Field of the Invention

The present invention relates to compounds for treating viral infections, and in particular to sialic acid derivatives that are capable of inhibiting influenza neuramidinases . The present invention also relates to compositions, methods and medical uses that employ such compounds .

Background of the Invention

Influenza is a major cause of human illness and death with an estimated 40,000 deaths annually in the US alone, and is responsible for considerable economic loss. Ongoing mutation in the surface antigens of the virus (antigenic drift) and the time required to produce vaccines make it difficult to provide annual protection against epidemic influenza, whilst the ability of influenza viruses to cross species (antigenic shift) , particularly birds, means there is an ever present threat of a new pandemic. More recently, the emergence of highly pathogenic avian H5N1 viruses has increased concerns for adaptation of this strain to humans and poses the next pandemic threat.

The surface proteins haemagglutinin (H) and neuraminidase (N) are important for the life cycle of the influenza virus. The haemagglutinin protein is responsible for recognising cell- surface carbohydrate receptors, and initiates infection by binding to these receptors and inducing absorption of the viral particle into the host cell. When newly formed viral particles are released from the infected cell, they initially re-attach to the infected cell through haemagglutinin binding to the cell- surface receptors. The neuraminidase protein is required at this stage to 'remove' the cell surface receptors so newly formed viral particles can be released from the cell and go on to infect other cells. Effective infection and replication of the virus requires a fine balance between the catalytic activity of its neuraminidase enzyme and the binding strength of its haemagglutinin protein. There are currently two neuraminidase inhibitors on the market recommended for the treatment of influenza infections, namely Zanamivir (Relenza, GSK) and Oseltamivir (Tamiflu, Roche) . These drugs are competitive inhibitors of influenza neuraminidase, and operate by affecting the release of viral particles. The inhibitory potency of both drugs is highly variable, depending on the particular strain of influenza virus being tested.

Zanamivir (Relenza) Oseltamivir (Tamiflu)

Relenza ^® and Tamiflu ^® are effective against all strains of influenza, and represent the current front-line in anti-influenza drugs. However, strains of avian influenza have already shown resistance to Tamiflu, while rates of resistance to annual influenza are estimated to be around 5-18%. In 2007 in the US, 73 (9.2%) of 797 influenza A (HlNl) viruses tested had mutations that confer resistance to Tamiflu. As many today consider that a new and devastating influenza pandemic is inevitable, there is an urgent need to develop new classes of anti-influenza drugs that possess activity across a range of influenza serotypes and will be systemically available, as well as being less susceptible to drug-induced resistance.

Summary of the Invention Broadly, the present invention relates to compounds for treating viral infections, such as influenza, and in particular to sialic acid derivatives that are capable of inhibiting influenza neuramidinases . Preferably, the compounds of the present invention are based on sialic acid derivatives that include modification at positions C4 and/or C7. Accordingly, in a first aspect, the present invention provides a compound represented by general formula ( I ) :

wherein :

Y ¹ is selected from -O- , -S-, or -NR-, wherein R is independently selected from H, C ₁ - _? alkyl, C _3-10 heterocyclyl, or C _5-20 aryl;

X ¹ is a leaving group;

R ¹ is -CO ₂ R, wherein R is as defined above;

X ² is H or an electron-withdrawing group;

R ² is selected from H or halide, wherein if X ² is H, R ² is selected from halide or OH;

R ³ and R ⁴ are each independently selected from H, -OR, -NR ₂ or

-ZMCH ₂ ) _H1 Z ² , where R is as defined above, Z ¹ is selected from -0-, -NR-, -CR ₂ - and -S-, m is from 0 to 5 and Z ² is selected from -OR, -NR ₂ or -CN;

R ⁵ is H;

R ⁶ is selected from Ci _-7 alkyl; Ci_ ₇ hydroxyalkyl , Ci _-7 amino alkyl or C _1-7 thioalkyl,-

R ⁷ is a group of formula: wherein Y ² is selected from N, O, S, and CH; Z ³ is selected from H, halide, C _1-7 alkyl, Ci _-7 aminoalkyl, Ci_ ₇ hydroxyalkyl , or Ci_ ₇ thioalkyl; R ⁹ and R ¹⁰ are independently selected from H, Ci_ ₇ alkyl, C _5- . ₂ o aryl, C(O)Z ⁴ , wherein Z ⁴ is selected from Ci_ ₇ alkyl or C ₅ _ ₂ o aryl, with the proviso that if Y ² is O or S, R ¹⁰ is absent;

or wherein R ⁴ is other than hydroxyl , R ⁷ may additionally be Ci- ₇ hydroxyalkyl ;

R ⁸ is hydrogen,-

or a dimer consisting of two molecules of formula (I) , linked via a Ci- ₁₂ alkylene linker; preferably via the Z ³ or R ⁹ substituents;

and isomers, salts, solvates, chemically protected forms, and prodrugs thereof.

In some embodiments, the compounds of formula I are defined by the proviso that R ³ and R ⁴ cannot both be H. In other embodiments, R ³ and R ⁴ are both H.

In a further aspect, the present invention provides a compound of formula (I) for use in therapy, in particular for the treatment of a viral infection. As will be explained further below, the therapy may be by way of prophylaxis or treatment of a viral infection.

In a further aspect, the present invention provides the use of a compound of formula (I) in the manufacture of a medicament for the treatment of viral infection.

In a further aspect, the present invention provides a method for treating a patient having a viral infection, the method comprising administering to the patient an effective amount of a compound of formula (I) .

In a further aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier or diluent.

In a further aspect, the present invention provides a kit comprising a first container comprising a compound of formula I and a second container comprising one or more additional antiviral agents, and more preferably one or more additional influenza treatments .

In all of the above aspects of the present invention, the compound represented by formula I may be administered with an additional therapeutic agent, either by being coformulated with the additional therapeutic agent or by being provided in the form of a kit containing each agent separately formulated for sequential or simultaneous administration. In a preferred embodiment, the additional therapeutic agent is a further antiviral agent, such as Zanamivir (Relenza, GSK) and/or Oseltamivir (Tamiflu, Roche) .

Embodiments of the present invention will now be described in more detail by way of example and not limitation with reference to the accompanying figures .

Brief Description of the Figures Figure IA shows the chemical mechanism for the hydrolysis reaction catalysed by Trypanosoma rangeli sialidase (Watts et. al., Can. J. Chem. (2004) 82, 1581-1588) .

Figure IB shows the covalent inactivation of neuraminidases by difluorosialic acids such that where ki»k ₂ there is an accumulation of the covalent intermediate. Figure 2 shows the binding interactions of 2_,_3-dehydroneuraminic acid (DANA) with conserved active site residues of an influenza A virus NA.

Figure 3 is the X-ray crystal structure of PIV HN co-crystallised with the difluoro inactivator (1) .

Figure 4 shows an inhibition of a panel of wild-type (WT) and drug resistant mutant influenza neuraminidases. Values given are IC 50 's (μM) determined using methylumbeliferyl N-acetyl neuraminic dcid as a fluorogenic substrate.

Detailed Description

Preferences The compounds of the invention are represented by general formula (I) . The following preferences for the substituents present in the compounds are may be present in any combination or permutation.

In the compounds of the invention, X ¹ is a leaving group and X ² is H or an electron-withdrawing group. While not wishing to be bound by any particular theory, the present inventors believe that a good leaving group at C2 selectively accelerates formation of the covalent intermediate (adduct with the tyrosine nucleophile in neuramidinase) , while the electron-withdrawing group at C3 destabilises the formation of the oxa-carbenium ion- like transition state for both breakdown (k _x ) and formation (k ₂ ) of the covalent intermediate. Groups X ¹ and X ² are therefore preferably chosen such that ki >> k ₂ , so that the covalent intermediate accumulates, inactivating the enzyme.

Preferably X ¹ is a leaving group selected from hydroxide, alkoxide or halide . More preferably X ¹ is halide, and mq_s_t preferably fluoride. X ² is preferably an electron-withdrawing group selected from halide, cyano, or hydroxyl . More preferably X ² is a halogen, and most preferably fluoride. In particularly preferred compounds of the present invention both X ¹ and X ² are F.

In the compounds of the invention, Y ¹ is a ring atom which may be -0-, -S-, or -NR-. In preferred embodiments, Y ¹ is 0.

R ¹ is a carboxylate group selected from -CO ₂ R, wherein R is preferably H, methyl, or ethyl. In some preferred embodiments, R ¹ is a carboxylate group protected as an ester, preferably an ethyl ester. This modification is used in Oseltamivir, which is a prodrug that is then converted to the active form of the drug (Oseltamivir carboxylate) by hepatic esterases.

R ² is selected from H or halide. Preferably R ² is H. In addition, in the case where X ² is H, R ² may be halide or OH.

R ³ and R ⁴ are each independently selected from H, -OR, -NR ₂ or

-Z ¹ (CH ₂ ) _m Z ² , where R is as defined above, Z ¹ is selected from -0-, -NR-, -CR ₂ - and -S-, m is from 0 to 5 and Z ² is selected from -OR, -NR ₂ , or-CN. In some embodiments R ³ and R ⁴ are not both H. In other embodiments, R ³ and R ⁴ are both H.

In some preferred embodiments, one of R ³ and R ⁴ is selected from -Z ¹ (CH ₂ ) _m Z ² , where Z ¹ is preferably 0 or NH. In these embodiments m is preferably 1 or 2. Z ² is preferably -NR ₂ or -CN. If Z ² is NR ₂ , each R may be the same or different, and is preferably H or methyl .

In some preferred embodiments, R ³ is H. In these embodiments the compound may mimic the configuration of the natural substrate (sialic acid) . However, in other preferred embodiments, R ⁴ is H, and the compound of the invention may therefore have a structure which is epimeric to the 'natural' stereochemistry at C4. In certain of these embodiments, R ⁴ may preferably be -NR ₂ , where each R may be the same or different and is preferably H or methyl, for example R ⁴ may be -NHMe or -NH ₂ .

R ⁷ is a group of formula:

wherein Y ² is selected from N, 0, S, and CH; Z ³ is selected from H, halide, C^ ₇ alkyl, Ci_ ₇ aminoalkyl, C ₁ ^ hydroxyalkyl, or C ₁ - ₇ thioalkyl; R ⁹ and R ¹⁰ are independently selected from H, Ci_ ₇ alkyl, C ₅ _ ₂₀ aryl, C(O)Z ⁴ , wherein Z ⁴ is selected from Ci_ ₇ alkyl or C5- ₂ 0 aryl, with the proviso that if Y ² is O or S, R ¹⁰ is absent

Preferably, Y ² is N or 0, most preferably N. Z ³ is preferably H. R ⁹ and R ¹⁰ are independently preferably selected from Cχ_ ₇ alkyl, most preferably methyl or ethyl. In particularly preferred compounds, R ⁷ is a group of formula:

wherein R ⁹ and R ¹⁰ are as previously defined.

In some embodiments, the present invention relates to compounds modified at the C4 position and in this case, where R ⁴ is other than hydroxyl, R ⁷ may additionally be Ci- ₇ hydroxyalkyl. In a preferred group of such compounds R ⁷ is C ₁ .-, hydroxyalkyl (e.g. - CH(OH) -CH(OH)-CH ₂ OH) , R ⁴ is H and R ³ is -NR ₂ .

