WO2016153432A1 - Drugs from substituted phenyl cinnamyl ketones - Google Patents

Abstract

The present invention relates to substituted phenyl cinnamyl ketones of Formula (I) for use in therapy: wherein R¹ to R¹¹ are defined as described in the specification, or a pharmaceutically acceptable salt, tautomer(s), solvate or polymorph thereof, that are used in the treatment on RNA virus related infections. The present invention also relates to use of the compounds as described in the specification for the manufacture of a medicament in the treatment of a disease associated with RNA virus infections. The present invention also relates a method of preventing or treating a disease associated with a RNA virus related infection comprising administering to a subject in need of treatment a compound as described in the specification, or a pharmaceutically acceptable salt thereof.

Description

Drugs from substituted Phenyl Cinnamyl Ketones

Cross-Reference to Related Applications

[0001] This application claims the benefit of priority of Singapore provisional application No. 10201502254Q, filed March 23, 2015, the contents of it being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The present invention generally relates to the field of biochemistry. In particular, it refers substituted phenyl cinnamyl ketones for use in therapy. It also relates to the use of the compounds in the manufacture of a medicament for the treatment of RNA virus related infections.

Background Art

[0003] RNA viruses are the source for many diseases. Notable human diseases caused by RNA viruses include Ebola hemorrhoragic fever, SARS, influenza, hepatitis C, West Nile fever, polio, measles and hand, foot and mouth disease (HFMD). One group within this virus family is group IV, the positive-sense ssRNA virus. For example, a notable family member of this virus group is the Enterovirus. Enterovirus 71 (EV71) is a small, non-enveloped, positive-sense RNA virus that is classified within the family Picornaviridae under the genus Enterovirus. As a major neurotropic causative agent of hand, foot and mouth disease (HFMD) that can be rapidly transmitted via the fecal-oral and droplet/aerosol routes, EV71 has replaced poliovirus as the most clinically relevant enterovirus following the global eradication of polioviruses. HFMD, as a typical disease associated with the RNA virus, is typically a benign and common self-limiting childhood disease that is characterized by rapidly ulcerating vesicles in the mouth and lesions on the hands and feet and typically affects young children below five years of age. While HFMD is caused by numerous members of the Enterovirus genus, HFMD caused by EV71 is considered to be the most virulent and has been known to develop potentially fatal neurological complications amongst children, manifesting as aseptic meningitis, poliomyelitis- like acute flaccid paralysis and brain stem encephalitis that leads on to cardiovascular collapse and pulmonary edema.

[0004] With no established clinical prophylaxis or antiviral treatment for EV71 infections or other diseases associated with RNA viruses, management of certain severe infections associated with RNA virus infections is mainly aimed at alleviating disease symptoms with supportive treatments. In the case of certain RNA associated diseases, such as EV71 infections, mechanical cardiopulmonary support systems and the administration of milrinone, a positive inotropic agent, were used to prevent cardiopulmonary failure and improve clinical outcome of patients. Administrations of intravenous immunoglobulin, interferon- alpha and corticosteroids were also associated with favorable clinical outcomes in patients.

[0005] With a currently limited antiviral repertoire against certain RNA virus infection, such as EV71, and the development of clinical antivirals against RNA viruses still in the process of being developed, it is essential to find ways and strategies to expand the drug discovery pipeline.

[0006] There is therefore a need to provide a compound for use in therapy that overcomes, or at least ameliorates, one or more of the disadvantages described above. There is also a need to provide use of a compound in the manufacture of a medicament for the treatment of diseases associated with RNA virus infections. Summary

[0007] In a first aspect, there is provided a compound of Formula (I) for use in therapy:

or a pharmaceutically acceptable salt, tautomer(s), solvate or polymorph thereof, wherein R¹ is hydrogen, hydroxyl or alkoxy; FT 2 and R 3^J are independently selected from hydrogen, hydroxyl, optionally substituted alkoxy, optionally substituted acyl, optionally substituted hydrocarbons selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, and optionally R 2 and R 3 are taken together to form a 4- to 7-membered heterocyclic ring;

R⁴ is hydrogen or a hydrocarbon;

R⁵ is selected from hydrogen, hydrocarbon, hydroxyl, oxygen, and acyl, and is optionally taken together with R⁶ or R⁷ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring;

R⁶ is selected from hydrogen, hydrocarbon or alkoxy or is optionally taken together with R⁵ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring;

R 7 is hydrogen or is optionally taken together with R 5 to form a 6-membered ring selected from a heterocyclic ring; and

R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from hydrogen, halogen, hydroxyl, an optionally substituted alkoxy, an optionally substituted acyl, an optionally substituted aryl, an optionally substituted acyloxy, an optionally substituted aryloxy and an optionally substituted hydrocarbon selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, and heteroaryl.

[0008] In a second aspect, there is provided the compound as disclosed above for use in the treatment of a disease associated with a RNA virus infection.

[0009] In a third aspect, there is provided use of a compound as disclosed above in the manufacture of a medicament for the prevention or treatment of a disease associated with a RNA virus infection.

[0010] In a fourth aspect, there is provided a method of preventing or treating a disease associated with a RNA virus related infection comprising administering to a subject in need of treatment a compound as disclosed above, or a pharmaceutically acceptable form or prodrug thereof.

Definitions

[0011] The following words and terms used herein shall have the meaning as indicated.

[0012] In the definitions of a number of substituents below it is stated that "the group may be a terminal group or a bridging group". This is intended to signify that the use of the term is intended to encompass the situation where the group is a linker between two other portions of the molecule as well as where it is a terminal moiety. Using the term alkyl as an example, some publications would use the term "alkylene" for a bridging group and hence in these other publications there is a distinction between the terms "alkyl" (terminal group) and "alkylene" (bridging group). In the present application no such distinction is made and most groups may be either a bridging group or a terminal group.

[0013] The term "alkyl" as used herein refers to monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 24 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1,2-dimethylpropyl, 1,1- dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3- methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1- methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2- dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2- trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. Alkyl groups may be optionally substituted.

[0014] The term "alkenyl" as used herein, unless otherwise specified, refers to a branched, unbranched or cyclic (e.g. in the case of C5 and C6) hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containing at least one double bond, such as ethenyl, vinyl, allyl, octenyl, decenyl, and the like.

[0015] The term "alkynyl" as used herein, unless otherwise specified, refers to a branched or unbranched hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containing at least one triple bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.

[0016] The term "acyl" means an R-C(=0)- group in which the R group may be an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl group as defined herein. Examples of acyl include acetyl, benzoyl and amino acid derived aminoacyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon. [0017] The term "alkyloxy" refers to an alkyl-O- group in which alkyl is as defined herein. Preferably the alkyloxy is a Ci-C₆alkyloxy. Examples include, but are not limited to, methoxy and ethoxy. The group may be a terminal group or a bridging group. The term alkyloxy may be used interchangeably with the term "alkoxy".

[0018] The term "alkoxy" or variants such as "alkoxide" as used herein refers to an -O-alkyl radical. Representative examples include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

[0019] The term "aromatic group", or variants such as "aryl" or "arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like. Aryl groups may be optionally substituted.

[0020] The term "aryloxy" refers to an aryl-O- group in which the aryl is as defined herein. Preferably the aryloxy is a C₆-C₁₈ aryloxy, more preferably a C₆-C₁₀ aryloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom. Examples of such groups include phenyloxy, biphenyloxy, naphthyloxy, and the like. Aryloxy groups may be optionally substituted.

[0021] The term "carboxy" refers to the group -C(0)OH.

[0022] The term "cycloalkyl" refers to a saturated monocyclic or fused or bridged or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. A cycloalkyl group typically is a C3-C12 alkyl group. The group may be a terminal group or a bridging group.

[0023] The term "cycloalkenyl" as used herein refers to a non-aromatic mono or multicyclic ring system comprising about 3 to about 10 carbon atoms which contains at least one carbon- carbon double bond. Non-limiting examples of suitable monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cyclohepta-1 ,3-dienyl, and the like. Non-limiting example of a suitable multicyclic cycloalkenyl is norbomylenyl, as well as unsaturated moieties of the examples shown above for cycloalkyl. Cycloalkenyl groups may be optionally substituted.

