INFECTION(S)

FIELD OF THE INVENTION

The present invention relates to compounds useful in methods of treating at least one microbial infection, such methods and uses of said compounds, pharmaceutical formulations comprising said compounds, and/or synthetic processes. BACKGROUND TO THE INVENTION

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Many disease-causing microorganisms possess several enzymes that play a part in infecting animals with the disease. One of the more common enzymes is neuraminidase (also known as sialidase) which is involved in the replication process of the microorganism. Some of the more important disease-causing microorganisms in man and/or animals which possess a neuraminidase include but are not limited to Vibrio cholerae, Clostridium perfringens. Streptococcus pneumoniae, Arthrobacter sialophilus, influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, equine influenza virus, Sendai virus and the like. For example, mortality due to influenza virus is a serious problem throughout the world. The disease is especially devastating to man, lower mammals and some birds. Although vaccines containing attenuated influenza virus are available, those vaccines only provide immunological protection towards a few influenza strains and are less effective in otherwise immunologically compromised populations such as the elderly, young children, and in those who suffer from chronic respiratory illness. The productivity loss from absence due to sickness from influenza virus infection has been estimated to be more than US$1 billion per year.

There are two major strains of influenza virus (designated A and B). Currently, there are only a few pharmaceutical products approved for treating influenza. These include amantadine and rimantadine, which are active only against the A strain of influenza viruses, and ribavirin, which suffers from dose-limiting toxicity. Mutant virus which is resistant to amantadine and rimantadine emerges quickly during treatment with these agents. Accordingly, there is always a need for new treatments for such microbial infections. In particular, there is a need for new compounds for use in treatment of such microbial infections, and new treatments of microbial infections using compounds which may be known for other purposes but have not been used to treat microbial infections.

The first influenza neuraminidase inhibitor to be approved was zanamivir. Although an effective drug against influenza, it can only be administered by inhalation and thus inconvenient for use. There is thus a continuing need for improved treatments and in particular, neuraminidase inhibitor(s) for both treatment and/or prevention of influenza infection amongst other infections.

To date, new neuraminidase inhibitors have largely been developed using conventional chemical synthesis from commercially available building blocks. Whilst these advances are impressive, efforts to identify useful neuraminidase inhibitors from natural sources which are expected to be more effective in treatment and have less side effects remains a challenge.

Natural products provide novel and structurally diverse chemical compounds often with potent biological activities, thus providing excellent starting points for drug discovery. Despite these advantages, very few pharmaceutical companies are involved in the screening of natural product compound libraries, let alone libraries for naturally occurring neuraminidase inhibitors. This may be due to high cost involved in isolation and identification of pure compounds, difficulty in collection, the complex nature of plants, absence of clear-cut regulatory guidelines for natural products and the like.

SUMMARY OF INVENTION

The present invention is defined in the appended independent claims. Some optional features of the present invention are defined in the appended dependant claims.

Thus, according to a first aspect of the invention, there is provided a use of a compound of formula I, a salt thereof or a solvate thereof for the manufacture of a medicament for treatment of at least one microbial infection. The compound has the general formula (I):

wherein

R1 is H, N0 ₂ , Br, I, NHC ₂ H ₄ OH, NHC(=NH)NH ₂ ; NH _2,

R2 is H, CH(CH ₃ ) ₂ , S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H4-OCH ₃ ;

R3 is H or N0 ₂ ; and

R4 is CHO or CH=CHC(0)OR5, wherein R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₃ , CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ (e.g. R1 is H, N0 _2> Br, I, NHC ₂ H ₄ OH, NHC(=NH)NH ₂ ; NH _2;

R2 is H, CH(CH ₃ ) _2l S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H ₄ -OCH ₃ ;

R3 is H or N0 ₂ ; and

R4 is CHO or CH=CHC(0)OR5, wherein R5 is H, CH _3> CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ ).

In one aspect of the invention, there is provided a compound of formula II:

a salt thereof or a solvate thereof, wherein

R4 is CHO or CH=CHC(0)OR5, wherein

when R4 is CH=CHC(0)OR5, the Carbon-Carbon double bond is in the (E) configuration and:

R1 is H or N0 ₂ ;

R2 is H, CH(CH ₃ ) ₂ , S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H ₄ -0CH ₃ provided that R2 is not H when R1 is H; and

R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ provided that R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably CH ₂ CH ₂ CH ₃ when R2 is H, and

when R4 is CHO:

R2 is H; and

R1 is NHC ₂ H ₄ OH, NHC(=NH)NH ₂ or NH ₂ . In one aspect of the present invention there is provided a compound of formula (II) or a salt or a solvate thereof as described herein, for use in medicine

In one aspect of the present invention there is provided a compound of formula (I) or a salt or a solvate thereof as described herein, or a compound of formula (II) or a salt or a solvate thereof as described herein, for use in the treatment of at least one microbial infection.

In one aspect of the present invention there is provided a method of treatment of at least one microbial infection comprising administering a compound of formula (I) or a salt or a solvate thereof as described herein, or a compound of formula (II) or a salt or a solvate thereof as described herein, to a subject in need thereof.

In one aspect of the present invention there is provided a pharmaceutical formulation including a compound of formula (I) or a salt or a solvate thereof as described herein, or a compound of formula (II) or a salt or a solvate thereof as described herein, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, the formulation being for use in the treatment of at least one microbial infection, wherein the pharmaceutical formulation optionally comprises one or more other therapeutic agents

In one aspect of the present invention there is provided a process for the preparation of a compound of formula II or a salt or a solvate thereof as described herein, which process comprises:

(i) for compounds of formula (II) when R4 is CH=CHC(0)OR5, R2 is H and R5 is

CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ ,

reacting a compound of formula (lla):

in which R6 is H or N0 ₂ and R7 is H, with a compound of formula R8-OH in the presence of an acid, wherein R8 is CH2CH2CH2CH ₃ , or more preferably CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ ;

(ii) for a compound of formula (II) when R4 is CH=CHC(0)OR5, R2 is CH(CH ₃ ) ₂ and R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH _3l reacting a compound of formula (Ha), in which R6 is N0 ₂ and R7 is CH2CH2CH2CH3, or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , with an isopropyl halide in the presence of a base and a phase transfer catalyst;

(iii) for compounds of formula (II) when R4 is CH=CHC(0)OR5, R2 is S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H ₄ -OCH ₃ and R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , reacting a compound of formula (lla), in which R6 is H and R7 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , with a compound of formula R9-X, wherein R9 is S0 ₂ -4-CeH ₄ F or S0 ₂ -4-C ₆ H ₄ -OCH ₃ and X is a halide;

(iv) for a compound of formula (II) when R4 is CHO and R1 is NHC ₂ H ₄ OH, reacting a compound of

in which R1 is a halide, with 2-ethanoiamine;

(v) for a compound of formula (II) when R4 is CHO and R1 is NH ₂ ,

reacting a compound of formula (lib), in which R1 is N0 ₂ , with hydrazine hydrate in the presence of glyoxylic acid; and

(vi) for a compound of formula (II) when R4 is CHO and R1 is NHC(=NH)NH ₂ , reacting a compound of formula (lib), in which R1 is NH ₂ , with cyanamide.

