Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4192—1,2,3-Triazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
  • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
  • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
  • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms

Definitions

• the present invention relates compounds that inhibit influenza A virus sialida ⁇ es and are therefore potential anti-influenza agents.
• influenza viruses in particular type A viruses, has had a significant impact on human health over the centuries, including three pandemics in the 20th century (Horimoto and Kawaoka, 2001) .
• Vaccines are available against influenza virus but are effective only against particular strains.
• Dntil recently the drugs of choice for the treatment of influenza A virus infection were the adamantane-based M2 ion channel protein
• sialidase nerve glycose
• NA neuropeptide-like protein
• the sialidase plays a key role in the life cycle of influenza viruses, facilitating the release of virus progeny from the infected cell surface by cleaving the cell-surface virus attachment ligands. Inhibition of the sialidase activity leads to clumping of the virus progeny at the cell surface, resulting in diminished propagation of infection (Falese and Compans, 1976) .
• Oseltamivir carboxylate is currently recommended by the WHO as the primary antiviral treatment for pharmacological management of influenza A(HlNl) virus infection (treatment and prophylaxis) (WHO Guidelines, August 2007), and has been stockpiled by governments around the world as part of their preparedness plans for an outbreak of pandemic influenza.
• strains of influenza virus resistant to oseltamivir carboxylate have been reported, both in oseltamivir- treated patients (reviewed in Reece, 2007) , and recently in circulating strains in wild bird populations.
• With the spectre of the decreased efficacy, through resistance development, of the most widely used sialidase inhibitor, work towards development of next generation sialidase inhibitors is of importance.
• influenza A virus sialidases There are two phylogenetically distinct groups of influenza A virus sialidases - group 1 (Nl, N4, N5, N8) and group 2 (N2, N3, N6, N7, N9) (Russell et al., 2006).
• Influenza A virus strains infecting humans in the 20th century carried either Nl (group 1) or N2 (group 2) sialidases (although there have been reports of a small number of people infected -with N7 viral strains) (Horimoto and Kawaoka, 2001) .
• An influenza A virus strain carrying a group 1 sialidase caused the most devastating influenza pandemic of the 20th century [1914-1918 (HlNl) .
• influenza virus sialidase inhibitors reported to date has been carried-out using the X-ray crystal structures of sialidases from influenza ⁇ virus group 2 (N2 and N9) sialidasea, and influenza B sialidase. These inhibitors show comparable inhibition of both influenza ⁇ virus group 1 and 2
• sialidases however none were designed to exploit binding to the structure of group 1 sialida ⁇ es with the 'open' conformation of the 150 loop.
• the present invention relates to novel compounds which bind to influenza A virus group 1 sialidases with the 150-loop in the 'open' conformation. Consistent with this observation; the compounds are selective inhibitors of influenza A virus group 1 sialidases.
• the present invention provides a compound of general formula (I) which is a selective inhibitor of influenza A virus group 1
• A is O, S or NR 1 ; where Ri is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted acyl or optionally substituted sulfonyl;
• Xi is CO 2 H, P(O)(OH) 2 , NO 2 , SO 2 H, SO 3 H, -C(O)NHOH or tetrazole;
• X 2 is alkyl, aralkyl, alkenyl, alkynyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted alkynyl, OR 2 , SR 2 , NR 2 Rz', or substituted triazole,
• R 2 and R 2 ' are selected independently from optionally substituted acyl, optionally substituted sulfonyl, alkyl, alkenyl, alkynyl, optionally substituted alkyl, or optionally substituted alkenyl,
• R 2 ' is hydrogen
• X3 and X 3 1 are selected independently from hydrogen, R 3 , halogen, CN, OR 3 , NR 3 R 3 1 , NHC (NR 3 ) N(R 3 ) 2 , N 3 , SR 3 , -0-CHa-C(O)-NR 3 R 3 ', -0-CH 2 -C(NH)-NR 3 R 3 1 , -0-CH 2 -C(S)- NR 3 R 3 1
• substituted sulfonyl alkyl, aralkyl, alkenyl, alkynyl, heteroalkyl, heterocy ⁇ lyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, -C(O)R 8 and -S(O) 2 R 8 ,
• R 8 is selected from optionally substituted alkyl and optionally substituted alkenyl
• X 4 is NR4R4 1 , OR 4 , SR 1 , CH 2 C(O)R 4 , CH 2 C(O)OR 4 , CH 2 C(O)NR 4 R 4 ', CHR 4 NO 2 , CKR 4 CN, CHR 4 R 4 ', or CH 2 NHR 4 ,
• R 4 and R 4 ' are selected independently from hydrogen, optionally substituted acyl, optionally
• substituted thioacyl optionally substituted sulfonyl, alkyl, alkenyl, alkynyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted heteroaryl, and optionally substituted heter ⁇ cyclyl;
• X 5 is optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(O)R 5 , - CO 2 R 5 / -C(O)NR 5 R 5 ', -P(O)(OR 5 )(OR 5 '), -P(O)(OR 5 )(NR 5 R 5 1 ), -P (O) (NR 5 R 5 ') 2» CN, ORg, azide, NHR 6 , NR 6 Re', SR 5 , or optionally substituted triazole,
• R 5 and R 5 • are independently selected from hydrogen, optionally substituted alkyl, optionally
• Re and R 6 1 are independently selected from optionally substituted acyl, optionally substituted sulfonyl, optionally substituted alkyl, optionally
• aralkyl optionally substituted alkenyl, optionally substituted aryl, heteroaryl, or heterocyclyl .
