Compounds for use against coronavirus infection

Abstract

The invention relates to compounds for use in the prevention or the treatment of infections caused by a coronavirus. In particular compounds extracted from Darksidea spp. are provided for use in the tre- atment of a coronavirus infection, in particular an infection by feline coronavirus or porcine coronavirus.

Description

[0001] COMPOUNDS FOR USE AGAINST CORONAVIRUS INFECTION FIELD OF THE INVENTION The invention relates to compounds for use in the prevention or the treatment of infections caused by a coronavirus. In particular compounds extracted from Darksidea spp. are provided for use in the treatment of a coronavirus infection, in particular an infection by feline coronavirus or porcine corona- virus. BACKGROUND OF THE INVENTION Coronaviruses (CoVs) have the potential to cause fatal enteric, respiratory or systematic disease in humans, domestic and wild animals. Besides, new CoV strains are recognized as a continuous threat to cause mild to severe disease in domestic and wild animals, poultry, rodents, carnivores and humans. CoVs are enveloped single-stranded positive-sense RNA viruses and are well-known in veterinary me- dicine. CoVs are sorted in four genera Alpha-, Beta-, Gamma-, and Delta-CoV. Almost all alpha-CoVs and beta-Covs have mammalian hosts, while gamma-CoVs and delta-CoVs are commonly found in avian hosts, even though some of them can also infect mammals (10.3390 / life11020123), doi: 10.1038 / s41579-018-0118-9) Members of species Alphacoronavirus 1 can infect different domestic animals. TGE (transmis- sible garstoenteritis) is a highly contagious enteric viral disease of pigs characterized by vomiting, severe diarrhea, and very high mortality rate (often 100%) in piglets less than 2 weeks of age. Porcine res- piratory coronavirus (PRCV) is a mostly apathogenic natural deletion mutant version of TGE that served as a naturel „vaccine” against TGE protecting most of the herds against the deadly disease. Canine and feline coronaviruses (CCoV, FCoV, respectvely) typically cause diarrhoea in young animals, however both viruses are able to cause severe, fatal multisystemic infections. A mutant version of CCoV named canine pantropic coronavirus CPCoV can infect a wide range of organs inducing a disease of high mor- tality, whereas the feline infectious peritonitis virus, a well known disease of cats, is also a natural mutant version of FCoV arising within the host due to prolonged infection gains access to the macrophages of the host and causes almost always fatal, disseminated, multisystemic infection. There are no effective vaccines on the market against FIP. Previous research on interspecies transmission of animal CoVs and wildlife reservoirs for CoVs showed that for example the porcine CoV, TGEV, and canine and feline CoVs can cross-infect pigs, dogs, and cats with variable disease expression (10.3390 / life11020123). As to the treatment of viral infections, two formally approved drugs could be regarded as natural product (i.e., the lignan podofilox and the flavonoid sinecatechin) [De Clercq, E. and Li, G., Approved antiviral drugs over the past 50 years. Clin Microbiol Rev, 2016.29: 695-747.]. Furthermore, more than 100 plant species were confirmed to express antiviral properties [Martinez, J.P., et al., Antiviral drug discovery: broad-spectrum drugs from nature. Nat Prod Rep, 2015.32: 29-48.]. Darksidea belong to the root-colonising fungi, dark septate endophytes (DSE) [Knapp, D.G., et al., Dark septate endophytic pleosporalean genera from semiarid areas. Persoonia, 2015.35: 87-100. ] To date, no medicinal use of Darksidea has been reported. Petasol (3S,4aR,5R,6R)-6-hydroxy-4a,5-dimethyl-3-prop-1-en-2-yl-3,4,5,6,7,8-hexahydronapht- halen-2-one) and S-petasin are eremophilane sesquiterpenoid compounds produced by e.g. Petasites formosanus and Penicillium sp., which have been tested for the treatment of e.g. malaria, cancer, HIV and migraine in humans. SUMMARY OF THE INVENTION In a first aspect a compound of general formula (I) for use in the prevention and / or treatment of a viral infection is provided. In a preferred embodiment the compound of general formula (I) is for use in the prevention and / or treatment of a disease caused by a viral infection. In a preferred embodiment a compound of general formula (I) for use in the treatment of a viral infection is provided, wherein in general the bond between carbon 1 and 6 is either a single bond or a double bond, the bond between carbon 13 of R1 and carbon 3 is either a single bond or a double bond, wherein R1 is isopropyl, if the bond between R1 and carbon 3 is a double bond, or isopropenyl, if the bond between R1 and carbon 3 is a single bond, X is O or nothing R2 is H or R2 has the following formula (i) wherein in formula (i) R4is thioalkyl or a C1-C7thi- oether, or a C1-C8alkyl or a C1-C8alkenyl substituted with a single -SH group, wherein said C1- C8 alkenyl is a straight or branched chain alkenyl; R3is H or R3has the following formula (i) wherein in formula thioalkyl or a C1-C7thi- oether, or a C1-C8alkyl or a C1-C8alkenyl substituted with a single -SH group, wherein said C1- C8alkenyl is a straight or branched chain alkenyl; Y is O or nothing. Preferably one of X and Y is O and preferably if X is nothing, then R3is H and if Y is nothing then R2is H. Preferably when X is nothing, Y is O, preferably when Y is nothing, X is O. In a preferred embodiment the compound has general formula (II) wherein in general formula (II) the bond between carbon 1 and 6 is either a single bond or a double bond, preferably a double bond, the bond between carbon 13 of R1and