WO2011134538A1 - Diyne compositions - Google Patents
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- WO2011134538A1 WO2011134538A1 PCT/EP2010/063161 EP2010063161W WO2011134538A1 WO 2011134538 A1 WO2011134538 A1 WO 2011134538A1 EP 2010063161 W EP2010063161 W EP 2010063161W WO 2011134538 A1 WO2011134538 A1 WO 2011134538A1
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- diyne
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- 0 CC[C@]1*[C@](C2)C[C@@]2C1 Chemical compound CC[C@]1*[C@](C2)C[C@@]2C1 0.000 description 3
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic 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
- C07D307/38—Heterocyclic 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
- C07D307/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, 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
- A01N43/04—Biocides, 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
- A01N43/06—Biocides, 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
- A01N43/08—Biocides, 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Definitions
- the present invention relates to diyne salt and in particular potassium 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.
- 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.
- This invention further encompasses diynes and diyne salts as Olel protein inhibitors, as well as methods for treating fungal infections in humans, animals and plants.
- 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 4 th largest cause of nosocomial blood stream infections.
- 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.
- the mortality due to Aspergillus fumigatus has increased 357% and is continuing to increase.
- 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 cosmetic, psychological and social implications. In some patient subsets it has serious medical consequences (e.g. foot amputations in diabetics).
- 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.
- Antifungals currently available for the treatment of systemic infections 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.
- AmBisome the less toxic lipid formulations
- the echinocandins which include Anidulafungin, Caspofungin, and Micafungin, all of which must be administered intravenously.
- the newer triazoles such as Posaconazole (oral) and Voriconazole (oral and intravenous) are FDA approved for the treatment and prevention of systemic Candida infections.
- Caspofungin resistance is still an uncommon occurrence with c. 8% of C. 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 reduced C. glabarata susceptibility developing during Caspofungin therapy.
- the target of Caspofungin is the enzyme l,3-P-D-glucan synthase, encoded by one of several FKS genes, depending on the species.
- Anti-fungals work by exploiting differences between mammalian and fungal cells to selectively inhibit growth or to kill the fungal organism preferably 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. Some of these side-effects can be life-threatening if the drug is not used properly.
- 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 also inhibit growth of fungal cells.
- yield losses caused by various fungal pathogens in crops and other plants are significant, particularly across the major groups of fungal diseases such as rust, rot (root and fruit), leaf spots, mildews and wilts.
- antifungal agents with one or more of the following properties, preferably all of the following properties:
- Anti-fungal activity preferably fungicidal activity
- Z is a carbon chain substituted with -COO " or a bioisostere thereof (preferably -COO " ) 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.
- the invention also provides pharmaceutical compositions comprising such diyne salts and one or more pharmaceutically acceptable excipients. These pharmaceutical compositions are in particular useful for treatment of an infection by a fungus in an individual, such as a mammal in need thereof.
- the invention furthermore provides use of the diyne salts for preventing or treating fungal infections of a plant.
- the present invention provides a new class of compounds with antifungal activity.
- diynes compounds disclosed herein generally have a broad spectrum (i.e. activity against a wide range of fungal infections), they generally have potent antifungal activity, including antifungal activity against A. fumigatus and other filamentous fungi and they are generally active via the oral and intravenous routes.
- the diynes disclosed herein have a novel mode of action, hence, their activity is generally unaffected by resistance to existing anti- fungal agents.
- 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- CoA desaturases (e.g. OLE-1) for viability.
- OLE-1 stearoyl- CoA desaturases
- the present invention discloses that for these fungi stearoyl-CoA desaturase is an interesting target for novel antifungal compounds.
- the present invention discloses diyne compounds that are capable of specifically inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in fungi.
- compositions comprising a diyne of formula ⁇ :
- R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions
- the diyne may be any of the diynes described herein below in the sections "Diyne” and “Particular diynes" [0045] wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus,
- said pharmaceutical composition is for treatment of an infection by a fungus dependent on activity of stearoyl-CoA desaturase (such as any 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”).
- a fungus dependent on activity of stearoyl-CoA desaturase such as any of the infections described herein below in the section "Fungal infection
- the individual may be any of the individuals described in the section "Individual in need of treatment”
- Z is a carbon chain substituted with -COOH or a bioisostere thereof and optionally also substituted with one or more additional substituents; and [0049] R 3 is a heterocyclic ring, which optionally may be substituted at one or more positions; and [0050] wherein said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus.
- 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 [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.
- 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;
- 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 preferably any of the diyne compounds described in the section "Particular diynes" herein below.
- 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;
- said diyne is capable of inhibiting conversion of a saturated fatty acid to a A9-monounsaturated fatty acid in a fungus
- 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 variety of fungal pathogens, and are thus capable of inhibiting fungal growth of fungi dependent on Olel protein function.
- the inventive thus contemplates antifungal compounds for formulations for treating a subject, and also for use in the agricultural setting.
- the invention thus provides a novel fungicide comprising an Olel protein inhibitor.
- the invention also provides novel diyne compounds, their salts, derivatives and analogs.
- a preferred embodiment of the invention are novel Olel protein inhibitors of structure II" a) (Z)-R 1 - C(0) - (C(R 2 ) 2 ) X - C 2 H 2 - C 4 - R4 II"
- Ri 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 or more substituents selected from the group consisting of a Ci_ 5 alkyl, a Ci_ 5 alkenyl, a Ci_5 alkoxy, a Ci_ 5 alcohol, a hydroxyl, an amine, a nitrate and a halogen; and x is an integer between 4 and 10, inclusive.
- R 4 is a pyrrole, furan, or thiophene ring.
- a preferred fungicide is a compound of structure III
- the invention also contemplates a compound of structure IX", a. R 1 - C(0) - (C(R 2 ) 2 ) X - C 2 H 4 - C4 - R4 IX"
- Ri 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 or more substituents selected from the group consisting of a Ci_ 5 alkyl, a Ci_ 5 alkenyl, a Ci_5 alkoxy, a Ci_ 5 alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive.
- R4 is a pyrrole, furan, or thiophene ring.
- Preferred compounds include those having the structure of one of X"- XII".
- the invention also contemplates a fungicide comprising an Olel protein inhibitor.
- Olel protein inhibitor is selected from the group consisting of compounds of structures II"-XII".
- the Olel inhibitor is a compound having the structure of
- One preferred Olel inhibitor is compound III
- fungicides comprise an Olel inhibitor having the structure of one of VI", VII" and VIII".
- Another preferred Olel inhibitor is a compound having the structure of one ofX"-XII.
- 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".
- 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 Olel protein inhibitor to a formulation, wherein the Olel inhibitor is a compound having the structure of one of II"- XII" .
- the subject is animal, preferably mammal, more preferably human.
- the compounds may also be used in co-therapy with one or more other therapeutically used classes of antifungal substances.
- 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.
- Trichophyton spp. for example T. mentagrophytes, T. rubrum or T. tonsurans
- Epidermophyton floccosum 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
- Madurella spp. for example M. mycetomatis or M. griseum
- Pneumocystis jirovecii Pneumocystis carinii,Ascomycota Botrytis cinerea
- M. mycetomatis or M. griseum Pneumocystis jiroveci
- Magnaporthe grisea Anamorph: Pyricularia oryzae Colletotrichum gleoesporioides- Chilli strain; Colletotrichum gleoesporioides- mango strain; Fusarium
- 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 inventive fungicides to a formulation.
- the formulation is used to combat a fungal pathogen in a plant, a grass or a field.
- 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.
- 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".
- the invention also contemplates the use of a fungicide comprising any of 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 Olel protein inhibitor.
- 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".
- Figure 1 shows XRPD diffracto grams of salts of (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid prepared using potassium ethoxide or sodium ethoxide as indicated in the figure.
- 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 , 13-diynoate.
- Figure 3 shows TGA and DSC analysis (as indicated in the figure) of potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate.
- Figure 4 shows a GVS analysis of the potassium salt of potassium (Z)-14- (furan-2-yl)tetradeca-9-en- 11 , 13-diynoate .
- Figure 5 shows an assay for synergistic effect between (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt and Amphotericin B.
- Figure 6 shows a time course of the fungicidal effect of (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid
- Figure 7 shows the morphological effect of (Z)-14-(furan-2-yl)tetradeca- 9-en-l 1,13-diynoic acid and Fluconazole on growing C. albicans.
- the % shown in the upper left corners indicates percent cell viability as assessed by MTT (metabolic dye staining).
- Figure 8 shows the dose-dependent inhibition of C. albicans germination by (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid (A), dose-dependent inhibition of vegetative growth by (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid (B) and dose dependent inhibition of germinated spores by (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid (C) [00104]
- Figure 9 shows 13 C-Acetate incorporation into Ergosterol in C.
- Figure 10 shows liver fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue.
- Figure 11 shows kidney fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue.
- Figure 12 show kidney fungal burden as determined by colony forming units (CFU) per gram of homogenised tissue.
- Figure 13 shows topical fungal burden as determined by culture positive hair removed from the site of infection.
- Figure 14 shows vaginal fungal burden as determined by colony forming units (CFU) from vaginal lavage fluid.
- Figure 15 shows regulation of OLEl transcriptional activation.
- 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".
- Figure 17a shows time dependent expressions of the OLEl gene in response to compound IV" in S. cerevisiae, numbers indicate time intervals in minutes. Expressions of OLEl are given as multiples of the expression at 10 min, condition a., and figure 17b shows time and concentration dependent expressions of the OLEl gene in response to compound IV" in C. albicans.
- Figure 18 shows (upper pictures, controls) normal hyphal growth as compared with changes in the plane of hyphal growth and abnormal thickening of the hyphae due to the antifungal effect of the potassium salt, XII", (two lower pictures).
- Figure 19 shows regulation of OLE1 transcriptional activation.
- Components of the GET complex putative ly mediate the insertion of Mga2/Spt23 into the ER membrane.
- the ERAD complex proteolytically activates Mga2/Spt23, which shuttles to the nucleus where it activates OLE1 transcription.
- Figure 20 shows oleic acid antagonises the sodium salt of (Z)-14-(furan-2- yl) tetradeca-9-en-l 1,13-diynoic acid.
- YPD agar plates were prepared containing 0, 0.5, 2 and 8 ⁇ g/ml of (Z)-14-(furan-2-yl) tetradeca-9-en-l 1,13-diynoic acid.