Examples of preferred compounds of the present invention includes compounds 10 to 24 described in the examples, and isomers, salts, solvates, chemically protected forms, and prodrugs thereof.

In some embodiments of the invention, the compound is a dimer consisting of two molecules of formula (I) , optionally connected by a linker group, for example a Ci- ₁₂ alkylene linker. In this case, the dimer_ may be represented by formula (I ¹ ):

wherein L is a Ci_i ₂ linker and R ^7' is a divalent connecting group derived from an R ⁷ group as previously defined. Preferably each R ^7' is -CH (Z ³ ) Y ² (R ¹¹ )-, where Z ³ and Y ² are as defined above. Preferably, L is a C ₃ _ ₇ alkylene linker, more preferably a C ₅ alkylene linker.

Definitions

Leaving group: the term "leaving group" is well known and commonly used in the art, and refers to an atom or functional group which can be expelled from a molecule in a chemical reaction. As used herein, the term "leaving group" refers to a group which is labile in a nucleophilic substitution reaction. Lability/leaving group ability of a particular functional group depends on the pK _a of its conjugate acid - generally speaking, the lower this is, the better the leaving group. Preferably, the leaving group is capable of supporting and stabilising a negative charge, i.e., the group is capable of leaving as an anion. Many such leaving groups are known in the art including, but not limited to, halides (F ^" , Cl ^" , Br ^" , I ^" ) , hydroxide (HO ^" ) , alkoxides (RO ^" , where R is an ether substituent as defined below) , carboxylates (RC(O)O ^" , where R is an acyloxy substituent as defined below,- e.g. AcO ^" ) , azide (N ₃ ^" ) , thiocyanate (SCN ^" ) , nitro (NO ₂ ^" ) , amine (NH ₂ ^" ) .

Electron-withdrawing group: the term "electron withdrawing group", or EWG, is also well known and commonly used in the art to refer to a group which withdraws electron density from the molecule to which it is attached, by either inductive/polar (σ) or conjugative/resonant (π) effects. Examples of electron withdrawing groups_include, but are not limited to, halogens (F, Cl, Br), hydroxyl (-0H), alkoxy (-0R), cyano (-CN), acyl (- C(O)R), carboxylic acids and derivatives (-C(O)OR, etc), fluoroalkyl (e.g. -CF ₃ ) etc.

Ci- ₇ alkyl : The term "Ci _-7 alkyl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a C ₁ - ₇ hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated.

Examples of saturated linear Ci _-7 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl) .

Examples of saturated branched Ci _-7 alkyl groups include, but are not limited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, and neo-pentyl .

Examples of saturated alicyclic Ci_ ₇ alkyl groups (also referred to as "C _3- . ₇ cycloalkyl" groups) include, but are not limited to, groups such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well as substituted groups (e.g., groups which comprise such groups), such as methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl , dimethylcyclobutyl, methylcyclopentyl, dimethylcyclopentyl, methylcyclohexyl , dimethylcyclohexyl, eye1opropylinethy1 and cyclohexylmethyl.

Examples of unsaturated Ci_ ₇ alkyl groups which have one or more carbon-carbon double bonds (also referred to as "C ₂ - ₇ alkenyl" groups) include, but are not limited to, ethenyl (vinyl, -CH=CH ₂ ) , 2-propenyl (allyl, -CH-CH=CH ₂ ), isopropenyl (-C (CH ₃ ) =CH ₂ ) , butenyl , pentenyl , and hexenyl .

Examples of unsaturated Ci_ ₇ alkyl groups which have one or more carbon-carbon triple bonds (also referred to as "C _2-7 alkynyl" groups) include, but are not limited to, ethynyl (ethinyl) and 2-propynyl (propargyl) .

Examples of unsaturated alicyclic (carbocyclic) Ci- ₇ alkyl groups which have one or more carbon-carbon double bonds (also referred to as "C ₃ _ ₇ cycloalkenyl" groups) include, but are not limited to, unsubstituted groups such as cyclopropenyl , cyclobutenyl, cyclopentenyl, and cyclohexenyl , as well as substituted groups (e.g., groups which comprise such groups) such as eye1opropenylinethy1 and cyclohexenylmethyl.

C3-20 heterocyclyl : The term "C ₃ _ ₂₀ heterocyclyl" , as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a C ₃ _ ₂₀ heterocyclic compound, said compound having one ring, or two or more rings (e.g., spiro, fused, bridged) , and having from 3 to 20 ring atoms, atoms, of which from 1 to 10 are ring heteroatoms, and wherein at least one of said ring (s) is a heterocyclic ring. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. Ring heteroatoms may preferably be selected from the group consisting of 0, N, S and P. "C _3-20 " denotes ring atoms, whether carbon atoms or heteroatoms. Similarly, the term "C ₃ - _I0 heterocyclyl" will be understood to pertain to an equivalent moiety of 3 to 10 ring atoms, and so on.

Examples of C ₃ - ₂ o heterocyclyl groups having one nitrogen ring atom include, but are not limited to, those derived from aziridine, azetidine, pyrrolidines (tetrahydropyrrole), pyrroline (e.g., 3- pyrroline, 2, 5-dihydropyrrole) , 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) , piperidine, dihydropyridine , tetrahydropyridine, and azepine.

Examples of C _3-20 heterocyclyl groups having one oxygen ring atom include, but are not limited to, those derived from oxirane, oxetane, oxolane (tetrahydrofuran) , oxole (dihydrofuran) , oxane

(tetrahydropyran) , dihydropyran , pyran (C ₆ ) , and oxepin. Examples of substituted C ₃ - ₂₀ heterocyclyl groups include sugars, in cyclic form, for example, furanoses and pyranoses, including, for example, ribose, lyxose, xylose, galactose, sucrose, fructose, and arabinose.

Examples of C ₃ _ ₂₀ heterocyclyl groups having one sulphur ring atom include, but are not limited to, those derived from thiirane, thietane, thiolane (tetrahydrothiophene) , thiane (tetrahydrothiopyran) , and thiepane.

Examples of C _3-20 heterocyclyl groups having two oxygen ring atoms include, but are not limited to, those derived from dioxolane, dioxane, and dioxepane.

Examples of C _3-20 heterocyclyl groups having two nitrogen ring atoms include, but are not limited to, those derived from imidazolidine, pyrazolidine (diazolidine) , imidazoline, pyrazoline (dihydropyrazole) , and piperazine.

Examples of C ₃ _ ₂ o heterocyclyl groups having one nitrogen ring atom and one oxygen ring atom include, but are not limited to, those derived from tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, and oxazine.

Examples of C ₃ _ ₂₀ heterocyclyl groups having one oxygen ring atom and one sulphur ring atom include, but are not limited to, those derived from oxathiolane and oxathiane (thioxane) .

Examples of C _3-20 heterocyclyl groups having one nitrogen ring atom and one sulphur ring atom include, but are not limited to, those derived from thiazoline, thiazolidine, and thiomorpholine.

Other examples of C ₃ _ ₂₀ heterocyclyl groups include, but are not limited to, oxadiazine and oxathiazine. Examples of heterocyclyl groups, which additionally bear one or more oxo (=0) groups, include, but are not limited to, those derived from:

C ₅ heterocyclics, such as furanone, pyrone, pyrrolidone (pyrrolidinone) , pyrazolone (pyrazolinone) , imidazolidone, thiazolone, and isothiazolone,-

C ₆ heterocyclics, such as piperidinone (piperidone) , piperidinedione, piperazinone, piperazinedione, pyridazinone, and pyrimidinone (e.g., cytosine, thymine, uracil), and barbituric acid; fused heterocyclics, such as oxindole, purinone (e.g., guanine), benzoxazolinone, benzopyrone (e.g., coumarin) ; cyclic anhydrides (-C (=0) -0-C (=0) - in a ring), including but not limited to maleic anhydride, succinic anhydride, and glutaric anhydride; cyclic carbonates (-0-C(=0)-0- in a ring), such as ethylene carbonate and 1, 2-propylene carbonate,- imides (-C (=0) -NR-C (=0) - in a ring), including but not limited to, succinimide, maleimide, phthalimide, and glutarimide,- lactones (cyclic esters, -0-C(=0)- in a ring), including, but not limited to, β-propiolactone, γ-butyrolactone, δ-valerolactone (2-piperidone) , and ε-caprolactone; lactams (cyclic amides, -NR-C(=O)- in a ring), including, but not limited to, β-propiolactam, γ-butyrolactam (2 -pyrrolidone) , δ-valerolactam, and ε-caprolactam,- cyclic carbamates (-0-C (=0) -NR- in a ring), such as

2-oxazolidone; cyclic ureas ( -NR-C (=0) -NR- in a ring), such as 2-imidazolidone and pyrimidine-2 , 4-dione (e.g., thymine, uracil) .

C ₅ - ₂ o aryl: The term "C ₅ ^ _2O aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of a C ₅ - ₂ o aromatic compound, said compound having one ring, or two or more rings (e.g., fused), and having from 5 to 20 ring atoms, and wherein at least one of said ring(s) is an aromatic ring. Preferably, each ring has from 5 to 7 ring atoms . The ring atoms may be all carbon atoms, as in "carboaryl groups", in which case the group may conveniently be referred to as a "C ₅ - ₂₀ carboaryl " group .

Examples of C ₅ - ₂₀ aryl groups which do not have ring heteroatoms (i.e. C _5-20 carboaryl groups) include, but are not limited to, those derived from benzene (i.e. phenyl) (C ₆ ), naphthalene (Ci ₀ ), anthracene (Ci ₄ ) , phenanthrene (C ₁₄ ) , naphthacene (Ci ₈ ) , and pyrene (Ci ₆ ) .

Examples of aryl groups which comprise fused rings, one of which is not an aromatic ring, include, but are not limited to, groups derived from indene and fluorene.

Alternatively, the ring atoms may include one or more heteroatoms, including but not limited to oxygen, nitrogen, and sulphur, as in "heteroaryl groups". In this case, the group may conveniently be referred to as a "C ₅ _ ₂ o heteroaryl" group, wherein "C ₅ - ₂₀ " denotes ring atoms, whether carbon atoms or heteroatoms.

Preferably, each ring has from 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms .

Examples of C ₅ . ₂ o heteroaryl groups include, but are not limited to, C ₅ heteroaryl groups derived from furan (oxole) , thiophene (thiole) , pyrrole (azole) , imidazole (1, 3-diazole) , pyrazole (1, 2-diazole) , triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, and oxatriazole,- and C ₆ heteroaryl groups derived from isoxazine, pyridine (azine) , pyridazine (1, 2-diazine) , pyrimidine (1, 3-diazine; e.g., cytosine, thymine, uracil), pyrazine (1, 4-diazine) , triazine, tetrazole, and oxadiazole (furazan) .

Examples of C ₅ - ₂₀ heterocyclic groups (some of which are C _5-20 heteroaryl groups) which comprise fused rings, include, but are not limited to, C ₉ heterocyclic groups derived from benzofuran, isobenzofuran, indole, isoindole, purine (e.g., adenine, guanine) , benzothiophene, benzimidazple,- C ₁₀ heterocyclic groups derived from quinoline, isoquinoline, benzodiazine, pyridopyridine, quinoxaline; C ₁₃ heterocyclic groups derived from carbazole, dibenzothiophene, dibenzofuran,- C ₁₄ heterocyclic groups derived from acridine, xanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine.