carbon 3 is either a single bond or a double bond, wherein R1is isopropyl, if the bond between R1and carbon 3 is a double bond, or isopropenyl, if the bond between R1and carbon 3 is a single bond, R2is H or R2has the following formula (i) wherein in formula (i) R4is thioalkyl or a C1-C7thi- oether, or a C1-C8 alkyl or a C1-C8 alkenyl substituted with a single -SH group, wherein said C1- C8 alkenyl is a straight or branched chain alkenyl. In a preferred embodiment the compound has general formula (III) wherein R1 is isopropyl, if the bond between R1 and carbon 3 is a double bond, or isopropenyl, if the bond between R1 and carbon 3 is a single bond, R2 is H or R2 has the following formula (i) wherein in formula (i) R4is thioalkyl or a C1-C7thioether, or a C1-C8alkyl or a C1-C8alkenyl substituted with a single -SH group, wherein said C1-C8alkenyl is a straight or branched chain alkenyl. Preferably when the bond between R1and carbon 3 is a single bond, the compound is the S isomer. In a preferred embodiment the compound is selected from the group consisting of compounds having general formulae IV.1, IV.2, IV.3, preferably general formulae IV.1, IV.2, wherein R2is as defined above. In a preferred embodiment R2is selected from the group consisting of In a preferred embodiment when the bond between C3 and R1 is a single bond, the compound is the S isomer. In a preferred embodiment the compound has general formula (III) wherein R1is isopropyl, if the bond between R1and carbon 3 (the ring carbon atom to which R1is attached) is a double bond, or isopropenyl, if the bond between R1and carbon 3 (the ring carbon atom to which R1is attached) is a single bond and the compound is the S isomer, and R2is H. In a preferred embodiment the compound is selected from the group consisting of petasol and esters thereof, isopetasol and esters thereof. In a preferred embodiment the compound is selected from the group consisting of petasol, peta- sin, S-petasin, isopetasol, isopetasin and S-isopetasin. In a preferred embodiment the compound is selected from the group consisting of petasol and isopetasol. In a preferred embodiment the compound is selected from the group consisting of petasol, peta- sin and S-petasin. In a preferred embodiment the viral infection is caused by a coronavirus. Preferably the corona- virus infects domesticated and / or wild animals, i.e. the coronavirus does not infect humans. Preferably viral infection is an animal viral infection, i.e. non-human viral infection. Preferably the subject to be treated with the compound is a non-human animal. Preferably the subject to be treated is a felid (i.e. belonging to Felidae), a canid (i.e. belonging to Canidae) or a pig (i.e. belonging to Suidae). Preferably the coronavirus is selected from Alphacoronavirus and Betacoronavirus. Preferably the coronavirus is Alphacoronavirus 1. In a preferred embodiment the virus is a coronavirus, preferably animal coronavirus, highly pre- ferably porcine transmissible gastroenteritis coronavirus or feline infectious peritonitis virus strain. In a preferred embodiment the compound is a pharmaceutically acceptable variant of the com- pound defined above (e.g. the compound according to formula I, II or III), such as a pharmaceutically acceptable solvent, salt or prodrug (e.g. an ester which can be cleaved by an esterase in vivo) thereof. In a second aspect a method for the prevention and / or treatment of a viral infection is provided, the method comprising administering to a subject in need thereof a compound of formula (I) defined above. In a third aspect a pharmaceutical or veterinary composition is provided, comprising the com- pound defined above. Preferred embodiments of the first aspect are also preferred embodiments of the second and third aspect. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. HPLC-UV (λ=250 nm) chromatogram of the methyl alcohol extract obtained from the in vitro culture of Darksidea alpha. Peaks at retentions 7.3, 7.6 and 8.5 min correspond to the compounds DA-1 (petasol), DA-2 (isopetasol) and DA-3 (neopetasol), respectively. Figure 2. HR-MS spectra of the compounds DA-1 (petasol) (A), DA-2 (isopetasol) (B) and DA- 3 (neopetasol) (C), obtained from the HPLC-MS chromatogram of Darkside alpha extract. Figure 3. UV spectra of petasol (A) isopetasol (B) and neopetasol (C) obtained from the HPLC- UV chromatogram of Darksidea alpha extract. Figure 4. Chemical structure of petasol (A), isopetasol (B) and neopetasol (C). Figure 5. Antiviral effect of the compounds against feline infectious peritonitis virus strain DF-2. The host cell was Fcwf-4 [Fcwf] (ATCC: CRL-2787) feline macrophage. Toxicity of the compounds on the host cells was also tested. Black bars represent the concentration values where the compound was cytotoxic to the host cell. Figure 6. Antiviral effect of the compounds against Porcine transmissible gastroenteritis corona- virus (strain Purdue). The host cell was PK-15 (ATCC: CCL-33) porcine kidney cells. Toxicity of the compounds on the host cells was also tested. Black bars represent the concentration values where the compound was cytotoxic to the host cell. DETAILED DESCRIPTION OF THE INVENTION The term “tioalkyl” as used herein refers to a saturated (i.e., S-alkyl) or unsaturated (i.e., S-alkenyl and S-alkynyl) group attached to the parent molecular moiety through a sulfur atom. In certain embodi- ments, the alkyl group contains 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl group contains 1- 4 aliphatic carbon atoms. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like. Petasol is an eremophilane sesquiterpenoid compound, having the structure (A):