[0024] The term "halogen" represents chlorine, fluorine, bromine or iodine. [0025] The term "heteroaryl" as used herein refers to an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. "Heteroaryl" may also include a heteroaryl as defined above fused to an aryl as defined above. Non- limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[l,2-a]pyridinyl, imidazo[2,l-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, benzothiazolyl and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like. Heteroaryl groups may be optionally substituted.

[0026] The term "heterocycloalkyl" refers to a saturated monocyclic, fused or bridged or spiro polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane. A heterocycloalkyl group typically is a C2-C12 heterocycloalkyl group. A heterocycloalkyl group may comprise 3 to 9 ring atoms. A heterocycloalkyl group may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S. The group may be a terminal group or a bridging group.

[0027] The term "heterocycloalkenyl" refers to a heterocycloalkyl as defined herein but containing at least one double bond. A heterocycloalkenyl group typically is a C2-C12 heterocycloalkenyl group. The group may be a terminal group or a bridging group.

[0028] The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups other than hydrogen provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a stable compound. Such groups may be, for example, halogen, hydroxy, oxo, cyano, nitro, alkyl, alkoxy, haloalkyl, haloalkoxy, arylalkoxy, aryloxy, alkylthio, hydroxyalkyl, alkoxyalkyl, cycloalkyl, cycloalkylalkoxy, alkanoyl, alkoxycarbonyl, alkylsulfonyl, alkylsulfonyloxy, alkylsulfonylalkyl, arylsulfonyl, arylsulfonyloxy, arylsulfonylalkyl, alkylsulfonamido, alkylamido, alkylsulfonamidoalkyl, alkylamidoalkyl, arylsulfonamido, arylcarboxamido, arylsulfonamidoalkyl, arylcarboxamidoalkyl, aroyl, aroyl- 4-alkyl, arylalkanoyl, acyl, aryl, arylalkyl, alkylaminoalkyl, a group R_xR_yN-, R_xOCO(CH₂)_m, R_xCON(R_y)(CH₂)_m, R_xR_yNCO(CH₂)_m, R_xRyNS0₂(CH₂)_m or R_xS0₂NR_y(CH₂)_m (where each of R_x and R_y is independently selected from hydrogen or alkyl , or where appropriate R_xR_y forms part of carbocylic or heterocyclic ring and m is 0, 1 , 2, 3 or 4), a group R_xR_yN(CH₂)_p- or R_xRyN(CH₂)_pO- (wherein p is 1 , 2, 3 or 4); wherein when the substituent is R_xR_yN(CH₂)_p- or R_xR_yN(CH₂)_pO, R_x with at least one CH₂of the (CH₂)_P portion of the group may also form a carbocyclyl or heterocyclyl group and R_y may be hydrogen, alkyl.

[0029] The term "cyclic ring system" refers to any combination of atoms, which represent a closed chain or ring of atoms.

[0030] The term "independently substituted" refers to the possible substitution pattern within the compound. It means that the referred to substituents may be different from each other and that substitution with one particular substituent does not affect the choice of substituent of another possible substitution site.

[0031] It is understood that included in the family of compounds of Formula (I) are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in "E" or "Z" configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.

[0032] Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and /or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the subject matter described and claimed.

[0033] Additionally, Formula (I) is intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

[0034] The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above-identified compounds, and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of Formula (I) may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, arylsulfonic In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds, agents and salts may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present disclosure and specified formulae.

[0035] The term "pharmaceutically acceptable excipient" may refer to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated.

[0036] The term "patient," as used herein, refers to an animal, preferably a mammal, and most preferably a human.

[0037] The term "subject" refers to a human or an animal.

[0038] The term "prevention" or "preventing a disease" refers to inhibiting completely or in part the development or progression of a disease, for example in a person who is exposed to the virus causing the disease.

[0039] The term "treatment" or "treating a disease" refers to a therapeutic intervention that inhibits, or suppresses the worsening of the disease and ameliorates at least one sign or symptom of a disease or pathological condition, or interferes with a pathophysiological process, after the disease or pathological condition has begun to develop.

[0040] The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state

[0041] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

[0042] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Brief description of drawings [0043] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0044] Figure 1 shows a HTS (high-throughput screening) procedure used to identify antivirals which act early during EV-71 infection and late after EV-71 infection. This schema of different procedures was used in order to identify antivirals which act prior and/ or post EV-71 infection.

[0045] Figures 2A & 2B show the dose dependent cytotoxicity profiles of 10 selected flavonoids, relative to the vehicle control (1% DMSO). Data points represent mean readings of triplicate experiments. Error bars represent + SD.

The compound codes correspond to the structures shown in the 'Detailed Description' .

[0046] Figure 3 shows a more detailed cell viability and virus inhibition profiles of (a) ST077124 and (b) ST024734. Cell viability results are expressed relative to the untreated control. 1% DMSO served as the vehicle control for both experiments. Data points represent mean readings of triplicate experiments. Error bars represent ± SD. Statistical analyses are for virus titre and were carried out with one-way ANOVA and Dunnett's post-test using Graphpad Prism 6.0. It can be seen that no significant inhibition was observed for both compounds and it is apparent that flavonoids were fairly well-tolerated by the RD cells (>80% relative cell viability) even at highest concentrations. [0047] Figure 4 shows the effects of (a) ST077124 and (b) ST024734 on viral RNA production. Viral RNA was detected and quantified via qRT-PCR and is expressed relative to the untreated, EV71 -infected control. Viral RNA was not detected in mock- infected samples. Data points represent mean readings of triplicate experiments. Error bars represent ± SD. It can be seen that decreases in the amount of viral RNA produced, relative to the untreated control, occurred across all dosages for both compounds. [0048] Figure 5 shows the effects of (a) ST077124 and (b) ST024734 on viral protein production. Actin bands (42kDa) served as internal loading controls and mouse monoclonal antibodies were used to detect viral protein VP2 (28kDa) and its precursor, VPO (35kDa). It was observed that dosage dependent inhibition of virus titre was accompanied by significant disruption of protein production. Taken together, the reduction in the amount of viral protein produced due to the flavonoids treatment correlates with the reduced amounts of virus RNA produced.

[0049] Figure 6 shows the inhibition profile of ST077124 and ST024734 after co- treatment. EV71 virus particles were treated with the flavonoids for lh prior to infection. Untreated samples represent the virus titre under normal infection circumstances. Data points represent mean readings of triplicate experiments. Error bars represent ± SD. It is deduced that both ST077124 and ST024734 do not appear to have any direct virucidal effects on EV71 and neither are capable of inhibiting the virus' ability to attach and enter RD cells. [0050] Figure 7 shows representative immunofluorescence images from time of addition and removal study of ST024734 and ST077124 (a) Between Ohpi to 12hpi, treatment was given in delayed 2h intervals with ST077124 (b) All infected cells were treated at Ohpi and between Ohpi to 12hpi, ST077124 was removed from the culture media at 2h intervals (c) Between Ohpi to 12hpi, treatment was given in delayed 2h intervals with ST024734 (d) All infected cells were treated at Ohpi and between Ohpi to 12hpi, ST024734 was removed from the culture media at 2h intervals. Cell nuclei are marked by DAPI (blue) and EV71 antigen positive cells are marked by anti-VP2 antibodies conjugated to FITC via secondary antibodies (green). It would appear that ST077124 retains its anti-viral effect if not washed out from the infected cells at an early time point, suggesting some ability by the compounds to stay within the treated cells. This strongly suggests that ST077124 works as an anti-EV71 agent by inhibiting viral replication processes occurring between 4-6hpi. [0051] Figure 8 shows (a) Nano-luciferase activity of EV71 replicon and (b) ratios of Firefly to Renilla Luciferase of EV71 bicistronic replicon upon different drug treatments or bioactive compounds at various concentrations. Data points represent mean readings of triplicate experiments. Error bars represent + SD. Statistical analyses were carried out with one-way ANOVA and Dunnett's post-test using Graphpad Prism 6.0. It can be concluded that both ST077124 and ST024734 can inhibit EV71 replication via direct inhibition of viral non-structural proteins, translation machinery or indirectly through host factors. [0052] Figure 9 shows the inhibition profiles of ST077124 and ST024734 against (a) EV71 H strain (b) CA16 (c) EV71 B5 subgroup (d) CHIKV-122508. Data points represent mean readings of triplicate experiments. Error bars represent + SD. Statistical analyses were carried out with one-way ANOVA and Dunnett's post- test using Graphpad Prism 6.0. It can be seen that 5μΜ of ST077124 and 50μΜ ST024734 were potent inhibitors against the other enteroviruses - EV71 H strain, EV71 B5 subgroup and CA16.