The above alkyl, heterocyclyl and aryl groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed and defined below.

Halo: -F, -Cl, -Br, and -I.

Hydroxy: -OH.

Ether: -OR, wherein R is an ether substituent, for example, a C ₁ - _? alkyl group (also referred to as a C _1-7 alkoxy group, discussed below) , a C _3-20 heterocyclyl group (also referred to as a C ₃ _ ₂ o heterocyclyloxy group) , or a C ₅ - ₂₀ aryl group (also referred to as a C _5-20 aryloxy group) , preferably a C _1-7 alkyl group.

C _1-7 alkoxy: -OR, wherein R is a C _1-7 alkyl group. Examples of C ₁ -- _? alkoxy groups include, but are not limited to, -OCH ₃ (methoxy) , -OCH ₂ CH ₃ (ethoxy) and -OC(CH ₃ ) ₃ (tert-butoxy) .

Oxo (keto, -one) : =0. Examples of cyclic compounds and/or groups having, as a substituent, an oxo group (=0) include, but are not limited to, carbocyclics such as cyclopentanone and cyclohexanone,- heterocyclics, such as pyrone, pyrrolidone, pyrazolone, pyrazolinone, piperidone, piperidinedione, piperazinedione, and imidazolidone,- cyclic anhydrides, including but not limited to maleic anhydride and succinic anhydride; cyclic carbonates, such as propylene carbonate,- imides, including but not limited to, succinimide and maleimide,- lactones (cyclic esters, -0-C(=0)- in a ring) , including, but not limited to, β-propiolactone, γ-butyrolactone, δ-valerolactone, and ε-caprolactone,- and lactams (cyclic amides, -NH-C (=0)- in a ring) , including, but not limited to, β-propiolactam, γ-butγrolactam (2-pyrrolidone) , δ-valerolactam, and ε-caprolactam.

Imino (imine) : =NR, wherein R is an imino substituent, for example, hydrogen, Ci- ₇ alkyl group, a C ₃ - ₂ oheterocyclyl group, or a C5- ₂₀ aryl group, preferably hydrogen or a Cj _. .. ₇ alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde) : -C(=O)H.

Acyl (keto) : -C(=O)R, wherein R is an acyl substituent, for example, a Ci_ ₇ alkyl group (also referred to as Ci_ ₇ alkylacyl or Ci_7 alkanoyl) , a C ₃ - ₂₀ heterocyclyl group (also referred to as C3-20 heterocyclylacyl) , or a C ₅ - ₂ o aryl group (also referred to as C _5-20 arylacyl), preferably a Ci- ₇ alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH ₃ (acetyl), -C(^O)CH ₂ CH ₃ (propionyl) , -C (=0)C (CH ₃ J ₃ (butyryl) , and -C(=O)Ph (benzoyl, phenone) .

Carboxy (carboxylic acid) : -COOH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl) :

-C(=O)OR, wherein R is an ester substituent, for example, a Cj _. - ₇ alkyl group, a C ₃ _ ₂ o heterocyclyl group, or a C ₅ - ₂₀ aryl group, preferably a Cj _. _ ₇ alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH ₃ , -C (=0) OCH ₂ CH ₃ , -C (=0)0C (CH ₃ ) _3/ and -C(=O)OPh.

Acyloxy (reverse ester) : -OC(=O)R, wherein R is an acyloxy substituent, for example, a C _x-7 alkyl group, a C ₃ - ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably a C _1-7 alkyl group. Examples of acyloxy groups include, but are not limited to,

-OC(=O)CH ₃ (acetoxy) , -OC (=0) CH ₂ CH ₃ , -OC (=0) C (CH ₃ J ₃ , -OC(=O)Ph, and -OC (=0) CH ₂ Ph. Amido (carbamoyl, carbamyl, aminocarbonyl , carboxamide) : -C (=0 JNR ¹ R ² , wherein R ¹ and R ² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH ₂ , -C (=0) NHCH ₃ , -C(=O)N(CH ₃ ) ₂ , -C (=0) NHCH ₂ CH ₃ , and -C (=O)N(CH ₂ CH ₃ ) ₂ , as well as amido groups in which R ¹ and R ² , together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl , morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Acylamido ( acy1amino ) : -NR ¹ C( _^ O)R ² , wherein R ¹ is an amide substituent, for example, hydrogen, a Ci- ₇ alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably hydrogen or a Ci_ ₇ alkyl group, and R ² is an acyl substituent, for example, a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably hydrogen or a Ci _-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC (=0) CH ₃ , -NHC (=0 JCH ₂ CH ₃ , and -NHC (=0) Ph. R ¹ and R ² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl and phthalimidyl .

Acylureido: -N(R ¹ JC(O)NR ² C(O)R ³ wherein R ¹ and R ² are independently ureido substituents, for example, hydrogen, a Ci _-7 alkyl group, a C ₃ _ ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably hydrogen or a Ci_ ₇ alkyl group. R ³ is an acyl group as defined for acyl groups. Examples of acylureido groups include, but are not limited to, -NHCONHC(O)H, -NHCONMeC(O)H, -NHCONEtC(O)H, -NHCONMeC(O)Me, -NHCONEtC(O)Et, -NMeCONHC(O)Et, - NMeCONHC(O)Me, -NMeCONHC(O)Et, -NMeCONMeC(O)Me, -NMeCONEtC(O)Et, and -NMeCONHC(O)Ph.

Carbamate: -NR ¹ -C (0) -OR ² wherein R ¹ is an amino substituent as defined for amino groups and R ² is an ester group as defined for ester groups. Examples of carbamate groups include, but are not limited to, -NH-C(O)-O-Me, -NMe-C(O)-O-Me, -NH-C(O)-O-Et, -NMe- C(O)-O-t-butyl, and -NH-C(O)-O-Ph. Thioamido (thiocarbamyl) : -Cf=S)NR ¹ R ² , wherein R ¹ and R ² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH ₂ , -C (=S) NHCH ₃ , -C ( =S) N (CH ₃ ) ₂ , and -C (=S JNHCH ₂ CH ₃ .

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

Amino: -NR ¹ R ² , wherein R ¹ and R ² are independently amino substituents, for example, hydrogen, a Ci _-7 alkyl group (also referred to as C _x - ₇ alkylamino or di-Ci- ₇ alkylamino) , a C _3-2 O heterocyclyl group, or a C ₅ - ₂₀ aryl group, preferably H or a C ₁ . ₇ alkyl group, or, in the case of a "cyclic" amino group, R ¹ and R ² , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of amino groups include, but are not limited to, -NH ₂ , -NHCH ₃ , -NHC (CH ₃ ) ₂ , -N(CH ₃ J ₂ , -N(CH ₂ CH ₃ J ₂ , and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino .

Imino: =NR, wherein R is an imino substituent, for example, for example, hydrogen, a Ci_ ₇ alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably H or a Ci _-7 alkyl group.

Amidine: -C(=NR)NR ₂ , wherein each R is an amidine substituent, for example, hydrogen, a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C ₅ - ₂₀ aryl group, preferably H or a Ci _-7 alkyl group. An example of an amidine group is -C(=NH)NH ₂ .

Carbazoyl (hydrazinocarbonyl) : -C(O)-NN-R ¹ wherein R ¹ is an amino substituent as defined for amino groups. Examples of azino groups include, but are not limited to, -C(O)-NN-H, -C(O)-NN-Me, -C(O)-NN-Et, -C(O)-NN-Ph, and -C(O)-NN-CH ₂ -Ph. Nitro: -NO ₂ .

Nitroso: -NO.

Azido: -N ₃ .

Cyano (nitrile, carbonitrile) : -CN.

Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato) : -SCN.

Isothiocyano (isothiocyanato) : -NCS.

Thio: (sulfhydryl, thiol, mercapto) : -SH.

Thioether (sulfide) : -SR, wherein R is a thioether substituent, for example, a Ci _-7 alkyl group (also referred to as a Ci- ₇ alkylthio group) , a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a Ci _-7 alkyl group. Examples of Ci _-7 alkylthio groups include, but are not limited to, -SCH ₃ and -SCH ₂ CH ₃ .

Disulfide: -SS-R, wherein R is a disulfide substituent, for example, a Ci _-7 alkyl group, a C ₃ _ ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably a Ci- ₇ alkyl group (also referred to herein as Ci_ ₇ alkyl disulfide) . Examples of Ci_ ₇ alkyl disulfide groups include, but are not limited to, -SSCH ₃ and -SSCH ₂ CH ₃ .

Sulfone (sulfonyl) : -S(=O) ₂ R, wherein R is a sulfone substituent, for example, a C _1-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a Ci _-7 alkyl group. Examples of sulfone groups include, but are not limited to, -S(=O) ₂ CH ₃ (methanesulfonyJL, mesyl) , -S(=O) ₂ CF ₃ (triflyl) , -S (=O.). ₂ CH ₂ CH ₃ , -S(=O) ₂ C ₄ F ₉ (nonaflyl), -S (=0) ₂ CH ₂ CF ₃ (tresyl) , -S(=O) ₂ Ph (phenylsulfonyl) , 4-methylphenylsulfonyl (tosyl) , 4-bromophenylsulfonyl (brosyl) , and 4-nitrophenyl (nosyl) .

Sulfine (sulfinyl, sulfoxide) : -S(=O)R, wherein R is a sulfine substituent, for example, a Ci_ ₇ alkyl group, a C ₃ - ₂₀ heterocyclyl group, or a C ₅ - ₂ o aryl group, preferably a Ci _-7 alkyl group. Examples of sulfine groups include, but are not limited to, -S(=O)CH ₃ and -Sf=O)CH ₂ CH ₃ .

Sulfonyloxy: -OS(=O) ₂ R, wherein R is a sulfonyloxy substituent, for example, a Ci_ ₇ alkyl group, a C ₃ _ ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably a Ci_ ₇ alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -OS(=O) ₂ CH ₃ and -OS( _^ O) ₂ CH ₂ CH ₃ .

Sulfinyloxy: -OS(=O)R, wherein R is a sulfinyloxy substituent, for example, a Ci _-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a Ci_ ₇ alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH ₃ and -OS (=0) CH ₂ CH ₃ .

SuIfamino: -NR ¹ S( _^ O) ₂ OH, wherein R ¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=0) ₂ 0H and -N(CH ₃ ) S (=0) ₂ 0H.

Sulfinamino: -NR ¹ Sf=O)R, wherein R ¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a Ci _-7 alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably a C _x-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=O)CH ₃ and -N(CH ₃ ) S (=0) C ₆ H ₅ .

Sulfamyl: -Sf=O)NR ¹ R ² , wherein R ¹ and R ² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include,__but are not limited to, -S(=O)NH _2/ -S (=0LNH (CH ₃ ) , -S(=O)N(CH ₃ ) ₂ , -S(^O)NH(CH ₂ CH ₃ ), -S (=O)N(CH ₂ CH ₃ ) ₂ , and -S (=0) NHPh.

Sulfonamino: -NR ¹ S(^O) ₂ R, wherein R ¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a Ci- ₇ alkyl group, a C ₃ _ ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably a Ci_ ₇ alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(^O) ₂ CH ₃ and -N(CH ₃ )Sf=O) ₂ C ₆ H ₅ .