[0002] and isomers isopetasol, having the structure (B): and neopetasol, having the structure (C): . Petasol may be synthesized by the method described in Neuenschwander, M., Neuenschwander, A. and Steinegger, E. (1979), Struktur der Sesquiterpene von Petasites hybridus (L.) G. M. et SCH.: Neopetasol‐Abkömmlinge. HCA, 62: 627-634. https: / / doi.org / 10.1002 / hlca.19790620229, while the synthesis of isopetasol was reported by Bohlmann and Otto (Natürlich vorkommende Terpen‐Derivate, 3831) Synthese des Isopetasols. Liebigs Ann. Chem., 1982: 186-190. https: / / doi.org / 10.1002 / jlac.198219820119) and Neuenschwander et al (supra), Yamakawa et al. (Chem. Pharm. Bull., 1979, 27, 331-340. b) Torii et al. (Bull. Chem. Soc. Jpn., 1979, 52, 861-866). More refe- rence to the synthesis of petasol and isopetasol are found in Pirrung et al. (The Total Synthesis of Natural Products: Bicyclic and Tricyclic Sesquiterpenes. John Wiley & Sons, 22. Sept.2009); Burow, Kenneth Wayne Jr., "Approaches to the synthesis of the petasin sesquiterpenes" (1973). Retrospective Theses and Dissertations.6138. https: / / lib.dr.iastate.edu / rtd / 6138); DE4447594A1. Petasol and its isomers may be isolated from a number of sources with methods well-known in the art. See e.g. Lin et al. (Eremophilanes from Petasites formosanus Kitamura. Chem. Pharm. Bull. 46(11) 1807-1809 (1998)) and Debrunner and Neuenschwander (Sesquiterpenes of Petasites hybridus ( L.) G.M. et Sch.: influence of locations and seasons on sesquiterpene distribution. Pharmaceutics Acta Helvetiae 70 (1995) 315-323) from Petasites formosanus; Jayasuriya et al. Identification of Diverse Microbial Metabolites as potent Inhibitors of HIV-1 Tat Transactivation. Chem Biodivers. 2005 Jan;2(1):112-22. doi: 10.1002 / cbdv.200490162.) from Penicillium sp. Isolation of petasol and its isomers from Darksidea alpha is described in the Examples. Identification of the isolated compounds can be performed by conventional methods, such as HPLC and NMR. An example of such methods is given in the Examples. Petasin (CAS: 26577-85-5) is an ester of angelic acid and petasol, having the structure (D) and the isomers isopetasin (CAS: 469-26-1), having the structure (E) and neopetasin (CAS: 70387-53-0), having the structure (F) Synthesis of petasin and isopetasin is described in e.g. Burns and Taylor Synthetic Approaches to Enantiomerically Enriched 4-Hydroxycyclohex-2-en-1-one - A Key Chiral Building Block in Complex Natural Product Synthesis; Synthesis 2011. No.5, pp0681-0707 and in DE4447594. Extraction of peta- sin is described e.g. in EP2485718, EP0281656, CH690355 and KR20150047814. S-petasin (CAS:70238-51-6) is a methylsulfanyl derivative of petasin, having the structure (G) and the isomers S-isopetasin with the structure and S-neopetasin (CAS: 87984-58-5) with the structure A method for producing an extract containing S-petasin and its isomers from cranberry is described in WO2009149288, from Petasites hybridus in WO2011042469 and by Aebi et al.. In- haltsstoffe von Petasites hybridus (L.) Fl. Wett. Pharmaceutica acta Helvetiae.1995;29:277–279 (con- taining petasin, isopetasin, S-petasin and S-isopetasin), Lin Y-L, Mei C-H, Huang S-L, Kuo Y-H. Four new sesquiterpenes from Petasites formosanus. Journal of Natural Products.1998;61(7):887–890. Up to date, seven species in the Darksidea genus have been characterized: D. alpha, beta, gamma, delta, epsilon, zeta and phi [Knapp, D.G., et al., Dark septate endophytic pleosporalean genera from semiarid areas. Persoonia, 2015. 35: 87-100.; Romero-Jiménez M.J,. et al., Darksidea phi, sp. nov., a dark septate root-associated fungus in foundation grasses in North American Great Plains. Mycologia, 2022.114:254-269.]. The genus has a worldwide distribution and has been detected in several different habitats, mainly grassland. The biggest amount of petasol (together with isopetasol and neopetasol) pro- duced was detected in D. alpha, however, isolates from other species also produced these compounds. The production of petasol and / or any of the compounds described herein by a plant or fungus species may be detected by well known methods (an example is given in the Examples section) and the extraction of petasol and / or any of the compounds for use according to the invention may be extracted from plants or fungus by known methods (see references above and the Examples). Petasol and isopeta- sol may be isolated from any of the Darksidea species by the method described in the Examples, comprising: (a) culturing an isolate of a Darksidea sp. under suitable conditions (such as a medium allowing growth of the culture (e.g. Potato agar dextrose), a temperature allowing growth of the culture (e.g. room temperature) and light conditions allowing growth of the culture (e.g. dark)) allowing the production of petasol by the cells, (b) obtaining an extract of the culture (either the complete culture comprising medium and fungi or the fungi alone or the medium alone) by a suitable method (e.g. by alcoholic extraction), (c) isolating the desired compound by suitable means (e.g. preparative HPLC). Culturing