 10 MFC, more preferably at a concentration of at the most 1.5 time MFC, such as at a concentration of at the most MFC of said particular diyne against a specific fungus, then chitin synthase and/or B-glucan synthase activity of said fungus is inhibited by no more than 30%, preferably no more than 20%. Inhibition of chitin synthase and/or B glucan synthase may for example be determined as described herein in Example 15. 15 B4. Pharmaceutical composition [003721 The pharmaceutical compositions comprising diyne compound according to the present invention may be in any suitable form depending on the fungal infection 20 to be treated. [003731 Thus, the pharmaceutical composition may be formulated for topical administration or for systemic administration. Typically, if the fungal infection is a local infection on a body surface, then the pharmaceutical composition is typically 25 formulated for topical administration. If the fungal infection is a disseminated infection and/or an infection of one or more inner organs, tissues or cells then the pharmaceutical composition is typically formulated for systemic administration. [003741 In addition, to said diyne compounds the pharmaceutical compositions 30 according to the invention will frequently comprise one or more pharmaceutically acceptable excipients. [003751 For therapeutic uses, the pharmaceutical compositions comprising dines may be administered systemically, for example, formulated in a pharmaceutically 84 WO 20111134538 PCT/EP2010/063161 acceptable buffer such as physiological saline. Treatment may be accomplished directly, e.g., by treating the individual (such as a human being) with a diyne compound or pharmaceutical composition comprising a dine compound of the invention. Preferable routes of administration include, for example, inhalation or 5 subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the diyne compounds in the patient. Treatment of human beings or other animals is carried out using a therapeutically effective amount of a diyne compound of the invention in a physiologically acceptable carrier. Suitable carriers and their formulation are described, for example, 10 in Remington: The Science and Practice of Pharmacy, (19th ed.) ed. A. R. Gennaro A R., 1995, Mack Publishing Company, Easton, Pa. [003761 In one preferred method, one or more diyne compounds of the invention are formulated in combination with a solid or a liquid dermatologically acceptable 15 carrier. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, alcohols, or glycols (or water-alcohol/glycol blends), in which the present compounds can be dissolved or dispersed at effective levels. Adjuvants (such as flavourings and/or fragrances), surfactants, and additional antimicrobial agents can be 20 added to optimize the properties for a given use. The compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. The liquid compositions can also be employed as eye drops, mouth washes, douches, etc. 25 [003771 Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user, which is mainly relevant for treating fungal infections of the skin. 30 [00378] In other medical applications, an antifungal compound can be added to materials used to make medical devices such as catheters, including but not limited to intravenous, urinary, intraperitoneal, ventricular, spinal, and surgical drainage catheters, in order to prevent colonization and systemic seeding by potential fungal 85 WO 20111134538 PCT/EP2010/063161 pathogens. Similarly, an antifungal compound may be added to the materials that constitute various surgical prostheses and to dentures to prevent colonization by fungal pathogens and thereby prevent more serious invasive infection or systemic seeding by these pathogens. 5 [00379] The amount of the diyne compound to be administered may vary depending upon the manner of administration, the age and body weight of the individual, and the type of fungal infection and extensiveness of the infection. 10 [00380] However, preferably the pharmaceutical compositions comprising diyne compounds according to the invention are formulated for administration of in the range of 0.001mg/Kg to 100mg/kg, preferably in the range of 0.001mg/Kg to 100mg/kg daily, in particular when the individual to be treated is a mammal, such as a human being. 15 [003811 The total concentration of one or more diyne compounds of the invention in the present compositions can be varied widely, and will depend on factors such as the compatibility of the active ingredient(s) with the vehicle, the potency of the active ingredient(s) and the condition to be treated. Generally, the concentration of the diyne 20 compound(s) in a composition for topical administration, such as a lotion, will be from about 0.01 to 25% by weight, such as from 0.1-25% by weight, preferably from about 0.5-10% by weight, and more preferably from about 0.5% to 5% by weight. In liquid formulations the concentration may be from 0.01 to 90%, preferably from 0.01 to 50%, such as from 0.01 to 25% by weight. The concentration in a semi-solid or 25 solid composition such as a gel or a powder will be about 0.01-99% by weight, such as from 0.01 to 50%, for example from 0.01 to 25% and preferably about 0.5-2.5% by weight. [003821 The pharmaceutical compositions according to the invention may in 30 addition to the diyne compounds also comprise one or more additional active agents. [003831 Said additional active agents may for example be antifungal agents. B5. Individual in need of treatment 86 WO 20111134538 PCT/EP2010/063161 [00384] The present invention relates to pharmaceutical compositions comprising diyne compounds for treatment of fungal infections in an individual in need thereof. 5 [00385] Said individual may be any individual suffering from a fungal infection, preferably an animal, more preferably a mammal, even more preferably a human being suffering from a fungal infection. In some preferred embodiments of the invention, the individual is however a non-human mammal suffereing from a fungal infection, preferably a mammal selected from the group consisting of horses, cattle, 10 dogs and cats. Said fungal infection may be any of the fungal infections described herein below in the section "Fungal Infections". [003861 In one embodiment, the individual is an individual susceptible to fungal infections, for example an immunocompromised individual, such as an 15 immunocompromised human being. Said immunocompromised individual may be immunocompromised for various reasons, for example the individual may receive immune suppressing medication (for example in connection with transplantation) or the individual may be suffering from an immunocompromising condition, such as HIV infection. 20 [00387] Interestingly, the present invention discloses that the diyne compounds of the invention does not reduce the activity of cytochromes P450 significantly. Whether a diyne compound is capable of reducing the activity of of cytochromes P450 may be determined by any suitable assay known to the skilled person, preferably by an assay 25 comprising the steps of: a) providing a cytochrome P450 b) providing a substrate for said cytochrome P450 c) incubating said cytochrome P450 with said substrate under conditions 30 allowing for cytochrome P450 activity in the presence and absence of the diyne compound d) determining a reduction in product formed in the presence of said diyne compared to in the absence of said diyne compound 87 WO 20111134538 PCT/EP2010/063161 [00388] A diyne compound is said to not "reduce the activity of cytochromes P450 significantly" if at least 60%, preferably at least 70% product is formed in the presence of 10 ptM of the diyne compound compared to in the absence of said diyne compound. Preferably, said diyne compound does not significantly reduce the activity 5 of at least 3, preferably at least 4, more preferably at least 5, even more preferably at least 6, such as in the range of 3 to 10, for example in the range of 5 to 10, such as 7 different cytochromes P450. Said substrate may be labelled, and the product formed may then be detected by detecting labelled product. However, frequently the detection may also be direct using for example chromatographic methods, for example HPLC 10 UV/VIS or HPLC-MS/MS. A preferred method for determining whether a compound reduces the activity of cytochromes P450 is described in Example 9 herein below, [003891 The substrate used is dependent on the particular cytochrome P450. Substrates useful for individual cytochromes P450 are well known to the skilled 15 person. For example the catalogue of Cerep, France (as of 10 July 2009) describes suitable substrates for various cytochromes P450. Useful substrates for some cytochrome P450s are also described in Example 9 herein below. [00390] Accordingly, the pharmaceutical compositions according to the invention 20 are particularly useful for treating individuals receiving one or more other active agents, in particular such active agents wherein the activity of said active agents is increased or even depending on the activity of cytochromes P450. Typically, immunocompromised individuals will receive such active agents, and the pharmaceutical compositions of invention are accordingly in particular useful for 25 treatment of immuno compromised individuals. [00391] Thus, in a preferred embodiment, the pharmaceutical compositions comprising diynes are in particular useful for treating fungal infections in individuals to whom one or more active agents selected from the following group is being 30 administered, has been or is foreseen to be administered, preferably is being administered, has been administered within the past 48 hours or is foreseen to be administered within the next 48 hours. 88 WO 20111134538 PCT/EP2010/063161 [00392] Said active agents, wherein the activity is increased or even depending on the activity of cytochromes P450 is preferably one or more selected from the group consisting of acetaminophen, alfentanil, alprazolam, alprenolol, aminophyllin, amiodarone, amitriptyline, amlodipine, amphetamine, amprenavir, aniline, 5 artemisinin, astemizole, atorvastatin, azelastine, azithromycin, barnidepine, benzene,bufuralol, bupropion, buspirone, bezafibrate, caffeine, carbamazepine, carisoprodol, carvedilol, celecoxib, cerivastatin, chlorpheniramine, chlorpromazine, chlorzoxazone, cimetidine, ciprofloxacin, cisapride, citalopram, clarithromycin, clemastine, clomipramine, clopidogrel, clozapine, cocaine, codeine, cyclobenzaprine, 10 cyclophosphamide, cyclosporine, dapsone, debrisoquine, delavirdine, desipramine, dexamethasone, dexfenfluramine, dextromethorphan, dextropropoxyphene, diclofenac, diazepam, diltiazem, N,N-dimethyl formamide, diphenhydramine, disulfiram, docetaxel, dofetilide, dolasetron, econazole, efavirenz, encainide, enflurane, enoxacin, ergotamine, estradiol, erythromycin, ethanol, ethinylestradiol, 15 etomidate, etoposide, felbamate, felodipine, fenofibrate, fentanyl, finasteride, flecainide, fluconazole, fluorouracil, fluoxetine, flurbiprofen, fluvastatin, fluvoxamine, gemfibrozil, glibenclamide, glipizide, glyburide,granisetron, growth hormone, halofantrine, haloperidol, halothane, hexobarbital, hydrocortisone, hydroxyzine, ibuprofen, ifosfamide, imipramine, indinavir, indoramine, insulin, 20 indomethacin, irbesartan, irinotecan, isoflurane, isoniazid, isradipine, itraconazole, ketoconazole, lansoprazole, lercanidipine, levomepromazine, lidocaine, lignocaine, loratadine, lornoxicam, losartan, lovastatin, meloxicam, mephenytoin, mephobarbital, mequitazine, mestranol, methadone, methoxsalen, methoxyamphetamine, methoxyflurane, metoclopramide, metoprolol, metronidazole, mianserin, mibefradil, 25 miconazole, midazolam, mifepristone, mirtazapine, mepyramine, methoxyamphetamine, metoclopramide,metyrapone, mexiletine, midazolam, minaprine, moclobemide, montelukast, naproxen, nefazodone, nelfinavir, nicardipine, nifedipine, nilutamide, nisoldipine, nitrendipine, norethindrone, norfloxacin, nortriptyline, omeprazole, ondansetron, orphenadrine, oxcarbazepine, pantoprazole, 30 paracetamol, paroxetine, pefloxacin, perhexiline, perphenazine, pethidine, pentobarbitone, phenacetin, phenformin, phenobarbitone, phenytoin, pimozide, piroxicam, prednisone, primidone, procainamide, progesterone, proguanil, promethazine, propafenone, propofol, propranolol, quanoxan, quinidine, quinine, ranitidine, rifabutin, rifampicin, riluzole, risperidone, ritonavir, ropinirole, 89 WO 20111134538 PCT/EP2010/063161 ropivacaine, rosiglitazone, salmeterol, saquinavir, secobarbital, selegiline, sildenafil, simvastatin, sertraline, sevoflurane, Snaproxen, sparteine, sufentanil, suprofen, sulphamethoxazole, sulphonamides (sulfonamides), tamoxifen, tacrine, tacrolimus, taxol,teniposide, terbinafine, terfenadine, terfenidine, testosterone, theophylline, 5 thiopental, thioridazine, ticlopidine, timolol, tirilazad, tobacco, tolbutamide, tolterodine, topiramate, torsemide, tramadol, tranylcypromine, trazodone, triazolam, trofosfamide, troglitazone, troleandromycin, tropisetron, valsartan, venlafaxine, verapamil, vesnarinone, vigabatrin, vinblastine, vincristine, warfarin, zafirlukast, zaleplon, zanamivir, zileuton, zolmitriptan, zolpidem, zonisamide, zotepine and 10 zuclopenthixol. B6. Fungal infection [00393] The present invention relates to pharmaceutical compositions comprising 15 diynes (such as any of the diynes described herein above in the section "Diynes") for treatment of infections by a fungus. The invention also relates to methods of treating an infection by a fungus by administering to an individual in need thereof a therapeutically effective amount of a diyne (such as any of the diynes described herein above in the section "Diynes"). 20 [00394] In general said fungus is a fungus dependent on the activity of stearoyl CoA desaturase (such as OLE-1). The fungus may also be a fungus dependent on the activity of Spt23p/Mga2p, preferably a fungus dependent on both the activity of stearoyl-CoA desaturase (such as OLE-1) and Spt23p/Mga2p. 25 [00395] In one embodiment of the invention it is preferred that the infection by said fungus is associated with formation of hyphae in said fungus, preferably the infection is dependent on formation of hyphae in said fungus. Fungi may form hyphae during infections, a process also referred to as hyphal morphogenesis. The hyphae may be 30 any kind of hyphae, for example hyphae selected from the group consisting of septate hyphae, pseudohyphae, aseptate or coenocytic hyphae, generative hyphae, skeletal hyphae and fusiform skeletal hyphae, for example from the group consisting of septate hyphae and pseudohyphae. Interestingly, the diyne compounds according to the present invention are capable of inhibiting formation of hyphae. Hyphae are long, 90 WO 20111134538 PCT/EP2010/063161 filamentous cell(s) of fungi, which may be identified by visual inspection, such as by visual inspection with the aid of a microscope. Example 13 herein below describes how inhibition of hyphal formation for example can be assessed in vitro. Because the diyne compounds of the invention are capable of inhibiting formation of hyphae, they 5 are in particular useful for treating infections by a fungus associated with or dependent on formation of hyphae. [00396] Thus, in one embodiment, the pharmaceutical composition comprising diyne compounds according to the invention may be prepared for inducing growth of 10 small, rounded, compact hyphal forms rather than the hyphal growth seen in the absence of pharmaceutical composition. [003971 In one embodiment of the invention it is preferred that the infection by said fungus is associated with clamydospores, for example the infection by said fungus 15 may be associated with formation of clamydospores of said fungus, preferably the infection may be dependent on formation of clamydospores of said fungus. The infection may also be initiated by germination of clamydospores or associated with germination of clamydospores, preferably the infection by said fungus may be dependent on germination of clamydospores. The infection by said fungus may also 20 be associated with conidia, for example the infection by said fungus may be associated with formation of conidia of said fungus, preferably the infection may be dependent on formation of conidia of said fungus. The infection may also be initiated by conidia, such as by germination of conidia or associated with germination of conidia, preferably the infection by said fungus may be dependent on germination of 25 conidia. [00398] Clamydospores are fungal spores, which may be formed asexually or sexually. They are usually essentially spherical (or spherical), and have a smooth surface. They may be multicellular and the cells may be connected by pores in septae between cells. Conidia may also be referred to as conidiospores or mitospores. 30 Conidia are generally asexual, non-motile spores of a fungus. [00399] Interestingly, the diyne compounds according to the present invention are capable of inhibiting formation of clamydospores and/or conidia. Furthermore, the diyne compounds of the invention may be capable of inhibiting germination of clamydospores and/or conidia. Formation and germination of clamydospores and/or 91 WO 20111134538 PCT/EP2010/063161 conidia may be detected by visual inspection, preferably by visual inspection with the aid of a microscope. Because the diyne compounds of the invention are capable of inhibiting formation of clamydospores and/or conidia, and/or capable of inhibiting germination of clamydospores and/or conidia, the compounds are in particular useful 5 for treating infections by a fungus associated with or dependent on formation and/or germination of clamydospores and/or conidia. [00400] Accordingly, the pharmaceutical composition comprising diyne compounds according to the invention may be prepared for inhibition of conidiation 10 and/or for inhibition of sporulation. [00401] In one embodiment of the invention the infection is associated with formation of a biofilm of said fungus (i.e. a fungal biofilm) and/or the infection causes formation of a biofilm of said fungus (i.e. a fungal biofilm). A fungal biofilm is a 15 layer of fungus, which is phenotypically different from suspended fungal cells. In general, biofilms have a significantly decreased susceptibility to antifungal agents, including Amphotericin B and Fluconazole. However, interestingly the present invention discloses that infections by fungus associated with biofilm formation may be treated using the diyne compounds disclosed herein. Biofilms may be formed by a 20 mixture of unicellular fungi, hyphae and/or pseudohyphae arranged in a layer structure, for example in a bilayer structure. Frequently, biofilms are formed on a solid surface, for example on indwelling medical devices (e.g. dental implants, catheters, heart valves, vascular bypass grafts, ocular lenses, artificial joints or central nervous system shunts). The biofilm bilayer may consist of a dense, basal fungus 25 layer that anchors the biofilm to a surface an overlying but more open, hyphal layer. An extracellular matrix typically surrounds the cells within a biofilm. [004021 Examples of fungi, which may form biofilms include Candida species, such as any of the Candida species mentioned herein below, for example Candida 30 albicans. [004031 In some embodiments of the invention the infection by fungus is associated with formation of macronodules, for example the infection may cause formation of macronodules of said fungus, preferably macronodules surrounded by a 92 WO 20111134538 PCT/EP2010/063161 perimeter of ground-glass opacity. Such macronodules and in particular macronodules surrounded by a perimeter of ground-glass opacity (also referred to as a "halo") may for example be identified using computed tomography (CT) scanning. Examples of fungi, which may form such macronodules include Aspergillus species, such as any of 5 the Aspergillus species mentioned herein below. Macronodules may in particularly be associated with invasive pulmonary infections by fungus, such as invasive pulmonary aspergillosis. Interestingly the present invention discloses that infections by fungus associated with macronodule formation may be treated using the diyne compounds disclosed herein. 10 [004041 In one embodiment of the invention the pharmaceutical compositions comprising diyne compounds according to the invention are prepared for induction of nuclear membrane collapse in one or more fungi. 15 [004051 In one embodiment the pharmaceutical composition is prepared for inhibition of white-opaque switching. [004061 In one embodiment, the pharmaceutical composition is prepared for inhibition of the morphogenetic switch between the hyphal growth form, the pseudo 20 hyphal growth form and the budding growth form. [004071 Another interesting feature of the diyne compounds according to the present invention is that they are capable of killing fungi, i.e. they have fungicidal activity as described in more detail herein above in the section "Properties of diyne 25 compounds". Accordingly, the pharmaceutical compositions comprising diyne compounds according to the invention are in particular suitable for treating infections by fungus, wherein it is desirable to kill the fungus, rather than just to inhibit growth of the fungus. Thus, the pharmaceutical compositions comprising diyne compounds according to the invention are particularly useful for treating recurrent infections by 30 fungus, such as an infection by a fungus, which is expected to be recurrent or an infection by fungus, which has re-occurred at least once, for example at least twice, such as at least 3 times. 93 WO 20111134538 PCT/EP2010/063161 [00408] Preferably, the pharmaceutical compositions comprising diyne compounds according to the invention are prepared for killing at least 50%, preferably at least 80%, more preferably at least 95% of the infecting fungus. 5 [00409] Another very interesting aspect of the present invention is that the pharmaceutical compositions comprising diynes according to the invention are particularly useful for treating infection by a fungus under hypoxic conditions. Without being bound by theory it is believed that this is based on OLE-i being particularly important for fungal growth under hypoxia. Thus, OLE-I transcript levels 10 are upregulated in fungi under hypoxia (for example in C. albicans). [00410] Accordingly, the infection by a fungus may preferably be an infection involving at least partly infection of tissue, organs or cells with hypoxic conditions, preferably the infection may be infection of tissues, organs or cells with hypoxic 15 conditions. Thus, said infection may at least partly involve infection of one or more inner organs, tissues or cells of a mammal, preferably a human being. More preferably, said infection may be infection of one or more inner organs, tissues or cells of a mammal, preferably a human being. 20 [004111 Said hypoxic condition is preferably an oxygen partial pressure (pO2) of at the most 140 mmHg, preferably at the most 110 mmHg, such as at the most 80 mmHg. Such conditions may in general be found in inner organs, for example in the liver, pancreas, gut, duodenum, skeletal muscles, brain, kidney or peritoneal cavity. 25 [004121 It is also comprised within the present invention that the pharmaceutical compositions comprising diynes according to the invention may be for treatment of a disseminated infection or a local infection. [004131 The infection by said fungus may also involve at least partly infection of a 30 body surface, for example infection of skin, nails or mucosal membranes of body surfaces. Thus, said infection may be infection of a body surface, for example infection of skin, nails or mucosal membranes of body surfaces. Body surfaces may include the oral cavity, the genital organs, nose or eyes. 94 WO 20111134538 PCT/EP2010/063161 [00414] Accordingly, the fungal infection may be one or more selected from the group consisting of oropharyngeal fungal infections (such as thrush, glossitis, stomatitis or angular cheilitis), cutaneous fungal infections (such as intertrigo, diaper candidiasis, paronychia or onychomycosis), paronychia, onychomycosis, 5 vulvovaginal fungal infection, balanitis, mucocutaneous fungal infection, neonatal fungal infection, congenital fungal infection, oesophageal fungal infection, gastrointestinal fungal infection, pulmonary fungal infection, peritonitis, urinary tract fungal infections, renal fungal infection, meningitis associated with fungi, hepatic fungal infection, hepatosplenic fungal infection, endocarditis, myocarditis, 10 pericarditis, ocular fungal infection, endophthalmitis and osteoarticular fungal infection. [004151 Interestingly, the diyne compounds according to the present invention are even useful for treating onychomycosis, i.e. fungal infection of the nails. 15 [004161 The infection by a fungus may be infection by one species of fungus or infection by more than one fungal species, such as two, for example 3, such as 4, for example 5, such as more than 5 different fungal species. 20 [004171 The fungus may be any fungus, but usually it is a pathogenic fungus, such as a fungus pathogenic in the individual to be treated. In one preferred embodiment of the invention, the individual to be treated is a mammal, preferably a human being, and then the fungus is a fungus pathogenic in mammals, preferably in human beings. 