WO1992006691A1

WO1992006691A1 - Anti-viral compounds that bind the active site of influenza neuramidase and display in vivo activity against orthomyxovirus and paramyxovirus

Info

Publication number: WO1992006691A1
Application number: PCT/AU1990/000501
Authority: WO
Inventors: Peter Malcolm Colman; Laurence Mark Von Itzstein; Jose Noozhumurray Varghese; Wen-Yang Wu; The Van Phan; Hume Forrest White
Original assignee: Biota Scientific Management Pty. Ltd.
Priority date: 1990-10-19
Filing date: 1990-10-19
Publication date: 1992-04-30

Abstract

A pharmacologically active composition of the invention comprises (i) a virus-inhibiting amount of a compound that binds the active site of influenza virus neuraminidase and that displays in vivo activity against orthomyxovirus or paramyxovirus; and (ii) a pharmaceutically-acceptable carrier for the compound which is preferably suitable for intranasal administration. In preferred embodiments, the compound possesses a Ki value, with respect to the active site, of less than 10-7 M. Preferably, the compound is a carbocyclic or heterocyclic compound comprised of a 5-, 6- or 7-membered ring carrying a substituent selected from a carboxylate moiety and an analogue thereof, the ring and the substituent being positioned in the same plane.

Description

Anti-viral compounds that bind the active site of influenza neuramidase and display in-vivo activity against orthomyxovirus paramyxovirus

Background of the Invention

The present invention relates to a new class of anti-viral compounds, exemplified by certain 2-deoxy and 2,3- dehydro analogues of α-D-neuraminic acid, and to their use, via inhibition of viral neuraminidases, for the prophylaxis and for the treatment of infections such as influenza,

Newcastle disease and fowl plague.

Enzymes with the ability to cleave N-acetyl

neuraminic acid (NANA), also known as sialic acid, from other sugars are present in many microorganisms. These include bacteria such as Vibrio cholerae, Clostridium perfringens, Streptococcus pneumoniae, and Arthrobacter sialophilus, and viruses such as influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, and Sendai virus. Host of these viruses are of the orthomyxovirus or paramyxovirus groups, and carry a neuraminidase activity on the surface of the virus particles.

Many of the neuraminidase-possessing organisms are major pathogens of man and/or animals, and some, such as influenza virus, Newcastle disease virus, and fowl plague virus, cause diseases of enormous economic importance.

It has long been thought that inhibitors of

neuraminidase activity might prevent infection by

neuraminidase-bearing viruses. But while several such inhibitors are known, none has been shown to possess

antiviral activity in vivo. Most of the known neuraminidase inhibitors are analogues of neuraminic acid, such as 2-deoxy- 2,3-dehydro-N-acetylneuraminic acid (DANA) and its

derivatives. See, e.g., Meindl et al., Virology 1974 58 457- 63. The most active of these is 2-deoxy-2,3-dehydro-N- trifluoracetyl-neuraminic acid (FANA), which inhibits multicycle replication of influenza and parainfluenza viruses in vitro. See Palese et al., Virology 1974 59490-498. Table 1 below presents a listing of known N- acetylneuraminic acid derivatives. Many of these compounds are active against neuraminidase from V. cholerae or

Newcastle disease virus as well as that from influenza virus. Neuraminidase in at least some strains of influenza or parainfluenza viruses is also inhibited by 3-aza-2,3,4- trideoxy-4-oxo-D-arabinoctonic acid δ-lactone and O-α-N- acetyl-D-neuraminosyl-(2--->3)-2-acetamido-2-deoxy-D-glucose

Zakstel'skaya et al., Vop. Virol. 197217 223-28.

Neuraminidase from Arthrobacter sialophilus is inhibited by the glycals 2,3-dehydro-4-epi-N-acetyl- neuraminic acid, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and 5-acetamido-2,6-anhydro-2,3,5-trideoxy-D-manno-non-2-en- 4-ulosonate, and by their methyl esters. See Kumar et al., Carbohydrate Res. 1981 94123-130; Carbohydrate Res. 1982 103 281-285.

The thio analogues 2-α-azido-6-thio-neuraminic acid and 2,3-dehydro-6-thioneuraminic acid. Mack & Brossmer, Tetrahedron Letters 198728 191-194, and the fluorinated analogue N-acetyl-2,3-difluoro-α-D-neuraminic acid, Nakajima et al., Agric. Biol. Chem. 198852 1209-1215, were reported to inhibit neuraminidase, although the type of neuraminidase was not identified. Schmid et al., Tetrahedron Letters 1988 29 3643-3646, described the synthesis of 2-deoxy-N-acetyl-α- D-neuraminic acid, but did not report its activity or

otherwise against neuraminidase.

R ₁ R₂ R₂' R₃ R₄ R₄ ' R₅ R₅' R₆

46 C₆H₅CH₂ H CH₃COO- CH₃CO H CH₃COO- CH₃COO- H CH₃COO-

Compounds 1 - 18 P. Meindl, G. Bodo, P. Palese, J. Schulman and H. Tuppy.

Inhibition of Neuraminidase Activity by Derivatives of

2-Deoxy-2,3,-dehydro-N-acetylneuraminic Acid.

Virology 58, 457-463( 1974).

Compounds 19-23 P.Meindl and H. Tuppy. Ueber

2-Desoxy-2,3-deshydro-sialinsaeuren 1. Mitt. : Synthese

und Eigenschaften von 2-Desoxy-2,3-deshydro-N-acylneuraminsaeuren und deren

Methylestern. Mh. Chem. 100 (4) 1295-1306 ( 1969)

Compounds 24-32 M. Flashner et al. Methyl-5-acetamido-2,6-anhydro-3,5

-dideoxy-D- manno-non-2-en-4-ulosonate. Carbohydrate

Research 103, 281 -285(1982)

Compounds 33-40 E. Zbiral et al. Synthesis of 2,7-, 2,8-, and 2,9-Dideoxy

and 2,4,7 -Trideoxy-2,3-didehydro-N-acetylneuraminic Acids

and Their Behavior Towards Sialidase from Vibrio cholerac

Licbigs Ann ( 'hem 1989, 159 165.

Compounds 41 -42 T. Ogawa and Y. Ito. An Efficient Approach to Stereo- selective Glycosylation of N-Acetylneuraminic Acid: Use

of Phenylselenyl Group as a Stereocontrolling Auxillary.

Tetrahedron Letters 28, (49), 6221-6224(1987).

Compounds 43-45 T. Goto et al. Synthesis of (α2-9) and (α2-8) Linked

Neuraminylneuraminic Acid Derivatives. Tetrahedron

Letters 27, (43), 5229-5232(1986).

Compound 46 H. Ogura et al. Studies on Sialic Acids XV. Synthesis of α and β-O- Glycosides of

3-Deoxy- D-glycero - D-galacto -2-nonulopyra nosonic Acid (KDN). Chem. Pharm. Bull.36, ( 12), 4807-4813( 1988)

Meindl and Tuppy, Hoppe-Seyler's Z. Physiol. Chem. 1969 350 1088, described hydrogenation of the olefinic double bond of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to produce the β-anomer of 2-deoxy-N-acetylneuraminic acid.

This β-anomer did not inhibit Vibrio cholerae neuraminidase.

The most potent in vitro inhibitors of viral neuraminidase have thus been identified as compounds that are based on the neuraminic acid framework, and these are thought by some to be transition-state analogues. Miller et al., Biochem. Biophys. Res. Comm. 1978 83 1479. But while many of the aforementioned neuraminic acid analogues are competitive inhibitors of neuraminidases, none is known to have antiviral activity in vivo. For example, although a half-planar, unsaturated 6-member ring system has been asserted to be important for inhibitory activity, see Dernick et al. in

ANTIVIRAL CHEMOTHERAPY (K.K. Gauri ed.) Academic Press, 1981, at pages 327-336, some compounds characterized by such a system, notably FANA, have been reported not to possess invivo anti-viral activity. See Palese and Schulman in

CHEMOPROPHYLAXIS AND VIRUS INFECTION OF THE UPPER RESPIRATORY TRACT, Vol. 1 (J.S. Oxford ed. ) CRC Press, 1977, at pages 189-205. Accordingly, the conventional wisdom has been that compounds exhibiting in vitro inhibition of viral

neuraminidase would not effect an in vivo blockade of virus infection.

