Antifungal compounds and methods for making the same

Abstract

The invention discloses 4-((1R, 2S, 7S)-4,7-dimethyl-1-((E)-prop-1-en-1-yl )-1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl) -7,8-dihydroxybenzofuro [3,2-c] pyridin-3(2H)-one (ilicicolin K), formulations and methods for production thereof.

Description

[0001] Antifungal compounds and methods for making the same

[0002] The present invention relates to new antifungal compounds and methods for making the same.

[0003] Since ancient times, mankind has valued natural products for their therapeutic potential. While plants have long been recognized for their medicinal value, fungi, often overlooked, harbor a remarkably abundant reservoir of potentially beneficial compounds .

[0004] The incidence of invasive fungal infections has been dramatically increasing over the years. In particular, Candida species (spp.) are the fourth most common cause of nosocomial bloodstream infections in the United States. Other fungal pathogens such as Aspergillus spp., Mucorales, Fusarium spp., Cryptococcus spp., Histoplasma spp., and Scedosporium spp. have become more common at causing invasive infections. There are multiple reasons for this increased incidence, including the use of broad-spectrum antibiotics, central venous catheters, and prosthetic devices. Additionally, patients with burns and neutropenia or those who are the recipients of parenteral nutrition, renal replacement therapy, immunosuppressive therapy, and antineoplastic agents can also be predisposed to fungal infections. Invasive fungal infections can have a significant impact on patient morbidity and mortality.

[0005] Azole antifungals have been used in clinical practice to treat various fungal infections. They are categorized into three distinct classes: the imidazoles, the tetrazoles, and the triazoles. The imidazoles include several agents, most notably clotrimazole, ketoconazole, and miconazole. The tetrazoles include oteseconazole and VT-1598. The triazoles include fluconazole, itraconazole, terconazole, voriconazole, isavuconazole, and posaconazole . As a class, they exert their effect by impairing the synthesis of ergosterol, a vital component in the fungal cellular membrane. This effect occurs through the inhibition of CYP450, which converts lanosterol to ergosterol, resulting in increased cellular permeability and leakage of cellular contents as well as inhibition of fungal growth. However, strains resistant to these antifungal agents have been identified. Given the increase in the occurrence of drug-resistant fungal strains, especially in the context of common species of Aspergillus spp., as well as rare and hard-to- treat molds including Fusarium spp., Scedosporium spp., and molds from the Mucorales order, there is still a need in the art for new antifungal therapies against these fungal pathogens.

[0006] Also in agriculture, the use of antifungal agents to kill or prevent the growth of undesirable plant pathogenic organisms has been studied extensively. Although a high number of antifungal agents exists, the existing antifungal agents used in the field have drawbacks. For example, they can be very toxic and difficult to handle and not environmentally friendly, which limits their use. In addition, also in agriculture the problem of fungicide resistance may occur. Fungicide resistance occurs when a product is no longer effective at controlling a disease due to a shift in the genetics of the target pathogen organism. Fungicide resistance is due to natural selection of spores with less sensitivity due to either mutation or sexual recombination. It can be a very serious problem where fungicide resistance develops in a plant pathogen population .

[0007] Among the antifungal agents derived from fungi, the natural product ilicicolin H (Fig. 1) was discovered in 1971 as an antibiotic from the imperfect fungus Cylindrocladium ilicicola.

[0008] Known as a potent and broad-spectrum antifungal compound that specifically inhibits the cytochrome bcl complex (reductase) , the agent was also applied in several in vitro studies for treatment of various cancer cell lines (DLD-1 colon adenocarcinoma and A549 non-small cell lung carcinoma, PC-3 and 22Rvl prostate carcinoma, Huh7 and HepG2 hepatocellular carcinoma) . Notably, ilicicolin H acts much more potent against fungi compared to mammalian cells, with half maximal inhibitory concentrations (IC50) of 2 - 3 ng mlr1for C. albicans MY1055 NADH : cytochrome bcl reductase compared to 2000 - 5000 ng mlr1for rat liver cytochrome bcl reductase!. Although ilicicolin H appeared as a promising treatment against yeast strains, it only showed modest efficacy in vivo mouse models, which was attributed to high plasma protein binding. Several attempts were undertaken to improve the efficacy by chemically modifying its structure, however, with limited success.

[0009] Ilicicolin H is produced by several fungi during standard cultivation, e.g. Cylindrocladium ilicicola ( strain MFC-870 ) , Gli- ocladium roseum, Nectria sp . B13, and Neonectria sp. DH2, and many additional fungi seem to possess the corresponding genes. The biosynthetic gene cluster (BGC) of ilicicolin H was discovered by two groups independently in 2019. Zhang et al. (J Am Chem Soc 2019, 141 (14) , 5659-5663) and Lin et al . (Molecules 2019, 24 (12) , 2267) heterologously expressed the BGCs from Talaromyces varlablle and Neonectria sp . DH2, respectively in Aspergillus nidulans. Recently, Shenouda et al. (Journal of Fungi 2021, 7 (12) , 1034) heterologously expressed the ilicicolin H BGC from Trichoderma reesei in A. oryzae. The BGCs of all three fungi contain similar genes with a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) core gene (Fig. 2 and Table 1) .

[0010] Table 1. Proteins in the ilicicolin H BGC, comparison to other strains .

[0011] Protein Enzyme JGI Protein Homolog in Homolog in designa- function ID in T. Neonectria T.s variation reesei QM6a / sp. DH2 (% bilis HXQ-H-

[0012] RutC30 identity) 1 (% identity)

[0013] TriliA PKS-NRPS 58285 / 128011

[0014] (59.8 %)

[0015] TriliB Enoyl re- 58289 / 74247 HIB (65.3 %) IccB ductase (68.6 % )

[0016] Trilic Cy- 58953 / 33667Ilic (70 > 7 %jIccCtochrome (69.4 %)

[0017] P450

[0018] TriliD Diels-Al- IliD (55.6 %) IccD

[0019] 75073 derase (51.8 % )

[0020] TriliE Epimerase76204

[0021] TrlllR Tran-72993 scription factor

[0022] All three groups obtained mostly matching results, resulting in the following model for the biosynthetic pathway: first, the polyketide synthase (PKS) part of IccA / IliA assembles the polyketide backbone, supported by the enoyl reductase IccB / IliB, and adds the methyl groups. The NRPS part of IccA / IliA then adds a tyrosine. Following a Dieckmann condensation, a tetramic acid intermediate is released. This moiety is converted to the pyridone part of ilicicolin H by the cytochrome P450 IccC / IliC via a ring expansion (Fig. 1, (3) ) . Next, the Diels-Alderase IccD / IliD is suggested to catalyze the intramolecular Diels-Alder reaction yielding the decaline moiety (Fig. 1, (2) ) of ilicicolin H. Up to this step, the results of the three groups concur.

[0023] Lin et al., 2019, and Shenouda et al., 2021, report that these four enzymes from Neonectria sp . DH2 and T. reesei, respectively, are sufficient to obtain ilicicolin H in their heterologous expression experiments, whereas Zhang et al., 2019, demonstrate that IccE is necessary to catalyze the epimerization of 8-epi-ilicico- lin H (2) to ilicicolin H (1) in vivo and in vitro. Lin et al., 2019, speculate that IccD and IliD differ, resulting in the formation of 8-epi-ilicicolin H and ilicicolin H, respectively. The epimerization appears to be pH-dependent and might thus be a result of different cultivation or extraction conditions. Further, the possibility may be considered that a host enzyme is responsible for the epimerization. It is unclear whether Zhang et al., 2019, and Lin et al., 2019, used the same A. nidulans strain, since Lin et al., 2019, did not provide a strain designation. Importantly, there were other differences observed in the studies of these two groups that might be the result of different hosts. Most prominently, the heterologous expression conducted by Lin et al., 2019, did not only produce ilicicolin H but also a shunt product with similar antifungal activities, ilicicolin J (Fig. 1, (4) ) . Simi- larily, Shenouda et al., 2021, reported on several acetylated shunt products during the heterologous expression of the T. reesei BGC in A. oryzae which they explained by unexpected shunt pathways as a result of the native metabolism of the production host.

[0024] Singh et al. (Tetrahedron Letters, 52 (2011) , 6190-6191) report the biotransformation of ilicicolin H. Liu et al. (Tetrahedron Letters 46 (2005) , 8009-8012) disclose a new method for the synthesis of 1 , 4 , 5-oxadiazocines and its application in the structure modification of natural products. Singh et al. (Med. Chem. Lett. 3 (2012) , 814-817) analyse the antifungal spectrum, in vivo efficacy, and structure-activity relationship of ilicicolin H. WO 2017 / 075527 Al discloses the use of ilicicolin H in agriculture. Zhang et al. (J. Am. Chem. Soc. 141 (2019) , 5659-5663) report the enzyme-catalysed inverse-electron demand Diels-Alder reaction in the biosynthesis of ilicicolin H. Shenouda et al. (J. Funghi 7 (2021) , 1034) report that T. reesei contains a biosynthetic gene cluster that encodes ilicicolin H. Hayakawa et al. (J. Antib. 24 (1971) , 653-654) describes the ilicicolin antibiotics from Cylindrocladium ilicicola. Kildgaard et al. (Mar. Drugs 15 (2017) , 253) report a dereplication and bioguided discovery approach to reveal new compounds from a marine-derived fungus Stilbella fime- taria. Suntar et al. (Biotechnol. Adv. 50 (2021) , 107768) refers to biotechnological aspects in the bioproduction process of natural products and biopharmaceuticals. In the agricultural industry there is a need to control plant pathogens that would otherwise destroy crops and reduce yields. Farmers have traditionally employed a variety of methodologies to control these pests, one of which has been the use of compounds with antimicrobial activity, called fungicides, to protect the plants and prevent crop damage. There are many types of plant diseases, and foreign microorganisms can be introduced into new areas causing disease outbreaks. Current fungicides have traditionally been chemically synthesized compounds that have limited spectrum of activity and often require repeated usage creating a cause for environmental concern. These concerns include contamination of foodstuffs, soil, surface and ground water and their impact on native microbial and insect populations. In addition, pathogens have been able to develop resistance to conventional fungicides, and as a result the industry is consistently searching for new chemical compounds with new modes of action to combat disease resistance. WO 2017 / 075527 Al disclose the use of ilicicolin H as an active ingredient in an agricultural composition with an activity against plant pathogenic fungi as an agricultural fungicide to treat, prevent, or control fungal infections in agricultural products, such as plants and seeds. WO 2021 / 202951 Al discloses an agricultural formulation comprising ilicicolin H, hydroxy ilicicolin H, ilicicolin I, or a lamellicolic anhydride .

[0025] It is an object of the present invention to provide pharmaceutical, veterinarian and agricultural preparations and formulations useful in the treatment of human, animal and plant diseases. Preferably, compounds with improved antifungal properties should be provided. It is a preferred object of the present invention to provide a new ilicicolin variant with antibiotic and / or antifungal properties .

[0026] Therefore, the present invention provides 4- ( ( 1R, 2S , 7S ) -4 , 7- dimethyl-1- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydro- naphthalene-2-carbonyl ) -7, 8-dihydroxybenzofuro [3, 2-c] pyridin-3 (2H) -one, a novel ilicicolin variant (also referred herein under the term "ilicicolin H variant according to the present invention" and "ilicicolin K") which may be extracted from Trichoderma reesei after high-yield expression of ilicicolin H by BGC activation. This variant according to the present invention may also be synthetically produced by straightforward organic chemistry.

[0027] This ilicicolin H variant according to the present invention (ilicicolin K) turned out to be useful in antifungal compositions that inhibit fungal growth which have both medical and non-medical utility and applications, especially in pharmaceutical and / or agricultural preparations and formulations useful, preferably in the treatment of human, animal and plant diseases caused by fungal pathogens. Within the present invention, the term "pharmaceutical" relates to the veterinarian field, unless the pharmaceutical disclosure is explicitly and exclusively related to the human pharmaceutical use. It is, however, clear that the use for human med- ical / pharmaceutical use is the preferred embodiment for each and every disclosure for pharmaceutical use. More specifically, the ilicicolin H variant according to the present invention turned out to ilicicolin H have improved anti-fungal properties compared to ilicicolin H as disclosed in WO 2017 / 075527 Al. The ilicicolin H variant according to the present invention is therefore a new ilicicolin variant with significant antibiotic and / or antifungal properties. The ilicicolin H variant according to the present invention is therefore useful as a pharmaceutical and / or an agricultural fungicide to treat, prevent, or control fungal infections in human or animal patients or in agricultural products, such as plants and seeds.

[0028] The present invention also relates to an antifungal preparation comprising 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) -prop-l-en-1- yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8-dihy- droxybenzofuro [3,2-c]pyridin-3 (2H) -one .

[0029] The present invention also relates to an antifungal preparation comprising 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) -prop-l-en-1- yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8-dihy- droxybenzofuro [3, 2-c] pyridin-3 (2H) -one, and an agriculturally or and a pharmaceutically acceptable carrier or excipient.

[0030] The present invention also relates to an agricultural composition comprising an agriculturally effective amount of 4- ( (lR,2S,7S)-4, 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8- dihydroxybenzofuro [3, 2-c] pyridin-3 (2H) -one and an agriculturally acceptable carrier.

[0031] The present invention also relates to a food composition comprising an antifungally effective amount of 4- ( ( 1R, 2S , 7S ) -4 , 7-di- methyl-1- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaph- thal ene- 2- carbonyl ) -7, 8 -dihydroxybenzofuro [3, 2-c] pyridin-3 (2H) - one and a food composition which needs to be conserved from fungal activity. .

[0032] According to a preferred embodiment, the preparation or composition according to the present invention further comprising one or more further antifungal agents, and / or one or more antibiotic agents, and / or one or more diluents, and / or one or more binders or binding agents, and / or one or more dispersing agents, and / or one or more emulsifying agents, and / or one or more surfactants or wetting agents, and / or one or more sticking agents, and / or one or more thickening agents, and / or one or more pH adjusters or buffering agents, and / or one or more nutrients, and / or one or more plant growth regulators, and / or one or more herbicides, and / or one or more pesticides, and / or one or more insecticides, and / or one or more acaricides, and / or physiologically acceptable fluids, especially water, physiological saline, balanced salt solutions, aqueous dextrose, and / or glycerol; and / or pharmaceutical grades of mannitol, lactose, starch or magnesium stearate, and / or one or more suspending agents, and / or one or more propelling agents, and / or one or more stabilizing agents.

[0033] According to another aspect, the present invention relates to ilicicolin K or the preparation or composition according to the present invention, for use in the treatment and prevention of a fungal disease, especially of the human or animal body or of plants, associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides , Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera.

[0034] The present invention therefore also concerns ilicicolin K for use in a therapeutic or prophylactic treatment of the human or animal body, preferably for the prevention or treatment of a fungal disease associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides , Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera.

[0035] According to another aspect, the present invention relates to a method of treating or preventing a fungal disease or disorder, preferably for the prevention or treatment of a fungal disease associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides, Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera, wherein an effective amount of ilicicolin K is administered to a human or animal patient, preferably a human patient, in an effective amount.