• a pharmaceutical composition comprising a compound of general formula (I) and a
• a method of preventing or treating influenza in a subject comprising administering to said subject a compound of general formula (I) .
• a compound of general formula (I) in the manufacture of a' medicament for the prevention or treatment of influenza.
• a compound of general formula (I) in the prevention or treatment of influenza.
• a seventh aspect of the present invention there is provided a method of preparing a compound of general formula (I)/ comprising the steps of:
• X 2 , X 3 , X 4 and Xs are as defined and may be protected by ' protecting groups
• X ⁇ is X x , or a functional group that can be modified to form Xi, where X 6 can be selected from, but is not limited to, CHO, CN, CH 2 OR 1 , thiazole, and
• Z is a group that can be activated to enable beta- elimination
• Z is a' halide and elimination is achieved under basic conditions
• Z is a halide and elimination is achieved in the presence of a heavy metal reagent
• Z is acyloxy and elimination is achieved under Lewis acidic conditions
• Z is alkoxy and elimination is achieved under acetolysis conditions
• Z is phosphite and elimination is achieved under Lewis acidic conditions.
• a compound of general formula IV where Z is halide can be formed by balogenation of a compound of the general formula VI where Q can be selected from, but is not limited to, -COOR' , -CN, -CH 2 OR' .
• E is a halogen.
• Xi is protected with an alkyl group, which can be removed by hydrolysis.
• Figure 1 A. Superimposition of influenza A virus N8 sialidase-inhibitor complexes of 3-allyl-NeuAc2en (7) (dark grey; complex obtained after 60 min. soak) and Neu5Ac2en (white; PDB: 2htr) .
• the 3-allyl-Neu5Ac2en complex maintains the 'open 1 conformation of the 150-loop seen in the apo structure (Russell et al., 2006)., in contrast to the complex with Neu5Ac2en where the 150-loop is 'closed 1 (Fig. 1C) .
• the N8-(9c) complex maintains an 'open' conformation of the 150-loop with the C-3 phenylallyl ⁇ ub ⁇ tituent extending into the 150-cavity.
• FIG. 3 Influenza A virus N8 sialidase- inhibitor complex of 3- (p-tolyl) allyl-Neu5Ac2en (9d) .
• Left panel 3- (p-tolyl) allyl-Neu5A ⁇ 2en (9d) in stick format;
• Right panel 3- (p-tolyl) allyl-Neu5Ac2en (9d) in CPK format.
• the N8- (9d) complex maintains an 'open' conformation of the 150 -loop with the C-3 (p-tolyl)allyl substituent extending well into the 150 -cavity.
• the invention discloses compounds that selectively inhibit influenza A virus group 1 sialidases and may therefore interrupt the infectious cycle of influenza A virus strains.
• the invention is concerned with compounds of general formula (I) ;
• A is O, S or NRi
• Ri is hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted acyl or optionally substituted sulfonyl;
• X 1 is CO 2 H, P(O)(OH) 2 , NO 2 , SO 2 H, SO 3 H, -C(O)NHOH or tetraz ⁇ le;
• X 2 is alkyl, aralkyl, alkenyl, alkynyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alk ⁇ nyl, optionally substituted alkynyl, OR 2 / SR 2 , NR 2 R 2 1 / or substituted triazole,
• R 2 and R 2 1 are selected independently from optionally substituted a ⁇ yl, optionally substituted sulfonyl, alkyl, alkenyl, alkynyl, optionally substituted alkyl, or optionally substituted alkenyl,
• R 2 1 is hydrogen
• X 3 and X 3 ' are selected independently from hydrogen, R 3 , halogen, CN, OR 3 , NR 3 R 3 ', NHC (NR 3 ) N (R 3 ) 2 , N 3 , SR 3 , -0-CH 2 -C(O)-NR 3 R 3 1 , -0-CH 2 -C(NH)-NR 3 R 3 1 , -0-CH 2 -C(S)- NR 3 R 3 '
• substituted sulfonyl alkyl, aralkyl, alkenyl, alkynyl, heteroalkyl, heterocyclyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, -C(O)R 8 and -S(O) 2 R 8 '
• Re is selected from optionally substituted alkyl and optionally substituted alkenyl
• X 4 is NR 4 R 4 1 , 0 R 4 , S R 4 , CH 2 C(O) R 4 , CH 2 C(O)OR 4 , CH 2 C(O)N R 4 R 4 1 , CH R 4 NO 2 , CH R 4 CN, CH R 4 R 4 1 , or CH 2 NHR,
• R 4 and R 4 1 are selected independently from hydrogen optionally substituted acyl, optionally
• substituted thioacyl optionally substituted sulfonyl, alkyl, alkenyl, alkynyl, optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;
• X 5 is optionally substituted alkyl, optionally substituted aralkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(O)R 5 , - CO 2 R 5 , -C (O) NR 5 R 5 ' , -P (0) (OR 5 ) (OR 5 ' ) , -P (O) (OR 5 ) (NR 5 Rs ' ) , -P(O) (NR 5 R 5 1 ) 2 .