Summary of the Invention

It is therefore an object of the present invention to provide improved inhibitors of neuraminidase which have anti-viral activity in vivo.

It is also an object of the present invention to provide medicinal compositions which can be used to prevent or ameliorate symptoms of viral infection.

It is a further object of the present invention to provide means for producing such medicinal compositions.

In achieving this object there has been provided, in accordance with one aspect of the invention, a

biologically active substance that binds the active site ("receptor") of influenza virus neuraminidase such that said substance displays anti-orthomyxovirus or paramyxovirus activity in an animal. In a preferred embodiment, the active substance displays (a) in vitro activity in an assay which measures binding of the active site of influenza virus neuraminidase; and (b) in vivo anti-orthomyxovirus or

paramyxovirus activity. Preferably, the in vivo activity is displayed in mice or ferrets challenged intranasally with influenza virus.

According to another aspect, the present invention provides a biologically active substance which possesses stereochemical complementarity to an enzyme active site comprised of amino acids positioned at atomic coordinates enumerated as part of Figure 1 below, or a subset thereof, and said substance displays in vivo activity against an orthomyxovirus or a paramyxovirus. Preferably, the

stereochemical complementarity is such that the compound has a K_i for said active site of less than 10^-7M. More

preferably, the K_i value is less than 0.5 x 10^-aM.

It is also preferred, according to either aspect of the present invention, that the substance be a carbohydrate comprising a non-mutarotatable anomeric carbon atom. More preferably, this carbon atom is optionally substituted by a functional group. Even more preferably, the functional group is carried on the C₂ carbon.

In one preferred embodiment the compound is a novel 2-deoxy derivative of α-D-neuraminic acid of general

structural formula I:

and pharmacologically acceptable salts or derivatives

thereof , wherein A denotes O ,

R denotes hydrogen, CN, CH-NHR⁶, CH₂OR⁶, CH₂F,

CH₃, Sn(R⁶)₃, Si(R⁶)₃ or SR⁷, where R⁷ is an alkyl group which has an alkyl chain of 1 to 6 carbons; or an aryl group wherein the aryl moiety is mono-, di- or tri-substituted with halogen, amino, hydroxyl or carboxyl,

R¹ denotes COOH, P(O)(OH)₂, NO₂, SOOH, SO₃H, tetrazole, CH₂CHO, CHO, CH(CHO)₂ or, where R²- is COOH,

P(O)(OH)₂, SOOH or SO₃H, an ethyl, methyl or pivaloyl ester thereof,

R² denotes H, OR⁶, F, Cl, Br, CN, NHR⁶, SR⁶ or CH₂X, wherein X is NHR⁶, halogen or OR⁶ and

R⁶ is hydrogen; an acyl group having 1 to 4 carbon atoms; a linear or cyclic alkyl group having 1 to 6 carbon atoms, or a halogen-substituted analogue thereof; or an unsubstituted aryl group or an aryl substituted by a halogen, an allyl group, an OH group, an NO₂ group, an NH₂ group or a COOH group,

R³ and R³' are the same or different, and each denotes hydrogen, N(R⁶)₂, SR⁶ or OR⁶,

O

R⁴ denotes NHC-R⁷, where R⁷ is an unsubstituted or halogen-substituted linear or cyclic alkyl group of 1 to 6 carbon atoms, or SR⁶, OR⁶, COOH or alkyl/aryl ester thereof,

NO₂, C(R⁶)₃, CH₂COOH or alkyl/aryl ester thereof, CH₂NO₂ or

CH₂NHR⁷, and

R⁵ denotes CH₂YR⁶, CHYR⁶CH₂YR⁶ or

CHYR⁶CHYR⁶CH₂YR⁶ where Y is O, S or H, and successive Y moieties in an R⁵ group are the same or different, subject to the provisos that

(i) when R³ or R³' is OR⁶ or hydrogen, then said compound cannot have both

(a) an R² that is hydrogen and

(b) an R⁴ that is NH-acyl,

(ii) R⁷ is not CH₃, CH₂CH₃, phenyl, glucosyl, galactosyl, mannosyl, acetyl, benzoyl, cyclohexyl or

substituted cyclohexyl and (iii) R⁶ represents a covalent bond when Y is hydrogen. The compound is preferably one selected from the grot consisting of methyl N-acetyl-4,7,8,9-tetra-O-acetyl-2- deoxy-2α-allylthioneuraminate, and sodium N-acetyl-2-deoxy- 2α-allylthioneuraminate.

In a second preferred embodiment, the compound has general formula II:

where A is oxygen and where R¹, R², R³, R³', R⁴ , R⁵ and R⁶ are as defined in general formula I above, subject to the

provisos that, in general formula II,

(i) when R³ or R³' is OR⁶ or hydrogen, then said compound cannot have both

(a) an R² that is hydrogen and

(b) an R⁴ that is NH-acyl, and

(ii) R⁶ represents a covalent bond when Y is hydrogen, and pharmaceutically acceptable salts or

derivatives thereof. Preferably, the compound is synthesized using an intermediate selected from the group consisting of 3,4,6-tri-0-acetyl-2-deoxy-β-L-arabinohexapyranosyl

thiophenoxide; 4-O-benzyl-3,6-bis (t-butylmethylsilyloxy)-2- deoxy-β-L-arabino-hexapyranosyl thiophenoxide; 4-O-benzyl- 3,6-bis(t-butylmethylsilyloxy)-2-deoxy-β-L-arabinohexa- pyranosyl phenylsulphone; α-carboxymethyl-β-phenysulphonyl-4- o-benzyl-3,6-bis(t-butyldimethylsilyloxy)-2-deoxy-L- arabinohexapyranose; methyl-4-O-benzyl-3,6-bis(t- butyldimethylsilyloxy)-2-deoxy-α-L-arabinohexapyranosyl- carboxylate and methyl-N-acetyl-4,7,8,9-tetra-O-acetyl-2β- chloro-2-deoxy-D-neuraminate.

According to a third aspect of the invention there is provided a method of synthesis of a compound according to general formula I, comprising the steps of providing an alkyl N-acetyl neuraminate, reacting said alkyl N-acetyl

neuraminate with an alcohol in the presence of an acid catalyst to yield the corresponding ester, acylating and halogenating the ester by reaction with an acyl halide, treating the halogenated and acylated ester with a

nucleophile to effect halogen-nucleophile exchange,

deacylating and deesterifying the resulting compound under hydrolytic conditions, and recovering the compound of general formula I.

In an alternative embodiment of this aspect of the present invention, there is provided a method of synthesis of a desired compound of general formula I which comprises the steps of:

(a) treating a glycal of formula (i) i

with hydrogen chloride and then with sodium thiophenoxide to form a thioglycoside of formula (ii) below i i

(b) oxidizing the thioglycoside with

metachloroperoxybenzoic acid to form a sulphone of formula (iii) i i

and either

(c) reacting the sulphone with lithium diisopropyl amide and then with dimethylcarbonate to form alpha and beta C-1 substituted sugars of formula (iv)

and

(c') isolating the C-1 substituted sugar.

or

(d) reacting the sulphone with lithium diisopropyl amide in the presence of lithium naphthalenide and a compound containing a COOH, P(O)(OH)₂ or SOOH group and

(d') isolating the desired compound of formula I.

In step (d) the compound containing the P(O)(OH)₂ group is preferably diethyl chlorophosphate. Other compounds of the present invention can be synthesized using the products of step (c') or step (d') as starting materials, as will be readily appreciated by those skilled in the art.

According to a fourth aspect, the invention

provides a pharmacologically active composition comprising (i) an orthomyxovirus or paramyxovirus-inhibiting amountn of a substance that binds the active site of influenza virus neuraminidase such that said substance displays anti- orthomyxovirus or paramyxovirus activity in an animal and (ii) a physiologically-compatible carrier diluent or

excipient for said substance. The substance is preferably a compound that conforms to general formula I or II except for the fact that the exclusionary provisos set out above do not apply.