[0036] With the present invention, a new ilicicolin variant is provided (ilicicolin K) . This enables this new compound also synthesized by organic chemistry. The present invention therefore also concerns a method for production of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) -7, 8-dihydroxybenzofuro [3, 2-c] pyridin-3 (2H) -one, wherein a suitable precursor of ilicicolin K is finished to ilicicolin K by a suitable chemical step. In doing so, the established chemistry of ilicicolin H synthesis can be analogously applied. The chemical total synthesis of ilicicolin H is disclosed e.g. in Williams et al. (J. Org. Chem. 50 (1985) , 2807-2809) . This method can also be applied for total synthesis of ilicicolin K according to the present invention and its novel molecular variants (see below) by specifically closing the furan ring at C4 of the pyridine ring, preferably after any one of decalin synthesis (13, 14 and 15) in Williams et al., 1985. Suitable derivatives may be synthesized preferable by the analogous application of the methods disclosed by Singh et al. (Tetrah. Lett. 52 (2011) , 6190-6191; Bioorg. Med. Chem. Lett. (2013) , 3018-3022) for ilicicolin H. For example, ilicicolin K can be delivered by biometric synthesis from ilicicolin H as disclosed in Figs. 10 and 11. The desired cyclization reaction and oxidative dearomatisation of the phenol is clearly responsible and is emulated for the key intramolecular C-0 bond formation. The full sequence from ilicicolin H to dearomatized, cyclised intermediate is possible in one-pot, by then changing reaction conditions to allow for elimination of the labile allylic ether norhydroxy-ilicicolin K can be accessed in 1 step. To reach ilicicolin K a regioselective functionalisation of the arene next to the phenol is required, there is a broad body of literature on the subject, and both direct oxidation protocols and multi-step tactics available. Exemplary reactions to be applied in this process where similar transformations were successfully achieved and applied in synthesis are disc in Organic Letters, 2024, vol. 26, # 12, p. 2376 - 2380, Journal of Organic Chemistry, 2019, vol. 84, # 1, p. 346 - 364, Journal of Organic Chemistry, 2018, vol. 83, # 12, p. 6776 - 6782, Journal of the American Chemical Society, 2013, vol. 135, # 18, p. 6774 - 6777, Journal of the American Chemical Society, 2010, vol. 132, # 35, p. 12203 - 12205, Tetrahedron, 2007, vol. 63, # 19, p. 4052-4060, and Tetrahedron Letters, 2004, vol. 45, # 11, p. 2293 - 2295.

[0037] According to another aspect, the present invention also relates to a method for production of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) -7, 8-dihydroxybenzofuro [3, 2-c] pyridin-3 (2H) -one (ilicicolin K) , wherein mycelium of an ilicicolin K-expressing fungi is lysed and extracted, preferably by a one-phase extraction, into an organic, preferably a polar, extraction agent, more preferred an extraction agent comprising acetonitrile, especially an extraction agent comprising acetonitrile, methanol and water; and / or extracted, preferably by a two-phase extraction, into diethyl ether, whereafter the extract is subjected to HPLC chromatography to enrich ilicicolin K, preferably to HPLC chromatography by a C-18 column, to obtain an ilicicolin K-comprising eluate.

[0038] Preferably, the method according to the present invention applies as ilicicolin K-expressing fungi Trichoderma reesei, especially Trichoderma reesei wherein the biosynthetic gene cluster (BGC) of ilicicolin is activatedby overexpressing the BGC transcription factor under the constitutive tefl promotor in T. reesei.

[0039] The present invention therefore also concerns a composition comprising ilicicolin K, preferably a pharmaceutical composition, especially a parenteral formulation, a tablet, powder, pill, or capsule, a liquid formulation, a lozenge, an aerosol composition, an ampule, a suppository, a retention enema, a cream, an ointment, a depot preparation, and / or a microsphere preparation; an agricultural composition, especially an emulsifiable concentrate, an emulsion concentrate, a suspension concentrate, a soluble liquid, an oil-based suspension concentrate, and / or a suspoemulsion; or a disinfectant composition, especially a surface or consumer product .

[0040] The present invention also relates to a pharmaceutical composition comprising ilicicolin K for use in the treatment and prevention of fungal disease or disorder, wherein the composition is administered via a route selected from oral, topical, vaginal, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, via transmucosal absorption, or via catheter delivery, wherein the amount of ilicicolin K in the composition is preferably from 10 ng to 10 g, more preferred from 10 mg to 8 g, especially from 500 mg to 5 g .

[0041] According to another aspect, the present invention relates to the use of ilicicolin K as an antifungal conserving agent for food product .

[0042] The present invention is therefore concerned with pharmaceutical and / or agricultural compositions of the ilicicolin H variant according to the present invention comprising an effective amount of the antifungal compound and a pharmaceutically or an agriculturally acceptable carrier. Such compositions may additionally comprise one or more excipients selected from the group consisting of (a) one or more diluents, (b) one or more binders or binding agents, (c) one or more dispersing agents, (d) one or more emulsifying agents, (e) one or more surfactants or wetting agents, (f) one or more sticking agents, (g) one or more thickening agents, and (h) a pH adjuster. In one embodiment the surfactant is an anionic surfactant. In another embodiment the surfactant is a nonionic surfactant.

[0043] The ilicicolin H variant according to the present invention and pharmaceutical, veterinarian and agricultural compositions thereof may also be used in combination with one or more other agents useful to treat, prevent, or control diseases, preferably diseases caused by fungal pathogens, especially human diseases caused by fungal pathogens, or agricultural pests in the field. In such combinations the compositions of the present invention and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of the other agent (s) . Examples of other active ingredients that may be administered in combination with compositions of the present invention, and either administered separately or in the same pharmaceutical or agricultural composition.

[0044] Preferred other active ingredients in pharmaceutical compositions according to the present invention include pharmaceutically useable carriers or excipients. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or, as the case may be, an animal without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable ben- efit / risk ratio. The pharmaceutically acceptable carriers useful in this disclosure are conventional. Accordingly, compositions and formulations suitable for pharmaceutical delivery of ilicicolin K are known to the person skilled in the art and will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms) , conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0045] For oral administration, the pharmaceutical compositions of ilicicolin K may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate) ; lubricants (e.g., magnesium stearate, talc or silica) ; disintegrants (e.g., potato starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulphate) . Tablets can be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats) ; emulsifying agents (e.g., lecithin or acacia) ; non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) ; and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active composition. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compositions for use according to the embodiments are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (propelling agents) , e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetraf luoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0046] The ilicicolin K containing compositions according to the present invention can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0047] The present compositions can also be formulated for rectal administration such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0048] The ilicicolin K containing compositions can also be formulated for vaginal administration such as suppositories or creams, e.g., containing conventional suppository bases such as cocoa butter or other glycerides or cream bases such as mineral oil combined with emulsion stabilizers or white soft paraffin.

[0049] The ilicicolin K containing compositions can be formulated for topical application to susceptible organisms, especially for human patients, such as topical creams and ointments in the case of animals, or as sprays, fogs, mists, powders and the like in the case of plants.

[0050] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation of ilicicolin K. Such long-acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0051] It is also possible to administer ilicicolin K containing microsphere preparations.

[0052] Fungal infections in humans can range from superficial and cutaneous to deeply invasive and disseminated. The management of disseminated fungal infections continues to be a major clinical problem. The limited spectrum of available antifungal agents contributes to both the development of acquired resistance and the rise in incidence of previously rare but intrinsically resistant pathogens. Consequently, there is an unacceptably high mortality rate in patients with fungal infections. Antifungal discovery is complicated by the similar cell biology of fungi and mammals. Therefore, there is a longstanding and unmet need for new anti- fungals for use in both human and veterinary medical fields. This need is addressed by the present invention.

[0053] Accordingly, the present invention provides the use of ilic- icolin K in methods of treating or preventing a fungal disease in a subject comprising administering an effective amount of ilicic- olin K alone or in combination with other antifungal therapies. In some aspects, ilicicolin K is administered via a route selected from oral, topical, vaginal, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, or via transmucosal absorption, such as by intravenous injection or catheter delivery. Therefore, the present invention also relates to a pharmaceutical composition comprising ilicicolin K for use in the treatment and prevention of fungal disease or disorder, wherein the composition is administered via a route selected from oral, topical, vaginal, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, via transmucosal absorption, or via catheter delivery, wherein the amount of ilicicolin K in the composition is preferably from 10 ng to 10 g, more preferred from 10 mg to 8 g, especially from 500 mg to 5 g. It is clear that the dose significantly depends on the mode of administration. Whereas an intravenous dose or a site-directed intramuscular or intradermal dose may be lower than a topical dose, the relative optimal amount also depends on the severity of the antifungal effect to be obtained from the application of ilicicolin K. Whereas in preventive use, the dose is usually kept lower, heavy fungal infections usually require larger doses and more frequent intervals between the administration treatments.

[0054] Ilicicolin K turned out to be an antifungal agent which is broadly applicable to various fungal pathogens. Accordingly, the present invention is used for treatment and prevention of a fungal disease associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides , Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera. The medical use of ilicicolin K or the methods of treating or preventing a fungal disease or disorder are applied to human or non-human mammalian sub j ects .

[0055] Preferred other active ingredients in agricultural compositions according to the present invention include :

[0056] ( 1 ) anti fungal agents , such as azoxystrobin and myclobutanil ;

[0057] ( 2 ) plant growth regulators , such as dikegulac-sodium and trinexapac-ethyl ;

[0058] ( 3 ) herbicides , such as prodiamine and glyphosate ;

[0059] ( 4 ) insecticides , such as bi fenthrin and malathion; and

[0060] ( 5 ) acaricides , such as avermectin and kelthane .

[0061] The agricultural compositions of the present invention may contain about 0 . 1 to 95 percent by weight of the il icicolin H variant according to the present invention .

[0062] It is another aspect of the present invention the ilicicolin H variant according to the present invention can be used in methods of treating, controlling, or preventing fungal infections on an agricultural product comprising applying to the agricultural product or plant the present agricultural compositions of the ilicicolin H variant according to the present invention . The compositions of the present invention may be applied using a variety of methodologies , including soil drench, seed treatment , granular and foliar spray . With regard to the treatment of seeds , the compositions may alternatively be applied directly to the seed before planting of the seed or applied to the locus or soil in which the seeds are sown prior to the planting of the seed .

[0063] In such methods of the present invention the concentration of the ilicicolin H variant according to the present invention to be applied to the agricultural product is about 0 . 001 to 1 percent by weight . In a class of this method the concentration of the ilicicolin H variant according to the present invention to be applied to the agricultural product is about 0 . 001 to 0 . 01 percent by weight .

[0064] Another aspect of the present invention relates to the total amount of the ilicicolin H variant according to the present invention to be applied per area of the field . In one class of this aspect about 2 to about 1000 grams of the ilicicolin H variant according to the present invention are to be applied per acre . In a subclass of this class about 5 to 500 grams of the ilicicolin H variant according to the present invention are to be applied per acre . In a second class of this aspect about 0 . 025 kilograms to about 5 kilograms of the ilicicolin H variant according to the present invention are to be applied per hectare . In a subclass of this second class about 0 . 05 kilograms to about 1 kilogram of the ilicicolin H variant according to the present invention are to be applied per hectare .

[0065] Another aspect of the present invention relates to the use of the ilicicolin H variant according to the present invention and the compositions of the present invention to treat , control , or prevent fungal infections on an agricultural product .

[0066] It is another aspect of the present invention to provide for the use of the ilicicolin H variant according to the present invention in the manufacture of a composition for use in treating, controlling, or preventing a fungal infection on an agricultural product .

[0067] The present invention also relates to a food, feed, or agricultural product treated with a composition of the present invention .

[0068] The present invention also relates to a process for the treatment of an agricultural product which comprises applying a composition of the present invention to such agricultural product or plant .

[0069] The present invention also relates to the use of a fermentation broth derived from strains of Tri choderma reesei , Cylindro- cladi um ili ci cola or Gli ocladi um roseum as a live cell or cell suspension to treat , control , or prevent a fungal infection on an agricultural product . Such fermentation broths contain the antifungal agent ( the ilicicolin H variant according to the present invention) which need not be isolated, puri fied, and re- formulated for use as an agricultural fungicide .

[0070] A preferred embodiment of the present invention is concerned with the use of the anti fungal ilicicolin H variant according to the present invention to control the growth of agricultural pests including, sphaerella ni s , Monog scl eroti orum, and Puccinia . The ilicicolin H variant according to the present invention can be used to control a variety of plant diseases and can be applied in a variety of methodologies , including soil drench, seed treatment , granular and foliar spray . It can be readily formulated into a variety of compositions , including emulsifiable concentrates, inverse emulsions, microemulsions, dispersible granules, wettable powders, dusts, and granules.

[0071] The ilicicolin H variant according to the present invention offers several advantages over existing synthetic fungicides. Since it is derived from natural sources, it already occurs in the environment and biological processes to degrade the compound are already present in the environment. Unlike many conventional fungicides, the ilicicolin H variant according to the present invention does not contain any heavy metals or halogens and therefore is totally biologically degradable. Moreover, the ilicicolin H variant according to the present invention has a different mechanism of action from existing commercial fungicides and therefore can be used to reduce fungicide resistance found in certain plant diseases .

[0072] The term "agricultural product" as used herein is to be understood in a very broad sense and includes cereals, e.g., wheat, barley, rye, oats, rice, sorghum, and the like; beets, e.g. sugar beet and fodder beet; pome and stone fruit and berries, e.g. apples, pears, plums, apricots, peaches, almonds, cherries, strawberries, raspberries and blackberries; leguminous plants, e.g. beans, lentils, peas, and soybeans; oleaginous plants, e.g. rape, mustard, poppy, olive, sunflower, coconut, castor-oil plant, cocoa, and ground-nuts; cucurbitaceae, e.g. pumpkins, gherkins, melons, cucumbers, squashes, and aubergines; fibrous plants, e.g. cotton, flax, hemp, and jute; citrus fruit, e.g. oranges, lemons, grapefruits, mandarins, and limes; tropical fruit, e.g. papayas, passion fruit, mangos, carambolas, pineapples, bananas, and kiwis; vegetables, e.g. spinach, lettuce, asparagus; brassicaceae, such as cabbage and turnips, carrots, onions, tomatoes, potatoes, seed potatoes, hot and sweet peppers; laurel-like plants, e.g. avocado, cinnamon, camphor tree; or products, such as maize, tobacco, nuts, coffee, sugarcane, tea, grapevines, hops, rubber plants, as well as ornamental plants, e.g. cut flowers, roses, tulips, lilies, Narcissus, crocuses, hyacinths, dahlias, gerbia, carnations, fuchsias, chrysanthemums, and flower bulbs, shrubs, deciduous trees and evergreen trees such as conifers, plants and trees in greenhouses. It includes plants and their parts, fruits, seeds, cuttings, cultivars, grafts, bulbs, tubers, root-tubers, rootstocks, cut flowers and vegetables.

[0073] The properties of the adjuvant per se will be understood to provide the same advantages for an agrochemical formulation comprising said adj uvant . Therefore , an agrochemical formulation is provided, when comprising the adj uvant of the present invention, having the advantages of the properties of the adj uvant per se .

[0074] Agrochemically active compounds , including insecticides and fungicides , require a formulation which allows the active compounds to be taken up by the plant / the target organisms .