Description of Embodiments

[0053] A solution to the above outlined problem is to focus on identifying promising drug candidates from a specific class of compounds. The search for these potential hit compounds begins by looking at one of Nature's largest compound libraries - flavonoids or related natural products bearing the structural element of substituted cinnamyl ketones.

[0054] Flavonoids, and their relatives, are a class of secondary plant metabolites that are ubiquitous in the plant kingdom. Under normal circumstances, flavonoids represent a common constituent of the human diet, with main dietary sources coming from fruits, vegetables and plant-derived beverages such as tea, coffee and red wine. The basic structure of flavonoids is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings (C6-C3-C6). Despite having relatively homogenous structures and differing only by the pattern of substitution around the three carbon rings, flavonoids have been regarded as 'nature's biological response modifiers' and possess a wide range of biological activity, with reported anti-viral, anti- oxidant, anti-cancer, anti-bacterial, anti-inflammatory and eukaryotic enzyme inhibition activities. Amongst all the reported biological functions, notable aspects of flavonoids remain their antioxidant activity and their ability to manipulate biological signaling pathways. Most flavonoids have an inherent ability to scavenge free radicals and reduce free radical formation and some flavonoids still retain such abilities when degraded upon ingestion to various phenolic acids. Another major emerging view of flavonoids is that they are able to exert modulatory actions on cellular systems through direct action on various signaling pathways and transcription factors such as phosphoinositide 3-kinase, Akt/protein kinase B, mitogen- activated protein kinase (MAPK), tyrosine kinases and protein kinase C.

[0055] It appears therefore possible, that suitable compounds for diseases associated with a RNA virus infection can be found and developed into effective clinical therapeutic agents from a compound class comprising substituted phenyl cinnamyl ketones.

[0056] The present invention refers to a compound of Formula (I) which may be used in therapy:

or a pharmaceutically acceptable salt, tautomer(s), solvate or polymorph thereof. In formula (I), the following substituents may be defined as follows.

R¹ may be hydrogen, hydroxy or alkoxy.

R 2" and R 3^J may be independently selected from hydrogen, hydroxyl, optionally substituted alkoxy, optionally substituted acyl, optionally substituted hydrocarbons selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, and optionally R and R are taken together to form a 4- to 7-membered heterocyclic ring.

R⁴ may be selected from hydrogen or a hydrocarbon;

R⁵ may be selected from hydrogen, hydrocarbon, hydroxyl, oxygen, and acyl, and may be optionally taken together with R⁶ or R⁷ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring.

R⁶ may be selected from hydrogen, hydrocarbon or alkoxy or may be optionally taken together with R⁵ to form a 5 -or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring. R 7 may be hydrogen or may be optionally taken together with R 4 to form a 6-membered ring selected from a heterocyclic ring.

R⁸, R⁹, R¹⁰ and R¹¹ may be independently selected from hydrogen, halogen, hydroxyl, an optionally substituted alkoxy, an optionally substituted acyl, an optionally substituted aryloxy, an optionally substituted aryl, an optionally substituted acyloxy and an optionally substituted hydrocarbon selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, and heteroaryl.

[0057] In one embodiment, the compound of the present invention may be represented by the following Formula (II)

Formula (II)

wherein the substituents R¹ to R³ and R⁵ to Rⁿ are as indicated above.

[0058] The double bond indicated in Formula I or Formula II may be present in the cis or in the trans configuration. In embodiments, wherein R⁵ is not taken together with either of R⁶ or R to form a bond, the double bond may be in the trans configuration.

Accordingly, Formula (III) refers to compounds wherein R⁵ is not taken together with either of R⁶ or R⁷ to form a bon

Formula (III),

wherein R⁵ may be selected from hydrogen, hydrocarbon and hydroxyl and the remaining substituents are as defined above. [0059] In embodiments, wherein R 5 and R 7 are taken together to form a 6-membered heterocyclic ring, the double bond may be in a cis configuration. Accordingly, Formula (IV) refers to compounds wherein R 5 is taken together with R 7 to form a bond:

Formula (IV)

wherein R⁵ may be selected from oxygen and acyl, and all the other substituents are as defined herein. In Formula (IV), R¹ may be alkoxy, for example methoxy.

[0060] R¹ may be hydrogen, hydroxyl or alkoxy. When R¹ is alkoxy, it may be selected from, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy. For example, it may be methoxy.

[0061] R 2 and R 3 may be independently selected from hydrogen, hydroxyl, substituted acyl, optionally substituted alkoxy, optionally substituted hydrocarbons selected from the group consisting of, but not being limited to, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, and optionally R 2 and R 3 may be taken together to form a 4- to 7-membered heterocyclic ring. The optionally substituted hydrocarbons may be selected from alkyl. Alkyl may be methyl, ethyl, 1 -propyl, isopropyl or 1 -butyl. For example, it may be methyl or isopropyl. The cycloalkyl substituent may be selected from a C3-C12 alkyl group, which forms a cyclic ring system. The heterocycloalkyl substituent may comprise 3 to 9 ring atoms. The heterocycloalkyl substituent may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S. For example, it may be a 5-membered ring containing one or two oxygen atoms. In certain embodiments, R 2 and R 3 may be taken together to form a 4- to 7-membered heterocyclic ring. The heterocyclic ring may be a heteroalkenyl ring.

Accordingly, R 2 and R 3 may be taken together to form a 5 to 6 membered heteroalkenyl ring. For example, they may form a 5-membered heteroalkenyl ring. The heteroatom may be selected from, but is not limited to, one or more oxygen atoms. For example, it may be 2 oxygen atoms. R 2 and R 3 may be selected such that the oxygen atoms may be directly bonded to the phenyl ring and the linking moiety between R 2 and R 3 may be chosen from alkyl, for example from a CH₂-group.

[0062] R⁴ may be selected from hydrogen or hydrocarbon.

[0063] R⁵ may be selected from hydrogen, hydrocarbon, hydroxyl, oxygen, and acyl, and may be optionally taken together with R⁶ or R⁷ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring. In embodiments, wherein R⁵ is taken together with R⁶, they may form a 5-membered ring. In these embodiments, R⁵ may be selected from oxygen and acyl. The resulting ring system may be a heterocycloalkenyl, wherein the hetero atom is oxygen, or a cycloalkenyl ring. Additionally, in embodiments, wherein R⁵ is selected from acyl, R 1 to R3 may be selected from hydrogen. In embodiments, wherein R 5 may be selected from hydrocarbon, the resulting ring system may be a 6-membered ring. The hydrocarbon may be selected from an alkyl. Alkyl may be methyl, ethyl, 1 -propyl, isopropyl or 1 -butyl. For example, it may be ethyl. In embodiments, wherein R⁵ is taken together with

R 7 , they may form a 6-membered ring. In these embodiments, R 5 may be selected from oxygen. The resulting ring system may be a heterocycloalkenyl, wherein the hetero atom is oxygen. For example, the heterocycloalkenyl ring may be a pyran. Additionally, in embodiments wherein R 5 is taken together with R 7 to form a 6-membered ring, R 1 to R3 may be selected from, but is not limited to, hydroxy, substituted acyl and alkoxy. Alkoxy may be selected from, for example, methoxy. Additionally, in embodiments wherein R⁵ is taken together with R 7 to form a 6-membered ring, R 2 and R 3 may not be taken together to form a 4- to 7-membered heterocyclic ring. Additionally, in embodiments wherein R⁵ is taken together with R⁷ to form a 6-membered ring, R⁶ may be selected from, but is not limited to, alkoxy. Alkoxy may be selected from, for example, methoxy. Accordingly, Formula (V) refers to compounds wherein R 5 is taken together with R 7 to form a bond:

Formula (V)

wherein R¹ to R³ may be selected from hydroxy, substituted acyl and alkoxy, R⁶ may be selected from alkoxy and the remaining substituents are as defined herein.