According to a fifth aspect, the invention provides a method of preventing or ameliorating the symptoms of an orthomyxovirus or paramyxovirus infection, comprising the step of administering to an animal a virus-inhibiting amount of a substance that binds the active site of influenza virus neuraminidase such that the substance displays anti- orthomyxovirus or paramyxovirus activity in an animal. The substance may be administered orally, intranasally, buccally or sublingually.

In each of these five aspects of the invention, the virus is preferably selected from the group consisting of influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, and Sendai virus. In the method according to the fourth aspect of the invention, it is particularly preferable that the virus either

(A) is selected from the group consisting of influenza virus, parainfluenza virus, Sendai virus and mumps virus, and the animal is a human, or

(B) is Newcastle disease virus or fowl plague virus, and the animal is a bird.

According to a sixth aspect, the invention provides novel glycosyl halides of general formula III, which are useful as intermediates in the synthesis of compounds of general formula I above:

I

wherein

R may be F, Cl or Br when R² is not H, F, Cl or Br; if R³, R³' is OR⁶ or H then R⁴ is NH-Acyl; and

A, R¹, R², R³, R³', R⁴, R⁵ and R⁶ are as defined in general formula I above. Formula III compounds can be used as glycosyl donor intermediates in the synthesis of compounds of general formula I.

According to a seventh aspect of the invention, there is provided an improved method of synthesis of glycosyl halides of general formula III comprising the step of treating the corresponding neuraminic acid analogue with excess acetyl halide at room temperature under a nitrogen atmosphere until no starting material is observable by thin layer chromatography, and recovering the desired glycosyl halide compound.

Brief Description of the Drawings

Figure 1 depicts an exemplary influenza-viral neuraminidase, that of A/Tokyo/3/67, in terms of refined atomic coordinates in Angstrom units (accuracy: ± 0.3 Å) for all amino-acid moieties, including the active site, of the enzyme molecule. The coordinates are in relation to a

Cartesian system of orthogonal axes.

Figure 2 is a detailed representation, provided in terms of refined atomic coordinates as in Figure l, of N- acetyl neuraminic acid as observed bound to influenza virus neuraminidase as described in Figure 1.

Figure 3 shows the atomic coordinates in Angstrom units of 3-fluoro-1,1,1,3,5,5,5-heptanitropentane in its predicted mode of binding to the active site of the influenza viral neuraminidase of Figure 1.

Figures 4 and 6 are schematic representations of a general scheme for the synthesis, respectively, of two subclasses of anti-viral agents within the present invention.

Each of Figures 5 and 7 represents schematically a particular synthesis according to Figures 4 and 6, respectively.

Detailed Description of Preferred Embodiments

A refined view of the three-dimensional structure of the active site of influenza virus neuraminidase has now been developed (with errors of the order of 0.3 Å) that enables the production of molecules which tightly bind the enzyme active site, something that heretofore could not have been accomplished based, for example, on extant information regarding the crystal structure of N2 influenza virus

neuraminidase soaked with neuraminic acid. See Varghese et al., Nature 1983 303 35-40. Notwithstanding expectations to the contrary regarding the import of neuraminidase-binding capability, it has also been discovered that compounds possessing high affinity for the enzyme active site are also prime candidates for in vivo anti-viral agents, which

property is routinely ascertainable by means of a

conventional animal assay, as described in greater detail below.

The mechanism or mechanisms underlying this beneficial correlation between neuraminidase affinity and in vivo anti-viral activity are not fully clarified. But the tight binding of the active site, preferably with an affinity on the order of 10^-8 M, is understood to arise from an

enhanced stereochemical complementarity, relative to known in vitro-effective neuraminidase inhibitors, between compounds of the present invention and the active site, which favors desolvation of the compound. Such enhanced complementarity is accomplished, in accordance with the present invention, by assuring that the structure of the receptor-binding molecule correlates, in the manner of the classic "lock-and-key" visualization of ligand-receptor interaction, with the critical features of the active site.

A molecule within the present invention can be designed, based on the atomic-coordinate information set out in Figure 1, so that selected portions of the molecule match surface residues positioned within the substrate binding site on the neuraminidase molecule. By "match" it is meant that the identified portions interact with the surface residues, for example, via hydrogen-bonding and by enthalpy-reducing Van der Waals interactions which promote desolvation of the molecule within the site, in such a way that retention of the molecule in the site is favored energetically.

Such stereochemical complementarity, pursuant to the present invention, is characteristic of a molecule that matches intra-site surface residues located in the vicinity of coordinate point (92, 92, 67 Å) in Figure l. The latter point is near tyrosine 406 of the neuraminidase molecule, and defines the site where sialic acid has been observed to bind. Tyrosine 406 is surrounded by residues including amino acids 118, 119, 151, 224, 276, 277, 292 and 371, that define a depression on the surface of the enzyme molecule and that do not vary from strain to strain, as illustrated by the

sequence alignments for neuraminidases from different strains of influenza virus. See Colraan & Ward, Curr. Topics

Microbiol. Immunol., 1985 114 177.

This surface depression represents the

neuraminidase active site which is highly conserved.

According to the present invention, therefore, the effort of matching portions of an anti-viral agent within the present invention should be directed to the invariant residues which define the active site. Chemical entities which are

complementary to the shape of an enzyme active site

characterized by the aforementioned invariant structural elements are able to bind to the active site and, when the affinity of binding is sufficiently strong -- as reflected by a K_i preferably on the order of 10^-7 or less -- will prohibit access of natural substrate to the site.

By way of illustration, for the compound 2-deoxy-N- acetyl-α-D-neuraminic acid (see Examples 1, 4, 18, 24 and 25), a carboxylate substituent on carbon C₂ interacts with the guanidinium moiety of arginine 371 in the neuraminidase active site, while the glycerol side chain makes (i) Van der Waals contacts with the hydrocarbon moiety of arginine 224 and (ii) hydrogen bonds with the carboxylate of glutamic acid 276. By the same token, the carboxylate substituent and glycerol side chain, respectively, of each of the compounds N-acetyl-neuraminic acid, 2,3-dehydro-N-acetyl-neuraminic acid and 2,3-dehydro-N-trifluroacetyl-D-neuraminic acid interact in similar fashion with the same residues of the active site.

In general, the design of a molecule possessing stereochemical complementarity can be accomplished by means of techniques that optimize, either chemically or

geometrically, the "fit" between a molecule and a target receptor. Known techniques of this sort are reviewed by Sheridan and Venkataraghavan, Acc. Che Res. 1987 20 322;

Goodford, J. Med. Chem. 198427557; Beddell, Chem. Soc.

Reviews 1985, 279; and Hoi, Angew. Chem. 1986 25 767, the respective contents of which are hereby incorporated by reference. See also Blundell et al., Nature 1987 326 347 (drug development based on information regarding receptor structure).

Thus, there are two preferred approaches to designing a molecule, according to the present invention, that complements the active site of influenza virus

neuraminidase. By the geometric approach, the number of internal degrees of freedom (and the corresponding local minima in the molecular conformation space) is reduced by considering only the geometric (hard-sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets" or "grooves" that form binding sites for the second body (the complementing molecule, as ligand). The second preferred approach entails an assessment of the interaction of respective chemical groups ("probes") with the active site at sample positions within and around the site, resulting in an array of energy values from which three-dimensional contour surfaces at selected energy levels can be generated.