25 [004181 The fungus may preferably be selected from the group consisting of wherein one or more fungus is selected from the group consisting of Candida spp., Aspergillus spp., Histoplasina capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp., Zygomycetes, Malassezia spp., Hyalohyphomycetes, Dermatophytes, Epiderinophyton floccosum, Microsporum spp, Blastomyces 30 derniatitidis, Sporothrix schenkii, Chromomycotic fungi and Madurella spp. [004191 Thus the fungus may be selected from the group consisting of Candida spp., preferably from the group consisting of C. albicans, C. krusei, C. glabrata, C. 95 WO 20111134538 PCT/EP2010/063161 tropicalis, C. parapsilosis, C. guillieriondii, C. haenmulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis. [00420] The fungus may also be selected from the group consisting of Aspergillus 5 spp., preferably from the group consisting of A. funigatus, A. flavus, A. niger and A. terreus. [00421] The fungus may also be selected from the group consisting of Cr)yptococcus spp., Preferably from the group consisting of C. neoformans, C. bidus, 10 C. laurentii, and C. fisarium. Said C. neoformans is preferably selected from the group consisting of var. neoformans and var. gattii. [004221 The fungus may also be selected from the group consisting of zygomycetes, preferably from the group consisting of Rhizopus oryzae, R. 15 inicropsorus, R. pusillus, Cunninghamelle bertholletiae, Saksenaea vasifonris, Mucor circinelloides, Al. rainosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus and Syncephalastruni racemosum. [00423] The fungus may also be selected from the group consisting of Malassezia 20 spp., preferably from the group consisting of M.fuifur and M. globosa. [004241 The fungus may also be selected from the group consisting of Hyalohyphomycetes, preferably from the group consisting of Fusarium solani and Scedosporium spp., wherein said Scedosporium spp. preferably is selected from the 25 group consisting of S. prohificans and S. apiospermun. [00425] The fungus may also be selected from the group consisting of Dermatophytes. This is in particular the case when the infection is partly or entirely an infection of the skin. Said Dermatophyte may preferably be selected from the group 30 consisting of Trichophyton spp., Epidermophyton floccosum, Aicrosporun spp and Trichosporon terrestre. Said Trichophvton spp. may preferably be selected from the group consisting of T. nentagrophy'tes, T. rubrum and T. tonsurans. Said Microsporun spp may preferably be selected from the group consisting of Al. cookei, M. canis, M. vanbreusegheinii, M. gallinae and M. gypseun. 96 WO 20111134538 PCT/EP2010/063161 [00426] The fungus may also be selected from the group consisting of Chromomycotic fungi, preferably from the group consisting of Fonsecaea pedrosoi, F. compacta, Cladophylophora carrionii and Phialophora verrucosa. 5 [00427] The fungus may also be selected from the group consisting of Madurella spp., preferably from the group consisting of M. mycetonatis and M. griseun. [004281 In embodiments of the invention wherein the individual is a non-human 10 animal, preferably a mammal, more preferably a mammal selected from the group consisting of horses, cattle, dogs and cats then the fungus may for example be selected from the group consisting of Aspergillus spp., Batrachochytrium dendrobatidis, Blastomyces spp., Branchiomyces spp., Candida spp., Cladosporiun spp., Coccidioides spp., Cryptococcus neoformans, Entoinophthora spp., Epidermophyton 15 spp., Fonsecaea spp., Geotrichum spp., Histoplasnia spp., Ichthyophonus hoferi, Lacazia loboi, Malassezia spp., Metarhiziun spp., Microsporum spp., Mucor spp., Ochroconis spp., Paecilomyces spp., Penicilliuni spp., Phialophora spp., Saprolegnia spp., Sporothrix schenckii, Trichophyton spp. and Wangiella spp. 20 B7. Resistant fungus [004291 Another very interesting aspect of the present invention is that the diyne compounds and diyne salts according to the present invention are capable of treating infections by fungi which are resistant to one or more conventional antifungal agents, 25 in particular antifungal agents, which are not capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus. [004301 Said fungi may be resistant for any reason. Thus, for example that particular species of fungus may be resistant to treatment with that particular 30 antifungal agent. Alternatively, the fungus may have aquired resistance, i.e. in general said fungal species is not resistant to treatment with the particular antifungal agent, but this particular fungus has become resistant. In a preferred embodiment of the invention, the fungus has aquired resistance to one or more conventional antifungal agents. 97 WO 20111134538 PCT/EP2010/063161 [00431] Thus, the pharmaceutical compositions comprising diynes or diyne salts according to the invention are useful for treating infection by a fungus, which is resistant to one or more antifungal agents, which are not of formula I'. In particular, 5 the pharmaceutical compositions comprising diynes or diyne salts according to the invention are useful for treating infection by a fungus, which is resistant to one or more antifungal agents capable of at least one of a) inhibiting ergosterol biosynthesis; b) binding to ergosterol; 10 c) inhibiting 1,3-p-glucan synthase; d) inhibiting epoxidase; e) inhibiting Leucyl-tRNA synthetase; and/or f) inhibition of elongation factor 2. 15 [004321 In particular, the pharmaceutical compositions comprising diynes according to the invention are useful for treating infection by a fungus, which is resistant to one or more antifungal agents selected from the group consisting of polyene antifungal agents, azole antifungal agents, allylamine antifungal agents and echinocandins. 20 [00433] Polyene antifungal agents are antifungal agents with multiple conjugated double bonds. Typically, polyene antifungal agents also comprise a heavily hydroxylated region. Non-limiting examples of polyenes include Natamycin, Rimocidin, Filipin, Nystatin, Amphotericin B or Candicin. 25 [00434] Azole antifungal agents may for example be imidazole or triazole or thiazole antifungal agents. Non-limiting examples of imidazole antifungal agents include miconazole, ketoconazole, clotromazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, seraconazole, sulconazole or tioconazole. 30 Non-limiting examples of triazole antifungal agents include fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole or terconazole. A non limiting example of a thiazole antifungal is abafungin. 98 WO 20111134538 PCT/EP2010/063161 [00435] Non-limiting examples of allylamine antifungals include Terbinafine, Amorolfine, Naftifine or Butenafine. [00436] Non-limiting examples of echinocandins include Anidulafungin, 5 Caspofungin or Micafungin. [004371 The pharmaceutical compositions comprising diynes according to the invention may also be useful for treating infection by a fungus, which is resistant to one or more antifungal agents selected from the group consisting of benzoic acid, 10 ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate. [004381 By the term "resistant to an antifungal agent", it is meant that said infection by fungus in said individual cannot be treated in a curable manner with said 15 antifungal agent. [004391 The pharmaceutical compositions for treating fungal infections according to the invention may in addition to one or more diyne compounds also comprise additional active agents, preferably one or more antifungal agents. 20 [00440] Thus said pharmaceutical compositions may in addition to one or more diynes also comprise one or more antifungal agents capable of at least one of a) inhibiting ergosterol biosynthesis; b) binding to ergosterol; 25 c) inhibiting 1,3--glucan synthase; d) inhibiting epoxidase; e) inhibiting Leucyl-tRNA synthetase; and/or f) inhibition of elongation factor 2. 30 [00441] Thus, the additional antifungal agent may be for example be selected from the group consisting of polyene antifungal agents (such as any of the polyene antifungal agents described herein above in the section), azole antifungal agents (such as any of the azole antifungal agents described herein above in the section), allylamine antifungal agents (such as any of the allylamine antifungal agents 99 WO 20111134538 PCT/EP2010/063161 described herein above in the section) and echinocandins (such as any of the echinocandins described herein above in the section). [00442] In particular, due to the synergistic effect the pharmaceutical compositions 5 of the invention may preferably comprise a diyne compound as described herein above and a polyene antifungal agent. [00443] Said polyene antifungal agent may preferably be Amphotericin B. 10 [00444] Due to the synergistic effect such pharmaceutical compositions be formulated for administration of very low levels of said polyene antifungal agent (such as Amphotericin B). Thus, such pharmaceutical compositions may be prepared for administration of less than 0.2 mg/kg, preferably less than 0.1 mg/kg, even more preferably less than 0.05 mg/kg of said polyene antifungal agent, preferably 15 Amphotericin B. [004451 Administration of low levels of polyene antifungal agent (such as Amphotericin B) is preferable, due to reduced toxicity compared to higher levels. As described in more details in the "Back ground" section, then polyene antifungal agent 20 (such as Amphotericin B) may have many adverse effects. B8. Fungal infection of plants [004461 The present invention also relates to methods of reducing the risk of an 25 infection or to methods of treating an infection in a plant by contacting said plant with a diyne compound according to the invention. [004471 Thus, in one aspect the invention relates to use of a diyne of the formula I': 30 Z-[C=C- C=C]-R 3 wherein Z and R 3 are as defined herein above in the section "Diynes" 100 WO 20111134538 PCT/EP2010/063161 such as any of the diyne compounds described herein above in the section "Diynes", for inhibiting or treating an infection by a fungus in a plant, preferably by a plant pathogenic fungus. Said infection by a fungus is preferably an infection by a fungus dependent on activity of stearoyl-CoA desaturase, more preferably an infection by a 5 plant pathogenic fungus dependent on the activity of stearoyl-CoA desaturase. [004481 Said diyne compound may in particular be a diyne of formula II',
R
4 -Y C C-C C-R 3 10 wherein R 4 , Y and R 3 are as described herein above in the section "Diyne". [004491 The diyne compound for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA 15 desaturase) may also be a diyne of formula III': C-Y C--C--C C- R3 R1 wherein R 1 , Y and R 3 is as described herein above in the section "Diynes", 20 [004501 The diyne compound for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may also be a diyne of formula IV', 25 R4-(C(R 2
)
2 )n-X-(C(R 2
)
2 )m-[C=C- C=C]-R 3 wherein R 4 , R 2 , n, m and R 3 are as described herein above in the section "Diyne" in relation to formula IV'. 101 WO 20111134538 PCT/EP2010/063161 [00451] The diyne compound for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may also be a diyne of formula V', C- (C(R 2 )2)n-X- -C-C- -R 3 5 R1 wherein R 1 , R 2 , n, X and R 3 are as described herein above in the section "Diyne" in relation to formula V'. 10 [004521 The diyne compound for treating or reducing the risk of infection by a fungus in a plant (preferably a fungus dependent on activity of stearoyl-CoA desaturase) may preferably be selected from the group consisting of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid and 14-(furan-2-yl)tetradeca- 11,13-diynoic acid, and salts thereof The salts thereof are preferably the salts described herein above in 15 the section diynes. [00453] Thus, for example the plant pathogenic fungus may be selected from the group consisting of Albugo spp., Alternaria spp., Anisogramma anomala, Apiosporina inorbosa, Ascochyta spp., Aureobasidiun zeae, Bipolaris spp., Bluneria spp., 20 Blumeriella jaapii, Botritis spp., Botryosphaeria dothidea, Ceratocystic paradoxa, Cercospora spp., Cercosporidiun spp., Cladosporiun spp., Cochiliobolus spp., Colletotrichuni spp., Corynespora cassiicola, Cristulariella ioricola, Diaporthe phaseolorun, Didynella bryoniae, Drechslera tritici, Entylona oryzai, Erysiphe spp., Fluvia fluva, Fusariun spp., Gaeunannonyces graninis, Gnomnonia spp., 25 Gremnieniella abietina, Helninthosporiun spp., Leptosphaerulina crassiasca, Leveillula taurica, Lophoderniun hypophyllun, Macrophonina phaseoli, Magnaporthe spp., Microdochiun spp., Microsphaera spp., Monilinia spp., Afycosphaerella spp., Myrothediuni roridun, Oidiopsis sicula, Passalora puncta, Penicilliun spp., Peronospora spp., Phaeocryptopus gaeunannii, Phakopsora spp., 30 Phona arachidicola, Phragnidiun potentillae, Phytophtora spp., Plasnopara spp., Plectosporiun tabacinurn, Pleospora herbarun, Podosphaera spp., 102 WO 20111134538 PCT/EP2010/063161 Pseudocercosporella spp., Pseudoperonospora cubensis, Puccinia spp., Pucciniastrun vaccinii, Pyrenophora spp., Piythiun spp., Ramularia cynarae, Rhizoctonia spp., Rhizosphaera spp., Rhynchosporiuni secalis, Sclerotinia spp., Sclerotium spp., Selenophoma spp., Septoria spp., Setosphaeria turcica, Sirococcus 5 conigenus, Sphaerotheca spp., Stagonospora nordorum, Stemphyllium botryosum, Taphrina deformans, Thielaviopsis spp., Tilletia barclayana, Tranzschelia discolor, Uncinula necator, Uromyces appendiculatus, Ustilaginoidea virens, Ustilago spp., Venturia spp., Verticilliuni spp. and Wilsonomyces carpophilus. 10 [00454] Treatment of infections by a fungus in a plant may be done by any suitable means, for example the diyne compounds may be applied as sprays or dusts on the foliage of plants, or in irrigation systems. Typically, the diyne compounds according to the invention are administered on the surface of the plant in advance of the pathogen in order to prevent infection. Seeds, bulbs, roots, tubers, and/or corms may 15 also be treated to prevent pathogenic attack after planting and for example thereby controlling pathogens carried on them or existing in the soil at the planting site. However, plants may also be treated once an infection is already present in order to eliminate or reduce the infection, preferably eliminate the infection. Similarly, seeds, bulbs, roots, tubers and/or corms may also be treated once an infection is already 20 present in order to eliminate or reduce the infection, preferably eliminate the infection. [00455] Soil to be planted with vegetables, ormementals, shrubs, or trees can also be treated with the diyne compounds of the invention for control of a variety of fungal 25 pathogens. Treatment is preferably done several days or weeks before planting. The diyne compounds can be applied by either a mechanized route, e.g., a tractor, or with hand applications. [004561 In most applications said diyne compounds are used with an agronomically 30 acceptable carrier. An "agronomically acceptable carrier" is a solid or liquid which is biologically, chemically and physically compatible with the diyne compounds of the present invention, and which may be used in agricultural applications. Agronomically acceptable carriers suitable for use in the method of the present invention include organic solvents, and finely divided solids, and aqueous solutions or suspensions. For 103 WO 20111134538 PCT/EP2010/063161 example, the diyne compounds for use in treatment or prevention of an infection by a fungus in a plant can be formulated as wettable powders, emulsifiable concentrates, dusts, granular formulations, aerosols, or flowable emulsion concentrates. In such formulations, the diyne compounds may be extended with a liquid or solid carrier and, 5 when desired, suitable surfactants may be incorporated. Optionally added components or additives, not required for fungicidal activity but useful or required for other properties, include, but are not limited to, adjuvants such as wetting agents, spreading agents, dispersing agents, stickers, adhesive and the like. 10 Such adjuvants are well known in the art. [00457] In general, the diyne compounds of this invention may be dissolved in solvents such as water or other aqueous solutions, acetone, methanol, ethanol, dimethylformamide, pyridine or dimethyl sulfoxide and such solutions can further be 15 diluted with water. The concentrations of the solution after dilution may vary from 1% to 90% by weight, with a preferred range being from 5% to 50%. [004581 For the preparation of emulsifiable formulations and concentrates of the diyne compounds of the present invention, the diyne compound can be dissolved in 20 suitable organic solvents, or a mixture of solvents, together with an emulsifying agent to enhance dispersion of the diyne compound in water. The concentration of the diyne compound in emulsifiable concentrates is usually from 10% to 90%, and in flowable emulsion concentrates, can be as high as 75%. 25 [004591 Wettable, powdered formulations suitable for spraying can be prepared by admixing the diyne compound with a finely divided solid, such as clays, inorganic silicates and carbonates, and silicas and incorporating wetting agents, sticking agents, and/or dispersing agents in such mixtures. The concentration of total active ingredients in such formulations is usually in the range of from 20% to 99% by 30 weight, preferably from 40% to 75%. A typical wettable powder is made by blending 50 parts of a diyne compound, 45 parts of a synthetic precipitated hydrated silicon dioxide, such as that sold under the trademark Hi-SilR, and 5 parts of sodium lignosulfonate. In another preparation a kaolin type (Barden) clay is used in place of the Hi-Sil in the above wettable powder, and in another such preparation part of the 104 WO 20111134538 PCT/EP2010/063161 Hi-Sil is replaced with a synthetic sodium silicoaluminate sold under the trademark Zeolex. RTM. 7 (J. M. Huber Corporation). [00460] Dusting formulations may be prepared by mixing the diyne compounds 5 with finely divided inert solids which can be organic or inorganic in nature. [004611 Materials useful for this purpose include botanical flours, silicas, silicates, carbonates and clays. One convenient method of preparing a dust is to dilute a wettable powder with a finely divided carrier. Dust formulations or concentrates 10 containing from 20% to 80% of the active ingredient are commonly made and are subsequently diluted to from 1% to 10% use concentration. [004621 The fungicidal compound and formulations may be applied as fungicidal sprays by methods commonly employed, such as conventional high-gallonage 15 hydraulic sprays, low-gallonage sprays, air-blast spray, aerial sprays and dusts. [004631 The dilution and rate of application will depend upon the type of equipment employed, the method of application, plants to be treated and diseases to be controlled. Generally, the diyne compounds of this invention will be applied in an 20 amount of from 0.06 to 60 kilograms (kg) per hectare and preferably from I to 28 kg per hectare of the active ingredient. [00464] As a seed protectant, the diyne formulation may be coated on the seed. The dosage rate may for example be from 3 g of diyne compound per hundred kg of seed, 25 to 1000 g per hundred kg of seed. As a soil fungicide the fungicidal formulation may be incorporated in the soil or applied to the surface for example at a rate of from 0.02 to 20 kg per hectare. As a foliar fungicide, the diyne compounds may be applied to growing plants for example at a rate of from 0.01 to 10 kg per hectare. 30 [00465] The diyne compounds of the present invention may be combined with other known fungicides. B9. Method for identification of fungitoxic compound 105 WO 20111134538 PCT/EP2010/063161 [00466] In one aspect the invention relates to methods for identifying a fungitoxic compound. Preferably, said method comprising the steps of, a) providing an indicator composition or cell comprising a gene encoding stearoyl-CoA desaturase and/or a stearoyl-CoA desaturase peptide 5 (preferably a wild type fungal stearoyl-CoA desaturase); b) contacting the indicator composition or cell with a test compound, wherein the test compound comprises a -COOH group or a bioisostere thereof; c) evaluating the activity of stearoyl-CoA desaturase in the presence and 10 absence of the dine compound; and d) selecting a diyne compound that down modulates the activity of stearoyl-CoA activity, thereby identifying a fungitoxic compound. 15 [004671 Interestingly, the present invention discloses that fungitoxic compounds capable of down modulating fungal stearoyl-CoA desaturase are superior to other clasees of antifungal agents. Several diyne compounds capable of downmodulating the activity of stearoyl-CoA desaturase are described in more detail herein above and several advantages connected with this class of compounds are also described. 20 [00468] The present invention discloses that in particular compounds comprising a -COOH group or a bioisostere thereof may be useful for down modulating fungal stearoyl-CoA desaturase activity. Thus, the invention provides methods for testing whether a test compound comprising a -COOH group or a bioisostere thereof may be 25 useful for down modulating fungal stearoyl-CoA desaturase activity. [00469] The biostere of -COOH may be any of the bioisosteres of -COOH described herein above in the section "Diynes". Preferably, the bioisostere is selected from the group consisting of -CO-R 1 , wherein R 1 is -OH or or a moiety that can be 30 replaced by a hydroxyl group in a hydrolysis reaction, tetrazoles, tetrazolates and salts thereof [004701 In one embodiment the test compound may be any of the diynes compounds described herein above in the section "Diynes". 106 WO 20111134538 PCT/EP2010/063161 [00471] However, the test compound may also be any other compound comprising a -COOH group or a bioisostere thereof Thus, the test compound may in addition to the -COOH group or a bioisostere thereof comprise one or more carbon chains, which 5 may be linear or branched and which may comprise one or more double bonds and one or more triple bonds. The carbon chain may also be a saturated carbon chain. Said carbon chain may be substistuted for example with one or more substituents selected from the group consisting of amines, alkoxy, carbonyl, alcohols, halogens, carboxylic acids, esters, ethers or combinations thereof The test compound may in addition to 10 the -COOH group or a bioisostere thereof also comprise one or more cyclic groups, which may be heterocyclic groups. The heterocyclic groups may be any of the R3 groups described herein above in the section "Diynes". The cyclic group may however also be a cycle only with carbon atoms, such as a 3 to 10 membered ring, for example a 3 to 7 membered ring. The cyclic group may also consist of several cycles, 15 i.e. it may be ring system. The cyclic group may be substistuted for example with one or more substituents selected from the group consisting of amines, alkoxy, carbonyl, alcohols, halogens, carboxylic acids, esters, ethers or combinations thereof. If the test compound comprises several cyclic groups these may be directly connected or connected via linkers, which typically may be carbon chains. 20 [00472] The invention also relates to methods for treating a fungal infection in an individual in need thereof, said method comprising administering a therapeutically effective amount of a fungitoxic compound identified according to the method described in this section. 25 C1. Olel Protein Inhibitors [00473] The present invention provides new class of fungicides that comprise an Ole1 protein inhibitor. 30 [00474] As such the novel fungicides of the invention act as potent antifungals against a wide variety of fungal pathogens that include novel diynes and their salts, derivatives and analogs. The inventive Ole1 protein inhibitors provide potent broad spectrum antifungal agents for the treatment of humans and animals against a wide 107 WO 20111134538 PCT/EP2010/063161 variety of fungal pathogens. In addition, the compounds provide effective fungicides against agricultural fungal pathogens. [00475] Their mode of action make the inventive compounds highly attractive 5 alternatives to currently available treatment regimes where the treatment drugs have undesirable side effects due to their mode of action. [00476] The inventors have shown that the inventive compounds inhibit oleic acid biosynthesis by inhibition of the Olel protein, a process that appears to be conserved 10 across the entire fungal kingdom. Moreover, the instant compounds were tested against the mode of action of existing antifungal drugs and were shown not to act via the targets of existing drugs. Thus, the inventive compounds provide a new mechanism of action with great promise for broad spectrum antifungal treatments. [00477] The inventive compounds exploit a mechanism of action that has so far not 15 been exploited in the development of fungicides to date, the inhibition of the Olel protein. The Olel protein is essential for the survival of the fungal organism. [004781 Provided herein therefore, are two new classes of antifungal compounds, both derived from a compound of general formula I", 20 a. R 1 - C(O) - (C(R2)2)x - R3 - C 4 - R4I [00479] wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, H or a monovalent 25 hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R 3 is C 2
H
2 or C 2
H
4 ; R 4 is a pyrrole, furan, or thiophene ring; and x is an integer between 4 and 10, inclusive. [004801 Provided in the invention therefore are new antifungal compounds based 30 on the cis-isomer (Z) of structure II": a. (Z)-R 1 - C(O) - (C(R 2
)
2 )x - C 2
H
2 - C 4 - R 4 II' 108 WO 20111134538 PCT/EP2010/063161 [00481] wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, H or a monovalent hydrocarbon moiety containing between 1 and 4 carbon atoms, inclusive; R4 is a heterocyclic ring, optionally substituted at one or more positions, preferably with one 5 or more substituents selected from the group consisting of a C1_s alkyl, a C 1
_
5 alkenyl, a C 1
_
5 alkoxy, a C1_s alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive. [004821 In one embodiment, R4 is a pyrrole, furan, or thiophene ring. In other 10 embodiments, R 4 may be an imidazole, oxazole, and cyclopentadiene. [00483] In other preferred embodiments, R4 is a heterocyclic ring substituted at one or more positions with one or more, preferably one or two selected from the group consisting of lower alkyl, lower alkenyl, lower alkoxy, lower alcohol, hydroxyl, 15 amine, NO 2 and halogen. A lower alkyl is preferably C 1
_
5 , more preferably C 1
-
3 , even more preferably C 1 alkyl. A lower alkenyl is preferably C 1
_
5 , more preferably C- 3 , even more preferably C1- 2 alkenyl. A lower alkoxy is preferably C 1
_
5 , more preferably C1_ 3 , even more preferably C 1 alkoxy. A lower alcohol is preferably C 1
_
5 , more preferably C 1
_