BRIEF DESCRIPTION OF FIGURES

Preferred embodiments will now be described by way of examples with reference to the accompanying figures in which:

Figure 1 is the 'H-NMR spectrum for compound FAD009, i.e. (E)-ethyl 3-(4-hydroxy-3- methoxy-5-nitrophenyl)acrylate.

Figure 2 is an image of viral inhibition assay plates containing A/Malaysia/Muar 33/2009 H1 N1 influenza virus treated with ferulic acid at concentrations 10 Mg/ml, 1 Mg/ml, 0.1 Mg/ml and 0.01 Mg/ml. A virus only well (positive control) and cell only well (negative control) are also included. 015 000067

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Figure 3 is a graph illustrating percentage (%) inhibition for A/Malaysia/Muar 33/2009 H1 1 versus log concentration in g/ml of ferulic acid. Figure 4 is an image of a viral inhibition assay plate. The A/chicken/Malaysia/5858/2004(H5N1) influenza virus was treated with Ferulic Acid at 10 concentrations 100 pg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/ml, 6.25 pg/ml, 3.125 pg/ml, 1.56 pg/ml, 0.78 pg/ml, 0.39 pg/ml and 0.2 Mg/ml. Non-treated virus well acted as the positive control and the MDCK cell line only well acted as the negative control.

Figure 5 are graphs showing cytotoxicity test results of ferulic acid. The Cytotoxicity Concentration (CC ₅ o) for the compound towards the cells line was determined via Graphpad Prism Version 5. Figure 6 provides inhibition assay graphs for compounds in Example 3, labelled according to Table 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

Compounds having a general formula, e.g. "formula I", as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter (in any aspect or embodiment of the invention) referred to together as the compounds of the general formula, e.g. "compounds of formula I". Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. 7

Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene- 2-sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L- ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor- sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2- hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, ^aminosalicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography. P T/MY2015/000067

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The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3. "Pharmaceutically functional derivatives" of compounds of formula I as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I. The term "prodrug" of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)). Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs" p. 1-92, Elsevier, New York-Oxford (1985).

Thus, according to a first aspect of the invention, there is provided a use of a compound of formula I, a salt thereof or a solvate thereof for the manufacture of a medicament for treatment of at least one microbial infection. The compound has the general formula (I):

R1 is H, NC-2, Br, I, NHC ₂ H ₄ OH, NHC(=NH)NH ₂ ; NH _2;

R2 is H, CH(CH ₃ ) ₂ , S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H ₄ -OCH ₃ ;

R3 is H or N0 ₂ ; and

R4 is CHO or CH=CHC(0)OR5, wherein R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₃ , CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ .

In particular, the compound of formula (I) may be such that

when R4 is CHO, then R2 is H and R1 is H, N0 ₂ , Br, I, NHC ₂ H ₄ OH,

NHC(=NH)NH ₂ or NH ₂ ; and

when R4 is CH=CHC(0)OR5, then R1 is H, N0 ₂ or NH ₂ .

Compounds of formula I may also be referred to in this specification as ferulic acid and/or a derivative thereof, and/or vanillin and/or a derivative thereof. Surprisingly, these compounds have been found to function as a neuraminidase inhibitor when used in the treatment of at least one microbial infection.

When R4 is CH=CHC(0)OR5, the compounds of formula I contain a Carbon-Carbon double bond (also denoted as C=C) and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about the double bond. Both isomers and mixtures thereof are included within the scope of any aspect of the invention. In certain embodiments, when R4 is CH=CHC(0)OR5, the Carbon-Carbon double bond may be in the (E) configuration.

For example, ferulic acid may have the following chemical structures:

Ferulic acid may exist as two isomers, the (Z)-isomer form is a yellow oily liquid and the (£)-isomer is a white crystalline solid. Accordingly, the compounds of formula I, having (E)- and (Z)- isomeric forms, may be used as a mixture of both (£)-isomer and (Z)-isomer or as only one of the two isomers. The mixture may be a 1 :1 mixture of these isomers, or it may be enriched in one isomer, e.g. having from 1 wt.% to 99 wt. % of one isomer with respect to the other isomer. Alternatively, the compounds of the invention may be a substantially pure (E) or (Z) form, e.g. 99.6% or above).

In any aspect of the present invention, the compounds of formula I may be selected from the group consisting of:



, salts thereof and solvates thereof.

In particular, ferulic acid and/or a derivative thereof may be a hydroxycinnamic acid, a type of organic compound. Hydroxycinnamic acids are a class of hydroxy derivatives of cinnamic acid and include coumaric acids, caffeic acid, diferulic acids, etc. Alternatively the ferulic acid and/or a derivative thereof (i.e. the compound of formula I) may be a vanillin derivative. The ferulic acid and/or a derivative thereof used in the present invention may be isolated from nature and/or may be chemically synthesised. Particularly, ferulic acid derivatives used in the present invention may be chemically synthesised from ferulic acid or vanillin according to any method known in the art, or as provided in Examples 3 and 4 below, and/or variations thereof.

More in particular, at least one ester of ferulic acid and/or a derivative thereof may also be used according to any aspect of the present invention.

Ferulic acid and/or a derivative thereof may be extracted from a plant or part thereof according to any method of extraction known in the art. Non limiting examples of plants include coffee, onion, Japanese radish, lemon, Angelicae radix, Cnidii Rhizoma, goldthread, asafetida, sugarcane, corn, barley and rice, with rice being particularly preferred. The term "rice" as used herein means raw or dry seeds of rice (Oryza sativa LINNE). More in particular, the plants from which ferulic acid and/or an ester thereof may be extracted may include but are not limited to Garcinia Mangostana, Ferula Communis, Smallanthus Sonchifolius, Centaurium Erythraea Phaseolus Vulgaris and the like. Esters of ferulic acid and/or a derivative thereof include those obtained by conversion upon extraction or fractionation of those originally contained in a natural substance, particularly, a plant; and to the chemically modified products thereof. For example, rice bran oil obtained from rice bran is separated using hydrous ethanol and hexane at room temperature under a weak alkaline condition and ferulate ester is available in the hydrous ethanol fraction. Ferulic acid can be obtained by hot hydrolysis of the ferulate ester obtained with sulfuric acid under pressure, followed by purification. It can also be obtained by culturing bacteria (Pseudomonas) in a broth containing a clove oil obtained by steam distillation of buds and leaves of Syzygium aromaticum MERRILL et PERRY or a broth containing eugenol available by purification of the clove oil, followed by separation of the resulting culture broth and purification. Chemical synthesis of ferulic acid and/or a derivative thereof may be carried out, for example, by condensation reaction of vanillin and/or a derivative thereof and malonic acid according to the method disclosed in Roger Adams et al (1952). Ferulic acid and/or a derivative thereof may have steric isomers. Any one of them may be used. A mixture of the isomers may also be usable.

In any aspect of the present invention, a plant or part thereof may comprise any plant or portion thereof, including but not limited to leaves, flowers, roots, seeds, pods, stems, fruits, seed coats, buds, and other parts of a plant. For the purpose of the present invention, the term "plant" also includes "herb". A part thereof may be a combination of different parts of the plant and/or may comprise different parts of different plants. In particular, the plant or part thereof may be useful in obtaining a high percentage of ferulic acid and/or a derivative thereof.

Alternatively, according to any aspect of the present invention, the compound of formula (I) may exclude ferulic acid and/or vanillin. In one embodiment, the use of a compound of formula (I) according to any aspect of the invention may be with the proviso that the compound of formula I is not ferulic acid, a salt thereof or a solvate thereof, and/or that the compound of formula I is not vanillin, a salt thereof or a solvate thereof.