may be performed in a bioreactor. Accordingly, a method for the production of petasol, isopetasol and / or neopetasol is provided, comprising steps (a)-(b) and optionally (c) defined above. In another aspect, use of a Darksidea sp., highly preferably Darksidea alpha, for the production of petasol, S-petasin is provided, comprising steps (a)-(b) and optionally (c) defined above. A pharmaceutical or veterinary, preferably veterinary composition comprising a compound ac- cording to formula (I) and a pharmaceutically acceptable excipient or an excipient suitable for veterinary use, respectively, is also provided. Preferably the pharmaceutical or veterinary, preferably veterinary composition is for use in the treatment of a virus infection, preferably a virus infection as defined above and in the claims. Preferably the pharmaceutical or veterinary, preferably veterinary composition is su- itable for oral administration, e.g. is in the form of a tablet, powder, capsule. In other embodiments the pharmaceutical or veterinary, preferably veterinary composition is suitable for parenteral administration, e.g. intravenous administration. In another aspect, an extract comprising a compound according to formula (I) derived from a plant or a fungus is provided for use in the treatment of a virus infection. Preferably the plant belongs to a Petasites sp. or the fungus belongs to a Darksidea sp., preferably to Darksidea alpha. Preferably the extract is enriched in the compound. Preferably the compound is selected from the group consisting of petasol and esters thereof, S-petasin. Preferably the compound is selected from the group consisting of petasol, petasin, S-petasin. Preferably the compound is selected from the group consisting of petasol and S-petasin. In another aspect the compound for use is a prodrug of petasol or S-petasin, which may be con- verted into petasol or S-petasin, respectively, upon administration to a subject in need thereof. A phar- maceutical or veterinary composition comprising the prodrug and a pharmaceutically acceptable exci- pient or an excipient suitable for veterinary use, respectively, for use in the treatment of a coronavirus infection is provided. The virus is preferably selected from coronaviruses (family Coronaviridae, genera Coronavirus: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, Deltacoronavirus), preferably respiratory co- ronaviruses, highly preferably Alphacoronavirus 1, preferably feline infectious peritonitis virus (FIPV), preferably porcine transmissible gastroenteritis virus. The virus may be e.g. bovine respiratory corona- virus, canine respiratory coronavirus, feline respiratory coronavirus, infectious bronchitis viruses, and coronaviruses causing mainly respiratory, enteric, hepatic and neurological symptomps, such as rabbit coronavirus, feline enteric coronavirus (FECV), feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhoea virus (PEDV), and porcine deltacoronavirus (PDCoV). Preferably the subject to be treated is a mammal or an avian subject, preferably a felid, a canid or a pig, preferably belonging to the family Felidae, preferably to the genus Felis, preferably a domestic cat, cheetah, lion, mountain lion, preferably belonging to the family Canidae, preferably to the genus Canis, preferably a domestic dog, or preferably belonging to the family Suidae, preferably to the genus Sus, preferably a domestic pig. Preferably the subject to be treated is a mammal or an avian subject, preferably a companion animal (such as a dog, cat, pig or rabbit), a domesticated or farm animal, such as cattle, swine, sheep, fowl or a wild animal, such as bats, big cats (lion, leopard etc.), hares or african wild dogs. Treatment as used herein preferably comprises preventive measures, e.g. prevention of an in- fection by the administration of a compound as defined herein. Pharmaceutical or veterinary composition The compound for use provided herein may be comprised in a pharmaceutical or preferably a veterinary composition, together with one or more pharmaceutically acceptable excipients or one or more excipients suitable for veterinary use, e.g. carriers. The compound for use provided herein and the pharmaceutical or veterinary composition may be formulated for administration via the oral, parenteral or topical route. For example, the compound or the pharmaceutical or veterinary composition may be administered to a subject in need thereof in the form of nasal drops or a nasal spray, oral dosage forms (such as tablets, powders, suspensions or solutions), via injection or via inhalation. Doses The skilled person will understand that the dose to be administered, the route to be chosen for administration and the duration of the treatment depend on the individual and the type and severity of infection to be treated, as well as on the specific compound used and the virus. A dose of e.g. 1-100 mg / kg body weight, e.g.5-50 mg / kg body weight, e.g.10-50 mg / kg body weight may be administered. The term animal viral infection preferably refers to an infection of animals other than humans, e.g. an infection that