[0064] R⁶ may be generally selected from hydrogen, hydrocarbon or alkoxy. Alternatively, it may be taken together with R⁵ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring. In certain embodiments, wherein R⁶ is selected from alkoxy, alkoxy may be selected from, but is not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy. For example, it may be methoxy. In certain embodiments, wherein R⁶ may be selected from hydrocarbon, it may be selected from alkyl. Alkyl may be methyl, ethyl, 1- propyl, isopropyl or 1 -butyl. For example, it may be ethyl and may form a 6-membered ring with R⁵. In certain embodiments, wherein R⁶ forms a 5-membered ring with R⁵, the substituents R 1 , R 2 and R 7 may be hydrogen. Additionally, in these embodiments, R 2 and R 3 may not be taken together to form a 4- to 7-membered heterocyclic ring. Accordingly, Formula (VI) refers to compounds wherein R⁵ is taken together with R⁶ to form a bond:

Formula (VI)

[0065] R 7 may be hydrogen or may be optionally taken together with R 5 to form a 6- membered ring selected from a heterocyclic ring. In certain embodiments, wherein R is selected from hydrogen, R¹ may be hydrogen. Accordingly, Formula (VII) refers to compounds wherein R is hydrogen:

Formula (VII) [0066] Alternatively, R 7 may be taken together with R 5 to form a 6-membered ring selected from a heterocyclic ring. The ring may be a 6-membered ring selected from a heterocyclic ring, wherein the heteroatom may be oxygen. The heterocyclic ring may be, for example, a pyran ring. In these embodiments, R⁶ may be selected from, but is not limited to, alkoxy, for example methoxy.

[0067] R⁸, R⁹, R¹⁰ and R¹¹ may be independently selected from hydrogen, halogen, hydroxyl, an optionally substituted alkoxy, acyl, an optionally substituted aryl, an optionally substituted acyloxy and an optionally substituted hydrocarbon selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, and heteroaryl. The optionally substituted hydrocarbons may be selected from alkyl. Alkyl may be methyl, ethyl, 1-propyl, isopropyl or 1-butyl. For example, it may be methyl. The optionally substituted alkyl may be substituted with an optionally substituted aryloxy. The optionally substituted aryloxy may be, for example, an optionally substituted phenyloxy. The optionally substituted phenyloxy may be, for example, a dialkylphenyloxy or a trichlorophenyloxy. The cycloalkyl substituent may be selected from a C3-C12 alkyl group, which forms a cyclic ring system. The heteroaryl substituent may comprise 3 to 9 ring atoms. The heteroaryl substituent may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S. For example, it may be a 5- membered ring containing one or two oxygen atoms. The aryl substituent may comprise 5 to 9 ring atoms. The aryl substituent may be, for example, phenyl.

[0068] R may be selected from, but is not limited to, hydrogen and alkoxy, for example from methoxy.

[0069] R⁹ may be selected from, but is not limited to, hydrogen, hydroxyl, an optionally substituted alkoxy, a substituted aryloxy, and an optionally substituted hydrocarbon selected from the group consisting of alkyl. Alkoxy may be, for example, methoxy. The substituted hydrocarbon may be substituted with, for example, an optionally substituted aryloxy. The substituted aryloxy may be substituted with, for example, alkyl, for example methyl. Alternatively, it may be substituted with, for example, halogen, for example chlorine.

[0070] R¹⁰ may be selected from, but is not limited to, hydrogen, alkoxy and a substituted acyloxy. Alkoxy may be, for example, methoxy. The substituted acyloxy may be substituted with, for example, an optionally substituted heteroaryl, for example a furan.

[0071] R¹¹ may be selected from, but is not limited to, hydrogen and alkoxy, for example from methoxy. [0072] In an embodiment, there is provided the compound according to the formula above, wherein a maximum of 4 cyclic ring systems may be present in the compound. For example, the compound may have 2, 3 or 4 cyclic ring systems. One of these cyclic ring systems may be the phenyl ring in a position to the ketone functionality, wherein the other cyclic ring system may be the phenyl ring directly connected to the double bond, i.e. in γ position to the ketone. A third ring system is optional and may be selected from a ring wherein R 2 and R 3 are taken together to form a 4-7-membered heterocyclic ring. Alternatively, it may be selected from a ring wherein R⁵ and R⁶ are taken together to form a 5-membered heterocyclic ring. Alternatively, it may be selected from a ring wherein R⁵ and R⁶ are taken together to form a 5-membered cycloalkenyl ring. Alternatively, it may be selected from a ring wherein R⁵ and R⁶ are taken together to form a 6-membered cycloalkenyl ring. Alternatively, it may be selected from a ring wherein R 5 and R 7 are taken together to form a 6-membered heterocyclic ring. A fourth ring system may be selected from optional substituents as defined for the variables R 2 , R 3 and R 8 to R 11. For example, the fourth ring system may be in the R 10 position. In embodiments, wherein R⁶ is selected from hydrogen, the compound may comprise 2 or 3 cyclic ring systems.

Accordingly, the compound of formula I may be defined such that of the four optional cyclic ring systems formed between R² and R³, R⁵ and R⁶ and R⁵ and R⁷, a maximum of one of those cyclic ring system ring is present in the compound.

[0073] In an embodiment, wherein R 2 and R 3 are independently selected from a substituted alkoxy, they may be substituted by one or more substituents independently selected from alkyl, alkoxy and acyl and optionally are taken together to form a 5-membered heterocyclic ring.

[0074] In an embodiment, wherein R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from a substituted alkyloxy and a substituted alkoxy, they may be substituted by one or more substituents independently selected from alkyl, acyl, aryl and heteroaryl.

[0075] In an embodiment, wherein R⁵ is hydrocarbon, oxygen or acyl, it may be attached to R⁶ or R⁷ to form a 5- or 6-membered heterocyclic or cycloalkenyl ring.

[0076] The compound according to Formula I may be selected from the following:

ST057770 _and ST070181

Compounds of the formula (Villa) (ST02473) and (Vlllb) (STOW 124) should be mentioned as a particular embodiment:

Formula (VHIb).

[0077] The present invention refers to the compound as disclosed above for use in the treatment of diseases associated with a RNA virus infection. Accordingly, the compound may have antiviral activity. The compound may have pre antiviral activity. Additionally or alternatively, the compound may have post antiviral activity. The compound may inhibit replication of the virus. The compound may inhibit translation of the virus. The compound may inhibit viral RNA synthesis. Accordingly, the compounds of the present disclosure may be used as effective antiviral compounds.

[0078] The compound, or a pharmaceutically acceptable form or prodrug thereof, may be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, diluent or excipient. The compounds, while effective themselves, may be typically formulated and administered in the form of their pharmaceutically acceptable salts as these forms are typically more stable, more easily crystallised and have increased solubility.

[0079] The compound may, however, typically be used in the manufacture of a medicament for the treatment of diseases associated with a RNA virus. Accordingly, they may be in the form of pharmaceutical compositions which are formulated depending on the desired mode of administration.

[0080] The present invention relates to a pharmaceutical composition comprising a compound as disclosed above, or a pharmaceutically acceptable form or prodrug thereof, and a pharmaceutically acceptable excipient. The composition may be prepared in manners well known in the art.

[0081] In using the compounds in the manufacture of a medicament for the treatment of a disease associated with a RNA virus infection, they may be administered in any form or mode which may make the compound bioavailable. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected, the condition to be treated, the stage of the condition to be treated and other relevant circumstances.

[0082] Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this disclosure may include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene - polyoxypropylene-block polymers, polyethylene glycol or wool fat.

[0083] Compositions as defined above may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra- articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this disclosure may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0084] For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0085] Pharmaceutically acceptable compositions as defined above may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0086] Pharmaceutical compositions for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0087] These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of micro-organisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin. [0088] If desired, and for more effective distribution, the compounds may be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

[0089] The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[0090] Alternatively, pharmaceutically acceptable compositions as defined above may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0091] Pharmaceutically acceptable compositions as defined above may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations may be readily prepared for each of these areas or organs.

[0092] Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically- transdermal patches may also be used.

[0093] For topical applications, the pharmaceutically acceptable compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of compounds as defined above may include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutically acceptable compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers may include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcohol and water.

[0094] For ophthalmic use, the pharmaceutically acceptable compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.

[0095] Pharmaceutically acceptable compositions as defined above may also be administered by nasal aerosol or inhalation. Such compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

[0096] Most preferably, pharmaceutically acceptable compositions as defined above may be formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions as defined above may be administered without food. In other embodiments, pharmaceutically acceptable compositions as defined above may be administered with food.

[0097] The amount of compound that may be combined with the carrier materials to produce a composition in a single dosage form may vary depending upon the host treated, the particular mode of administration. Preferably, the compositions should be formulated so that a dosage of between 1 mg - 100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.