- the plates were either supplemented with 28 ⁇ g/ml oleic acid, 28 ⁇ g/ml stearic acid or 0.2 % Tergitol alone as control required for solubilisation of the fatty acids.
- Figure 21 shows antagonistic effects between the potassium salt of (Z)-14- (furan-2-yl) tetradeca-9-en-l 1,13-diynoic acid and fatty acids as sodium salt in C. albicans. Compounds were combined in 96-well plates and inoculated with C.
- Figure 22 shows antagonistic effects between oleic acid and the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-l 1,13-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 were grown for 48 h and growth determined through visual inspection of growth wells.
- 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-l 1,13-diynoic acid in Candida albicans. Numbers indicate time intervals in minutes. Expression values are normalised to TUB1 and given as multiples of OLE1 expression at 10 minutes in ethanol.
- Figure 24 shows the liver tissue fungal load of rats infected with C.
- 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-l 1,13-diynoic acid, sodium salt or untreated.
- the present invention relates to diyne compounds, in particular diyne salts as well as to use of the diyne salts - mainly in the treatment of fungal infections.
- the diyne salts according to the present invention are diyne salts of formula I: 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 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.
- 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.
- Z may preferably be selected from the group consisting of alkyl and alkenyl, which is substituted with R 4 , wherein R 4 is - COO " or a bioisostere thereof, preferably -COO " .
- 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 defined herein below in relation to diynes of formula II.
- Z is a C 6 -2o, preferably a C 6-15 , more preferably C 6-12 , even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkyl or alkenyl substituted with -COO " or a bioisostere thereof (preferably with -COO " )and 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 .
- Z is -COO " -(C6_ 2 o alkyl or alkenyl)-, such as -COO " -(C9_ 2 o alkyl or alkenyl)-, for example -COO " -(C9_i5 alkyl or alkenyl)-, such as -COO " -(C 6-12 alkyl or alkenyl)-.
- the diyne salt is a diyne salt of the formula II:
- 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 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds.
- each carbon atom is linked to none, one or two R 2 groups.
- 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 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.
- 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.
- the diyne salt is a diyne salt of the formula III:
- 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 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 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.
- each carbon atom is linked to none, one or two R 2 groups.
- 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 atoms in the carbon chain by double bonds will not be linked to any R 2 groups.
- Y may be a linear C 6 -2o, preferably a C 6-15 , more preferably C 6 -i2, even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkyl, preferably a linear C7_n alkyl, yet more preferably a linear Cs-io alkyl, even more preferably a linear C9-alkyl.
- Y may also be a linear C 6 -2o, preferably a C 6 -i5, more preferably C 6 -i2, even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkenyl, preferably a linear C7_n alkenyl, yet more preferably a linear Cs-io alkenyl, even more preferably a linear C9- 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 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.
- Y may be a linear C 6 -i2, preferably a linear C7_n alkenyl, yet more preferably a linear Cs-io alkenyl preferably a linear C9 alkenyl, wherein all double 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 , C9 OT Cio position, preferably at the C9 position (the C in the carbonyl group being Ci), even more preferably at least one double bond in the cis conformation is situated at the C 8 , C9 or Cio position, preferably at the C9 position (the C in the carbonyl group being Ci).
- the diyne salt is a compound of the formula IV:
- R4 is -COO " or a bioisostere thereof, preferably however R4 is -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; and
- the diyne salt is a compound of formula V:
- 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.
- 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, -N0 2 and halogen.
- Lower alkyl is preferably Ci_ 5 , more preferably Ci_ 3 , even more preferably Ci alkyl.
- Lower alkenyl is preferably Ci_ 5 , more preferably Ci_ 3 , even more preferably Ci_ 2 alkenyl.
- Lower alkoxy is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkoxy.
- Lower alcohol is preferably Ci_ 5, more preferably Ci_ 3 , even more preferably Ci 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 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.
- R 3 of the diyne salt of formula I, II, III, IV or V is preferably an aromatic heterocyclic ring.
- 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, even more preferably a 5 membered heterocyclic ring.
- the heterocyclic ring may be aromatic or non-aromatic.
- 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.
- 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 group consisting of S, N and O.
- 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.
- 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 the group consisting of methyl, methoxy, hydroxyl, amine and halogen, preferably methyl.
- R 3 is furan
- the heterocyclic ring is 2-substituted with the -[C ⁇ C- C ⁇ C]-Z chain.
- 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.
- 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 -[C ⁇ C- C ⁇ C]-Z chain.
- Each R 2 of a diyne salt 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 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 Ci_ 4 alkyl, Ci_ 4 alkenyl or Ci_ 4 alkynyl. It is however also possible that two neighbouring 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 R 2 carbons).
- the ring system may be aromatic or none aromatic, for example the ring system may be an 6 membered aromatic ring. However, preferably neighbouring R 2 groups are not connected.
- 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, II, III, IV or V is -H.
- 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 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.
- R 2 groups are connected to 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 Cn, preferably on C 9 and C 10 position (the C in the carbonyl group being Ci).
- a preferred diyne compound according to the invention is potassium (Z)- 12-(furan-2-yl)dodeca-7-en-9,l l-diynoate.
- Another preferred diyne compound according to the invention is potassium (Z)- 13-(furan-2-yl)trideca-8-en- 10,12-diynoate.
- Yet another preferred diyne compound according to the invention is potassium (E)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate.
- Yet another preferred diyne compound according to the invention is the diyne compound potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate.
- Another preferred diyne compound according to the invention is the diyne compound potassium 14-(furan-2-yl)tetradeca-l 1,13-diynoate.
- the diyne compound may be selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt; (Z)-14-(5-methylfuran-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt; 8-(2-(4-(furan-2-yl)buta-l,3-diynyl)phenyl)octanoic acid, potassium salt; (Z)-14-(4,5-dimethylfuran-2-yl)tetradeca-9-en-l l,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-l l,13-diynoate.
- 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, which is hereby incorporated by reference.
- the diyne carboxylic acid is then mixed with a base in a useful solvent.
- the diyne carboxylic acid is dissolved in solvent and the base is dissolved in solvent prior to mixing.
- the base is a base comprising a potassium cation, such as potassium ethoxide.
- the solvent may be any useful solvent.
- the solvent is a water miscible organic solvent, such as a solvent selected from the group consisting of IP A, Dioxane, EtOH and acetone.
- 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 carboxylic acid and base is allowed to cool slowly, for example to a temperature in the range of 0 to 30°C, such as to 15-25°C, for example to 18°C.
- a heating step such as heating to in the range of 40 to 80°C, such as to 50°C.
- a cooling step is employed - typically, the heating solution comprising diyne carboxylic acid and base is allowed to cool slowly, for example to a temperature in the range of 0 to 30°C, such as to 15-25°C, for example to 18°C.
- Diyne salt may then precipitate as solids.
- the solvent or part thereof may also be allowed to evaporate.
- 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.
- 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.
- 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 acid in a fungus comprises the steps of
- stearoyl-CoA 9 desaturase such as OLE-1
- 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.
- 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 acid to palmitoleic acid in a fungus comprises the steps of
- stearoyl-CoA 9 desaturase such as OLE-1
- 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.
- 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).
- stearoyl-CoA 9 desaturase is mainly located in the membranes of the endoplasmatic reticulum and accordingly it is preferred that said fungal extract comprises endoplasmatic reticulum or parts thereof.
- 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.
- 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.
- 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.
- Example 6 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 conversion of palmitic acid to palmitoleic acid, by exchanging the substrate provided.
- 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 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.
- 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.
- the activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1) may be inhibited by different means by said diyne compound.
- the diyne may directly inhibit the enzymatic activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1).
- the diyne compound may preferably be an inhibitor of the fatty acid desaturase activity of OLE-1 polypeptide.
- Whether said diyne salt is capable of directly inhibiting the activity of fungal stearoyl-CoA 9 desaturase may for example be determined using an in vitro assay for fungal stearoyl-CoA 9 desaturase (such as OLE-1) activity.
- 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 salt to said reaction mixture preferably significantly inhibits the formation of oleic acid and/or palmitoleic acid or activated forms thereof.
- 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 mixture in a purified form or as part of a crude extract, for example a fungal extract or as prepared in vitro. .
- Mammalian desaturases are significantly different to fungal stearoyl-CoA 9 desaturases (such as OLE-1), for example mammalian desaturases lacks an integral cytochrome bs 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.
- the diyne salt according to the invention is a selective inhibitor of a fungal stearoyl-CoA 9-desaturase (such as OLE- 1).
- the diyne compound is capable of inhibiting the 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.
- the diyne compounds according to the invention are capable of reducing 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% 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.
- said diyne compound in a similar in vitro assay 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 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).
- 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 fungal stearoyl-CoA 9 desaturase (such as OLE-1) in a fungus.
- 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 9 desaturase (such as OLE-1).
- the diyne compound may down modulate the expression of the OLE-1 polypeptide by modulating the activity of a transcriptional regulator of the gene encoding the OLE-1 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-1 promoter or enhancer region.
- Spt23p/Mga2p is a fungal transcriptional regulator that amongst others controls the expression of fungal stearoyl-CoA 9 desaturase (such as OLE-1).
- the diyne salt may be capable of inhibiting the activity of Spt23p/Mga2p.
- the MIC minimal Inhibitory Concentration
- the MIC is the minimal concentration of diyne salt required for inhibiting essentially 100%, such as 100% growth of a fungi.
- 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 most 20 ng/ml, such as at the most 10 ng/ml in relation to at least 3 different fungi.
- 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.
- the diyne compound has a minimum fungicidal concentration (MFC) of at the most 100 ⁇ g/ml, preferably at the most 50 ⁇ g/ml, yet more preferably at the most 10 ⁇ g/ml, even more preferably at the most 2 ⁇ g/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.
- said MFC for a given fungus is determined in a method comprising the steps of a) cultivating said fungus in vitro
- step e) determining growth of said fungus in said another in vitro culture medium
- 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.
- contacting a fungus with said diyne 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
- 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 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.
- 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.
- 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 ⁇ g/ml, preferably at the most 50 ⁇ / ⁇ 1, yet more preferably at the most 10 ⁇ / ⁇ 1, even more preferably at the most 2 ⁇ . Inhibition of growth may for exampe be determined as described herein below in Example 7. d. Solubility
- the antifungal compound is soluble in water.