The geometric approach is illustrated by Kuntz et al., J. Mol. Biol. 1982 161 269, the contents of which are hereby incorporated by reference, whose algorithm for ligand design is implemented in a commercial software package distributed by the Regents of the University of California and further described in a document, provided by the

distributor, which is entitled "Overview of the DOCK Package, Version 1.0,", the contents of which are hereby incorporated by reference. Pursuant to the Kuntz algorithm, the shape of the cavity represented by the neuraminidase active site is defined as a series of overlapping spheres of different radii. One or more extant data bases of crystallographic data, such as the Cambridge Structural Database System maintained by Cambridge University (University Chemical

Laboratory, Lensfield Road, Cambridge CB2 1EW, U.K.) and the Protein Data Bank maintained by Brookhaven National

Laboratory (Chemistry Dept. Upton, NY 11973, U.S.A.), is then searched for molecules which approximate the shape thus defined. Molecules identified in this way, on the basis of geometric parameters, can then be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions and Van der Waals interactions. For example, the compound 3-fluoro- 1,1,1,3,5,5,5-heptanitropentane (FHNP) is represented by the structural formula

and has been identified, pursuant to the Kuntz algorithm, as a molecule that complements, as represented according to the aforementioned geometric definition. Based on the

orientation predicted using the above-mentioned software package, modifications in the FHNP molecule would be made in order to adjust localized hydrophilicity or

hydrophobicity and, thereby, improve the degree of

stereochemical complementarity. For example, from the predicted orientation shown in Figure 3 it is apparent that replacement of the nitro group N13, 026, 027 by a methylene amino group could improve the hydrogen bonding

complementarity to glutamic acid 277 on the neuraminidase.

The chemical-probe approach to ligand design is described, for example, by Goodford, J. Med. Chem. 198528 849, the contents of which are hereby incorporated by reference, and is implemented in several commercial software packages, such as GRID (product of Molecular Discovery Ltd., West Way House, Elms Parade, Oxford OX2 9LL, U.K.). Pursuant to this approach, the chemical prerequisites for a site- complementing molecule are identified at the outset, by probing the active site (as represented via the atomic coordinates shown in Fig. 1) with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen, and a hydroxyl. Favored sites for interaction between the active site and each probe are thus determined, and from the resulting three-dimensional pattern of such sites a putative complementary molecule can be generated.

The chemical-probe approach is especially useful in defining variants of a molecule known to bind the target receptor. Since sialic acid is such a molecule, vis-a-vis the neuraminidase active site, crystallographic analysis of sialic acid bound to neuraminidase provides useful

information regarding the interaction between an archetype ligand and the active site of interest. In particular, it has been found that sialic acid binds to neuraminidase in a distorted conformation, with the carboxylate group pushed into the plane of the sugar (see Figure 2).

Since this carboxylate-planar feature is inherent in the DANA molecule and molecules that are "DANA-like" by virtue of having an sp²-hybridized system at C₂/C₃, no

distortion is needed for such molecules to fit -- that is, to possess stereochemical complementarity with relation to -- the active site. The resulting increased complementarity of DANA and DANA-like molecules is reflected, for example, in a Ki value for DANA that is significantly lower (indicating higher active-site affinity) than the corresponding values for sialic acid and its derivatives. As described in greater detail below, the increased complementarity is also evidenced by in vivo anti-viral activity of DANA.

Accordingly, a preferred subgroup of anti-viral agents suitably used in pharmaceutical formulations of the present invention includes DANA-like molecules, especially those with a K_i of greater than 10^-7. More generally, 5-, 6- and 7-membered carbocyclic and heterocyclic compounds that possess the structural feature of carboxylate-planarity are preferred candidates for anti-viral agents to use in

accordance with the present invention. Exemplary of such compounds are the molecules represented, respectively, by formula II. These molecules comprise a carboxylate moiety that is positioned in the plane of the ring nucleus by virtue of the sp²-hybridized system which includes the heteroatom or C₃, as the case may be, and the carbon that bears the

carboxylic-acid moiety or an analogue thereof, where "analogue" denotes a moiety that can interact either

ionically (say, charge-charge interaction) or covalently (via a Schiff reaction, for instance) with a reactable amino moiety in the active site, such as is presented by arginine 371 corresponding to the coordinates for the atoms ARG NH1 371 and ARG NH2 371 (see Figure 1).

Another group of preferred candidate anti-viral molecules is comprised of heterocyclic compounds wherein the heteroatom is oxygen, a ring carbon is present that is

"anomeric", or positioned for substituent dipole:dipole interactions with the heterooxygen, and the anomeric carbon carries A-face substituents that are not subject to

anomerization, i.e., substituents around this carbon atom are "non-mutarotatable." It has been found that heterocyclic compounds comprising such an anomeric carbon, which cannot undergo anomerization under physiological conditions, are more likely to possess (or to be amenable, as described above, to modifications effecting) stereochemical

complementarity with the neuraminidase active site. In addition, such non-mutarotatable compounds are expected to be less susceptible to the influence of neuraminic acid- degradation pathways than known in vitro inhibitors of viral neuraminidase.

Exemplary of such heterocyclic compounds are molecules represented by formula I. In this vein, the fact that neuraminic acid has a binding affinity in the millimolar range for viral neuraminidase, and that an equilibrium mixture of neuraminic acid is mostly A-neuraminic acid

(beta:alpha = 98:2), see Kitajiraa et al., Biochemistry 1984 23310, indicates that the actual affinity of the alpha form of a formula I molecule (where substituent R on the anomeric carbon extends into the plane of the paper) is on the order of 50 times greater than that of the beta form. Accordingly, a preferred subgroup of anti-viral candidate molecules within formula I includes α-neuraminic acid analogues that are substituted at the C₂ and C₃ carbons, respectively, so that the anomeric carbon cannot mutarotate due to steric

interference or interactions between substituents and active- site moieties which favor the non-mutarotated form.

Additional modifications can also be made, for example, at C₃, C₄ or C₅.

It is known that single amino-acid changes can cause major changes in activity of influenza virus

neuraminidase which are not predictable on the basis of any theoretical method. Insofar as it may not be necessary for the complementarity between compound and active site to extend over all residues of the active site, compounds that bind atoms comprising fewer than all of the residues of the active site are encompassed by the present invention.

In summary, the general principles of receptor- based drug design can be applied by persons skilled in the art, using the crystallographic data presented above, to produce compounds having sufficient stereochemical

complementarity to produce a high-affinity binding of the active site of influenza virus neuraminidase.

The present invention is further described below by reference to the following, non-limiting examples. Example 1 2-Deoxy-N-acetyl-a-D-neuraminic acid (DANA)

The simplest method of preparing this compound is by catalytic hydrogenation of 2,3-dehydro-N-acetylneuraminic acid using methods previously described by T.W. Greene,

PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Wiley and Sons

(1981), at pages 29-31. See Example 4 infra. Although it is possible to prepare 2-deoxy-α-D-neuraminic acid in a one-pot reaction, analogues of the general formula (I) are not so readily synthesized from this template.

Example 2 General Synthesis of Compounds of Formula I

A general synthetic route to this class of compound is described in Scheme 1, shown in Figure 4. The starting point for the preparation of C-1 substituted sugars is the glycal structure which upon treatment with hydrogen chloride followed by reaction with sodium thiophenoxide results in the formation of the thioglycoside. The thioglycoside (structure III) is converted to the corresponding sulphone (structure IV) by oxidation with metachloroperoxybenzoic acid. The sulphone is the key intermediate in the preparation of C-1 substituted sugars, because the C-1 position is now activated towards electrophiles. Treatment of the sulphone IV with lithium diisopropyl amide followed by reaction with dimethyl carbonate yields respectable quantities of the isolable alpha and beta C-1 substituted sugars la.

We have extended this synthesis to the preparation of C-1 phosphorus sugars lb by treating sulphone IV with the electrophile diethyl chlorophosphate in the presence of base. This entry into these classes of compounds provides us with very "user-friendly" templates and allows one to

functionalize various centers around the carbohydrate ring. Other electrophiles may also be used, for example to make sulphur-based compounds Ic.

Example 3 Specific synthetic strategy according to Scheme 1

Figure 5 summarizes a flow sheet for synthesis of specific compounds according to the invention, utilizing the general strategy set out in Scheme 1 (Example 2 above and Figure 4). Abbreviations used are as follows:

DMF H,N-dimethylformamide

TBDMS tertiary butyldimethylsilyl

Ph phenyl

Bn benzyl

The following examples represent typical syntheses utilizing

Scheme 1. Roman numerals refer to

Figure 5.