Phosphoramidite: -OP(OR ¹ ) -NR ² ₂ , where R ¹ and R ² are phosphoramidite substituents, for example, -H, a (optionally substituted) Ci _-7 alkyl group, a C ₃ _ ₂₀ heterocyclyl group, or a C _5-20 aryl group, preferably -H, a Ci_ ₇ alkyl group, or a C _5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to,

-OP (OCH ₂ CH ₃ ) -N(CH ₃ ) ₂ , -OP (OCH ₂ CH ₃ ) -N(i-Pr) ₂ , and -OP(OCH ₂ CH ₂ CN) -N (i- Pr) ₂ .

Phosphoramidate : -OP (=0) (OR ¹ ) -NR ² ₂ , where R ¹ and R ² are phosphoramidate substituents, for example, -H, a (optionally substituted) Ci_ ₇ alkyl group, a C _3-20 heterocyclyl group, or a C _5-20 aryl group, preferably -H, a Ci_ ₇ alkyl group, or a C _5-20 aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP (=0) (OCH ₂ CH ₃ ) -N(CH ₃ ) ₂ , -OP (=0) (OCH ₂ CH ₃ ) -N (i-Pr) ₂ , and -OP (=0) (OCH ₂ CH ₂ CN)-Nd-Pr) ₂ .

In many cases, substituents may themselves be substituted. For example, a Ci _-7 alkoxy group may be substituted with, for example, a Ci _-7 alkyl (also referred to as a Ci _-7 alkyl-Ci_ ₇ alkoxy group) , for example, cyclohexylmethoxy, a C _3-20 heterocyclyl group (also referred to as a C _5-20 aryl-Ci_ ₇ alkoxy group) , for example phthalimidoethoxy, or a C _5-20 aryl group (also referred to as a C _5-20 aryl-C _1-7 alkoxy group) , for example, benzyloxy.

C _1-I2 Alkylene: The term "Ci _-12 alkylene", as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of an aliphatic linear hydrocarbon compound having from 1 to 12 carbon atoms (unless otherwise specified) , which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term "alkylene" includes the sub- classes alkenylene, alkynylene, etc., discussed below.

Examples of saturated Ci- _I2 alkylene groups include, but are not limited to, -(CH ₂ J _n - where n is an integer from 1 to 12, for example, -CH ₂ - (methylene) , -CH ₂ CH ₂ - (ethylene) , -CH ₂ CH ₂ CH ₂ - (propylene) , -CH ₂ CH ₂ CH ₂ CH ₂ - (butylene) , and -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - (pentylene) .

Examples of partially unsaturated Ci- _I2 alkylene groups include, but is not limited to, -CH=CH- (vinylene ), -CH=CH-CH ₂ -, -CH ₂ - CH=CH ₂ -, -CH=CH-CH ₂ -CH ₂ -, -CH=CH-CH ₂ -CH ₂ -CH ₂ -, -CH=CH-CH=CH- and -CH=CH-CH=CH-CH ₂ - .

Alkylene groups may optionally be substituted with one or more substituents including but not limited to those listed above. The Ci_i ₂ alkylene chain may be interrupted with one or more divalent heteroatom groups such as, for example oxygen, nitrogen (which may be substituted with e.g. Ci_ ₇ alkyl) , or sulfur.

Where a particular label or definition (e.g. R) is applied to more than one substituent in one or more compounds, each incidence of that substituent is independent of the others, and may be the same as or different to any other substituent with that label.

Includes Other Forms: Included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid ( -COOH) also includes the anionic (carboxylate) form (-COO ^" ) , a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N ⁺ HR ¹ R ² ) , a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference_._to a hydroxy! group also includes the anionic form (-0 ^" ) , a salt or solvate thereof, as well as conventional protected forms of a hydroxyl group .

Isomers, Salts, Solvates, Protected Forms, and Prodrugs: Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms,- C-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms,- D- and L- forms; d- and 1-forms; (+) and (-) forms; keto-, enol-, and enolate- forms; syn- and anti-forms; synclinal- and anticlinal- forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair- forms,- and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms " ) .

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space) . For example, a reference to a methoxy group, -OCH ₃ , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH ₂ OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures or to a general formula includes structurally isomeric forms falling within that class or formula (e.g., Ci _-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta- , and para-methoxyphenyl) and, except where specifically stated or indicated, all possible conformations and configurations of the compound (s) herein are intended to be included in the general formula (e) .

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate- forms, as in, for example, the following tautomeric pairsj keto/enol (illustrated below) , imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxγazo, and nitro/aci-nitro . keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹ H, ² H (D) , and ³ H (T) ; C may be in any isotopic form, including ¹² C, ¹³ C, and ¹⁴ C; 0 may be in any isotopic form, including ¹⁶ O and ¹⁸ O,- and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof . Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al . , J. Pharm. Sci., 66, 1-19 (1977) .

For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ^" ), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not_JLimited to, ammonium ion (i.e., NH ₄ ⁺ ) and substituted ammonium ions (e.g., NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ) . Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dieyelohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is cationic, or has a functional group which may be cationic (e.g., -NH ₂ may be -NH ₃ ⁺ ), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulphuric, sulphurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic , phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicyclic, sulfanilic, 2-acetyoxybenzoic, fumaric, phenylsulfonic, toluenesulfonic, methanesulfonic, ethanesulfonic, ethane disulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic, and gluconic. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono- hydrate, a di-hydrate, a tri-hydrate, etc. It may be convenient or desirable _t_o _. prepare, purify, and/or handle the active compound in a chemically protected form. The term "chemically protected form", as used herein, pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions, that is, are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group) . By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group,- the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, 'Protective Groups in Organic Synthesis' (T. Green and P. Wuts, Wiley, 1999) .

For example, a hydroxy group may be protected as an ether (-0R) or an ester (-OC(=O)R), for example, as: a t-butyl ether,- a benzyl, benzhydryl (diphenylmethyl) , or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether,- or an acetyl ester (-OC(=O)CH ₃ , -OAc) .

For example, an aldehyde or ketone group may be protected as an acetal or ketal, respectively, in which the carbonyl group (>C=0) is converted to a diether (>C(OR) ₂ ) , by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid.

For example, an amine group may be protected, for example, as an amide or a urethane, for example, as: a methyl amide (-NHCO-CH ₃ ),- a benzyloxy amide (-NHCO-OCH ₂ C ₆ H ₅ , -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH ₃ ) ₃ , -NH-Boc) ; a 2-biphenyl-2-propoxy amide ( -NHCO- OC (CH ₃ J ₂ C ₆ H ₄ C ₆ H ₅ , -NH-Bpoc) , as a 9-fluorenylmethoxy amide (-NH- Fmoc) , as a 6-nitroveratryloxy amide (-NH-Nvoc) , as a 2- trimethylsilylethyloxy amide (-NH-Teoc) , as a 2,2,2- trichloroethyloxy amide (-NH-Troc) , as an allyloxy amide (-NH-Alloc), as a 2 (-phenylsulphonyl) ethyloxy amide (-NH-Psec); or, in suitable cases, as an N-oxide (>NO$) .

For example, a carboxylic acid group may be protected as an ester for example, as: an C _1-7 alkyl ester (e.g. a methyl ester,- a t- butyl ester); a Ci_ ₇ haloalkyl ester (e.g., a Ci_ ₇ trihaloalkyl ester) ; a triCi_ ₇ alkylsilyl-Ci- ₇ alkyl ester,- or a C ₅ - ₂ o aryl-C].--? alkyl ester (e.g. a benzyl ester,- a nitrobenzyl ester); or as an amide, for example, as a methyl amide.

It may be convenient or desirable to prepare, purify, and/or handle the active compound in the form of a prodrug. The term "prodrug", as used herein, pertains to a compound which, when metabolised (e.g. in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound (e.g. a physiologically acceptable metabolically labile ester) .

During metabolism, the ester group (-C(=O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Examples of such metabolically labile esters include those wherein R is Ci _-7 alkyl (e.g. -Me, -Et); Ci- ₇ aminoalkyl (e.g. aminoethyl,- 2-(N, N- diethylamino) ethyl,- 2- (4-morpholino) ethyl) ; and acyloxy-Ci_ ₇ alkyl (e.g. acyloxymethyl ,- acyloxyethyl ,- e.g. pivaloyloxymethyl ,- acetoxymethyl ,- 1-acetoxyethyl,- 1- (1-methoxy-l-methyl) ethyl- carbonxyloxyethyl ,- 1- (benzoyloxy) ethyl; isopropoxy- carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; cyclohexyl- carbonyloxymethyl ,- 1-cyclohexyl-carbonyloxyethyl ,- cyclohexyloxy- carbonyloxymethyl ; 1-cyclohexyloxy-carbonyloxyethyl,- (4- tetrahydropyranyloxy) carbonyloxymethyl; l-(4- tetrahydropyranyloxy) carbonyloxyethyl ,- (4-tetrahydropyranyl ) carbonyloxymethyl ;__and 1- (4-tetrahydropyranyl)carbonyloxyethyl) .

Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Synthesis

The compounds of the invention may be synthesised using methodology known to the skilled person. Suitable methods include, but are not limited to, those discussed herein. Suitable modifications and developments of the methods described in this application will be known to the skilled person.

Compounds of the invention may be synthesised using methods analogous to the literature procedure for the synthesis of compounds (1) and (2) (Watts et. al . , Can. J. Chem. (2004) 82, 1581-1588) . For example, an aldolase reaction between a compound of formula (II) and a β-substituted pyruvate may be performed in the presence of an aldolase enzyme (e.g. Neu5Ac aldolase) :

This may be followed by protection of the resulting compound (IV) , for example by acetylation of free hydroxyl groups and esterification of the carboxylic acid group, before subsequent introduction of a leaving group (X ¹ ) , preferably using a nucleophilic reagent (for example, a nucleophilic fluorination reagent, such as DAST: (diethylamino) sulfur trifluoride) to give a compound of formula (V) .

A compound of formula (V) can be deprotected and/or further modified to produce compounds of the invention.

For example, an oxidative cleavage reaction may be carried out on the R ⁷ triol group in the compounds shown above, to produce an aldehyde (VI) , which may then be further reacted, as is known in the art, to produce a range of substituents at the C7 position. Particularly preferred compounds of the invention may be produced from a reductive amination reaction on such an aldehyde, using a primary amine (RNH ₂ ) and a mild reducing agent (such as sodium cyanoborohydride ) . Resultant amine (VII) may optionally be further alkylated, for example using an alkyl halide.

Alternatively or additionally a compound of formula (IV) may be converted to an acetonide (VIII) and alkylated. Alkylation is preferably performed regioselectively, for example alkylation at the C4 position may be performed (e.g. with chloroacetonitrile) to produce intermediate (IX) , prior to protection of the compound and introduction of the X ¹ group as before. Resultant compound (X) may then be further modified (e.g. by reduction of the nitrile group) .

Other compounds of the invention may be synthesised from an oxazoline such as compound (XI ) , which may itself be synthesised from sialic acid .