[0098] It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present disclosure in the composition will also depend upon the particular compound in the composition.

[0099] The activity of a compound as an inhibitor may be assayed in vitro or in vivo or in a cell line. The cell may be in vitro.

[00100] In vitro assays may include assays that determine post-treatment activity and/or pre treatment activity.

[00101] The compounds may be used in the prevention and/or treatment of RNA virus infections, selected from, but not limited to dsRNA virus infections, (+) ssRNA virus infections and (-) ssRNA virus related infections. Examples for dsRNA viruses may include, but are not limited to, the species Amalgaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megabirnaviridae, Partitiviridae, Picobornaviridae, Reoviridae, Totiviridae and Quadriviridae. Examples for (+) ssRNA virus may include, but are not limited to, the orders Nidovirales, Picomavirales and Tymovirales. Examples for (-) ssRNA virus may include, but are not limited to, the families Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae and Orthomyxoviridae.

[00102] In one embodiment, the compound may be used in the prevention and/or treatment of (+) ssRNA virus infections. It may be used, for example, in the treatment of virus infections associated with the Family Picornaviridae. Examples for virus' of the Family Picornaviridae include, but are not limited to, the geni Aphthovirus, Aquamavirus, Avihepatovirus, Cardiovirus, Dicivirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Megrivirus, Parechovirus, Piscevirus, Salivirus, Sapelovirus, Senecavirus, Teschovirus and Tremovirus. For example, the compound may be used in the prevention and/or treatment of Enterovirus related infections.

[00103] In one embodiment, the compound may be used in the prevention and/or treatment of Enterovirus related infections. Examples for virus' of the Enterovirus species include, but are not limited to, the geni Enterovirus A, Enterovirus B, Enterovirus C, Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H and Enterovirus J. For example, the compound may be used in the prevention and/or treatment of Enterovirus A related infections. The compound may be used in the prevention and/or treatment of certain serotypes of the Enterovirus related A infections. These serotypes may include, but are not limited to, serotypes of the Enterovirus types, such as EV-A71, EV-A76, EV-A89, EV-A90, EV-A91, EV-A92, EV-A114, EV-A119, SV19, SV43, SV46 and BA13. Alternatively, they may include, but are not limited to, serotypes of the Coxsackievirus, such as CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A7, CV-A8, CV-A10, CV-A12, CV-A14 and CV-A16. For example, the compound may be used in the prevention and/or treatment of Enterovirus 71 (EV-A71) related infections. Alternatively, they may be used in the treatment of Coxsackie virus A16 (CV-A16) related infections.

[00104] The present invention refers to use of the compounds as disclosed above in the manufacture of a medicament for the prevention or treatment of RNA virus infections. It is understood, that this use comprises all of the embodiments as described above in relation to the selection of the virus to be prevented or treated.

[00105] Enterovirus 71 and Coxsackie virus A16 cause, inter alia, hand, foot and mouth disease. Accordingly, the present invention may also refer to compounds for the treatment and/or prevention of hand, foot and mouth disease (HFMD). The present invention may provide medicaments for the treatment and/or prevention of HFMD. The compounds may be used for simultaneous prevention and treatment of hand, foot and mouth disease.

[00106] The present invention may provide medicaments for the simultaneous prevention and treatment of HFMD. Accordingly, the present invention may provide "dual action" medicaments for the simultaneous prevention and treatment of HFMD.

[00107] The compound of the present invention may be administered as a dual action medicament for prevention and treatment of HFMD.

[00108] The compound may be an RNA virus inhibitor. The compound may be a (+)

RNA virus inhibitor. The compound may be a (+) ssRNA virus inhibitor. The compound may be a Picomavirus inhibitor. The compound may be an Enterovirus inhibitor. The compound may be an Enterovirus 71 inhibitor. Alternatively, the compound may a Coxsackie virus A16 inhibitor.

[00109] The compound as disclosed above may be used in the manufacture of a medicament in the treatment of meningitis, encephalitis, pulmonary edema, herpangina, pompholyx or haemorrhage, acute flaccid paralysis and myocarditis as a result of Enterovirus 71 infection.

[00110] In a fifth aspect, the present invention refers to a formulation comprising the compound as disclosed above, wherein the compound is incorporated into mouthwashes, sweets, edible films and as a topical cream or other acceptable forms of drug delivery methods.

[00111] The compound may be administered with an additional therapeutic agent, wherein said compound is to be administered in combination or alteration with the additional therapeutic agent.

[00112] In one embodiment, the present invention refers a method of preventing or treating a disease associated with a RNA virus or RNA virus related infection comprising administering to a subject in need of treatment a compound as disclosed above, or a pharmaceutically acceptable form or prodrug thereof. [00113] A method of preventing or treating in a cell may comprise administering to a cell a compound as disclosed above, or a pharmaceutically acceptable form or prodrug thereof, a composition or a formulation as disclosed above.

[00114] In one embodiment of the before mentioned method, the RNA virus related infection is an infection with Enterovirus 71.

[00115] In one embodiment of the before mentioned method, the Enterovirus 71 infection is hand, foot and mouth disease.

[00116] The method as disclosed above may further comprise the step of administering an additional therapeutic agent in the subject.

[00117] The present invention refers to a kit providing for the convenient application of the compound as disclosed above. The kit may comprise the compound as disclosed above, the pharmaceutical composition as disclosed above, or the formulation as disclosed above.

[00118] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00119] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00120] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Application

[00121] Diseases associated with RNA virus infections are a major cause for disruption of a fulfilled life, can sometimes have serious neurological implications or even cause death. The compounds disclosed in the present invention have been found to be suitable for use in therapy against diseases associated with these viruses, for example they have been found to be useful in therapy against Enterovirus related infections. Among the group of RNA viruses, Enterovirus-71 (EV-71) is a strain of (+) ssRNA virus, genus Enterovirus of the Picornaviridae family. EV-71, as a representative member of RNA viruses, is one of the most common causative agents of hand, foot and mouth disease (HFMD). EV- 71 infection causes fever, sore throat, headache and more evidently ulcers in the throat, mouth and tongue and a rash with vesicles (small blisters, 3-7 mm) on hands and feet. HFMD affects predominantly young children below 5 years of age but adults are also susceptible. HFMD is spread from person to person by direct contact with the nasal discharge, saliva, faeces, and fluids from the rash of an infected person. EV-71 epidemics have long occurred in many countries such as USA, Bulgaria, Hungary. More recently, EV-71 outbreaks have been reported in the Asia-Pacific region like Taiwan, Australia, Singapore, Malaysia, China, Vietnam and Cambodia where the infection is not only mild HFMD, but is also associated with severe neurological complications such as aseptic meningitis and brainstem encephalitis, which may lead to cardiopulmonary failure and death. According to data from the Chinese Center for Disease Control and Prevention (CDC), more than 2,100,000 cases of EV71 infection and 569 deaths were reported in China in 2012, which is higher than in 2011 with 1,600,000 reported cases and 509 deaths.

[00122] Since the start of 2014 till the 11 Oct 2014, there are 17711 HFMD cases thus far and many affected childcare centres have been forced to shut down their operations for clean-up before resuming their services. This has caused much disruption to the parents' work as they would have to take leave to take care of their ill child. Up till now, there was no licensed prophylactic or therapeutic for clinical use.

[00123] In this invention, there is described a portfolio of natural derived compounds which has been identified to be able to inhibit EV-71 infection. With this discovery, these compounds when used individually or in combination may stop the spread of EV-71 infection.

[00124] As the disease affects many countries and many individuals and is also still spreading, the application potential of this invention is very likely. Big and small pharmaceutical companies and even natural herbal medicine companies may realize the invention into a product which could be sold over the counter (OTC). As these are small molecules, they can be easily incorporated into mouthwashes, sweets or edible films for use in treating mouth blisters in EV-71 infected kids. Topical creams can be easily formulated for use on hand and foot blisters or other areas to prevent and get rid of EV-71 infection. Examples

[00125] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1

[00126] A cell-based, high throughput indirect immunofluorescence screening assay using a 500 Flavonoid derivative compound library (Timtec) was used to identify potential antivirals against EV-71. From the studies, 3 compounds (ST074529, ST074528 and ST076546) were found to be able to prevent early EV-71 infection, while 3 2 other compounds (ST024734 and ST077124) were found to be able to prevent late EV-71 infection. 3 compounds (ST078115, ST079167 and ST070204) were identified to possess both pre and post antiviral activity (i.e. prevent early and late EV-71 infection- dual action). The materials and methods used in this initial screening are now being described in detail.