Example 4 2-Deoxy-N-acetyl-a-D-neuraminic acid

The compound 2,3-dehydro-N-acetyl-D-neuraminic acid (5.8 mg) was dissolved in methanol (5 ml) and treated with PtO₂ (3 mg). The mixture was hydrogenated at 1 atmosphere and room temperature. The reaction proceeded quantitatively to yield the title compound, which had Rf on thin layer chromatography in propanol:water (3:1) of approximately 0.3. The ¹H and ¹³C NMR data were consistent with the proposed structure (i.e., no definite olefinic proton observed, H_3axial nd H_3equatorial δ 1,8 and 2,3, respectively).

Example 5 3,4,6-tri-O-acetyl-2-deoxy-β-L-arabino- hexopyranosyl thiophenoxide

Tri-O-acetyl-L-glucal (10.64 g) was dissolved in toluene (150 ml) and cooled to -5ºC. Dry HCl gas was bubbled through the solution until the starting material had been consumed, as indicated by thin layer chromatography. The solution was evaporated and the residue dissolved in N.N- dimethylformamide (DMF)(100 ml), and treated dropwise with a solution of sodium thiophenoxide (11.38 g) in DMF (60 ml) at 0ºC. The mixture was refrigerated overnight and the DMF removed under high vacuum. The residue was partitioned between ice water (200 ml) and CH₂Cl₂ (200 ml). The organic layer was washed with ice water (3 x 200 ml), dried, and evaporated to give an orange oil (17 g). The crude product VIII was purified by flash chromatography in two 8.5 g batches on a 6 x 15 cm column, eluting with ethyl

acetate:hexane 3:7 and taking 150 ml fractions. Those fractions with a single spot at R_f:0.27 (in the same solvent) were combined and evaporated to give a yellow oil which crystallized on standing (8.16 g, 54%). ¹H-NMR (CDCl₃): δ 1.78 (m, 1H, H_2a; 2.03 (m, 9H, 3xCH₃); 2.52 (m, 1H, H_2e); 3.68 (m, 1H, H₅); 4.18 (m, 2H, H₆x2) 4.79 (m, 3H, H₁, H₃, H₄); 7.18 (m, 5H, ArH).

Example 6 2-Deoxy-β-L-arabinohexopyranosylthiophenoxide

(Compound IX)

Compound VIII (9.6 g) was dissolved in dry methanol (200 ml) and treated with sodium (0.1 g). The mixture was left at room temperature for 2 hours and then CO₂ was bubbled through the mixture for 15 minutes. The solvent was removed and the residue crystallized. The solid was isolated by filtration with the aid of some diethyl ether, and dried under vacuum to give a light yellow solid (5.05 g, 78%).

1H-NMR (D₂O): δ 1.92 (m, 2H, H_2a, H_2e) , 3.70 (m, 5H, H₃, H₄, H₅, H₆x2), 4.95 (m, 1H, H₁), 7.40 (m, 5H, ArH). Example 7 4,O-Benzyl-3,6 bis(t-butyldimethylsilyloxy)-2- deoxy-β-L-arabinohexopyranosyl thiophenoxide (Compound XI)

The hydroxy compound. Compound IX (3 g) was

dissolved in DMF (60 ml) and treated with imidazole (3.51 g) and t-butyldimethylsilylchloride (3.87 g), and stirred overnight at room temperature. The solvent was removed under high vacuum and the residue partitioned between CH₂Cl₂ (150 ml) and ice water (100 ml). The organic layer was washed with ice water (3 x 100 ml), dried and evaporated to give a yellow oil (6.01 g). The oil is 3,6 bis (t-butyldimethyl- silyloxy)-2-deoxy-β-L-arabino-hexopyranosyl thiophenoxide) (Compound X).

This intermediate (5.2 g), dissolved in DMF (30 ml), was added to a suspension of NaH (0.37 g) in DMF (30 ml). The mixture was stirred for 30 min. then benzyl bromide (1.9 ml) in DMF (20 ml) was slowly added. An equivalent amount of benzyl chloride could also suitably be used. The resulting solution was stirred at room temperature for 2 hours. The solvent was removed under high vacuum and the residue dissolved in CH₂Cl₂ (150 ml) and washed with ice water (3x80 ml). The organic solution was dried and

evaporated to give a yellow oil. The oil was purified by flash chromatography (6 x 12 cm), eluting with 3% ethyl acetate in hexane and taking 100 ml fractions. Those

fractions with a single spot at R_f = 0.67 (10% ethyl acetate in hexane) were combined and evaporated to give a colorless oil (4.75 g, 81% overall). The intermediate hydroxy Compound X (1.48 g) was also recovered from the column. (R_f 0.41 10% Ethyl acetate in hexane)

1H-NMR δ 0.10 (m, 12H, SiCH₃ x 4); 0.89 (m, 18H, SitBu X 2); 1.70 (ddd, 1H, J_2a,1a 11.7, J_2a,2e 12.0, J_2a,3 5.14, H_2a); 2.22 (ddd, 1H, J_2a,1e 1.72, J_2e,2a 12.0, J_2e,3 5.14, H_2e);

3.32 (m, 2H, H₄, H₅); 3.78 (m, 3H, H₃, H₆x2); 4.63 (d, 1H, J_11.07, CH₂Ph); 4.77 (dd, 1H, J_1a,2a 11.7, J_1a,2e 1.72, H_1a); 4.88 (d, 1H, J_11.07, CH₂Ph); 7.37 (m, 10H, SPh, CH₂Ph). Example 8 4-O Benzyl-3,6 bis (t-butyldimethylsilyloxy)-2- deoxy-β-L-arabinohexopyranosylphenylsulphone (Compound XII)

The sulphide, Compound XI (4.75 g) was dissolved in CH₂Cl₂ (50 ml) and added dropwise to a suspension of m- chloroperoxybenzoic acid (3.8 g) and NaHCO₃ (7.6 g) in CH₂Cl₂ (50 ml) at 0°C. The mixture was stirred for 1 1/2 hours and extracted with ice water (100 ml), 5% Na₂S₂O₃/ saturated

NaHCO₃ 1:1 (100 ml) and ice water (100 ml). The organic solution was dried and evaporated to give an oil that

crystallized on standing (4.62 g, 92%).

¹Η-NMR (CDCl₃): δ -0.03 (m, 12H, SiCH₃x4); 0.83 (m, 18H, SitBu x 2); 1.75 (ddd, 1H, J_2a,2e 12.0, J_2a,1a 12.1, J_2a,3 12.0, H_2a); 2.39 (ddd, 1H, J_2e,1a 12.0, J_2e,1a 2.0, J_2e,3 5.0, H_2e); 3.13 (m, 1H, H₅), 3.33 (dd, 1H, J_4,5 9.09, J_4,3 9.09, H₄), 3.69 (m, 3H, H₃, H₆X2), 4.34 (dd, 1H, J_1a-2a 12.1, J_1a,2e 2.0, H_1a), 4.55 (d, 1H, J_gem 10.9, CH₂Ph), 4.78 (d, 1H, J_gem 10.9, CH₂Ph), 7.47 (m, 10H, SPh, CH₂Ph).

Example 9 α-carboxymethyl-β-phenylsulphonyl-4-O-benzyl- 3,6 bis (t-butyldimethyl-silyloxy)-2-deoxy-L- arabinohexopyranose (Compound XIII)

The sulphone, Compound XII (0.5 g), was dissolved in tetrahydrofuran (3 ml) and cooled to -78°C under argon, then treated with lithium diisopropyl amide solution (0.8 ml, 1.24 M) and stirred for 5 minutes. The mixture was treated with dimethylcarbonate (1 ml) and allowed to warm to room temperature over 1 hour, then treated with saturated NH₄Cl solution (5 ml). Ether (100 ml) was added and the mixture extracted with saturated NaCl solution (2X20 ml). The organic solution was dried and evaporated. The crude

material was purified on a chromatatron eluting with 5% ethyl acetate in hexane. One main band eluted from the plate after several minor bands. This band was evaporated to give the carboxy compound (0.404 g, 73%) as a colorless oil which crystallized on standing.