is caused by a virus that does not infect humans or does not cause a disease when infecting humans. The animal infected is preferably a vertebrate, more preferably a bird or a mammal. EXAMPLES Fungal isolates The fungal isolates from which DA-1 (petasol), DA-2 (isopetasol) and DA-3 (neopetasol) were isolated, represent the widespread root endophytic fungi Darksidea alpha (Ascomycota, Pleosporales). The isolates are in the fungal root endophyte strain collection at the Mycological Research Group (De- partment of Plant Anatomy, Institute of Biology, Eötvös Loránd University). Albeit several isolates were screened and they produced the compounds, the most efficient producers were the isolates DSE7 / 1, DSE7 / 15 and DSE7 / 20 (Table 1-2). All those isolates belong to our collection based on which we described the genus and species [Knapp, D.G., et al., Dark septate endophytic pleosporalean genera from semiarid areas. Persoonia, 2015. 35: 87-100.]. We used phylogenetic analyses of sequences of seven nuclear DNA loci for the species identification of the isolates, nrDNA ITS: internal transcribed spacer regions of the nrDNA and intervening 5.8S nrDNA; LSU: partial 28S large subunit of the nrRNA gene; SSU: partial 18S small subunit of the nrRNA gene; ACT: partial actin gene; TUB: partial beta-tubulin gene; CAL: partial calmodulin gene; TEF: partial translation elongation factor 1-alpha gene. All of the strains were isolated from surface sterilized healthy root segments of healthy-looking host plants [Knapp, D.G., et al., Dark septate endophytic pleosporalean genera from semiarid areas. Persoonia, 2015. 35: 87-100.; Knapp, D.G., A. Pintye, and G.M. Kovacs, The dark side is not fastidious - dark septate endophytic fungi of native and invasive plants of semiarid sandy areas. PLoS One, 2012. 7: e32570.]. Tables 1-2. The host, geographic origin and GenBank accession numbers of DNA sequences of the three Darksidea alpha isolates, which produced the highest amounts of the three compounds. Isolate Host Location ITS LSU DSE7 / 1 Bromus tectorum Fülöpháza, Hungary KP183966 KP184005 DSE7 / 15 Ailanthus altissima Tatárszentgyörgy, Hungary KP183970 KP184037 DSE7 / 20 Festuca vaginata Fülöpháza, Hungary KP183994 KP184015 Isolate SSU ACT TUB CAL TEF DSE7 / 1 KP184005 KP184093 KP184192 KP184122 KP184160 DSE7 / 15 KP184055 KP184086 KP184205 KP184124 KP184163 DSE7 / 20 KP184066 KP184084 KP184210 KP184128 KP184164 Preparation of fungal extracts for analysis and isolation Isolates of Darksidea alpha were grown in Petri dishes (60 mm) on Potato dextrose agar (PDA) medium (VWR, Hungary) at room temperature in dark for 30 days. Complete in vitro cultures containing the medium and fungal mycelium were lyophilized and pulverized. These powders were extracted three times. First, they were extracted with 5 mL of methyl alcohol held under reflux at boiling point for 30 min. Thereafter, the insoluble, centrifuged material was extracted for a second time and a third time, as before. The combined supernatants were dried by a rotary vacuum evaporator at 40 °C. The dried ext- racts were dissolved in 5 mL of methanol. These solutions were used 1) subsequent to their dilution with methyl alcohol to identify the compounds by analytical high- performance liquid chromatography (HPLC) hyphenated with ultraviolet (UV) and high-resolution mass spectrometry (HR-MS) detections and 2) to isolate compounds by preparative HPLC. Analytical HPLC hyphenated with UV and HR-MS detections A Dionex Ultimate 3000 UHPLC system (3000RS diode array detector (DAD), TCC-3000RS column thermostat, HPG-3400RS pump, SRD-3400 solvent rack degasser, WPS-3000TRS autosamp- ler), hyphenated with a Orbitrap Q Exactive Focus Mass Spectrometer equipped with electrospray ioni- zation (ESI) (Thermo Fischer Scientific, Waltham, MA, USA) was used for chromatographic separation and high resolution mass spectral analysis. The HPLC separations were performed on a Kinetex C18 column (75 × 3 mm; 2.6 μm) (Phenomenex, USA). Eluents: eluent A, 0.1% v / v formic acid, eluent B, acetonitrile:0.1% v / v formic acid (80:20, v / v). Linear gradient: 0.0 min, 20% B; 10.0 min, 70% B; flow rate: 0.3 mL / min; column temperature: 25 °C; injected volume: 1.0–5.0 μL. The ESI source was opera- ted in positive ionization mode and operation parameters were optimized automatically using the built- in software. The working parameters were as follows: spray voltage, 3500 V (+); capillary temperature 256 °C; sheath-, auxiliary- and spare-gases (N2): 47.50, 11.25 and 2.25 arbitrary units, respectively. The resolution of the full scan was of 70,000 and the scanning range was between 100–1000 m / z units. DAD spectra were recorded between 230 and 600 nm. Preparative HPLC A Pharmacia LKB HPLC (Uppsala, Sweden) system (2248 pumps, VWM 2141 UV detector) was connected to a preparative HPLC column: Gemini, 5 µm, C6-Phenyl, 100 × 21.2 mm (Phenomenex, USA). The eluents were the same as described above. Linear gradient: 0.0 min, 10% B; 20.0 min, 70% B; flow rate: 5.0 mL / min; column temperature: ambient; injected volume: 500 μL. Nuclear magnetic resonance (NMR) spectroscopy NMR spectra of the isolated compounds were recorded in chloroform-d at 25 °C on a Varian DDR spectrometer (599.9 MHz for 1H and 150.9 MHz for 13C) equipped with a dual 5 mm inverse detection gradient (IDPFG) probe-head. Chemical shifts were referenced relative to the appropriate sol- vent resonances. Compounds Compounds to be tested were dissolved in DMSO at 20 mM concentration and stored at -20 oC. Stock solutions were diluted with DMEM medium to result 100 µM concentration. The DMSO content was 0.5% in the highest concentration containing wells. Cells and viruses. Felis catus whole fetus 4 (FCWF-4) and PK-15 cells were used for virus propagation and titration. The cell lines were maintained as monolayer cultures in Eagle’s Minimum Essential Medium (Sigma- Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 0.2 mM glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 μg / ml amphotericin B, 1 mM sodium pyruvate, and 1% nonessential amino acids (Sigma-Aldrich). For the determination of the growth kinetics of the viruses in the cell line, cells were infected at multiplicity of infection (MOI) of 0.1. After 1 h of adsorption at 37°C, cells were washed twice with the medium and incubated at 37°C with 5% CO2. Titers of the extracellular and intracellular viruses were determined by the titration of cell culture supernatants collected at different time points from CrFK cells. Intracellular virus titers were determined after washing monocytes five times with RPMI 1640, followed by two freeze-thaw cycles. Supernatant was collected after low-speed centrifugation (3,000 × g for 10 min) and added to the original culturing volume with RPMI 1640. The titers were calculated as 50% tissue culture infectious doses (TCID50) / ml with the Reed and Muench method (Reed LJ, Muench H. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg.27:493–497). TGEV Purdue and FIPV DF-2 strains were used (kindly provided by Berndt Klingeborn (SVA, Uppsala, Sweden)). Cytotoxicity assay In vitro cytotoxicity testing of the compounds was performed by the Alamar Blue cell viability assay. Briefly, the cells were plated in a 96-well flat bottom tissue culture plates in 10% FBS containing EMEM media (5000 cells / 100 µL) one day prior to experiment. Before the test, 50 µL supernatant was removed and replaced with 50 µL EMEM serum free (SFM) medium. Compounds were serially diluted with SFM EMEM medium and added to the cells (final con-centration range: 0.02 - 100 µM, final FBS concentration: 2.5%). Two days after, 20 µL Alamar Blue (resazurin sodium salt, Merck) solution (0.15 mg / mL in PBS, filtered 0,45 µ M Merck) was added to each well and after 3-3.5 hrs of incubation the fluorescence was measured at λEx = 530 / 30 and at λEM = 610 / 10 nm using a Synergy H4 multi-mode microplate reader (BioTek). All measurements were performed in quadruplets and the mean IC50 values together with SEM were represented on the graphs. Virus titration and antiviral effect of the compounds The cells were plated in 96-well flat bottom tissue culture plates (TPP) in 10% FBS containing EMEM medium (5000 cells / 100 µL) 24 hrs prior to experiment. After removing the supernatant, cells were treated for 1 hour with the serial dilution of the compounds in EMEM media. As negative control DMEM containing 0.5% DMSO medium was applied. After treatment, the cells were infected with TGEV or FIPV, respectively at a multiplicity of infection (MOI) of 0.05, then cells were incubated with the viruses for further 1.5 hrs. After the removal of the supernatant from the wells, infected cells were incubated with 200 µL serially diluted compound solutions in EMEM containing 5% FBS. The inoculated cultures were growing in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effect (CPE) daily. Microscopic images were taken 2 days after infection. When CPEs were observed, typically 48 hrs after infection, 50 µL of virus-containing supernatants were transferred to 96-well plates to determine the infective titer and kept in -80°C until further use. To estimate the effect of the treatment by infective titration we determined the TCID50 / mL values from each dilutions from the supernatant. The supernatant were diluted in 10-fold serial dilution in a dilution plate and were added to a the cell monolayers in 96-well cell culture plate. The plates were incubated for 5days, the supernatant were removed and the plates were inactivated by 10% formal- dehyde in PBS solution for 30 minutes, washed with tap water and stained with 0.5% crystal violet in ethanol for 15 minutes at room temperature. Clear CPE were visualized and the viral titers were meas- ured by determining the TCID50 / mL using TCID50 calculator v2.1 [Binder M. TCID50 Calculator (v2.1-20-01-2017_MB) [(accessed on 10 June 2020)]; available online: https: / / www.klinikum.uni-hei- delberg.de / fileadmin / inst_hygiene / molekulare_virologie / Downloads / TCID50_calculator_v2_17-01- 20_MB.xlsx.]