Treatment of (XI) with an amine (RNH ₂ ) in the presence of a catalyst (preferably a palladium(O) catalyst) opens the oxazoline to yield a C4 amino product (XII) which may then be reacted further to introduce groups X ¹ and X ² , for example by treatment with a fluorinating agent such as XeF ₂ , followed by deprotection, to yield a compound (XII) .

Use of the compounds of the invention

The compounds of the present invention are active compounds, specifically active neuramidinase inhibitors. The term "active", as used herein, pertains to compounds which are capable of inhibiting neuramidinase activity, and specifically includes both compounds__with intrinsic activity (drugs) as well_as prodrugs of such compounds, which prodrugs may themselves exhibit little or no intrinsic activity.

Assays which may be used in order to assess the inhibition and/or antiviral activity offered by a particular compound is described in the examples below.

The present invention provides active compounds for use in a method of treatment of the human or animal body. Such a method may comprise administering to such a subject a therapeutically- effective amount of an active compound, preferably in the form of a pharmaceutical composition.

The term "treatment", as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications) , in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a preventative measure, i.e. prophylaxis, is also included.

The term "therapeutically-effective amount" as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

The present invention provides compounds which are antiviral agents. The compounds of the invention are of use in therapy, specifically in the treatment of a viral infection. In particular, the compounds are of use in the treatment of influenza. In some preferred embodiments, the viral infection may be caused by a virus strain with reduced sensitivity to oseltamivir and/or zanamivir. It should be noted that in all of the above aspects of the present invention, the compound represented by formula I may be administered with an additional therapeutic agent, either by being coformulated with the additional therapeutic agent or by being provided in the form of a kit containing each agent separately formulated for sequential or simultaneous administration. In a preferred embodiment, the additional therapeutic agent is a further anti -viral agent, such as Zanamivir (Relenza, GSK) and/or Oseltamivir (Tamiflu, Roche) . This may be advantageous as it is presently believed that the mechanism of action of the compounds of the present invention differs from the mechanism of Zanamivir and Oseltamivir. This may provide a wider spectrum of activity against different viral (e.g. influenza) strains, depending on which compound has the better activity against any particular strain.

Administration

The active compound or pharmaceutical composition comprising the active compound may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion) ; topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneousIy or intramuscularly .

The subject may be a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse) , a primate, simian (e.g. a monkey or_ape) , a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon) , or a human.

Formulations

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, 'Remington's Pharmaceutical Sciences', 18th edition, Mack Publishing Company, Easton, Pa., 1990. The formulations may- conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product .

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, losenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols .

Formulations suitable for oral administration (e.g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free- flowing form such as a powder or granules, optionally mixed with one or more binders (e.g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose) ,- fillers or diluents (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate) ; lubricants (e.g. magnesium stearate, talc, silica) ; disintegrants (e.g. sodium starch glycolate, cross-linked povidone, cross- linked sodium carboxymethyl cellulose) ; surface-active or dispersing or wetting agents (e.g. sodium lauryl sulfate); and preservatives (e.g. methyl, p-hydroxybenzoate, propyl p- hydroxybenzoate, sorbic acid) . Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Formulations suitable for topical administration (e.g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or diluents.

Formulations suitable for topical administration in the mouth include losenges comprising the active compound in a flavoured basis, usually sucrose and acacia or tragacanth,- pastilles comprising the active compound in an inert basis such as gelatin and glycerin, or sucrose and acacia,- and mouthwashes comprising the active compound in a suitable liquid carrier.

Formulations suitable for topical administration to the eye also include eye drops wherein the active compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active compound.

Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a .liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

Formulations suitable for administration by inhalation include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro- tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for topical administration via the skin include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water-miscible ointment base.

Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol , i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1, 3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent) , or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier (s) with or without stabiliser (s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol

CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs . Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to__about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets . Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs .

Dosage

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. ^' Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the_purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Design and Mode of Action

The structures of the competitive inhibitors Oseltamivir and Zanamivir are designed to mimic the 'transition-state' species formed during the chemical reaction carried out by the neuraminidase enzyme, and form strong interactions with amino acid residues in the active site. This transition-state species involves the sugar adopting a ^λ flattened-ring' conformation, which is mimicked in the structure of Zanamivir and Oseltamivir which each contain such a 'flattened ring' structure, which does not change shape upon binding .

Scheme 1, below, depicts the currently accepted literature mechanism for the hydrolysis of sialylglycosides by neuraminidases. This mechanism was used as the basis for the design of both Zanamivir and Oseltamivir.

Scheme 1 ; Currently accepted mechanism for neuraminidase catalysed hydrolysis of sialylglycosides . The present inventors have recently found, however, that influenza neuramidinases actually operate through the involvement of a covalent enzyme-substrate intermediate. A new class of compounds has therefore been developed for therapeutic use against influenza and similar viral infections. The new class of therapeutic has the potential to overcome many of the limitations of current therapeutics, by combining the properties of oral bioavailability with a reduced susceptibility for developing drug-induced resistance.

The 2, 3-difluorosialic acid derivative (1) was previously synthesized and used as an inactivator of sialidases from the parasites Trypanosoma cruzi (Watts et. al . , J. Am. Chem. Soc. (2003) 125, 7532-7533) and Trypanosoma rangeli. (Watts et. al., Can. J. Chem. (2004) 82, 1581-1588) . This initial work led to the discovery that these sialidases operate through the involvement of a covalent sialosyl-enzyme intermediate (Figure 1), and established that compounds such as (1) acted as time- dependent covalent inactivators of Trypanosomal sialidases.

Subsequently it was shown that the 2,3-difluoro neuraminic acid derivative (2), which possesses a hydroxyl group at C-5 rather than the natural N-acetyl group, also acts as a covalent inactivator of T. rangeli sialidase, but displays different kinetic behaviour (k _inact and k _react ) to the original inhibitor (1) (Watts et. al., J. Biol. Chem. (2006) 281, 4149-4155) .

Sialidase enzymes are classified into different sequence-based families (Henrisatt et. al, Biochem. J, 316, 695-696) . The Trypanosomal sialidase enzymes in the studies discussed above belong to family 33. Influenza virus neuraminidases belong to ^family 34, for which no data on mechanism _operating through a covalent intermediate has been published. As discussed above, the currently accepted literature mechanism for influenza neuraminidases remains the tight 'ion-pair' transition-state used in the rational design of Zanamivir and Oseltamivir.

The present inventors have obtained a first crystallographic structure showing the existence of a covalent intermediate for an influenza neuramidinase . This has enabled the inventors to design further inactivators, such as the compounds of the invention.

The compounds of the invention are 'mechanism-based' inhibitors of neuramidinases . They are slow substrates for neuramidinases, and inactivate the enzyme by forming a relatively 'stable' covalent adduct with a tyrosine nucleophile. Without wishing to be bound by theory, it is proposed that the introduction of the electron-withdrawing group at C3 inductively destabilizes the formation of the oxa-carbenium ion-like transition state for both the formation (ki) and breakdown (k ₂ ) of the covalent intermediate (Figure IB) , while the good leaving group at C2 selectively accelerates the formation of the covalent intermediate (k _x ) . If k _x >>k ₂ , accumulation of the covalent intermediate and potent inactivation of the enzyme is observed.

Two important characteristics of ^λ mechanism-based' difluoro sialic acid inactivators compared to traditional competitive inhibitors like Zanamivir or Oseltamivir can be seen from the results disclosed herein. Firstly, the inhibition properties of the compound (Ki) can be controlled, and improved, by removing key binding interactions with the enzyme. Secondly, these inactivators specifically target the catalytic nucleophile of the neuramidinase, a residue which is essential for enzyme activity. As such, the neuraminidase is unable to tolerate any mutation to this residue and so the key mode of action for these inactivators does not impart evolutionary pressure on the virus. Without .wishing to be bound by theory, the compounds of the present invention may operate by forming relatively stable covalent linkages to a specific amino acid in the active site of a neuramidinase (NA) . The more stable this linkage is, the more potent the inhibitor may be. To increase the stability of this linkage to the NA, therefore, it may be preferred that the ability for the NA to stabilize the transition state for its breakdown is reduced. In preferred embodiments of the invention, this may be achieved by removing hydrogen bonds formed between the inhibitor and active site amino acids (as compared to the natural substrate) . Preferably, hydrogen bonding interactions at hydroxyls OH-4,7,8,9 are modified relative to the natural substrate. Modifications at these positions may contribute to binding and catalysis (transition-state stabilisation) .

Preferably, the compounds of the invention include pharmacophores which make specific binding interactions with active site residues, to increase the rate of forming the Michaelis complex, and hence the rate of forming the covalent intermediate. In certain preferred embodiments, pharmacophores are introduced at carbons C4 and/or C7. In the compounds of the invention, at least one, preferably both, of the C4 and C7 positions is modified relative to compounds (1) and (2) . At C7, preferred embodiments have an amino alkyl group or other group which takes advantage of acidic residues in close proximity to this position. Preferably the compounds of the invention minimize the chance for introducing pressure on the virus to evolve resistance. In some embodiments, this may be accomplished by introducing favourable binding interactions with essential active site residues, as these are unlikely to produce active neuraminidase if mutated.

It is preferred that the compounds of the invention are orally bioavailable, e.g. for ease of administration to patients. In some preferred embodiments, this may be achieved by reducing the total polar surface area, compared to compound (1) , as well as reducing the number of hydrogen bonding groups and increasing hydrophobicity . Compounds of the present invention preferably have oraLbioavailability characteristics (logP)_ comparable to or better than those of Oseltamivir, which is known to be orally bioavailable.

In preferred embodiments, the compounds of the invention may have some or all of the desired properties listed below:

• steady-state inactivation within 20-40 minutes

• covalent intermediate with a half-life of 6-8 hours

• IC50 around 1-10 nM • >40% bioavailability

• plasma half-life of 2-4 hours

As discussed above, in some preferred embodiments, the compound mimics the configuration of the natural substrate (sialic acid) . However, in some preferred embodiments, the compound of the invention is epimeric to the 'natural' stereochemistry at C4. There is literature precedent that this stereochemical arrangement is tolerated by influenza NA. Without wishing to be bound by theory, it is thought it may take advantage of an acid residue well conserved in influenza NA' s, but which is not present in human NA's (i.e., selectivity) . DANA-like inhibitors with equivalent stereochemistry at C4 were found to inhibit influenza NA 10-fold lower than the corresponding equatorial (α) epimer. However, the DANA-like inhibitors adopt a flattened boat conformation, which moves the group away from the acidic residue. On the other hand, the compounds of the invention adopt a stable conformation when trapped as a covalent intermediate, which will place the β substituent in an axial position, significantly closer to the acidic glutamate residue in the enzyme. Furthermore, this favourable conformation is only adopted once the inhibitor has formed the covalent intermediate (i.e. has inhibited the NA) . As breakdown of the covalent intermediate would now involve the loss of this binding interaction, the presence of the β group may stabilise the intermediate and improve inhibition. It is worth^noting that the compounds of the invention^ inhibit very differently to competitive inhibitors. Without wishing to be bound by theory, a major difference may be that the compounds of the invention undergo considerable conformational ring changes during the inactivation process, whereas the ring conformation remains essentially unchanged in competitive inhibitors. A consequence of this is that there is no evidence to show that a particular pharmacophore will impart similar kinetic properties on both competitive and mechanism-based inhibitors in the same way.