According to a further aspect of the invention, there is provided a compound of formula II:

(Π) a salt thereof or a solvate thereof, wherein

R4 is CHO or CH=CHC(0)OR5, wherein

when R4 is CH=CHC(0)OR5, the Carbon-Carbon double bond is in the (E) configuration and:

R1 is H or N0 ₂ ;

R2 is H, CH(CH ₃ ) ₂ , S02-4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H4-OCH ₃ provided that R2 is not H when R1 is H; and

R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ provided that R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably CH ₂ CH ₂ CH ₃ when R2 is H, and

when R4 is CHO:

R2 is H; and

R1 is NHC ₂ H ₄ OH, NHC(=NH)NH ₂ or NH ₂ . Particularly, there are provided the following compounds, i.e. the compound of formula II may be:

; or more preferably

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solvate thereof. In one aspect of the present invention there is provided a compound of formula (II) or a salt or a solvate thereof as described herein, for use in medicine

In any aspect of the present invention, Ferulic acid and/or a derivative thereof, i.e. a compound of formula I, may be used as an effective and efficient neuraminidase inhibitor. In particular, these compounds may be capable of 100%, 90%, 80%, 75%, 60%, 50%, 45%, 40%, 35% inhibition of the neuraminidase in the microbes infecting a subject. More in particular, ferulic acid and/or a derivative thereof may be capable of at least 10% neuraminidase inhibition of the microbe. The ferulic acid and/or a derivative thereof may be capable of completely hindering the activity of neuraminidase or may be capable of only reducing the activity of neuraminidase by 5%.

Neuraminidase has functions that aid in the efficiency of virus release from cells. Neuraminidase cleaves terminal neuraminic acid (also called sialic acid) residues from carbohydrate moieties on the surfaces of infected cells. This promotes the release of progeny microbes from infected cells. In particular, neuraminidase also cleaves sialic acid residues from proteins, for example viral proteins, thus preventing aggregation of viruses. Administration of inhibitors of neuraminidase as a treatment thus may be capable of limiting the severity and spread of several microbial infections. As used herein, a microbial infection may be an infection caused by at least one microbe/ microorganism which includes, but is not limited to a bacterium, an archaebacterium, a virus, a yeast, a fungus a protist and the like. In particular, the microbe may be selected from the non-limiting group consisting of Vibrio cholerae, Clostridium perfringens, Streptococcus pneumoniae, Arthrobacter sialophilus, influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, equine influenza virus, Sendai virus and the like. More in particular, the microbial infection according to any aspect of the invention may be from at least one virus. The virus may be at least one influenza virus.

The term "influenza virus" as used in the context of the invention includes all subtypes of influenza viruses that fall under the categories of "avian influenza viruses" and "human influenza viruses". The influenza viruses may be influenza A, B or C viruses. In particular, the influenza A virus may include but are not limited to H1N1 , H3N2, H5N1 , H5N2, H5N8, H5N9, H7N2, H7N3, H7N4, H7N7, H9N2 and the like. Particularly, the at least one influenza virus according to any aspect of the invention may be of type H1N1 and/or H1N5. More particularly, the at least one influenza virus according to any aspect of the invention may be of type H1 1.

When the at least one influenza virus according to any aspect of the invention is of type H1N1, the compound of formula I may be selected from the group consisting of:

, salts thereof and solvates thereof.

Compounds of formulas I and II mentioned in the above-mentioned aspects of the invention may thus be utilised in medical treatment or a method of medical treatment. Accordingly, in a further aspect of the present invention there is provided a compound of formula (I) or a salt or a solvate thereof, or a compound of formula (II) or a salt or a solvate thereof, both as described herein, for use in the treatment of at least one microbial infection.

In yet another aspect of the present invention there is provided a method of treatment of at least one microbial infection comprising administering a compound of formula (I) or a salt or a solvate thereof, or a compound of formula (II) or a salt or a solvate thereof, both as described herein, to a subject in need thereof.

It will be appreciated that the amount administered to a subject is a therapeutically effective amount of the compound, e.g. of formula (I) or of formula (II).

For the avoidance of doubt, in the context of the present invention, the term "treatment includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment of patients which are susceptible to the relevant disease states.

The terms "subject", "patient and "patients" as used in the context of the invention refers to any animal, including a human, non-human animal, plant or insect that may be infected by a microorganism. In particular, the subject is any animal, including humans and non- human animals that may be infected by a microorganism against which a compound of formula I may be active, i.e. have an inhibitory effect.

The term "effective amount" refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the bacterial infection). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

Likewise, the amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person. For the avoidance of doubt, references herein to compounds of formula I include, where the context permits, references to any of compounds of formula I and II. Further, references to any of compounds of formula I and II include references to such compounds per se, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

Thus, there is provided in a further aspect of the invention, a pharmaceutical formulation including a compound of formula (I) or a salt or a solvate thereof, or a compound of formula (II) or a salt or a solvate thereof, both as described herein, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, the formulation being for use in the treatment of at least one microbial infection. The pharmaceutical formulation may optionally comprise one or more other therapeutic agents.

Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration. Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w w) active ingredient; from 0 to 99% (w w) diluent or filler; from 0 to 20% (w w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer. A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of the compound, e.g. a compound of formula I or formula II.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

Some compounds of formula I and formula II may be known and/or may be commercially available. Other compounds of formula I and formula II (e.g. that are not known, or not commercially available) may be prepared in accordance with techniques as described hereinafter.

In a further aspect of the invention there is provided a process for the preparation of a compound of formula II or a salt or a solvate thereof as described herein, which process comprises:

(i) for compounds of formula (II) when R4 is CH=CHC(0)OR5, R2 is H and R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , reacting a compound of formula (lla):

in which R6 is H or N0 ₂ and R7 is H, with a compound of formula R8-OH in the presence of an acid, wherein R8 is CH2CH2CH2CH3, or more preferably CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ ;

(ii) for a compound of formula (II) when R4 is CH=CHC(0)OR5, R2 is CH(CH ₃ ) ₂ and R5 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH2CH2CH3, reacting a compound of formula (lla), in which R6 is N0 ₂ and R7 is CH ₂ CH ₂ CH ₂ CI-I ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , with an isopropyl halide in the presence of a base and a phase transfer catalyst;

(iii) for compounds of formula (II) when R4 is CH=CHC(0)OR5, R2 is S0 ₂ -4-C ₆ H ₄ F or S0 ₂ -4-C ₆ H ₄ -OCH ₃ and R5 is CH ₂ CH ₂ CH ₂ CH _3l or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , reacting a compound of formula (lla), in which R6 is H and R7 is CH ₂ CH ₂ CH ₂ CH ₃ , or more preferably H, CH ₂ CH ₃ or CH ₂ CH ₂ CH ₃ , with a compound of formula R9-X, wherein R9 is S0 ₂ -4-C ₆ H ₄ F or S0 ₂ - -C ₆ H ₄ -OCH ₃ and X is a halide;

(iv) for a compound of formula (II) when R4 is CHO and R1 is NHC ₂ H ₄ OH, reacting a compound of

in which R1 is a halide, with 2-ethanolamine;

(v) for a compound of formula (II) when R4 is CHO and R1 is NH ₂ ,

reacting a compound of formula (lib), in which R1 is N0 ₂ , with hydrazine hydrate in the presence of glyoxylic acid; and

(vi) for a compound of formula (II) when R4 is CHO and R1 is NHC(=NH)NH ₂ , reacting a compound of formula (lib), in which R1 is NH ₂ , with cyanamide. Substituents, such as R1 , R2, R3, R4, R5 in final compounds of formula I (or precursors thereto and other relevant intermediates) may be modified one or more times, after or during the processes described above by way of methods that are well known to those skilled in the art. Examples of such methods include substitutions, reductions (e.g. carbonyl bond reductions in the presence of suitable and, if necessary, chemoselective, reducing agents such as LiBH ₄ or NaBH ₄ ), oxidations, alkylations, acylations, hydrolyses, esterifications, and etherifications. The precursor groups can be changed to a different such group, or to the groups defined in formula I, at any time during the reaction sequence. The same applies to compounds of formula II, precursors thereto and other relevant intermediates.