IR (neat): 2980, 1770, 1335, 1280, 1160, 1115, 860, 800 cm^-1. ¹H-NMR (CDCl₃) δ -0.04 (m, 12H, SiCH₃x4); 0.86 (m, 18H, SitBu x 2); 1.18 (dd, 1H, J_2a,2e 7.2, J_2a,3 7.2, H_2a); 2.50 (m, 1H, H_2e), 2.97 (m, 1H, H₅); 3.38 (s, 3H, OCH₃); 3.39 - 4.88 (m, 6H, H₆x2, H₄, H₃, CH₂Ph); 7.26 (m, 10H, SPh, CH₂Ph). Example 10 Methyl 4-Q-benzyl-3,6 bis (t-butyldimethyl- silyloxy)-2-deoxy-β-L-arabinohexopyranosyl carboxylate (Compound XIV)

Lithium naphthalenide solution was prepared as follows:

Naphthalene (1 g) was added to a suspension of lithium clippings (0.1 g) in tetrahydrofuran (20 ml) under argon. The mixture was stirred vigorously for 18 hours.

[LiNap] = 0.393 M.

(a) From the carboxy sulphone (two step method). The second sulphone Compound XIII (0.35 g) s dissolved in tetrahydrofuran (10 ml), cooled to

-90ºC and treated with the LiNap solution (3.5 ml). After 10 minutes the mixture was treated with methanol (0.2 ml) at - 78°C and allowed to warm to room temperature over 1 hour, then saturated NH₄Cl solution (2 ml) was added. Ether (100 ml) was added to the mixture and the solution was extracted with sat. NaCl (2x20 ml). The organic extract was dried and evaporated to give a viscous yellow oil. The crude product was purified by flash chromatography (2x15 cm) eluting with 5% ethyl acetate in hexane.

Those fractions with a single spot at R_f - 0.23 were combined and evaporated to give the alpha methyl

carboxylate (0.112 g, 40%). Those fractions with a single spot at R_f = 0.11 (5% ethyl acetate in hexane) were combined and evaporated to give the beta methylcarboxylate (0.05 g, 18%).

alpha carboxy: ¹Η-NMR (CDCl₃) δ -0.01 (m, 12H, SiCH₃x4); 0.80 (m, 18H, SitBu X 2); 1.76 (ddd, 1H, J_2a,2e 13.2, J_2a,1e 5.3, J_2a,3a 10.4, H_2a), 2.27 (ddd, 1H, J_2e,2a 13.2, J_2e,1e 3.2, J_2e,3a 3.6, H_2a); 3.32 (dd, 1H, J_4a,3a 8.2, J_4a,5a 8.2, H_4a); 3.58 (m, 1H,

H_5a); 3.66 (s, 3H, OCH₃) 3.73 (m, 3H, H_3a, H₆x2); 4.40 (dd, 1H, J_1e,2a 5.3, J_1e,2e 3.2, H_1e); 4.56 (d, 1H, J_gem 11.13, CH₂Ph); 4.74 (d, 1H, J_gem 11.13' CH₂Ph); 7.21 (m, 5H, CH₂Ph).

beta carboxy: ¹Η-NMR δ -0.02 (m, 12H, SiCH₃x4); 0.78 (m, 18H, SitBu x 2); 1.62 (ddd, 1H, J_2a,2e 11.5, J_2a,1a 12.1, J_2a,3a 11.5, H_2a); 2.10 (ddd, 1H, J_2e,2a 11.5, J_2e,1a 2.17, J_2e,3a 5.0,

H_2e); 3.15 (ddd, 1H, J_5a,4a 9.4, J_5a,6a 3.0, J_5a,6b 3.0, H_5a); 3.29 (dd, 1H, J_4a,3a 9.4, J_4a,5a 9.4, H_4a); 3.67 (s, 3H, OCH₃); 3.74 (m, 3H, H_3a, H₆X2); 4.79 (d, 1H, J_gem 10.9, CH₂Ph), 7.21 (m, 5H, CH₂Ph). Example 11 4-O-benzyl-3 ,6 bis ( t-butyldimethylsilyloxy-2- deoxy-α-L-arabinohexepyranosyl carboxylate

As will be readily appreciated by those skilled in the art, the alpha and beta forms of Compound XIV can be deesterified by treatment with base, utilizing conditions previously described. See Greene, op. cit., at pages 158- 159.

Example 12 Alternative method of synthesis

The C-1 carbanion generated by reduction of the corresponding C-l chloro compound can be quenched with an appropriate electrophile to produce a desired compound of general formula I. An exemplary synthesis along these lines is illustrated below:

Compound II --------------------> Compound VII

(Fig. 4) HCl gas (Fig. 5)

Compound VII -------------------> Li salt of VII

Li naphthalenide

Li salt of VII --------------------> 2-deoxy-α-D- electrophile neuraminic acid

Example 13

(a) Preparation of Methyl N-acetyl-D- neuraminate ( 2 )

N-acetylneuraminic acid (100 mg, 0.32 mmol) was stirred in anhydrous methanol (25 ml) containing Dowex 50X8 (H⁺) (25 mg) at room temperature for 16 hours. Thin layer chromatography of the reaction mixture (ethyl

acetate/methanol/water: 10/4/1 ) indicated that the reaction was complete (product R_f 0.50). The reaction mixture was filtered and the resin washed with methanol (10 ml x 2). The filtrate and washings were combined and concentrated to dryness to afford a white crystalline powder (102 mg, 98%).

1H-NMR (D₂O) δ 3.82 (s, 3H, COOCH₃).

The rest of the spectrum was identical to that previously reported. See Ogura et al. (1986), OP. cit.

(b) Preparation of Methyl N-Acetyl-4,7,8,9- tetra-Q-acetyl-2β-chloro-2-deoxy-D- neuraminate (3)

Compound (2) (100 mg, 0.32 mmole) was stirred with acetyl chloride (5 ml) at room temperature for 60 hours. The solution was evaporated to dryness, taken up in anhydrous benzene (20 ml x 3) and concentrated to a white foam powder (130 mg, 0.255 mole).

Η-NMR indicated the title compound to be the only product present and to be identical with that previously reported by Ogura et. al., Carbohydr. Res. 1986 158 37. The literature also describes other methods for the preparation of certain other glycosyl halides, and these methods are adequate to obtain reasonable amounts of those compounds. See, e.g., Kuhn et al., Chem. Ber. 1966 99611; Warner & O'Brien, Biochemistry 197918 (13) 2783; Ogura et al., loc. cit.; Okamoto et al., Bull. Chem. Soc. Japan 1987 60 631.

(c) Preparation of Methyl N-acetyl-4,7,8,9- tetra-Q-acetyl-2-deoxy-2α-allylthio- neuraminate (4)

Compound (3) (500 mg, 0.98 mmole) was dissolved in anhydrous N,N-dimethylformamide (5 ml), treated with sodium allylthiolate (136 mg, 1.08 mmole), and stirred at room temperature under nitrogen for 48 hours. The reaction mixture was concentrated to dryness under high vacuum. The residue was partitioned between ethyl acetate (50 ml) and 5% sodium hydrogen carbonate solution (25 ml). The organic phase was separated and washed with 10% sodium chloride solution, dried over anhydrous sodium sulphate, then

evaporated to dryness to afford a crude product which was purified by flash-column chromatography (silica gel, ethyl acetate as eluting solvent) to give the title compound (3) (200 mg, 37.3%) ¹Η-NMR (CDCl₃): δ 1.86-2.16 (dd, 5₈, H_3ax,, NAC, 4xAc, 16H, J_3ax,eq 12.7 Hz, R_{3ax, 4} 11.4 Hz), 2.72 (dd, 1H, J_3eq,3ax 12.7 Hz, H_3eq,4 4.68 Hz, H_3eq); 3.34 (m, 2H, SCH₂); 3.79 (s, 3H, OCH₃); 3.89 (dd, 1H, J_6,510.57 Hz, H_6,7 2.04 Hz, H₆); 4.07 (ddd, 1H, J_5,6 10.57 Hz, H_5,4 11.4 Hz, H_5,NH 9.95 Hz, H₅); 4.13 (dd, 1H, J₉,₈ 5.46 Hz, H_9,9, 12.52 Hz, H₉); 4.35 (dd, 1H, J₉,_,9 12.52 Hz, H₉,_,82.53 Hz, H₉,); 4.86 (ddd, 1H, J_4,3ax 11.4 Hz, H_4,5 11.4 Hz, H_4,5 4.68 Hz, H₄); 5.09 (d, 1H, olefinic J_cis 9.92 Hz); 5.2 (dd, 1H, olefinic J_trans 16.97 Hz, allylic 1.43 Hz); 5.33 (dd, 1H, J_7,6 1.9 Hz, H_7,8 7.8 Hz, H₇); 5.39 (m, 1H, H₈); 5.59 (d, 1H, J_NH,5 9.95 Hz, NH); 5.76 (m, 1H J_CK2, olefinic, 6.4 Hz)

(d) Preparation of Sodium N-acetyl-2-deoxy-2α- allylthioneuraminate (5)

Compound (4) (200 mg, 0.36 mmole) was dissolved in anhydrous methanol (20 ml) containing sodium methoxide (20 mg, 0.37 mmole). The solution was stirred at room

temperature for two hours before a mixture of mixed-bed resin AG 501X 8 (50 mg) and Dowex 50X 8 (H⁺) (25 mg) was added.