3 , even more preferably C 1 alcohol comprising one or more OH groups, 20 preferably only one OH group. Halogen may be any halogen, but is preferably F. It is however preferred that R 4 is a heterocyclic ring, which is not substituted or that R 4 is a heterocyclic ring substituted with a small substituent, preferably a small substituent selected from the group consisting of methyl, methoxy, hydroxyl, CH 2 -OH, amine and halogen, and preferably methyl. 25 [00484] In other embodiments, R 4 is preferably a 3 to 7-membered heterocyclic ring, more preferably a 5 to 6-membered heterocyclic ring, even more preferably a 5 membered heterocyclic ring. The heterocyclic ring may be aromatic or non-aromatic. In one embodiment the heterocyclic ring is a 3 to 7-membered aromatic heterocyclic 30 ring, more preferably a 5 to 6-membered aromatic heterocyclic ring, even more preferably a 5-membered aromatic heterocyclic ring. [004851 The heterocyclic ring may comprise one or more heteroatoms, preferably in the range of 1 to 3 heteroatoms, more preferably in the range of I to 2 heteroatoms, 109 WO 20111134538 PCT/EP2010/063161 yet more preferably 1 heteroatom, preferably selected from the group consisting of S, N and 0. [00486] For example, preferred compounds of the invention are of structure (Z) 5 14-(furan-2-yl) tetradeca-9-en-1 1, 13-diynoic acid, III", and its potassium and sodium salts. The substantially pure cis-isomer, (Z)-14-(furan-2-yl) tetradeca-9-en-11, 13-diynoic acid: H H ICOOH }} 10 (Z) as well as its potassium salt, H COO ,K* IV'' 15 (Z) 110 WO 20111134538 PCT/EP2010/063161 are preferred embodiments of the present invention. Production of the cis, or Z, isomer compounds is a regioselective Wittig reaction stage which determines the cis/trans structure and produces predominantly the cis-isomer in a 98:2 cis:trans ratio 5 (see Example 21 below). Equally, a preferred salt compound is the sodium salt of (Z)- 1 4-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid, V": H H Z isomer a. (Z) COO-,Na' V" 10 [00487] These preferred cis (or Z) isomer compounds are highly water soluble salts, chemically distinct from any known class of antifungal agents. [00488] Also provided are chemical analogs of III", such as (Z)-14-(4,5 dimethylfuran-2-yl) tetradeca-9-en- 11, 13-diynoic acid (VI"), 15 O COOH VI" 8-(2-(4-(furan-2-yl) buta-1,3-diynyl) phenyl) octanoic acid (VII") 20 111 WO 20111134538 PCT/EP2010/063161 0 COOH ViP' [004891 A trans analog of genetic formula I" is (E)-14-(furan-2-yl) tetradeca-9-en 11, 13-diynoic acid (VIII"), unsubstituted or substituted as outlined for compound 5 II". COOH a) (E) 10 [004901 This novel compound class is related to the enediyne compounds general structure R 1
-C(O)-(C(R
2
)
2 )x-C 2
H
2 - C 4 - R 3 and disclosed in US Patent 6,541,506, incorporated herein in entirety. That patent disclosed a structure Ri-C(O)-(C(R 2
)
2 )x
C
2
H
2 - C 4
-R
3 as a mixture of isomers and which the disclosed synthesis described as being predominantly the trans isomer. That patent did not disclose nor suggest 15 isolating a substantially pure cis-compound. Although the patent stated that antifungal properties attended the disclosed compounds, poor solubility led them to be unsuitable for formulation. Nor did that patent disclose any compound in which the C 2
H
2 group is an alkyl moiety. 20 [00491] Also provided in the invention is another new class of compounds similar to the compound of structure I" above, but in which the group R, is an alkyl moiety, having a single bond instead of a double bond at the position of R3. This class has also been shown to also provide potent antifungal activity. 25 [00492] Thus, preferred compounds of the instant invention include analogs, IX", 112 WO 20111134538 PCT/EP2010/063161 a) R 1 - C(O) - (C(R 2
)
2 )x - C 2 H4 - C 4 - R 4 IX'' wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, H or a monovalent hydrocarbon 5 moiety containing between 1 and 4 carbon atoms, inclusive; R4 is a heterocyclic ring, optionally substituted at one or more positions, preferably with one or more substituents selected from the group consisting of a C1_s alkyl, a C1_s alkenyl, a C1_s alkoxy, a C 1
_
5 alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive. 10 [004931 In one embodiment, R 4 is a pyrrole, furan, or thiophene ring. In other embodiments, R 4 may be an imidazole, oxazole, and cyclopentadiene. [00494] In other preferred embodiments, R4 is a heterocyclic ring substituted at 15 one or more positions with one or more, preferably one or two selected from the group consisting of lower alkyl, lower alkenyl, lower alkoxy, lower alcohol, hydroxyl, amine, -NO 2 and halogen. A lower alkyl is preferably C 1
_
5 , more preferably C 1
-
3 , even more preferably C 1 alkyl. A lower alkenyl is preferably C 1
_
5 , more preferably C 1
_
3 , even more preferably C1- 2 alkenyl. A lower alkoxy is preferably C 1
_
5 , more preferably 20 C 1
-
3 , even more preferably C 1 alkoxy. A lower alcohol is preferably C1_ 5 , more preferably C 1
-
3 , even more preferably C 1 alcohol comprising one or more OH groups, preferably only one OH group. Halogen may be any halogen, but is preferably F. It is however preferred that R 4 is a heterocyclic ring, which is not substituted or that R4 is a heterocyclic ring substituted with a small substituent, preferably a small substituent 25 selected from the group consisting of methyl, methoxy, hydroxyl, CH 2 -OH, amine and halogen, and preferably methyl. [004951 In other embodiments, R 4 is preferably a 3 to 7-membered heterocyclic ring, more preferably a 5 to 6-membered heterocyclic ring, even more preferably a 5 30 membered heterocyclic ring. The heterocyclic ring may be aromatic or non-aromatic. In one embodiment the heterocyclic ring is a 3 to 7-membered aromatic heterocyclic ring, more preferably a 5 to 6-membered aromatic heterocyclic ring, even more preferably a 5-membered aromatic heterocyclic ring. 113 WO 20111134538 PCT/EP2010/063161 [00496] The heterocyclic ring may comprise one or more heteroatoms, preferably in the range of 1 to 3 heteroatoms, more preferably in the range of 1 to 2 heteroatoms, yet more preferably 1 heteroatom, preferably selected from the group consisting of S, N and 0. 5 [00497] Thus, contemplated in the present invention is the unknown undisclosed acid, single-bonded analog of compound III" above, the compound 14-(furan-2-yl) tetradeca-11,13-diynoic acid, X", COOH 10 0 X" [004981 Both 14-(furan-2-yl) tetradeca-11, 13-diynoic acid, and its salts, for example the sodium salt, XI", COO-,Na' 15 0 XI" and potassium salt XII", are effective and potent Olel protein inhibitors, and show potency and effectiveness in defeating or lessening agricultural fungal pathogens, providing effective and potent fungicides for use in the agricultural setting. 20 [004991 In preferred embodiments, the inventive compounds and salts, being highly soluble and suitable for formulation, provide highly effective components for formulations as fungicides for a variety of fungal pathogens in humans and animals. 25 [00500] Thus, methods are described herein that use the inventive compounds derived from or based on compound II", such as salts, acids and analogs, as effective and potent antifungal agents for use in formations and other forms of the compounds suitable for a wide variety of recipients and delivery modes. Their potent antifungal 114 WO 20111134538 PCT/EP2010/063161 capability, through the mechanism of Ole1 protein inhibition, offers a superior alternative to current antifungal drug treatments and fungicides. [00501] A preferred diyne compound according to the invention is potassium (Z) 5 12-(furan-2-yl) dodeca-7-en-9, 1 1-diynoate. [005021 Another preferred diyne compound according to the invention is potassium (Z)-13-(furan-2-yl) trideca-8-en-10, 12-diynoate. 10 [00503] Yet another preferred diyne compound according to the invention is potassium (E)-14-(furan-2-yl) tetradeca-9-en-11, 13-diynoate. [005041 Yet another preferred diyne compound according to the invention is the diyne compound potassium (Z)- 14-(furan-2-yl) tetradeca-9-en- 11, 13 -diynoate. 15 [005051 Another preferred diyne compound according to the invention is the diyne compound potassium 14-(furan-2-yl)tetradeca-11,13-diynoate. [00506] In yet another embodiment of the invention the diyne compound may be 20 selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, potassium salt; (Z)-14-(5-methylfuran-2-yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt; 8-(2-(4-(furan-2-yl)buta-1,3-diynyl)phenyl)octanoic acid, potassium salt; (Z)-14-(4,5-dimethylfuran-2-yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-11,13-diynoic acid, potassium salt. 25 C2. Mechanism of inhibition of Olel protein [00507] An Olel protein inhibitor is inherently fungicidal because the Olel protein is an essential protein to the fungal organism. In the biosynthesis of lipids, the Olel protein converts stearic acid to oleic acid. Oleic acid is an essential component of 30 lipids and thus essential to the fungal organism - without oleic acid the organism fails to survive due to collapse of the nuclear membrane. [005081 The inventors tested yeast knock-out strains, the results of which indicated that the Ole1 protein might be the target of the inventive compounds in 115 WO 20111134538 PCT/EP2010/063161 Saccharomyces cerevisiae. See Example 17 below. "Competition" experiments showed that in both Saccharonyces cerevisiae and Candida albicans oleic acid is an antagonist of the inventive compounds, but that stearic acid was not, indicating that the inventive compounds were most likely inhibitors of the biosynthesis of oleic acid 5 from stearic acid. See Example 18 below. The conclusion that the inventive compounds are inhibitors of the biosynthesis of oleic acid from stearic acid in both Saccharomyces cerevisiae and Candida albicans meant that the inventive compounds either inhibited the Ole 1 protein itself, or inhibited the transcriptional activators of the OLE1 gene, the Mga2 and Spt23 proteins. The inventors subsequently determined that 10 the addition of the inventive compounds to a culture of both Saccharomyces cerevisiae and Candida albicans, increased the expression of the OLE1 gene, indicating that the inventive compounds targeted the Ole1 protein and not the Mga2 and Spt23 proteins (because otherwise a transcriptional down-regulation of the OLE1 gene would have been seen). See Example 19 below. 15 [005091 An Ole1 protein inhibitor is a broad spectrum fungicidal. The inventive diyne compounds are found to inhibit the Olel protein in many species. The Olel protein is conserved across the fungal kingdom, sequence homologs to the Candida albicans OLE1 gene were identified in the following organisms: Candida glabrata, 20 Candida tropicalis, Candida parapsilosis, Candida guillermondii, Candida lusitaniae, Aspergillusfumigatus, Aspergillus terreus, Aspergillus nidulans, Coccidioides iinmitis, Histoplasma capsulatum and in Cryptococcus neoformans. Wilson, R. A. et al., 2004 and Kraus, P. R. et al., 2004, have shown that the OLE1 gene is essential to pathogenic fungal species such as Aspergillus and Cryptococcus, 25 and in vitro susceptibility data for compound III" and compound IV" on a set of clinical isolates of human fungal pathogens are provided as follows. C3. Antifungal efficacy in vitro [005101 The efficacy of compound IV" was investigated on 563 clinical isolates of 30 33 human fungal pathogens. The efficacy was determined according by broth micro and macro dilution. For species with n > 10, the MIC 9 o (MIC, minimal inhibitory concentration) is given as ng/ml. For species with n < 10, the MIC range is given as ng/ml. 24h and 48h reads are indicated (Table 1). 116 WO 20111134538 PCT/EP2010/063161 Table 1. Susceptibility of human fungal pathogens to compound IV" and compound III". Test MIC 90 / MIC range Strain (# of isolates)Copud 2h Compound 24 h 48 h C. albicans (n-=20) IV" - 2.5 C. glabrata (n=25) IV" - 20 C. tropicalis (n=35) IV" - 5 C. dubliniensis (n=25) IV" - 2.5 C. krusei (n=27) IV" - 20 C. lusitaniae (n=24) IV" - 128 C. parapsilosis (n=37) IV" - 1024 C. albicans, Fluconazole, Voriconazole, Itraconazole IV" - 2.5 and/or Caspofungin resistant isolates (n=20) C. glabrata, Fluconazole, Voriconazole, Itraconazole IV" - 20 and/or Caspofungin resistant iso lates(n=2 1) A. fumigatus (n=40) IV" 160 1280 A. terreus (n=5) IV" 10-20 10 - >5120 A. niger (n=6) IV" 40 - >5120 80 - >5120 A. flavus (n=5) IV" 640 - >5120 >5120 A. versicolor (n=4) IV" 80- 2560 160-5120 A. nidulans (n=5) IV" 320 - >5120 1280 - >5120 Coccidioides spp. (n=30) IV" - 30 B. dermatitidis (n=30) IV" - 30 H. capsulatum (n=20) IV" - 30 C. neoformans (n=18) IV" - 2000 Zygomycetes (n=20) IV" - 16000 Fusarium solani (n=10) IV" - >64000 Scedosporium (n=10) IV" - 2048- >64000 Sporothrix schenckii (n=10) IV" - 32 - 5120 117 WO 20111134538 PCT/EP2010/063161 Fonsecaea pedrosi (n=4) IV" - 80 - 2560 Phialophora verrucosa (n=4) IV" - 40 - 640 Trichophyton tonsurans (n= 15) III" - <0.06 T. mentagrophytes (n=15) III" - <0.06 Microsporum canis (n=15) III" - <0.06 T. rubrum (n=15) III" - <0.06 Epidermophyton floccosum III" - <0.06 (n= 15) T. rubrum, Terbinafine resistant III" - <0.06 isolates (n=9) M. cookie (n=1) III" - 1000 M. vanbreuseghemii (n=1) III" - 30 M. gypseum (n=1) III" - <0.06 T. terrestre (n=1) III" - <0.06 M. gallinae (n=1) III" - <0.06 [005111 The in vitro efficacy of compounds III", IV", VI", VII", VIII" and XII" was investigated on 8 Candida reference strains. The efficacy was determined according by broth micro dilution and the MIC given as ng/ml (see Table 2). 5 Table 2. Susceptibility of reference Candida strains to compounds III", IV", VT", VII", VIII" and XII". Strain name Compound III" IV", VI"l VII"l VIII"I XII"' >1024 >1024 C. albicans ATCC-24433 1.25 1.25 - 10 0 0 >1024 >1024 C. albicans ATCC-90028 1.25 1.25 - 10 0 0 >1024 >1024 C. glabrata ATCC-90030 10 20 0 0 - 320 C. krusei ATCC-6258 10 20 >1024 >1024 - 640 118 WO 20111134538 PCT/EP2010/063161 0 0 C. parapsilosis ATCC- >1024 >1024 >1024 2560 2560 22019 0 0 0 C. parapsilosis ATCC- >1024 >1024 2560 2560 - 5120 90018 0 0 >1024 >1024 C. tropicalis ATCC-750 2.5 5 - 10 0 0 >1024 >1024 C. albicans SC5314 0.5 1.25 150 10 0 0 C4. Antifungal efficacy in vivo [00512] The efficacy of compound V" and compound IV" in systemic C. albicans infections in small mammals is demonstrated as follows. 5 [00513] When compound V" was administered to rats infected systemically with C. albicans as a single intravenous infusion at 12 mg/kg, liver fungal burden decreased 93% after 24h (see figure 10 showing liver fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue). 10 [005141 When compound V" was administered to rats infected systemically with C. albicans as a single oral dose at 17 mg/kg, kidney fungal burden decreased by 57% after 6h (see figure 11 showing kidney fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue). 15 [005151 Mice were infected systemically with C. albicans. Compound IV" was administered orally twice daily for 3 days at 5 mg/kg or 10 mg/kg per dose. Kidney fungal burden decreased by 88% and 94% respectively, compared to the untreated control (as shown in figure 12 showing kidney fungal burden as determined by colony 20 forming units (CFU) per gram of homogenised tissue). [005161 The efficacy of compound III" in skin infections of small mammals was shown in guinea pigs infected with Trichophyton mentagrophytes on abrased skin. Compound III" was administered topically at 0. 1% or 1% once daily for seven days. 119 WO 20111134538 PCT/EP2010/063161 Fungal burden decreased by 82% and 98% respectively, compared to the untreated control (see figure 13 showing topical fungal burden as determined by culture positive hair removed from the site of infection). 5 [00517] The efficacy of compound III" in vaginal infections of small mammals has been shown. Mice were vaginally infected with Candida albicans. Compound III" was administered topically at 0.01% daily for five days. Fungal burden decreased 86% compared to the untreated control (see figure 14 showing vaginal fungal burden as determined by colony forming units (CFU) from vaginal lavage fluid). 10 C5. Agricultural fungal infection [00518] The Olel protein inhibitors of the instant invention provide potent broad spectrum antifungal agents for a wide variety of agricultural purposes. Preferred embodiments comprise any of the inventive compounds disclosed herein based on 15 and/or derived from compound I", its salts and analogs. The inventive Olel protein inhibitors are suitable and efficacious for treating a fungal infection in the agricultural setting, including reducing the risk of a fungal infection, and in particular may be used for methods of treating an infection in a plant, or a grass, by contacting a plant with an Olel protein inhibitor according to the invention. Plants include trees, crops, grasses, 20 and flowering plants. [005191 Thus, contemplated in the present invention is a pesticide composition comprising and plant propagation material comprising any of the inventive compounds described herein, its derivatives, salts and analogs. 25 [00520] In a preferred embodiment, a pesticide composition is provided comprising compound X". In other preferred embodiments, the pesticide composition is provided comprising 14-(furan-2-yl) tetradeca-11,13-diynoic acid, compound X" or its derivatives, especially its salts, such as compound XI", or its analogs, providing 30 effective and potent Olel protein inhibitors, in defeating or lessening agricultural fungal pathogens, providing effective and potent compounds for use in the agricultural setting. 120 WO 20111134538 PCT/EP2010/063161 [00521] In other embodiments are provided, incorporating the same inventive compounds, a plant propagation material, a system for protecting a plant from disease caused by a given susceptible fungus, and methods of controlling or preventing fungal infestation in plants, parts of plants, seeds, or at their locus of growth. 5 [00522] Also contemplated is a system for protecting a plant from disease caused by a given susceptible fungus, comprising inventive compounds X", its derivatives, salts and analogs. 10 [00523] Further, the present invention contemplates methods for preventing or controlling fungal infections in plants, parts of plants, seeds, or at their locus of growth. C6. Efficacy in Agricultural species 15 [005241 Compounds based on structure IX", X", XI" and XII" and analogs and derivatives have been tested on various agricultural setting fungal pathogens (see Example 20 below). Conidia/spores are the major source of spreading diseases, and if the sporulation is affected, disease spread in the farming field is contained. Thus, inhibiting sporulation is an indirect way of conducting disease control. Moreover, if 20 sporulation is affected, the emergence of disease resistance is minimised, because the genetic changes which make the pathogen to adopt for the fungicide will not be carried to the next generation. The asexual fruit body of the plant pathogenic fungus Colletotrichum gloeosporioides is called acervulus. Acervuli, visible to the naked eye and salmon colour, are produced in concentric circles. When the potassium salt of 25 compound X" was loaded on a sterile paper disc in the path of pathogen growth, the growth of the mycelia is arrested. Although the pathogen continues growth somewhat, there is no sporulation observed in mycelia grown around the region where the disc is loaded with test compound. The mycelia grown in the region diffused with the test compound was weak and did not differentiate into conidiophores - no sporulation 30 observed. [005251 Malformation and inhibition takes place in the spore germination in M. grisea. 121 WO 20111134538 PCT/EP2010/063161 [00526] Spore germination in the M. grisea control starts with small beak-like germination, which extends into long germ tube and culminates into an appressorium. The appressorium will be densely melanized to withstand the high turgor pressure created during penetration of infection peg through the plant cell wall. The germ tube 5 starts from one or two terminal cells of a three-celled spore, and the process is completed in 8hrs. When the spores were incubated in sterile water containing different concentrations of test compound (potassium salt of compound X") , more than 50% of spores did not germinate, and where small beak-like germination started in germinated spores: in some more spores, though the appressorium is formed, they 10 were not melanised enough to withstand the pressure; breakage in the germ tube near the formation of appressoria was observed; some appressoria burst due to turgor pressure; and in some spores germ tubes formed from the middle cell, instead of from the terminal cells. 15 a. Olel protein inhibitor compound solubility [005271 For desired antifungal activity in a clinical setting it is preferable that the inventive fungacides are soluble in water. In the preparation of pharmaceutical compositions comprising antifungal compounds to reach the site of disease and be functional there, water solubility is important, particularly the case with infections by 20 a fungus which involve infection of inner organs or disseminated infections, but also in relation to treatment or reduction of risk of a fungal infection in agriculture where water solubility is crucial. Accordingly, the inventive compounds preferably have a water solubility of at least 50 mg/ml, preferably at least 60 mg/ml, more preferably at least 70 mg/ml, yet more preferably at least 80 mg/ml, even more preferably at least 25 90 mg/ml. b. Crystalline compound [005281 An exemplary crystalline compound is the potassium (Z)-14-(furan-2-yl) tetradeca-9-en-1 1, 13-diynoate, having a high resolution XPRD pattern comprising 30 peaks at least at the following 20 angles: 2.420 and 4.78'. In preferred embodiments, the potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 20 angles: 7.140, 9.520 and 11.890, or at the following 20 angles: 2.42' and 4,78', 7.140, 9.52 and 11.890, or at the following 20 angles: 2.420, 4,780, 7.140, 9.52', 9.520, 16.450, 17.270, 122 WO 20111134538 PCT/EP2010/063161 18.410, 19.110, 19.680, 21.27, 21.950, 23.06, 23.860, 24.90, 26.98, 27.82, 28.68, 28.860 and 38.770. Preferably, this high resolution XPRD pattern does not comprises any other peaks with an intensity of >5%. 5 c. Moisture uptake [00529] It is preferred that the fungacides according to the invention do not take up much moisture from the surroundings, and that the weight change of a compound is less than 5%, preferably less than 4%, more preferably less than 3%, even more preferably less than 2%, for example even less than 1% at a humidity of 60% RH 10 compared to a humidity of 10% RH, or that the weight change of the compound is less than 5%, preferably less than 4% at a humidity of 70% RH compared to a humidity of 10% RH, or that if a crystalline compound is exposed to humidity higher than 80%, then upon return to a lower humidity, when moisture is lost its crystalline form is kept/regained. 15 d. Melting temperature and stability [005301 It is also preferred that the inventive fungacides have a sufficiently high melting temperature to allow handling during manufacture of pharmaceutical compositions and storage at ambient temperature. Thus, it is preferred that the melting 20 point of the compounds is at least 100 C, preferably at least 1 10 C, more preferably at least 120'C, yet more preferably at least 130'C, even more preferably at least 140'C, and are stable upon storage. e. Formulations 25 [005311 The inventive compounds disclosed herein should be understood to include any pharmaceutically acceptable salts encompassing either salts with inorganic acids or organic acids like hydrohalogenic acids, e.g. hydrochloric or hydrobromic acid; sulfuric acid, phosphoric acid, nitric acid, citric acid, formic acid, acetic acid, maleic acid, tartaric acid, methanesulfonic acid, p-toluenesulfonic acid and the like or in case 30 the compound is acidic in nature with an organic base such as, for example, triethylamine, triethanolamine, tert-butylamine, or an inorganic base like an alkali or earth alkali base, e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide etc. 123 WO 20111134538 PCT/EP2010/063161 [00532] Because of their ability to inhibit a wide variety of fungal pathogens occurring in humans and/or animals, occurring both systemically and topically, the described compounds can be used for the treatment of diseases which are associated with an infection by such type of pathogens. They are valuable antifungal treatments. 5 [00533] The compounds can be administered orally, rectally, parenterally e.g. by intravenous, intramuscular, subcutaneous, intrathecal or transdermal administration or sublingually or as ophthalmic preparation or administered as aerosol. Examples of applications are capsules, tablets, orally administered suspensions or solutions, 10 intravenous solutions, suppositories, injections, eye-drops, ointments or aerosols/nebulizers. [005341 Preferred applications are oral or i.v. systemic formulations and ointment, pellet, liquid or liquid suspension topical formulations. The dosage used depends upon 15 the type of the specific active ingredient, the age and the requirements of the patient and the kind of application. The preparations with the inventive compounds can contain inert or as well pharmacodynamically active excipients. Tablets or granules, for example, could contain a number of binding agents, filling excipients, carrier substances or diluents. 20 [00535] These pharmaceutical compositions may contain the compounds of the invention as well as their pharmaceutically acceptable salts in combination with inorganic and/or organic excipients which are usual in the pharmaceutical industry like lactose, maize or derivatives thereof, talcum, stearinic acid or salts of these 25 materials. [00536] For gelatine capsules vegetable oils, waxes, lipids, liquid or half-liquid polyols etc. may be used. For the preparation of solutions and syrups e.g. water, polyols, saccharose, glucose etc. are used. Injectables are prepared by using e.g. 30 water, polyols, alcohols, glycerin, vegetable oils, lecithin, liposomes etc. Suppositories may be prepared by using natural or hydrogenated oils, waxes, fatty acids (fats), liquid or half-liquid polyols etc. 124 WO 20111134538 PCT/EP2010/063161 [00537] The compositions may contain in addition preservatives, stabilisation improving substances, viscosity improving or regulating substances, solubility improving substances, sweeteners, dyes, taste improving compounds, salts to change the osmotic pressure, buffer, antioxidants etc. 5 f. Resistant fungus infections [00538] Fungal infections may be resistant to treatment for many reasons, resistant to treatment with a particular antifungal agent, or because of acquired resistance. 10 Thus, a further aspect of the invention is the use of the inventive compounds for treating infection by a fungus resistant to one or more alternative treatment and that acts via: a) inhibiting ergosterol biosynthesis; 15 b) binding to ergosterol; c) inhibiting 1, 3-p-glucan synthase; d) inhibiting epoxidase; e) inhibiting Leucyl-tRNA synthetase; and/or f) inhibition of elongation factor 2. 