- the antifungal compound is soluble in water.
- 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.
- the solubility in water is preferably determined as described in Example 1 herein below.
- e. Crystal For pharmaceutical applications it is furthermore generally preferred that the active compound is provided in a crystalline form.
- 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).
- XRPD X- Ray Powder Diffraction
- Example 1 Crystalline compounds gives rise to a XRPD pattern with distinctive peaks, whereas an amorphous compound gives rise to an XPRD pattern without distinctive peaks.
- an XPRD pattern for a crystalline compound (potassium (Z)-14-(mran-2-yl)tetradeca-9-en-l 1,13-diynoate) with distinctive peaks and an amorphous compound (sodium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13- diynoate) without distinctive peaks is shown in figure 1.
- the diyne salt is potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate.
- said potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate is in the crystalline form.
- the high resolution XPRD pattern may be obtained using any suitable method known to the skilled person, but in one preferred embodiment it is prepared as described herein below in Example 2.
- the potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13- diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 2 ⁇ angles: 2.42° and 4.78°.
- 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 2 ⁇ angles: 7.14°, 9.52° and 11.89°.
- the potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 2 ⁇ angles: 2.42° and 4,78°, 7.14°, 9.52° and 11.89°.
- the potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 2 ⁇ angles: 2.42°, 4,78°, 7.14°, 9.52°, 9.52°, 16.45°, 17.27°, 18.41°, 19.1 1°, 19.68°, 21.27°, 21.95°, 23.06°, 23.86°, 24.90°, 26.98°, 27.82°, 28.68°, 28.86° and 38.77°.
- the high resolution XPRD pattern does not comprises any other peaks with an intensity of >5%.
- the potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate crystals have a high resolution XPRD pattern as shown herein in figure 2B
- the diyne salt according to the invention does not take up to much moisture from the surroundings.
- 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.
- 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.
- 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.
- 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
- the diyne salt has a sufficiently high melting temperature in order to allow handling during manufacture of pharmaceutical compositions and storage at ambient temperature.
- the melting point of said compound is at least 100°C, preferably at least 110°C, more preferably at least 120°C, yet more preferably at least 130°C, even more preferably at least 140°C.
- the melting point of a compound may be determined using any suitable 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
- the diyne salt according to the present invention is stable upon storage.
- the diyne salt when stored as a solid, is stable 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 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.
- 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 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 temperature of 25°C and a humidity of in the range of 10 to 60%, such as 30 to 60%>, for example 60%.
- 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%>.
- 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 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°C, for example at a temperature of 40°C and a humidity of in the range of 60 to 75%, such as 70 to 75%, for example 75%.
- 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%.
- compositions comprising diyne salts according to the present invention are useful for treating fungal infections in an individual in need thereof.
- the pharmaceutical compositions may be in any suitable form depending on the fungal infection to be treated.
- the pharmaceutical composition may be formulated for topical administration or for systemic administration.
- the fungal infection is a local infection on a body surface
- the pharmaceutical composition is formulated for topical administration.
- 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 administration or oral administration.
- compositions according to the invention will frequently comprise one or more pharmaceutically acceptable excipients.
- 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 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 of a diyne compound of the invention in a physiologically-acceptable carrier.
- one or more diyne salts of the invention are formulated in combination with a solid or a liquid dermato logically acceptable carrier.
- Useful solid carriers include finely divided solids such as talc, clay, micro crystalline 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 such as surfactants, and additional antimicrobial agents
- 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 using pump-type or aerosol sprayers.
- the liquid compositions can also be employed as eye drops, mouth washes, douches, etc.
- 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.
- diyne salts 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 pathogens.
- 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.
- 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.
- compositions comprising diyne salts according to the invention are formulated for administration of in the range of O.OOlmg/Kg to lOOmg/kg, preferably in the range of O.OOlmg/Kg to lOOmg/kg daily, in particular when the individual to be treated is a mammal, such as a human being.
- 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.
- concentration of the diyne 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.
- 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 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.
- the pharmaceutical compositions according to the invention may in addition to the diyne salts also comprise one or more additional active agents.
- Said additional active agents may for example be antifungal agents.
- A5. Individual in need of treatment may for example be antifungal agents.
- the present invention relates to pharmaceutical compositions comprising diyne salts for treatment of fungal infections in an individual in need thereof.
- 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.
- the individual is however a non-human mammal suffereing from a fungal infection, preferably a mammal selected from the group consisting of horses, cattle, dogs and cats.
- Said fungal infection may be any of the fungal infections described herein below in the section "Fungal Infections".
- the individual is an individual susceptible to fungal infections, for example an immunocompromised individual, such as an 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.
- 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 any suitable assay known to the skilled person, preferably by an assay comprising the steps of: a) providing a cytochrome P450
- 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 ⁇ of the diyne salt compared to in the absence of said diyne salt.
- said diyne salt does not significantly reduce the activity 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-UV7VIS 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,
- the substrate used is dependent on the particular cytochrome P450.
- Substrates useful for individual cytochromes P450 are well known to the skilled 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.
- 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.
- immunocompromised individuals will receive such active agents, and the pharmaceutical compositions of invention are accordingly in particular useful for treatment of immunocompromised individuals.
- 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 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.
- 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, 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, clomipra
- the present invention in one aspect relates to pharmaceutical compositions 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").
- a diyne salt such as any of the diyne salts described herein above in the section "Diyne salts”
- 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.
- the diyne salts according to the present invention are capable of killing fungi, i.e. they have fungicidal activity.
- 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.
- 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 once, for example at least twice, such as at least 3 times.
- 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.
- 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 particularly important for fungal growth under hypoxia. Thus, OLE-1 transcript levels are upregulated in fungi under hypoxia (for example in C. albicans).
- 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 conditions.
- 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.
- Said hypoxic condition is preferably an oxygen partial pressure (p0 2 ) 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.
- compositions comprising diyne salts according to the invention may be for treatment of a disseminated infection or a local infection.
- 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.
- 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.
- 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,
- oropharyngeal fungal infections such as thrush, glossitis, stomatitis or angular cheilitis
- cutaneous fungal infections such as intertrigo, diaper candidiasis, paronychia or onychomycosis
- paronychia paronychia
- onychomycosis 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 fungal infection, hepatosplenic fungal infection, endocarditis, myocarditis,
- pericarditis pericarditis, ocular fungal infection, endophthalmitis and osteo articular fungal infection.
- the diyne compounds according to the present invention are even useful for treating onychomycosis, i.e. fungal infection of the nails.
- 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.
- the fungus may be any fungus, but usually it is a pathogenic fungus, such as a fungus pathogenic in the individual to be treated.
- 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.
- 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., Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp., Zygomycetes, Malassezia spp., Hyalohyphomycetes, Dermatophytes, Epidermophyton floccosum, Microsporum spp, Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi and Madurella spp.
- 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. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis.
- Candida spp. preferably from the group consisting of C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis.
- the fungus may also be selected from the group consisting of Aspergillus spp., preferably from the group consisting of A. fumigatus, A. flavus, A. niger and A. terreus.
- the fungus may also be selected from the group consisting of Cryptococcus spp., Preferably from the group consisting of C. neoformans, C. bidus, C. laurentii, and C. fusarium. Said C. neoformans is preferably selected from the group consisting of var. neoformans and var. gattii.
- the fungus may also be selected from the group consisting of zygomycetes, preferably from the group consisting of Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle berthoUetiae, Saksenaea vasiformis, Mucor circinelloides, M. ramosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus and Syncephalastrum racemosum.
- the fungus may also be selected from the group consisting of Malassezia spp., preferably from the group consisting of M. furfur and globosa.
- 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 group consisting of S. prolificans and S. apiospermum.
- 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 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. rubrum and T. tonsurans.
- Said Microsporum spp may preferably be selected from the group consisting of M. cookei, M. canis, M. vanbreuseghemii, M. gallinae and M. gypseum.
- 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.
- the fungus may also be selected from the group consisting of Madurella spp., preferably from the group consisting of mycetomatis and M. griseum.
- the fungus may for example be selected from the group consisting of Aspergillus spp., Batrachochytrium dendrobatidis, Blastomyces spp., Branchiomyces spp., Candida spp., Cladosporium spp., Coccidioides spp., Cryptococcus neoformans, Entomophthora spp., Epidermophyton spp., Fonsecaea spp., Geotrichum spp., Histoplasma spp., Ichthyophonus hoferi, Laca
- the diyne 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.
- Said fungi may be resistant for any reason.
- particular species of fungus may be resistant to treatment with that particular antifungal agent.
- 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.
- the fungus has acquired resistance to one or more conventional antifungal agents.
- 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, which are not of formula I.
- 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
- 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 polyene antifungal agents, azole antifungal agents, allylamine antifungal agents and echinocandins.
- Polyene antifungal agents are antifungal agents with multiple conjugated double bonds. Typically, polyene antifungal agents also comprise a heavily
- Non-limiting examples of polyenes include Natamycin,
- Azole antifungal agents may for example be imidazole or triazole or thiazole antifungal agents.
- 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- limiting example of a thiazole antifungal is abafungin.
- Non-limiting examples of allylamine antifungals include Terbinafme, Amorolfine, Naftifme or Butenafme.
- Non-limiting examples of echinocandins include Anidulafungin,
- 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, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate.
- antifungal agents selected from the group consisting of benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate.
- 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.
- compositions may in addition to one or more diynes also comprise one or more antifungal agents capable of at least one of
- 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 described herein above in the section) and echinocandins (such as any of the echinocandins described herein above in the section).
- 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
- echinocandins such as any of the echinocandins described herein above in the section.
- compositions of the invention may preferably comprise a diyne compound as described herein above and a polyene antifungal agent.
- Said polyene antifungal agent may preferably be Amphotericin B. A8. Fungal infection of plants
- 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.
- a diyne salt of the formula I a diyne salt of the formula I:
- 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 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.
- Said diyne salt may in particular be a diyne salt of formula II, + F3 ⁇ 4 Y C- ⁇ C C- ⁇ C R 3
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may also be a diyne of formula III:
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may also be a diyne salt of formula IV, K + R4-(C(R 2 ) 2 ) n -X-(C(R 2 ) 2 ) m -[C ⁇ C- C ⁇ C]-R 3 wherein R4, R 2 , n, m and R 3 are as described herein above in the section "Diyne salts" in relation to formula IV.