Example la - Materials and Methods

Cell lines and viruses

[00127] Human rhabdomyosarcoma (RD) (CCL-136™, ATCC) cells were cultured in

Dulbecco's Modified Eagle's Medium (DMEM; Sigma- Aldrich) supplemented with 10% fetal calf serum (FCS) and maintained in a 37°C, 5% C02, humidified incubator (Thermo Scientific). [00128] The entereovirus virus strain which causes Hand, Foot and Mouth Disease

(HFMD) used in the study was HEV-71 strain H (VR-1432™, ATCC), with an infectious titre of 2.2x106 PFU/ml. Flavonoid Derivative Library (Timtec) and screening for bioactive antivirals

The FL-500 Flavonoid Derivatives (Timtec) were used in the screening for antivirals against EV-71. This library consists of 500 individual chemically characterized flavonoids compounds assembled from 9 core flavonoid structures. Flavonoids make one of the most preferred groups of natural chemotypes tested for various biological activities. Flavonoids have low toxicity and are widely available in plants which are edible. This group of compounds has been shown to possess anti-inflammatory, anticancer, anti-oxidant activities.

RD cells were plated onto 384- well plates and grown for 12h prior to the screening procedure using the 500 flavonoid derivatives compound library. The screening method was carried out as previously described and validated by Low et al. (2011). Briefly, RD cells were infected with EV-71 at a multiplicity of infection (MOI) of 1 and incubated at 37°C for 1 h to allow virus adsorption. The cells were then incubated with DMEM containing 20μ^ηύ of each Flavonoid derivative compound (Procedure A) and subsequently processed using anti-EV-71 specific antibody for immunofluorescence assay as previously described by Low et ah, (2011). Procedure A selects for antiviral inhibitors that act post virus infection. For screening of antivirals which act prior to exposure to virus particles, a similar screening method was used with the exception that compounds were added 2h before the cells were exposed to EV- 71 virus (Procedure B) (Fig. 1). Inhibition of EV-71 infection by the various compounds, relative to the DMSO-treated control was expressed as the percentage of EV-71 antigen positive cells. The positive hits that displayed greater than 75% inhibition against EV-71 were then selected for further validation using secondary assays.

Cell viability assay

[00129] AlamarBlue® cell viability assay (Invitrogen, Carlsbad, CA, USA) was used to measure the cell viability of the various cell lines following treatment with various concentrations of compounds following the manufacturer's instructions. 1% DMSO were used as experimental controls. The fluorescence intensity was measured by the Infinite M200 microplate reader (Tecan) at an excitation and emission wavelengths of 570 nm and 585 nm, respectively.

Example lb - Dose-dependent studies

Infection of cells

[00130] RD cells were seeded on 24-well plates at 2 x 105 cells per well one day prior to infection. The cells were then infected at MOI of 1 for 1 h to allow virus adsorption. Following infection, the cells were rinsed with PBS before treatment with compounds ST024734, STOW 124 and ST024703 (Control) at doses of 0.02 - 20 μ ηιΐ. 12 h post compound treatment, the supernatant of the treated wells were collected and clarified for viral plaque assay as described below to determine the viral titre after each treatment.

Viral plaque assay

[00131] EV-71 virus stock was serially diluted in 10-fold dilutions (10 ¹ to 10^"7) and each dilution set was used to infect one well of cells on a 24-well plate (section 2.2.2). After lh virus adsorption incubation, the cells were washed with PBS and then 1ml DMEM with 1% carboxymethylcellulose (CMC) and 2% FCS was used as the overlay medium. After 4 days of incubation, the overlay medium was removed and 1ml of crystal violet staining solution were added to each well to stain and fix the cells overnight on an orbital shaker at room temperature. The staining solution was then removed and the wells were rinsed under running water before drying. Clearings in the cell monolayer represent plaques formed by an initial virion infection and the mean number of visible plaques from the least diluted set would be multiplied by its dilution factor to determine the infectious virus titre of a sample, expressed as plaque forming units per ml (PFU/ml).

Example lc - Secondary assays used to investigate effects of compounds on viral replication- Strand specific real time, reverse transcription polymerase chain reaction (qRT-PCR)

[00132] RD cells in 24-well plate were first infected with EV71 (MOI=l) as previously described followed by treatment with the appropriate compound dosages (0.2, 2 and 20ug/ml). At 6 hours post infection (hpi) , RNA from the samples was extracted using a commercial kit (RNeasy Mini Kit, Qiagen). Each sample was then treated with DNAse (Promega) before being reversed transcribed for 30 minutes at 44°C with either the forward or reverse primer in two reaction tubes using M-MLV Reverse Transcriptase (Promega) to generate cDNA of either positive-sense or negative- sense viral RNA. Following reverse transcription, a 2-minute 94°C heating step inactivates the reverse transcriptase. The cDNA was then quantitatively assayed in a StepOne Plus Real-Time PCR System (Applied Biosystems) using Maxima® SYBR Green/ROX qPCR Master Mix (2X), containing Maxima™ Hot Start Taq DNA Polymerase, dNTPs and SYBR green supplemented with ROX passive reference dye. The thermal cycling conditions are as follows: 40 cycles consisting of 15 seconds denaturation at 94°C and 60 seconds annealing and extension at 60°C. The primer pair, forward and reverse listed in Table 1, identifies the presence of viral RNA by targeting conserved regions in the VP1 gene for amplification. At the end of the run, the copy number of the positive-sense and negative-sense viral RNA in each sample was derived from the cycle threshold value (normalized using actin CT values of the samples) referenced against a lab-established EV71 viral RNA standard curve generated using the same set-up.

Table 1. Primer pair used for amplification of EV71 viral RNA.

Sequence

Forward 5 ' -TTTGGATCCGGCC ATGGTTCAAGG-3 '

(SEQ. ID NO 1)

Reverse 5 ' - C AGACTGCAGACCGAATTTCCGAG-3 '

(SEQ. ID NO 2)

[00133] In Example 1, flavonoids and derivatives from a 500-compound library

(Timtec) were screened for post-treatment and pre-treatment anti-EV71 activity using a cell- based, fluorescence-detection high throughput screening (HTS) assay to generate an extensive list of hits. Many of the high scoring post-treatment hits were validated with secondary assays as promising novel drug candidates against EV71.

[00134] The results of this screening are shown in the below Table 2,Table 3 & Table

4: Table 2

[00135] From Table 2, it can be observed that the cell pre-treated with substituted phenyl cinnamyl ketones ST074529, ST074528 and ST0765464 show significant inhibition of EV71 infection. This showed that the compounds can be used to prevent the host from getting EV71 virus infection. Table 3

[00136] The substituted phenyl cinnamyl ketones ST078115, ST079167 and ST070204 are able to prevent and eliminate EV71 infection. Hence, they are working in dual action . Table 4

[00137] From Table 4. it can be observed that exposure of the substituted phenyl cinnamyl ketones compounds ST024734 and ST077124 after EV71 infection can indeed inhibit the EV-71 infection from spreading. This strongly indicated that these compounds when administered to infected EV-71 individuals can cure them from the disease (therapeutic).

[00138] Example 2 follows up and further investigates two such post-treatment hits -

ST024734 and ST077124. Example 2

Example 2a- Results

Assessment on-Bioactive Compounds cytotoxicity.

[00139] Cytotoxicity for most of the bioactive compounds in Table 2, 3 and 4 were evaluated by using AlamarBlue cell viability assays with a wide dosage range, up to an allowed maximum of 1% DMSO used as vehicle. No significant inhibition was observed for compounds ST074529, ST074528, ST078115, ST070204 (Fig. 2A) and ST024703 (Control), ST024734 and ST077124 (Fig 2B). Compounds ST0765464 and ST079167 were not tested. Apparently all the flavonoids were fairly well-tolerated by the RD cells (>80% relative cell viability) even at highest concentrations tested.

Effects on viral replication.