• CN OR 6 , azide, NHR 6 , NR 6 R*;', SR 6 , or
• R 5 and Rs• are independently selected from hydrogen, optionally substituted alJcyl, optionally
• R 6 and R 5 ' are independently selected from optionally substituted acyl, optionally substituted sulfonyl, optionally substituted alkyl, optionally
• substituted aralkyl optionally substituted alkenyl, optionally substituted aryl, heteroaryl, or heterocy ⁇ lyl .
• X 5 denotes CH 2 YR 7 , CHYR 7 CH 2 YR 7 or CHYR 7 CHY R 7 CH 2 YR 7 ,
• Y is 0, S, or NR 7 ', and successive Y moieties in an X 5 group are the 1 same or different, or
• two adjacent YR 7 groups together form part of a ring structure which optionally includes at least one heteroatom selected from 0, S and N and is optionally substituted; in particular, an epoxide, aziridine, 5 or 6 membered cyclic ether group,
• R 7 and R 7 * are independently selected from hydrogen, optionally substituted acyl, optionally
• the compounds are of general formula (II) , with the stereochemistry as shown;
• A is 0.
• an embodiment Xi is CO 2 H or P(O) (OH) 2 or an ester thereof.
• the ester will readily hydrolyse in vivo into the free acid.
• Xi is CO 2 H.
• X 3 ' is H and X 3 is selected from
• R 3 halogen, CN, OR 3 , NR 3 R 3 1 , NHC (NR 3 )N(R 3 ) 2/ N 3 , SR 3 and optionally substituted triazole,
• R 3 and R 3 1 are independently selected from alkyl, alkenyl, alkynyl, optionally substituted alkyl, optionally substituted alkenyl, -C(O)R 8 or -S(O) 2 R 8 /
• R 8 is selected from optionally substituted alkyl and optionally substituted alkenyl.
• X 4 is - NR 4 R 4 1 .
• R 5 is optionally substituted acyl and R 5 1 is hydrogen, typically acyl such as acetyl.
• the compounds are of general formula (III) , with the stereochemistry as shown;
• one of Xs and X 8 1 is hydrogen
• one of X 9 and X 9 ' is hydrogen, .and
• X 7 , X 7 1 , Xe, Xs', X 9 , and X 9 1 are the same or different, and are selected from H, OR 7 , NR 7 R 7 ', SR 7 , or optionally substituted triazole, or
• the compounds are selected from the group consisting of:
• alJcyl used either alone or in a compound word such as “optionally substituted alkyl” or “optionally substituted cycloalkyl” denotes straight chain, branched or mono- or poly- cyclic alkyl.
• straight chain and branched C alkyl examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1, 1-dimethylpropyl, hexyl , 4 -methylpentyl, 1-raethylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2- dim ⁇ thylbutyl, 3,3-dimethylbutyl, 1, 2-dimethylbutyl, 1,3- dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2- trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2 , 2-dimethylpentyl, 3,3-dimethylpentyl, 4,4- dimethylpentyl, 1,2-d
• alkyl is C1-C5 alkyl.
• alkenyl used either alone or in compound words such as “alkenyloxy” denotes groups formed from straight chain, branched or cyclic alkenes including ethylenically mono-, di- or poly-unsaturated alkyl or cycloalkyl groups as defined above.
• alkenyl examples include allyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1- hexenyl, 3-hexenyl, cyclohexenyl , 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3- cyclohexadienyl, 1,4-cyclohexadienyl, 1,3,- cycloheptadienyl, 1,3, 5-cycloheptatrienyl and 1,3,
• an acyl group may include between 1 and 30 carbon atoms but more commonly is an aliphatic C1-C5 acyl such as acetyl.
• acyl include straight chain or branched alkanoyl such as forroyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2- dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl,
• nonanoyl decanoyl, undecanoyl, dodecanoyl, tridecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as cyclopropylcarbonyl ⁇ yclobutylcarbonyl,
• cyclopentylcarbonyl and cyclohexylcarbonyl cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutan ⁇ yl, phenylisobutyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g.
• naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl ; aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl) ; hetero ⁇ ycliccarbonyl;
• phenylalkenoyl e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl
• naphthylalkenoyl e.g. naphthylprop
• heterocyclicalkanoyl such as thienylacetyl
• thienylpropanoyl 'thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; and h ⁇ terocyclicalkenoyl such as heterocycli ⁇ propenoyl, h ⁇ t ⁇ rocyclicbutenoyl,
• sulfonyl used either alone or in compound words such as “optionally substituted sulfonyl” denotes one of the groups -S(O) 2 Rs wherein each R 9 is independently H, optionally substituted alkyl or
• the group in its entirety may be, for example, a sulfonate ester or amide, depending on the. context, such as -0-S(O) 2 R 9 Or -NR 4 -S- (O) 2 R 9 .