[0075] The term "agrochemical formulation" as used herein refers to compositions including an active agrochemical , and is intended to include all forms of compositions , including concentrates and spray formulations . I f not speci fically stated, the agrochemical formulation of the present invention may be in the form of a concentrate , a diluted concentrate , or a sprayable formulation .

[0076] The ilicicolin H variant according to the present invention of the present invention may be combined with other components in order to form an agrochemical formulation comprising at least one agrochemical active .

[0077] Accordingly, agrochemical active compounds may be formulated as an emulsi fiable concentrate (EC ) , emulsion concentrate (EW) , suspension concentrate ( SC ) , soluble liquid ( SL ) , as an oil-based suspension concentrate ( CD) , and / or suspoemulsions ( SE ) .

[0078] In an EC formulation and in an SL formulation, the active compound may be present in dissolved form, whereas in an CD, SC, EW, or SE formulations the active compound may be present as a solid or emulsi fied liquid . It is envisaged that the adj uvant of the present invention will particularly find use in a EC, EW, SC, SL, CD, or SE formulation .

[0079] Agrochemical concentrates are agrochemical compositions , which may be aqueous or non-aqueous , and which are designed to be diluted with water ( or a water-based liquid) to form the corresponding spray formulations . Said compositions include those in liquid form ( such as solutions , emulsions , or dispersions ) and in solid form ( especially in water dispersible solid form) such as granules or powders .

[0080] Spray formulations are aqueous agrochemical formulations including all the components which it is desired to apply to the plants or their environment . Spray formulations can be made up by simple dilution of concentrates containing desired components (other than water ) , or by mixing of the individual components , or a combination of diluting a concentrate and adding further individual components or mixtures of components . Typically, such end use mixing is carried out in the tank from which the formulation is sprayed, or alternatively in a holding tank for filling the spray tank . Such mixing and mixtures are typically termed tank mixing and tank mixtures .

[0081] The ilicicolin H variant according to the present invention may therefore be incorporated into the formulation of a further agrochemical active compound ( in-can / built-in formulation) as an adj uvant or be added after dilution of the concentrated formulation of the spray liquor ( tank-mix ) . To avoid dosage errors and to improve user safety during application of agrochemical products , it is advantageous to incorporate the ilicicolin H variant according to the present invention into the formulation . This also avoids the unnecessary use of additional packaging material for the tankmix products .

[0082] According to the needs of the customer, concentrates thus formed may comprise typically up to 95 wt . % agrochemical actives . Said concentrates may be diluted for use resulting in a dilute composition having an agrochemical active concentration of about 0 . 5 wt . % to about 1 wt . % . In said dilute composition ( for example , a spray formulation, where a spray application rate may be from 10 to 500 l . ha- 1 ) the agrochemical active concentration may be in the range from about 0 . 001 wt . % to about 1 wt . % of the total formulation as sprayed .

[0083] The ilicicolin H variant according to the present invention will typically be used in an amount proportional to the amount of the active agrochemical in the formulation . In agrochemical formulation concentrates , the proportion of the ilicicolin H variant according to the present invention will depend on the solubility of the components in the liquid carrier . Typically, the concentration of the ilicicolin H variant according to the present invention in such a concentrate will be from 1 wt . % to 99 wt . % . Preferably, the concentration of the ilicicolin H variant according to the present invention in such a concentrate will be from 1 wt . % to 70 wt . % . More preferably, the concentration of the ilicicolin H variant according to the present invention in such a concentrate will be from 3 wt . % to 50 wt . % .

[0084] Upon dilution to form, for example , a spray formulation, the ilicicolin H variant according to the present invention will typically be present at a concentration of from 0 . 01 wt . % to 2 wt . % , more usually from 0.03 wt . % to 0.5 wt . % of the spray formulation. Further preferably, the adjuvant will be present at a concentration of from 0.12 wt . % to 0.4 wt . % of the spray formulation.

[0085] The ratio of the ilicicolin H variant according to the present invention to (other) active agrochemical in the agrochemical formulation is preferably from about 1:40 to about 1:1. More preferably, the ratio of the ilicicolin H variant according to the present invention to active agrochemical in the agrochemical formulation is from about 1:20 to about 1:1. Further preferably, the ratio of the ilicicolin H variant according to the present invention to active agrochemical in the agrochemical formulation is from about 1:5 to 1 about 1:1. This ratio range will generally be maintained for concentrate forms of formulations (e.g. where the ilicicolin H variant according to the present invention is included in a dispersible liquid concentrate or dispersible solid granule formulation) , and in the spray formulations.

[0086] When concentrates (solid or liquid) are used as the source of an active agrochemical and / or the ilicicolin H variant according to the present invention, the concentrates will typically be diluted to form the spray formulations. The dilution may be with from 1 to 10,000, particularly 10 to 1,000, times the total weight of the concentrate of water to form the spray formulation.

[0087] Where the agrochemical active is present in the aqueous end use formulation as solid particles, most usually it will be present as particles mainly of active agrochemical.

[0088] However, if desired, the active agrochemical can be supported on a solid carrier e.g. silica or diatomaceous earth, which can be a solid support, filler or diluent material as mentioned above.

[0089] The spray formulations will typically have a pH within the range from moderately acidic (e.g. about 3) to moderately alkaline (e.g. about 10) , and particularly near neutral (e.g. about 5 to 8) . More concentrated formulations will have similar degrees of acidity / alkalinity, but as they may be largely non-aqueous, pH is not necessarily an appropriate measure of this.

[0090] The agrochemical formulation may include solvents (other than water) such as monopropylene glycol, oils which can be vegetable or mineral oils such as spray oils (oils included in spray formulations as non-surf actant adjuvants) , associated with the ilicicolin H variant according to the present invention and co-adj uvants . Such solvents may be included as a solvent for the ilicicolin H variant according to the present invention, and / or as a humectant, e.g. especially propylene glycol. When used, such solvents will typically be included in an amount of from 5 wt . % to 500 wt.%, desirably 10 wt.% to 100 wt.%, by weight of the adjuvant. Such combinations can also include salts such as ammonium chloride and / or sodium benzoate, and / or urea especially as gel inhibition aids .

[0091] In an alternative embodiment, either the ilicicolin H variant according to the present invention, or the organism comprising the ilicicolin H variant according to the present invention may be included in a seed coating composition suitable for applying to seeds. Preferably, the ilicicolin H variant according to the present invention may be included in the seed coating composition. The ilicicolin H variant according to the present invention are suitably present in the seed coating composition at a concentration in the range from 0.5 to 25 wt.%, preferably 2 to 18 wt.%, more preferably 5 to 15 wt.%, in particular 8 to 12 wt.% based on the total weight of the composition. The coating may include film coating, pelleting, and encrusting or a combination of these techniques as known in the art. It is envisaged that the present invention applies to all said coating types, preferably to film coating. The seed coating composition of the invention may be applied to the seed in conventional manners.

[0092] The seed may be primed or not primed (having been subjected to a treatment to improve the germination rate, e.g. osmopriming, hydropriming, matrix priming) .

[0093] In a preferred embodiment, the seed is not provided with artificial layers prior to applying the seed coating composition of the invention, for example primer layers comprising a binder, such as a polymer. Accordingly, the seed coating composition is preferably applied directly on the natural outer surface of the seed. Nonetheless, it is possible that the seed surface has undergone a surface treatment prior to applying the seed coating composition .

[0094] Preferably, the seed coating composition is applied as a liquid composition and / or emulsion and / or dispersion and / or latex composition and thereafter solidified (including cured and / or dried) to form a seed coating. The term "liquid coating composition" according to the present invention is meant to include coating compositions in the form of a suspension, emulsion, and / or dispersion, preferably a dispersion . Conventional means of coating may be employed for coating the seeds . Various coating machines are available to the person skilled in the art . Some well-known techniques include the use of drum coaters , fluidised bed techniques , rotary coaters (with and without integrated drying) , and spouted beds . Suitably, the seed coating composition is applied to the seed by a rotary coater, a rotary dry coater, a pan coater or a continuous treater . The seed coating composition can, for instance , be applied by film coating, spraying, dipping, or brushing of the seed coating composition . Preferably, the method comprises applying the seed coating composition to form a film or seed coating layer . Seed coating typically involves forming on the surface of the seeds a firmly adhering, moisture permeable coating . The process typically comprises applying a liquid seed coating composition to the seeds before planting .

[0095] An additional film coat layer may optionally be applied over the top of the coating layer of the invention to provide additional benefits , including cosmetics , coverage , actives , nutrients , and processing improvements such as faster drying, seed flow, durability and the like .

[0096] The agrochemical formulation or seed coating composition may also include other components as desired . These other components may be selected from those including : binders , particularly binders which are readily water soluble to give low viscosity solutions at high binder concentrations , such as polyvinylpyrrolidone ; polyvinyl alcohol ; carboxymethyl cellulose ; gum arable ; sugars e . g . sucrose or sorbitol ; starch; ethylene-vinyl acetate copolymers , sucrose and alginates ; diluents , absorbents or carriers such as carbon black; talc ; diatomaceous earth; kaolin; aluminium, calcium or magnesium stearate ; sodium tripolyphosphate ; sodium tetraborate ; sodium sulphate ; sodium, aluminium and mixed sodium-aluminium silicates ; and sodium benzoate ; disintegration agents , such as surfactants , materials that swell in water, for example carboxy methylcellulose , collodion, polyvinylpyrrolidone and microcrystalline cellulose swelling agents ; salts such as sodium or potassium acetate , sodium carbonate , bicarbonate or sesquicarbonate , ammonium sulphate and dipotassium hydrogen phosphate ; wetting agents such as alcohol ethoxylate and alcohol ethoxylate / propoxylate wetting agents; dispersants such as sulphonated naphthalene formaldehyde condensates and acrylic copolymers such as the comb copolymer having capped polyethylene glycol side chains on a polyacrylic backbone; emulsifiers such as alcohol ethoxylates, ABA block co polymers, or castor oil ethoxylates; antifoam agents, e.g. polysiloxane antifoam agents, typically in amounts of 0.005 wt . % to 10 wt . % of the formulation; viscosity modifiers such as commercially available water soluble or miscible gums, e.g. xanthan gums, and / or cellulosics, e.g. carboxy- methyl, ethyl or propylcellulose; and / or preservatives and / or anti-microbials such as organic acids, or their esters or salts such as ascorbic e.g. ascorbyl palmitate, sorbic e.g. potassium sorbate, benzoic e.g. benzoic acid and methyl and propyl 4-hydroxybenzoate, propionic e.g. sodium propionate, phenol e.g. sodium 2-phenylphenate ; 1 , 2-benzisothiazolin-3-one ; or formaldehyde as such or as paraformaldehyde; or inorganic materials such as sulphurous acid and its salts, typically in amounts of 0.01 wt . % to 1 wt . % of the formulation.

[0097] The agrochemical formulation or seed coating composition according to the present invention may also contain components, such as surfactant materials which form part of the emulsifier system. Said surfactants may include surfactant dispersants. Examples include alkylpolysaccharides (more properly called alkyl oligosaccharides) ; fatty amine ethoxylates e.g. coconut alkyl amine 2EO; and derivatives of alk(en)yl succinic anhydride, in particular those described in WO 94 / 00508 Al and WO 96 / 16930 Al.

[0098] The f ormulation / composition may comprise one or more additional biologically active ingredients (including plant enhancing agents, in particular plant protective products (also referred to as PPPs) ) . Suitable examples of active ingredients, in particular plant enhancing agents, are further fungicidal agents, bactericidal agents, insecticidal agents, nematicidal agents, mollusci- cidal agents, biologicals, acaricides or miticides, pesticides, and biocides. Further possible active ingredients include disinfectants, microorganisms, rodent killers, weed killers (herbicides) , attracting agents, (bird) repellent agents, plant growth regulators (such as gibberellic acid, auxin or cytokinin) , nutrients (such a potassium nitrate, magnesium sulphate, iron chelate) , plant hormones, minerals, plant extracts, germination stimulants, pheromones, biological preparations, etc.

[0099] Suitable agrochemical actives for use in the formulations or seed coating composition according to the invention are all agro- chemically active compounds that may be solid or liquid at room temperature. The ilicicolin H variant according to the present invention has a broad applicability to all types of agrochemical actives. Agrochemical actives refer to biocides which, in the context of the present invention, are plant protection agents, more particular chemical substances capable of killing different forms of living organisms used in fields such as medicine, agriculture, forestry, and mosquito control. Also counted under the group of biocides are so-called plant growth regulators. Biocides for use in agrochemical formulations or seed coating compositions of the present invention are typically divided into two sub-groups: pesticides, including fungicides, herbicides, insecticides, algicides, molluscicides, miticides and rodenticides; and antimicrobials, including germicides, antibiotics, antibacterials, antivirals, antifungals, antiprotozoals and antiparasites.

[0100] In particular, biocides selected from insecticides, fungicides, or herbicides may be particularly preferred.

[0101] The term "pesticide" is understood to refer to any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest. A pesticide may be a chemical substance or biological agent (such as a virus or bacteria) used against pests including insects, plant pathogens, weeds, molluscs, birds, mammals, fish, nematodes (roundworms) and microbes that compete with humans for food, destroy property, spread disease or are a nuisance.