20 [00539] Particularly, such resistant antifungal treatments may be benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate or may be polyenes, azoles, allylamines or echinocandins. Polyene antifungal agents have multiple conjugated double bonds, and typically, also 25 comprise a heavily hydroxylated region, exemplified by Natamycin, Rimocidin, Filipin, Nystatin, Amphotericin B or Candicin. Azole antifungal agents may for example be imidazole or triazole or thiazole antifungal agents. Imidazole antifungal agents may for example include miconazole, ketoconazole, clotromazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, seraconazole, 30 sulconazole or tioconazole. Triazole antifungal agents may for example include fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and abafungin. Allylamine antifungals include Terbinafine, Amorolfine, Naftifine or Butenafine. Non-limiting examples of echinocandins include Anidulafungin, Caspofungin or Micafungin. 125 WO 20111134538 PCT/EP2010/063161 D1. The Mechanism of Action of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoic acid and its salts 5 [00540] The potassium salt of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid (aka (9Z)-14-(2-furyl)tetradeca-11,13-diynoic acid) is shown as follows: H H Z isomer COO ,K* 10 [005411 The mechanism of action of the sodium salt was investigated in a chemical-genetic screen in S. cerevisiae. The screen made use of the S. cerevisiae knock-out collection, which consists of 4917 individual strains each deleted for one defined gene. All 4917 strains were tested for increased or decreased susceptibility to this sodium salt. The rationale of the screen was chemical enhancement genetics. In 15 biological systems, pathway redundancies ensure robust signaling of essential processes. [005421 Given that a certain gene deletion strain is more susceptible to this sodium salt, it it possible that the sodium salt acts on a redundant pathway. Inhibition of the 20 two redundant pathways, one through the gene deletion, the other through the action of the sodium salt, will lead to cell death if both pathways regulate one common essential function. It is also possible that the sodium salt interferes with a complex that is already destabilized by the absence of the gene product of the deleted gene. 126 WO 20111134538 PCT/EP2010/063161 [00543] Together, the screen for gene deletion strains with increased or decreased susceptibility to the sodium salt highlights biological processes affected by sodium (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoate. 5 [00544] From 4917 analyzed strains of the S. cerevisiae knock-out collection, 44 had an increased susceptibility to the sodium salt. No strain with a decreased susceptibility was identified (Knechtle, P. and Greve, K., 2008). The S. cerevisiae GO-Slim vocabulary was used to map the identified genes to higher level biological processes. The GO-Slim vocabulary consists of higher level gene ontology terms used 10 to describe the biology of a gene product. From a total of 39 biological processes available, the 44 genes identified mapped to 29 processes, from which "lipid metabolic process" (p<0.00 4 ) and "organelle organization and biogenesis" (p<0.0 4 ) were significantly overrepresented. 15 [005451 Among the genes identified was MGA2, which encodes a transcriptional activator of the OLE1 A9-fatty acid desaturase. MGA2 is a duplicated gene in S. cerevisiae with SPT23 being its homolog. The Olel protein converts stearic to oleic acid and palmitic to palmitoleic acid, respectively, and is an essential gene in S. cerevisiae. Another six genes from the identified set of 44 genes could be 20 assembled together with MGA2 to a pathway likely to regulate OLE1 transcriptional activation. This pathway included components of the ERAD (endoplasmatic reticulum associated protein degradation) complex required for the proteolytic activation of Mga2and genes coding for GET complex components, which is putatively required to insert Mga2 into the membrane of the endoplasmatic reticulum (see Figure 19). 25 [00546] Mutations in genes of this three-step pathway most likely decrease the expression of the OLE1 gene. Consequently, less Olel protein is available in these mutant cells and thus the levels of oleic acid decreased, suggesting that decreased levels of endogenous oleic acid increases the susceptibility to the sodium salt. 30 [00547] Indeed, the addition of exogenous oleic acid counteracted the increased sensitivity to the sodium salt of (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid of the identified mutants (2). In the wild-type, the addition of oleic acid decreased the sensitivity to the sodium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid. 127 WO 20111134538 PCT/EP2010/063161 Stearic acid, however, decreased the sensitivity to Na-(Z)-14-(furan-2-yl)tetradeca-9 en-i 1,13-diynoic acid only marginally (see Figure 20). [00548] This direct competition between oleic acid and Na-(Z)-14-(furan-2 5 yl)tetradeca-9-en- 11,13-diynoic acid suggests that Na-(Z)-14-(furan-2-yl)tetradeca-9 en-11,13-diynoic acid is an inhibitor of oleic acid biosynthesis. Inhibition is taking place at the conversion of stearic to oleic acid, since stearic acid does not compete with Na-(Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid. 10 [00549] Mechanistically, it is likely that Na-(Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid inhibits the biosynthesis of oleic acid, either through direct inhibition of the A9 fatty acid desaturase Olel or through inhibition of Mga2 dependent transcriptional activation of OLE1, thereby inducing an oleic acid auxotrophy to the cell as it is observed for cells deleted for the OLE1 gene (Stukey, J. 15 E. et al., "Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae", J Biol Chem 264(28), 16537-16544 (1989)). [00550] OLE 1 transcriptional levels were determined to distinguish between these 20 two hypotheses, and to generally confirm the oleic acid biosynthesis inhibitory model. [005511 Assuming that the potassium salt of (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid directly inhibited the Olel protein, a compensating up-regulation of the OLE1 transcriptional level for the potassium salt of (Z)-14-(furan-2-yl) 25 tetradeca-9-en-11,13-diynoic acid treated cells would be expected. If the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid inhibited Mga2 or any other component required for OLE1 transcriptional activation such as the GET complex or the CDC48 proteasome, a down-regulation of OLE1 for the potassium salt treated cells would be expected. potassium salt independent transcription of OLEI 30 would indicate another mechanism of action and suggested that the antagonistic effect of oleic acid with potassium salt was indirect. 128 WO 20111134538 PCT/EP2010/063161 [00552] S. cerevisiae was cultured in the presence of 1 and 10 [g/ml of the potassium salt, 10 [tg/ml oleic acid and 1 [tg/ml of the potassium salt combined with 10 [tg/ml oleic acid. The calculated doubling times are shown in Table 24 below. 5 Table 24. Doubling times of S. cerevisiae in the presence of the potassium salt of (Z) 14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid and/or oleic acid condition oleic acid Doubling time in h A 2.0 B 1 pLg/ml 2.4 C 10 pg/ml - 4.2 D - 10 pg/ml 2.0 E 1 tg/ml 10 [g/ml 2.1 [005531 The potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid increased the doubling times in a concentration dependent manner, which was 10 reversed by the addition of oleic acid. [005541 The expression of OLE1 was determined by real time PCR 10, 60, 120 and 240 min after compound addition (Knechtle, P. and Greve, K., 2009). OLE1 transcript levels were calibrated to the expression of the tubulin gene (TUBI) and normalized to 15 the OLE1 expression at 10 min without compound addition (Figure 17(a)). [00555] Expression levels of OLE1 in cultures without the potassium salt of (Z) 14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid did not vary significantly over the time scale investigated. The addition of 1 pg/ml of the potassium salt of (Z)-14 20 (furan-2-yl) tetradeca-9-en-11,13-diynoic acid increased the expression level of OLE1 about 3-5 fold (4.9 fold at 120 min, p<0.023) and 10 pg/ml increased the expression level about 7-19-fold (18.7 fold at 120 min, p<0.018). As reported elsewhere, the addition of 10 pg/ml oleic acid alone decreased the expression level of OLE1 about 6 fold, which was confirmed in the experiment (condition "d"). 10 pg/ml oleic acid 25 compensated for the OLEl increase caused by 1 pg/ml of the Potassium salt of (Z) 14-(furan-2-yl) tetradeca-9-en- 11,13-diynoic acid alone. 129 WO 20111134538 PCT/EP2010/063161 [00556] The potassium salt (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid dependent up-regulation of OLEl strongly favours the first hypothesis with the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-1 1,13-diynoic acid as an inhibitor of the Olel protein. The antagonistic effect of oleic acid, which was found in the 5 susceptibility assays, is reflected mechanistically in this experiment by a down regulation of the target Olel through oleic acid. [00557] For all described genes above, a sequence homologue had been identified in C. albicans, which indicates that the identified process is conserved in at least these 10 two organisms. For S. cerevisiae MGA2/SPT23 and OLE1, even functional homologues have been described in C. albicans (Krishnamurthy, S. et al., "Dosage dependent functions of fatty acid desaturase Oleip in growth and morphogenesis of Candida albicans.", Microbiology 150(Pt 6), 1991-2003 (2004); Martin, C. E. et al., "Regulation of long chain unsaturated fatty acid synthesis in yeast.", Biochim. 15 Biophys.Acta 1771(3), 271-285 (2007); Oh, C. S. et al., "Candida albicans Spt23p controls the expression of the Oleip Delta9 fatty acid desaturase and regulates unsaturated fatty acid biosynthesis.", J.Biol.Chem. 281(11), 7030-7039 (2006)). [00558] Sequence homologues were further identified in Aspergillus and 20 Cryptococcus (Kraus, P. R. et al., "Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray.", Eukaryot.Cell 3(5), 1249-1260 (2004); Wilson, R. A. et al., "Two Delta9-stearic acid desaturases are required for Aspergillus nidulans growth and development", Fungal.Genet.Biol. 41(5), 501-509 (2004)). 25 [00559] Competition assays were done in C. albicans to investigate the antagonistic effect of oleic acid to the potassium salt of (Z)-14-(furan-2-yl) tetradeca 9-en-11,13-diynoic acid. 30 [00560] As in S. cerevisiae, oleic acid but not stearic acid antagonized the effect of the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid in C. albicans (see Figure 21). We have also identified linoleic acid and palmitoleic (but not palmitic acid) as antagonists of the potassium salt of (Z)-14-(furan-2-yl) tetradeca 9-en-Il, 1 3-diynoic acid in C. albicans, which are all A9-unsaturated fatty acids. In 130 WO 20111134538 PCT/EP2010/063161 S. cerevisiae, different A9-unsaturated fatty acids including palmitoleic and linoleic acid, but not saturated fatty acids, have been shown to complement the oleic acid auxotrophy of OLE1 deletion strains (1), which further supports our model. 5 [00561] In contrast, oleic acid did not decrease the susceptibility of C. albicans to Voriconazole, AmphotericinB or Caspofungin, which represent the relevant classes of clinical antifungal compounds. This corroborates that the oleic acid auxotrophy induced by the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-1 1,13-diynoic acid is not a general stress response of the cell but specific to the potassium salt of (Z)-14 10 (furan-2-yl) tetradeca-9-en-1 1,13-diynoic acid (Figure 22). [00562] Further, the expression of OLE1 in C. albicans was investigated in response to the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid (Knechtle, P. and Greve, K., "EV-086-3314K up-regulates OLEl expression in 15 Candida albicans,"in REP-S-EV-086-3314-0092 ed.(Evolva, 2009)). In the presence of 250 ng/ml of the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en- 1,13-diynoic acid, OLE1 transcript levels were increased 4-fold after 120 min incubation time compared to the drug free control (Figure 23). These results demonstrate a conservation of the mechanism of action between S. cerevisiae and C. albicans. 20 [00563] From a structural point of view the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid is similar to oleic acid. The molecules share a A9 monounsaturated decanoic acid group characteristic for unsaturated fatty acids in fungi. (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid might therefore be 25 recognized as a fatty acid and incorporated as an acyl chain into membrane lipids. The rapid incorporation of exogenous non S. cerevisiae fatty acids into lipids has previously been shown (8). (Z)-14-(furan-2-yl) tetradeca-9-en-1 1,13-diynoic acid-oyl lipids might then inhibit the Ole1 protein. Inhibition, however could also take place through free (Z)-14-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid. 30 [00564] Thus, there is strong evidence that the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-11,13-diynoic acid inhibits oleic acid biosynthesis through inhibition of the Ole1 protein. The process is likely to be conserved across the entire fungal 131 WO 20111134538 PCT/EP2010/063161 kingdom and it represents a novel mechanism of action that has so far not been described for any antimicrobial compound in clinical development or on market. 132 WO 20111134538 PCT/EP2010/063161 EXAMPLES [00565] The invention is furthermore illustrated by the following non-limiting examples. 5 Example 1 [005661 This example describes the characterisation of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid (herein after in this example designated "the material"). 10 [005671 An X-Ray Powder Diffraction (XRPD) analysis was performed to determine crystallinity of the material. X-Ray Powder Diffraction patterns were collected on a Siemens D5000 diffractometer using Cu Ka radiation (40kV, 40mA), 0-0 goniometer, divergence of V20 and receiving slits, a graphite secondary 15 monochromator and a scintillation counter. The software used for data collection was Diffrac Plus XRD Commander v2.3.1 and the data were analysed and presented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2. [00568] Samples run under ambient conditions were prepared as flat plate 20 specimens using the material. Approximately 35 mg of the sample was gently packed into a cavity cut into polished, zero-background (510) silicon wafer. The sample was rotated in its own plane during analysis. The details of the data collection are: Angular range: 2 to 42 020 Step size: 0.05 020 25 Collection time: 4 s.step-1 [00569] The analysis shows that the material has good crystallinity. Material having the XRPD pattern similar to the material prior to any subsequent treatment is designated "Form 1". 30 [005701 A Differential Scanning Calorimetry (DSC) was performed. DSC data were collected on a TA Instruments Q2000 equipped with a 50 position auto-sampler. The instrument was calibrated for energy and temperature calibration using certified 133 WO 20111134538 PCT/EP2010/063161 indium. Typically 0.5 mg of each sample, in a pin-holed aluminium pan, was heated at 10 C.min-l from 25 'C to 300 'C. A nitrogen purge at 50 ml.min- 1 was maintained over the sample. 5 [00571] The instrument control software was Advantage for Q Series v2.8.0.392 and Thermal Advantage v4.8.3 and the data were analysed using Universal Analysis v4.3A. [005721 The DSC trace shows a melting temperature with an onset of 45 'C, then 10 an exotherm of degradation with an onset of 171 'C. [00573] Also a Thermo-Gravimetric Analysis (TGA) was performed to determine the melting temperature and heat stability of the material. TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16 position auto-sampler. The 15 instrument was temperature calibrated using certified Alumel. Typically 5-30 mg of each sample was loaded onto a pre-tared platinum crucible and aluminium DSC pan, and was heated at 10 C.min-l from ambient temperature to 350 'C. A nitrogen purge at 60 ml.min- 1 was maintained over the sample. 20 [005741 The instrument control software was Advantage for Q Series v2.8.0.392 and Thermal Advantage v4.8.3 [00575] The TGA trace of the material showed no weight loss until the start of the degradation at approximately 160 'C. 25 [00576] Solubility in water was determined. This was carried out by transferring 10 mg of the material into 1.5 ml HPLC vials. Then 10 pl (ivol) of water was added. The sample was then isothermally held at 50 'C with stirring for 30mins. The process was continued up to 50 pl by 10 tl increments with heating to 50'C in between. The 30 material however still did not dissolve. Example 2 134 WO 20111134538 PCT/EP2010/063161 [00577] Bases used for preparation of salts of (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid are shown in Table 3. Table 3 Base Class pKa Solution. Potassium ethoxide 1 14.00 1M EtOH Sodium ethoxide 1 14.00 1M EtOH L-Arginine 1 13.20 0.5M H20 L-Lysine 1 10.79 0.5M H20 Ammonium hydroxide 1 9.27 2M EtOH Dimethylaminoethanol 1 8.83 1M EtOH N-Methylglucamine 1 8.03 1MH20 5 [005781 Several of the bases employed were only soluble in water and water miscible solvents were therefore employed. To this end the use of IPA, Dioxane, EtOH and acetone were selected. 10 [005791 (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid (25mg) was dissolved in solvent (250pl, 10 volumes) and the mixture warmed to 50'C. The base solution was added (1.1eq) and the reactions were then allowed to cool. Reactions that did not produce any solid were allowed to cool to 18'C and if no solid had formed they were then allowed to slowly evaporate. 15 [005801 The results using IPA as solvent are shown in Table 4 Table 4. Experiment Base RT Cool - Evaporation 18 0 C DJP401-006- Potassium ethoxide IM Solid n/a n/a 1 in EtOH 97p1l filtered off DJP401-006- Sodium ethoxide IM in Solid at n/a n/a 2 EtOH 971 addition, 135 WO 20111134538 PCT/EP2010/063161 filtered off DJP401-006- L-Arginine 0.5M inH20 Solution Solution Gum 3 194pl DJP401-006- L-Lysine 0.5M in H20 Solution Solution Gum 4 194p1l DJP401-006- Ammonium hydroxide Solution Solution Gum 5 2M in EtOH 48p1l DJP401-006- Dimethylaminoethanol Solution Solution Gum 6 1 M in EtOH 97pl [00581] The results using dioxane, ethanol or acetone as solvents are shown in Table 5. 5 Table 5 Experiment Solvent Base RT Cool - Evaporation 18 0 C DJP401-009- Dioxane Potassium ethoxide IM Solid n/a n/a 1 in EtOH 97pl formed upon cooling DJP401-009- Dioxane Sodium ethoxide IM in Solid n/a n/a 2 EtOH 97pl formed upon addition DJP401-009- Dioxane L-Arginine 0.5M in Solution Frozen Gum 3 H20 194pl solid DJP401-009- Dioxane L-Lysine 0.5M in H20 Solution Frozen Gum 4 1941 solid DJP401-009- Dioxane Ammonium hydroxide Solution Frozen Gum 5 2M in EtOH 4 8 t 1 solid DJP401-009- Dioxane Dimethylaminoethanol Solution Frozen Gum 6 IM in EtOH 971 solid 136 WO 20111134538 PCT/EP2010/063161 DJP401-009- Dioxane N-MethylglucaminelM Solution Frozen gum 7 in H20 97[i solid DJP401-009- EtOH Potassium ethoxide IM Solid n/a n/a 8 in EtOH 97pl formed upon cooling DJP401-009- EtOH Sodium ethoxide IM in Solid n/a n/a 9 EtOH 97pl formed upon addition DJP401-009- EtOH L-Arginine 0.5M in Solution Solution Gum 10 H20 194d DJP401-009- EtOH L-Lysine 0.5M in H20 Solution Solution Gum 11 194pl DJP401-009- EtOH Ammonium hydroxide Solution Solution Gum 12 2M in EtOH 48tt1 DJP401-009- EtOH Dimethylaminoethanol Solution Solution Gum 13 1M in EtOH 97[d DJP401-009- EtOH N-MethylglucaminelM Solution Solution Gum 14 in H20 97p DJP401-009- Acetone Potassium ethoxide IM Solid n/a n/a 15 in EtOH 97pl formed upon cooling DJP401-009- Acetone Sodium ethoxide IM in Solid n/a n/a 16 EtOH 97pl formed upon addition DJP401-009- Acetone L-Arginine 0.5M in Solution Solution Gum 17 H20 194ptl DJP401-009- Acetone L-Lysine 0.5M in H20 Solution Solution Gum 18 1 94 [d DJP401-009- Acetone Ammonium hydroxide Solution Solution Gum 137 WO 20111134538 PCT/EP2010/063161 19 2M in EtOH 4 8pi DJP401-009- Acetone Dimethylaminoethanol Solution Solution gum 20 1M in EtOH 97pl DJP401-009- Acetone N-MethylglucaminelM Solution Solution Gum 21 in H20 97pl [00582] The reactions only yielded gums for most of the bases tried. The only exception was the reactions with potassium ethoxide and the sodium ethoxide. These 5 solids were analysed by XRPD (XRPD performed as described herein above in Example 1). The potassium salts were all crystalline and appeared to be the same solid form and the sodium salts were all amorphous but there were some hints of crystallinity. 10 [00583] The potassium and sodium salts were scaled up to a 100mg scale of the free acid to provide more material for characterisation. In an attempt to allow the sodium salt to crystallise the sodium reaction was carried out in a more dilute fashion and also both reactions were carried out at 70'C. The experiments are summarised in Table 6. 15 Table 6 Experiment Solvent Base + amount Result DJP401-007-1 IPA (1ml) KOEt (388pl, IM Solid crystallised in EtOH) while still warm. DJP401-007-1 IPA (2ml) NaOEt (388ptl, Im Solid precipitated IN EtOH) upon addition of base. [005841 The solids from these reactions were analysed by XRPD and the diffractograms are shown in figure 1. This X-Ray Powder Diffraction pattern was 20 collected on a Bruker AXS C2 GADDS diffractometer using Cu Ka radiation (40 kV, 40 mA), automated XYZ stage, laser video microscope for auto-sample positioning and a HiStar 2-dimensional area detector. X-ray optics consists of a single G6bel 138 WO 20111134538 PCT/EP2010/063161 multilayer mirror coupled with a pinhole collimator of 0.3 mm. The beam divergence, i.e. the effective size of the X-ray beam on the sample, was approximately 4 mm. A 0 0 continuous scan mode was employed with a sample - detector distance of 20 cm which gives an effective 20 range of 3.2 - 29.7 '. Typically the sample would be 5 exposed to the X-ray beam for 120 seconds. The software used for data collection was GADDS for WNT 4.1.16 and the data were analysed and presented using Diffrac Plus EVA v 9.0.0.2 or v 13.0.0.2. [005851 Samples run under ambient conditions were prepared as flat plate 10 specimens using powder. Approximately 1-2 mg of the sample was lightly pressed on a glass slide to obtain a flat surface. [005861 As can be seen in figure 1 the potassium salt was crystalline and the sodium salt was amorphous with some hints of crystallinity. 15 [005871 The potassium salt was analysed by various techniques in order to fully understand its solid form behaviour. A high resolution XRPD diffractogram was prepared. These X-Ray Powder Diffraction patterns were collected on a Bruker D8 diffractometer using Cu K radiation (40kV, 40mA), 0-20 goniometer, and divergence 20 of V4 and receiving slits, a Ge monochromator and a Lynxeye detector. The instrument is performance checked using a certified Corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRD Commander v2.5.0 and the data were analysed and presented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2. 25 Samples were run under ambient conditions as flat plate specimens. Approximately 20 mg of the sample was gently packed into a cavity cut into polished, zero background (510) silicon wafer. The sample was rotated in its own plane during analysis. The details of the data collection are: Angular range: 2 to 420 20 30 Step size: 0.05' 20 Collection time: 0.5 s.step' [005881 The result is shown in figure 2A. 139 WO 20111134538 PCT/EP2010/063161 [00589] The free acid (100mg) was dissolved in THF (iml) and warmed to 50'C before the addition of potassium ethoxide (388ml, IM in EtOH). The reaction was then cooled and the solid produced filtered and dried under suction and then analysed by high resolution XRPD on a Bruker D8 diffractometer as described above. The 20 5 angles were calculated at Cu-Ka, k =1.54173. The diffractogram with the 20 angles of the peaks with an intensity >5% are shown in figure 2B. The 20 Angle, d value, A and intensity of peaks are indicated in Table 7. Table 7 Peak Intensity, No. 2 E Angle d value, A % 1 2.42 36.56055 87.50 2 4.78 18.47136 100.00 3 7.14 12.36523 27.90 4 9.52 9.28642 7.30 5 11.89 7.43965 9.50 6 16.45 5.38417 5.70 7 17.27 5.13092 15.10 8 18.41 4.81433 4.30 9 19.11 4.64095 5.20 10 19.68 4.50779 4.50 11 21.27 4.17455 18.30 12 21.95 4.04675 4.60 13 23.06 3.85315 3.40 14 23.86 3.72571 13.10 15 24.90 3.57343 5.30 16 26.98 3.30246 27.80 17 27.82 3.20449 5.10 18 28.68 3.10984 5.50 19 28.86 3.09123 6.00 20 38.77 2.32077 3.50 140 WO 20111134538 PCT/EP2010/063161 [00590] The potassium salt was also analysed by TGA (TGA performed as 5 described herein above in Example 1) and DSC (DSC performed as described herein above in Example 1) and the data is shown in figure 3. [00591] The material shows only a small loss in the TGA until decomposition start at approximately 170'C. The DSC shows a melting temperature with an onset of 10 159'C followed by gross decomposition peaking at 192.5 0 C. [00592] The potassium salt was furthermore submitted to a solubility determination. Aqueous solubility was determined by suspending sufficient compound in water to give a maximum final concentration of >100 mg.ml-1 of the 15 parent free-form of the compound. The suspension was equilibrated at 25 'C for 24 hours then the pH was measured. The suspension was then filtered through a glass fibre C filter into a 96 well plate. The filtrate was then diluted by a factor of 101. Quantification was by HPLC with reference to a standard solution of approximately 0.1 mg.ml-1. in DMSO. Different volumes of the standard, diluted and undiluted 20 sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection. Analysis was performed on an Agilent HP 1100 series system equipped with a diode array detector and using ChemStation software vB.02.01-SRI. 25 [00593] The aqueous solubility of potassium (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoate was >96.5 mg/ml. [005941 The potassium salt was also analysed by GVS. 30 [00595] Sorption isotherms were obtained using a Hiden IGASorp moisture sorption analyser, controlled by CFRSorp software. The sample temperature was maintained at 25 'C by a Huber re-circulating water bath. The humidity was controlled by mixing streams of dry and wet nitrogen, with a total flow rate of 250 141 WO 20111134538 PCT/EP2010/063161 ml.min-1. The relative humidity (RH) was measured by a calibrated Vaisala RH probe (dynamic range of 0-95 %RH), located near the sample. The weight change, (mass relaxation) of the sample as a function of %RH was constantly monitored by the microbalance (accuracy +0.001 mg). Typically 10-20 mg of sample was placed in a 5 tared mesh stainless steel basket under ambient conditions. The sample was loaded and unloaded at 40 %RH and 25 'C (typical [005961 room conditions). A moisture sorption isotherm was performed. The software uses a least squares minimisation procedure together with a model of the mass 10 [00597] relaxation, to predict an asymptotic value. The measured mass relaxation value must be within 5% of that predicted by the software, before the next %RH value is selected. The sample was recovered after completion of the isotherm and re analysed by XRPD. 15 [005981 The result of the GVS analysis is shown in figure 4. As shown the potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate takes up very little moisture until it reaches approximately 70-80% RH at which point the sample almost certainly deliquesces due to the massive uptake of 28% in weight at 90% RH. The sample does however loose this moisture upon going to lower RH levels. The sample 20 was analysed by XRPD post GVS and the diffractogram was similar to the diffractogram pre GVS. Example 3 25 [005991 The example describes attempts to prepare crystalline sodium, arginine or lysine salts of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid. [006001 Free acid (300mg) was suspended in water (30ml) and leq of base was added. After 5 minutes of stirring, a clear solution was obtained. The solution was 30 frozen and dried via lyophilisation. For the maturation of the amorphous sodium, arginine and lysine salts approximately 10mg of the amorphous salt was treated with 200 l of solvent and then placed in a maturation chamber that cycled between 50'C and ambient with four hours spent under each condition. The experiments were maturated for four days and then any solids produced were analyzed by XRPD. The 142 WO 20111134538 PCT/EP2010/063161 results are shown in Table 8 (sodium salt), Table 9 (arginine salt) and Table 10 (lysine salt). 