- the diyne salt for treating or reducing the risk of infection by a fungus in a plant may also be a diyne salt of formula V,
- the diyne salt for treating or reducing the risk of infection by a fungus in a plant may preferably be selected from the group consisting of potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate and potassium 14-(furan-2-yl)tetradeca-l 1,13-diyne- diynoate.
- the plant pathogenic fungus may be selected from the group consisting of Albugo spp., Alternaria spp., Anisogramma anomala, Apiosporina morbosa, Ascochyta spp., Aureobasidium zeae, Bipolaris spp., Blumeria spp., Blumeriella jaapii, Botritis spp., Botryosphaeria dothidea, Ceratocystic paradoxa, Cercospora spp., Cercosporidium spp., Cladosporium spp., Cochiliobolus spp., Colletotrichum spp., Corynespora cassiicola, Cristulariella moricola, Diaporthe phaseolorum, Didymella bryoniae, Drechslera tritici, Entyloma oryzai, Erysiphe spp.,
- Treatment of infections by a fungus in a plant may be done by any suitable means, for example the diyne salts may be applied as sprays or dusts on the foliage of plants, or in irrigation systems.
- 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 infection and for example thereby controlling pathogens carried on them or existing in the soil at the planting site.
- plants may also be treated once an infection is already present in order to eliminate or reduce the infection, preferably eliminate the infection.
- 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 eliminate the infection.
- 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 diyne salts can be applied by either a mechanized route, e.g., a tractor, or with hand applications.
- agronomically acceptable carrier is a solid or liquid which is 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.
- 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.
- Optionally added components or additives 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.
- 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.
- concentrations of the solution after dilution may vary from 1% to 90% by weight, with a preferred range being from 5% to 50%.
- the diyne compound can be dissolved in suitable organic solvents, or a mixture of solvents, together with an emulsifying agent to enhance dispersion of the diyne compound in water.
- 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%.
- Wettable, powdered formulations suitable for spraying can be prepared by 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.
- 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 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.
- 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 synthetic sodium silicoaluminate sold under the trademark Zeolex. RTM. 7 (J. M. Huber Corporation).
- Dusting formulations may be prepared by mixing the diyne salts with finely divided inert solids which can be organic or inorganic in nature.
- 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 subsequently diluted to from 1% to 10%> use concentration.
- 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.
- 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 1 to 28 kg per hectare of the active ingredient.
- 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.
- 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.
- the diyne compounds may be applied to growing plants for example at a rate of from 0.01 to 10 kg per hectare.
- the diyne salts of the present invention may be combined with other known fungicides.
- the present invention relates to diyne compounds per se as well as to the use of the diyne compounds.
- the present invention relates to pharmaceutical compositions 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.
- the diynes according to the present invention are diynes of formula ⁇ : Z-[C ⁇ C- C ⁇ C]-R 3 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 ;
- 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 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.
- Z may preferably be selected from the group consisting of alkyl and alkenyl, which is substituted with R 4 , wherein R 4 is -COOH or a bioisostere thereof.
- 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 defined herein below in relation to diynes of formula IF.
- Said bioisostere of -COOH may for example be -CO-Ri, wherein Ri preferably is as defined herein below in relation to diynes of formula IF.
- the bioisostere of -COOH may also preferably be selected from the group consisting of tetrazoles, preferably from the group consisting of tetrazoles, tetrazolates and salts thereof.
- bioisostere of-COOH may be selected from the group of azoles, preferably the bioisostere may be a 1 ,2,4- oxadiazoie heterocycle .
- Z is a C 6 -2o, preferably a C 6-15 , more preferably C 6-12 , even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkyl or alkenyl substituted with -COOH or a bioisostere thereof and optionally also substituted at one or more positions with R 2 , preferably substituted with one or more selected from the group consisting of -CO-Ri and R 2 .
- Z is Ri-CO-(C6_ 2 o alkyl or alkenyl)-, such as Ri-CO-(C 9 _ 2 o alkyl or alkenyl)-, for example Ri-CO-(C 9 _i 5 alkyl or alkenyl)-, such as Ri-CO-(C6_i 2 alkyl or alkenyl)-.
- the diyne is a diyne of the formula IF:
- 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 F, 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 carbon atoms, even more preferably 9 carbon atoms with up to three double bonds.
- each carbon atom is linked to none, one or two R 2 groups.
- 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 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.
- 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.
- the diyne is a diyne of the formula ⁇ :
- Ri 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 4 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, 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 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.
- each carbon atom is linked to none, one or two R 2 groups.
- 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 atoms in the carbon chain by double bonds will not be linked to any R 2 groups.
- Y may be a linear C 6 - 2 o, preferably a C 6-15 , more preferably C 6-12 , even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkyl, preferably a linear C 7-1 1 alkyl, yet more preferably a linear C 8-10 alkyl, even more preferably a linear C9-alkyl.
- Y may also be a linear C 6 - 2 o, preferably a C 6-15 , more preferably C 6-12 , even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkenyl, preferably a linear C 7-1 1 alkenyl, yet more preferably a linear C 8-10 alkenyl, even more preferably a linear C9- 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 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.
- Y may be a linear C 6-12 , preferably a linear C 7-1 1 alkenyl, yet more preferably a linear C 8-10 alkenyl preferably a linear C9 alkenyl, wherein all double 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 , C9 OT Cio position, preferably at the C9 position (the C in the carbonyl group being Ci), even more preferably at least one double bond in the cis conformation is situated at the C 8 , C9 or Cio position, preferably at the C9 position (the C in the carbonyl group being Ci).
- the diyne compound is a compound of the formula IV :
- R4 is -COOH or a bioistere thereof
- 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 ⁇
- 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
- R 3 is a heterocyclic ring, preferably R 3 is any of the R 3 groups described in this section below.
- the diyne is a compound of formula V :
- the diyne is a compound of formula ⁇ , 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.
- Ri of the diyne either of formula IF, IIP or V or of formula ⁇ , 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 consisting of amines and lower linear or branched alkoxy groups. Lower alkoxy are preferably Ci_ 6 , more preferably Ci_ 3 , even more preferably Ci_ 2 alkoxy, preferably linear alkoxy.
- Ri of the diyne either of formula IF or formula ⁇ may be selected from the group consisting of -OH, -NH 2 , -OCH 3 and -OC 2 H 5 , preferably Ri is -OH.
- R 3 of the diyne of formula F, IF, IIF, 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, -N0 2 and halogen.
- Lower alkyl is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkyl.
- Lower alkenyl is preferably Ci_ 5 , more preferably Ci_ 3 , even more preferably Ci_ 2 alkenyl.
- Lower alkoxy is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkoxy.
- Lower alcohol is preferably Ci_ 5, more preferably Ci_ 3 , even more preferably Ci 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 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.
- R 3 of the diyne of formula ⁇ , IF, III', IV or V is preferably an aromatic heterocyclic ring.
- R 3 of the diyne of formula F, IF, ⁇ , 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 heterocyclic ring, even more preferably a 5 membered aromatic heterocyclic ring.
- 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 group consisting of S, N and O.
- 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 consisting of imidazole, oxazole, cyclopentadiene and triazole.
- 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 the group consisting of methyl, methoxy, hydroxyl, amine and halogen, preferably methyl.
- R 3 is furan.
- heterocyclic ring is 2-substituted with the -[C ⁇ C- C ⁇ C]- Z chain.
- 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.
- 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 -[C ⁇ C- C ⁇ C]-Z chain.
- Each R 2 of a diyne of formula IV or V as well as each R 2 when contained in diynes of formula ⁇ , IF or ⁇ 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 Ci_ 4 alkyl, Ci_ 4 alkenyl or Ci_ 4 alkynyl. It is however also possible that two neighbouring 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 R 2 carbons).
- the ring system may be aromatic or none aromatic, for example the ring system may be an 6 membered aromatic ring.
- 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 ⁇ , IF, ⁇ , IV or V is -H.
- R 2 groups of a diyne compound of formula ⁇ , IF, ⁇ , IV or V except for in the range of 0 to 5 R 2 groups preferably all R 2 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 ⁇ , IF, ⁇ , IV or V is -H.
- R 2 groups are connected to 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 Cn, preferably on C 9 and C 10 position (the C in the carbonyl group being Ci).
- a preferred diyne compound according to the invention is (Z)-12-(furan-2- yl)dodeca-7-en-9,l 1-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof.
- 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.
- Yet another preferred diyne compound according to the invention is (E)- 14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof.
- Yet another preferred diyne compound according to the invention is the diyne compound (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof.
- Another preferred diyne compound according to the invention is the diyne compound (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid or a salt thereof, preferably a pharmaceutically acceptable salt thereof.
- Yet another preferred diyne compound according to the invention is (Z)- methyl 14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate or a salt thereof, preferably a pharmaceutically acceptable salt thereof.
- the diyne compound may be selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid, (Z)-14-(5-methylfuran-2-yl)tetradeca-9-en-l 1,13-diynoic acid, 8-(2-(4-(furan-2- yl)buta-l,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-l 1,13-diynoic acid.
- the diyne compounds according to the invention may be prepared essentially as described in US patent US6, 541,506, which is hereby incorporated by reference.
- the diyne compound is a diyne compound of formula ⁇ :
- 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- (furan-2-yl)dodeca-7-en-9, 11 -diynoic acid.
- the diyne compound is not a compound according to formula VII',
- Ri 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 thiophene ring
- X is an integer between 4 and 10, inclusive.
- 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.
- the diyne compound of these embodiments may also be a diyne of formula VIIF,
- R4, 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.
- the diyne compound of these embodiments may be a diyne of formula IX':
- Ri 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 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.
- the diyne compound of these embodiments may also be a diyne of formula IV,
- the diyne compound of these embodiments may also be a diyne of formula V,
- R l s R 2 , n, X and R3 are as described herein above in the section "Diyne" in relation to formula V, with the proviso that the diyne compound is not any of the above mentioned compounds, which are preferably excluded.
- the diyne is a diyne of the formula ⁇ , wherein Z is alkyl, which optionally may be substituted.
- Z may for example be substituted with one or more selected from the group consisting of -CO-Ri and R 2 , wherein Ri and R 2 preferably are as defined herein above in relation to diynes of formula IF.