As mentioned above, the compounds of the invention are expected to have a lower propensity for inducing the evolution of drug resistance than competitive inhibitors, as they specifically target amino acid residues unable to be mutated by the viral neuraminidase. Furthermore, the compounds of the invention may exist in a ground state conformation analogous to the natural substrates for the neuraminidase and haemagglutinin proteins, introducing the potential for these molecules to 'inhibit' both proteins and, as such, operate by affecting both attachment and release of viral particles.

Examples

Inhibition of human parainfluenza virus hemagglutinin- neuraminidase

The difluoro sialic acid (1) has been co-crystallised with human parainfluenza virus (PIV) hemagglutinin-neuraminidase (HN), and the structure of the complex determined to 1.9A. PIV HN is an interesting enzyme in that it is a bi- functional enzyme, performing both the functions on neuraminidase and hemagglutinin binding. The compound was observed to form a covalent intermediate with the catalytic tyrosine residue in the neuraminidase site, and was also observed to bind (in the ground state 2C5 conformation) in the hemagglutinin binding site (Figure 2) . This is an extremely important observation as it suggested to the present inventors that difluoro sialic acids may have the potential to inhibit influenza virus by simultaneously targeting both the neuraminidase and hemagglutinin proteins . This _biτ_ functional property is an attribute of the ring conformation of difluoro sialic acids which adopt a similar conformation to the natural sialic acid substrates of both neuraminidase and hemagglutinin, a property which does not exist for transition state analogues such as Zanamivir and Oseltamivir.

The results of these experiments can be summarised in the following important points: • The present inventors have found that influenza neuraminidases actually operate through the involvement of a covalent intermediate.

• The modified sialic acids are slow substrates for neuraminidases, inactivating them through formation of a covalent intermediate on the basis of mechanism.

• Inhibitory activity is a combination of both competitive and covalent binding properties .

• Inhibitory activity can be improved by removing binding interactions necessary for transition-state stabilization. • The sugar ring undergoes a dramatic conformational change upon covalent binding.

• The modified sialic acids in solution adopt a ground state conformation similar to the natural substrates of the neuraminidase and haemagglutinin proteins . • The modified sialic acids are able to bind to both the neuraminidase and haemagglutinin proteins .

Synthesis Examples

Synthesis of the original 2, 3-difluorosialic acid inhibitor was carried out according to literature procedure (Watts et. al . ,

Can. J. Chem. (2004) 82, 1581-1588) . A summary of this sequence is given below and lists the yields and reagents /solvents for each step (Scheme 2) . The parent 2 , 3-difluorosialic acid compound may be used as a starting point for certain modifications.

N-Acetyl mannosamine β-fluoropyruvate, Na salt Chemical Formula C ₁₁ H ₁₈ FNO ₉ Chemical Formula C ₈ H ₁₅ NO ₈ Chemical Formula C ₃ H ₃ FNaO ₃ Molecular Weight 3273 Molecular Weight 221 2 Molecular Weight 129 0

pyπdιne/Ac ₂ O

Yield: 85%

Chemical Formula C ₁₂ H ₂₀ FNO ₉ Molecular Weight 341 3

hydrazine acetate DAST methanol dichloromethane

Yield: >95% Yield: 45%

NaOMe/MeOH Chemical Formula C ₅₂ H ₇₄ F ₅ N ₃ O ₃₃ Molecular Weight 1364 1

Yield. >95% Total Yield: >31.6%

Scheme 2 - Literature Sequence for 2 , 3 -dif luorosialic acid

Modifications at C7 were introduced by performing an oxidative cleavage of the glycerol side chain to give the C7 aldehyde , followed by a reductive amination using sodium cyanoborohydride and the respective primary amine ( Scheme 3 ) .

Scheme 3 - Synthetic route for C7 modified dif luorosialic acids

Methyl 5-N-acetamido-4, 7, 8, 9-tetra-O-acetyl-2-O-benzyl-3 , 5- dideoxy-3-fluoro-D-erythro-α-L-manno-2-nonulopyranosonate (3) Sodium hydride (54.00 mg, 2.23 mmol) was added to a stirred solution of methyl 5-i\7-acetamido-4, 7, 8, 9-tetra-O-acetyl-3, 5- dideoxy-3-fluoro-D-erythro-α-L-manno-2-nonulopyranosonate (948.70 mg, 1.86 mmol) and benzyl bromide (270 μL, 2.23 mmol) in DMF (28 mL) . The reaction was stirred for 2h at room temperature. The reaction was quenched with methanol (1 mL) . The mixture was then concentrated in vacuo and then subjected to a standard work up (EtOAc) and the residue purified by flash chromatography (EtOAc/Petroleum ether, 9 : 1 -> EtOAc) . After the purification the compound was dissolved in warm EtOAc and left to cold down in the fridge to give the benzyl glycoside 3 as a white solid (786.90 mg, 70%) . ¹ H NMR (400 MHz, CDCl ₃ ) : δ 7.39 - 7.26 (m, 5H, Ph); 5.46 - 5.38 (m, 2H, H-4, 7); 5.36 - 5.31 (m, IH, H-8) 5.22 (d, IH, J^ ₅ = 10.2 Hz, NHAc) ; 5.05 (dd, IH, J ₃ , ₄ = 2.3, J ₃ , _F3 = 49.3 Hz, H-3); 4.91 (dd, IH, J _{9, 8} = 2.7, J _9ι9 . = 12.5 Hz, H-9) ; 4.65 (d, IH, J _CH2 , _CH2 > = 11.7 Hz, CH ₂ ) ; 4.46 (d, IH, J _CH2' , _CH2 = H -7 Hz, CH ₂ ' ) ; 4.37 (q, IH, Js _1NHAc = 10.6, J ₅ , ₄ = 21.1 Hz, H-5) ; 4.15 (dd, IH, J ₉ , _η ^, = 7.8, J ₉ ., _g = 10.9 Hz, H-9' ) ; 4.10 (dd, IH, J ₆ , ₇ = 1.6, J ₆ , ₅ = 10.9 Hz, H- 6) ; 3.73 (s, 3H, OMe) ; 2.16, 2.08, 2.04, 1.96 (4s, 12 H, Oac) ; 1.88 (S, 3H, NHAc) . ¹³ C NMR (100.59 MHz, CDCl ₃ ) : δ 20.68, 20.79, 20.81, 20.87 (OAc) ; 23.21 (NHAc) ; 45.34 (d, J ₅ , _F3 = 2.3 Hz, C-5) ; 52.98 (OMe) ; 62.36 (C-9) ; 66.17 (CH ₂ ) ; 68.19 (C-7) ; 69.21 (d, J ₄ , _F3 = 17.6 Hz, C-4) ; 71.53, 71.60 (C-6,8) ; 87.40 (d, J ₃ , _F3 = 184.0 Hz, C-3) ; 98.22 (d, J _{2, F3} = 26.8 Hz, C-2) ; 127.67, 128.28, 128.59 (CH, Ph) ; 135.63 (C, Ph) ; 165.27 (C-I) ; 170.00 (NHAc) ; 170.38, 170.59, 170.66, 170.73 (OAc) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -205.84 (dd, IF, J _F3,4 = 29.0, J _F3,3 = 48.8 Hz, F-3) . m/z found (ES+) : 600 . 2061 [M+H] \ C ₂₇ H ₃₅ FiNiO ₁₃ -requires 600 . 2092 ; 622 . 1869 [M+Na] ⁺ , C ₂₇ H ₃₄ F ₁ N ₁ O ₁₃ Na requires 622 . 1912 .

Methyl 5-N-acetamido-2-0-benzyl-3 , 5-dideoxy-3-fluoro-D-erytiiro-oc- L-manno-2-nonulopyranosonate (4)

Catalytic sodium methoxide (180 μL) was added to a solution of the peracetylated benzyl glycoside derivative 3, (786.90 mg, 1.31 mmol) in methanol (18 mL) at 0 ⁰ C and the mixture left to warm to room temperature (Ih) . The mixture was then neutralized (Dowex 50 WX8 H ⁺ form) , filtrated and concentrated in vacuo. Then the mixture was dissolved in warm methanol and left to cold down in the fridge to give 4 as a white solid (533.20 mg, 95%) . ¹ H NMR (400 MHz, CDCl ₃ ) : δ 7.34 (m, 5H, CH Ph); 4.91 (dd, IH, J ₃ , ₄ = 2.3, J ₃ , _F3 = 50.5 Hz, H-3); 4.71 (d, IH, Jαα.αα- = 10-6 Hz, CH ₂ ); 4.30 (dd, IH, J _CH2' , _CH2 = 11.0 Hz, CH ₂ '); 4.19 (t, IH, J ₅ ,^ = 10.6, J ₅ , ₄ = 21.1 Hz, H-5); 4.06 (m, 2H, H-4, 6); 3.90 (m, IH, H-8) ; 3.79 (m, IH, H-9); 3.74 (s, IH, OMe); 3.61 (m, 2H, H-7, 9'); 1.96 (s, 3H, NHAc) . ¹³ C NMR (100.59 MHz, CDCl ₃ ) : δ 22.06 (NHAc); 47.11 (d, J ₅ , _F3 = 1.5 Hz, C-5); 53.77 (OMe); 63.32 (C-9); 65.99 (CH ₂ Ph); 67.49 (C-7); 67.68 (d, J _{4, F3} = 2.3 Hz, C-4) ; 69.88 (C-8) ; 70.75 (C-6) ; 89.60 (d, J ₃ , _F3 = 177.9 Hz, C-3); 98.10 (d, J ₂ , _F3 = 21.6 Hz, C-2); 128.80 (CH, Ph); 135.42 (C, Ph); 168.41 (C-I) ; 174.93 (NHAc) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -209.06 (dd, IF, J ^" _F3 , ₄ = 30.5, J _F3 , ₃ = 48.8 Hz, F-3) . m/z found (ES+) : 432.1644 [M+H] ⁺ , C ₁₉ H ₂₇ F ₁ N ₁ O ₉ requires 432.1670; 454.1458 [M+Na] ⁺ , C ₁₉ H ₂₈ F ₁ N ₁ O ₉ Na requires 454.1489.

Methyl 5-N-acetamido-2-0-benzyl-7-N-ethylamine-3 , 5, 7-trioxy-3- fluoro-D-erythro-cc-L-manno-2-heptapyranosonate (5) Sodium periodate 0.4 M (6.50 mL, 2.60 mmol) was added to a solution of 4 (533.20 mg, 1.24 iranol) in H ₂ O (6.5 mL) in a cover flask at room temperature (30 min) . The mixture was then evaporated in vacuo and the aldehyde formed was used without further purification. Ethylamine 70% (1.50 mL, 26.50 mmol) was dissolved in methanol (4 mL) and the pH was adjusted to 5 with AcOH. This solution was then added to a solution of the aldehyde in methanol (4 mL) . The mixture was left to equilibrate for Ih at room temperature. Sodium cyanoborohydride (776.70 mg, 12.36 mmol) dissolved in methanol (4 mL) was added to the reaction. The mixture was stirring overnight . The reaction mixture was then absorved in silica, concentrated in vacuo and the residue purified by flash chromatography (EtOAc —> EtOAc/MeOH 9:1) . In terms of characterisation it was decided to acetylate the molecule and proceed to the fully characterisation then.