5 Table 8 Experiment Solvent Observation DJP401-022-1 Ethyl acetate Amorphous DJP401-022-2 Isopropyl acetate Amorphous DJP401-022-3 Ethanol Amorphous DJP401-022-4 Methanol In solution DJP401-022-5 IPA Amorphous DJP401-022-6 1-Butanol Amorphous DJP401-022-7 Acetone Amorphous DJP401-022-8 Butanone Amorphous DJP401-022-9 MIBK Amorphous DJP401-022-10 Toluene Amorphous DJP401-022-11 Dioxane Amorphous DJP401-022-12 THF Amorphous DJP401-022-13 MeCN Amorphous DJP401-022-14 IPA + 5% H20 Amorphous DJP401-022-15 MeCN + 5% H20 Amorphous DJP401-022-16 Ethanol + 5% H20 Amorphous DJP401-022-17 Dioxane + 5% H20 Amorphous DJP401-022-18 Acetone + 5% H20 Gum DJP401-022-19 THF + 5% H20 Oiled out DJP401-022-20 Methanol + 5% H20 In solution Table 9 Experiment Solvent Observation DJP401-023-1 Ethyl acetate Amorphous DJP401-023-2 Isopropyl acetate Amorphous DJP401-023-3 Ethanol Amorphous DJP401-023-4 Methanol In solution 143 WO 20111134538 PCT/EP2010/063161 DJP401-023-5 IPA Oiled out DJP401-023-6 1-Butanol Amorphous DJP401-023-7 Acetone Oiled out DJP401-023-8 Butanone In solution DJP401-023-9 MIBK Amorphous DJP401-023-10 Toluene In solution DJP401-023-11 Dioxane Amorphous DJP401-023-12 THF In solution DJP401-023-13 MeCN Amorphous DJP401-023-14 IPA + 5% H20 Oiled out DJP401-023-15 MeCN + 5% H20 Oiled out DJP401-023-16 Ethanol + 5% H20 Oiled out DJP401-023-17 Dioxane + 5% H20 In solution DJP401-023-18 Acetone + 5% H20 In solution DJP401-023-19 THF + 5% H20 Oiled out DJP401-023-20 Methanol + 5% H20 In solution Table 10 Experiment Solvent Observation DJP401-024-1 Ethyl acetate Amorphous DJP401-024-2 Isopropyl acetate Amorphous DJP401-024-3 Ethanol Amorphous DJP401-024-4 Methanol Solution DJP401-024-5 IPA Amorphous DJP401-024-6 1-Butanol Amorphous DJP401-024-7 Acetone Amorphous DJP401-024-8 Butanone Amorphous DJP401-024-9 MIBK Amorphous DJP401-024-10 Toluene Amorphous DJP401-024-11 Dioxane Amorphous DJP401-024-12 THF Amorphous DJP401-024-13 MeCN Amorphous 144 WO 20111134538 PCT/EP2010/063161 DJP401-024-14 IPA + 5% H20 Amorphous DJP401-024-15 MeCN + 5% H20 Amorphous DJP401-024-16 Ethanol + 5% H20 Amorphous DJP401-024-17 Dioxane + 5% H20 Oiled out DJP401-024-18 Acetone + 5%1 H20 Oiled out DJP401-024-19 THF + 5% H20 Oiled out DJP401-024-20 Methanol+ 5% H20 Oiled out [00601] No crystalline sodium, arginine or lysine was obtained. Example 4 5 [006021 This example describes preparation of a potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate. [006031 (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid may be prepared by 10 any useful method, for example as described in US6,541,506. [006041 300mg of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid were dissolved in IPA [006051 (3ml) and warmed to 50'C before the addition of potassium ethoxide 15 (1066pl of IM solution in EtOH 1.01 equivalents). The reaction was allowed to cool to room temperature and it was then filtered and dried under suction and then placed in a vacuum oven at 25'C / 5mbar for two and a half days. The yield was 298mg (88%). 20 Example 5 Stability of diyne salts [006061 Potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate was kept as a 25 crystalline solid for 6 month at 2-8'C. The content of potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate was determined by HPLC against a reference sample 145 WO 20111134538 PCT/EP2010/063161 kept at -20'C. After 6 months of storage characteristics such as colour, water content, XRPD profile, of the solid had not changed and it was still a beige solid. Furthermore, the content of potassium (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoate had not significantly changed. Also no change in the impurity profile was observed after 6 5 months storage. [006071 Potassium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate was also kept as a crystalline solid for 6 month at 25C, 60%RH. Analysis was performed as described above. After 6 months of storage characteristics of the solid has not 10 changed and it was still a beige solid. Furthermore, the potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate content had not changed significantly, as analysed by HPLC. Also no change in the impurity profile was observed after 6 months storage. 15 [006081 Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate was also kept as a crystalline solid for 6 month at 40C, 75%RH. Analysis was performed as described above. After 6 months of storage the appearance of the solid had changed to a brown solid. Furthermore, a decrease in the content of potassium (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoate to 96% was observed as analysed by HPLC. 20 [00609] Sodium (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13 -diynoate was kept as an amorphous solid at room temperature. After short term storage the colour darkened. Furthermore the salt was dissolved in an aqueous solution and already after few days the content of sodium (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate was reduced 25 by 35%, thus proving strong instability of the amorphous salt form. Example 6 [006101 Biochemical assay for the measurement of OLE-I inhibition by potassium 30 (Z)- I 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoate [006111 Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate dependent inhibition of the OLE-I protein is investigated in a cell free assay. Fungi are grown on oleic acid free medium to enrich for cellular OLE-1 protein. Spheroplasts are 146 WO 20111134538 PCT/EP2010/063161 generated by enzymatic digestion and lysis through polycarbonate filters. Differential centrifugation is used to enrich for endoplasmatic reticulum fractions. [00612] Fractions are supplemented with radiolabelled stearyl-CoA and co-factors 5 (stearyl-CoA is the activated form of stearic acid). OLE-i dependent conversion from stearyl-CoA to oleoyl-CoA is determined photoradiographically from chromatographic separations of stearyl-CoA and Oleoyl-CoA or by tandem mass spectrometry. [006131 Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate dependent 10 inhibition of OLE-1 is determined by the addition of different amounts of potassium (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoate to the reaction mixture. Example 7 Anti-fungal Activity 15 [006141 Potassium (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoate was tested against Aspergillus fumigatus and various Candida species alone to determine antifungal activity and in various combinations with Amphotericin B to determine if any synergistic or antagonistic interactions may occur. Combinations resulting in 20 significantly decreased MICs of the combined compounds are defined as synergistic; those resulting in increased MICs of one or both compounds are antagonistic. [00615] Amphotericin B and potassium (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoate were added to an in vitro culture of different fungi in various concentrations 25 and the culture was visually inspected. If there is inhibition of growth the culture will remain optically clear, whereas growth results in a hazy culture. The results are shown in fig. 5, where 0 means optically clear (i.e. no growth), 1 means slightly hazy, 2 means prominent decrease (at least 50%) in visible growth, 3 means slight reduction in visible growth and 4 means no reduction in visible growth. 30 [00616] Potassium (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate clearly inhibits growth of both Candida glabrata, Candida parapsilosis and Candida albicans at a concentration of 2 pg/ml and even at lower concentrations (see fig. 5). 147 WO 20111134538 PCT/EP2010/063161 [00617] Synergism was observed for potassium (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoate with Amphotericin B on Candida glabrata and on Aspergillus funigatus and to a lesser extent on Candida parapsilosis. Thus, AmphothericinB/pot a s s i u m ( Z )-14-(furan-2-yl)tetradeca-9-en-11,13-diynoate 5 combinations with the concentrations 250/2.5 ng/ml or 125/10 ng/ml clearly inhibit growth of Candida glabrata, whereas the individual compounds at that concentration do not or just slightly. Example 8 10 Minimum Inhibitory Concentration (MIC) Testing [00618] MIC determinations of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-11,13-diynoic acid against fungi were performed according to the NCCLS M27A standard (NCCLS. Reference Method 15 for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. NCCLS document M27-A. NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA. 19087-1898. 1997). A ten-dilution range of each drug was prepared in RPMI-1640 and dispensed into 96-well microtiter plates. 20 [006191 The inoculum size was 0.5 - 2.5 x 103 colony-forming units (CFU)/ml. Incubation time and temperature were 350 C and 24 hrs. [006201 The MIC endpoint was 100% inhibition as compared to the growth control for all strains. The results are shown in Table 11. 25 148 WO 20111134538 PCT/EP2010/063161 Table 11 MICs (100% inhibition) potassium (Z)-14- 14-(furan-2 (furan-2- yl)tetradeca Organism ID yl)tetradeca-9-en- 11,13-diynoic 11,13-diynoate acid C. albicans 2.5 20 ATCC 24433 C. albicans 5 20 ATCC 90028 C. glabrata 60 2560 ATCC 90030 C. krusei 60 1280 ATCC 6258 C. parapsilosis 10240 >10240 ATCC 22019 C. parapsilosis >10240 >10240 ATCC 90018 C. tropicalis 7.5 80 ATCC 750 5 Example 9 [006211 The inhibition of CYP450 enzymes by (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid, potassium salt was tested. Peak areas corresponding to the metabolite of each substrate were recorded. The percent of control activity was then 10 calculated by comparing the peak area obtained in the presence of the diyne potassium salt and in the absence. A range of concentrations of (Z)-14-(furan-2 yl)tetradeca-9-en-1l,13-diynoic acid, potassium salt (1, 3, 10, 30 and 100 tM) were tested and IC 5 o values were determined 15 Table 12. Inhibition of cytochrome P450 enzymes by (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid, potassium salt 149 WO 20111134538 PCT/EP2010/063161 Cytochrome Substrate IC 5 o (tM) % control at 10 P450 tM 1A ethoxyresorufin 59 92 2B6 bupropion 24 82 2C8 paclitaxel > 100 84 2C9 diclofenac 18 74 2C19 omeprazole 32 84 2D6 dextromethorphan > 100 88 3A midazolam 45 85 3A testosterone 57 90 5 Abbreviations AcN, ACN acetonitrile, methyl cyanide n-BuOAc n-butyl acetate s-BuOAc s-butyl acetate DCE Dichloroethane DCM Dichloromethane, methylene chloride DIPE di-isopropylether DMA dimethyl acetamide DMF dimethyl formamide EtOAc ethyl acetate EtOH ethanol, ethyl alcohol
H
2 0 water - distilled or HPLC grade IPA iso-propyl alcohol, propan-2-ol i-PrOAc iso-propyl acetate MEK methyl ethyl ketone, butanone MeOH methanol, methyl alcohol 150 WO 20111134538 PCT/EP2010/063161 MTBE tertiary butyl methyl ether NMP n-methyl pyrrolidone t-BME / TBME t-butyl methyl ether THF Tetrahydrofuran Example 10 5 [00622] This example shows the antifungal activity of various diynes according to the invention against various yeast and dermatophyte strains. The inhibition is partly measured by minimum inhibitory and minimum fungicidal concentration (MIC and MFC, respectively) and partly as IC 5 o (i.e. concentration required for 50% inhibition). 10 [00623] The antifungal activity was compared to the activity of for example Fluconazole (FLU) and Terbinafine (TERB) for yeasts and dermatophytes, respectively (herein also named comparators). a. Test isolates 15 [006241 The fungal isolates tested in this study to evaluate the antifungal activity of (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoic acid, 14-(furan-2-yl)tetradeca- 11,13 diynoic acid, (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt and comparators are clinical isolates collected from patients and deposited at the 20 culture collection of the Center for Medical Mycology. Tested isolates included: Candida albicans, C. glabrata, C. guilliermondii, C. krusei, C. lipolytica, C. lusitaniae, C. parapsilosis, C. tropicalis and Malassezia furfur. Dermatophyte strains included Trichophyton rubruin, T. mentagrophytes, T. tonsurans, T. terrestre, Epidermophytonfloccosum, Microsporumn canis, M. cookie, M. gallinae, M. gypseum, 25 and M. vanbreuseghenii. b. Minimum Inhibitory Concentration (MIC) Testing [006251 MIC determinations of various diyne compounds against yeasts were performed according to the NCCLS M27A standard (NCCLS. Reference Methodfor 151 WO 20111134538 PCT/EP2010/063161 Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. NCCLS document M27-A. NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA. 19087-1898. 1997) with dermatophytes tested in a modified microdilution method. (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid and TERB were dissolved in 5 dimethyl sulfoxide (DMSO), while FLU was dissolved in sterile water. A ten-dilution range of each drug was then prepared in RPMI-1640 and dispensed into 96-well microtiter plates. [006261 For yeast strains, the inoculum size was 0.5 - 2.5 x 103 colony-forming 10 units (CFU)/ml. Incubation time and temperature were 35' C and 24 hrs. (The Malassezia strain required a week's incubation with the addition of olive oil to obtain adequate growth for visual reading). For dermatophytes, the inoculum size, temperature, and time were 1-3 x 10 3 CFU/ml, 350 C and 4 days, respectively. The MIC endpoint was 90% inhibition as compared to the growth control for all strains. 15 c. Minimum Fungicidal Concentration (MFC) Testing [006271 For MFC testing, 20 pl from each visibly clear well from the MIC assay were subcultured onto the center of potato dextrose agar plates and allowed to dry overnight. The plates were then streaked with an inoculating loop to remove the cells 20 from the drug source. The MFC was defined as the lowest concentration to inhibit 100% of fungal growth upon subculture. a. Activity of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid against yeasts 25 [006281 Table 13 lists the MIC and MFC data for Candida and Malassezia strains obtained as described above. As can be seen (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoic acid MIC range for all Candida strains was <0.00006 - 4.0 pg/ml, as compared with 0.125 - 16 pg/ml for FLU. The MIC of (Z)-14-(furan-2-yl)tetradeca 9-en-1l,13-diynoic acid against the Malassezia strain was <0.00006 ug/ml, as 30 compared to 0.125 pg/ml for FLU. Importantly, the (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid MIC against C. krusei, a strain with known innate resistance to FLU, was 0.0005 pg/ml (corresponding FLU MIC = 16 ptg/ ml). The MFC range for (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid against yeasts was <0.00006 32.0 pg/ml. 152 WO 20111134538 PCT/EP2010/063161 b. Activity of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid against dermatophytes [00629] Table 14 is a summary of the activity of (Z)-14-(furan-2-yl)tetradeca-9-en 5 11,13-diynoic acid against dermatophytes. As can be seen, the MIC range against all dermatophyte strains was <0.00006-1.0 pg/ml, as compared to 0.002 - 4 ptg/ml for TERB. (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid demonstrated potent activity against the nine T. rubrum strains with elevated TERB MICs (4.0 pg/ml) tested. The MFC range for (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid 10 against dermatophytes was <0.00006 -16 pg/ ml. [00630] (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid demonstrated potent activity against all fungal isolates tested. The MICs for (Z)-14-(furan-2-yl)tetradeca 9-en-11, 1 3-diynoic acid would be within the susceptible range established for FLU vs. 15 Candida strains (< 8 pig/ml). Importantly (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoic acid showed activity against the FLU-resistant (MIC = 16 pig/ml) C. krusei and also against the T. rubrum strains with elevated TERB MICs (MIC = 4.0 pg/ml). [00631] The data shows (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid 20 possesses potent activity against yeast and dermatophytes. This activity was superior to FLU (against yeast) and TERB (against dermatophytes). The data indicate that (Z) 14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid have utility in the treatment of yeast and dermatophyte infections. 153 WO 20111134538 PCT/EP2010/063161 Table 13. In vitro antifungal activity vs. yeasts (in tg/ml). (Z)- 14-(furan-2-yl)tetradeca-9-en- FLU Organism 11,13-diynoic acid MIC MFC MIC Candida albicans (n = 8) <0.00006 <0.00006 1.0 C. glabrata (n=1) 0.008 0.5 2.0 C. lusitaniae (n=1) <0.00006 <0.00006 0.125 C. lipolytica (n=1) <0.00006 <0.00006 0.125 C. guilliermondii (n=1) <0.00006 0.0005 4.0 C. parapsilosis (n=2) 4.0 32 1.0 C. krusei (n=1) 0.0005 0.001 16 Malassezia furfur (n=1) <0.00006 <0.00006 0.125 5 Table 14. In vitro antifungal activity vs. dermatophytes (in pg/ml). (Z)-14-(furan-2-yl)tetradeca- TERB Organism 9-en-11,13-diynoic acid MIC MFC MIC Trichophyton tonsurans (n=15) <0.00006 <0.00006 0.008 T. mentagrophytes (n=15) <0.00006 0.0001 0.008 Microsporuin canis (n=15) <0.00006 0.0005 0.015 T. rubrum (n=15) <0.00006 <0.00006 0.002 Epiderniophyton floccosum <0.00006 0.015 0.015 (n=15) T. rubrum (TERB-resistant) <0.00006 <0.00006 4.0 (n=9) M. cookie (n=1) 1.0 2.0 0.008 M. vanbreusegheniii (n=1) 0.03 16 0.015 M. gypseum (n=1) <0.00006 16 0.004 154 WO 20111134538 PCT/EP2010/063161 T. terrestre (n=1) <0.00006 16 0.008 M. gallinae (n=1) <0.00006 4.0 0.008 [00632] In contrast to its potent anti-fungal activity, (Z)-14-(furan-2-yl)tetradeca-9 en-11,13-diynoic acid has minimal antibacterial (Table 15) or mammalian cytotoxic activities in vitro (Table 16); in many cases, the drug is 10-20,000-fold more toxic to 5 fungal organisms than to bacteria and mammalian cells. These data suggest a very high therapeutic index for treatment of fungal infections in mammals. 10 Table 15 In vitro Antibacterial Activity MIC9o, gg/ml (Z)- 14-(furan-2 Organism Strain yl)tetradeca-9-en- Standard Agent 11,13-diynoic acid Staphylococcus aureus 29213 50 Gentamicin -0.78 Enterobacterfaecalis 29212 12.5 Ampicillin - 0.78 Escherichia coli 700 >200 Ampicillin - 3.125 15 Table 16 In vitro Mammalian Cell Cytotoxicity of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid
CC
5 o, ptg/ml Vero Cells 46 HFF Cells 75 20 155 WO 2011/134538 PCT/EP2010/063161 [00633] IC 50 against two different S. cerevisiae strains and two differens C. albicans strains by a number of diyne compounds was determined and the results are shown in Table 17. 5 Table 17 S. cerevisiae IC50 C. albicans ICso (pg/ml) (pg/ml) Compound MW ADI-9 2229-5C DSY1024 SC5314 (Z)-12-(furan-2-yl)dodeca-7-en- 256 ~6.25 ~50 1,8 50 9,11-diynoic acid (Z)-13-(furan-2-yl)trideca-8-en- 270 ~50 >200(25%) 0,5 >200 10,12-diynoic acid (E)-14-(furan-2-yl)tetradeca-9- 284 0,4 2,7 0,004 3 en-11,13-diynoic acid (Z)-methyl1 14-(furan-2 298 >0.4(6%) >0.4(4%) 0,19 0,4 yl)tetradeca-9-en-1 1,13 diynoate (Z)-ethyl 14-(furan-2 312 0,7 8,9 - 9 yl)tetradeca-9-en-11,13 diynoate [00634] Table 18 shows the minimal inhibitory concentrations of (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid, potassium salt and (Z)-14-(furan-2-yl)tetradeca 10 9-en-i1,13-diynoic acid versus reference Candida strains. Table 18 156 WO 20111134538 PCT/EP2010/063161 (Z)-14-(furan-2 1 4-(furan-2 yl)tetradeca-9-en Organism ID 11,13-diynoic yletrdcca 1 1,13-diynoic acid, potassium acid salt C. albicans ATCC 24433 2.5 ng/ml 20 ng/ml C. albicans ATCC 90028 5 ng/ml 20 ng/ml C. glabrata 60 ng/ml 2560 ng/ml ATCC 90030 C. krusei ATCC 6258 60 ng/ml 1280 ng/ml 10240 ng/ml >10240 ng/ml ATCC 22019 C. parapsilosis >10240 ng/ml >10240 ng/ml ATCC 90018 C. tropicalis 7.5 ng/ml 80 ng/ml ATCC 750 Example 11 5 In vitro Fun2icidal Activity vs. Candida albicans [00635] The fungicidal activity by (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid was more exhaustively examined with respect to C. albicans. MIC 9 os were determined following a 24 hr incubation period. Aliquots of growth-negative samples 10 were both subcultured by dilution into fresh media and filtered and washed extensively to remove remaining drug. The filters were placed on the surface of nutrient agar plates and incubated for 24 hours. (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid gave greater than 99.9% reduction of the original inoculum 157 WO 2011/134538 PCT/EP2010/063161 (<5cfu/ml) indicating that it had killed the organisms rather than merely retarding growth. Several standard anti-fungal compounds were tested side-by-side with (Z) 14-(furan-2-yl)tetradeca-9-en-I 1,13-diynoic acid and the results are shown in Table 19. 5 Table 19 In vitro fungicidal activity vs. Candida albicans Amphotericin 5 (ng/ml) Diyne* B Cycloheximide Ketoconazole fluorocytosin e 24 h 0.4 200 1,600,000 50,000 98
MIC