Compounds of the invention may be isolated from their reaction mixtures using conventional techniques (e.g. recrystallisation, column chromatography, preparative HPLC, etc.).

In the processes described above and hereinafter, the functional groups of intermediate compounds may need to be protected by protecting groups. The protection and deprotection of functional groups may take place before or after a reaction in the above- mentioned schemes.

Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter. For example, protected compounds/intermediates described herein may be converted chemically to unprotected compounds using standard deprotection techniques.

The type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.

The use of protecting groups is fully described in "Protective Groups in Organic Chemistry", edited by J W F McOmie, Plenum Press (1973), and "Protective Groups in Organic Synthesis", 3 ^rd edition, T.W. Greene & P.G.M. Wutz, Wiley-lnterscience (1999).

A person skilled in the art will appreciate that the present invention may be practised without undue experimentation according to the method given herein. The methods, techniques and chemicals are as described in the references given or from standard protocols. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

7

26

EXAMPLES

EXAMPLE 1

Cytotoxicity Test

The determination of drug cytotoxicity concentration towards Madin-Darby canine kidney (MDCK) cell lines (CC ₅₀ ) was done using 9 different concentrations for each compound. These concentrations were prepared by 2 fold dilutions starting from 100 pg/ml.

200 μΙ of MDCK cells 0.5 x 10 ⁵ cells/ml were seeded in each well of 96 well-plate and incubated in humidified C0 ₂ incubator at 37°C overnight to produce monolayer cell lines. A potential neuraminidase inhibitor, ferulic acid was dissolved in 100% DMSO and filtered using 0.22 μΜ filters. Series of dilutions 100 pg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/rnl, 6.25 pg/ml, 3.125 pg/ml, 1.5625 pg/ml, 0.78125 pg/ml and 0.390625 pg/ml were then prepared and used in the cytotoxity test. All the compounds were incubated with cells in humidified C0 ₂ at 37°C for 24h. The WST-1 cell proliferation kit (Roche) was used to determine the cells viability and results were read by 96 well-plate reader (450 nm). The data obtained were analyzed using Prism Ver.5 (GraphPad Software) to determine the cytotoxicity concentration CC ₅₀ of each compound towards the cells line.

The ferulic acid data indicated no toxicity with CC5 ₀ 5.639 x 10 ¹⁸ pg/ml as shown in Figure 5.

EXAMPLE 2

Viral Inhibition Assay

Viral inhibition assays have been done using ferulic acid against A chicken/Malaysia/5858/2004 H5N1 and A/Malaysia/Muar 33/2009 H1N1. In the assay for A/Malaysia/Muar 33/2009 H1N1 , the results of which are shown in Figure 2, 4 different concentrations were used based on ferulic acid CC ₅₀ value (5.639 x 10 ¹⁸ pg/ml).

Table 1 showed that ferulic acid can inhibit 90% of viral propagation at 10 pg/ml. For A/chicken/Malaysia/5858/2004(H5N1) the results of which are shown in Figure 4, 10 different concentrations were used with the highest concentration of 100 μg ml as seen in Table 2. From Figure 3, it can be seen that the IC ₅₀ for A/Malaysia/Muar 33/2009 H1 1 was 8.21pg/ml and there was no IC ₅₀ for A/chicken/Malaysia/5858/2004(H5N1) that was analyzed using Prism Ver.5 (GraphPad Software).

For viral inhibition assay of A/Malaysia/Muar 33/2009 H1 N1 , the ferulic acid was dissolved in 100% DMSO and filtered with 0.45 μηι membrane filters but for viral inhibition assay of A/chicken/Malaysia/5858/2004(H5N1), ferulic acid was dissolved in 25% DMSO and filtered with 0.22 μιτι membrane filters. There was a possibility that PHDM5 did not dissolve properly in 25% DMSO and could not be obtained through the 0.22 m filters.

Ferulic acid Number of Plaques Percentage of Inhibition (%) concentrations

10 Mg/ml 10 90

1 Mg/ml 100 0

0.1 Mg/ml 100 0

0.01 Mg/ml 100 0

Virus non-treated 100 0

TABLE 1 The number of A/Malaysia/Muar 33/2009 H1N1 plaques existed for each PHDM5 concentration. Percentage of inhibition by PHDM5 treatment towards the A/Malaysia/Muar 33/2009 H1N1 virus is 90% at 10 Mg/ml and no viral inhibition occurred for the rest of concentrations

Ferulic Acid Number of Plaques Percentage of concentrations inhibition (%)

(Mg/ml) (Plate A) (Plate B) (Plate A) (Plate B)

100 22 15 45 51.6

50 43 44 -7.5 -41.9

25 18 51 55 -64.5

12.5 36 37 10 -19.4

6.25 46 35 -15 -12.9

3.125 23 27 42.5 12.9

1.56 36 33 10 -6.5

0.78 33 26 17.5 16.1

0.39 30 27 25 12.9

0.2 40 37 0 -19.4

Virus 40 31 0 0 non-treated

TABLE 2 The number of A/chicken/Malaysia/5858/2004(H5N1) plaques existed and percentage of inhibition for each concentration of Ferulic Acid for duplicate assays

ICso experimental protocols used in Examples 3 and 4

Method (MUNANA Assay modified from Potier er a/., 1979).

The procedure of H1 N1 neuraminidase assay was carried out by preparing the reaction mixture containing assay buffer, tested samples (at concentration 125 Mg/mL in 12.5 μΙ of DMSO-Buffer), and constant 0.3 unit of neuraminidase were pre-incubated at 37°C for 30 minutes with 200 rpm. Then after the addition of 100 μΜ of substrates, the reaction assays were incubated at 37°C for 60 minutes with 200 rpm and then add 100 μΙ of stop solution. The assays were triplicated. The 15 000067

28 fluorescence intensities of NANA product were measured by Modulus Microplate

Reader with UV optical kit at λ 340/440 nm.

M. Potier, L.M., M. Belisleb, L. Dallaireb, and S. B. Melancon, Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-a-lmage -N-acetylneuraminate) substrate. Analytical Biochemistry, 1979. 94 (2): p. 287-296

EXAMPLE 3

Synthesis of Ferulic acid derivatives from Ferulic acid, with NMR data and IC50 data Provided below are some synthetic routes that may be used to obtain ferulic acid derivatives from ferulic acid. NMR, IC ₅₀ and other characterising data are also provided. The IC ₅₀ experimental protocols are as specified above. Table 3 at the end of this example provides a summary of the structures and IC50 values of each compound as analyzed using Prism Ver.5 (GraphPad Software).