- Z may be a C 6 -2o alkyl, preferably a C 6 -i5, more preferably C6-12, even more preferably a C9-20, yet more preferably a C9-15, such as a C9-12 alkyl, optionally substituted, preferably substituted with one or more selected from the group consisting of -COOH, bioisosters of-COOH and R 2 , more preferably from the group consisting of -CO-Ri and R 2 , yet more preferably substituted with one group selected from the group consisting of -COOH and bioisoters of -COOH and with one or more selected from the group consisting of R 2 .
- bioisosters of - COOH may be any of the bioisosters of-COOH described herein above in the section “Diynes” and R 2 is as described herein above in the section “Diynes” in relation to formula IV and V.
- Z is R4-(C 6 - 2 o alkyl)-, for example R4-(C 6 -i5- alkyl), such as R4-(C6_i 2 -alkyl), for example R4-(C 9 _ 2 o-alkyl), such as R4-(C 9 _i5-alkyl), for example R4-(C 9 _i 2 -alkyl), wherein R4 is as described herein above in the section "Diynes" in relation to formula IF.
- the present invention relates to diyne compounds 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 particularly in the section “Diynes", but also in the section “Particular diynes”.
- the diynes according to the invention in particular the diyne compounds contained in the pharmaceutical compositions of the invention also have the functional properties described in detail in this section.
- 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.
- 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.
- 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 acid in a fungus comprises the steps of
- stearoyl-CoA 9 desaturase such as OLE-1
- 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.
- 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 acid to palmitoleic acid in a fungus comprises the steps of
- stearoyl-CoA 9 desaturase such as OLE-1
- 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.
- 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).
- the stearoyl-CoA desaturase is mainly located in the membranes of the endoplasmatic reticulum and accordingly it is preferred that said fungal extract comprises endoplasmatic reticulum or parts thereof.
- 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.
- fractions or extracts comprising endoplasmatic reticulum may also be obtained by other useful methods known to the skilled person.
- stearoyl- CoA desaturase may be obtained by in vitro assembly by methods known to the skilled person.
- 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.
- 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.
- Example 12B 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 substrate provided.
- 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 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.
- 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.
- the activity of said fungal stearoyl-CoA 9 desaturase may be inhibited by different means by said diyne compound.
- the diyne may directly inhibit the enzymatic activity of said fungal stearoyl-CoA 9 desaturase (such as OLE-1).
- the diyne compound may preferably be an inhibitor of the fatty acid desaturase activity of OLE- 1 polypeptide.
- Whether said diyne is capable of directly inhibiting the activity of fungal stearoyl-CoA 9 desaturase may for example be determined using an in vitro assay for fungal stearoyl-CoA 9 desaturase (such as OLE-1) activity.
- 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 preferably significantly inhibits the formation of oleic acid and/or palmitoleic acid or activated forms thereof.
- a diyne compound preferably added 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 mixture in a purified form or as part of a crude extract, for example a fungal extract or as prepared in vitro.
- Mammalian desaturases are significantly different to fungal stearoyl-CoA 9 desaturases (such as OLE-1), for example mammalian desaturases lacks an integral cytochrome bs 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.
- the diyne compound according to the invention is a selective inhibitor of a fungal stearoyl-CoA 9-desaturase (such as OLE-1).
- the diyne compound is capable of inhibiting the 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.
- the diyne compounds according to the invention are capable of reducing 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% 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.
- said diyne compound in a similar in vitro assay 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 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).
- the diyne may also indirectly inhibit the activity of said fungal stearoyl- Co A 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.
- 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 9 desaturase (such as OLE-1).
- the diyne compound may down modulate the expression of the OLE-1 polypeptide by modulating the activity of a transcriptional regulator of the gene encoding the OLE-1 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-1 promoter or enhancer region.
- Spt23p/Mga2p is a fungal transcriptional regulator that amongst others controls the expression of fungal stearoyl-CoA 9 desaturase (such as OLE-1).
- the diyne compound may be capable of inhibiting the activity of Spt23p/Mga2p.
- the diyne compounds according to the present 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
- Induction of germ tube formation from blastospores may be determined by a method comprising the steps of
- 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.
- the diyne compound is said to be capable of reducing formation and/or growth of hyphal filaments or formation of germ tubes.
- the diyne compound is said to be capable of inhibiting formation of hyphal filaments or to be capable of inhibiting hyphal growth.
- 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:
- 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.
- the diyne compound is said to be capable of reducing formation of clamydospores.
- the diyne compound is said to be capable of inhibiting formation of clamydospores.
- the MIC is the minimal concentration of diyne compound required for inhibiting essentially 100%, such as 100% growth of a fungi.
- 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.
- 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.
- the diyne compound has a minimum fungicidal concentration (MFC) of at the most 100 ⁇ g/ml, preferably at the most 50 ⁇ g/ml, yet more preferably at the most 10 ⁇ g/ml, even more preferably at the most 1 ⁇ g/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.
- said MFC for a given fungus is determined in a method comprising the steps of a) cultivating said fungus in vitro
- 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.
- the MFC may preferably be determined using the assay described herein below in Example 10.
- contacting a fungus with said diyne 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
- step e) determining the CFU/ml in said fungal culture [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, 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 fungus.
- step e) 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.
- said predetermined amount of time is at least 3 hours, such as 3 hours.
- rapid loss of viability is described in Example 11.
- 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.
- the diyne compounds according to the invention are capable of inhibiting growth of fungi.
- the IC50 indicates the concentration where 50% growth inhibition is obtained.
- 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 least 1 , preferably at least 3 different fungi.
- 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 mammals.
- 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. 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.
- 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
- Coccidioides immitis 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.
- C. neoformans for example var. neoformans or var. gattii
- C. bidus for example var. neoformans or var. gattii
- C. bidus for example var. neoformans or var. gattii
- C. bidus for example var. neoformans or var. gattii
- C. bidus for example var. neoformans or var. gattii
- vanbreuseghemii M. gallinae or M. gypseum
- 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).
- the diyne compound substantially does not significantly inhibit ergosterol synthesis; 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 ergosterol synthesis.
- 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 ergosterol produced in the absence of said diyne compound is produced in the presence of said diyne compound.
- 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 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 acetate and determining the ratio of 13 C labelled to unlabelled ergosterol.
- ergosterol synthesis may be determined as described herein in Example 16.
- the diyne compound does not significantly inhibit chitin synthase and/or ⁇ -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 ⁇ - glucan synthase.
- chitin synthase and/or ⁇ -glucan synthase activity of said fungus is inhibited by no more than 30%, preferably no more than 20%.
- Inhibition of chitin synthase and/or ⁇ - glucan synthase may for example be determined as described herein in Example 15.
- compositions comprising diyne compound according to the present invention may be in any suitable form depending on the fungal infection to be treated.
- the pharmaceutical composition may be formulated for topical administration or for systemic administration.
- the fungal infection is a local infection on a body surface
- the pharmaceutical composition is typically formulated for topical administration.
- 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.
- compositions according to the invention will frequently comprise one or more pharmaceutically acceptable excipients.
- the pharmaceutical compositions comprising diynes 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 diyne compound or pharmaceutical composition comprising a diyne compound 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 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.
- one or more diyne compounds of the invention are formulated in combination with a solid or a liquid dermato logically acceptable carrier.
- Useful solid carriers include finely divided solids such as talc, clay, micro crystalline 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 such as surfactants, and additional antimicrobial agents can be added to optimize the properties for a given use.
- 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.
- 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.
- 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 pathogens.
- 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.
- 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.
- the pharmaceutical compositions comprising diyne compounds according to the invention are formulated for administration of in the range of O.OOlmg/Kg to lOOmg/kg, preferably in the range of O.OOlmg/Kg to lOOmg/kg daily, in particular when the individual to be treated is a mammal, such as a human being.
- 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.
- concentration of the diyne compound(s) in a composition for topical administration such as a lotion
- 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 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.
- compositions according to the invention may in addition to the diyne compounds also comprise one or more additional active agents.
- Said additional active agents may for example be antifungal agents.
- the present invention relates to pharmaceutical compositions comprising diyne compounds for treatment of fungal infections in an individual in need thereof.
- 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.
- the individual is however a non-human mammal suffereing from a fungal infection, preferably a mammal selected from the group consisting of horses, cattle, dogs and cats.
- Said fungal infection may be any of the fungal infections described herein below in the section "Fungal Infections".
- the individual is an individual susceptible to fungal infections, for example an immunocompromised individual, such as an 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.
- 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 comprising the steps of: a) providing a cytochrome P450
- 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 ⁇ of the diyne compound compared to in the absence of said diyne compound.
- said diyne compound does not significantly reduce the activity 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- UV7VIS 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,
- the substrate used is dependent on the particular cytochrome P450.
- Substrates useful for individual cytochromes P450 are well known to the skilled 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.
- 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.
- 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.
- immunocompromised individuals will receive such active agents, and the pharmaceutical compositions of invention are accordingly in particular useful for treatment of immunocompromised individuals.
- 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 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.
- 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, 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, clomipra
- the present invention relates to pharmaceutical compositions comprising 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”).
- 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.
- 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 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.
- the diyne compounds according to the present invention are capable of inhibiting formation of hyphae.
- Hyphae are long, 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 are in particular useful for treating infections by a fungus associated with or dependent on formation of hyphae.
- the pharmaceutical composition comprising diyne compounds according to the invention may be prepared for inducing growth of small, rounded, compact hyphal forms rather than the hyphal growth seen in the absence of pharmaceutical composition.
- the infection by said fungus is associated with clamydospores, for example the infection by said fungus 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 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 conidia.
- 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.
- Conidia are generally asexual, non-motile spores of a fungus.
- 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 conidia may be detected by visual inspection, preferably by visual inspection with the aid of a microscope.
- 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 for treating infections by a fungus associated with or dependent on formation and/or germination of clamydospores and/or conidia.
- the pharmaceutical composition comprising diyne compounds according to the invention may be prepared for inhibition of conidiation and/or for inhibition of sporulation.
- 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 layer of fungus, which is phenotypically different from suspended fungal cells.
- bio films have a significantly decreased susceptibility to antifungal agents, including Amphotericin B and Fluconazole.