9 0 48 h 0.4 800 12,800,000 >800,000 195 Fungicidal 0.4 800 12,800,000 >800,000 1563 Concentration * The diyne used in this example is (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid. 10 [006361 The fungicidal effect of (Z)-14-(firan-2-yl)tetradeca-9-en-11,13-diynoic acid is also rapid. As seen in Figure 6, (Z)-1 4-(furan-2-yl)tetradeca-9-en-1 1,13 diynoic acid produced > 3-log reduction in C. albicans CFUs after 3 hours of incubation. 15 [006371 C. albicans at 5 X 10 3 cfu/ml was incubated with 200 ng/ml (~100-fold MIC) of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid (A) or with various concentrations of (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid within 0.25 to 2-fold the MIC (B). Samples were removed periodically, filtered, washed and plated 20 onto nutrient agar. 158 WO 20111134538 PCT/EP2010/063161 Example 12A 1 Genes encoding the regulation of OLE-i expression are conserved between 5 S. cerevisiae and C albicans [006381 44 S. cerevisiae gene deletion strains with increased sensitivity to (Z)-14 (furan-2-yl)tetradeca-9-en-11,13-diynoic acid, sodium salt have been identified. Analysis of the deleted genes allowed the assembly of a putative pathway critical for 10 (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, sodium salt sensitivity. The pathway regulates the expression of OLE-1, which encodes a A9 fatty acid desaturase that converts stearic acid to oleic acid. [00639] Genes encoding components of the assembled pathway in S. cerevisiae 15 were searched for homologues in C. albicans. For all of the genes, an orthologue was identified in C. albicans (Table 20). MGA2 and SPT23 are duplicate genes in S. cerevisiae but only one orthologue was identified in C. albicans, and OLE-i is present as single copy gene in S. cerevisiae whereas two copies were identified in C. albicans. 20 [006401 This suggests that the pathway identified in S. cerevisiae is conserved in C. albicans. Table 20. S. cerevisiae - C. albicans orthologues S. cerevisiae gene Candida homologue Standard Systematic Standard Systematic Name Name Name Name MGA2 1 YIR033W CaSPT23 orfl9.751 SPT23' YKLO20C UBX2 YML013W orfl9.3135 SSM4 YILO30C orfl9.5175 UBC7 YMR022W orfl9.7329 GETI YGLO20C orfl9.2101 GET2 YER083C orfl9.4839 159 WO 20111134538 PCT/EP2010/063161 GET3 YDL100C orfl9.2965 CaOLE-1 orfl9.5117 OLE-i YGL055W CaOLE2' orf. 19.2264 Example 12B [00641] Biochemical assay for the measurement of OLE-I inhibition by (Z)-14 5 (furan-2-yl)tetradcca-9-en-11,13-diynoic acid, potassium salt and other diyne compounds [00642] (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, potassium salt dependent inhibition of the OLE-i protein is investigated in a cell free assay. Fungi 10 are grown on oleic acid free medium to enrich for cellular OLE-i protein. Spheroplasts are generated by enzymatic digestion and lysis through polycarbonate filters. Differential centrifugation is used to enrich for endoplasmatic reticulum fractions. [00643] Fractions are supplemented with radiolabelled stearyl-CoA and co-factors 15 (stearyl-CoA is the activated form of stearic acid). OLE-i dependent conversion from stearyl-CoA to oleoyl-CoA is determined photoradiographically from chromatographic separations of stearyl-CoA and Oleoyl-CoA or by tandem mass spectrometry. [006441 by_(Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt 20 dependent inhibition of OLE-i is determined by the addition of different amounts of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt to the reaction mixture. 25 Example 13 Effect on the Morphology of Growing Candida albicans Cultures [006451 Cultures of C. albicans were grown in the presence and absence of inhibiting concentrations of (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid and 30 Fluconazole, a standard anti-fungal agent. Periodically, the cultures were examined 160 WO 20111134538 PCT/EP2010/063161 microscopically and photographed and an aliquot was tested for cell viability using the MTT metabolic dye method. Figure 7 shows the morphology of normal growing yeast and yeast growing in the presence of either anti-fungal agent. (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid was fungicidal, totally preventing cell growth. 5 Furthermore, (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid also prevented hyphal tube formation. By contrast, Fluconazole (a fungistatic agent) permitted fungal growth (albeit at a reduced rate) and did not affect hyphal tube formation. Example 14 10 Effect on the Germination of C. albicans [00646] To assess the effect of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid on fungal spore germination, C. albicans spores were incubated with or without (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid for 5 hours, washed and then 15 incubated with a metabolic dye (MTT) for a further 24 hours to assess cell growth and viability. The IC 50 of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid for germination inhibition (Figure 8 A) was essentially identical to its IC 50 for inhibition of vegetative growth (Figure 8 B). In addition, (Z)-14-(furan-2-yl)tetradeca-9-en 11,13-diynoic acid effectively inhibited the subsequent growth of spores that were 20 allowed to germinate in the absence of drug (spores were incubated for 3 hours, a sufficient period to allow germination before the addition of (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid ), (Figure 8 C). Thus, (Z)-14-(furan-2 yl)tetradeca-9-en-11,13-diynoic acid is capable of acting at multiple stages of the fungal growth cycle. 25 Example 15 Effect on Fungal Cell Wall Synthetic Enzymes [006471 The inhibitory activity of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic 30 acid on two enzymes essential for cell wall biosynthesis was determined. Specific inhibitors of each enzyme served as positive controls (Cilofungin, a P-glucan synthase inhibitor and nikkomycin Z, a chitin synthase inhibitor). As shown in Table 21, (Z) 14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid only inhibited these enzymes at 161 WO 20111134538 PCT/EP2010/063161 very high concentrations (>100 ptg/nil) which exceed effective anti-fungal concentrations by 5 orders of magnitude. This indicates that inhibition of p-glucan synthase or chitin synthase is not the primary mechanism of anti-fungal activity of (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid 5 Table 21 Inhibition of fungal cell wall synthesis enzymes Concentration % Inhibition, % Inhibition, CompoundI pg/ml Chitin Synthase (1,3)p-Glucan Synthase 500 97 (Z)-14-(furan- 400 - 90 2-yl)tetradeca 9-en-11,13 diynoic acid 100 85 73 10 16 0 Cilofungin 20.6 - 51 Nikkomycin Z 9.9 61 Example 16 10 Inhibition of Sterol Biosynthesis [006481 Many therapeutically important anti-fungal agents are known to inhibit the biosynthesis of essential membrane sterols, primarily ergosterol. For instance, azole anti-fungal agents, such as Ketoconazole and Fluconazole, do so principally by 15 inhibition of cytochrome P450 14 a-demethylase (P45014DM). Terbinafine and other allylamines inhibit ergosterol biosynthesis at the penultimate step of lanosterol biosynthesis, squalene epoxidase. A stable-isotopic method was developed to probe the metabolic effects of anti-fungal agents in yeast. Exponentially growing cultures of Candida albicans were incubated with "C-labeled acetate and the resulting labeled 20 sterols evaluated by API-LC-MS. The ratio of labeled to unlabeled sterol (e.g., ergosterol) was a quantitative indication of the net biosynthesis of the sterol from 162 WO 20111134538 PCT/EP2010/063161 acetate. Representative results are shown in Figure 9. After the first 15 minutes, drugs were added to the incubation medium at concentrations approximating their respective MICs; the control contained only DMSO vehicle. As expected, both Terbinafine and Fluconazole inhibited the incorporation of acetate into ergosterol. 5 However, (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid did not significantly inhibit the net biosynthesis of ergosterol (i.e. the level of "3C incorporation into ergosterol in the presence of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid was nearly identical to the control level). 10 Example 17 [006491 The mechanism of action of the sodium salt of (Z)-14-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid, V", was investigated in a chemical-genetic screen in S. cerevisiae. The screen made use of the S. cerevisiae knock-out collection consisting of 4917 individual strains each deleted for one defined gene. All 4917 15 strains were tested for increased or decreased susceptibility to compound V". From 4917 analyzed strains, 44 had an increased susceptibility to compound V". No strain with a decreased susceptibility was identified. The S. cerevisiae Gene Ontology Slim vocabulary was used to map the identified genes to higher level biological processes. From a total of 39 biological processes available, the 44 genes identified mapped to 20 29 processes, from which "lipid metabolic process" (p<O. 004) and "organelle organization and biogenesis" (p<0.0 4 ) were significantly overrepresented. [00650] Among the genes identified was MGA2, which encodes a transcriptional activator of the OLE1 A9-fatty acid desaturase gene. MGA2 is a duplicated gene in 25 S. cerevisiae with SPT23 being its homolog. The Ole1 protein converts stearic acid to oleic acid and is an essential gene in S. cerevisiae. Another six genes from the identified set of 44 genes could be assembled together with MGA2 to a pathway likely to regulate OLE1 transcriptional activation. This pathway included components of the ERAD (endoplasmatic reticulum associated protein degradation) complex 30 required for the proteolytic activation of the Mga2 protein and genes coding for GET complex components, which is putatively required to insert Mga2 into the membrane of the endoplasmatic reticulum. Figure 15 illustrates the regulation of OLEI transcriptional activation. Components of the GET complex putatively mediate the insertion of the Mga2/Spt23 proteins into the ER membrane. The ERAD complex 163 WO 20111134538 PCT/EP2010/063161 proteolytically activates Mga2/Spt23, which shuttles to the nucleus where it activates OLE1 transcription). These data indicate that compound V", interferes with the regulatory pathway for OLE1 gene expression, or the Olel protein itself 5 Example 18 [00651] The Ole1 protein is a A9-fatty acid desaturase, which converts stearic acid to oleic acid. Deletion of the OLE1 gene induces an oleic acid auxotrophy, which is lethal to the fungal cell. In S. cerevisiae, oleic acid (but not stearic acid) had an antagonistic effect on the activity of compound V", demonstrating that compound V" 10 inhibits the conversion from stearic acid to oleic acid. See figure 16a which shows that when Saccharomyces cerevisiae was cultured on nutrient agar supplemented with increasing amounts of compound V", addition of oleic acid decreased the susceptibility of S. cerevisiae to compound V", whereas stearic acid did not, indicating that oleic acid is an antagonist of compound V", but not stearic acid. 15 Similarly in C. albicans, oleic acid (but not stearic acid) had an antagonistic effect on compound IV". Figure 16 b. and c. show that when Candida albicans was cultured in nutrient broth and supplemented with combinations of compound IV" and b) oleic acid, sodium salt or c) stearic acid, sodium salt, oleic acid, sodium salt, had an antagonistic effect on compound IV", whereas stearic acid, sodium salt, did not, 20 demonstrating that oleic acid, sodium salt, is an antagonist of compound IV", but not stearic acid, sodium salt. Example 19 [006521 Compound V" was thus seen to inhibit the biosynthesis of oleic acid, via a 25 mechanism that is either through direct inhibition of the A9 fatty acid desaturase Ole1 protein, or through inhibition of Mga2 protein dependent transcriptional activation of OLE1 gene expression, thereby inducing an oleic acid auxotrophy to the cell. To distinguish between these two hypotheses, OLEI transcriptional levels were determined. Assuming that compound IV" directly inhibited the Ole 1 protein, a 30 compensating up-regulation of the OLE1 transcriptional level for compound IV" treated cells would be expected. If compound IV" inhibited the Mga2 protein or any other component required for OLE 1 transcriptional activation such as the GET complex or the CDC48 proteasome, a down-regulation of OLE1 for compound IV" treated cells would have been expected. Compound IV" independent transcription of 164 WO 20111134538 PCT/EP2010/063161 the OLE1 gene would indicate another mechanism of action and suggested that the antagonistic effect of oleic acid with compound IV" was indirect. [00653] When S. cerevisiae was cultured in the presence of 1 and 10 jig/mIl 5 compound IV", the doubling times increased to 2.4 h and 4.2 h, respectively, compared to 2 h for the untreated control. The addition of 10 jig/ml oleic acid, sodium salt, to 1 [Lg/ml compound IV" reversed the doubling time to 2h as observed for the untreated control or 10 [tg/ml oleic acid, sodium salt alone. 10 [00654] Doubling times found are shown in Table 22. Table 22. Doubling times of S. cerevisiae in the presence of compound IV" and/or oleic acid condition compound IV" oleic acid doubling time in h a 2.0 b 1 pLg/ml 2.4 c 10 jig/ml 4.2 d - 10 pig/ml 2.0 e 1 Vg/ml 10 ptg/ml 2.1 15 [00655] The expression of the OLE1 gene was determined by real time PCR 10, 60, 120 and 240 min for the conditions a.-e., as indicated in Table 3 above. OLE1 transcript levels were calibrated to the expression of the tubulin gene (TUB 1) and normalized to the OLE 1 expression at 10 min without compound addition. See figure 20 17a showing time dependent expressions of OLE 1 in response to compound IV". Conditions a.-e. are listed in Table 3 above, numbers indicate time intervals in minutes. Expressions of OLE1 are given as multiples of the expression at 10 min, condition a. Expression levels of OLE1 in cultures without compound IV" did not vary significantly over the time scale investigated. The addition of 1 pg/ml compound 25 IV" increased the expression level of OLE1 about 3-5 fold (4.9 fold at 120 min, p<0.023) and 10 pig/ml increased the expression level about 7-19-fold (18.7 fold at 120 min, p<0.0 18). 10 pig/ml oleic acid compensated for the OLE1 expression 165 WO 20111134538 PCT/EP2010/063161 increase caused by 1 pg/ml compound IV alone. Compound IV" dependent up regulation of OLE1 expression thus confirms that compound IV" is acting as an inhibitor of the Olel protein. The antagonistic effect of oleic acid found in the susceptibility assays, is reflected mechanistically in this experiment by a down 5 regulation of the target Olel protein through oleic acid. [006561 Similarly, the expression of OLE1 in C. albicans was investigated in response to compound IV". In the presence of 250 ng/ml Compound IV, OLE1 transcript levels were increased 4-fold after 120 min incubation time compared to the 10 drug free control. Figure 17b shows time and concentration dependent expressions of OLE1 in response to compound IV" in C. albicans. a: compound free control; b: 250 ng/ml compound IV"; c: 5000 ng/ml compound IV"; time intervals in minutes. Expression values are normalised to TUBI and given as multiples of OLEI expression at 10 minutes in ethanol. 15 Example 20 [006571 Compounds IX" and X" of the invention were screened against the following 15 agricultural pathogens: Ascomycota Botrytis cinerea; Magnaporthe grisea anamorph: Pyricularia oryzae; Colletotrichum gleoesporioides- Chilli strain; 20 Colletotrichum gleoesporioides- mango strain; Fusarium verticillioides; Fusarium oxysporum; Alternaria solani; Uncinula necator Syn Ervsiphe necator; Macrophomina phaseolinaSyn. Sclerotium bataticola and Rizoctonia bataticola; Botrvodiplodia theobromae; Basidiomycota Sclerotium rolfsii; Rhizoctonia solani; Puccinia arachidis; Oomycota Pythium aphanidermatum; Plasmopara viticola Syn. 25 Personopora viticola, selection being based on i) yield losses and disease severity caused on crops and other plants (for example, ornamental and amenity grasses); ii) host infected by fungi; iii) difficulty in providing control measures with existing fungicides; representation across major classes of pathogenic fungi; and representation across major groups of fungal diseases viz., rust, rot (root and fruit), 30 leaf spots, mildews and wilts. Agriculture Assays: [006581 In vitro mycelial growth inhibition assays - poisoned plate: 166 WO 20111134538 PCT/EP2010/063161 [00659] Growth of fungi was carried out in potato dextrose agar media at 40-45'C, and test compound was added at different concentrations and at pH 5.8 for compound X" and at pH 7.0 for its potassium salt, compound X11", with a water control instead of test compound. Radial mycelial growth was measured at regular intervals 4, 8, 12 5 and 16 days for slow growing fungi and at 3, 6 and 8 days for fast growing fungi. When the mycelia reached the end of the plate, measurements were stopped. Morphological changes in the hyphal growth and sporulation patterns were also observed. For dose response studies a range of concentrations and a range of inhibition obtained falling below and above 50% inhibition (see Table 23 below) 10 [006601 Spore germination studies for plant fungal pathogens- hanging drop method: [006611 Spore/conidial suspension of 5-10x10 3 spores per ml (for larger spores) and 5-10 x 105 for smaller spores, was tested against different concentrations of the 15 potassium salt of compound X". Spore germination was carried out in sterile distilled water, under a moist chamber, in cavity slides. After over night incubation the spores were observed. Where applicable, the solvent dimethyl sulfoxide was added to both test and control. Spore suspensions were prepared from good sporulating field isolates and grown in appropriate media, generally potato dextrose agar, or for Magnaporthe 20 grisea oatmeal agar. For specific sporulating structure, spore suspensions were prepared devoid of mycelial bits. Spore number was adjusted using haemocytometer. Final spore concentration of spores was 5-10 X 10 3 spores per ml. Photographic recordings were made of perfect/good germination; recording of any malformation such as disintegration, shrinking of germ tubes or spores. Inhibition was calculated by 25 comparison with the control germination (inhibition = [(% of spore germination in control with DMSO - % of spore germination in treated with compound)/( [(% of spore germination in control with DMSO)]. Leaf disk assay: 30 [00662] Leaf disk assay was carried out by the cavity well plate method for powdery mildew disease of grapes. Leaf disks of 14 mm in diameter were cut with a cork borer from the healthy leaves (second and third from the tip) of grapevine plants, and were dipped in 100pl of each test compound at different concentrations for two minutes, as in treatment details shown below. The control leaf disks were dipped only 167 WO 20111134538 PCT/EP2010/063161 in sterile water for two minutes. The compound treated leaf disks were placed, abaxial side up, in TC-24 well plates containing water agar medium. The disks were inoculated by placing 2 0pl of inoculum (1-5 x 10 6 spores/ml) on the centre of the disk. After inoculation, the cavity well plates were incubated at 20'C for 10 days. After 5 incubation, the powdery mildew lesions on the leaf disks were rated to 0-9 scale, in which, 0 was no visible symptoms and 9 represented more than 50% leaf area with mildew growth/lesion. Percent disease index (PDI) was calculated as follows: a) PDI = Sum of individual ratings x 100 * Total no. of leaf disk maximum disease 10 grade e observed [00663] After the observation, the conidia were washed from the leaf disks in known volume of a fixative solution of ethanol-formaldehyde-acetic acid (90:5:5, v/v/v) and counted with a hemocytometer. 15 Table 23. Mycelial growth inhibition Compound XII", potassium salt S. Quantity %0 MyeClial growth No. Pathogen pg/mi nhibition 1 Botrytis cinerea 2.5 76 2 Magnaporthe grisea 5 27 Colletotrichum gloeosporioides 3 (mango strain) 5 28 Colletotrichun gloeosporioides 4 (chili strain) 5 10 5 Alternaria solani 50 42 Compound X" Serial % Mycelial No Pathogen Quantity IgmI Growth Inhibition 6 Rhizoctonia solani 50 19 7 Botrvodiplodia theobromae 50 15 168 WO 20111134538 PCT/EP2010/063161 8 Sclerotium roifsii 50 5 9 Macrophonina phaseolina 100 15 10 Pythium aphanidernatum 50 No inhibition 11 Fusariun verticillioides 50 14 Disc diffusion 12 Fusariun oxysporuin No inhibition assay Compound XII", potassium salt S. Quantity Reduction in 00 disease No. Pathogen g/ml index /germination 13 Plasmnopara viticola 01 93 (spore germiantion) Uncinula necator 10 65 14 (leaf disc assay) 20 100 Puccinia arachidis 15 (leaf disc assay) 10 67 20 75 [00664] Changes in the hyphal tips of Botrytis cinerea using potassium salt of 14 (furan-2-yl) tetradeca-1 1,13-diynoic acid, XII", were investigated to measure the 5 effect of the Olel inhibitor upon growth and thriving. The compound was found to be toxic to the fungus Botrytis cinerea as described in the following. Apical dominance is an important criterion for growth of hyphae of fungi: at the apical tip of the mycelia branching is not seen near the growing tip. Apical dominance is maintained but branching of hyphae will start at sub-apical point, a distance away from the growth 10 point. Under abnormal conditions of stress, apical dominance is lost, extensive branching begins, resulting in the growth of the fungal mycelia being arrested. When the test compound is incorporated in the media in which the fungi is present, hyphal tip splitting and branching is seen with loss of apical dominance and polarity. Figure 18 shows (in the two lower pictures) the changes in the plane of hyphal growth and 15 abnormal thickening of the hyphae when the mycelia are inoculated in the plates with 169 WO 20111134538 PCT/EP2010/063161 potassium salt of 14-(furan-2-yl) tetradeca- 11, 13-diynoic acid (XII"), 2 tg/ml). Hyphae from the inoculated disc start growing against gravity and are thicker than the normal hyphae seen in the control (the two upper pictures). Abnormal thickening of the hyphae shows the stress created by the presence of the Ole 1 inhibitor. 5 Example 21 Synthesis of (Z)-14-(furan-2-yl) tetradeca-9-en-1 1, 13-diynoic acid (III") [006651 (Z)-14-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid was prepared in a required isomeric ratio of at least 95:5 cis:trans, as determined by HPLC and NMR. 10 Additional purification steps were performed in order to increase the purity of the coupling fragments. The route provided involves a convergent synthesis of intermediate 8 from the two fragments 2 and 7. The final product is synthesized by hydrolysis of the methyl ester to provide (Z)-14-(furan-2-yl) tetradeca-9-en-11, 13 diynoic acid 9 (compound III") 15 170 WO 20111134538 PCT/EP2010/063161 [00666] Fragment synthesis: 0 Br Br Br 1 2 (i) PPh 3 , CBr 4 , TEA, DCM; (ii) NaHMDS, Et 2 O, -78C TMS MeO 2 C MeO 2 C MeOC MeOC 72 2 77 4 5 6 7 (iii) NaHMDS, HMPA, CH2I+PPh 3 1-; (iv) Pd(PPh 3
)
4 , Cul, TMS acytlene; (v) TBAF, THF, OC 5 [006671 CoLpling Steps: (vi) 0 0
-
Br MeO 2 C ( MeO 2 C 7 2 7 8 (vii) 0 HO 2 C 7 9, compound III" (vi) CuCI, NH 2 OH.HCI, EtNH 2 , H 2 0, MeOH, 2; (vii) LiOH.H 2 0, THF, H 2 0 171 WO 20111134538 PCT/EP2010/063161 [006681 Preparation of fragment 2: 0(i 0 0 Br Br Br 1 2 (i) PPh 3 , CBr 4 , TEA, DCM; (ii) NaHMDS, Et 2 O, -78C [006691 Synthesis of fragment 2 began from commercially available furylfuran, 5 which underwent an in situ Wittig reaction, with carbon tetrabromide, to afford compound 1 in 66% yield after column chromatography. [006701 Schema for fragment 7: TMS MeO 2 C MeO 2 C ( MMeOC C 1MeO2C 2 : 7 2 4 5 6 7 (iii) NaHMDS, HMPA, CH21PPh 3 l-; (iv) Pd(PPh 3
)
4 , Cul, TMS acytlene; (v) TBAF, THF, OC 10 [006711 Synthesis of intermediate 5 is based on a Wittig olefination and the required salt was prepared from diiodomethane and triphenyl phosphine in 41% yield. The Wittig reaction of 9-oxononanoic acid methyl ester was initially carried out on a 10g scale to afford the desired product in 88% yield. The column chromatography 15 separated a close running impurity, which has been tentatively identified as triphenylphosphine, which presumably arises from decomposition of the excess phosphonium salt. The 1 H NMR of the final product also showed the presence of a second impurity (based on a triplet at 6.6 ppm in the 1 H NMR). The cis:trans ratio of the alkene was determined by 1 H NMR, and shown to be 98:2. 20 172 WO 20111134538 PCT/EP2010/063161 [00672] Schema for (Z)-14-(furan-2-yl) tetradeca-9-en-11, 13-diynoic acid (I1"): +(vi) 0 +~ \Z SBr MeO 2 C 7 MeO 2C 7 2 7 8 (vii) 0
HO
2 C 7 9 (vi) CuCI, NH2OH.HCI, EtNH 2 , H20, MeOH, 2; (vii) LiOH.H 2 0, THF, H 2 0 [00673] The final steps towards (Z)-14-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid involved the coupling of the two key building blocks 2 and 7, followed by the 5 hydrolysis of the ester 8 to yield the final compound. Compound 8 was prepared via a Cadiot-Chodkiewicz coupling of an ether solution of furan 2 to acetylene 7 under standard conditions (CuCl, HONH 2 .HCl, EtNH 2 , MeOH). The reaction was carried out on a test scale of 0.5g of compound 7 and gave a recovery of 0.27g in 37% yield. The final scale up of the coupling reaction was undertaken in two discrete batches and 10 the results are summarized below: [006741 Synthesis of compound 8": Experiment Scale, 7 (g) Product (g) Yield (%) HPLC Purity (%) CM/291/18 0.51 0.27 37 92 CM/291/27 lOg 8.28 58 92 CM/291/29 lOg 8.24 58 91 [00675] The scale up of the coupling reaction proceeded well, presumably due to 15 the high purity of fragment 7. Column chromatography separated the product from several unidentified impurities. Hydrolysis of ester 8 was carried out in a 6:1:1 mixture of THF:water:MeOH with lithium hydroxide monohydrate at room temperature for five hours. The reaction mixture was acidified to pH 2 with 2N HCl 173 WO 20111134538 PCT/EP2010/063161 and the partially precipitated solid was extracted with ethyl acetate. The reaction was initially carried out on an 0.4 g scale to afford the final product in high purity after dry flash column chromatography followed by precipitation from cold heptanes. The two batches from the coupling reactions were hydrolyzed separately to afford 13.7g of 5 (Z)-14-(furan-2-yl) tetradeca-9-en-1 1, 13-diynoic acid after purification: Experiment Scale (g) Product (g) Yield (%) HPLC Purity (%) CM/291/15 0.38 0.3 83 92 CM/291/31 8.28 6.5 82 87 CM/291/32 8.24 7.2 91 89 [00676] HPLC analysis of the crude product showed the presence of an impurity, arising from the coupling step, and although the levels were reduced after purification, 10 they were still high. The two batches were therefore combined and slurried in the minimal amount of heptanes at room temperature for one hour. Cooling to 0 0 C before filtration and washing with cold heptanes afforded 10.5g of (Z)-1 4-(furan-2-yl) tetradeca-9-en- 11, 13-diynoic acid with an HPLC purity of 96% by area. Experimental 15 2-(2,2-Dibromovinyl)Furan (1): [006771 A solution of carbon tetrabromide (241.6g, 0.73 mol) in anhydrous DCM (1000 mL) was cooled to -20'C under nitrogen and triphenylphosphine (19 1.1 g, 0.73 mol) in anhydrous DCM (1000 mL) was added drop wise. After twenty minutes of stirring, the reaction was cooled to -60'C, and then a mixture of furfural (30 mL, 0.36 20 mol) and triethylamine (50.5 mL, 0.36 mol) in anhydrous DCM (375 mL) were added drop wise. The mixture was brought to room temperature and diethyl ether (500 mL) was added with stirring. The reaction was filtered and filtrate was concentrated in vacuo. Column chromatography (SiO 2 , heptanes) yielded the title compound (58.1 g, 63%) as a brown oil: 25 [006781 H-NMR (400 MHz, CDCl 3 ) 6 6.46 (s, 1H), 6.94 (s, 1H), 7.40 (s, 1H), 7.43 (s, 2H). 2-Bromoethynylfuran (2): 174 WO 20111134538 PCT/EP2010/063161 [00679] Sodium Hexamethyldisilazane (NaHMDS, 111 mL, 0.22 mol, 2M in THF) was added drop wise to a solution of compound 1 (56 g, 0.22 mol) in anhydrous diethyl ether (1120 mL) at -78'C and the resulting solution stirred for fifteen minutes. Aqueous ammonium chloride (500 mL, sat.) was added at -78C and the reaction 5 allowed to warm to room temperature. More aqueous ammonium chloride (200 mL, sat.) was added along with diethyl ether (200 mL). The layers were separated and the aqueous phase was extracted with diethyl ether (2 x 200 mL). The combined organic layers were washed with brine (500 ml) before being dried (MgSO 4 ) and filtered. The solvent was removed by atmospheric distillation to afford the title compound (20.8 g, 10 55%) in a condensed solution. lodomethylenetriphenyphosphonium iodide: [006801 Diiodomethane (150 mL, 1.86 mmol) and triphenylphosphine (425 g, 1.62 mmol) was dissolved in toluene (500 mL) and the resulting solution heated to 50'C 15 for eighteen hours. The solution was allowed to cool to room temperature before being filtered. The precipitate was washed with toluene (2 x 500 mL) before being dried under vacuo to yield the title compound (404 g, 41% yield) as a white solid: [006811 'H NMR (400 MHz, CDCl3) 6 5.05 (m, 2H), 7.79-7.87 (m, 15H). 20 10-lododec-9(Z)-enioc acid methyl ester (5): [00682] NaHMDS (285 mL, 0.57 mol, 2M in THF) was added drop wise to a suspension of CH 2 IPPPh 3 I- (299 g, 0.56 mol) in anhydrous THF (1.5 L) at room temperature. After stirring for five minutes the solution was cooled to -78'C and HMPA (139 mL, 0.77 mol) was added drop wise. 9-oxononanoic acid methyl ester 4 25 (75.0 g, 0.40 mol) was dissolved in anhydrous THF (375 mL) and added drop wise at -78'C. The resulting solution was allowed to warm to room temperature and stirred for sixteen hours. Ethyl acetate (1000 niL) and water (500 mL) were added and the layers separated. The aqueous phase was extracted with ethyl acetate (2 x 500 mL). The combined organic layers were washed with water (2 x 500 mL) and brine (500 30 mL) before being dried (MgSO 4 ), filtered and concentrated in vacuo. The resulting brown oil was dry loaded onto silica (~1 volume) and purified by column chromatography (SiO 2 , 10% diethyl ether in heptanes) to afford the title compound (64.8 g, 52%) as a yellow oil. The mixed fractions were combined and concentrated 175 WO 20111134538 PCT/EP2010/063161 before being purified by column chromatography (SiO 2 , 10% DCM in heptanes) to afford compound 5 (18.