[00140] Using concentration determined by the cytotoxicity assay, the two compoundsST0765464 and ST079167 were used to treat EV71 -infected cells to validate them as anti-EV71 compounds via viral plaque assays. RD cells were infected with EV71 at a MOI of 1 and given the relevant treatments before harvesting at 12hpi. Compared to the untreated and vehicle controls, it is clear that both compounds are potent inhibitors of EV71 replication that exhibit dose- dependent effects on virus titres, with ST077124 reducing infectious titres by about 4 log 10 PFU/ml and ST024734 reducing infectious titres by about 3.5 loglO PFU/ml (Fig. 3). The effects that ST077124 and ST024734 on viral RNA production were also examined using qRT-PCR. RD cells were infected with EV71 at a MOI of 1 and given the relevant treatments before total RNA was harvested at 6hpi. It could be seen that decreases in the amount of viral RNA produced, relative to the untreated control, occurred across all dosages for both compounds (Fig. 4) and were accompanied by significant disruption of viral production in a similar dose -dependent manner (-0.3% of untreated control). It can also be seen that viral RNA production was disrupted even at treatment concentrations that did not result in any decrease in infectious virus titres. It may be postulated that at the lower, less effective concentrations of the flavonoid treatments, while the replication cycle was still able to 'complete' by 12hpi, viral replication slowed down without any biologically significant decreases in virus progeny produced.

In parallel to RNA production, viral protein productions were also examined via SDS-PAGE and Western blot analysis. RD cells were infected with EV71 at a MOI of 1 and given the relevant treatments before total cell lysates were harvested at 12hpi (Fig. 5). Precursor viral protein, VP0 as well as structural protein, VP2 were detected using the mouse monoclonal antibody and it was observed that dosage dependent inhibition of virus titre were accompanied by significant disruption of protein production. Taken together, the reduction in the amount of viral protein produced due to the flavonoids treatment correlates with the reduced amounts of virus RNA produced. Co-treatment Assay to determine presence of virucidal activity.

[00141] In order to determine if any of the compounds have virucidal effect, co- treatment assay is performed by incubating diluted virus (MOI 1) with two drugs and thereafter inoculating the mixture onto RD cell (Fig. 6). It is deduced that both ST077124 and ST024734 do not appear to have any direct virucidal effects on EV71 and neither are capable of inhibiting the virus' ability to attach and enter RD cells.

Time of addition and removal study of ST077124 and ST024734.

[00142] Times of addition and removal experiments were used to identify the critical time periods in the virus replication cycle during which ST077124 and ST024734 exerts its anti-EV71 properties. Visualization via indirect immunofluorescence is done on infected (MOI 1) and treated RD cells at various time points (Fig. 7). As treatment with ST077124 was delayed during time of addition experiment, an apparent large jump in the number EV71- infected cells is observed between 4hpi treatment and 6hpi treatment. As for the time of removal immunofluorescence image analysis, it would appear that ST077124 retains its anti- viral effect if not washed out from the infected cells at an early time point, suggesting some ability by the compound flavonoid to stay within the treated cells. This strongly suggests that ST077124 works as an anti-EV71 agent by inhibiting viral replication processes occurring between 4-6hpi.

[00143] ST024734 is most efficacious when administered within the first 2h of infection. This is reflected in the time of removal immunofluorescence images, which shows potent inhibition of EV71 by the compound if it is not removed from the infected cells within the first 2hpi. These data strongly suggest that ST024734 might target EV71 replication processes occurring between Ohpi to 2hpi. Inhibition of EV-71 replicon.

[00144] Nano-luciferase reporter construct of EV71 is used to determine if the drug affect the general replication process. The n-Luc gene is inserted in between of IRES and non structural gene of EV71. Inhibition of viral non- structural proteins such as 2C and 3D or IRES will result in lower transcript level and in turns reduces luciferase activity. Treatment with both STOW 124 and ST024734 shows significant reduction in luciferase signal compared to DMSO carrier control (Fig. 8A). Therefore it can be concluded that both drugs inhibits EV71 replication via direct inhibition of viral non-structural proteins, translation machinery or indirectly through host factors.

Drugs' effect on translation efficiency.

[00145] To decipher if the two drugs affect translation machinery of the viral replication process via inhibition of EV71 promoter IRES, bicistronic reporter assay is carried out. The reporter construct, which contains two cinstrons (Firefly luciferase driven by CMV, cap dependent promoter and Renilla luciferase driven by EV71-26M IRES, cap independent promoter) is transfected into RD cell. Transfected cells are treated with the two drugs and thereafter luciferase activities were measured. Amantadine was used as positive control, which is known to inhibit IRES specifically using 0.5 mg/ml. Two luciferase activities are shown in term of ratios (CMV to IRES) (Figure 8B). While amantadine shows significant inhibition of IRES at 0.5 mg/ml both compounds of interest do not seems to target IRES as the relative promoter activities do not defer significantly through increasing concentration of 5 to 50 μΜ.

Spectrum of Antiviral activities of ST077124 and ST024734.

[00146] In order to determine if antiviral effects are specific to EV71 or are more broad- spectrum, plaque assays on ST077124-treated or ST024734-treated samples infected with one of the four other viruses are performed. The four viruses picked were - EV71 H strain, EV71 B5 subgroup, CA16 and CHIKV- 122508 . EV71 H strain and B5 subgroup were chosen to examine if the compounds have any strain specific effects, while CA16, a less virulent HFMD-causing species of enteroviruses, was chosen to examine if the flavonoids have similar antiviral effects on related enteroviruses. CHIKV- 122508 is an alphavirus from the Togaviridae family that was chosen to investigate the broad- spectrum antiviral activity of the compounds. [00147] Given the data, it can be seen that 5μΜ of ST077124 and 50μΜ ST024734 were potent inhibitors against the other enteroviruses - EV71 H strain, EV71 B5 subgroup and CA16 (Fig. 9).

Example 2b - Methods

Cells lines and viruses

[00148] Human rhabdomyosarcoma (RD) (CCL-136™, ATCC) cells were cultured with 15ml sodium hydrogen carbonate buffered Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS; PAA). SJCRH30 (SJ) (CRL-2061™, ATCC) and BHK-21 (BHK) (CCL-10™, ATCC) cells were cultured with 15ml sodium hydrogen carbonate buffered Roswell Park Memorial Institute 1640 medium (RPMI-1640; Sigma-Aldrich) supplemented with 10% fetal calf serum. The main EV71 virus strain used in the study was EV71 virus strain 41 (5865/S IN/00009) with an infectious titre of 1.3x107 plaque-forming units per ml (PFU/ml). Other EV71 strains used include - EV71 strain H (VR-1432™, ATCC) with an infectious titre of 2.2x 106 PFU/ml and EV71 B5 genotype (NUH0083/SIN/08) with an infectious tire of 1.4x107 PFU/ml. The Coxsackie A virus used in the study was Coxsackie A16 (CA16) with an infectious titre of 1.2x109 PFU/ml. The Chikungunya (CHIKV) virus strain used in the study was CHIKV- 122508 with an infectious titre of 1x107 PFU/ml.

Preparation of compounds for Antiviral Screening.

[00149] Dimethyl sulfoxide (DMSO) was used as the vehicle for flavonoid delivery in this study. ST077124 (lOmg) and ST024734 (lOmg) were purchased from TimTec and diluted in DMSO to achieve 25mM and lOmM stock solutions respectively and stored at 4°C. Subsequent dilutions to working concentrations were performed with assay diluent to minimize exposure of cells to DMSO. Vehicle controls of DMSO were used in the relevant experiments to determine compound- specific effects.

Cell viability and post-treatment assay.

[00150] 96-well plates seeded with RD cells or SJ cells were infected with

EV71/CA16 and CHIKV respectively. After the adsorption period (lh for EV71 and CA16, 1.5h for CHIKV), ΙΟμΙ of the flavonoid at working concentration and 90μ1 maintenance medium were added to wells to achieve the target concentration in a final volume of ΙΟΟμΙ media per well. After incubation (12hpi for EV71 and CA16, 24hpi for CHIKV), the plates and cells were then frozen at -80°C before subsequent thawing and quantification via viral plaque assays. To determine viability RD or SJ cells seeded onto 96-well plates were treated with the respective flavonoids at varying dosages in ΙΟΟμΙ of maintenance media and incubated for 24h. At the end of the incubation, the cells were subjected to two washes with PBS before the addition of ΙΟΟμΙ of maintenance medium and ΙΟμΙ of AlamarBlue® reagent (Invitrogen) per well. The plates were further incubated for 4h before being analyzed for absorbance using a plate reader (Tecan) at an excitation wavelength of 570nm and emission wavelength of 585nm to determine relative cell viability against relevant controls.

Time of addition and removal assay.