[0102] A fungicide is a chemical control of fungi. Fungicides are chemical compounds used to prevent the spread of fungi in gardens and crops. Fungicides are also used to fight fungal infections. Fungicides can either be contact or systemic. A contact fungicide kills fungi when it comes into contact with the fungicide retained on leaf surfaces. A systemic fungicide is absorbed into plant tissues and kills the fungus when it attempts to invade the host. Examples for preferred fungicides to be used in combination with the ilicicolin H variant according to the present invention, encompass the following species: ( 3-ethoxypropyl ) mercury bromide, 2- methoxyethylmercury chloride, 2-phenylphenol, 8-hydroxyquinoline sulphate, 8-phenylmercuri oxyquinoline, acibenzolar, acylamino acid fungicides, acypetacs, aldimorph, aliphatic nitrogen fungicides, allyl alcohol, amide fungicides, ampropylfos, anilazine, anilide fungicides, antibiotic fungicides, aromatic fungicides, aureofungin, azaconazole, azithiram, azoxystrobin, barium polysulphide, benalaxyl-M, benodanil, benomyl, benquinox, bentaluron, benthiavalicarb, benzalkonium chloride, benzamacril, benzamide fungicides, benzamorf, benzanilide fungicides, benzimidazole fungicides, benzimidazole precursor fungicides, benzimidazolylcarbamate fungicides, benzohydroxamic acid, benzothiazole fungicides, bethoxazin, binapacryl, biphenyl, bitertanol, bithionol, blasti- cidin-S, Bordeaux mixture, boscalid, bridged diphenyl fungicides, bromuconazole, bupirimate, Burgundy mixture, buthiobate, butylamine, calcium polysulphide, captafol, captan, carbamate fungicides, carbamorph, carbanilate fungicides, carbendazim, carboxin, carpropamid, carvone, Cheshunt mixture, chinomethionat , chloben- thiazone, chloraniformethan, chloranil, chlorf enazole, chlorodinitronaphthalene, chloroneb, chloropicrin, chlorothalonil, chlorquinox, chlozolinate, ciclopirox, climbazole, clotrimazole, conazole fungicides, conazole fungicides (imidazoles) , conazole fungicides (triazoles) , copper (II) acetate, copper (II) carbonate, basic, copper fungicides, copper hydroxide, copper naphthenate, copper oleate, copper oxychloride, copper (II) sulphate, copper sulphate, basic, copper zinc chromate, cresol, cufraneb, cuprobam, cuprous oxide, cyazofamid, cyclafuramid, cyclic dithiocarbamate fungicides, cycloheximide, cyf luf enamid, cymoxanil, cypendazole, cyproconazole, cyprodinil, dazomet, DBCP, debacarb, decafentin, dehydroacetic acid, dicarboximide fungicides, dichlofluanid, dichlone, dichlorophen, dichlorophenyl, dicarboximide fungicides, dichlozoline, diclobutrazol , diclocymet, diclomezine, dicloran, diethofencarb, diethyl pyrocarbonate, dif enoconazole, diflumeto- rim, dimethirimol , dimethomorph, dimoxystrobin, diniconazole, dinitrophenol fungicides, dinobuton, dinocap, dinocton, dinopenton, dinosulphon, dinoterbon, diphenylamine, dipyrithione, disulphi- ram, ditalimfos, dithianon, dithiocarbamate fungicides, DNOC, do- demorph, dodicin, dodine, donatodine, drazoxolon, edifenphos, epoxiconazole, etaconazole, etem, ethaboxam, ethirimol, ethoxyquin, ethylmercury 2 , 3-dihydroxypropyl mercaptide, ethylmercury acetate, ethylmercury bromide, ethylmercury chloride, ethylmercury phosphate, etridiazole, famoxadone, fenamidone, f enaminosulph, fenapanil, fenarimol, f enbuconazole, fenfuram, fenhexamid, fenitropan, fenoxanil, fenpiclonil, fenpropidin, f enpropimorph, fentin, ferbam, ferimzone, fluazinam, fludioxonil, flumetover, f luopicolide, fluoroimide, f luotrimazole, f luoxastrobin, fluquin- conazole, flusilazole, f lusulphamide, flutolanil, flutriafol, folpet, formaldehyde, fosetyl, fuberidazole, furalaxyl, fu- rametpyr, furamide fungicides, furanilide fungicides, furcarbanil, furconazole, furconazole-cis , furfural, furmecyclox, furophanate, glyodin, griseofulvin, guazatine, halacrinate, hexachlorobenzene, hexachlorobutadiene, hexachlorophene, hexaconazole, hexylthiof os , hydrargaphen, hymexazol, imazalil, imibenconazole, imidazole fungicides, iminoctadine, inorganic fungicides, inorganic mercury fungicides, iodomethane, ipconazole, iprobenfos, iprodione, iprov- alicarb, isoprothiolane, isovaledione, kasugamycin, kresoxim-me- thyl, lime sulphur, mancopper, mancozeb, maneb, mebenil, mecarbin- zid, mepanipyrim, mepronil, mercuric chloride, mercuric oxide, mercurous chloride, mercury fungicides, metalaxyl, metalaxyl-M, metam, metazoxolon, metconazole, methasulphocarb, methfuroxam, methyl bromide, methyl isothiocyanate, methylmercury benzoate, methylmercury dicyandiamide, methylmercury pentachlorophenoxide, metiram, metominostrobin, metrafenone, metsulphovax, milneb, morpholine fungicides, myclobutanil , myclozolin, N- ( ethylmercury ) -p- toluenesulphonanilide, nabam, natamycin, nitrostyrene, nitrothal- isopropyl, nuarimol, OCH, octhilinone, ofurace, organomercury fungicides, organophosphorus fungicides, organotin fungicides, orysastrobin, oxadixyl, oxathiin fungicides, oxazole fungicides, oxine copper, oxpoconazole, oxycarboxin, pefurazoate, penconazole, pencycuron, pentachlorophenol, penthiopyrad, phenylmercuriurea, phenylmercury acetate, phenylmercury chloride, phenylmercury derivative of pyrocatechol, phenylmercury nitrate, phenylmercury salicylate, phenylsulphamide fungicides, phosdiphen, phthalide, phthalimide fungicides, picoxystrobin, piperalin, polycarbamate, polymeric dithiocarbamate fungicides, polyoxins, polyoxorim, polysulphide fungicides, potassium azide, potassium polysulphide, potassium thiocyanate, probenazole, prochloraz, procymidone, pro- pamocarb, propiconazole, propineb, proquinazid, prothiocarb, prothioconazole, pyracarbolid, pyraclostrobin, pyrazole fungicides, pyrazophos, pyridine fungicides, pyridini tril , pyrifenox, pyrimethanil , pyrimidine fungicides, pyroquilon, pyroxychlor, py- roxyfiir, pyrrole fungicides, quinacetol, quinazamid, quinconazole, quinoline fungicides, quinone fungicides, quinoxaline fungicides, quinoxyfen, quintozene, rabenzazole, salicylanilide, silthiofam, simeconazole, sodium azide, sodium orthophenylphenoxide, sodium pentachlorophenoxide, sodium polysulphide, spiroxamine, streptomycin, strobilurin fungicides, sul- phonanilide fungicides, sulphur, sultropen, TCMTB, tebuconazole, tecloftalam, tecnazene, tecoram, tetraconazole, thiabendazole, thiadifluor, thiazole fungicides, thicyofen, thif luzamide, thiocarbamate fungicides, thiochlorf enphim, thiomersal, thiophanate, thiophanate-methyl , thiophene fungicides, thioquinox, thiram, ti- adinil, tioxymid, tivedo, tolclof os-methyl , tolnaftate, tol- ylfluanid, tolylmercury acetate, triadimefon, triadimenol, tri- amiphos, triarimol, triazbutil, triazine fungicides, triazole fungicides, triazoxide, tributyltin oxide, trichlamide, tricyclazole, trif loxystrobin, trif lumizole, triforine, triticonazole, unclassified fungicides, undecylenic acid, uniconazole, urea fungicides, validamycin, valinamide fungicides, vinclozolin, zarilamid, zinc naphthenate, zineb, ziram, zoxamide, and mixtures thereof.

[0103] A herbicide is a pesticide used to kill unwanted plants. Selective herbicides kill specific targets while leaving the desired crop relatively unharmed. Some of these act by interfering with the growth of the weed and are often based on plant hormones. Herbicides used to clear waste ground are non-selective and kill all plant material with which they come into contact. Herbicides are widely used in agriculture and in landscape turf management. They are applied in total vegetation control (TVC) programs for maintenance of highways and railroads. Smaller quantities are used in forestry, pasture systems, and management of areas set aside as wildlife habitat. Preferred herbicides may be selected from the group comprising: aryloxycarboxylic acid e.g. MCPA, aryloxyphenoxypropionates e.g. clodinafop, cyclohexanedione oximes e.g. sethoxydim, hydroxybenzonitriles e.g. bromoxynil, sulphonylureas e.g. nicosulphuron, triazolopyrimidines e.g. penoxsulam, trike- tiones e.g. mesotriones, triazine herbicides such as metribuzin, hexaxinone, or atrazine; sulphonylurea herbicides such as chlor- sulfuron; uracils such as lenacil, bromacil, or terbacil; urea herbicides such as linuron, diuron, siduron, or neburon; acetanilide herbicides such as alachlor, or metolachlor; thiocarbamate herbicides such as benthiocarb, triallate; oxadiazolone herbicides such as oxadiazon; isoxazolidone herbicides, phenoxyacetic acids; diphenyl ether herbicides such as fluazifop, acifluorfen, bifenox, or oxyfluorfen; dinitro aniline herbicides such as trifluralin; organophosphonate herbicides such as glufosinate salts and esters and glyphosate salts and esters; and / or dihalobenzonitrile herbicides such as bromoxynil, or ioxynil, benzoic acid herbicides, dipyridilium herbicides such as paraquat; and other herbicides such as clomazone, carf entrazone, saf luf enacil , and pyroxasul- phone . Particularly preferred herbicides may be selected from 2,4- dichlorophenoxyacetic acid (2,4-D) , atrazine, dicamba as benzoic acid, glyphosate, glufosinate, imazapic as imidazolinone, metolachlor as chloroacetamide, picloram, clopyralid, and triclopyr as pyridinecarboxylic acids or synthetic auxins, their respective water soluble salts and esters, and mixtures thereof.

[0104] An insecticide is a pesticide used against insects in all developmental forms, and include ovicides and larvicides used against the eggs and larvae of insects. Insecticides are used in agriculture, medicine, industry and the household. Preferred insecticides may include those selected from: chlorinated insecticides such as, for example, Camphechlor, DDT, Hexachloro- cyclohexane, gamma-Hexachlorocyclohexane, Methoxychlor, Pentachlorophenol, TDE, Aldrin, Chlordane, Chlordecone, Dieldrin, Endosul- phan, Endrin, Heptachlor, Mirex and their mixtures; organophosphorous compounds such as, for example, Acephate, Azinphos-methyl , Bensulide, Chlorethoxyf os , Chlorpyrifos, Chlorpyriphos-methyl , Diazinon, Dichlorvos (DDVP) , Dicrotophos, Dimethoate, Disulphoton, Ethoprop, Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Malathion, Methamidophos , Methidathion, Methyl-parathion, Mevinphos, Naled, Omethoate, Oxydemeton-methyl , Parathion, Phorate, Phosalone, Phosmet, Phostebupirim, Pirimiphos-methyl , Profenofos, Terbufos, Tetrachlorvinphos , Tribufos, Trichlorfon and their mixture; carbamates such as, for example, Aldicarb, Carbofuran, Carbaryl, Methomyl, 2— (1— Methylpropyl ) phenyl methylcarbamate and their mixtures; pyrethroids such as, for example, Allethrin, Bifenthrin, Deltamethrin, Permethrin, Resmethrin, Sumithrin, Tet- ramethrin, Tralomethrin, Transf luthrin and their mixtures; plant toxin derived compounds such as, for example, Derris (rotenone) , Pyrethrum, Neem (Azadirachtin) , Nicotine, Caffeine and their mixture; neonicotinoids such as imidacloprid; abamectin e.g. emamac- tin; oxadiazines such as indoxacarb; and / or anthranilic diamides such as rynaxypyr. Miticides are pesticides that kill mites . Antibiotic miticides , carbamate miticides , formamidine miticides , mite growth regulators , organochlorine , permethrin and organophosphate miticides all belong to this category . Molluscicides are pesticides used to control molluscs , such as moths , slugs and snails . These substances include metaldehyde , methiocarb and aluminium sulphate . A nematicide is a type of chemical pesticide used to kill parasitic nematodes ( a phylum of worm) .

[0105] Most preferably, a further active component present in the agrochemical formulation or seed coating composition of the present invention is selected from triazoles fungicides , strobilurins fungicides , or a combination thereof . In particular, tebuconazole , flutriafol , carbendazim, azoxystrobin, kresoxim-methyl , cyprocon- azole , or pyraclostrobin .

[0106] Nutrients may be present in addition to , or as an alternative to , agrochemical actives . In such f ormulations / compositions the nutrient is typically in a dry form . The nutrients may preferably be a solid phase nutrients . Solid nutrients are to be understood in the present invention as meaning substances whose melting point is above 20 ° C ( at standard pressure ) . Solid nutrients will also include insoluble nutrient ingredients , i . e . nutrient ingredients whose solubility in water is such that a signi ficant solid content exists in the concentrate after addition . Nutrients refer to chemical elements and compounds which are desired or necessary to promote or improve plant growth . Suitable nutrients generally are described as macronutrients or micronutrients . Suitable nutrients for use in the concentrates according to the invention are all nutrient compounds . Micronutrients typically refer to trace metals or trace elements , and are often applied in lower doses . Suitable micronutrients include trace elements selected from zinc, boron, chlorine , copper, iron, molybdenum, and manganese . The micronutrients may be in a soluble form or included as insoluble solids , and may be salts or chelated . Macronutrients typically refer to those comprising nitrogen, phosphorus , and potassium, and include fertilisers such as ammonium sulphate , and water conditioning agents . Suitable macro nutrients include fertilisers and other nitrogen, phosphorus , potassium, calcium, magnesium, sulphur containing compounds , and water conditioning agents . It is envisaged that inclusion of the nutrient would be dependent upon the speci fic nutrient , and that micronutrients would typically be included at lower concentrations whilst macronutrients would typically be included at higher concentrations.

[0107] Suitable fertilisers include inorganic fertilisers that provide nutrients such as nitrogen, phosphorus, potassium or sulphur. Fertilisers may be included in diluted formulations at relatively low concentrations or as more concentrated solutions, which at very high levels may include solid fertiliser as well as solution.

[0108] Biostimulant component may be added to the formulation or seed coating composition to promote growth of a crop plant. The biostimulant component may comprise or consist of one or more biostimulants. Preferred examples of useful biostimulants include plant growth hormones and plant growth regulators, such as cytokinins, auxins, gibberellins, ethylene, abscisic acid. Other biostimulants include, protein hydrolysate derivatives, seaweed extracts, amino acids, botanical extracts, chitosan derivatives, biopolymers, inorganic compounds, humic substances, microbial inoculants and microbial products, or mixtures thereof.

[0109] The ilicicolin H variant according to the present invention may be provided as adjuvant in the formulations of the present invention so as to provide adjuvancy to the agrochemical formulation of (another) active agent in which it is comprised, and particular may find application providing fungicide adjuvancy.

[0110] As used herein, the term "adjuvant" or "adjuvancy" refers to compounds which when added to an agrochemical formulation will improve the agrochemical's desired effect. The adjuvant may affect the diluent, the mixture, the active, or the target by its improvements of the active's performance. An adjuvant can be used to adhere the pesticide on the area where the pesticide is functional, change the epidermal layer of the leaf surface permitting pesticide entry, and / or sensitise the target pest to the active pesticide in an agrochemical formulation. Specific adjuvancy effects may include surfactants, emulsifiers (dispersants and suspending agents) , oils, emulsifiable oils, compatibility agents, buffering and conditioning agents, defoaming agents, deposition agents, drift control agents, thickeners, spreaders (wetters) , stickers (builders and extenders) , plant penetrants, translocators, soil penetrants, stabilising agents (UV filters) , and / or pest sensitisation to the active pesticide.

[0111] Preferably, the ilicicolin H variant according to the present invention used as an adjuvant may find use as either the sole component or principal functioning agent in adjuvants formulated either for tank-added use, or formulated directly into pesticide concentrates. As a measure of the adjuvant activity in relation to the activity of the fungicide alone (e.g. pyraclostrobin) to Bo- trytis cinerea a value of percent inhibition (adjuvant and fungicide) divided by percent inhibition (fungicide) can be defined, with higher values desired. A value of 1 would therefore represent equal activity of the adj uvant / fungicide combination to the fungicide alone, whereas a value above 1 would represent higher activity with the adj uvant / fungicide combination than the fungicide alone. The actives of the present invention may have a value greater than 1. Preferably, the actives of the present invention have a value greater than 1.5, most preferably greater than 2.