• aryl used either alone or in compound words such as “optionally substituted aryl”, “optionally substituted aryloxy” or “optionally substituted
• heteroaryl denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons ("carbocyclic aryl” or “carboaryl”) or aromatic heterocyclic
• heteroaryl ring systems.
• carbocyclic aryl include phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, napthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl.
• heteroaryl examples include pyridyl, 4- phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazolyl, pyrrolydinyl, pyrldinyl, piperidinyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoguinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like.
• a carbocyclic aromatic ring system contains 6-10 carbon atoms and an aromatic heterocyclic ring system contains 1 to 4 heteratoms independently selected from N, 0 and S and up to 9 carbon atoms in the like.
• heterocyclic used either " alone or in compound words such as “"optionally substituted saturated or unsaturated heterocyclyl” denotes monocyclic or polycyclic
• heterocyclyl groups containing at least one heteroatom atom selected from nitrogen, sulphur and oxygen.
• Suitable heterocyclyl groups include N-containing heterocyclic groups, such as, unsaturated 3 to 6 membered
• heteromonocyclic groups containing 1 to 4 nitrogen atoms for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl , pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; - ⁇
• unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom such as, oxiranyl, pyranyl or furyl;
• unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, oxazolyl, isoxazolyl or oxadiazolyl;
• unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, benzoxazolyl or benzoxadiazolyl;
• unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl;
• unsaturated condensed heterocyclic group containing 1 to 2 sulphur atoms and 1 to' 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl.
• carbohydrate denotes a carbohydrate residue or a functionalised or deoxygenated carbohydrate residue, and includes monosaccharides and
• a carbohydrate residue is an acyclic polyhydroxy-aldehyde or ketone, or one of their cyclic tautomers, and includes a compound resulting from
• Oxygen atoms may be replaced by hydrogen or bonds to a halogen, nitrogen, sulfur or carbon atoms, or carbon- oxygen bonds such as in ethers or esters may be
• carbohydrates include but are not limited to D-galactose, D-galactofuranose, N-acetyl-D- galactofuranose, D-galactopyranose, N-acetyl-D- galactopyranose, D-glucose, D-glucofuranose, N-acetyl-D- glucofuranose, D-glucopyranose and N-acetyl-D- glucopyranose, D-mannose, D-mannofuranose, D- mannopyranose, N-acetyl-D-mannopyranoae, D- arabinofuranose, D-arabinopyranose, L-rhamnopyranose, D- ribose, D-fucose, N-acylneuraminic acid, 2-keto-3-deoxy- nonulosonic acid, 2-keto-3-deoxy- ⁇ ctulosonic acid, D- galacturonic acid, D-glu
• ⁇ optionally substituted means that a group may or may not be further substituted with one or more functional groups such as alkyl, alkenyl. alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro,
• nitroalkyl nitroalkenyl, nitroalkynyl, nifcroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alky ⁇ ylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl,
• alkynylacyl arylacyl, acylamino, diacylamino, a ⁇ yloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocy ⁇ loxy, heterocyclamino, halohetero ⁇ yclyl,
• any of the moieties whose length is defined in terms of the number of carbon atoms present may possess any number of carbon atoms within the specified range.
• the compounds of the invention may be prepared by manipulation of carbohydrate structures to introduce the functional groups as described in the general formulae.
• An extensive array of methodologies has been developed to manipulate different positions on carbohydrate templates as disclosed, for example, in Ernst, Hart & Sinay, 2000;
• neuraminic acid template have been developed as disclosed for example in Zbiral 1992; von Itzstein and Thomson,
• halohydrin formation can be achieved using N-bromosuccinimide, as described for example in Okamoto et al., 1987. Radical reaction of the bromohydrin can be employed to introduce a carbon-linked ⁇ ubstituent X 2 using Bu 3 Sn(Z 2 )/ as described for example in Paulsen and Mats ⁇ hulat, 1991.
• Chlorination or bromination at the alpha position and subsequent elimination of HX can be employed to give the beta- substituted alpha,beta-unsaturated derivative.
• Direct introduction of a carbon-linked s ⁇ bstituent X 2 can be achieved through transition metal-mediated radical
• sulfuric acid, acetic acid and acetic anhydride such as described in Kok et al., 1999, can be employed to form the beta-substituted alpha,beta-unsaturated derivative.
• bromination alpha to the carboxylate (path C) followed by elimination of HBr can be employed to form the beta- substituted alpha,beta-un ⁇ aturated derivative.
• G-2 Hoveyda-Grubbs 2nd generation
• HG-2 Hoveyda-Grubbs 2nd generation
• Grela's catalyst Grela's catalyst
• the multivalent template is selected from the group consisting of, but not limited to, polystyrene nanopartides, ceramic nanoparticles, coated gold particles / di-, tri- and tetra-ante ⁇ nary structures and dendrimers (as described for example in Roy 1997) , liposomes, micelles, and virus hybrid systems.
• Multivalent arrays of influenza virus sialidase inhibitors are selected from the group consisting of, but not limited to, polystyrene nanopartides, ceramic nanoparticles, coated gold particles / di-, tri- and tetra-ante ⁇ nary structures and dendrimers (as described for example in Roy 1997) , liposomes, micelles, and virus hybrid systems.