The mixture was stirred for a further 30 minutes and then was filtered. The resins were washed with methanol

(5 ml X 2) and the filtrate and washings were combined and concentrated to dryness. The residue was taken up in water (10 ml), adjusted to Ph 13 by the addition of 0.1N NaOH and stirred for 2 hours at room temperature. The solution was then adjusted to pH 6.5 by stirring with Dowex 50 X 8 (H⁺) resin. Following filtration the reaction mixture was

lyophilized to afford the title compound (120 mg, 85%).

1H-NMR (D₂O) δ 1.79 (dd, 1H, J_3ax,3eq 12.6 Hz, J_3ax,4 11.4 Hz, H_3ax), 2.02 (s, 3H, N-Ac), 2.79 (dd, 1H, J_3eq,3ax 12.6 Hz, H_3eq,4 4.57 Hz, H_3eq), 3.37 (m, 2H, SCH₂); 3.5-3.89 (m, 7H, H₄, H₅, H₆, H₇, H₈, H₉, H₉,), 5.10 (d, 1H, olefinic J_cis 9.94 Hz); 5.22 (dd, 1H, olefinic J_trans17 Hz, allylic 1.35 Hz);

5.84-6.0 (m, 2H, NH, H, olefinic).

This procedure is summarized in Figure 6. Example 14 Second General Reaction Scheme

Example 13 represents a specific instance of the general reaction scheme which is summarized in Figure 3, in which the substituents R¹ to R⁶ are as defined in general formula I, R in compound 3 is as defined in general formula III, while R in compounds 4 and 5 is as defined in general formula I. Designations of compounds in Examples 15 to 17 are as in Figure 5.

The scheme comprises the steps of:

preparing an alkyl N-acetyl neuraminate, reacting said alkyl N-acetyl neuraminate with an alcohol in the presence of an acid catalyst to yield the corresponding ester,

acylating and halogenating the ester by reaction with an acyl chloride,

treating the halogenated and acylated ester with a nucleophile to effect halogen-nucleophile exchange,

deacylating and de-esterifying the resulting compound, and recovering the compound of general formula I.

Thus, in Example 13 the treatment of compound (1) with an alcohol in the presence of an acid catalyst yielded the corresponding ester in good yield (compound (2)).

Acylation and halogenation of compound (2) was achieved through reaction with the appropriate acyl chloride,

resulting in the formation of compound (3). Halogen- nucleophile exchange was achieved by treatment of compound (3) with the appropriate nucleophile to yield compound (4). Deacylation and deesterification by treatment of compound (4) under hydrolytic conditions resulted in the formation of compound (5).

Example 15 Preparation of Methyl N-Acetyl-4 7,8,9-tetra-

Q-acetyl-2-deoxy-2α-fluoro-D-neuraminnte (4 ) Compound (3) (130 mg, 0.255 mmole) was dissolved in anhydrous acetonitrile (50 ml), treated with silver fluoride (130 mg, 1.025 mmole), stirred at room temperature under nitrogen, and protected from light for 72 hours. Two major components were isolated from the reaction mixture (thin layer chromatography; ethyl acetate, R_t 0.45 and 0.30) by flash chromatography. Compound (3) was identified as the slower moving compound by NMR spectroscopy.

1H-NMR (CDCl₃), δ 1.7 (m,1H,H_3ax), 2.0-2.2

(m,15H,acetyl-CH₃ X5), 2.6 (m,1H,H_3eq), 3.72

(s,3H,COOCH₃), 4.10-4.20 (m,2H,H₅ and H₉), 4.20-4.30 (m,1H,H₆), 4.45-4.55

(m,1H,H₉,), 5.05-5.10 (m,1H,H₄), 5.35-5.45 (m,3H,H₇,H₈, and NH)

1⁹F-NMR (CDCl₃, δ 1,1',2,2'-tetrachloro-3,3',

4,4'-'etrafluorocyclobutane as external reference) -2.5 - -3.0

Example 16 Preparation of Sodium N-acetyl-2-deoxy-2α- fluoro-neuraminate ( 5)

Compound (4) was dissolved in anhydrous methanol (5 ml) containing sodium methoxide (2 mg). The solution was stirred at room temperature for 40 min before a mixture of mixed-bed resin AG 501X 8 (5 mg) and Dowex 50X 8 (H⁺) (2.5 mg) was added. The mixture was stirred for a further 30 minutes and then was filtered. The resins were washed with methanol (2 ml x 2) and the filtrate and washings were combined and concentrated to dryness. The residue was taken up in water (10 ml), adjusted to pH 11.8 by the addition of 0.1N NaOH and stirred for 1 hour at room temperature. The solution was then adjusted to pH 6.5 by stirring with Dowex 50X 8 (H⁺) resin. Following filtration the reaction mixture was lyophilized to afford the title compound (5 mg) as a white powder.

1H-NMR (D₂O) δ 1.7-1.9 (m,1H,H_3ax), 2.1 (s, 3H, acetyl- Ch₃), 2.9-3.0 (m,1H,H_3eq), 3.5-4.1

(m, 7H,H₄,H₅,H₆,H₇,H₈,H₉,H₉')

Example 17 Third general method of synthesis

Catalytic hydrogenation of the β-chlorosialic acid can be achieved, as described in Example 18 for a typical case. The β-chloroneuraminic acid is prepared along the lines of Example 13(b) above. The method is modified from that of Schmid, Christian and Zbiral, Tetrahedron Letters 198829 3643-3646. The N-acetylneuraminic acid or analogues thereof used as starting materials for preparation of the β- chloro compounds may be synthesized using N-acetylneuraminic acid aldolase (E.C.4.1.3.3) See, e.g., Bednarski et al., J. Am. Chem. Soc. 1987 109 1283; Augé et al., Tetrahedron

Letters 1984 254663-4664.

Example 18 Preparation of 2-deoxy-N-acetyl-α-D-neuraminic acid by catalytic hydrogenation

Methyl-N-acetyl-4,7,8,9-tetra-O-acetyl-2 chloro-2- deoxy-D-neuraminate (2.0 g) was dissolved in toluene (30 ml) and Pd/C (10%, 0.91 g) and pyridine (0.6 ml) were added. The mixture was hydrogenated at 50 psi for 18 hrs. Insoluble solid was filtered off and washed with toluene (40 ml x 3) and methanol (40 ml x 2). The combined filtrate and washings were evaporated to dryness. The residue was dissolved in ethyl acetate (150 ml), and this solution washed with 5% sodium chloride solution (50 ml), dried over calcium chloride and evaporated affording the crude compound (1.76 g).

Purification by column chromatography using ethyl acetate as solvent gave 1.0 g of 2-deoxy-N-acetyl-α-D-neuraminic acid.

Example 19 Preparation of Sodium 2,3-dideoxy-α-D-galacto- 2-octulosonate

This compound was prepared using catalytic hydrogenation as described in Examples 17 and 18, followed by deacylation/deesterification as broadly described in Example 13(d).