[0112] According to another aspect of the present invention, ilic- icolin K may be provided in disinfectant compositions. Such compositions are provided comprising ilicicolin K formulated for application to a surface or a consumable product. Fungal growth on indoor and outdoor surfaces is a major environmental concern today affecting home, work and recreational environments. Not only can fungus (e.g., mold, mildew) be unsightly on exposed surfaces, it can destroy wood, fiber and other materials if left untreated, causing severe damage to buildings and other structures and equipment. Paints and paint films or coatings are known to be vulnerable to mold contamination due to the presence of common organic components that act as cellulosic thickeners, surfactants and defoamers, and which can also serve as a source of food for fungus cells. Some of these components are casein, acrylic, polyvinyl and other carbon polymers. For example, latex is a water-dispersed binder comprising a carbon polymer. Inside the paint can, certain fungi (e.g., yeasts) can convert enough carbon-containing food sources to CO2 to swell or even explode the can. Fungi can also discolour and reduce the viscosity of the paint and produce foul odors. Both in-can preservation of paints and protection of the end use paint films, and the surfaces they cover, from mold, mildew and yeasts is necessary. To combat fungi, a variety of coating materials may be formulated which include organic or inorganic chemicals to discourage or prevent the growth of mildew on the paint film. Ideally, these chemical fungicides or mildewcides slowly leach out of the paint to the surface and maintain their inhibitory properties for the life of the paint film, causing little or no harm to the environment. According to the present invention, an ilicicolin K containing composition may be in the form of a paint or coating. For example, the coating may be an architectural coating, an industrial coating, or a specification coating. This composition may also be part of a multicoat system, preferably comprising a binder, such as a thermoplastic binder, a thermosetting binder, or a combination thereof. In this connection, ilicicolin K may be used in methods for inhibiting fungal growth on a surface or in a consumable product comprising contacting the composition with a surface or consumable product. This method of inhibiting fungal growth on a surface or in a consumable product may comprise contacting the composition with a surface or consumable product, for example a foodstuff or a liquid. This disinfectant or antifungal composition may also be included in kits comprising ilicicolin K with other antifungal components or means for applying ilicicolin K.

[0113] All of the features described herein may be combined with any of the above aspects, in any combination.

[0114] According to another aspect, the present invention also relates to novel molecular variants of ilicicolin K, preferably novel molecular variants of ilicicolin K with similar antifungal properties as ilicicolin K, especially novel molecular variants of ilicicolin K with improved antifungal properties as ilicicolin K. As already disclosed above, the novel molecular variants of ilicicolin K may be synthesized by finishing a suitable precursor of ilicicolin K to the novel molecular ilicicolin K variant by a suitable chemical step. In doing so, the established chemistry of ilicicolin H synthesis can, again, be analogously applied. The chemical total synthesis of ilicicolin H is disclosed e.g. in Williams et al. (J. Org. Chem. 50 (1985) , 2807-2809) . This method can also be applied for total synthesis of ilicicolin K according to the present invention and its novel molecular variants (see below) by specifically closing the furan ring at C4 of the pyridine ring, preferably after any one of decalin synthesis (13, 14 and 15) in Williams et al., 1985. Suitable derivatives may be synthesized preferable by the analogous application of the methods disclosed by Singh et al. (Tetrah. Lett. 52 (2011) , 6190-6191; Bioorg. Med. Chem. Lett. (2013) , 3018-3022) for ilicicolin H.

[0115] For example, basic 4 '-esters, especially imidazole and imidazolyl esters, and moderately polar N- and O-alkyl derivatives, especially acetyloxy and cyclohexoyloxy derivatives, are preferred derivatives of ilicicolin K according to the present invention with comparable antifungal activities as ilicicolin K. 4',8-Diac- etate and 8-cyclopropyl acetate (substituting the 4' / 7 OH group at the dihydroxybenzofuro-ring) are specifically preferred derivatives of ilicicolin K according to the present invention with comparable antifungal activity and selectivity.

[0116] Specifically preferred ilicicolin K variants according to the present invention are

[0117] 4- ( (lR,2S,7S) -7 -hydroxymethyl- 4 -methyl- 1- ( (E) -prop-l-en-1- yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-di- hyrdoxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8A) , 4- ( (lR,2S,7S) -7 -carboxy- 4 -methyl- 1- ( (E) -prop- 1-en- 1-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-di- hyrdoxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8B) ,

[0118] 4- ( (lR,2S,7S)-7- (acetyloxy) hydroxymethyl- 4 -methyl- 1- ( (E)- prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-car- bonyl ) - ( 7 , 8-dihyrdoxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig.

[0119] 80) ,

[0120] 4- ( (lR,2S,7S)-74- ( (lR,2S,7S) - 6 -hydroxy- 7 -hydroxymethyl- 4 -me - thyl-1- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphtha- lene- 2- carbonyl ) - (7, 8-dihyrdoxybenzofuro) [3, 2-c] pyridine-3 (2H) - one ( Fig . 8D) ,

[0121] 4- ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-diace- tyloxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8E) , - ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-cyclo- hexoyloxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8F) , ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -

[0122] 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-hydrazone ) - (7, 8-dihy- droxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8G) ,

[0123] ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-methylhydrazone ) — (7,8— dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8H) , ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- (hydroxyethyl) hydrazone )- ( 7 , 8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig.

[0124] 81) ,

[0125] ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-oxime- (7, 8-dihy- droxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8J) , 4- ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-di- methoxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8K) , 4- ( (lR,2S,7S) - 4 -bromo- 4 , 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1,2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 -carbonyl ) - (6, 9-di- bromo ) - ( 7 , 8-dimethoxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8L) , and

[0126] 4- ( (lR,2S,7S) - 4 -bromo- 4 , 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1,2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 - carbony 1 ) - (6, 9-di- bromo) - (7, 8-diimidazoylbenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8M) .

[0127] The present invention is further described by the following examples and the figures, yet without being limited thereto.

[0128] Fig. 1 shows prior art ilicicolin H products. (1) , (2) and (3) from Zhang et al., 2019, (4) from Lin et al., 2019.

[0129] Fig. 2 shows the ilicicolin BGCs of T. reesei QM6a, T. vari- abilis HXQ-H-1, and Neonectria sp . DH2 were compared using the clinker tool [https: / / academic.oup.com / bioinformatics / arti- cle / 37 / 16 / 2473 / 6103786 ] . The input .gbk files are provided in the Suppiementals. The T. variabilis contains a second gene for an epimerase, but no information is available. The Neonectria epimerase gene is comparatively larger, maybe due to a mistake of gene model (prediction contains a TIM-like epimerase domain as well as a DnaJ-class molecular chaperone domain) .

[0130] Fig. 3 shows A, significant upregulation of all five enzymes involved in ilicicolin H production on protein-level, OE vs. WT (FDR: 0.05, s: 0.1) . B, RCA clustering of the four strains. C, extracted ion current chromatograms (EICCs) for ilicicolin H (C27H31NO4, [M+H]+434.2326 ± 0.02) of the four constructed strains. D, MSI Peak areas of ilicicolin species, including all four replicates .

[0131] Fig. 4 shows ion-identity molecular networking using MZmine shows the relationships between previously known ilicicolin H compounds (1) , (2) and (3) and reveals the presence of several related compounds. Grey colors indicate ionization adducts (+H, +Na, +H- H2O) . Ice-blue: M = 433.2252 Da, assuming an ilicicolin H compound, but eluting much later than (1) , (2) and (3) and being only visible when ion-mobility is turned off. Green: m / z 432.2169, potentially ilicicolin J, red: m / z 450.2273, a potentially oxidized ilicicolin H derivative, yellow: m / z 448.2114, the novel ilicicolin product.

[0132] Fig. 5 shows fragmentation spectra of ilicicolin H (1) , 8- epi -ilicicolin H (2) , the bi s-diene (3) and the novel ilicicolin H compound with the sum formula C27H29NO5. Structure is depicted in Fig. 6.

[0133] Fig. 6 shows the chemical formula of 4- ( ( 1R, 2S , 7S ) -4 , 7-dime- thyl-1- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphtha- 1 ene- 2- carbonyl ) -7, 8-dihydroxybenzofuro [3,2-c]pyridin-3 (2H) -one (ilicicolin K) .

[0134] Fig. 7 shows the antifungal effect of ilicicolin K compared to ilicicolin H in S. cerevisiae (A) and A. nidulans (B) The test organims were cultivated in the defined medium RPMI 1640 containing Ilicicolin H and K at the indicated concentrations in a 96 well plate microtiter plate. The optical density of the cultures were measured after 25 hours (FDR: 0.05, s: 0.1) .

[0135] Fig. 8 discloses preferred ilicicolin variants according to the present invention: 4- ( ( 1R, 2S, 7S ) -7-hydroxymethyl-4-methyl-l- ( (E) -prop-l-en-l-yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) - (7, 8-dihyrdoxybenzofuro ) [3, 2-c] pyridine -3 (2H) -one (Fig. 8A) ; 4-( (lR,2S,7S) -7 -carboxy- 4 -methyl- 1- ( (E) -prop-l-en-l-yl ) -

[0136] 1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-dihyrdoxybenzofuro) [3, 2-c] pyridine-3 (2H) -one (Fig. 8B) ; 4-

[0137] ( (lR,2S,7S)-7- (acetyloxy) hydroxymethyl-4-methyl-l- ( (E) -prop-l- en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - ( 7 , 8-dihyrdoxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8C) ; 4-

[0138] ( (lR,2S,7S)-74- ( (1R,2S,7S) - 6 -hydroxy- 7 -hydroxymethyl- 4 -methyl- 1- ( (E) -prop-l-en-l-yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) - (7, 8-dihyrdoxybenzofuro) [3, 2-c] pyridine-3 (2H) -one (Fig. 8D) ; 4- ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -

[0139] 1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-diace- tyloxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8E) ; ( (1R,2S,7S)- 4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahy- dronaphthalene-2-carbonyl ) - (7, 8-cyclohexoyloxybenzofuro ) [3, 2- c] pyridine-3 (2H) -one (Fig. 8F) ; ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) - prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-hydra- zone )- ( 7 , 8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8G) ; ( (lR,2S,7S)-4, 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) -

[0140] 1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-methylhydrazone ) — (7,8— dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8H) ; ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- (hydroxyethyl) hydrazone )- ( 7 , 8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 81) ; ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -

[0141] 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-oxime- (7, 8-dihy- droxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8J) ; 4-

[0142] ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-di- methoxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8K) ; 4-

[0143] ( ( 1R, 2S , 7S ) - 4 -bromo- 4 , 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) - 1,2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 -carbonyl ) - (6, 9-di- bromo ) - ( 7 , 8-dimethoxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8L) ; and 4- ( ( 1R, 2S , 7S ) -4-bromo-4 , 7-dimethyl-l- ( (E ) -prop-l-en-l- yl)-!, 2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 -carbonyl ) - (6, 9-di- bromo) - (7, 8-diimidazoylbenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8M) .

[0144] Fig. 9 shows the minimal inhibitory concentrations of ilic- icolin H and ilicicolin K as determined in microdilution tests according to the EUCAST guidelines (EUCAST E. Def 7.4 October 2023) by cultivating C. auris in RPMI 2% G medium with different concentrations of ilicicolin H and ilicicolin K; optical density at 530 nm was measured after 24 hours at 35°C.

[0145] Figs. 10 and 11 show the synthesis of ilicicolin K from ilicicolin H by biometric synthesis.

[0146] Examples

[0147] Herein, we describe the high-yield expression by BGC activation of ilicicolin H in a native host for the first time. To this end, we overexpressed the BGC transcription factor under the constitutive tefl promotor in T. reesei. Further, we deleted the genes triliA and triliE to elucidate their roles in the ilicicolin H biosynthesis in the native host T. reesei. Notably, we obtained slightly different results compared to the previous heterologous expression experiments (Zhang et al., 2019) . Overexpression of the cluster resulted in the production of three isobaric products, namely ilicicolin H (1) , 8-epi-ilicicolin H (2) and the bis-diene (3) , and shed further light on the production process and the roles of the enzymes involved. During this analysis, we uncovered the new ilicicolin compound according to the present invention. 1. Material and Methods

[0148] 1.1. Material

[0149] All chemicals used in this study were sourced as follows: unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) . Phenol was purchased from Ap- pliChem (Darmstadt, Germany) . Malt extract, peptone, isoamylalko- hol, FeSO4, COC12*6H20, Na2Mo04*H20, (NH4)SO4and KH2PO4were acquired from Merck (Darmstadt, Germany) . Isopropanol, MgSO4*7H2O, CaCl2*2H2O, Na2HPO4*2H2O and uridine were obtained from Carl Roth (Karlsruhe, Germany) . Agar and chloroform were purchased from VWR (Radnor, PA, USA) . LC-MS grade acetonitrile (ACN) was acquired from VWR chemicals (Radnor, PA, USA) . LC-MS grade methanol (MeOH) was obtained from Honeywell (Muskegon, MI, USA) . LC-MS grade 2- propanol (IPA) was obtained from Fisher Scientific (Hampton, NH, USA) . HPLC grade Diethyl ether was obtained from Sigma-Aldrich (St. Louis, MO, USA) . Water (H20) was purified in-house using a Barnstead™ Smart2Pure™ Water Purification System from Thermo Fisher Scientific (Waltham, MA, USA) . Ilicicolin H (CAS #12689- 26-8) standard, Catalog #10-3243 (Lot #X107453) , was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA) .

[0150] Mycelium samples were lysed using the Bead Mill Max Homogenizer in combination with Tough Microorganism Lysing Mix Glass Beads (both VWR International, Radnor, PA, USA) . Sonication was carried out using a Branson SFX550 sonifier from Emerson (Ferguson, MO, USA) . Isolation of ilicicolin H from the medium was conducted using the Supelclean™ LC-18 SPE Tubes (Merck, Darmstadt, Germany) .

[0151] Four independent biological replicates of fungal mycelium or culture supernatant were processed for each of the constructed and utilized strains, resulting in a total of four samples per experimental condition, unless otherwise noted.

[0152] 1.2. Strains and cultivation conditions

[0153] All T. reesei strains (Table 2) were maintained on malt extract (MEX) plates (3 % malt extract, 0.1 % peptone, 1.5 % agar) . 5 mM uridine or 25 pL per 100 mL Hygromycin B (Millipore, 400051) were added if required. For liquid cultivations 109spores L-1were inoculated in Mandels Andreotti (MA) medium15(KH2PO42 g L-1, (NH4)2SO41.4 g L-1, Urea 0.3 g L-1, FeSO4-7H2O 0.005 g L-1, MnSO4-H2O 0.0016 g L-1, ZnSO4-7H2O 0.0014 g L-1, CoCl20.002 g L-1, MgSO4-7H2O 0.3 g CaC12 0.3 g peptone 0.75 g carbon source 10 g LT) and incubated at 30 °C at 180 rpm.

[0154] Table 2. Utilized strains.

[0155] Strain designation Genotype Source

[0156] QM6a Amus53, Emus53 Steiger et al.

[0157] "wildtype"

[0158] QM6a Apyr4 Emus53 Epyr4 Derntl et al.

[0159] QM6a OETriliR hmus53 This study

[0160] Ptef: : triliR: : Tcbh2 (upstream of pyr4)

[0161] QM6a ATriliA A triliA: : hygR This study in QM6a OETriliR

[0162] QM6a ATriliE EtriliE: : hygR This study in QM6a OETriliR

[0163] Steiger et al. (Appl Environ Microbiol 2011, 77 (1) , 114-121) ; Derntl et al. (Appl Environ Microbiol 2015, 81 (18) , 6314-6323)

[0164] 1.3. Genetic constructions

[0165] All PCRs were performed with the Q5 High-Fidelity DNA Polymerase (NEB) according to the manufacturer's instructions. For cloning purposes, the Escherichia coii strain ToplO (Invitrogen) and the Saccharomyces cerevisiae strain WW-YH10 (ATCC 208405) were used. All plasmids and genetic constructs were verified by sequencing at Microsynth.