- infections by fungus associated with biofilm formation may be treated using the diyne compounds disclosed herein.
- Bio films may be formed by a mixture of unicellular fungi, hyphae and/or pseudohyphae arranged in a layer structure, for example in a bilayer structure.
- 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 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.
- fungi which may form biofilms
- Candida species such as any of the Candida species mentioned herein below, for example Candida albicans.
- the infection by fungus is associated with formation of macronodules
- the infection may cause formation of macronodules of said fungus, preferably macronodules surrounded by a perimeter of ground-glass opacity.
- macronodules and in particular macronodules surrounded by a perimeter of ground-glass opacity may for example be identified using computed tomography (CT) scanning.
- CT computed tomography
- fungi which may form such macronodules include Aspergillus species, such as any of the Aspergillus species mentioned herein below.
- Microdules may in particularly be associated with invasive pulmonary infections by fungus, such as invasive pulmonary aspergillosis.
- fungus such as invasive pulmonary aspergillosis.
- present invention discloses that infections by fungus associated with macronodule formation may be treated using the diyne compounds disclosed herein.
- the pharmaceutical compositions comprising diyne compounds according to the invention are prepared for induction of nuclear membrane collapse in one or more fungi.
- the pharmaceutical composition is prepared for inhibition of white-opaque switching.
- the pharmaceutical composition is prepared for inhibition of the morphogenetic switch between the hyphal growth form, the pseudo- hyphal growth form and the budding growth form.
- diyne compounds according to the present invention are capable of killing fungi, i.e. they have fungicidal activity as described in more detail herein above in the section "Properties of diyne 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.
- the pharmaceutical compositions comprising diyne compounds 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 once, for example at least twice, such as at least 3 times.
- 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.
- 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-1 being particularly important for fungal growth under hypoxia. Thus, OLE-1 transcript levels are upregulated in fungi under hypoxia (for example in C. albicans).
- 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 conditions.
- 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.
- Said hypoxic condition is preferably an oxygen partial pressure (p0 2 ) of at the most 140 mmHg, preferably at the most 110 mmHg, such as at the most 80 mmHg.
- compositions comprising diynes according to the invention may be for treatment of a disseminated infection or a local infection.
- 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.
- 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.
- 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,
- oropharyngeal fungal infections such as thrush, glossitis, stomatitis or angular cheilitis
- cutaneous fungal infections such as intertrigo, diaper candidiasis, paronychia or onychomycosis
- paronychia paronychia
- onychomycosis 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 fungal infection, hepatosplenic fungal infection, endocarditis, myocarditis,
- pericarditis pericarditis, ocular fungal infection, endophthalmitis and osteo articular fungal infection.
- the diyne compounds according to the present invention are even useful for treating onychomycosis, i.e. fungal infection of the nails.
- 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.
- the fungus may be any fungus, but usually it is a pathogenic fungus, such as a fungus pathogenic in the individual to be treated.
- 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.
- 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., Histoplasma capsulatum, Coccidioides immitis, Coccidioides posadasii, Cryptococcus spp., Zygomycetes, Malassezia spp., Hyalohyphomycetes, Dermatophytes, Epidermophyton floccosum, Microsporum spp, Blastomyces dermatitidis, Sporothrix schenkii, Chromomycotic fungi and Madurella spp.
- 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. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis.
- Candida spp. preferably from the group consisting of C. albicans, C. krusei, C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii, C. haemulonii, C. lusitaniae, C. lipolytica, C. norvegensis, C. viswanathii, C. kefyr and C. dubliniensis.
- the fungus may also be selected from the group consisting of Aspergillus spp., preferably from the group consisting of A. fumigatus, A. flavus, A. niger and A. terreus.
- the fungus may also be selected from the group consisting of Cryptococcus spp., Preferably from the group consisting of C. neoformans, C. bidus, C. laurentii, and C. fusarium. Said C. neoformans is preferably selected from the group consisting of var. neoformans and var. gattii.
- the fungus may also be selected from the group consisting of zygomycetes, preferably from the group consisting of Rhizopus oryzae, R. micropsorus, R. pusillus, Cunninghamelle berthoUetiae, Saksenaea vasiformis, Mucor circinelloides, M. ramosissimus, Absidia corymbifera, Apophysomyces elegans, Cokeromyces recurvatus and Syncephalastrum racemosum.
- the fungus may also be selected from the group consisting of Malassezia spp., preferably from the group consisting of M. furfur and M. globosa.
- 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 group consisting of S. prolificans and S. apiospermum.
- 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 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. rubrum and T. tonsurans.
- Said Microsporum spp may preferably be selected from the group consisting of M. cookei, M. canis, M. vanbreuseghemii, M. gallinae and M. gypseum.
- 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.
- the fungus may also be selected from the group consisting of Madurella spp., preferably from the group consisting of mycetomatis and M. griseum.
- the fungus may for example be selected from the group consisting of Aspergillus spp., Batrachochytrium dendrobatidis, Blastomyces spp., Branchiomyces spp., Candida spp., Cladosporium spp., Coccidioides spp., Cryptococcus neoformans, Entomophthora spp., Epidermophyton spp., Fonsecaea spp., Geotrichum spp., Histoplasma spp., Ichthyophonus hoferi, Lacazia loboi, Malassezia spp., Metarhizium spp., Microsporum spp., Mucor spp., Ochroconis s
- 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, 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.
- Said fungi may be resistant for any reason.
- that particular species of fungus may be resistant to treatment with that particular antifungal agent.
- 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.
- the fungus has aquired resistance to one or more conventional antifungal agents.
- 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 ⁇ .
- 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
- 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.
- Polyene antifungal agents are antifungal agents with multiple conjugated double bonds. Typically, polyene antifungal agents also comprise a heavily
- Non-limiting examples of polyenes include Natamycin,
- Azole antifungal agents may for example be imidazole or triazole or thiazole antifungal agents.
- 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- limiting example of a thiazole antifungal is abafungin.
- Non-limiting examples of allylamine antifungals include Terbinafine, Amorolfine, Naftifine or Butenafine.
- Non-limiting examples of echinocandins include Anidulafungin,
- 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, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate.
- antifungal agents selected from the group consisting of benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine, griseofulvin, haloprogin and sodium bicarbonate.
- 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 antifungal agent.
- 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.
- compositions may in addition to one or more diynes also comprise one or more antifungal agents capable of at least one of
- 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 described herein above in the section) and echinocandins (such as any of the echinocandins described herein above in the section).
- 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
- echinocandins such as any of the echinocandins described herein above in the section.
- compositions of the invention may preferably comprise a diyne compound as described herein above and a polyene antifungal agent.
- Said polyene antifungal agent may preferably be Amphotericin B.
- Said polyene antifungal agent may preferably be Amphotericin B.
- 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 Amphotericin B.
- polyene antifungal agent such as Amphotericin B
- polyene antifungal agent such as Amphotericin B
- polyene antifungal agent such as Amphotericin B
- the present invention also relates to methods of reducing the risk of an infection or to methods of treating an infection in a plant by contacting said plant with a diyne compound according to the invention.
- the invention relates to use of a diyne of the formula ⁇ : Z-[C ⁇ C- C ⁇ C]-R 3 wherein Z and R 3 are as defined herein above in the section "Diynes” 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 plant pathogenic fungus dependent on the activity of stearoyl-CoA desaturase.
- Said diyne compound may in particular be a diyne of formula IF,
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may also be a diyne of formula ⁇ :
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may also be a diyne of formula IV, R 4 -(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 R3 are as described herein above in the section "Diyne" in relation to formula IV.
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may also be a diyne of formula V,
- the diyne compound for treating or reducing the risk of infection by a fungus in a plant may preferably be selected from the group consisting of (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid and 14-(furan-2-yl)tetradeca-l 1,13-diynoic acid, and salts thereof.
- the salts thereof are preferably the salts described herein above in the section diynes.
- the plant pathogenic fungus may be selected from the group consisting of Albugo spp., Alternaria spp., Anisogramma anomala, Apiosporina morbosa, Ascochyta spp., Aureobasidium zeae, Bipolaris spp., Blumeria spp., Blumeriella jaapii, Botritis spp., Botryosphaeria dothidea, Ceratocystic paradoxa, Cercospora spp., Cercosporidium spp., Cladosporium spp., Cochiliobolus spp., Colletotrichum spp., Corynespora cassiicola, Cristulariella moricola, Diaporthe phaseolorum, Didymella bryoniae, Drechslera tritici, Entyloma oryzai, Erysiphe spp.,
- 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 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 present in order to eliminate or reduce the infection, preferably eliminate the infection.
- 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 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.
- 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.
- 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, when desired, suitable surfactants may be incorporated.
- Optionally added components or additives 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.
- 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 diluted with water.
- concentrations of the solution after dilution may vary from 1% to 90% by weight, with a preferred range being from 5% to 50%>.
- the diyne compound can be dissolved in suitable organic solvents, or a mixture of solvents, together with an emulsifying agent to enhance dispersion of the diyne compound in water.
- 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%.
- 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.
- 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 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.
- 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 synthetic sodium silicoaluminate sold under the trademark Zeolex. RTM. 7 (J. M. Huber Corporation).
- Dusting formulations may be prepared by mixing the diyne compounds with finely divided inert solids which can be organic or inorganic in nature.
- 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 subsequently diluted to from 1% to 10%> use concentration.
- the fungicidal compound 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.
- 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 1 to 28 kg per hectare of the active ingredient.
- 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, to 1000 g per hundred kg of seed.
- 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.
- the diyne compounds may be applied to growing plants for example at a rate of from 0.01 to 10 kg per hectare.
- the diyne compounds of the present invention may be combined with other known fungicides.
- Method for identification of fungitoxic compound relates to methods for identifying a fungitoxic compound.
- said method comprising the steps of,
- an indicator composition or cell comprising a gene encoding stearoyl-CoA desaturase and/or a stearoyl-CoA desaturase peptide (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;
- fungitoxic compounds capable of down modulating fungal stearoyl-CoA desaturase are superior to other clasees of antifungal agents.
- 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.
- 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.
- the invention provides methods for testing whether a test compound comprising a -COOH group or a bioisostere thereof may be useful for down modulating fungal stearoyl-CoA desaturase activity.
- the biostere of -COOH may be any of the bioisosteres of -COOH described herein above in the section "Diynes".