6 Methyl 5-N-acetamido-2-0-benzyl~ 7-N-ethylacetamido-4-mono-0- acetyl -3 , 5, 7-trioxy-3-fluoro-D-erythro-cc-L-manno-2- heptapyranosonate ( 6 )

Acetic anhydride (1.37 g, 13.39 mmol) was added to a solution of 5 (533.20 mg, 1.34 mmol) in pyridine (11 mL) at room temperature and the reaction was left overnight. The mixture was then concentrated in vacuo and residual pyridine removed by azeotropic distillation with toluene to give a yellow oil. The oil was subjected to a normal work up (EtOAc) and purified by flash chromatography ( EtOAc/Hexane, 9:1 -→ EtOAc) to give the acetylated derivateve 6, as a white solid (215.20 mg, 36% over three steps) . ¹ H NMR (400 MHz, CDCl ₃ ) : δ 7.36 - 7.28 (m, 5H, CH Ph); 6.06 (d, IH, J _NHAC15 = 9 Hz, NHAc); 5.33 (ddd, IH, J ₄ , ₃ = 2.3, J ₄ , ₅ = 10.6, J ₄ , _F3 = 29 Hz, H-4); 4.98 (br dd, IH, J ₃ , ₄ = 2.3, J ₃ , _F3 = 49.5 Hz, H- 3); 4.42 (d, IH, J _CH2 .C _H2 > = 11 Hz, CH ₂ ); 4.25 (d, IH, J _C H2',CH2 = 11 Hz, CH ₂ ' ) ; 4.23 - 4.07 (m, 2H, H- 5_, 6_)_; 3.95 (d, IH, J _NCH2 , _NCH2 > = 14.5 Hz, NCH ₂ ) ; 3.74 (s, 3H, OMe) ; 3.67 - 3.44 (m, 2H, CH ₃ CH ₂ N) ; 3.14 (dd, IH, J _NCH2' , ₆ = 8.6, J _NC H _2' ,N _C H ₂ = 14.1 Hz, NCH ₂ ' ) ; 2.09 (s, 3H, OAc) ; 2.07 (s, 3H, NAc) ; 1.95 (s, 3H, NHAc) ; 1.24 (t, 3H, J _CH3 . _CH2 = 7.4, J _CH3 . _CH2 - = 14.5, CH ₃ CH2 ) . ¹³ C NMR (100.59 MHz, CDCl ₃ ) : δ 13.92 (CH ₃ CH ₂ ) ; 20.74 (OAc) ; 21.23 (NAc) ; 23.31 (NHAc) ; 45.79 (CH ₃ CH ₂ ) ; 47.60 (NCH ₂ ) ; 47.69, 72.21 (C-5,6) ; 52.86 (OMe) ; 65.96 (CH ₂ Ph) ; 69.30 (d, J _{4, F3} = 17.3 Hz, C-4) ; 87.60 (br d, J _3ιF3 = 181.7 Hz, C-3) ; 97.83 (d, J ₂ , _F3 = 27.6 Hz, C-2) ; 128.28, 128.42, 128.58 (CH Ph) ,- 135.38 (C Ph) ; 165.87 (C-I) ; 170.74, 170.77,

170.84 (Ac) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -205.98 (dd, IF, J _F3ii ÷ 29.0, Jp _{3, 3} = 48.8 Hz, F-3) . m/z found (ES+) : 483.2143 [M+H] ⁺ , C ₂₃ H ₃₂ F ₁ N ₂ O ₈ requires 483.2143,- 505.1955 [M+Na] ⁺ , C ₂₃ H ₃₁ F ₁ N ₂ O ₈ Na requires 505.1962.

7

Methyl 5-N-acetamido-7-N-ethylacetamido-4-inono-0 -acetyl -3 , 5, 7- trioxy-3-fluoro-D-erythro-a-L-manno-2-heptapyranosonate (7 ) Acetic acid was added to a solution of 6 (100.00 mg, 0.21 mmol) in THF (10 mL) until pH reached 5. Palladium on charcoal 10% (30.00 mg) was added then to the reaction and the reaction was put under an hydrogen gas atmosphere (3 days) . The solution was filtered over Celite and evaporate to dryness. The residue was purified by flash chromatography (EtOAc → EtOAc/MeOH, 9:1) to afford 7 as a white solid (39.40 g, 48%) . ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.50 (d, IH, J _ΛHACS = 8.6 Hz, NHAc); 5.44 - 5.35 (m, IH, H-4); 4.94 (br dd, IH, J _{3, 4} = 2.3, J ₃ , _F3 = 50.1 Hz, H-3); 4.28 - 4.22 (m, IH, H-6); 3.90 (d, IH, J _N C _H2 , _NCH2' = 14.1 Hz, NCH ₂ ); 3.83 (s, 3H, OMe); 3.65 - 3.35 (m, 3H, H-5, CH ₃ CH ₂ ) ; 3.08 (dd, IH, J _NCH2 -, _S =

8.2, J _NCH2' , _NCH2 = 14.1 Hz, NCH ₂ ' ) ; 2.10, 2.06, 1.99 (3s, 9H, 3xAc) ; 1.18 (t, 3H, J _CH3 , _CH2 = 7.4, J _CH3 , _CH2 . =■ 14.5 Hz, CH ₃ CH ₂ ) . ¹³ C NMR (100.59 MHz, CDCl ₃ ) : δ 13.64 (CH ₃ CH ₂ ) ,- 20.79, 21.04, 23.18 (Ac) ; 46.18 (CH ₃ CH ₂ ) ; 47.61 (C-5) ; 48.25 (NCH ₂ ) ; 53.10 (OMe) ; 69.53 (d, Ji _{1 F3} = 17.6 Hz, C-4) ; 71.24 (C-6) ; -8-7_..32_ (br d, J ₃ , _F3 = 184.8 Hz, C-3) ; 94.21 (d, J _{2, F3} = 25.3 Hz, C-2) ; 167.62 (C-I) ; 171.07, 171.12, 171.29 (Ac) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -205.09 (dd, IF, JF ₃ , ₄ = 29.0, J _F3 , ₃ = 50.3 Hz, F-3) . m/z found (ES+) : 393.1674 [M+H] ⁺ , Ci ₆ H ₂₆ F ₁ N ₂ O ₈ requires 393.1673; 415.1484 [M+Na] ⁺ , C ₁₆ H ₂₅ F ₁ N ₂ O ₈ Na requires 415.1493.

8 Methyl 5-N-acetamido-2, 3 , 5, 7-tetradeoxy-4-mono-0-acetyl-2, 3- difluoro-7-ethylacetamido-α-L-manno-2-heptapyranosonate (8) DAST (20 μL, 0.15 iranol) was added dropwise to a stirred solution of the 7, (39.40 mg, 0.10 mmol) in DCM (2 mL) at -30 ² C and the mixture held at this temperature (30 min) . The reaction was quenched by the addition of methanol (40 μL) then subjected to a Standard work up (DCM) and the residue purified by flash chromatography ( EtOAc /Hexane, 9:1 —> EtOAc) to give the difluorosialic acid derivative 8, as a white solid (19.00 mg, 50%) . ¹ H NMR (400 MHz, CDCl ₃ ) : δ 6.33 (d, IH, J _mc , _s = 7.4 Hz, NHAc) ; 5.47 - 5.38 (m, IH, H-4) ; 5.13 (ddd, IH, J ₃ , ₄ = 2.3, J ₃ , _F2 = 3.9, J _{3, F3} = 50.5 Hz, H-3) ; 4.08 - 4.04 (m, 2H, H-5, 6) ; 3.88 (s, 3H, OMe) ; 3.81 (d, IH, J _NCH2 , _NCH2' = 14.5 Hz, NCH ₂ ) ; 3.48 (q, 2H, JcH2,CH3 = 5.4, J _CH2 , _CH2 - = 13.7 Hz , CH ₃ CH ₂ ) ; 3.30 (dd, IH, J _NCH 2-,6 = 6.3, J _NCH2' , _NCH2 = 14.5 Hz, NCH ₂ ') ; 2.11, 2.11, 1.96 (3s, 9H, Ac) ; 1.18 (t, 3H, , J _CH3 , _CH2 = 7.0, Jcm.cm- = 14.5 Hz, CH ₃ CH ₂ ) . ¹³ C NMR (100.59 MHz, CDCl ₃ ) : δ 14.18 (CH ₃ CH ₂ ) ; 20.68, 21.24, 23.36 (Ac) ; 45.82 (CH ₃ CH ₂ ) ; 47.44 (d, J _5,F3 = 3.1 Hz, C-5) ; 47.50 (NCH ₂ ) ; 53.63 (OMe) ; 68.71 (dd, J _{4, F2} = 6.1, J _{4, F3} = 16.9 Hz, C-4) ; 74.16 (d, J _6/F2 = 3.1 Hz, C-6) ; 85.52 (dd, J _{3, F2} = 19.9, J _{3, F3} = 192.5 Hz, C-3) ; 104.73 (dd, J _2;F3 = 17.6, J _2,F2 = 223.1 Hz, C-2) ; 164.58 (dd, J ₁ , _F3 = 3.8, J _{1, F2} = 30.7 Hz, C-I) ; 170.33, 171.00, 171.54 (Ac) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -122.37 (d, J _F2,F3 = 12.2 Hz, F2 ) ; -217.11 (ddd, J _F3/F2 = 12.2, J _F3 , ₄ = 25.9, J _F3,3 = 50.4 Hz, F-3) . m/z found ( ES+ ) : 395 . 1620 [M+H] ⁺ , C ₁₆ H ₂₅ F ₂ N ₂ O ₇ requires . 395 . 1630 ; 417 . 1437 [M+Na ] ⁺ , C ₁₆ H ₂₄ F ₂ N ₂ O ₇ Na requires 417 . 1449 .

Methyl 5-N-acetamido-2, 3 , 5, 7-tetradeoxy-2, 3-difluoro-7- ethylacetamido-α-L-manno-2-heptapyranosonate (9 ) Catalytic sodium methoxide was added to a solution of the difluorosialic acid derivative 8, (26.10 mg, 0.07 mmol) in methanol (1 mL) at room temperature (2h) . The mixture was then neutralised (Dowex 50 WX8, H ⁺ form), filtrated and concentrated in vacuo. The residue was purified by flash chromatography (EtOAc —» EtOAc/MeOH, 9:1) to give 9, as a white solid (15.60 mg, 57%) . ¹⁹ F NMR (376.42 MHz, CDCl ₃ ) : δ -123.80 (d, J _F2 , _F3 = 12.2 Hz, F2); - 220.72 (ddd, J _F3 , _F2 = 12.2, J ^" _F3i4 = 27.5, J ^" _F3 , ₃ = 50.4 Hz, F-3) . m/z found (ES+) : 375.1338 [M+Na] ⁺ , C ₁₄ H ₂₂ F ₂ N ₂ O ₆ Na requires 375.1344.