[00151] 96-well plates and 24-well plates were seeded with RD cells and infected with EV71. The infected cells were then treated with the respective flavonoids at 2h intervals from Ohpi up till the end point of 12hpi for time of addition assay. At 2h intervals from Ohpi to 12hpi, the EV71-infected treated cells were washed twice with PBS before maintenance medium was added for time of removal assay. At the 12hpi end point, the 96-well plates and infected cells were frozen at -80°C before subsequent thawing and quantification via viral plaque assays while the 24-well plates were processed for indirect immunofluorescence visualization.

Real time, reverse transcription polymerase chain reaction (qRT-PCR)

[00152] At 6hpi, total RNA from samples was extracted using a commercial kit (RNeasy Mini Kit, Qiagen). Each sample was then treated with DNAse (Promega) before being quantitatively assayed using a 1-step reaction mix, Maxima® SYBR Green/ROX qPCR Master Mix (2X), containing Maxima™ Hot Start Taq DNA Polymerase, dNTPs and SYBR green supplemented with ROX passive reference dye (Thermo Scientific) along with M- MLV Reverse Transcriptase (Promega). The primer pair used to detect EV71 viral RNA identifies the presence of viral RNA by targeting the 5'-UTR region of enteroviruses for amplification and is as follow:

Sequence Forward 5'-CAAGCACTTCTGTTTCCCCGG-3' (SEQ. ID NO 3)

Reverse 5 ' - ATTGTC ACC ATAAGC AGCC A -3' (SEQ. ID NO 4)

[00153] Control wells quantifying the amount of actin RNA were also run alongside to normalize the total amount of RNA present for relative quantification. Cycling conditions for the 1-step quantification are as follows: 59 minutes of reverse transcription at 42°C, before inactivation/hot start for 10 minutes at 95 °C. This is followed by 40 cycles of amplification consisting of 15 seconds of denaturation at 95 °C and 1 minute of annealing and extension at 60°C. At the end of the run, melt curve analyses were carried out to ensure the amplification of the appropriate amplicons. Using the actin CT values of the samples the amount of viral RNA present within each sample can then be normalized and relatively quantified against the control samples.

SDS-PAGE and Western blot analysis.

[00154] Cell lysates from EV71 -infected 24-well plate samples with appropriate compound treatments were harvested at 12hpi. The samples were subject to two washes with ice-cold PBS before 50μ1 of freshly prepared cell lysis buffer were added to each well. After 5 minutes, the cell lysates were collected in micro -centrifuge tubes.

[00155] The cell lysates were then mixed with an appropriate volume of 4X SDS- PAGE loading buffer and heated for 10 minutes at 95°C. 20μ1 of each heated sample and 7μ1 of protein ladder (PageRuler, Thermo Scientific) were then loaded into a 10-well, 12% polyacrylamide, tris-glycine gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then carried out at 120 volts using the Mini-PROTEAN Tetra cell electrophoresis system (Bio-rad) with PowerPAC™ HC Power Supply (Bio-rad).

[00156] After 3.5h, Western blotting was performed to transfer the separated protein contents from the polyacrylamide gel onto a nitrocellulose membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad).

[00157] Following the transfer, the membrane was blocked with 5% skim milk

(Anlene) for lh on a tabletop orbital shaker at room temperature. Following which the membrane was probed for lh at room temperature with 5ml of skim milk containing 1:2500 diluted mouse anti-EV71 antibody (MAB979, Millipore) and 1:5000 diluted mouse anti-actin antibody (MAB 1501, Millipore). After probing with the primary antibodies, the membrane was subjected to four 5-minute washes with Tris-buffered saline and 0.1% Tween-20 (TBST) before a lh incubation at room temperature with 5ml of 5% skim milk containing 1:2500 diluted polyclonal goat anti-mouse IgG (H+L) antibody conjugated with horseradish peroxidase (HRP). After incubation with the secondary antibodies, the membrane was put through five 5-minute washes with TBST before enhanced chemiluminescent substrate (Thermo Scientific) was added and incubated for five minutes at room temperature. Subsequently, the protein bands were visualized and digitally captured using the C-DiGit™ Blot Scanner. The primary anti-EV71 antibody used was a mouse monoclonal antibody specific to an epitope of the VP2 protein, a structural protein of EV71.

[00158] Luciferease assay. RD cells are transfected with nano-lucif erase or bicistronic luciferase constructs using JetPREVIE transfection reagent (Polyplus). Appropirate drugs are added at 12-hour post infection. At 24-hour post infection cells are lysed and luciferase activities are measured using Nano-Glo or Dual-Glo assay systems from Promega.

Claims

Claims

1. A compound of Formula (I) for use in therapy:

Formula (I) or a pharmaceutically acceptable salt, tautomer(s), solvate or polymorph thereof, wherein R¹ is hydrogen, hydroxyl or alkoxy;

2 3

R and R are independently selected from hydrogen, hydroxyl, optionally substituted alkoxy, acyl, optionally substituted hydrocarbons selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo alkyl, and optionally R² and R³ are taken together to form a 4- to 7-membered heterocyclic ring;

R⁴ is selected from hydrogen or a hydrocarbon;

R⁵ is selected from hydrogen, hydrocarbon, hydroxyl, oxygen, and acyl, and is optionally taken together with R or R to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring;

R⁶ is selected from hydrogen, hydrocarbon or alkoxy or is optionally taken together with R⁵ to form a 5- or 6-membered ring selected from a cycloalkenyl ring or a heterocyclic ring; R⁷ is hydrogen or is optionally taken together with R⁵ to form a 6-7-membered ring selected from a heterocyclic ring; and

R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from hydrogen, hydroxyl, halogen, an optionally substituted alkoxy, an optionally substituted aryloxy, an optionally substituted acyl, an optionally substituted aryl, an optionally substituted acyloxy and an optionally substituted hydrocarbon selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, and heteroaryl.

2. The compound according to claim 1, wherein a maximum of 4 cyclic ring systems is present in the compound.

3. The compound according to claims 1 or 2, wherein of the 3 optional ring systems which may be formed between R² and R³, R⁵ and R⁶ and R⁵ and R⁷, only a maximum of one ring is present in the compound.

4. The compound according to any of the preceding claims, wherein when R and R are independently selected from a substituted alkoxy, they are substituted by one or more substituents independently selected from alkyl, alkoxy and acyl and optionally are taken together to form a 5-membered heterocyclic ring.

5. The compound according to any of the preceding claims, wherein when R⁸, R⁹, R¹⁰ and R¹¹ are independently selected from a substituted alkyloxy and a substituted alkoxy, they are substituted by one or more substituents independently selected from alkyl, acyl, aryl and heteroaryl.

6. The compound according to any one of the preceding claims, wherein when R⁵ is hydrocarbon, oxygen or acyl, it is attached to R⁶ or R⁷ to form a 5- or 6-membered heterocyclic or cycloalkenyl ring.

7. The compound according to any one of the preceding claims selected from the group consisting of:

8. The compound according to any one of the preceding claims for use in the treatment of a disease associated with a RNA virus infection.

9. The compound according to claim 8, wherein the RNA virus is a (+) ssRNA virus.

10. The compound according to claim 9, wherein the (+) ssRNA virus is a Picomavirus.

11. The compound according to claim 10, wherein the Picomavirus is an Enterovirus.

12. The compound according claim 1 1, wherein the Enterovirus is an Enterovirus 71.

13. The compound according to claim 11, wherein the Enterovirus is a Coxsackie virus

A16.

14. The compound according to claim 8 for the treatment or prevention of hand, foot and mouth disease.

15. The compound according to claim 14, whereby the compound is any one of

, or

ST079167 for use in preventing or treating hand, foot and mouth disease.

16. The compound according to claim 12 in the treatment of meningitis, encephalitis, pulmonary edema, herpangina, pompholyx or haemorrhage, acute flaccid paralysis and myocarditis as a result of Enterovirus 71 infection.

17. Use of the compound according to any one of claims 1 to 8 in the manufacture of a medicament for the prevention or treatment of diseases associated with a RNA virus infections.

18. The use according to claim 17 in the treatment of meningitis, encephalitis, pulmonary edema or haemorrhage, herpangina, pompholyx, acute flaccid paralysis and myocarditis as a result of Enterovirus 71 infection.

19. A method of preventing or treating a disease associated with a RNA virus related infection comprising administering to a subject in need of treatment a compound according to the any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.