- the bioisostere is selected from the group consisting of -CO-Ri, wherein Ri is -OH or or a moiety that can be replaced by a hydroxyl group in a hydrolysis reaction, tetrazoles, tetrazolates and salts thereof.
- the test compound may be any of the diynes compounds described herein above in the section "Diynes”.
- the test compound may also be any other compound comprising a -COOH group or a bioisostere thereof.
- the test compound may in addition to the -COOH group or a bioisostere thereof comprise one or more carbon chains, which 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 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, i.e.
- 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.
- 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.
- the present invention provides new class of fungicides that comprise an Olel protein inhibitor.
- 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.
- inventive Olel protein inhibitors provide potent broad spectrum antifungal agents for the treatment of humans and animals against a wide variety of fungal pathogens.
- the compounds provide effective fungicides against agricultural fungal pathogens.
- inventive compounds inhibit oleic acid biosynthesis by inhibition of the Olel protein, a process that appears to be conserved 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.
- the inventive compounds exploit a mechanism of action that has so far not 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.
- Ri 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; 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.
- Ri 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 or more substituents selected from the group consisting of a Ci_ 5 alkyl, a Ci_ 5 alkenyl, a Ci_5 alkoxy, a Ci_ 5 alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive.
- R 4 is a pyrrole, furan, or thiophene ring. In other embodiments, R 4 may be an imidazole, oxazole, and cyclopentadiene.
- 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, amine, N0 2 and halogen.
- a lower alkyl is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkyl.
- a lower alkenyl is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci_ 2 alkenyl.
- a lower alkoxy is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkoxy.
- a lower alcohol is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci 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 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.
- 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.
- 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.
- 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 O.
- preferred compounds of the invention are of structure (Z)- 14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoic acid, III", and its potassium and sodium salts.
- (Z) 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 (see Example 2J_ below).
- a preferred salt compound is the sodium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoic acid, V":
- 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 II".
- This novel compound class is related to the enediyne compounds general structure Ri -C(0)-(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(0)-(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 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.
- preferred compounds of the instant invention include analogs, IX", a) Rj - C(O) - (C(R 2 ) 2 ) X - C 2 H 4 - C 4 - R4 IX" wherein Ri 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 or more substituents selected from the group consisting of a Ci_ 5 alkyl, a Ci_ 5 alkenyl, a Ci_ 5 alkoxy, a Ci_ 5 alcohol, a hydroxyl, an amine, a nitro group and a halogen; and x is an integer between 4 and 10, inclusive.
- R 4 is a pyrrole, furan, or thiophene ring. In other embodiments, R 4 may be an imidazole, oxazole, and cyclopentadiene.
- 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, amine, -N0 2 and halogen.
- a lower alkyl is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkyl.
- a lower alkenyl is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci_ 2 alkenyl.
- a lower alkoxy is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci alkoxy.
- a lower alcohol is preferably C 1-5 , more preferably Ci_ 3 , even more preferably Ci 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 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.
- 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.
- 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.
- 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 O.
- contemplated in the present invention is the unknown undisclosed acid, single-bonded analog of compound III" above, the compound 14-(furan-2-yl) tetradeca-l l,13-diynoic acid, X",
- 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.
- inventive compounds derived from or based on compound II such as salts, acids and analogs
- their potent antifungal capability through the mechanism of Olel protein inhibition, offers a superior alternative to current antifungal drug treatments and fungicides.
- a preferred diyne compound according to the invention is potassium (Z)- 12-(furan-2-yl) dodeca-7-en-9, 11-diynoate.
- Another preferred diyne compound according to the invention is potassium (Z)-13-(furan-2-yl) trideca-8-en-lO, 12-diynoate.
- Yet another preferred diyne compound according to the invention is potassium (E)-14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoate.
- Yet another preferred diyne compound according to the invention is the diyne compound potassium (Z)-14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoate.
- Another preferred diyne compound according to the invention is the diyne compound potassium 14-(furan-2-yl)tetradeca-l 1, 13-diynoate.
- the diyne compound may be selected from the group consisting of (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt; (Z)-14-(5-methylfuran-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt; 8-(2-(4-(furan-2-yl)buta-l,3-diynyl)phenyl)octanoic acid, potassium salt; (Z)-14-(4,5-dimethylfuran-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-l 1,13-diynoic acid, potassium salt.
- 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 lipids and thus essential to the fungal organism - without oleic acid the organism fails to survive due to collapse of the nuclear membrane.
- 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 Olel protein itself, or inhibited the transcriptional activators of the OLEl gene, the Mga2 and Spt23 proteins.
- the inventors subsequently determined that the addition of the inventive compounds to a culture of both Saccharomyces cerevisiae and Candida albicans, increased the expression of the OLEl gene, indicating that the inventive compounds targeted the Olel protein and not the Mga2 and Spt23 proteins (because otherwise a transcriptional down-regulation of the OLEl gene would have been seen). See Example 19 below.
- An Olel 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 homo logs to the Candida albicans OLEl gene were identified in the following organisms: Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida guillermondii, Candida lusitaniae, Aspergillus fumigatus, Aspergillus terreus, Aspergillus nidulans,
- 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).
- 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).
- 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 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, and flowering plants.
- contemplated in the present invention is a pesticide composition
- a pesticide composition comprising and plant propagation material comprising any of the inventive compounds described herein, its derivatives, salts and analogs.
- a pesticide composition comprising compound X".
- 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 effective and potent Olel protein inhibitors, in defeating or lessening agricultural fungal pathogens, providing effective and potent compounds for use in the agricultural setting.
- inventive compounds X its derivatives, salts and analogs.
- the present invention contemplates methods for preventing or controlling fungal infections in plants, parts of plants, seeds, or at their locus of growth.
- Acervuli visible to the naked eye and salmon colour, are produced in concentric circles.
- the potassium salt of 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 observed.
- M. grisea Malformation and inhibition takes place in the spore germination in M. grisea.
- 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 starts from one or two terminal cells of a three-celled spore, and the process is completed in 8hrs.
- the inventive fungacides are soluble in water.
- water solubility is important, particularly the case with infections by 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.
- 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 90 mg/ml.
- An exemplary crystalline compound is the potassium (Z)-14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoate, having a high resolution XPRD pattern comprising peaks at least at the following 2 ⁇ angles: 2.42° and 4.78°.
- the potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1, 13-diynoate crystals have a high resolution XPRD pattern comprising peaks at least at the following 2 ⁇ angles: 7.14°, 9.52° and 11.89°, or at the following 2 ⁇ angles: 2.42° and 4,78°, 7.14°, 9.52° and 11.89°, or at the following 2 ⁇ angles: 2.42°, 4,78°, 7.14°, 9.52°, 9.52°, 16.45°, 17.27°, 18.41°, 19.11°, 19.68°, 21.27°, 21.95°, 23.06°, 23.86°, 24.90°, 26.98°, 27.82°, 28.68°, 28.86° and 38.77°.
- this high resolution XPRD pattern does not comprises any other peaks with an intensity of >5%.
- 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 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.
- Melting temperature and stability 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, or that the weight change of the compound is less than 5%, preferably less than 4% at a humidity of 70%
- the inventive fungacides have a sufficiently high melting temperature to allow handling during manufacture of pharmaceutical compositions and storage at ambient temperature.
- the melting point of the compounds is at least 100°C, preferably at least 110°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.
- 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 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.
- hydrohalogenic acids e.g. hydrochloric or hydrobromic acid
- 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.
- 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.
- parenterally e.g. by intravenous, intramuscular, subcutaneous, intrathecal or transdermal administration or sublingually or as ophthalmic preparation or administered as aerosol.
- applications are capsules, tablets, orally administered suspensions or solutions, intravenous solutions, suppositories, injections, eye-drops, ointments or
- Preferred applications are oral or i.v. systemic formulations and ointment, pellet, liquid or liquid suspension topical formulations.
- the dosage used depends upon 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.
- 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 materials.
- Suppositories may be prepared by using natural or hydrogenated oils, waxes, fatty acids (fats), liquid or half-liquid polyols etc.
- 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.
- Fungal infections may be resistant to treatment for many reasons, resistant to treatment with a particular antifungal agent, or because of acquired resistance.
- 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;
- 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 comprise a heavily hydro xylated 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, sulconazole or tioconazole.
- Triazole antifungal agents may for example include fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole, and abafungin. Allylamine antifungals include Terbinafme, Amorolfme, Naftifine or Butenafme. Non-limiting examples of echinocandins include
- 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 biological systems, pathway redundancies ensure robust signaling of essential processes.
- 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 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 ⁇ 0.004) and "organelle organization and biogenesis” (p ⁇ 0.04) were significantly overrepresented.
- 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 homo log.
- 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 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).
- ERAD endoplasmatic reticulum associated protein degradation
- OLE1 transcriptional levels were determined to distinguish between these two hypotheses, and to generally confirm the oleic acid biosynthesis inhibitory model.
- OLEl transcript levels were calibrated to the expression of the tubulin gene (TUB1) and normalized to the OLEl expression at 10 min without compound addition ( Figure 17(a)).
- OLE1 in C. albicans was investigated in response to the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-l 1,13-diynoic acid (Knechtle, P. and Greve, K., "EV-086-3314K up-regulates OLE1 expression in Candida albicans " REP-S-EV-086-3314-0092 ed.(Evolva, 2009)).
- the potassium salt of (Z)-14-(furan-2-yl) tetradeca-9-en-l 1, 13-diynoic acid is similar to oleic acid.
- the molecules share a ⁇ 9- monounsaturated decanoic acid group characteristic for unsaturated fatty acids in fungi.
- (Z)-14-(furan-2-yl) tetradeca-9-en-l 1,13-diynoic acid might therefore be 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).
- Example 1 The invention is furthermore illustrated by the following non-limiting examples.
- Example 1 The invention is furthermore illustrated by the following non-limiting examples.
- XRPD X-Ray Powder Diffraction
- Step size 0.05 °2 ⁇
- DSC Differential Scanning Calorimetry
- the DSC trace shows a melting temperature with an onset of 45 °C, then an exotherm of degradation with an onset of 171 °C.
- TGA Thermo-Gravimetric Analysis
- Solubility in water was determined. This was carried out by transferring 10 mg of the material into 1.5 ml HPLC vials. Then 10 ⁇ (lvol) of water was added. The sample was then isothermally held at 50 °C with stirring for 30mins. The process was continued up to 50 ⁇ by 10 ⁇ increments with heating to 50°C in between. The material however still did not dissolve.