[0166] For the overexpression of TriliR the plasmid pRP4-OETriliR was constructed. First, a Notl-free coding region of triliR was constructed by a SOE-PCR using the primers TriliR_fwd-Af III, TriliR _MRev_SOE, TriliR _MFwd_SOE, and TriliR _rev-SpeI and chromosomal DNA of T. reesei QM6a Amus53 as template yielding a coding region with a silent mutation at R227. This modified coding region was inserted into pRP4-TX(WT) (Derntl et al., Biotechnol Biofuels 2019, 12 (1) , 231) via digestion with Aflll and Spel (both NEB) and ligation with T4 DNA Ligase (NEB) . Approx. 20 pg of the plasmid were linearized with Notl, precipitated with sodium acetate and ethanol and dissolved in 15 pL ddH2O before the transformation into T. reesei .

[0167] For the construction of the triliA deletion cassette, a yeast recombinational cloning was performed using the lithium acetate method (Schiestl et al., Curr Genet 1989, 16 (5-6) , 339-346) for the yeast transformation. The plasmid pRS426 (Christianson et al., Gene 1992, 110 (1) , 119-122) was linearized by digestion with Kpnl and Hindlll. The flanking regions were amplified by PGR with the primers 5_ TriliA _fwd-pRS426 and 5_ TriliA _fwd-pRS426 or 3_ TriliA _fwd-hph and 3_TriliA_rev-pRS426 using chromosomal DNA of T. reesei QM6a Amus53 as template. The hph gene was amplified by PGR with the primers hph_fwd-5_ TriliA and hph_rev-3_ TriliA using the plasmid pAN7-l (Punt et al., Gene 1987, 56 (1) , 117-124) as template. The plasmid was extracted from yeast using the Zymoprep Yeast Plasmid Miniprep Kit (Zymo Research) and transformed into E. coll ToplO for amplification. To delete triliA, a split marker approach was used. To this end, a fusion of the 5' flank and a part of the hygR were amplified by PGR using the primers 5_ TriliA _fwd- pRS426 and hph_MR and the plasmid as template. Accordingly, the remaining part of the hph gene was amplified together the 3' flank using the primers hph_MF and 3_TriliA_rev-pRS426. Several PGR reactions were pooled, precipitated with ethanol and dissolved in 15 pL ddH2O.

[0168] For the deletion of triliE, also a split marker approach was used, but the fusion products were directly constructed by SOE- PCRs . For the hph gene (from pAN7-l (Punt et al., 1987) ) fragments, the primers PgpdA_fwd, hph_MR, hph_MF, and TtrpC_rev were used. The flanking regions were amplified by PGR with the primers triliE, the primers TriliE_5fwd, TriliE _5rev-hph, TriliE _3fwd-hph, and TriliE _3rev using chromosomal DNA of T. reesei QM6a Amus53 as template. The fusion PGR products were cloned into pJET1.2 using the CloneJET PGR Cloning Kit (Thermo Scientific) . Before transformation the fusion products were amplified by PCR, several PCR reactions were pooled, precipitated with ethanol and dissolved in 15 pL ddH2O.

[0169] 1.4. Fungal transformation

[0170] T. reesei was transformed using a polyethylene glycol-medi- ated transformation protocol of protoplasts. Spores of the recipient strain were plated on sterile cellophane sheets laid on MEX plates at 30 °C overnight. The mycelium was scraped off and transferred into 15 mL Buffer A (1.2 M sorbitol, 100 mM KH2PO4, pH 5.6) containing 600 mg Vinotaste Pro (Novozymes) and 0.5 mg chitinase from Streptomyces griseus (Sigma-Aldrich C6137) . This mixture was incubated in a sterile petri dish in an orbital shaker at 60 rpm and 30 °C for approx. 2-3 hours until the mycelium was completely disintegrated. The suspension was filtered through a 70 pm cell sieve and incubated on ice for 5 min. The suspension was filled up to 40 mL with ice-cold 1.2 M sorbitol and centrifuged at 2,500 g at 4 °C for 10 min. The pellet was resuspended in 30 mL 1.2 M sorbitol and again centrifuged. The protoplasts were finally resuspended in 1 mL ice-cold Buffer B (1 M sorbitol, 25 mM CaC12, 10 mM Tris. Cl, pH 7.5) . Next, the DNA (either 20 pg linearized plasmid or 5 pg of fusion BCR products each for the split marker approach) was filled up to 150 pL with ice-cold Buffer B, carefully mixed with 100 pL of the protoplast suspension and 100 pL 20 % PEG (mixture of 6.7 mL Buffer B and 3.3 mL 60% PEG (60 g PEG 4000, 1 mL 1 M Tris-HCl pH 7.5, 1 mL 1 M CaCl2, 38 mL ddH2O) ) in a 50 mL reaction tube. This mixture was incubated on ice for 30 min before "60 % PEG" was added in steps (50 pL, 200 pL, 500 pL) . Next, the tube was incubated at room temperature for 20 min, and finally, Buffer C was added in steps (200 pL, 400 pL, 1 mL, 2.5 mL) . For plating, the tube was filled up to 50 mL with molten, 50 °C-warm selection medium (containing 1 M sucrose) and poured into a 14.5 cm petri dish. For insertion of the TriliR overexpression construct together with pyr4 a minimal medium was used (MA medium with glucose without peptone, pH 5.8) . For the deletion of triliA and triliE by replacement with the hygR, MEX medium with hygromycin was used. The plates were incubated at 30 °C under light until colonies were visible (up to a week) . The candidates were then homokaryon selected by spore streaking on selection plates.

[0171] 1.5. DNA extraction and genotyping

[0172] Mycelium was harvested and pressed dry between two sheets of filter paper. Approx. 50 mg were lysed in 1 mL CTAB buffer (1.4 M NaCl, 100 mM Tris-HCl pH 8.0, 10 mM EDTA, 2 % CTAB, 1% polyvinylpyrrolidone) with 0.37 g small glass beads, 0.25 g medium glass beads and one large glass bead in a 2 mL screw cap reaction tube using a Fast-Prep-24 (MP Biomedicals, Santa Ana / , CA, USA) at 6 m s-1for 30 sec. The samples were incubated at 65 °C for 20 min and finally centrifuged at 12,000 g for 10 min. The supernatant was transferred to a 2 mL reaction tube and the DNA was purified by a phenol-chlorof orm-isoamyl alcohol extraction, followed by two chloroform extractions. The samples were then treated with RNase A (Thermo Fisher Scientific) according to the manufacturer's instructions and the DNA finally precipitated using isopropanol and dissolved in 10 mM Tris-HCl pH 8.0.

[0173] All PCR reactions for genotyping were performed with the On- eTaq DNA Polymerase (NEB) according to the manufacturer's instructions .

[0174] 1.6. RNA extraction and RT-qPCR analyses

[0175] Mycelium was harvested and pressed dry between two sheets of filter paper, frozen in liquid nitrogen, and stored at -80 °C for up to a week. Approx. 50 mg of mycelium were disrupted in 1 mL RNAzol RT with 0.37 g small glass beads, 0.25 g medium glass beads and one large glass bead in a 2 mL screw cap reaction tube using a Fast-Prep-24 (MP Biomedicals) at 6 m s-1for 30 sec. The samples were centrifuged at 12,000 g for 10 min, the supernatant was transferred to a 1.5 mL reaction tube and mixed with ethanol 1:1. The RNA was purified using the Direct-zol RNA MiniPrep Kit (Zy- moreasearch) according to the manufacturer's instructions. Notably, this kit contains a DNase treatment step. The total RNA was reverse transcribed using the LunaScript RT SuperMix Kit (NEB) according to the manufacturer's instructions. The cDNA was diluted 1:50 in ddH2O and 2 pL used as a template in a 15 pL reaction using the Luna Universal qPCR Master Mix (NEB) on a Rotor-Gene Q (Qiagen) . Primers were added and PCR reaction conditions were chosen according to the manufacturer's instructions. To calculate the relative transcript abundance, we used the Pfaffl method (Pfaffl et al., Nucleic Acids Res 2001, 29 (9) , e45-e45) and the actl and sari genes as reference genes (Steiger et al . , J Biotechnol 2010, 145 (1) , 30-37) .

[0176] 1.7. Sample preparation for HPLC(-MS / MS) and NMR analysis

[0177] 1.7.1. Ilicicolin H standard

[0178] 100 pg of ilicicolin H standard (Focus Biomolecules) were dissolved in 100 pL of DMSO for a final concentration of 1 pg pin A 1 pL of this stock was diluted with 99 pL of 50 % ACN, to obtain a final concentration of 0.01 pg plr1. For LC-MS analysis, 1 pL was inj ected .

[0179] 1.7.2. Polar extracts of mycelium

[0180] 100 mg of frozen mycelium sample were weighed into glass bead- milling tubes and 1 mL of polar extract composed of a mixture of acetonitrile / methanol / water (40:40:20) were added. Lysis was conducted by bead-milling (4x 30 sec, 6 m s-1) and subsequent sonication (30 s, 10 % intensity) . The lysed samples were centrifuged for 10 min at 20, 000 g and 20 °C and the supernatant was transferred into fresh Eppendorf tubes. The supernatants were centrifuged again, for 1 min at 20, 000 g. For analysis, 10 pL of the supernatant were taken and diluted in 90 pL of 50 % ACN. From that, 1 pL was injected for LC-MS analysis.

[0181] 1.7.3. Quantification of ilicicolin H in medium

[0182] For quantification of ilicicolin H in the media, a matrix- matched external calibration curve was utilized. For that, 4 mL of culture medium of each wildtype and ATriliA quadruplicate (n = 8 in total) were pooled, which do not contain ilicicolin H in detectable quantities (below LOD of 21.4 ng mL-1) . 6x 2 mL were taken out and each spiked with precise amounts of ilicicolin H standard (50 ng, 100 ng, 200 ng, 500 ng, 1000 ng and 2000 ng) . These standards and the culture supernatants of the other 8 samples (4x OETriliR, 4x ATriliE, 50 mL each) were cleaned up using SPE columns. The elution buffer was constituted of 10 % acetonitrile in isopropanol supplemented with 15 mM ammonium formate. The eluates were centrifuged for 10 min at 20,000 g before vacuum-drying them and reconstitution in 100 pL 50 % ACN each. From that, 1 pL was directly injected for LC-MS analysis.

[0183] 1.7.4. Proteomics

[0184] For proteomics analysis, 100 mg of frozen mycelium sample were weighed into glass bead-milling tubes and 1 mL of an in-house reducing and alkylating buffer (100 mM TRIS HC1; pH = 8.5, 1 % sodium dodecyl sulphate, 10 mM tris (2-carboxyethyl ) phosphine, 40 mM 2-chloroacetamide ) were added. Lysis was conducted by bead-milling (2 min, 6 m s-1) and subsequent sonication (10 s, 10 % intensity) . The lysed samples were spun down for 12 min at 20,000 g and 20 °C and the supernatant was transferred into fresh Eppendorf tubes. The supernatants were heated to 95 °C for 10 min at 330 rpm to perform reduction and alkylation of the proteins. 100 pg of protein per sample (after bicinchoninic acid assay protein estimation (BCA) , Thermo Fisher Scientific, reducing agent compatible) were subjected to acetone precipitation by adding NaCl to a final concentration of 10 mM and letting it incubate for 5 min at room temperature. Subsequently, 4x volumes of acetone were added and incubated for 2 min. After centrifugation (15 min at 15,000 g) the supernatant was removed. Dried samples were dissolved in 25 % trifluoroethanol in 100 mM Tris-HCl (pH = 8.5) and subjected to vor- texing and sonication until completely dissolved. For protein digest, samples were diluted to 10 % trifluoroethanol using 100 mM ammonium bicarbonate. Trypsin (Promega, Fitchburg, WI) was added in a 1:50 enzyme to protein ratio and digest was performed overnight at 37 °C and 500 rpm. The following day, 500 ng of digested sample were loaded on Evotips, according to the protocol of the manufacturer (Evosep, Odense, DK) .

[0185] 1.7.5. Scaled up extraction for NMR analysis

[0186] Around 5 g of mycelium of both OETriliR and ATriliE strains were extracted by first grinding in liquid nitrogen and then beadmilling in diethyl ether. The diethyl ether extracts were washed two times with ddH2O, dried under a nitrogen stream, and reconstituted in 60 % AON in 15 mM ammonium formate for subsequent HPLC separation and fractionation.

[0187] 1.8. HPLC(-MS / MS) analysis

[0188] 1.8.1. Polar extracts of mycelium

[0189] After 1:10 dilution of the polar extracts, ilicicolin H (C27H31NO4) was measured employing an untargeted lipidomics workflow in positive polarization mode on a Bruker timsTOF Pro equipped with a VIP-HESI source (Bruker Corporation, Billerica, MA, USA) . The frontend was a Thermo Fisher Scientific Vanquish H UHPLC with a Waters Acquity BEH C18 column (150 mm x 1 mm ID, 1.7 pm; Waters Corporation, Milford, MA, USA) . Mobile phase A was 60 % acetonitrile in 15 mM ammonium formate; mobile phase B was 10 % acetonitrile in isopropanol supplemented with 15 mM ammonium formate. The following gradient was employed at 50 °C: 0 min; 2 % B; 100 pL min-

[0190] 15 min; 100 % B; 70 pL min- 22 min; 100 % B; 70 pL min-1, followed by 5 min re-equilibration to starting conditions (2 % B; 100 pL min-1) . The timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) was operated in positive ionization mode with enabled trapped ion mobility spectrometry (TIMS) at 100 % duty cycle (100 ms ramp time) . Source capillary voltage was set to 4500 V and dry gas flow to 8 L min-1at 230 °C. Sheath Gas Flow was set to 4.0 L mirr1at 100 °C, with active exhaust being activated. Scan mode was set to parallel accumulation serial fragmentation (PASEF) with a scan range from 300 to 1300 m / z, using a mobility (1 / K0) window from 0.8 to 1.81 V*s err2. Collision Energy was set to 45.00 eV.

[0191] To obtain fragmentation spectra for all possible features without pre-filtering by ion mobility, measurements for molecular networking were conducted without trapped ion mobility spectrometry (TIMS off) . The LC eluents and gradient remained unaltered. The timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) was operated in positive ionization mode, with the same settings as described above. Only the Sheath Gas Flow temperature was changed to 200 °C. Scan mode was set to Auto MS / MS with 12.00 Hz MS spectra rate and 16.00 Hz MS / MS spectra rate, resulting in a total cycle time of 0.5 s. Scan range was set from 20 to 1300 m / z .

[0192] 1.8.2. Quantification of ilicicolin H in medium HPLC-MS / MS measurement of the prepared standards and extracts were precisely carried out as described in chapter 1.8.1.