4 g, 67% combined yield): [006831 'H NMR (400 MHz, Benzene-d6) 6 0.97-1.15 (in, 10H), 1.46-1.49 (m, 2H), 1.93-2.10 (in, 2H), 3.39 (s, 3H), 5.74 (q, 1H, J= 7.0, 13.9 Hz), 5.92 (dt, 1H, J= 5 7.3, 1.1 Hz). 12-Trimethylsilanyldodec-9(Z)-en-1 1-ynoic acid methyl ester (6): [00684] 10-Iododec-9-enioc acid methyl ester 5 (64.8 g, 0.21 mmol) and trimethylsilylacetylene (35.4 mL, 0.25 mol) was dissolved in anhydrous DMF (285 10 ml) and cooled to 0 0 C. Triethylamine (34.75 mL, 0.25 mol), copper (I) iodide (10.3 g, 0.05 mol) and tetrakis(triphenylphosphine)palladium (14.5 g, 0.01 mol) were added. The resulting solution was stirred at 0 0 C for eighteen hours before being allowed to warm to room temperature. Water (200 mL) was added and the mixture was extracted with diethyl ether (2 x 500 mL). The combined organic layers were 15 washed with brine (500 ml) before being dried (MgSO 4 ), filtered and concentrated in vacuo. The dark brown residue was dissolved in heptanes (500 mL) before being filtered and concentrated in vacuo. The residue was passed through a silica pad (SiO 2 , 5% ethyl acetate in heptanes) to afford the title compound (45.0 g, 77%) as a yellow oil: 20 [006851 1H NMR (400 MHz, CDCl3) 6 0.20 (s, 9H), 1.50-1.20 (m, 8H), 1.70-1.60 (m, 2H), 2.40-2.20 (m, 4H), 3.65 (s, 3H), 5.9-5.7 (m, 1H), 5.5-5.4 (m, IH). 9(Z)-Dodecen-1 1-ynoic acid methyl ester (7): [006861 Tetrabutylammonium fluoride, TBAF (177 mL, 0.18 mol, IM in THF) 25 was added dropwise to a solution of 12-trimethylsilanyldodec-9-en-1 1-ynoic acid methyl ester (6) (45.0 g, 0.16 mol) in anhydrous THF (950 mL) at 0 0 C. The resulting solution was stirred for thirty minutes before being allowed to warm to room temperature. The THF was removed in vacuo and the resulting dark oil was passed through a silica pad (SiO 2 , 10% ethyl acetate in heptanes) to afford the title compound 30 (20.0 g, 60%) as a yellow oil: [006871 'H NMR (400 MHz, CDCl3) 6 1.31-1.40 (m, 10H), 1.58-1.61 (in, 2H), 2.29-2.32 (m, 2H), 3.06 (s, 1H), 3.66 (s, 3H), 3.44 (m, IH), 5.99 (m, IH). 176 WO 20111134538 PCT/EP2010/063161 Methyl (9Z)- 14-(2-furyl)tetradeca- 11,13-diyneoate (8): [00688] Copper (I) chloride (0.52 g, 5.27 mmol), hydroxylamine hydrochloride (1.67 g, 24.00 mmol) and ethylamine (144 mL, 1.78 mol, 70% in H 2 0) were dissolved in methanol (120 mL) and cooled to 0 0 C. Dodec-9-en-1 1-ynoic acid methyl ester 7 5 (10 g, 0.048 mmol) in methanol (85 mL) was added drop wise to the above solution. 2-Bromoethynylfuran 2 (66 g, 0.06 mol, 16% THF/diethyl ether solution) was added drop wise to the above solution. The resulting solution was stirred at OC for two hours before water (150 mL) and diethyl ether (150 mL) were added and the layers separated. The aqueous phase was extracted with diethyl ether (3 x 1 OOmL) and 10 combined organic layers were washed with brine (250 mL) before being dried (MgSO 4 ), filtered and concentrated in vacuo. Column chromatography (SiO 2 , 2-5% ethyl acetate in heptanes) afforded the title compound (8.28 g, 58% yield) as a yellow oil: [006891 'H NMR (400 MHz, CDCl3) 6 1.50-1.20 (m, 8H), 1.70-1.60 (m, 2H), 15 2.42-2.30 (m, 4H), 3.60 (s, 3H), 5.59 (d, 1H, .J= 10.5 Hz), 6.19-6.14 (m, 1H), 6.48 6.38 (m, 1H), 6.70 (d, 2H,.1 = 3.0 Hz), 7.45-7.40 (m, 1H). (9Z)-14-(2-furyl)tetradeca- 11,1 3-diynoic acid (9)(11): [00690] Lithium hydroxide monohydrate (3.49 g, 0.083 mol) was added to a 20 solution of methyl ester 8 (8.28g, 0.028 mmol) in THF:water:methanol (160 mL, 6:1:1) at 0 0 C. The solution was allowed to warm to room temperature and stirred for eighteen hours before water (100 mL) was added. The pH of the solution was adjusted to pH2 with 2N aqueous hydrochloric acid (-45 mL). Ethyl acetate (100 mL) was added and the layers separated. The aqueous phase was extracted with ethyl acetate (2 25 x 50 mL) and the combined organic layers were washed with brine (100 mL) before being dried (MgSO 4 ), filtered and concentrated in vacuo. The residue was passed through a silica pad (SiO 2 , 10% ethyl acetate in heptanes) before being dissolved in heptanes (~15 volumes) and left in the freezer overnight. The yellow precipitate was filtered and washed with cold heptanes before being dried to afford the title compound 30 (7.5g, 910%) as a pale yellow solid: [006911 'H NMR (400 MHz, CDCl3) 6 1.5-1.2 (m, 8H), 1.7-1.6 (m, 2H), 2.4-2.3 (m, 4H), 5.59 (d, 1H, J= 10.5 Hz), 6.19-6.14 (m, IH), 6.48-6.38 (m, IH), 6.7 (d, 2H, J= 3.0 Hz), 7.45-7.4 (m, IH). 177 WO 20111134538 PCT/EP2010/063161 [00692] m.p. 49.8-52.3C; [006931 Synthesis of potassium (IV") and sodium (V") salts of (9Z)-14-(2 furyl)tetradeca-11, 13-diynoic acid (9)(III"): 5 [00694] Compound III" (25 mg) was dissolved in 250 pl of solvent (dioxane, acetone or ethanol) and warmed to 50'C. A solution of potassium or sodium ethoxide (IM in ethanol, 1.1 equivalents) was added and the reactions were then allowed to cool to room temperature. Salts (IV" and V" respectively) precipitated out and were filtered off and dried. 10 Example 22 [00695] Synthesis of 8-(2-(4-(furan-2-yl) buta-1,3-diynyl) phenyl) octanoic acid (VII") Scheme 1 BrTPP
CO
2 Et n-BuLi
CO
2 Et Pt2 H2 atm 1 CHO /Br 2 rt, 6 h, 95% Br 0 OC, 16 h 48%
CO
2 Et \ CO 2 Et THF, TBAF Br 3 Pd(PPh) 4 , CUil 4 rt, 3 h, 86% DMF, DIPA, TPP Si 120 oC, 0.5 h, 92% COOEt 6 Br, 2N KOH, EtOH Et 2 NH, EtOH | H 2 0, reflux, 3 h EtOOCI H 5 CuCI, NH2OH.HCI1 20% 7 HOOC Target 15 178 WO 20111134538 PCT/EP2010/063161 179 WO 2011/134538 PCT/EP2010/063161 Example 23 Synthesis of 14-(2-furyl)tetradeca- 1,13-diynoic acid (X") [00696] Laboratory scale synthesis of approximately 1Og of 14-(2-firyl)tetradeca 5 11,13-diynoic acid gave good overall yield of high purity from relatively cheap and readily available starting materials. The route involved the synthesis of two building blocks followed by the convergent synthesis of the target molecule. [006971 Fragment synthesis: Br-.--COOH a I COOH COOH CO 2 Me 2 3 4 (iv ) B r C 2
CO
2 Me 5 (i) Nal, acetone (ii) Li acetylide-EDA, HMPA (iii) MeOH, H 2 So 4 (iv) NBS, acetone (V) - 1 (vi) t s (vii) 0tins 0 6 7 8 (v) nBuLi, Et 2 0 (vi) TMS acetylene, Pd(CI) 2 (PPh 3
)
2 , Cul, TEA (iii) K 2 CO3 10 [00698] Coupling Steps: Br C 2Me + (viii) 5CO2Me 58 9 9 I (ix) 0 \ CO 2 H Compound X" (vii) Cul, pyrrolidine (ix) LiOH.H 2 0, THF/MeOH/H 2 0 180 WO 20111134538 PCT/EP2010/063161 [00699] Schema for decanoic acid fragment (5): Br COOH I COOH COOH CO 2 Me 2 3 4 (iv) BrC 2Me 9 5 (i) Nal, acetone (ii) Li acetylide-EDA, HMPA (iii) MeOH, H2SO4 (iv) NBS, acetone 5 [00700] Schema for 2-ethynylfuran building block (8): (V) (vi) ms (vii) 0 0 I o z tins 0 6 7 8 (v) nBuLi, Et 2 O (vi) TMS acetylene, Pd(CI) 2 (PPh 3
)
2 , Cul, TEA (iii) K 2
CO
3 [007011 Schema for 14-(2-furyl)tetradeca- 11,13-diynoic acid: 10 Br CO 2 Me + (viii) O 5 Q0 Zz 02
CO
2 Me 8 9 9 (ix) Z:Zzz ' CO 2 H Compound X" (vii) Cul, pyrrolidine (ix) LiOH.H 2 0, THF/MeOH/H 2 0 Experimental 10-Iododecanoic acid (2): 15 [007021 To a mechanically stirred solution of 10-bromodecanoic acid (50g, 0.2mol) in IL of acetone under nitrogen was added sodium iodide (238.7g, 1.59mol) and the resulting heterogeneous reaction mixture was allowed to stir at room temperature for 18h. The thick reaction mixture was filtered through a pad of Celite 521, concentrated in vacuo to one-fifth volume and diluted with IL of brine. This was 181 WO 20111134538 PCT/EP2010/063161 extracted with 4 X 250 mL of hexanes. The combined organic extracts were washed with 2 X 250mL of freshly prepared 10% aqueous sodium thiosulfate, dried over sodium sulfate, concentrated in vacuo and vacuum dried to give iodo acid 2 (57.5g, 98%) as a white solid: 5 [00703] 'H NMR (400 MHz, CDCl3): 1.23-1.41 (m, 1OH), 1.60-1.67 (in, 2H), 1.80-1.85 (in, 2H), 2.34 (t, J= 7.3Hz, 2H), 3.17 (t, J=7.32Hz, 2H), 11.45 (br s, OH). I1-Dodecynoic acid (3) (see De Jarlais, et al., Synth. Comm. 1980, 10, 653): [007041 A suspension of lithium acetylide-ethylenediamine complex (90% w/w, 10 58.7g, 573mmol) under nitrogen in 330mL of HMPA was mechanically stirred at room temperature for 45min and then cooled to -5 degrees C. A solution of iodo acid 2 (57.00g, 191mmol) in 130mL of HMPA was then added via pressure equalized addition funnel to the cooled acetylide suspension with stirring at such a rate as to maintain the internal temperature between 0 and 5 degrees C. The reaction mixture 15 was allowed to stir an additional 30min at -3 degrees C and then was carefully quenched in portions into 2L of ice. The yellowish ice mixture was acidified to pH 2.5 with 5M aqueous sulfuric acid, divided into two portions and the tan solids in each portion were extracted with 3 X 250mL of diethyl ether. The combined ether layers were washed with 4 X 50mL of water, dried over sodium sulfate, concentrated in 20 vacuo, and vacuum dried to give omega acetylenic acid 3 (37.6g, 100%) as an orange solid: [007051 'HNMR (400 MHz, CDCl3): 1.24-1.42 (m, 1OH), 1.48-1.66 (m, 4H), 1.92 (t, J= 2.56Hz, 1H), 2.16 (dt, Jl=2.56Hz, J 2 =7.69Hz, 2H), 2.39 (t, J=7.69Hz, 2H). 25 Methyl 11-dodecynoate (4): [00706] To a stirring solution of acetylenic acid 3 (37g, 188mmol) in 1.IL of methanol was added 2.2 mL of concentrated sulfuric acid, and the resulting reaction mixture was heated under reflux for 16h. Upon cooling to room temperature, the reaction mixture was concentrated to one third volume in vacuo, diluted with 1 L of 30 2:1 hexanes/diethyl ether, and washed with 2 X 50mL of saturated aqueous sodium bicarbonate. The combined aqueous washes were further extracted with 2 X 150mL of diethyl ether, treated with 50 mL of brine, combined with the previous organic extract and dried sodium sulfate. Concentration in vacuo gave a biphasic residue that 182 WO 20111134538 PCT/EP2010/063161 was partitioned with 500mL of hexanes and 50mL of brine. The aqueous layer was further extracted with 2 X 50mL of hexanes, the combined organic layers were dried over sodium sulfate, concentrated in vacuo, and vacuum dried to give methyl ester 4 (38.