FAD000. (£)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid was purchased from Sigma without further purification.

¹ H-NMR (CDCI ₃ ) δ 3.81 (3H, s, OCH ₃ ), 6.36 (1H, d, J trans - 16 Hz, Ar-CH=), 6.79 (1 H, d, J ortho = 8 Hz, Ar-H), 7.08 (1 H, dd, J _ortho = 8.5 Hz, J _meta = 2 Hz, Ar-H), 7.28 (1H, d, J _meta = 1.5 Hz, Ar-H), 7.49 (1 H, d, J _frans = 16 Hz, =CHCO)

Please refer to Fig. 6a for IC5 ₀ graph.

Log IC5 ₀ (GraphPad) = 1.436 (R ² = 0.9790)

IC ₅ o = 140 pM. -nitrophenyl)acrylic acid.

Ferulic acid (FAD000, 41 mmol) was dissolved into 58 mL of glacial acetic acid while warmed up, and then cooled it down at a room temperature. Fuming nitric acid (1.45 mL) was carefully added into the cool solution, and stirred up over a period of 60 minutes to get a brown mixture. The mixture was dropped gently into a bulk volume of ice water until the yellow solid precipitated out. These were filtered, washed with water and allowed to dry and then purified using column chromatography (Silica gel, n-hexane: EtOAc (2:2)) Yellow powder; yield ; 82%; _ crrf ¹ (KBr), 1618 (C=C), 1532 (C=0); ¹ H-NMR (DMSO-

De) δ 3.91 (1H, s, OH), 3.94 (3H, s, OCH ₃ ), 6.60 (1 H, d, J tra s - 16 Hz, Ar-CH=), 7.57 (1 H, d, J t^s = 16 Hz, Ar-CH=), 7.64 (1 H, d, J ^ = 1.5 Hz, Ar-H), 7.75 (1 H, d, J .ζ> Hz), 12.34 (1 H, br, s, COOH); δ ₀ (DMSO) 56.11 (OCH ₃ ), 111.51 (C=C), 115.96 (Ar-C), 116.13 (Ar-C), 123.24 (Ar-C), 126.22 (Ar-C), 144.94 (Ar-C), 148.35 (C=C), 149.45 (Ar-C), 168.56 (COOH).

Please refer to Fig. 6b for IC ₅₀ graph.

Log IC ₅₀ (GraphPad) = 1.483 (R ² = 0.9962)

IC ₅₀ = 127 pM.

General procedure for the preparation of FAD002 and FAD003

FAD002 and FAD003 were collected during purification of FAD001 using column chromatography. The ortho substitution toward 3-methoxy was yielded as the minor product.

67

30

FAD002. (E)-3-(4-hydroxy-3-methoxy-2-nitrophenyl)acrylic acid

Yellow powder; yield 9%.; __ _max / cm ¹ (KBr), 3469 (OH), 1618 (C=C), 1532 (C=0);

¹ H-NMR (CDCI ₃ ) δ 3.95 (3H, s, OCH ₃ ), 6.03 (1H, s, OH), 6.97 (1H, d, J ^ = 8.5 Hz, Ar- H), 7.14 (1H, dd, J _ortho = 10 Hz, J _a , = 1.5 Hz, Ar-H), 7.51 (1H, d, J ^ = 14 Hz, Ar- CH=), 7.95 (1H, d, J ^ = 14 Hz, =CHCO); 5 _C (DMSO) 56.30 (OCH ₃ ), 112.72 (C=C), 116.24 (Ar-C), 116.24 (Ar-C), 122.02 (Ar-C), 126.38 (Ar-C), 135.50 (Ar-C), 140.76 (C=C), 148.62 (Ar-C), 151.73 (COOH).

Please refer to Fig. 6c for IC50 graph.

Log IC ₅₀ (GraphPad) = 2.070 (R ² = 0.9958)

IC ₅₀ = 489 μΜ.

FAD003. (E)-3-{3-amino-4-hydroxy-5-methoxyphenyl)acrylic acid

Dark brown powder; yield 9%; El _max / cm ^'1 (KBr), 1618 (C=C), 1532 (C=0); ¹ H-NMR

(DMSO) δ 3.81 (3H, s, OCH ₃ ), 6.27 (1 H, d, J _(rans = 15.5 Hz, =CHCO), 7.16 (1 H, s, Ar-H), 7.23 (1 H, d, J te„ _s = 16 Hz), 7.70 (1H, s, Ar-H).

Please refer to Fig. 6d for IC ₅₀ graph.

Log IC5 ₀ (GraphPad) = 1.490 (R ² = 0.

IC ₅₀ = 147 μΜ.

General procedure for the preparation of ferulic acid ester.

FAD000 or FAD001 (4.18 mmol) was dissolved into the corresponding alcohol (8 mL) while cooling down the rounded bottom flask in the ice water. Concentrated H ₂ S0 ₄ was added carefully into the cool solution and allowed to reflux at 60-70°C for 24 hours. The mixture was neutralized with 25 mL of NaHC0 ₃ 10% and then extracted using ethyl acetate (2x 25 mL) followed by washing it with 2x 25 mL of water. The ethyl acetate was dried over anhydrous MgS0 ₄ and then concentrated under reduced pressure. The crude product was subjected to the preparative thin layer chromatography (Silica gel, n-hexane: EtOAc (2:2)) to afford the ester product.

FAO004. (£)-methyl 3-(4-hydroxy-3-methoxyphenyl)acrylate

Colorless semi solid; Yield 61%; 0 _max / cm ^"1 (KBr), 1618 (C=C), 1532 (C=0);

¹ H-NMR (CDCI ₃ ) δ 3.79 (3H, s, OCH ₃ ), 3.92 (3H, s, COOCH ₃ ), 5.89 (1 H, s, OH), 6.29 (1 H, of, J _irans = 16 Hz, Ar-CH=), 6.91 (1H, d, J _orth o = 8.5 Hz, Ar-H), 7.02 (1H, d, J _meta = 2 Hz, Ar-H), 7.06 (1 H, d, J _meta = 2 Hz, Ar-H), 7.08 (1 H, d, J _meta = 2 Hz, Ar-H), 7.62 (1 H, d, J trans = 6 Hz, =CHCO); 6 _C (DMSO) 51.69 (COOCH ₃ ), 56.16 (OCH ₃ ), 111.75 (Ar-C), 114.62 (C=C), 115.97 (Ar-C), 123.58 (Ar-C), 125.96 (Ar-C), 145.57 (Ar-C), 148.39 (C=C), 149.98 (Ar-C), 167.56 (COOCH ₃ ).

Please refer to Fig. 6e for IC ₅₀ graph.