. Experiments were done in quadruplets and repeated at least three times. The assay was validated by a negative and a positive control (medium treated virus control and chloroquine treated control). RESULTS Isolation and Identification of the compounds The DNA sequence information based on which the Darkside alpha as species was described [Knapp, D.G., et al., Dark septate endophytic pleosporalean genera from semiarid areas. Persoonia, 2015.35: 87-100.; Romero-Jiménez M.J,. et al., Darksidea phi, sp. nov., a dark septate root-associated fungus in foundation grasses in North American Great Plains. Mycologia, 2022. 114:254-269.] make the unambiguous identification of the isolates possible. Although we worked from our strain collection, whenever confirmation of the identity of the isolates was needed, the nrDNA ITS region was amplified and sequenced. The culture extracts of the fungus Darksidea alpha contained three main compounds (Fig. 2, peaks at 7.3, 7.6 and 8.5 min retention times). Based on the HR-MS spectra of these compounds (Fig.3), they can be identified using the same molecular formula C15H22O2 (Table 3), which refers to isomeric structures. Table 3. High-resolution mass-spectral (positive ion mode) data for compounds detected in Darksidea alpha culture extract. Compound Formula Detected ion Detected formula Calculated m / z Found m / z diff (ppm) RtaName 7.3 petasol C15H22O2 [M+H]+C15H23O2 235.16926 235.16867 -2.494 7.6 isopetasol C15H22O2[M+H]+C15H23O2235.16926 235.16875 -2.154 8.5 neopetasol C15H22O2 [M+H]+C15H23O2 235.16926 235.16875 -2.154 a Retention times of compounds correspond to those in Fig.1. The UV spectrum of compound DA-2 shows a bathochromic shift relative to the comparable UV spectra of compounds DA-1 and DA-3, suggesting a more extensive conjugated systems in compound DA-2 than in compounds DA-1 and DA-3 (Fig.4). Based on these results and the NMR data of the compounds DA-1–3 identical to those reported previously for petasol isomers [Sugama, K., et al., Sesquiterpenoids from petasites fragrans. Phytoc- hemistry, 1983. 7: 1619-22.; Neuenschwander, M., Neuenschwander, A., and Steinegger, E., Struktur der Sesquiterpene von Petasites hybridus (L.) G. M. et SCH.: Neopetasol‐Abkömmlinge. Helvetica Chi- mica Acta, 1979.; Le, D.H., et al., Eremophilane-type sesquiterpenes from cultured lichen mycobionts of Sarcographa tricosa. Phytochemistry, 2013. 91: 242-8.], Darksidea alpha compounds DA-1, DA-2 and DA-3 were identified as petasol, isopetasol and neopetasol, respectively (Fig.5). The effective compound petasol is a rarely occurring natural metabolite, determined earlier only in four fungal species [Le, D.H., et al., Eremophilane-type sesquiterpenes from cultured lichen mycobionts of Sarcographa tricosa. Phytochemistry, 2013.91: 242-8.; Bunkers, G.J. and Strobel, G.A., A proposed mode of action for green island induction by the eremophilane phytotoxins produced by Drechslera gigantea. Physiol Mol Plant Pathol, 1991.5: 313-23.; Jayasuriya, H., et al., Identification of diverse microbial metabolites as potent inhibitors of HIV-1 Tat transactivation. Chem Biodivers, 2005. 2: 112-22.; Chen, Y., et al., Bioactive sesquiterpene derivatives from mangrove endophytic fungus Pho- mopsis sp. SYSU-QYP-23: Structures and nitric oxide inhibitory activities. Bioorg Chem, 2021. 107: 104530.] and two plants (i.e., Petasites formosanus and P. fragrans) [Sugama, K., et al., Sesquiterpenoids from petasites fragrans. Phytochemistry, 1983.7: 1619-22.; Lin, Y.L., Eremophilanes from Petasits for- mosanus KITAMURA. Chem Pharm Bull, 1998.46: 1807-9.]. Among these plants accumulating peta- sol, P. formosanus is also used as medicinal plant [Lin, Y.L., Eremophilanes from Petasits formosanus KITAMURA. Chem Pharm Bull, 1998.46: 1807-9.] thus, suggesting the possibility of the safe use of petasol. The isolates of Darksidea alpha, a new source of the metabolites, are abundant sources of peta- sol, allowing the high-yield isolation of this compound through preparative HPLC. Namely, starting from 1.0 g lyophilized Darksidea alpha culture, 3.6 mg petasol could be isolated (average yield, obta- ined from four independently grown fungal cultures; differences could be characterized by a relative standard deviation (RSD) value of 23%). In addition to petasol, significant amount of isopetasol (1.0 mg) and neopetasol (0.33 mg) can also be isolated from 1.0 g lyophilized Darksidea alpha culture (ave- rage yields, obtained from four independently grown fungal cultures; differences could be characterized by the RSD values of 54 % (isopetasol) and 13% (neopetasol)). In Vitro Evaluation of the Compounds To validate our antiviral test system, antiviral activity of two drugs (as reference compounds) were determined, namely Nitazoxanide and Molnupiravir. Both compounds showed antiviral effect on feline and porcine CoVs (Fig.5-6) Next, the test compounds were measured in 0.3-100 µM concentration range. Data revealed that Petasol and S-Petasin are very effective against the feline and porcine CoVs. Petasol showed a visible CPE (cytophatologic effect) at 6.25 µM concentration, while S-Petasin at 0.78 µM on DF-2 virus infec- ted host cells. Besides, Petasol and S-Petasin was not cytotoxic to the PK-15 host cells up to 50 uM.