2 g, 96%) a golden-brown oil: 5 [007071 'H NMR (400 MHz, CDCl3): 1.25-1.36 (m, 1OH), 1.41-1.65 (in, 4H), 1.92 (t, J=2.56Hz, IH), 2.16 (dt, J 1 =2.56Hz, J 2 =7.32Hz, 2H), 2.31 (t, J=7.32Hz, 2H), 3.64 (s, 3H). Methyl 12-bromo- 11 -dodecynoate (5): 10 [00708] To a mechanically stirred solution of ester 4 (37g, 176mmol) in 700mL of acetone under nitrogen was added N-bromosuccinimide (34.4g, 194mmol) followed by silver nitrate (2.7g, 18mmol). The reaction mixture was allowed to stir at room temperature in the absence of light for 21h. The thick, pale yellow reaction mixture was filtered through a sintered glass funnel, concentrated in vacuo to one third 15 volume, and diluted with 500mL of water. This was extracted with 4 x 250mL of hexanes, and the combined organic layers were washed with 100mL of brine, dried over sodium sulfate, concentrated in vacuo, and vacuum dried to give bromoacetylene 5 (47.8 1g, 94%) as a yellow oil: [00709] 1H NMR (400 MHz, CDCl3): 1.25-1.40 (in, 1OH), 1.46-1.59 (in, 4H), 2.17 20 (t, J=6.96Hz, 2H), 2.27 (t, J=7.32Hz, 2H), 3.64 (s, 3H). 2-lodofuran (6): [007101 To a mechanically stirred, cooled (-78 0 C) solution of furan (60.0mL, 825mmol) in 500 mL of dry ether under nitrogen was added a solution of n 25 butyllithium in cyclohexane (2.OM, 412mL, 825mmol) via pressure-equalized addition funnel over 60 min. The metalation was allowed to proceed at -78'C for 5h, and at room temperature for 2h. The thick, yellow reaction mixture was again cooled to -78'C, and iodine (209g, 825mmol) was added in two portions with good stirring (a slight exotherm was noted). The reaction mixture was allowed to warm to room 30 temperature overnight. Upon warming to room temperature, the mixture was treated with 2 X 250mL of freshly prepared 10% aqueous sodium thiosulfate and 250mL each of saturated aqueous sodium bicarbonate and brine, and dried over sodium 183 WO 20111134538 PCT/EP2010/063161 sulfate. The organic layer was concentrated in vacuo at 35 torr to give iodide 6 (38 2 g, contaminated with cyclohexane, 30% w/w 6, 72% yield) as a red oil: [007111 'H NMR (400 MHz, CDCl3): 6.35 (s, 1H), 6.53 (s, 1H), 7.53 (s, 1H). 5 2-(Trimethylsilylethynyl)furan (7): [007121 To a stirring solution of 6 (30% w/w, 79g, 407mmol) in 600mL of triethylamine under nitrogen was added trimethylsilylacetylene (44g, 448mmol), dichlorobis(triphenylphosphine)palladium(II) (10g, 14mmol), and copper (I) iodide (5.lg, 27mmol). The reaction mixture was allowed to stir at room temperature in the 10 absence of light for 16 h. The thick reaction mixture was diluted with 100 mL of diethyl ether and passed through a pad of Celite 521. The pad was washed with 2 X 100 mL of diethyl ether and the combined filtrates were concentrated in vacuo at 50 torr. The residue was purified by flash chromatography on silica gel (hexanes) to give TMS-protected ethynylfuran 7 (56.9g, 85%) as a yellow oil: 15 [007131 1H NMR (400 MHz, CDCl3): 0.22-0.28 (br(s), 9H), 6.37 (t, 1H), 6.61 (d, 1H), 7.36 (d, 1H). 2-Ethynylfuran (8): [007141 To a mechanically stirred, ice-water bath-cooled solution of 7 (54g, 20 329mmol) in 540mL of methanol under nitrogen was added potassium carbonate (104.5g, 756mmol). The resulting heterogeneous reaction mixture was stirred for 18 h as the vessel came to room temperature. The reaction mixture was diluted with 1500mL of water and extracted with 4 X 500mL of diethyl ether. The combined organic layers were washed with 3 X 150mL of water, 200mL of brine, dried over 25 sodium sulfate and the ether was removed by fractional distillation at atmospheric pressure to give 8 (88.3g, contaminated with diethyl ether and silanol, 23% w/w 8, 68% yield) as a red oil. [007151 1 H NMR (400 MHz, CDCl3): 3.35 (s, 1H), 6.32 (s, 1H), 6.60 (s, 1H), 7.35 (s, 1H). 30 Methyl 14-(2-furyl)tetradeca-11,13-diynoate (9) (see Ferri, Tetrahedron Lett., 1996, 37, 2763): 184 WO 20111134538 PCT/EP2010/063161 [00716] A mechanically stirred solution of bromoacetylene 5 (35g, 121mmol) and ethynylfuran 8 (23% w/w, 16.7g, 182mmol) in 400mL of pyrrolidine under nitrogen was cooled via ice-water bath. Copper (I) iodide (3.69g, 19mmol) was added in one portion and after 5 min the cooling bath was removed and the homogeneous mixture 5 was stirred an additional 1 h. The dark red reaction was quenched by addition of 850mL of water with stirring. The orange-yellow suspension was extracted with 4 X 300ml of diethyl ether. The combined organic layers were washed with 3 X lOOmL of water and 1OmL of brine, dried over sodium sulfate, and concentrated in vacuo to a dark red oil. The residue was resuspended in lOOmL of 10% ethyl acetate in 10 hexanes and passed through a pad of silica gel in a fitted funnel. The pad was washed with an additional 4 X 50mL of solvent and concentrated in vacuo to give 21.0g of crude 9 as a dark red oil. [007171 'H NMR (400 MHz, CDCI3): 1.26-1.43 (m, 1OH), 1.53-1.66 (m, 4H), 2.31 (t, 2H), 2.37 (t, 2H), 3.65 (s, 3H), 6.39 (s, 1H), 6.65 (s, 1H), 7.38 (s, 1H). 15 14-(furan-2-yl) tetradeca- 11,13-diynoic acid (X") [00718] To a solution of crude ester 9 (10g) in 200 mL of THF:water:methanol (150:25:25) was added with stirring 4.2g Lithium hydroxide (3eq). The atmosphere was replaced with nitrogen and the mixture was stirred for 5h and then cooled via ice 20 water bath. The golden-brown mixture was slowly acidified with 2N aqueous hydrochloric acid to pH2. The partially precipitated acid was extracted in two portions with 4 X 250mL of ethyl acetate. The combined organic layers were washed with 2 X 75mL of water and 75mL of brine, dried over sodium sulfate, and concentrated in vacuo. The dark brown solid was partially purified by suction 25 filtration column chromatography on silica gel (0 to 30% ethyl acetate in heptane) to give a yellow solid. This solid was resuspended in heptane and cooled in an ice bath. The mixture was filtered to yield a fine tan-yellow powder (6.88g, 72 %). [007191 m.p. 62.6-63.5'C [00720] 'H NMR (400 MHz, CDCl3): 1.25-1.45 (m, 10H), 1.53-1.60 (in, 2H), 30 1.60-1.68 (m, 2H), 2.34-2.40 (m, 4H), 6.37 (d, 1H), 6.65 (d, IH), 7.36 (s, 1H) [007211 "C NMR (100MHz, CDCl3): 19.72, 24.72, 28.15, 28.87, 29.21, 29.24, 29.39, 29.46, 34.26, 64.30, 64.74, 79.19, 87.80, 111.07, 117.44, 136.77, 144.22, 180.45. 185 WO 20111134538 PCT/EP2010/063161 [00722] Potassium 14-(furan-2-yl) tetradeca-11,13-diynoate (XII"): [007231 To a solution of 1 mmol 14-(furan-2-yl) tetradeca-11,13-diynoic acid in water, 1 mmolar aqueous potassium hydroxide was added at room temperature with 5 stirring. After 2-3 hours the reaction mixture was concentrated under reduced pressure. The crude product was dissolved in acetone and heated to 40'C for 10 min, then the solid product filtered off and washed several times with acetone. The resultant solid product was evaporated under reduced pressure yielding potassium 14 (furan-2-yl) tetradeca-11,13-diynoate. The sodium salt 14-(furan-2-yl) tetradeca 10 11,13-diynoate (XI") was achieved in like manner from 14-(furan-2-yl) tetradeca 11,13-diynoic acid. Example 24 15 Minimum Inhibitory Concentration (MIC) Testing [007241 The in vitro efficacy of antifungal compounds is determined in susceptibility assays, which are indicative for the spectrum and potency of a compound. Susceptibilities are reported as minimal inhibitory concentrations (MIC, as 20 ng/ml or pig/ml) required to inhibit growth to a defined degree compared to the drug free control. The MIC 90 defines the MIC required to inhibit growth for >90% of the strains of a population tested. MIC of (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt versus 25 reference Candida strains [00725] (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-11,13-diynoic acid were tested against seven clinical Candida reference strains according to the CLSI reference method M27-A3. MIC break points were read at a growth inhibition of 100% compared to the drug free 30 control. The inoculum size was 0.5 - 2.5 x 103 colony-forming units (CFU)/ml. Incubation temperature and time were 350 C and 24 hrs. [007261 Table 25 lists the MIC breakpoints in ng/ml obtained for the indicated reference strains. 186 WO 20111134538 PCT/EP2010/063161 Table 25 (Z)-14-(furan-2-yl) 14-(furan-2 Organism ID tetradeca-9-en-11,13-diynoic yl)tetradeca-11,13 acid, potassium salt diynoic acid C albicans 2.5 20 ATCC 24433 C. albicans 5 20 ATCC 90028 C. glabrata 60 2560 ATCC 90030 C. krusei 60 1280 ATCC 6258 C. parapsilosis 10240 >10240 ATCC 22019 C. parapsilosis >10240 >10240 ATCC 90018 C. tropicalis 7.5 80 ATCC 750 5 MICqo of (Z)- 14-(furan-2-yl1tetradeca-9-en- 11,1 3-diynoic acid, potassium salt versus clinical Candida isolates [007271 (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, potassium salt was tested head-to-head against AmphotericinB, Voriconazole and Caspofungin versus 193 clinical Candida isolates according to the CLSI reference method M27-A3. For 10 (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoic acid, potassium salt and Voriconazole, MIC break points were read at a growth inhibition of 50% compared to the drug free control and for Amphotericin, MIC breakpoints were read at a growth inhibition of 100%. For Caspofungin, the minimum effective concentration (MEC) was determined according to the CLSI reference method M27-A3. The inoculum size 15 was 0.5 - 2.5 x 103 colony-forming units (CFU)/ml. Incubation temperature and time were 350 C and 24 hrs. The MIC 9 0 was calculated for the indicated species. 187 WO 20111134538 PCT/EP2010/063161 [00728] Table 26 lists the MIC 90 values in ng/ml obtained for the indicated Candida species tested versus (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt, AmphotericinB, Voriconazole and Caspofungin. 5 Table 26 (Z)-14-(furan-2 Organism ID yl)tetradeca-9-en- Ampho- Voricon- Caspo (# of isolates) 11,13-diynoic acid, tericin B azole fungin potassium salt C. albicans (n=20) 2.5 500 30 n.d. C. glabrata (n=25) 20 2000 125 2000 C. tropicalis (n=35) 5 1000 30 1000 C. dubliniensis (n=25) 2.5 1000 16 1000 C. krusei (n=27) 20 4000 500 2000 C. lusitaniae (n=24) 128 1000 30 2000 C. parapsilosis (n=37) 1024/128* 500 125 8000 *calculated MIC 90 and MIC 50 value, respectively [007291 In parallel, a panel of clinical C. albicans and C. glabrata isolates with 10 resistances versus Fluconazole, Itraconazole, Voriconazole and Caspofungin were tested for susceptibility versus (Z)-14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, potassium salt, AmphotericinB, Voriconazole and Caspofungin. [00730] Table 27 lists the MIC 90 values in ng/ml obtained for the resistant Candida 15 species tested versus (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt, AmphotericinB, Voriconazole and Caspofungin. Table 27 Organism ID EV-086- Ampho- Voricon- Caspo (# of isolates) 3314K tericin B azole fungin C. albicans resistant' (n=20) 2.5 500 >500 n.d. C. glabrata resistant 2 (n=21) 20 2000 500 2000 188 WO 20111134538 PCT/EP2010/063161 'including clinical isolates with resiscances against Fluconazole, Itraconazole and Caspofungun 2 including clinical isolates with resiscances against Fluconazole, Itraconazole, Voriconazole and Caspofungin 5 MIC of (Z)-14-(furan-2-yl)tetradeca-9-en-11,13-diynoic acid, potassium salt versus clinical Aspergillus isolates [007311 (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, potassium salt was 10 tested head-to-head against AmphotericinB and Voriconazole versus 39 clinical Aspergillus isolates. Susceptibilities were determined according to the CLSI reference method M38-A2. MIC break points were read at a growth inhibition of 50% compared to the drug free control. Incubation time was 24h and MIC 50 values were calculated. 15 [007321 Table 28 lists the MIC 50 values in ng/ml obtained for the indicated Aspergillus species tested versus (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, potassium salt, AmphotericinB and Voriconazole. 20 Table 28 Organism ID (Z)-14-(furan-2-yl)tetradeca-9-en- Ampho- Voricon (# of isolates) 11,13-diynoic acid, potassium salt tericin B azole A. fumigatus (n=20) 80 500 250 A. terreus (n=5) 10 2000 250 A. niger (n=6) 2560 1000 1000 A. flavus (n=2) >5120 2000 500 A. versicolor (n=3) 1280 2000 250 A. nidulans (n=3) 5120 2000 125 189 WO 20111134538 PCT/EP2010/063161 MIC of (Z)-14-(furan-2-yltetradeca-9-en-11,13-diynoic acid, potassium salt versus clinical dimorphic fungi [007331 (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid, potassium salt was tested head-to-head against AmphotericinB, Voriconazole and Caspofungin versus 80 5 clinical isolates of dimorphic fungi including Coccidioides spp., B. derinatitidis and H. capsulatun according to the CLSI reference method M38-A2. MIC break points were defined as a growth reduction of 50% compared to the drug free control [007341 Table 29 lists the MIC 90 values in ng/ml obtained for the indicated 10 dimorphic fungal species tested versus (Z)-14-(furan-2-yl)tetradeca-9-en-11,13 diynoic acid, potassium salt, AmphotericinB, Voriconazole and Caspofungin. Table 29 (Z)- 14-(furan-2 Organism ID Ampho- Voricon- Caspo (# of isolates) yl)tetradeca-9-en-11,13- tericinB azole fungin diynoic acid, potassium salt Coccidioides spp. 30 500 125 500 (n=30)' B. dermatitidis (n=30) 2 30 250 250 4000 H. capsulatum (n=20) 3 30 125 125 2000 MIC read at '48h, 2 96h, '120 h 15 Example 25 Antifungal activity of single (Z)- 14-(furan-2-yl)tetradeca-9-en- 11,1 3-diynoic acid, 20 sodium salt infusion in a rat model of systemic candidiasis [00735] The efficacy of (Z)-14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, sodium salt was investigated in an in vivo rat model system for invasive candidiasis. The rats were infected systemically by injection of 1x10 7 C. albicans blastospores via a surgically implanted catheter in the jugular vein. A single 12 mg/kg infusion of (Z) 25 14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, sodium salt was given as a 2 hour intravenous infusion through the same cannula starting 2 hours after infection. Livers 190 WO 20111134538 PCT/EP2010/063161 were collected 24 hours after the end of the infusion and fungal burdens were determined in the tissues. (Z)-14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, sodium salt reduced the tissue burden (cfu/g tissue) by 90% compared with vehicle treated animals at 24 hours. The comparator Fluconazole reduced the tissue burden 5 (cfu/g tissue) by 93% compared with vehicle treated animals at 24 hours. [007361 Figure 24 shows the liver tissue fungal load of rats infected with C. albicans blastospores and treated with a single infusion of (Z)-14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid, sodium salt, Fluconazole or untreated. 10 Antifungal activity of a single oral administration of (Z)-14-(furan-2-yl)tetradeca-9 en-i 1,13-diynoic acid, sodium in a rat model of systemic candidiasis [007371 The efficacy of (Z)-14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, sodium salt was investigated in an in vivo rat model system for invasive candidiasis. 15 The rats were infected systemically by injection of 1x10 7 C. albicans blastospores via a surgically implanted catheter in the jugular vein. A single 17 mg/kg infusion of (Z) 14-(furan-2-yl)tetradeca-9-en-1 1,13-diynoic acid, sodium salt was administered by oral gavage 2 hours after infection. Kidneys were collected 6 hours after the end of the infusion and fungal burdens were determined in the tissues. (Z)-14-(furan-2 20 yl)tetradeca-9-en- 11,13-diynoic acid, sodium salt reduced the tissue burden (cfu/g tissue) by 57% compared with vehicle treated animals at 6 hours. The comparator Fluconazole reduced the tissue burden (cfu/g tissue) by 90% compared with vehicle treated animals at 24 hours. 25 [007381 Figure 25 compares the kidney tissue fungal load of rats infected with C. albicans blastospores and treated with a single oral administration of (Z)- 14-(furan-2 yl)tetradeca-9-en- 11,13-diynoic acid, sodium salt or untreated. 191

Claims (24)

1. A crystalline diyne compound of formula I: K+ Z-[C=C- C=C]-R 3 wherein Z is a carbon chain substituted with -COO or a bioisostere thereof and optionally also substituted with one or more additional substituents; and R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions.
2. The compound according to claim 1, wherein the compound has a solubility in water of at least 50 mg/ml, preferably at least 60 mg/ml, more preferably at least 70 mg/ml, yet more preferably at least 80 mg/ml, even more preferably at least 90 mg/ml.
3. The compound according to claim 1 or 2, wherein the melting point of said compound is at least 100 C, preferably at least 1 10 C, more preferably at least 120'C, yet more preferably at least 130'C, even more preferably at least 140'C.
4. A compound of formula V': C -(C(R 2 ) 2 )n-X-C=C -C=C -R 3 R1 wherein, R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; n is an integer between 4 and 10, inclusive; X is -CH 2 CH 2 - and R 3 is a 3 to 7 membered aromatic heterocyclic ring, which optionally may be substituted at one or more positions. 192 5181067_1 (GHMatters) P89262.AU
5. The compound according to any one of claims 1-3, wherein the diyne is a diyne of formula IV: K+ R4-(C(R 2 ) 2 )n-X-(C(R 2 ) 2 )m-[C=C- C=C]-R 3 wherein R 4 is -COO- or a bioistere thereof, preferably -COO-; n is an integer, preferably an integer in the range of 4 to 10, inclusive, preferably in the range of 5 to 9, even more preferably in the range of 6 to 8, yet more preferably n is 7; m is an integer, preferably an integer in the range of 0 to 10, such as in the range of 0 to 8, for example in the range of 0 to 6, such as in the range of 0 to 4, for example in the range of 0 to 2, such as 0; each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; X is -CH 2 -CH 2 - or -CH=CH-; and R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions.
6. The compound according to any one of claims 1-5, wherein R 3 is furan.
7. The compound according to any one of claims 5-6, wherein X is -CH=CH- in the cis conformation.
8. The compound according to any one of claims 5-6, wherein X is -CH 2 -CH 2 -.
9. The compound according to any one of claims 1-3, wherein the diyne is potassium (Z)
14-(furan-2-yl)tetradeca-9-en- 11,13-diynoate. 10. The compound according to any one of claims 1-3, wherein the diyne is potassium 14 (furan-2-yl)tetradeca- 11,13-diynoate. 11. A pharmaceutical composition comprising the diyne compound according to any one of claims 1-10 and one or more pharmaceutically acceptable excipients. 193 5181067_1 (GHMatters) P89262.AU 12. The pharmaceutical composition according to claim 11, wherein the composition further comprises at least one antifungal polyene, such as amphotericin B. 13. A method of treating a fungal infection in an individual in need thereof, said method comprising administering a therapeutically effective amount of the diyne compound according to any one of claims 1-10 to said individual. 14. The method according to claim 13, wherein the fungal infection is infection by one or more fungi selected from the group consisting of Candida spp. (for example C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr or C. dubliniensis), Aspergillus spp. (for example A. fumigatus, A. flavus, A. niger or A. terreus), Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp. (for example C. neoformans (for example var. neoformans or var. gattii), C. bidus, C. laurentii, or C. fusarium), Zygomycetes (such as Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle bertholletiae, Saksenaea vasiformis, Mucor circinelloides, M. ramosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus or Syncephalastrum racemosum), Malassezia spp. (for example M. furfur or M. globosa), Hyalohyphomycetes (for example Fusarium solani or Scedosporium spp., such as S. prolificans or S. apiospermum), Dermatophytes (for example Trichophyton spp. (for example T. mentagrophytes, T. rubrum or T. tonsurans), Epidermophyton floccosum, Microsporum spp (for example M. cookei, M. canis, M. vanbreuseghemii, M. gallinae orM. gypseum) or Trichosporon terrestre), Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi (for example Fonsecaea pedrosoi, F. compacta, Cladophylophora carrionii or Phialophora verrucosa)and Madurella spp. (for example M. mycetomatis or M. griseum), Pneumocystisjirovecii, Pneumocystis carinii, Botrytis cinerea; Magnaporthe grisea; Anamorph: Pyricularia oryzae Colletotrichum gleoesporioides- Chilli strain; Colletotrichum gleoesporioides- mango strain; Fusarium verticillioides; Fusarium oxysporum; Alternaria solani; Uncinula necator Syn Erysiphe necator; Macrophomina phaseolina; Syn. Sclerotium bataticola and Rizoctonia bataticola; Botryodiplodia theobromae; Basidiomycota Sclerotium rolfsii; Rhizoctonia solani; Puccinia arachidis; Oomycota Pythium aphanidermatum; and Plasmopara viticola Syn. Personopora viticola. 194 5181067_1 (GHMatters) P89262.AU
15. The method according to claim 13, wherein said individual is an immunocompromised individual.
16. Use of a diyne compound according to any one of claims 1-10 for the preparation of a medicament for treatment of a fungal infection in an individual in need thereof
17. Use of the diyne compound according to any one of claims 1-10 for preventing or treating fungal infections of a plant.
18. A method for reducing the risk of infection by a fungus or treating infection by a fungus in a plant, said method comprising contacting said plant with the diyne compound according to any one of claims 1-10.
19. The method according to claim 18, wherein the method comprises contacting said plant with the aqueous solution comprising the compound according to any one of claims 1-10.
20. A method of treating an infection by a fungus dependent on activity of stearoyl-CoA desaturase in an individual in need thereof, said method comprising administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of a diyne compound of the formula I': Z-[C=C- C=C]-R 3 wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; and wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus, and wherein the infection is one or more of the following: i) is a recurrent fungal infection; 195 5181067_1 (GHMatters) P89262.AU ii) is an infection involving at least partly infection of tissue, organs or cells with hypoxic conditions.
21. The method according to claim 20, wherein said infection is an infection involving at least partly infection of a body surface, for example infection of skin, nails or mucosal membranes of body surfaces.
22. The method according to claim 20, wherein one or more fungus is selected from the group consisting of Candida spp. (for example C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr or C. dubliniensis), Aspergillus spp. (for example A. fumigatus, A. flavus, A. niger or A. terreus), Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp. (for example C. neoformans (for example var. neoformans or var. gattii), C. bidus, C. laurentii, or C. fusarium), Zygomycetes (such as Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle bertholletiae, Saksenaea vasiformis, Mucor circinelloides, M. ramosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus or Syncephalastrum racemosum), Malassezia spp. (for example M. furfur or M. globosa), Hyalohyphomycetes (for example Fusarium solani or Scedosporium spp., such as S. prolificans or S. apiospermum), Dermatophytes (for example Trichophyton spp. (for example T. mentagrophytes, T. rubrum or T. tonsurans), Epidermophytonfloccosum, Microsporum spp (for example M. cookei, M. canis, M. vanbreuseghemii, M. gallinae or M. gypseum) or Trichosporon terrestre), Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi (for example Fonsecaea pedrosoi, F compacta, Cladophylophora carrionii or Phialophora verrucosa)and Madurella spp. (for example M. mycetomatis or M. griseum), Pneumocystis jirovecii, Pneumocystis carinii, Botrytis cinerea; Magnaporthe grisea; Anamorph: Pyricularia oryzae Colletotrichum gleoesporioides- Chilli strain; Colletotrichum gleoesporioides- mango strain; Fusarium verticillioides; Fusarium oxysporum; Alternaria solani; Uncinula necator Syn Erysiphe necator; Macrophomina phaseolina; Syn. Sclerotium bataticola and Rizoctonia bataticola; Botryodiplodia theobromae; Basidiomycota Sclerotium rolfsii; Rhizoctonia solani; Puccinia arachidis; Oomycota Pythium aphanidermatum; and Plasmopara viticola Syn. Personopora viticola. 196 5181067_1 (GHMatters) P89262.AU
23. The method according to claim 20, wherein the diyne is a diyne of formula III': C-Y C=C-C=C-R 3 R1 wherein R 1 is a hydroxyl group or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction; Y is a carbon chain of 6 to 20 carbon atoms and up to three double bonds, wherein each carbon of said alkyl or alkenyl is linked to none, one or two R 2 groups, wherein each R 2 independently is -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; or a pharmaceutically acceptable salt of said diyne.
24. The method according to claim 20, wherein the diyne is a diyne of formula IV': R4-(C(R 2 ) 2 )n-X-(C(R 2 ) 2 )m-[C=C- C=C]-R 3 wherein R 4 is -COOH or a bioistere thereof; n is an integer, preferably an integer in the range of 4 to 10, inclusive, preferably in the range of 5 to 9, even more preferably in the range of 6 to 8, yet more preferably n is 7; m is an integer, preferably an integer in the range of 0 to 10, such as in the range of 0 to 8, for example in the range of 0 to 6, such as in the range of 0 to 4, for example in the range of 0 to 2, such as 0; each R 2 is, independently, -H, -OH or a hydrocarbon moiety containing between 1 and 6 carbon atoms, inclusive; X is -CH 2 -CH 2 - or -CH=CH- or phenyl; and R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions.
25. The method according to claim 20, wherein the diyne is selected from the group consisting of (Z)- 1 4-(furan-2-yl)tetradeca-9-en- 11,13 -diynoic acid, (Z)- 1 4-(furan-2-yl)tetradeca 9-en-11,13-diynoic acid and pharmaceutically acceptable salts thereof 197 5181067_1 (GHMatters) P89262.AU
26. The method according to claim 20, wherein the diyne is the compound according to any one of claims 1-10.
27. Use of a diyne of the formula I': Z-[C=C- C=C]-R 3 wherein Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus, for the preparation of a pharmaceutical composition for treatment of an infection by a fungus dependent on activity of stearoyl-CoA desaturase in a individual in need thereof, and wherein the infection is one or more of the following: i) is a recurrent fungal infection; ii) is an infection involving at least partly infection of tissue, organs or cells with hypoxic conditions.
28. A crystalline diyne compound of formula I as defined in claim 1, a compound of formula V as defined in claim 4, a pharmaceutical composition comprising the diyne compound of formula I, a method of treating a fungal infection with the diyne compound of formula I, use of the diyne compound of formula I, a method for reducing the risk of infection by a fungus or treating infection by a fungus in a plant with the diyne compound of formula I, a method of treating an infection by a fungus dependent on activity of stearoyl-CoA desaturase with a diyne compound of formula I', substantially as herein described with reference to any one of the examples of the invention. 198 5181067_1 (GHMatters) P89262.AU
AU2010352387A 2009-07-10 2010-09-08 Diyne compositions Ceased AU2010352387B2 (en)

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