• the compounds of the invention interrupt the infectious cycle of influenza A virus strains, and therefore are useful in the prevention or treatment of influenza in a subject, particularly a human subject when administered in a therapeutically effective amount.
• terapéuticaally effective amount means an amount of a compound of the present invention effective to yield a desired therapeutic response, for example to prevent or treat a disease by administration of a pharmaceutically-active agent.
• the specific "therapeutically effective amount” will, obviously, vary with such factors as the particular condition being treated, the physical condition and clinical history of the subject, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the compound or its
• a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering the compound of general formula (X) to the subject.
• the carrier may be liquid or solid, and is selected with the planned manner of administration in mind.
• compositions may include any solvate, hydrate or any other compound or prodrug which, upon administration to a subject, is capable of providing (directly or indirectly) a compound of formula I or an antiviralIy active metabolite or residue thereof.
• the pharmaceutically acceptable salts include acid addition salts, base addition salts, salts of pharmaceutically acceptable esters and the salts of quaternary amines and pyridiniums.
• the acid addition salts are formed from a compound of the invention and a pharmaceutically acceptable inorganic or organic acid including but not limited to hydrochloric, hydrobromic, sulphuric, phosphoric, methanesulfonic, toluenesulphonic, benzenesulphonic, acetic, propionic, ascorbic, citric, malonic, fumaric, maleic, lactic, salicyclic, sulfamic, or tartaxtic acids.
• the counter ion of quaternary amines and pyridiniums include chloride, bromide, iodide, sulfate, phosphate, methansulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartate.
• the base addition salts include but are not limited to salts such as sodium, potassium, calcium, lithium, magnesium, ammonium and alkylammonium.
• basis nitrogen-containing groups may be quaternised with such agents as lower alkyl halides, such a ⁇ methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
• the salts may be made in a known manner, for example by treating the compound with an appropriate acid or base in the presence of a suitable solvent.
• the compounds of the invention may be in crystalline form either as the free compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention.
• solvate is a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents preferably do not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of salvation are
• pro-drug is used in its broadest sense and encompass those derivatives that are converted in vivo to the compounds of the invention. Such derivate ⁇ would readily occur to those skilled in the art, and include, for example, compounds where a free hydroxy1 group is converted into an ester derivative or a ring nitrogen atom is converted to an N-oxide. Examples of ester derivatives include alkyl esters, phosphate esters and those formed from amino acids, preferably valine. Any compound that is a prodrug of a compound of the invention is within the scope and spirit of the invention. Conventional
• the compound of general formula (I) may be administered in any convenient form including orally, topically, or parenteralIy in dosage unit formulations containing conventional non-toxic pharmaceutically
• compositions can be administered, for in vivo application, parenteralIy by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intra-arterial, intraperitoneally,
• inhalation may be by way a dry powder inhaler, a metered dose inhaler or nebulizer as described, for example, in WO99/16421, the contents of which are incorporated herein by reference.
• agents may be added or dissolved in an
• treating covers any treatment of, or prevention of infection in a vertebrate, a mammal, particularly a human, and includes: preventing the infection from occurring in a subject that may have been exposed to an influenza virus, but has not yet been diagnosed as affected; inhibiting the infection, ie., arresting its development; or relieving or
• compositions of the invention comprise a pharmaceutically acceptable carrier
• Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, trehalose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives such as hydroxypropylraethyl cellulose, polymers such as
• Intravenous ' vehicles include fluid and nutrient replenishers.
• Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.
• Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co., 1405-1412,1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference.
• compositions are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed.). When desired the formulations may be adapted to give sustained release of the active ingredient.
• compositions are preferably prepared and administered in dosage units.
• Solid dosage unite include tablets, capsules and suppositories.
• the administration of the daily dose can be carried out both by single administration in the form of an Individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
• compositions according to the invention may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the microbial infection and the weight and general state of the subject. Typically/ dosages used in vitro may provide useful guidance in the amounts useful for in situ
• Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be. in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
• Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension.
• excipients may be suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example,
• heptade.caethylenoxycetanol (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol
• the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
• This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as those mentioned above.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• the acceptable vehicles and solvents which may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed, including synthetic mono-or diglycerides .
• fatty acids such, as oleic acid find use in the preparation of
• Liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
• Liposomes can be formed from a variety of phospholipids, such as
• cholesterol cholesterol, stearylamine, or phosphatidylcholines.
• Compounds of general formula (I) may also be administered in combination with cyclodextrins for enhanced aqueous solubility.
• the compounds of the invention may be administered by any of the methods and formulations employed in the art for intranasal administration.
• the compounds may be administered in the form of a solution or a suspension or as a dry powder.
• Solutions and suspensions will generally be aqueous, for example prepared from water alone (for example sterile or pyrogen-free water) , or water and a physiologically acceptable co-solvent (for example ethanol, propylene glycol, and polyethylene glycols such as PEG 400) .
• a physiologically acceptable co-solvent for example ethanol, propylene glycol, and polyethylene glycols such as PEG 400.