[0193] 1.8.3. Proteomics

[0194] 500 ng of the digest were separated on the Evosep One equipped with an lonopticks Aurora Series UHPLC C18 column (15 cm x 75 pm ID, 1.7 pm; lonopticks, Fitzroy, VIC, Australia) . The LC-method Whisper_40SPD was used with solvent A being 0.1 % formic acid in water and solvent B acetonitrile containing 0.1 % formic acid while maintaining the column at 40 °C. The timsTOF HT mass spectrometer (Bruker Daltonics, Germany) was operated in positive mode with enabled trapped ion mobility spectrometry (TIMS) at 100 % duty cycle (100 ms ramp time) . Source capillary voltage was set to 1500 V and dry gas flow to 3 L min-1at 180 °C. Scan mode was set to data independent parallel accumulation-serial fragmentation (diaPASEF) using parameters previously optimized with py_diAID (Skowronek et al., Molecular & Cellular Proteomics 2022, 21 (9) , 100279) . In brief, 24 isolation windows from m / z 300 to 1,200 and 1 / K0 0.7 to 1.35 were defined. After MSI scan, 2 isolation windows were fragmented per TIMS ramp resulting in an overall DIA cycle time of 1.38 s.

[0195] 1.8.4. Scaled up extraction for NMR analysis Separation and fractionation of the mycelial diethyl ether extracts was conducted on a Thermo Fisher Scientific Vanquish F UHPLC with a Waters XBridge BEH C18 column (150 mm x 4.6 mm ID, 2.5 pm; Waters Corporation, Milford, MA, USA) . Mobile phase A was 60 % acetonitrile in 15 mM ammonium formate; mobile phase B was 10 % acetonitrile in isopropanol supplemented with 15 mM ammonium formate. The following gradient was employed at 50 °C and a flowrate of 1 mL min-1, for OETriliR samples: 0 min; 2% B, 10 min; 45% B, 11 min; 98% B; 15 min; 98 % B, followed by 5 min re-equi- libration to starting conditions (2% B; 1 mL min-1) . For ATriliE samples, the gradient was slightly adapted for better isomer separation: 0 min; 2 % B, 7 min; 33.5 % B, 10 min; 40 % B; 11 min; 98 % B, 15 min; 98 % B, followed by 5 min re-equilibration to starting conditions. Flow-rate and separation temperature remained unchanged (1 mL min-1, 50 °C) . Fractions were manually collected, united, and dried under vacuum. Dried extracts were quantified gravimetrically and 6.73 mg ilicicolin H were extracted from the 5 g dried OETriliR mycelium employed for NMR analysis.

[0196] To purify ilicicolin H and K for the microbial tests, diethyl ether extracts from OETriliR mycelia were fractionated on an Autopurification system of Waters using an ACQUITY QDa Detector in combination with a 2998 Photodiode Array Detector, equipped with a XSELECT OSH 018 OBD Prep column (150 mm x 30 mm ID, 5 pm; Waters Corporation, Milford, MA, USA) . The following gradient was employed at room temperature (25 °C) and a flow-rate of 20 mL min-1: 1 min; 2 % B, 21 min; 45 % B, 23.40 min; 98 % B; 36 min; 98 % B, 37 min; 2 % B, followed by 13 min re-equilibration to starting conditions (2 % B; 20 mL min-1) . The PDA detector was set to a wavelength range from X = 200 nm to A = 450 nm, with a resolution of 1.2 nm and a sampling rate of 10 points s-1.

[0197] 1.9. NMR analysis

[0198] All samples were measured on a Bruker Avance III 600 MHz spectrometer with Prodigy nitrogen cryo BBFO{H-F} inverse probe head. Spectra were recorded at 298 K and are referenced to the residual solvent signal.

[0199] 1.10. Data analysis

[0200] 1.10.1. General utilized software

[0201] MarvinSketch 22.6.0 (Chemaxon, www.chemaxon.com) was used for drawing, displaying and characterizing chemical structures, substructures and reactions. For fragmentation spectra analysis, SIRIUS (version 5.8.6) was used: SIRIUS (Duhrkop et al., Nat Methods 2019, 16 (4) , 299-302) for molecular formula identification and CSI:FingerID (Hoffmann et al., bioRxiv 2021, 2021.03.18.435634; Duhrkop et al., Proceedings of the National Academy of Sciences 2015, 112 (41) , 12580-12585) for structure database search and substructure annotation. Mzmine version 4.0.3 (https: / / zio.io / ) was used feature finding and to conduct molecular networking (Schmid et al., Nature Biotechnology 2023 41:4 2023, 41 (4) , 447- 449; Nothias et al., Nature Methods 2020 17:9 2020, 17 (9) , 905- 908; Schmid et al., Nature Communications 2021 12:1 2021, 12 (1) , 1-12) ) .

[0202] 1.10.2. Polar Extracts

[0203] Ilicicolin H (C27H31NO4) was quantified on MSI level employing the open-source application Skyline version 22.2 (Pino et al., Mass Spectrom Rev 2020, 39 (3) , 229-244) .) Mass-to-charge, retention time, and CCS were manually matched to an authentic standard.

[0204] 1.10.3. Quantification of ilicicolin H in medium

[0205] Ilicicolin H (C27H31NO4) was quantified in standards and samples on MSI level employing the open-source application Skyline version 22.2 (Pino et al., Mass Spectrom Rev 2020, 39 (3) , 229- 244) , too. As usual, mass-to-charge, retention time, and CCS were manually matched to an authentic standard. Using the areas and known concentrations of the matrix matched standards, an external calibration curve was created.

[0206] 1.10.4. Proteomics

[0207] Proteomics data was analyzed using the software DIA-NN 1.8.1 (Demichev et al., Nature Methods 2019 17:1 2019, 17 (1) , 41-44; Demichev et al., Nature Communications 2022 13:1 2022, 13 (1) , 1- 8; . For Trichoderma reesei (Hypocrea j ecorina, taxonomy ID 51453, 19,246 entries) , all reviewed (Swiss-Prot) and unreviewed (trEMBL) FAS TA files were downloaded from UniProtKB (https: / / www.uni- prot.org / ) on the 26thof June 2023. The database was manually extended with common contaminants (https : / / www. thegp . org / crap / ) resulting in 19,366 entries, and used for a library-free search with FDR set to 1 % . Deep learning-based spectra, retention time and ion mobility prediction were enabled, minimum fragment ion m / z was set to 200 and maximal ion fragment m / z to 1800. Trypsin was employed as protease and the maximum number of missed cleavages was configured with 2. Peptide length was adjusted to a range from 7 to 30 amino acids. Cysteine carbamidomethylation was set as a fixed and methionine oxidation as a variable modification, allowing a maximum of one variable modification. DIA-NN optimized the mass accuracy automatically using the first run in the experiment.

[0208] This resulted in a list of 6, 039 proteins with their corresponding LFQ values. Data processing using protein group quantities was done with Perseus software version 2.0.11 (Tyanova et al., Nature Methods 2016 13:9 2016, 13 (9) , 731-740) . Intensities were log2 transformed and contaminants removed. The matrix was then filtered to contain 100% valid values in at least one group. This reduced the matrix to 5,977 proteins, and remaining missing values were imputed from normal distribution (downshift 1.8, width 0.3) . Principal component analysis was performed without category enrichment. Volcano blots of protein expression differences were generated using the following criteria: p-value of 0.05, SO of 0.1 and permutation-based FDR set to 5% to correct for multi-testing with 250 randomizations.

[0209] 1.11. Antifungal Activity Assays

[0210] Antifungal activity assays

[0211] A. nidulans

[0212] To determine the broth dilution minimum inhibitory concentration (MIC) of the isolated substances, we performed microdilution tests according to the EUCAST guidelines (https: / / www.eu- cas t , orq / as to t tun i / me thods mant i tun g l suscept xbil xty tes ing; EUCAST E.Def 7.4 October 2023 and EUCAST E.DEF 9.4 March 2022) . In brief, ilicicolin H and ilicicolin K were dissolved in DMSO to a concentration of 25.6 mg / ml. These stock solutions were serially diluted 1:2 to obtain the working solutions 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 mg / ml. All solutions were added 1:100 to double strength RPMI 2% G supplemented with Tween-20 (RPMI 1640 with L-glutamine without Sodium Bicarbonate (Thermo Scientific, Catalog number: 31800089) , 20.8 g / 1; MOPS, 69.06 g / 1; glucose, 36 g / 1; Tween-20, 0.004 % (v / v) ; pH adjusted to 7.0 with NaOH) . For the S. cerevisiae cultivations, uracil was added to a concentration of 40 mg / 1 to the double strength RPMI 1640 2% G. DMSO was added 1:100 to RPMI 2% G to obtain a 0.0 mg / ml control. To prepare the S. cerevisiae inoculum, the strain CEN.PK113-5D (Sikorski et al., Genetics 122 (1989) , 19-27) was incubated on YPD plates (yeast extract, 10 g / 1; peptone, 20 g / 1; glucose, 20 g / 1) at 30°C for 48 hours until individual colonies were obtained. Several colonies were suspended in sterile distilled water to a density of 0.5 McFarland. To the A. nidulans inoculum the strain FGSC A4 (CBS 112.46) was incubated on potato dextrose agar (PDA) plates at 30°C for a week. Spores were harvested by rolling a sterile cotton swab on the colony and resuspending them in sterile distilled water. The spore suspension was filtered through glass wool and adjusted to a density of 0.5 McFarland. Finally, 100 pl of the supplemented media were mixed with 100 pl of inoculums in a well of a sterile, 96-well microdilution plate with flat-bottom wells, resulting in the test concentrations 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 pg / ml of ilicicolin H and K. The assay was performed in technical triplicates, and the medium was mixed with sterile water instead of the inoculums used as blank. The well plates were incubated at 30°C and the optical density at 560 nm was measured using a plate reader (Promage ProMax) . We consider a reduction of the optical density of at least 50% in comparison to the untreated growth control samples as "inhibited".

[0213] C. auris

[0214] To determine the broth dilution minimum inhibitory concentration (MIC) of the isolated substances, we performed microdilution tests according to the EUCAST guidelines (https: / / www.eu-

[0215] CAST E.Def 7.4 October 2023) . In brief, ilicicolin H and ilicicolin K were dissolved in DMSO to a concentration of 20.0 mg / ml. These stock solutions were then diluted to obtain the working solutions with concentrations of 15.0, 10.0, 5.0, 2.0, 1.0, 0.5, 0.2, 0.1, 0.05 and 0.02 mg / ml. All solutions were added 1:100 to double strength RPMI 2% G supplemented with Tween-20 (RPMI 1640 with L- glutamine without Sodium Bicarbonate (Thermo Scientific, Catalog number: 31800089) , 20.8 g / 1; MOPS, 69.06 g / 1; glucose, 36 g / 1; Tween-20, 0.004 % (v / v) ; pH adjusted to 7.0 with NaOH) . DMSO was added 1:100 to RPMI 2% G to obtain a 0.0 mg / ml control. The inoculum was prepared by incubating the C. auris DSM 21092 strain on potato dextrose agar at 35 °C for 24 hours. Several distinct colonies (> 1 mm) were suspended in distilled water, homogenized for 15 seconds, and the cell density adjusted to X = 530 nm = 0.1 by adding sterile water. This resulted in a suspension with 1-5 x 106CFU / ml, equivalent to 0.5 McFarland. The C. auris suspension was further diluted 1:10 in distilled water providing a working solution with 1-5 x 105CFU / ml. Finally, 100 pl of the supplemented media were mixed with 100 pl of inoculums in a well of a sterile, 96-well microdilution plate with flat-bottom wells, resulting in the test concentrations 100, 75, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.1 pg / ml of Ilicicolin H or Ilicicolin K. The assay was performed in technical quadruplicates, and the medium was mixed with sterile water instead of the inoculums used as blank. The well plates were incubated at 35°C and the optical density at 530 nm was measured using a plate reader (Tecan INFINITE 200pro plate reader) .

[0216] 1.12. Biomimetic synthesis of ilicicolin K from ilicicolin H

[0217] Ilicicolin K can be delivered by biometric synthesis from ilicicolin H as disclosed in Figs. 10 and 11. The desired cyclization reaction and oxidative dearomatisation of the phenol is clearly responsible and is emulated for the key intramolecular C- 0 bond formation. The full sequence from ilicicolin H to dearomatized, cyclised intermediate is possible in one-pot, by then changing reaction conditions to allow for elimination of the labile allylic ether norhydroxy-ilicicolin K can be accessed in 1 step. To reach ilicicolin K a regioselective functionalisation of the arene next to the phenol is required, there is a broad body of literature on the subject, and both direct oxidation protocols and multi-step tactics available. Exemplary reactions to be applied in this process where similar transformations were success-fully achieved and applied in synthesis are disc in Organic Letters, 2024, vol. 26, # 12, p. 2376 - 2380, Journal of Organic Chemistry, 2019, vol. 84, # 1, p. 346 - 364, Journal of Organic Chemistry, 2018, vol. 83, # 12, p. 6776 - 6782, Journal of the American Chemical Society, 2013, vol. 135, # 18, p. 6774 - 6777, Journal of the American Chemical Society, 2010, vol. 132, # 35, p. 12203 - 12205, Tetrahedron, 2007, vol. 63, # 19, p. 4052-4060, and Tetrahedron Letters, 2004, vol. 45, # 11, p. 2293 - 2295. 2. Results

[0218] 2.1. Activation of the ilicicolin H BGC by overexpression of TriliR

[0219] The ilicicolin H BGC contains, next to the biosynthetic genes trlliA-E, also a gene encoding for a transcription factor (Protein ID 72993) . The JGI gene model (Martinez et al., Nature Biotechnology 2008 26:5 2008, 26 (5) , 553-560) suggests a coding region with 1,389 bp encoding for a protein with 462 aa containing only a partial fungal transcription factor middle homology region (FTFMHR, cdl2148 of the conserved domain database (Wang et al., Nucleic Acids Res 2023, 51 (DI) , D384-D388) ( at residues 16-231 with an E-value of 4.82e-15 but no DNA-binding domain (Marchler- Bauer et al., Nucleic Acids Res 2017, 45 (DI) , D200-D203) ) ) . An alternative gene model (Protein ID 74475) is suggested for T. reesei (Koike et al., https: / / home.liebertpub.com / ind 2013, 9 (6) , 352-367) (Lit. No.39) ; this model does not contain any introns, consists of 2,364 bp encoding for a protein with 787 aa, and it contains a full-length FTEMHR at residues 270-733 (E-value 1.34e- 27) and a GAL4-like zinc cluster DNA binding domain at residues 11-47 (E-value 9.28e-10) . Consequently, we used the RutC-30 gene model for our molecular biological work and refer to the gene and protein as triliR and TriliR, respectively.

[0220] To overexpress TriliR, we put an altered genomic sequence (silent mutation at R227) under the control of the constitutive promoter of tefl and inserted it in front of the pyr4 locus into the T. reesei strain QM6a Apyr4 as previously described (Derntl et al., 2015) . We could detect substantially higher transcript levels of all biosynthetic genes in the ilicicolin H BGC in the strain OETriliR (approx. 400 to 1,400 times higher compared to the control strain) .