Claims

CLAIMS 1. A compound of general formula (I) for use in the treatment of an infection caused by an Alpha- coronavirus in a subject,wherein in general formula (I) the bond between carbon 1 and 6 is either a single bond or a double bond, the bond between carbon 13 of R1and carbon 3 is either a single bond or a double bond, wherein R1is isopropyl, if the bond between R1and carbon 3 is a double bond, or isopropenyl, if the bond between R1and carbon 3 is a single bond, X is O or nothing R2is H or R2has the following formula (i) wherein in formula (i) R4isthioalkyl or a C1-C7thioether, or a C1-C8alkyl or a C1-C8alkenyl substituted with a single -SH group, wherein said C1-C8alkenyl is a straight or branched chain alkenyl; R3is H or R3has the following formula (i) wherein in formula (i) R4 isthioalkyl or a C1-C7 thioether, or a C1-C8 alkyl or a C1-C8 alkenyl substituted with a single -SH group, wherein said C1-C8 alkenyl is a straight or branched chain alkenyl; Y is O or nothing.

2. The compound for use according to claim 1, wherein the compound has general formula (III)wherein R1is isopropyl, if the bond between R1and the ring carbon atom to which it is attached is a double bond, or isopropenyl, if the bond between R1and the ring carbon atom to which it is attached is a single bond, R2is H or R2has the following formula (i)wherein in formula (i) R4is a C1-C8alkyl or a C1-C8alkenyl or a C1-C7thioalkyl or a C1-C7thioether, or a C1-C8alkyl or a C1-C8alkenyl substituted with a single -SH group, wherein said C1-C8alkenyl is a straight or branched chain alkenyl.

3. The compound for use according to claim 1 or 2, wherein when R1is isopropenyl and the ring carbon atom to which it is attached is C3, the compound is the S isomer.

4. The compound for use according to any one of the preceding claims, wherein R1is isopropyl, if the bond between R1and the ring carbon atom to which it is attached is a double bond, or isopropenyl, if the bond between R1and C3 to which it is attached is a single bond and the compound is the S isomer, and R2is H.

5. The compound for use according to any one of the preceding claims, wherein said compound is selected from the group consisting of petasol and esters thereof.

6. The compound for use according to claim 4 wherein said compound is selected from the group consisting of petasol and S-petasin.

7. The compound for use according to any of the previous claims wherein the Alphacoronavirus belongs to Alphacoronavirus 1.

8. The compound for use according to any one of the preceding claims, wherein the virus is se- lected from feline infectious peritonitis virus (FIPV) and porcine Transmissible Gastroenteritis Virus (TGEV).

9. The compound for use according to any of the previous claims, wherein the subject belongs to Felidae, Canidae or Suidae.

10. A pharmaceutical or veterinary composition comprising a compound as defined in any one of claims 1-5 and a pharmaceutically acceptable excipient, additive or carrier or an excipient, additive or carrier suitable for veterinary use, respectively.

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