Log IC50 (GraphPad) = 1.600 (R ² = 0.9984)

IC ₅₀ = 191 μΜ. -ethyl 3-(4-hydroxy-3-methoxyphenyl)acrylate

Colorless semi solid; Yield 68%; 0 _max / cm ^"1 (KBr), 1618 (C=C), 1532 (C=0);

¹ H-NMR (CDCI ₃ ) δ 1.33 (3H, t, J = 7 Hz, CH ₃ ), 3.92 (3H, s, OCH ₃ ), 4.25 (2H, q, J = 7 Hz, CH ₂ ), 5.85 (1 H, s, OH), 6.28 (1 H, d, J ^ = 16.5 Hz, Ar-CH=), 6.91 (1 H, d, J _ortho = 8.5 Hz, Ar-H), 7.03 (1H, d, J _meta = 2 Hz, Ar-H), 7.07 (1 H, dd, J _or tho = 9 Hz, J _meta = 2.5 Hz, Ar- H), 7.61 (1 H, d, J tens = 16 Hz, =CHCO). δ ₀ (DMSO) 14.72 (OCH ₂ CH ₃ ), 56.15 (OCH ₂ CH ₃ ), 60.15 (OCH ₃ ), 11 1.67 (Ar-C), 1 15.08 (C=C), 1 15.95 (Ar-C), 123.56 (Ar-C), 126.04 (Ar-C), 145.37 (Ar-C), 148.39 (C=C), 149.78 (Ar-C), 167.09 (COOC ₂ H ₅ ).

Please refer to Fig. 6f for IC50 graph.

Log IC ₅₀ (GraphPad) = 1.957 (R ² = 0.9965)

IC ₅₀ = 407 μΜ

FAD006. (£)-propyl 3-(4-hydroxy-3-methoxyphenyl)acrylate

Colorless semisolid; Yield 53%,

¹ H-N R (CDCI ₃ ) δ 0.89 (2H, m, CH ₂ ), 0.99 (3H, t, J = 7.5 Hz, CH ₃ ), 3.92 (3H, s, OCH ₃ ), 4.15 (2H, t, J = 7 Hz, OCH ₂ ), 5.85 (1 H, s, OH), 6.29 (1 H, cf, J _ftwls = 16 Hz, Ar-CH=), 6.91 (1 H, d, J ortho = 8.5 Hz, Ar-H), 7.03 (1 H, d, J _meta = 2 Hz, Ar-H), 7.07 (1 H, dd, J _ortho = 8 Hz, J _meta = 1 .5 Hz, Ar-H), 7.61 (1 H, d, J _tra ns = 16 Hz, =CHCO). ); 6 _C (DMSO) 10.80 (OCHzCHzCHa), 22.14 (OCH ₂ CH ₂ CH ₃ ), 56.16 (OCH ₃ ), 65.67 (OCH ₂ CH ₂ CH ₃ ), , 1 1 1 .64 (Ar-C), 1 14.98 (C=C), 1 15.94 (Ar-C), 123.60 (Ar-C), 126.05 (Ar-C), 145.40 (Ar-C), 148.39 (C=C), 149.78 (Ar-C), 167.19 (COOC ₃ H7).

Please refer to Fig. 6g for IC ₅₀ graph.

Log IC50 (GraphPad) = 2.028 (R ² = 0.9972)

IC ₅₀ = 451 μΜ -methyl 3-{4-hydroxy-3-methoxy-5-nitrophenyl)acrylate

Yellow powder; yield 18%; S _max / cm ¹ (KBr), 3424 (OH), 1718 (C=0), 1540 (C=C);

¹ H-NMR (CDCI ₃ ) δ 3.82 (3H, s, COOCH ₃ ), 3.99 (3H, s, OCH ₃ ), 5.51 (1/2H, br, s, OH), 6.39 (1 H, d, J trans = 16 Hz, Ar-CH=), 7.20 (1 H, d, J _mefa = 1.5 Hz, Ar-H), 7.60 (1 H, d, J trans = 16.5 Hz, =CHCO), 7.86 (1 H, d, J _m eta = 1 -5 Hz, Ar-H), 10.92 (1/2H, br, s, OH); δ ₀ (DMSO) 51.88 (COOCH ₃ ), 57.18 (OCH ₃ ), 110.59 (Ar-C), 117.19 (C=C), 122.11 (Ar-C), 123.79 (Ar-C), 132.81 (Ar-C), 137.57 (Ar-C), 140.55 (C=C), 143.75 (Ar-C), 167.26 (COOCH ₃ ).

Please refer to Fig. 6h for IC50 graph.

Log IC50 (GraphPad) = 2.197 (R ² = 0.9871)

IC ₅₀ = 621 μΜ -methoxy-5-nitrophenyl)acrylate

Yellow powder; yield ; m.p.; GW cm ¹ (KBr), 3465 (OH), 1614 (C=C), 1536 (C=0); ¹ H-NMR (CDCI ₃ ) δ 1.27 (3H, t, J = 7 Hz, CH ₃ ), 3.91 (3H, s, OCH ₃ ), 4.21 (2H, q, J = 8 Hz, OCH ₂ ), 6.32 (1 H, d, J trans = 16 Hz, Ar-CH=), 7.19 (1 H, s, Ar_H), 7.52 (1 H, d, J trans = 16 Hz, =CHCO), 7.79 (1 H, s, Ar-H), 10.85 (1 H, br, s, OH); 6 _C (DMSO) 14.67 (COOC 2CH3), 57.27 (COOCH ₂ CH ₃ ), 60.42 (OCH ₃ ), 113.94 (Ar-C), 117.98 (C=C), 118.34 (Ar-C), 137.70 (Ar-C), 143.42 (Ar-C), 150.47 (Ar-C), 166.73 (COOCH ₂ CH ₃ ).

Please refer to Fig. 6i for IC50 graph.

Log IC ₅₀ (GraphPad) = 1.818 (R ² = 0.9759)

ICso = 246 μΜ

FAD010. (£)-propyl 3-(4-hydroxy-3-methoxy-5-nitrophenyl)acrylate

Yellow powder; yield 67%; m.p.; S _max / cm ^"1 (KBr), 3428 (OH), 1699 (C=0), 1532 (C=C);

¹ H-NMR (CDCI ₃ ) δ 1 .00 (3H, t, J = 7.5 Hz, CH ₃ ), 1.73 (2H, m, CH ₂ ), 3.99 (3H, s, OCH ₃ ), 4.18 (2H, t, J = 6.5 Hz, OCH ₂ ), 6.39 (1 H, d, J _frans = 15 Hz, Ar-CH=), 7.27 (1 H, d, J _meta = 2 Hz, Ar-H), 7.59 (1 H, d, J _trans = 15 Hz, =CHCO), 7.86 (1 H, d, J _meta = 1.5 Hz, Ar-H), 10.92 (1 H, br, s, OH) ); 5 _C (DMSO) 10.80 (OCH ₂ CH ₂ CH3), 22.10 (OCH2CH ₂ CH ₃ ), 57.33 (OCH ₃ ), 65.93 (OCH ₂ CH ₂ CH ₃ ), 1 14.18 (Ar-C), 1 18.21 (C=C), 125.13 (Ar-C), 130.02 (Ar- C), 137.78 (Ar-C), 143.35 (C=C), 150.28 (Ar-C), 166.79 (COOC3H7).

Please refer to Fig. 6j for IC ₅₀ graph.