[0221] 2.2. Proteomics & targeted ilicicolin H analysis

[0222] This successful overexpression on mRNA level was also confirmed on proteomic level, with all five enzymes TriliA - E being significantly upregulated in the OETriliR strain versus the T. reesei wildtype strain, as depicted in Fig. 3A. Principal component analysis (PCA, Fig. 3B) revealed that the conducted genetical modifications resulted in the emergence of three distinct clusters. Specifically, sole overexpression of the ilicicolin H cluster (OETriliR) leads to a marked change in the protein expression profile, evident from its clustering distant from the wildtype samples. Samples with ilicicolin-BGC overexpression and simultaneous knockout of the epimerase TriliE (ATriliE) , the enzyme catalyzing the last step of ilicicolin H formation, demonstrate an overlap with the OETriliR cluster, suggesting minimal proteomic changes following the TriliE knockout. On the contrary, upon overexpression and knockout of TriliA (ATriliA) , which initiates ilicicolin H formation, a more pronounced proteomic change is induced, apparent by clustering apart from the others yet closer to the wildtype.

[0223] Targeted metabolomics analysis of ilicicolin H revealed that the T. reesei wildtype strain does not produce ilicicolin H in detectable quantities (LOD: 21.4 ng mL-1) under basal conditions, indicating that the ilicicolin H biosynthetic gene cluster (BGC) is silent under common laboratory conditions. Upon overexpression of TriliR (OETriliR) , the BGC is successfully activated and ilicicolin H gets produced in high yields, as seen in the extracted ion current chromatograms in Fig.3C and D. This production is abolished upon knockout of TriliA, underlining the necessity of this enzyme to initiate ilicicolin H biosynthesis. Overall, more product can be found in the mycelium than in the supernatant, with values of 1.346 pg ilicicolin H per mg mycelium (chapter 11.7.5) vs. around 6 ng of ilicicolin H in the medium per mg mycelium (see Supplementary Information, factor ~225 less) .

[0224] 2.3. Ilicicolin H isomers in detail

[0225] Extracted ion current chromatograms of iliH reveal three isomers at the expected m / z (Fig. 3C) , with peak (1) representing the main product, whereas peak (2) represents 8-epi-ilicicolin H and peak (3) the bfs-diene, respectively. The epimerase TriliE catalyzes the last step in the ilicicolin H biosynthesis, namely the epimerization from 8-epi-ilicicolin H (2) to ilicicolin H (1) (Zhang et al., 2019) . (2) in turn is a product of a putative S- adenosylmethionine (SAM)- dependent Diels-Alderase (Shenouda et al., 2021) , that catalyzes the transformation from bfs-diene (3) to 8-epf-ilicicolin H (2) . Knockout of the epimerase TriliE leads to a strong decrease in the production of (1) , and instead, (2) and (3) increase drastically in their abundance (Fig. 3C and D) . This is in contrast to the previous report by Zhang et al., 2019, where deletion of the epimerase-encoding gene yielded 8-epf- ilicicolin H and the in the biosynthesis initially formed tetramic acid (C27H33NO4, exact mass: 435.2410 Da) . A possible explanation for that could be that the ring-expanding cytochrome P450 (IccC (Zhang et al., 2019) , TriliC (Shenouda et al., 2021) ) , responsible for the conversion of the initially formed tetramic acid to the bis-diene (Zhang et al., 2019) , is more efficient in the native Trichoderma reesei host system. This might be either be caused by higher expression in the engineered BGC or, more likely, by a better cofactor supply by a matched P450 reductase (Lah et al., Fungal Genetics and Biology 2008, 45(4) , 446-458) .

[0226] Molecular networking of the untargeted metabolomics data combined several features to an "ilicicolin" network (Fig. 4) . Apart from the three ilicicolin H pathway (intermediate) products (1) , (2) and (3) , a fourth compound with mz 434.2325 was detected. Like the other three, it shows the same ionization adducts, namely [M+H]+, [M+H-H20] and [M+Na]+, but elutes far later, only be matched by RT but at Despite similar fragmentation spectra and m / z ratio, this feature is unlikely to be ilicicolin H or one of its measured intermediate products because of a vastly different retention time (RT = 8.23 min vs 5.84 min for (1) , 6.31 min for (2) and 6.57 min for (3) ) and the feature not being visible when ion mobility is turned on, in the applied mobility (1 / K0) window from 0.8 to 1.81 V*s cnr2. To (1) and (2) , three additional compounds were clustered: mz 432.2169, mz 450.2273 and mz 448.2117. Molecular formula prediction using SIRIUS revealed for mz 432.2169 the sum formula C27H29NO4, which corresponds to ilicicolin J (4) . For mz 450.2273, C27H31NO5 was proposed implying an oxidation variant of ilicicolin H. For mz 448.2117, C27H29NO5 was proposed, which equivalents a neutral mass of 447.2040 Da.

[0227] This unusual modification - an oxidation with simultaneous loss of two hydrogen atoms - caught our attention and we conducted further investigation by comparing its fragment spectra to those of (1) , (2) and (3) in Fig. 5. Although (1) , (2) and (3) are isomeric compounds with the sum formula C27H31NO4 and an exact mass of 433.2253 Da, their fragmentation spectra show certain differences (Fig. 5) . Whereas (1) and (2) , the two epimers, show nearly identical fragments with very similar intensities, (3) differs in regard to fragments which are part of the open chain (decalin unit) of the molecule. This becomes apparent by the intense fragment with m / z 189.1639, which is far less pronounced in (1) and (2) , and the fragment m / z 272.0909. The first one constitutes the fragmented decalin unit, which fragments more readily in open confirmation than when having undergone the IEDDA cycloaddition. The second one is the phenyl-pyridone unit with a part of the decalin unit attached. Overall, all three compounds fragment preferably directly between these two moieties, resulting in the most pronounced ion with m / z 230.0449. The novel, unknown compound shows a characteristic shift of + 13.98 Da for many of the fragments, compared to the spectrum of (1) . Judging from the location of the shift, the modification is most likely located in the phenyl- pyridone moiety of the molecule. The exact structure was determined by NMR analysis and is depicted in Fig. 6.

[0228] 2.4. Antifungal activity assays

[0229] The results of the antifungal assays are disclosed in Fig. 7A (for S. cerevisiae) , in Fig. 7B (for A. nidulans) , and Fig. 9 (for C. auris) . This comparison showed that the antifungal effect of ilicicolin K is significantly increased compared to the antifungal activity of ilicicolin H against S. cerevisiae and A. nidulans . In this experiment, ilicicolin K inhibited the growth of S. cerevisiae at a concentration of 1 to 2 pg / ml, which is approx. 64-fold lower than the minimal inhibition concentration of Ilicicolin H (approx. 64 - 128 pg / ml) . Ilicicolin K inhibited the growth of A. nidulans at a concentration of 16 to 32 pg / ml, which is at least 4-fold lower than the minimal inhibition concentration of ilicicolin H (above 128 pg / ml) . Against C. auris, ilicicolin H was active at lower concentrations than ilicicolin K (at approx. 1 pg / ml versus approx. 50 pg / ml) . The results of the antifungal assays are disclosed in Fig. 9. This comparison showed that ilicicolin K has moderate antifungal activity against C. auris compared to the ilicicolin H. In this experiment, ilicicolin H was active at lower concentrations than Ilicicolin K (at approx. 1 pg / ml versus approx. 50 pg / ml) .

[0230] Table 3. Utilized primer for cloning, genotyping and RT-qPCR.

Claims

Claims :

1. 4- ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8-dihy- droxybenzofuro [3, 2-c] pyridin-3 (2H) -one (ilicicolin K) .

2. Antifungal preparation comprising 4- ( ( 1R, 2S , 7S ) -4 , 7-dime- thyl-1- ( (E) -prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphtha- lene- 2 -carbonyl ) -7 , 8 -dihydroxybenzo f uro [3,2-c]pyridin-3 (2H) -one .

3. Antifungal preparation comprising 4- ( ( 1R, 2S , 7S ) -4 , 7-dime- thyl-1- ( (E) -prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphtha- lene- 2- carbonyl ) -7, 8 -dihydroxybenzo f uro [3, 2-c] pyridin-3 (2H) -one, and an agriculturally or and a pharmaceutically acceptable carrier or excipient.

4. An agricultural composition comprising an agriculturally effective amount of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) -prop-l-en-l- yl) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8-dihy- droxybenzofuro [3, 2-c] pyridin-3 (2H) -one and an agriculturally acceptable carrier, preferably in an invention to control the growth of agricultural pests especially an agricultural pest selected5. A food composition comprising an antifungally effective amount of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) -7, 8-dihy- droxybenzofuro [3, 2-c] pyridin-3 (2H) -one and a food composition which needs to be conserved from fungal activity.

6. The preparation or composition according to any one of claims 2 to 5, further comprising one or more further antifungal agents, and / or one or more antibiotic agents, and / or one or more diluents, and / or one or more binders or binding agents, and / or one or more dispersing agents, and / orone or more emulsifying agents, and / or one or more surfactants or wetting agents, and / or one or more sticking agents, and / or one or more thickening agents, and / or one or more pH adjusters or buffering agents, and / or one or more nutrients, and / or one or more plant growth regulators, and / or one or more herbicides, and / or one or more pesticides, and / or one or more insecticides, and / or one or more acaricides, and / or physiologically acceptable fluids, especially water, physiological saline, balanced salt solutions, aqueous dextrose, and / or glycerol; and / or pharmaceutical grades of mannitol, lactose, starch or magnesium stearate, and / or one or more suspending agents, and / or one or more propelling agents, and / or one or more stabilizing agents.

7. Ilicicolin K or the preparation or composition according to any one of claims 2 to 5, for use in the treatment and prevention of a fungal disease, especially of the human or animal body or of plants, associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides , Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera.

8. Ilicicolin K for use in a therapeutic or prophylactic treatment of the human or animal body, preferably for the prevention or treatment of a fungal disease associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides, Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera .

9. A method of treating or preventing a fungal disease or disorder, preferably for the prevention or treatment of a fungaldisease associated with, caused by, or the result of organisms in the Candida, Aspergillus, Histoplasma, Cryptococcus, Coccidioides , Paracoccidioides, Blastomyces, Mucor, Rhizopus, Scedosporium, Pneumocystis, Penicillium, Fusarium, Tinea, Malassezia, Trichophyton, Microsporum, or Epidermophyton genera, wherein an effective amount of ilicicolin K is administered to a human or animal patient, preferably a human patient, in an effective amount.

10. Method for production of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) - prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-car- bonyl) -7, 8-dihydroxybenzofuro [3,2-c]pyridin-3 (2H) -one, wherein a suitable precursor of ilicicolin K is finished to ilicicolin K by a suitable chemical step.

11. Method for production of 4- ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) - prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-car- bonyl) -7, 8-dihydroxybenzofuro [3,2-c]pyridin-3 (2H) -one (ilicicolin K) , wherein mycelium of an ilicicolin K-expressing fungi is lysed and extracted, preferably by a one-phase extraction, into a polar extraction agent, more preferred an extraction agent comprising acetonitrile, especially an extraction agent comprising acetonitrile, methanol and water; and / or extracted, preferably by a two- phase extraction, into diethyl ether, whereafter the extract is subjected to HPLC chromatography to enrich ilicicolin K, preferably to HPLC chromatography by a C-18 column, to obtain an ilicicolin K-comprising eluate.

12. Method according to claim 11, wherein the ilicicolin K-ex- pressing fungi is Trichoderma reesei, especially Trichoderma reesei wherein the biosynthetic gene cluster (BGC) of ilicicolin is activated by overexpressing the BGC transcription factor under the constitutive tefl promotor in T. reesei.

13. A composition comprising ilicicolin K, preferably a pharmaceutical composition, especially a parenteral formulation, a tablet, powder, pill, or capsule, a liquid formulation, a lozenge, an aerosol composition, an ampule, a suppository, a retention enema, a cream, an ointment, a depot preparation, and / or a microsphere preparation; an agricultural composition, especially an emulsifiable concentrate, an emulsion concentrate, a suspensionconcentrate, a soluble liquid, an oil-based suspension concentrate, and / or a suspoemulsion; or a disinfectant composition, especially a surface or consumer product.

14. A pharmaceutical composition comprising ilicicolin K for use in the treatment and prevention of fungal disease or disorder, wherein the composition is administered via a route selected from oral, topical, vaginal, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, via transmucosal absorption, or via catheter delivery, wherein the amount of ilicicolin K in the composition is preferably from 10 ng to 10 g, more preferred from 10 mg to 8 g, especially from 500 mg to 5 g.

15. A novel molecular variant of ilicicolin K according to claim1, an antifungal or agricultural preparation or composition of this variant as defined for ilicicolin K in any one of claims 2 to 8 and 13 to 14, especially wherein the ilicicolin variant is selected from the group 4- ( ( 1R, 2S , 7S ) -7-hydroxymethyl-4-methyl-l- ( (E) -prop-l-en-l-yl ) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) - (7, 8-dihyrdoxybenzofuro ) [3, 2-c] pyridine -3 (2H) -one (Fig. 8A) ; 4-( (lR,2S,7S) -7 -carboxy- 4 -methyl- 1- ( (E) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-dihyrdoxybenzofuro) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8B) ; 4-( (lR,2S,7S)-7- ( acetyloxy) hydroxymethyl-4-methyl-l- ( (E) -prop-l- en-l-yl) -1,2, 4a, 5, 6,7, 8, 8a-octahydronaphthalene-2-carbonyl ) -(7, 8-dihyrdoxybenzofuro) [3, 2-c] pyridine-3 (2H) -one (Fig. 8C) ; 4-( (lR,2S,7S)-74- ( (1R,2S,7S) - 6 -hydroxy- 7 -hydroxymet hyl- 4 -methyl- 1- ( (E) -prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- carbonyl) - (7, 8-dihyrdoxybenzofuro) [3, 2-c] pyridine-3 (2H) -one (Fig. 8D) ; 4- ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-diace- tyloxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8E) ; ( (1R,2S,7S)- 4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahy- dronaphthalene-2-carbonyl ) - (7, 8-cyclohexoyloxybenzofuro ) [3, 2- c] pyridine-3 ( 2H) -one (Fig. 8F) ; ( ( 1R, 2S , 7S ) -4 , 7-dimethyl-l- ( (E ) - prop-l-en-l-yl) -1,2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-hydra- zone ) - ( 7 , 8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8G) ; ( (lR,2S,7S)-4, 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-methylhydrazone ) - (7,8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8H) ;( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2- (hydroxyethyl) hydrazone )- ( 7 , 8-dihydroxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 81) ; ( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) -1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-oxime- (7, 8-dihy- droxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8J) ; 4-( (lR,2S,7S)-4, 7 -dimethyl- 1- ( (E) -prop-l-en-l-yl ) - 1, 2, 4a, 5, 6, 7, 8, 8a-octahydronaphthalene-2-carbonyl ) - (7, 8-di- methoxybenzofuro ) [ 3 , 2-c] pyridine-3 ( 2H) -one (Fig. 8K) ; 4-( ( 1R, 2S , 7S ) - 4 -bromo- 4 , 7-dimethyl-l- ( (E) -prop-l-en-l-yl ) - 1,2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 -carbonyl ) - (6, 9-di- bromo ) - ( 7 , 8-dimethoxybenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8L) ; and 4- ( ( 1R, 2S , 7S ) -4-bromo-4 , 7-dimethyl-l- ( (E ) -prop-l-en-l- yl)-!, 2, 4a, 5, 6, 7, 8, 8 a-octahydronaphthalene- 2 -carbonyl ) - (6, 9-di- bromo) - (7, 8-diimidazoylbenzofuro ) [3, 2-c] pyridine-3 (2H) -one (Fig. 8M) .

Images