- a ⁇ - ⁇ continuous scan mode was employed with a sample - detector distance of 20 cm which gives an effective 2 ⁇ range of 3.2 ° - 29.7 °. Typically the sample would be 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.
- Samples run under ambient conditions were prepared as flat plate specimens using powder. Approximately 1-2 mg of the sample was lightly pressed on a glass slide to obtain a flat surface.
- 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), ⁇ -2 ⁇ goniometer, and divergence 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. Samples were run under ambient conditions as flat plate specimens.
- Step size 0.05° 2 ⁇
- 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 159°C followed by gross decomposition peaking at 192.5°C.
- 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 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 sample solutions were injected.
- 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 HPl 100 series system equipped with a diode array detector and using ChemStation software VB.02.01-SR1.
- 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 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).
- 10-20 mg of sample was placed in a tared mesh stainless steel basket under ambient conditions.
- the sample was loaded and unloaded at 40 %RH and 25 °C (typical
- the example describes attempts to prepare crystalline sodium, arginine or lysine salts of (Z)-14-(furan-2-yl)tetradeca-9-en-l l,13-diynoic acid.
- DJP401-024- 13 MeCN Amorphous DJP401-024- 14 IPA + 5% H20 Amorphous
- This example describes preparation of a potassium (Z)-14-(furan-2- yl)tetradeca-9-en- 11 , 13-diynoate.
- (Z)-14-(furan-2-yl)tetradeca-9-en-l l,13-diynoic acid may be prepared by any useful method, for example as described in US6,541 ,506.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate was kept as a crystalline solid for 6 month at 2-8°C.
- the content of potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate was determined by HPLC against a reference sample 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)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate had not significantly changed. Also no change in the impurity profile was observed after 6 months storage.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate was also kept as a crystalline solid for 6 month at 25°C, 60%RH. Analysis was performed as described above. After 6 months of storage characteristics of the solid has not changed and it was still a beige solid. Furthermore, the potassium (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoate content had not changed significantly, as analysed by HPLC. Also no change in the impurity profile was observed after 6 months storage.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate was also kept as a crystalline solid for 6 month at 40°C, 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-l 1,13-diynoate to 96% was observed as analysed by HPLC.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate dependent inhibition of the OLE-1 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 generated by enzymatic digestion and lysis through polycarbonate filters. Differential centrifugation is used to enrich for endoplasmatic reticulum fractions.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate dependent inhibition of OLE-1 is determined by the addition of different amounts of potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate to the reaction mixture.
- Amphotericin B and potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13- diynoate were added to an in vitro culture of different fungi in various concentrations 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.
- Potassium (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoate clearly inhibits growth of both Candida glabrata, Candida parapsilosis and Candida albicans at a concentration of 2 ⁇ g/ml and even at lower concentrations (see fig. 5).
- 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 fumigatus and to a lesser extent on Candida parapsilosis.
- MIC determinations of (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt and 14-(furan-2-yl)tetradeca-l 1,13-diynoic acid against fungi were performed according to the NCCLS M27A standard (NCCLS. Reference Method 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.
- the inoculum size was 0.5 - 2.5 x 10 3 colony- forming units (CFU)/ml. Incubation time and temperature were 35° C and 24 firs.
- Example 10 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 50 (i.e. concentration required for 50% inhibition).
- MIC and MFC minimum inhibitory and minimum fungicidal concentration
- IC 50 i.e. concentration required for 50% inhibition.
- FLU Fluconazole
- TERB Terbinafine
- 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 rubrum, T. mentagrophytes, T. tonsurans, T. terrestre, Epidermophyton floccosum, Microsporum canis, M. cookie, M. gallinae, M. gypseum, and vanbreuseghemii.
- MIC Minimum Inhibitory Concentration
- the inoculum size was 0.5 - 2.5 x 10 3 colony- forming units (CFU)/ml. Incubation time and temperature were 35° C and 24 firs. (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, 35° C and 4 days, respectively. The MIC endpoint was 90% inhibition as compared to the growth control for all strains. c. Minimum Fungicidal Concentration (MFC) Testing
- Table 13 lists the MIC and MFC data for Candida and Malassezia strains obtained as described above.
- (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13- diynoic acid MIC range for all Candida strains was ⁇ 0.00006 - 4.0 ⁇ g/ml, as compared with 0.125 - 16 ⁇ g/ml for FLU.
- the MIC of (Z)-14-(furan-2-yl)tetradeca- 9-en-l 1,13-diynoic acid against the Malassezia strain was ⁇ 0.00006 ⁇ g/ml, as compared to 0.125 ⁇ g/ml for FLU.
- Table 14 is a summary of the activity of (Z)-14-(furan-2-yl)tetradeca-9-en- 11,13-diynoic acid against dermatophytes.
- the MIC range against all dermatophyte strains was ⁇ 0.00006- 1.0 ⁇ g/ml, as compared to 0.002 - 4 ⁇ g/ml for TERB.
- (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid demonstrated potent activity against the nine T. rubrum strains with elevated TERB MICs (4.0 ⁇ g/ml) tested.
- the MFC range for (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid against dermatophytes was ⁇ 0.00006 - 16 ⁇ g/ ml.
- (Z)-14-(furan-2-yl)tetradeca-9-en-l 1,13-diynoic acid demonstrated potent activity against all fungal isolates tested.
- the MICs for (Z)-14-(furan-2-yl)tetradeca- 9-en-l 1,13-diynoic acid would be within the susceptible range established for FLU vs.
- Candida strains ⁇ 8 ⁇ g/ml.
- Table 18 shows the minimal inhibitory concentrations of (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid, potassium salt and (Z)-14-(furan-2-yl)tetradeca- 9-en- 11 , 13-diynoic acid versus reference Candida strains.
- the diyne used in this example is (Z)-14-(furan-2- yl)tetradeca-9-en-l 1,13-diynoic acid.
Abstract
Description
Claims
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EP10754715A EP2563769A1 (en) | 2010-04-30 | 2010-09-08 | Diyne compositions |
AU2010352387A AU2010352387B2 (en) | 2009-07-10 | 2010-09-08 | Diyne compositions |
CA2797001A CA2797001A1 (en) | 2010-04-30 | 2010-09-08 | Diyne compositions |
US13/345,300 US8722910B2 (en) | 2009-07-10 | 2012-01-06 | Diyne compositions |
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US61/346,381 | 2010-05-19 |
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WO2023001905A1 (en) | 2021-07-20 | 2023-01-26 | Selmod Gmbh | Novel antifungal compounds |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US6541506B1 (en) | 1999-10-07 | 2003-04-01 | Phytera Incorporated | Antifungal enediynes |
WO2011006061A1 (en) * | 2009-07-10 | 2011-01-13 | Evolva Sa | Diyne compositions |
-
2010
- 2010-09-08 CA CA2797001A patent/CA2797001A1/en not_active Abandoned
- 2010-09-08 WO PCT/EP2010/063161 patent/WO2011134538A1/en active Application Filing
- 2010-09-08 AU AU2010352387A patent/AU2010352387B2/en not_active Ceased
- 2010-09-08 EP EP10754715A patent/EP2563769A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6541506B1 (en) | 1999-10-07 | 2003-04-01 | Phytera Incorporated | Antifungal enediynes |
WO2011006061A1 (en) * | 2009-07-10 | 2011-01-13 | Evolva Sa | Diyne compositions |
Non-Patent Citations (10)
Title |
---|
A. R. GENNARO A R.,: "Remington: The Science and Practice of Pharmacy(19th ed.)", 1995, MACK PUBLISHING COMPANY |
GRANDJEAN ET AL: "First total synthesis of optically pure methyl (2Z, 8S, 9R)-8,9-epoxydeca-4,6-diyn-2-en-10-hydroxy-1-oate and its acetate, two naturally-occurring antifeedants", TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 33, no. 37, 8 September 1992 (1992-09-08), pages 5355 - 5358, XP022084186, ISSN: 0040-4039, DOI: DOI:10.1016/S0040-4039(00)79091-5 * |
KRAUS, P. R. ET AL.: "Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray", EUKARYOT.CELL, vol. 3, no. 5, 2004, pages 1249 - 1260 |
KRISHNAMURTHY ET AL., MICROBIOLOGY, vol. 150, 2004, pages 1991 - 2003 |
KRISHNAMURTHY, S. ET AL.: "Dosage- dependent functions of fatty acid desaturase Olelp in growth and morphogenesis of Candida albicans", MICROBIOLOGY, vol. 150, 2004, pages 1991 - 2003 |
MARTIN, C. E. ET AL.: "Regulation of long chain unsaturated fatty acid synthesis in yeast", BIOCHIM. BIOPHYS.ACTA, vol. 1771, no. 3, 2007, pages 271 - 285 |
MOGGIO I ET AL: "Vibrational properties of novel diacetylenic monomers", JOURNAL OF THE CHEMICAL SOCIETY, PERKIN TRANSACTIONS 2: PHYSICAL ORGANIC CHEMISTRY, ROYAL SOCIETY OF CHEMISTRY, no. 10, 1 January 1998 (1998-01-01), pages 2249 - 2254, XP002595203, ISSN: 0300-9580 * |
OH, C. S. ET AL.: "Candida albicans Spt23p controls the expression of the Olelp Delta9 fatty acid desaturase and regulates unsaturated fatty acid biosynthesis", J.BIOL.CHEM., vol. 281, no. 11, 2006, pages 7030 - 7039 |
STUKEY, J. E. ET AL.: "Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae", J BIOL CHEM, vol. 264, no. 28, 1989, pages 16537 - 16544 |
WILSON, R. A ET AL.: "Two Delta9-stearic acid desaturases are required for Aspergillus nidulans growth and development", FUNGAL.GENET.BIOL., vol. 41, no. 5, 2004, pages 501 - 509 |
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WO2023001905A1 (en) | 2021-07-20 | 2023-01-26 | Selmod Gmbh | Novel antifungal compounds |
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EP2563769A1 (en) | 2013-03-06 |
CA2797001A1 (en) | 2011-11-03 |
AU2010352387A1 (en) | 2012-02-02 |
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