Log IC ₅₀ (GraphPad) = 1.792 (R ² = 0.8929)

IC ₅₀ = 220 μΜ -butyl 3-(4-hydroxy-3-methoxy-5-nitrophenyl)acrylate

¹ H-NMR (CDCU) δ 0.97 (3H, t, J = 7 Hz, CH ₃ ), 1.45 (2H, m, CH ₂ ), 1.69 (2H, m, CH ₂ ), 3.99 (3H, s, OCH ₃ ), 4.22 (2H, f, J = 7 Hz, OCH ₂ ), 6.39 (1H, d, J trans = 16 Hz, Ar-CH=), 7.27 (1H, d, J meta = 1.5 Hz, Ar-H), 7.58 (1H, of, J trans = 16 Hz, =CHCO), 7.86 (1H, d, J meta = 1.5 Hz, Ar-H), 10.92 (1H, br, s, OH)

Log IC50 (GraphPad) = 5.669 (R ² = 0.9434)

IC50 = 1 ,580,358 μΜ (1.581 M) - no figure provided.

FAD012. (£)-ethyl 3-(4-isopropoxy-3-methoxy-5-nitrophenyl)acrylate

FAD009 (1.87 mmol) and Na ₂ C0 ₃ ( mmol) was mixed in 5 mL of DMF and then added by the mixture of isopropyl bromide (3.74 mmol), tetrabutylammonium Iodide (TBAI; 3.74 mmol) in 5 mL of DMF. The reaction was carried out at room temperature for 6 hours and then the mixture was diluted with 50 mL of water. The dilute mixture was extracted using 2x 50 mL of ethyl acetate and followed by washing it using 2x 100 mL of water. The ethyl acetate phase was dried over anhydrous MgS0 ₄ and then concentrated under reduced pressure. The crude product was then subjected to the preparative thin layer chromatography (silica gel F ₂₅₄ , n-hexane:ethyl acetate (50:50)) to afford the pure product.

Dark brown powder; yield 9%; B _max / cm ^"1 (KBr), 3436 (OH), 1597 (C=0), 1552 (C=C); ¹ H-NM (CDCI3) δ 1.18 (3H, t, J = 7 Hz, CH ₃ ), 1.23 (6H, d, J = 7 Hz, (CH ₃ )), 1.24 (1 H, m, CH), 3.67 (3H, s, OCH ₃ ), 4.13 (2H, q, J = 8 Hz, OCH ₂ ), 6.06 (1 H, d, J trans = 16 Hz, Ar- CH=), 6.82 (1 H, s, Ar_H), 7.40 (1 H, d, J ^ = 16 Hz, =CHCO), 7.59 (1 H, d, J^ts = 1.5, Ar-H); 5 _C (DMSO) 13.96 (COOCH ₂ QH ₃ ), 19.68 (CH(CH ₃ ) ₂ ), 23.53 (OCH ₃ ), 57.99 (COOCH ₂ CH ₃ ).

Please refer to Fig. 6k for IC5 ₀ graph.

Log ICso (GraphPad) = 1.447 (R ² = 0.9573)

IC ₅₀ = 90 μΜ

General procedure for the preapartion of benzenesulfanylatedferulic acid.

FADOOO (2.57 mmol) was dissolved in 3 mL of pyridine at 0°C and then dropped carefully by 4-fluorobenzene-1-sulfonyl chloride. The mixture was stirred over a period of 5 hours until the white precipitates were coming and then neutralized using HCI 10%. The precipitates were then filtered, washed with water and recrystallised from chloroform to afford the pure product.

FAD013. (E)-3-(4-(4-fluorobenzenesulfonoyl)-3-methoxyphenyl)acrylic acid

White powder; Yield 24%; El _max / cm ^'1 (KBr) 3432 (OH), 1691 (C=0), 151 1 (C=C);

¹ H-NMR (CDCI ₃ ) δ 3 94 (3H, s, OCH ₃ ), 6.29 (1 H, d, J trans = 16, Ar-CH=), 6.93 (1 H, d, J ortno = 8 Hz, Ar-H), 7.58 (1 H, d, J _meta = 1 .5, Ar-H), 7.1 1-7.24 (4H, m, Ar-H), 7.70 (1 H, d, J _t r _ans = 16 Hz, =CHCO), 7.90 (1 H, dd, J _ort „ ₀ = 8.5, J _meta = 2.5, Ar-H). 5 _C (DMSO) 21 .12 (CH ₃ ), 56.27 (OCH ₃ ), 1 12.31 (Ar-C), 1 12.93 (C=C), 121 .58 (Ar-C), 124.16 (Ar-C), 125.84 MY2015/000067

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(Ar-C), 128.63 (Ar-C), 129.70 (Ar-C), 135.12 (Ar-C), 135.93 (Ar-C), 139.07 (Ar-C), 139. (Ar-C), 143.14 (C=C), 152.03 (Ar-C), 180.69 (COOH).

Please refer to Fig. 6I for IC ₅₀ graph.

Log ICso (GraphPad) = 2.009 (R ² = 0.9982)

IC ₅₀ = 289 μΜ -3-(4-(4-methoxy benzenesulf onoyl)-3-methoxyphenyl)acry lie acid

White powder; Yield 54%; m.p. ; EW cm ^-1 (KBr) 3007 (OH), 1728 (C=0), 1630 (C=C);

¹ H-NMR (CDCI ₃ ) δ 3.88 (3H, s, OCH ₃ ), 3.89 (3H, s, OCH ₃ ), 6.39 (1 H, d, J _tens = 16 Hz, =CHCO). 6.96-7.82 (8H, m, Ar-H). ); δ ₀ (DMSO) 56.30 (OCH ₃ ), 56.41 (OCH ₃ ), 112.88 (Ar-C), 115.07 (C=C), 121.54 (Ar-C), 124.16 (Ar-C), 124.19 (Ar-C), 126.61 (Ar-C), 131.16 (Ar-C), 134.53 (Ar-C), 139.10 (Ar-C), 139.66 (Ar-C), 141.14 (Ar-C), 143.04 (Ar-C), 145.98 (C=C), 152.02 (Ar-C), 164.69 (COOH).

Please refer to Fig. 6m for IC ₅₀ graph.

Log IC50 (GraphPad) = 2.196 (R ² = 0.9408)

IC ₅ o = 431 μΜ

Summary of structures and ICso values for compounds of Example 3

Table 3 below provides a summary of the structures of compounds of Example 3 and their IC ₅₀ values against H1 1 NA. Y2015/000067

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Example 4

VANOOO

VANOOO. 4-hydroxy-3-methoxybenzaldehyde was purchased from Sigma without further purification. White crystal; m.p. 80°C; ¹ H-NMR. (CDCI ₃ ). δ 3.97 (3H, s, OCH ₃ ), 6.16 (1 H, s, Ar-H) 7.65 (1 H, dd, J _ortfto =8.5 Hz, J _meta = 2.4 Hz, Ar-H), 8.23 (1 H, dd, J _orth0 = 6.8 Hz, J _meta = 1.8 Hz, Ar-H), 9.89 (1 H, s, H-aldehyde).

Please refer to Fig. 7a for IC ₅₀ graph.

Log IC ₅₀ (GraphPad) = 1.514 (R ² = 0.9935)

ΙΟ ₅₀ = 214 μΜ.

4-hydroxy-3-methoxy-5-nitrobenzaldehyde was purchased from Sigma- Aldrich, and was used as purchased.

Please refer to Fig. 7b for IC ₅₀ graph.

ICso = 272 μΜ.

VAN002

3-bromo-4-hydroxy-5-methoxybenzaldehyde was purchased from Sigma- Aldrich, and was used as purchased.

Please refer to Fig. 7c for IC ₅₀ graph.