5-N-acetamido-2, 3 , 5-trideoxy-7-ethylacetamido-2 , 3-difluoro-D- eτythro-α-L-manno-2- heptapyranosonate acid (10) Sodium hydroxide 0.5 M was added dropwise to a solution of 9 (15.60 mg, 0.04 mmol) in methanol (0.5 mL) and water (0.5 mL) until pH was basic, 12, at 0 ⁰ C (30 min) . The mixture was concentrated in vacuo and then lyophilised affording compound 10 as a white solid (16.10 mg, 100%) . m/z found (ES+) : 337.1217 [M- H] ^" , C ₁₃ H ₁₉ F ₂ N ₂ O ₆ requires 337.1211.

Methyl-5 -N-acetamido-3 , 5 -dideoxy-3 - fluoro- 8 , 9 - O- isoproylidene-D- eryfc.hro-/7-L-manno-2-nonulopyranosonate 18 :

18

To a solution of methyl-5-i\T-acetamido-3 , 5-dideoxy-3-fluoro-D- eryth.ro-L-manno-2-nonulopyranosonate (2 g, 5.9 mmol) in acetone (150 mL) , p-toluenesulfonic acid (39 mg, 0.21 mmol) , and 2,2- dimethoxypropane (3.6 mL, 29.3 mmol) were added. The mixture was allowed to stir at 50 ⁰ C for 5 hours. Triethylamine (Et ₃ N) (139 μL, 1 mmol) was then added. The crude mixture was concentrated in vacuo and purified by column chromatography (EtOAc/MeOH 20:1) to give 18. Colorless solid (1.949 g, 87%) . ¹ H-NMR (400 MHz, CD ₃ OD) δ 1.31 (S, 3H, Me ₂ C), 1.37 (s, 3H, Me ₂ C), 1.99 (s, 3H, NHAc), 3.52 (d, J = 7.4 Hz, IH, H-7) , 3.82 (s, 3H, OMe) , 3.97 - 4.25 (m, 6H, H-4, H-5, H-6, H-8, H-9, H-9'), 4.81 (dd, J = 49.3, 2.7 Hz, IH, H-3) .

Methyl-5-Jf-acetamido-3 , 5-dideoxy-3- f luoro- 8 , 9- 0-isopropylidene-4- O- (phenoxy) thiocarbonyl-D- eryt±ro-/?- L-manno-2 -nonulopyranosonate 19 :

19

To a solution of 18 (1.949 g, 5.1 mmol) in 70 mL dry DCM and 35 mL pyridine, phenylchlorothionoformate (761 μL, 5.6 mmol) was added at -40 ⁰ C and stirred for 30 minutes at this temperature. The mixture was then brought to room temperature and stirred for further 4 hours. MeOH (5 mL) was then added and after 10 minutes the -crude mixture was concentrated in .vacuo. and purified by column chromatography (EtOAc/MeOH 100:1) to give 19. Colorless solid (2.065 g, 76%) . ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.35 (s, 3H, Me ₂ C), 1.40 (s, 3H, Me ₂ C), 2.35 (s, 3H, NHAc), 3.49 (m, IH, H-5), 3.86 (s, 3H, OMe), 4.07 (dd, J = 21.5, 10.5 Hz, IH, H-9) , 4.37 (m, IH, H-9'), 4.45 (d, J = 4.9 Hz, IH, H-8), 4.58 (q, J = 2.7 Hz, IH, H-6), 5.16 (dd, J = 25.4, 1.7 Hz, IH, H-3), 5.69 (d, J = 3.6 Hz, IH, H-7), 6.11 (ddd, J = 8.7, 23.1 Hz, IH, H-4) , 6.25 (d, J = 8.2 Hz, IH, NHAc), 7.06 - 7.45 (m, 5H, OPh) .

Methyl-5-Jf-acetamido-3 , 4 , 5-trideoxy-3-fluoro-8 , 9-0- isopropylidene-D-erythrσ-β-L-manno-2-nonulopyranosonate 20.

A mixture of dry dioxane (6 mL) , tributyltin hydride (509 μL, 1.89 mmol) , and 2 , 2-bis ( tert-butylperoxy) -butane (123 μL, 0.23 mmol) was prepared and added dropwise to a solution of 19 (265 mg, 0.51 mmol) in dry dioxane (12 mL) at 100 ⁰ C over 2 hours. The mixture was stirred for 2 hours and was then cooled to room temperature. The crude mixture was filtered of, concentrated in vacuo and purified by column chromatography (EtOAc) to give 20. Colorless solid (152 mg, 81%) . ¹ H-NMR (400 MHz, CD ₃ OD) δ 1.32 (s, 3H, Me ₂ C), 1.38 (s, 3H, Me ₂ C), 1.95 (s, 3H, NHAc), 2.09 - 2.23 (m, 2H, H-4, H-4'), 3.53 (d, J = 7.6 Hz, IH, H-7), 3.80 (s, 3H, OMe), 4.02 (m, 2H, H-5, H-9), 4.12 (m, IH, H-9'), 4.27 (m, 2H, H-6, H- 8), 4.74 (m, IH, H-3), 5.49 (m, IH, NHAc) .

Methyl-5-ZV-acetamido-2 ,7,8, 9-tetra-O-acetyl-3 , 4 , 5-trideoxy-3- fluoro-ϋ-erytiiro- _/ ff-L-manno-2-nonulopyranosonate 21.

21

A solution of 20 (183 mg, 0.5 itimol) in 80% Ac0H/H ₂ 0 (10 inL) was stirred at 60° for 2 hours. The crude mixture was then concentrated in vacuo three times with toluene and DCM. The colorless solid was redissolved in pyridine (15 mL) and after adding Ac ₂ O (0.565 mL, 6 mmol) the mixture was heated to 40 ⁰ C for 65 hours. The mixture was then concentrated in vacuo, subjected to a Standard work up (DCM) and the residue was purified by column chromatography (EtOAc/MeOH 10:1) to give 21. Colorless solid (134 mg, 54% (2 steps)) . ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.94 (s, 3H, NHAc), 2.03 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.14 (s, 3H, OAc), 2.16 (s, 3H, OAc), 2.43 (m, 2H, H-4, H-4'), 3.82 (s, 3H, OMe), 3.9 - 4.01 (m, IH, H-6), 3.96 (m, 2H, H-8, H-9), 4.58 (dd, J = 12.5, 1.7 Hz, IH, H-9'), 4.81 (dt, J = 46.5, 5.5, 3.1 Hz, IH, H-3), 5.12 (m, IH, H-5), 5.34 (dd, J = 7.1, 2.3 Hz, IH, H-7) , 5.39 (bd, J = 7.4 Hz, IH, NHAc) .

Methyl-5-.fi/-acetamido-7, 8, 9-tri-O-acetyl-3 , 4, 5-trideoxy-3-fluoro- D-erythro-β-L-manno-2-nonulopyranosonate 22 :

22

To a solution of 21 (132 mg, 0.27 mmol) in DCM (4 mL) was added a solution of hydrazine acetate (99 mg, 1.07 mmol) in MeOH (2 mL) at 0 ⁰ C and left to stand at this temperature for 20 hours. The mixture was then concentrated in vacuo, subjected to a standard work up (DCM) and the residue was purified by column chromatography^ _( EtOAc to EtOAc/MeOH 10:1) to give _22_.__ Colorless solid (62 mg, 51%) . ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.93 (s, 3H, NHAc), 2.04 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.15 (s, 3H, OAc), 2.26 (m, 2H, H-4, H-4'), 3.85 (s, 3H, OMe), 4.10 (m, IH, H-9), 4.18 (m, IH, H-6), 4.27 (in, IH, H-8) , 4.69 -4.86 (m, 2H, H-3, H-9'), 5.29 (m, IH, H-5), 5.35 (m, IH, H-7), 5.53 (bd, J = 8.7 Hz, IH, NHAc) .

Methyl-5-iV-acetamido-7 , 8 , 9-tri-O-acetyl-2 ,3,4, 5-tetradeoxy-2 , 3- difluoro-D-eryt.hro-α-L-manno-2-nonulopyranosonate 23 :

23

To a solution of 22 (134 mg, 0.3 mmol) in dry DCM (5 mL) , DAST (58 μL, 0.45 mmol) was added at -40 ⁰ C and stirred for 45 minutes. The temperature was increased to -10 ⁰ C. Then MeOH (0.1 mL) and NaHCO ₃ (0.1 mL) were added and the crude mixture was concentrated in vacuo, subjected to a Standard work up (DCM) and purified by column chromatography (EtOAc to EtOAc/MeOH 100:1) to give 23. Colorless solid (53 mg, 39%) . ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.96 (s, 3H, NHAc), 2.04 (s, 3H, OAc) , 2.09 (s, 3H, OAc), 2.15 (s, 3H, OAc), 2.38 (m, 2H, H-4, H-4'), 3.86 (s, 3H, OMe), 3.95 (m, IH, H- 6), 4.20 (m, IH, H9), 4.36 (m, 2H, H-8, H-9'), 5.10 (m, IH, H3 ) , 5.29 (m, IH, H-5), 5.33 (m, IH, H-7) , 5.69 (bd, J = 8.7 Hz, IH, NHAc) .

5-iV-acetamido-2 , 3 , 4 , 5-tetradeoxy-2 , 3-dif luoro-D-eryt-hro-α-L- manno-2-nonulopyranosonic acid 24 :

24 To a solution of 23 (53 mg, 0.12 iranol) in dry MeOH (5 mL) , 50 μL 0.5 M NaOMe solution was added and stirred for 3 hours at room temperature. The mixture was neutralised with Amberlite IR 120 H+ form, filtered and concentrated in vacuo. The residue was redissolved in water (0.5 mL) and 0.5 M NaOH solution (0.2 mL) was added and stirred for 30 minutes at room temperature. The mixture was neutralised with Amberlite IR 120 H+ form, filtered and freeze dried to give 24 in quantitative yield. Colorless solid ¹ H-NMR (400 MHz, D ₂ O) δ 1.91 (s, 3H, NHAc) , 2.01 (m, IH, H- 4), 2.38 (m, IH, H-4'), 3.50 (m, IH, H-6) , 3.55 (m, IH, H-9), 3.77 (m, 2H, H-8,H-9'), 3.83 (m, IH, H-7), 4.28 (m, IH, H5), 5.12 (m, IH, H-3) .

Compounds of the invention which may be synthesised using this method include, but are not limited to, compounds 11-17 below:

11 12 13

16 17 Modifications at the_C4 position are performed on the 3-fluoro__ acetonide as shown in Scheme 4. Alkylation occurs selectively at C4 to give the nitrile derivative, which is then converted to the difluoro derivative and finally deprotected. The nitrile group is then reduced to give the primary amine modification.

pyndine

acetate

Scheme 4 - Alkylation route to C4 modified 2 , 3-difluorosialic acids

C4 modified targets containing an epimeric amino group, are synthesised according the sequence given in Scheme 5. The oxazoline (synthesised in 2 steps from sialic acid) is treated with methyl amine in the presence of a palladium(O) catalyst to yield the C4 amino product. This is then fluorinated with XeF ₂ to simultaneously introduce fluorines at both C3 and C2 with the desired stereo chemistry.

Scheme 5 - Synthesis of epimeric C4 dif luorosialic acids References

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes .

Watts et al, Can. J. Chem. (2004) 82, 1581-1588.

Watts et. al, J. Am. Chem. Soc. (2003) 125, 7532-7533.

Watts et al, J ^" . Biol. Chem. (2006) 281, 4149-4155.

Henrisatt et al, Biochem. J, 316, 695-696.