• preservatives such as benzalkoniuxn chloride
• solubilising agents/surfactants such as polysorbate ⁇ (e.g. Tween 80, Span 80, benzalkonium chloride) , buffering agents, isotonicity-adjusting agents (for example sodium chloride), absorption enhancers and viscosity enhancers.
• Suspensions may additionally contain suspending agents (for example microcrystalline cellulose, carboxymethyl cellulose sodium) .
• Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray, or metered dose inhaler.
• the formulations may be provided in single or multi-dose fashion. In the latter case a means of dose metering is desirably provided.
• a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension.
• a spray this may be achieved for example by means of a metering atomising spray pump.
• Intranasal administration may also be achieved by means of an aerosol formulation in which the compound is provided in a pressurised pack with a suitable
• propelIant such as a chlorofluorocarbon (CFC) , for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetraflu ⁇ roroethane, carbon dioxide or other suitable gas.
• CFC chlorofluorocarbon
• the aerosol may conveniently also contain a surfactant such as lecithin.
• the dose of drug may be controlled by provision of a metered valve.
• the compounds may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVF) .
• a powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVF) .
• the powder carrier will form a gel in the nasal cavity.
• the powder composition may be presented in unit dose form, for example in capsules or cartridges of e.g. gelatin or blister packs from which the powder may be administered by means of an inhaler.
• the intranasal formulations the compound will generally have a small particle size, for example of the order of 5 microns or. less. Such a particle size may be obtained by means known in the art, for example by micr ⁇ nisation.
• Dosage levels of the compound of general formula (X) of the present invention will usually be of the order of about 0.05mg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.05mg to about lOmg per kilogram body weight per day (from about 0. Ig to about 3g per patient per day) .
• the amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration.
• a formulation, intended for oral administration to humans may contain about img to Ig of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition.
• Dosage unit forms will generally contain between from about 5mg to 500mg of active ingredient.
• the compounds of the invention may additionally be combined with other compounds to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically-active agents, as long as the combination does not eliminate the activity of the compound of general formula (I) of this invention. In an embodiment are used in combination with other therapeutic agents, for example other anti-infective agents. In particular the compounds of the invention may be employed with other antiviral agents.
• the invention thus provides in a further aspect a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt or derivative thereof together with another therapeutically active agent, in particular an antiviral agent.
• Suitable therapeutic agents for use in such combinations include other anti-infective agents, in particular anti-bacterial and anti-viral agents such as those used to treat respiratory infections.
• anti-infective agents in particular anti-bacterial and anti-viral agents such as those used to treat respiratory infections.
• anti-bacterial and anti-viral agents such as those used to treat respiratory infections.
• other compounds effective against influenza viruses such as amantadine, rimantadine and ribavirin, may be included in such combinations .
• each compound when the compounds of the invention are used with a second therapeutic agent active against the same virus, the dose of each compound may either be the same as or differ from that employed when each compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.
• halide can be introduced beta to the
• halohydrin formation for example using N-bromo ⁇ uccinir ⁇ ide, as described for example in Okamoto et al., 1987.
• Radical reaction of the bromohydrin can be employed to introduce a carbon-linked substituent Z 2 using Bu 3 Sn (Z 2 ) / as described for example in Paulsen and Matschulat, 1991 (described in Example 1) .
• the hydroxyl group alpha to the carboxylate is then converted to a leaving group suitable to enable beta-elimination.
• Methods of beta-elimination include activation of the position alpha to the carboxylate of the beta-substituted ester with halogen, phosphite [as described for example in
• manipulation of the allyl group can be achieved using a range of reagents, for example using Grubbs catalyst as exemplified in Scheme 2 (described in Examples 5-18) , and borane reagents as exemplified in Scheme 3 (described in Examples 19 and 20) .
• Scheme 6 selective alkylation of the C-4 hydroxyl group of a suitably protected precursor can be achieved using an alkyl halide in the presence of Ag 2 O or a hydride reagent (as exemplified in Scheme 6) [a ⁇ described for example in Tindal, D.J. et al., Bioorg. Med. Cheat.
• the introduced alkyl group can be further modified [as described for example in Ikeda, K. et al., CarJbohydr. Res. (2001) 330. 31-41] .
• Scheme 6
• a substituent such as azide (as exemplified in Scheme 8) or thiolacetate at C-4 [as described for example in von Xtzstein, M. et al.,
• the introduced azide group can be further modified [as described for example in: Chandler, M. et al. J. Chem. Soc. Perkin Trans. I (1995) 1173-1180; Lu and Gervay-Hague, Carbohydr. Res. (2007) 342, 1636- 1650] .
• Scheme 8 Reagents and conditions: (a) BF 3 .Et 2 O, dry MeOH, dry CH 2 Cl 2/ rt, 20 h; (b) Azidotrimethylsilane, anhydrous 11 BuOH, 80 "C, 24 h. Exemplary methods of preparing the compounds of the invention, where X 2 is linked through oxygen to the scaffold, are shown in Schemes 9 and 10 (described in Examples 31-43) .
• a hydroxy1 group can be introduced beta to a carboxylate by manipulation of an alpha-beta
• the epoxide may be opened to introduce an alkyl group [as described for example in Okamoto et al. (1987)] or an acyl group [using a method such as described for example in Timmers, CM. et al., J. Carbohydr, Chem. (1998) 17, 471-487] .