IC ₅₀ = 166 μΜ.

VAN003

4-hydroxy-3-iodo-5-methoxybenzaldehyde was purchased from Sigma-Aldrich, and was used as purchased. Please refer to Fig. 7d for IC _S0 graph.

VAN004

VAN002 VAN004

Synthesis of VAN004 (Koley et a/., 2011 ).

VAN002 (9 mmol) was dissolved in DCM while adding 2-ethanolamine (13.5 mmol) and 1.8 ml_ of DIPEA. The mixture was stirred gently overnight at room temperature until the yellow precipitate formed and then followed by filtering them to collect the precipitate as the crude product. The filtrate in DCM was washed using water, 10% Na ₂ C0 ₃ to collect the DCM phase and followed by evaporating them in vacuo to get a yellow residue. This residue was combined with the earliest precipitate as VAN004.

Yellowish green powder; Yield 52%; m.p. 82-84°C; e cm ^"1 (KBr), 3328 (OH),

1675 (C=0), 1589 (C=C); 2.66 (2H, f, J = 5.5 Hz, CH ₂ OH), 3.43 (2H, r, J = 5.5 Hz, NHCH ₂ ), 3.73 (3H, s, OCH ₃ ), 7.21 (1 H, d, J _mefa = 1.5 Hz, Ar-H), 7.59 (1 H, d, J _meta = 2 Hz, Ar-H), 7.97 (1 H, s, H-aldehyde); 5 _C 43.70 ( NHCH ₂ ), 56.02 (OCH ₃ ), 15 000067

41

61.24 (CH ₂ OH), 61.98 (Ar-C), 79.64 (Ar-C), 88.36 (Ar-C), 109.36 (Ar-C), 134.71 (Ar-C), 148.90 (Ar-C), 160.39 (C-aldehyde).

Please refer to Fig. 7e for IC ₅₀ graph.

Log ICso (GraphPad) = 1.739 (R ² = 0.9643)

ICso = 259 μΜ.

VAN005

This compound is available from Sigma-Aldrich.

White powder; m. p. °C; H-NMR (DMSO-D ₆ ). δ 3.83 (3H, s, OCH ₃ ), 7.06 (1 H, d, J _meta = 1.8 Hz, Ar-H), 7.03 (1 H, d, J _me ta = 1.75 Hz, Ar-H), 9.79 (1 H, s, H-aldehyde) ; δο 56.39 (OCH ₃ ), 105.40 (Ar-C), 111.29 (Ar-C), 127.76 (Ar-C), 141.50 (Ar-C), 146.33 (Ar-C), 148.90 (Ar-C), 191.76 (C-aldehyde).

Please refer to Fig. 7f for IC ₅₀ graph.

Log ICso (GraphPad) = 1.058 (R ² = 0.9983)

IC ₅₀ = 68 μΜ.

VAN006

4-hydroxy-5-iodo-3-methoxy-2-nitrobenzaldehyde.

VAN003 _VAN006

Brown powder; Yield 43%; m. p. 152-155°C; ¹ H-NMR (CDCI ₃ ) δ _Η 4.03 (3H, s, CH ₃ ), 7.65 (1H, s, OH), 8.23 (1H, s, H), 9.90 (1H, s, C(O)H)); QTOF-MS m/z calcd for C ₈ H ₆ IN0 ₅ [M] ⁺ 323.0414, found 323.2141.

Please refer to Fig. 7g for IC ₅₀ graph.

ICso = 144 μΜ.

3-amino-4-hydroxy-5-methoxybenzaldehyde.

VAN001 VAN007

Nitro reduction of VAN001 (synthesis of VAN007) (Raju er a/., 2009)

Nitrovanillin (2.53 mmol) is dissolved in methanol/dichloromethane 2 ml, 1 :1 and 0.1 mg of the mixture of hydrazine hydrate-glyoxylic acid (10%). The mixture is stirred at room temperature. After the product is completed, it is filtered and the solid product is washed using cold methanol. The product is collected and then dried under fumehood. Light yellow powder; yield 55%; m.p. 273-276°C; H-NMR (DMSO-D ₆ ) δ _Η 3.91 (3H, s, CHs), 7.63 (1H, d, J _meta = 1.5 Hz, HC=C-C(0)H), 7.93 (1H, d, J _meta = 1.5 Hz, HC=C-OCH ₃ ), 8.66 (1H, s, C(O)H); QTOF-MS m/z calcd for C ₈ H ₉ N0 ₃ [M] ⁺ 167.1619, found 167.0923.

Please refer to Fig. 7h for IC ₅₀ graph.

ICso = 151 μΜ.

2-{5-formyl-2-hydroxy-3-methoxyphenyl)guanidine

VAN007 VAN008

Synthesis of VAN008 (Chand er a/., 1997)

Cyanamide (50.27 mmol) was mixed with water and then gradually added VAN007 in 2.5 mL of HCl 1 N. The mixture is then refluxed at 80-90°C for 4 hours. After completed, the mixture is cooled down at room temperature and followed by filtering. The formed precipitated is then collected, washed withcold methanol and then dried up under fumehood to afford the final product.

Brown powder; yield 48%; m.p. 284-286°C; B cm ^"1 (KBr), 3203 and 3077

(NH ₂ ), 1630 (C=C), 1548 (C=0); Ή-NMR (DMSO-D ₆ ) δ 1 .61 (2H, s, NH ₂ ), 3.66 (3H, s, OCH ₃ ), 6.82 (1H, d, J _meta = 2 Hz, Ar-H), 7.96 (1H, d, J _meta = 2 Hz, Ar-H), 9.39 (1H, s, H-aldehyde); δ ₀ 25.83 (C=N); 55.54 (OCH ₃ ), 116.08 (Ar-C), 135.38 (Ar-C), 156.33 (Ar-C), 165.45 (Ar-C), 175.16 (Ar-C), 188.30 (C-aldehyde).

Please refer to Fig. 7i for IC ₅₀ graph.

Log IC ₅₀ (GraphPad) = 1.022 (R ² = 0.9920)

IC ₅₀ = 50 μΜ.

Summary of structures and ICM values for compounds of Example 4

Table 4 below provides a summary of the structures of VAN000 - VAN0008 and their IC5 ₀ values against H1N1 NA.

Example 5

Selected compounds of Examples 3 and 4 were tested with oseltamivir carboxylate as a positive control in the:

• H1 N1 neuramidinase assay described above and used in Examples 3 and 4; and • H1 N1 assay described above in Example 2 (results provided below).

The obtained results are listed below, along with the SI values, in Table 5. The CC ₅₀ values of the compounds listed below were obtained using the methodology outlined in Example 1. The selectivity index (SI) was calculated using the IC ₅₀ of the H1N1 viral plaque reduction assay and is the ratio of the CC ₅₀ to IC ₅₀ values.

TABLE 5

The CC ₅₀ molar values presented in Table 5 are based upon an CC ₅₀ concentration of >100 pg/mL unless otherwise stated (VAN005 had a CC ₅₀ concentration of 24.16 pg/mL).

While FAD011 appears to be inactive in inhibiting neuraminidase, it nevertheless showed anti-viral activity in the viral plaque assay suggesting that the compound may act through a different mechanism of action.

As shown in Table 5, the compounds display a large selectivity index, meaning that they may well be of therapeutic benefit in humans and animals. REFERENCES

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