• the beta-hydroxyl group can be alkylated using an alkyl halide in the presence of Ag 2 O or a hydride reagent (described in Examples 34 and 39) .
• the substituent alpha to the carboxylate is then converted to a leaving group suitable to enable beta-elimination.
• this substituent is p-methoxybenzyloxy
• the p- methoxybenzyl group can be removed for example by
• Beta-elimination of HBr to form the beta- substituted alpha-beta-carboxylate functionality can be performed using for example a base such DBD or
• Scheme 10 the side chain introduced at C-3 can be further modified according to known procedures.
• X 2 is -0-CH 2 CN
• further manipulation of the ⁇ yano group can be achieved, for example, through
• Example 42 reduction to the amine (described in Example 42), and subsequent conversion of the amine to an azide (described in Example 42).
• X 2 is -0-CH 2 CH 2 NH 2 [for example (38)] the amine can be further modified by acylation under standard conditions.
• Scheme 11 an exemplary method for manipulation of the side -chain X 2 through elaboration of an azido group to a substituted triazole is shown in Scheme 11 (described in Examples 44 and 45 ) .
• an azide can be reacted with a substituted alkyne to produce a substituted triazole [as described for example in Lu and Gervay-Hague, Carbohydr. Res . (2007 )
• general precursors for the preparation of compounds of general formula (I) are compounds of general formula (IV) , where Z is a group that, in conjunction with the hydrogen beta to X 6 , is removed from (XV) to form an alpha,beta-unsaturated compound (VXX) , in which X 6 is Xi, or is a functional group that can be subsequently modified to obtain X x .
• Z is a group that, in conjunction with the hydrogen beta to X 6 , is removed from (XV) to form an alpha,beta-unsaturated compound (VXX) , in which X 6 is Xi, or is a functional group that can be subsequently modified to obtain X x .
• Xe is a functional group that can be modified to form X 1
• X 6 can be selected from, but is not limited to, CHO, CH 2 OR 1 , CN, or a thiazole, where R' is a protecting group.
• CHO and CH 2 OR 1 can be converted to Xi, where Xi is a carboxylate function, using oxidation methods.
• Xn general, CN can be converted, to X 1 , where Xi is a carboxylate function, by reaction under acidic or basic conditions.
• a thiazole can be converted to Xi, where X 1 is a carboxylate function, by a series of
• Formation of (VII) from (IV) when Z is halide can be performed, for example, by the use of a base [as described for example in Blattner, R. et al., «7. Chem.
• Formation of (VII) from (IV) when Z is alkoxy can be performed, for example, under acetolysis condition's [as described for example in Kok, G.B. et al., Chem. Commun. (1996) 2017]. Formation of (VII) from (IV) when Z is phosphite can be performed, for example, by the use of a Lewis acid [as described for example in Stolz, F. et al., J. Org. Chem. (2004) 69, 665- 679] .
• Compounds of general formula (IV) where Z is a halide can be formed, as described and exemplified in the Methods section.
• Compounds of general formula (IV) where Z is a halide can also be formed by halogenation of a compound of the general formula (VI) where Q can be selected from, but is not limited to, -COOR', -CN, and -
• MeOH (50%, 4 mL) at 5 "C or room temperature is adjusted to pH 13 using ag. NaOH (1 H) .
• the solution is stirred at a temperature of 5 'C or room temperature and the progress of reaction is monitored by TIiC analysis (EtOAc/MeOH/H 2 O, 7:2:1).
• TIiC analysis EtOAc/MeOH/H 2 O, 7:2:1.
• Amberlite ⁇ IR-120 (H + ) resin is added to adjust pH 3
• the reaction mixture is filtered, the resin is washed with MeOH/H 2 O 1:1 (25 mL) and the filtrate is concentrated to dryness under vacuum.
• the crude product is dissolved in water, the pH of the solution is adjusted to pH 7 using aq. NaOH (1 M) , and the solution is
• the product can be purified by reverse phase HPLC.
• bromohydrin (2) (1.55 g, 2.71 nunol) [prepared from (1) according to tlie method of
• the reaction mixture was concentrated under reduced pressure and azeotroped with toluene (3 x 20 mL) to yield glycosyl chloride (5) as an off-white foam.
• the crude chloride was taken up in dxy dichloromethane (10 mL), to which DBU (232 microL, 1.56 mmol, 4 mole equiv. ) was added, and the reaction was left to ⁇ tir at room temperature under N 2 for 8 h.
• the reaction mixture was evaporated to dryness, taken up in chloroform and washed successively with satd aq. NH 4 Cl, H 2 O, and satd aq. NaCl.
• the organic phase was dried (anhydrous Na 2 SO 4 ) , filtered and evaporated under reduced pressure, and the residue purified by flash chromatography on silica gel
• reaction mixture was stirred at 50 'C for 12 h.
• the crude boronic acid (10) was treated with hydrogen peroxide (2 mL) and aq. NaOH solution (0.2 mL, 1 N) at 0 'C and the reaction mixture was stirred at room temperature for 30 min.
• the reaction mixture is diluted with ethyl acetate and washed with aq. NaCl.
• the organic phase was dried (Na 2 SOe) r filtered and evaporated under reduced pressure.
• the crude product was dissolved in dry acetonitrile under N 2 and to it was added acetic anhydride (1 mL) followed by DMAP (5 mg) .
• the reaction mixture was stirred at room for 24 h after which it was concentrated under reduced
• the deacetylated compound was deprotected according to the general procedure at rt for 3 h.
• the crude product was purified by reverse phase HPLC and then lyophilized to give title compound (16) as white soild (29 mg, isolated yield 71%) .
• reaction mixture was then quenched with 0.1 mL of dry MeOH and, after a workup consisting of evaporation of DMF and aqueous extraction, the crude product was chroraatographed using 5 i 1 BtOAc/hexanes as eluent to give the desired product (20) as an off-white foam (25 mg, 47%) .
• the deprotection steps involved the initial removal of the i ⁇ opropylidene group protecting the C- 8 and C-9 hydroxyl groups followed by the de-esterification of the C-I carboxylic acid.
• De-isopropylidination of (20) was carried-out by the use of 80% AcOH at 80 0 C for 1 hr.
• After evaporation of the AcOH 4 de-esterification of (21) was carried out according to the general procedure at 0 * C-rt, 12 h.
• the crude product was purified by reverse phase HPLC and then lyophilized to give the title compound (22) as a white solid (18 mg, 83%) .
• the reaction mixture was diluted with ethyl acetate (50 mL) and the organic layer was washed with hydrochloric acid (0.1 N, 4 mL) and water (2 x 5 mL) .
• the combined aqueous layer was extracted with ethyl acetate (2 x 5 mL ⁇ .
• the combined organic extracts were then dried (anhydrous Na 2 SO 4 ) , and evaporated under reduced pressure to give crude product which was purified by column chromatography on silica (Acetone-Hexane, 30:70) to yield the title compound (24) as a white foam (105 mg, 31%) .
• Example 40 Methyl 5-acetamido-4,7, 8, 9-tetra-O-acetyl-3, 5- dodeoxy-3-O-acetonitrile-c-erythro- ⁇ -r.-g"luco-non-2- ulopyranosonate (36) ;
• Enzyme inhibition Inhibition data against influenza A virus Nl and N2 sialida ⁇ es for compounds (7) , (9b-d) and (21) , compared to parent template Neu5Ac2en, is described in Table 1. Sialida ⁇ e inhibition assays were carried-out on MES- ⁇ -dodecyl-D-maltoside cell extracts prepared from 293T cells transiently expressing the viral enzyme according to the known method (Rameix-Welti et al . , 2006) . Enzymatic activity was measured using the
• Cal Results are given as means ⁇ SD for at least three independent determinations for duplicate samples.
• Neu5Ac2en is described in Table 2.
• the H274Y, N294S and Q136K mutations were each introduced into a.plasmidic clone encoding the Nl of A/Hong K ⁇ ng/156/97, according to the known method (Raraeix-Welti et al., 2006). Sialidase Inhibition assays were carried-out as described in Example 46.
• the 50% effective concentration (EC 50 ) was determined graphically as the concentration of inhibitor that induced a 50% reduction in the average plaque diameter.
• the compounds with the C-3 side- chain (X 2 ) (7) and (9d) selectively inhibit growth of the influenza viruses that express an Nl sialidase (HlNl) , compared to the N2-expressing virus (H3N2) .
• TcLbIe 3. Xn vitro sensitivity of influenza virus isolates to (7) and (9d) , in comparison to standard Neu5Ac2en.
• Neu5Ac2en showed a similar decrease in plaque size for both viruses size moving from 1 to 10 to 100 microM concentration.
• N8 sialidase-inhibitor complex ⁇ group 1 (N8) influenza A virus sialidase crystal, prepared as previously- described (Russell et al., 2006), was soaked in li ⁇ H solution of compound (7) for 60 minutes.
• the N8/(7) complex ( Figures IA and IB) has the 'open' conformation of the 150-loop, in contrast to the N8 complex with C-3 unsubstituted Neu5Ac2en ( Figure 1C) where the 150-loop is 'closed 1 (Russell et al., 2006).
• the 3-allyl-Neu5Ac2en complex maintains the Open" conformation of the ' 150-loop seen in the apo structure (Russell et al., 2006), with the C-3 allyl side-chain of (7) bound into the 150-cavity as anticipated.
• Example 50
• influenza A virus N8 sialidase-inhibitor complex A group 1 (N8) influenza A virus sialidase crystal, prepared as previously
• left and right panels has the 'open* conformation of the 150-loop with the C-3 (p-tolyl) allyl substituent extending well into the 150-cavity.
• the dihydropyran ring and C-2, C-4, C-5, and C-6 substituents of (9d) and Neu5A ⁇ 2en have very similar positions in the active site.
• the phenyl ring of (9d) is positioned adjacent to Asp-151 in the open 150-loop conformation indicating the potential for interaction with this residue by suitable functionality (X 2 ) extending from the C-3 ppsition of Neu5Ac2en or the corresponding position of other compositions of the invention.

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