¹H-NMR (D₂O, DSS as internal standard)

δ (ppm): 1.77 (ddd, 1H, J_3a,3e -12.0, J_3a,4 11.7, J_3a,2 6.4,

H_3a); 2.44 (dd, 1H, J_3e,3a -12.0, J_3e,4 2.6, H_3e);

3.4-4.1 (m, 5H, H₅, H₆, H₇, H₈ & H₈,); 4.32 (d, 1H, J_1e,3a 6.4, H₁). Example 20 Preparation of Sodium 2,3,5-trideoxy-5- acetamido-α-D-galacto-2-octulosonate

1H-NMR (D₂O, DSS as internal standard).

δ (ppm): 1.82 (ddd, 1H, J_3a,3e -13.1, J_3a,4 11.9, J_3a,e 6.2,

H_3a); 2.02 (s, 3H, CH₃CO); 2.49 (dd, 1H, J_3a,3e - 13.1, J_3e,4 4.2, H_3e); 3.5-3.9 (m, 5H, H₅, H₆, H₇,

H₈ & H₈,); 4.44 (d, 1H, J_2,3a 6.2, H₂).

Example 21 Inhibition of influenza virus neuraminidase

An in vitro bioassay of the above-described compounds against N2 influenza virus neuraminidase was conducted, following Warner and O'Brien, Biochemistry 197918 2783-2787. For comparison, with the same assay the K_i for the compound of Example 1, 2-deoxy-N-acetyl-α-D-neuraminic acid, was determined to be 3 X 10^-4 M.

Values for K_i were measured via a spectrofluorometric technique which uses the fluorogenic substrate 4- methylumbelliferyl N-acetylneuraminic acid (MUN), as

described by Meyers et al., Anal. Biochem. 1980 101 166-174. For both enzymes, the assay mixture contained test compound at several concentrations between 0 and 2 mM, and

approximately 1 mU enzyme in buffer (32.5 mM MES, 4 mM CaCl₂, pH 6.5 for N2; 32.5mM acetate, 4 mM CaCl₂, pH 5.5 for V.

cholerae neuraminidase).

The reaction was started by the addition of MUN to final concentrations of 75 or 40 mM. After 5 minutes at 37°C, 2.4 ml 0.1 M glycine-NaOH, pH 10.2 was added to 0.1 ml reaction mixture to terminate the reaction. Fluorescence was read at excitation 365 nm, emission 450 nm, and appropriate MUN blanks (containing no enzyme) were subtracted from readings. The K_i was estimated by Dixon plots

(1/fluorescence versus compound concentration). Results are summarized in Table 2. Table 2

Inhibition of influenza virus neuraminidase in vitro

Compounds K_i(M)

2-deoxy-N-acetyl-α-D-neuraminic acid 3 x 10^-4 sodium 2,3-dideoxy-α-D-galacto-

2-octulosonate 1 x 10^-3 sodium 2,3,5-trideoxy-5-acetamido-α-D-galacto-

2-octulosonate 5 x 10^-5

2,3-dideoxy-α-D-glycero-D-galacto-2- nonulosonic acid 2 x 10^-2

2-α-fluoro-N-acetylneuraminic acid 4 x 10^-5 sodium N-acetyl-2-deoxy-2α-allyl- thioneuraminate 1 x 10^-5

Example 22 In vivo anti-viral activity

The compound DANA (2-deoxy-N-acetyl-α-D-neuraminic acid), which was shown in Example 23 to have anti- neuraminidase activity in vitro, was tested for anti-viral activity in an in vivo assay. When administered intranasally to mice before and during challenge with influenza A virus, this compound reduced the titre of virus in lung tissue 1 to 3 days after infection.

Mice were infected intranasally with 5011 of 10³ TCID₅₀ units/mouse of H2N2 influenza A virus (A/Sing/1/57).

The compound was administered intranasally at a dose rate of either 25 mg/kg body weight or 100 mg/kg body weight (50 ll of aqueous solution/mouse) as follows: 24 hours and 3 hours before infection; 3 hours after infection; then twice daily on each of days 1, 2 and 3 after infection.

The mice were sacrificed on days 1, 2 and 3 after infection, their lungs removed and virus titres in the lungs measured. The titres were plotted graphically and expressed as the areas under the curves (AUC). Results are summarized below.

Table 3

Dose of compound Virus titre

(mg/kg body weight) (AUC) compared to

untreated infected mice 25 57%

100 19%

In light of the fact that FANA was hitherto thought to be inactive in vivo, see Palese and Schulman, op. cit., the high antiviral activity revealed when DANA was

administered intranasally to mice is especially surprising. It appears that the route of administration may be

significant in this regard, since DANA is rapidly excreted when given by other routes. See Nhle et al., Eur. J.

Biochem. 1982 126 543-48. Pharmaceutical Compositions

A pharmaceutical formulation within the present invention combines, with an active agent that binds the viral neuraminidase active site and displays in vivo anti-viral activity, a carrier for the active agent which is

pharmaceutically acceptable. A pharmaceutically acceptable carrier is a solid, liquid or gaseous material that can be used as a vehicle for administering a medicament because the material is inert or otherwise medically acceptable, as well as compatible with the active agent, in a particular context of administration. In addition to a suitable excipient, a pharmaceutically acceptable carrier can contain conver tional additives like diluents, adjuvants, antioxidants, dispersing agents and emulsifiers, anti-foaming agents, flavor

correctants, preservatives, solubilizing agents and

colorants.

The nature of the excipient used with an anti-viral agent, pursuant to the present invention, is largely a function of the chosen route of administration, as discussed, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (E.W.

Martin ed.) and in PHARMACEUTICAL DOSAGE FORMS AND THEIR USE (H. Hess ed.) Hans Huber Publ., 1985, the respective contents of which are hereby incorporated by reference. Preferably, the pharmaceutical compositions of the present invention are provided in a unitary-dosage form which is suitable for administration intranasally, orally, buccally or

sublingually.

In accordance with the present invention, a

pharmaceutical composition is advantageously delivered to the throat, nasal cavity or lungs, the intranasal route of administration being especially preferred. Delivery of an active agent to the nasal cavity can be achieved with

preparations of the present invention that take the form, for example, of an aerosol or vapor, a nasal spray or nose drops, or an inhalation powder. For these applications, it may be appropriate for the active agent to be micronized, for example, to a particle size on the order of 5 microns or less. Suitable means for effecting delivery by direct application to the mucosal lining or via inhalation are well known to the art, for example, in the context of treating asthma. In this category are squeeze-bottle devices

(nebulizers) and pressurized packs, for delivering a solution of the active agent as a spray into the nose, and

conventional insufflators like the Spinhaler turbo-inhaler and liquid aerosol "puffers" (Spinhaler is a registered trade mark of Fisons Corporation), which deliver metered doses of a pharmaceutical preparation.

If the active agent is delivered from solution, as would typically be the case for a nasal spray or nose drops, the carrier preferably comprises distilled water that is both sterile and substantially free of fever-inducing (pyrogenic) substances, thereby to minimize the incidence of medical complications relating to contamination. Suitable

propellants to comprise carriers for use in administration by pressurized aerosol are well known, including halogenated fluorocarbon gases, carbon dioxide, and nitrogen. See, e.g., Lachman et al. in THE THEORY AND PRACTICE OF INDUSTRIAL

PHARMACY (Lea and Febiger, Philadelphia), 1976. In addition, a carrier for administration via intranasal delivery or insufflation may contain oleic acid or some other

pharmaceutically acceptable stabilizer, as well as a surface- active agent, e.g., a detergent like Tween 80 or Span 80, in order to enhance uptake of the active agent.

Conventional forms which are favored for oral administration include lozenges and pastilles, sublingual and buccal tablets, and oral sprays. Numerous carriers suitable for these forms are known, including solid pulverulent carriers comprising a simple sugar or corresponding alcohol (lactose, saccharose, sorbitol, mannitol, etc), a starch such as potato starch, corn starch or amylopectin, cyclodextrin, a cellulose derivative, and gelatine. Liquid carriers can also be employed to form suspensions, syrups, elixirs and

solutions containing the active agent. Non-aqueous vehicles which are suitable as liquid carriers in this regard include almond oil and other edible oils, fractionated coconut oil, oily esters, propylene glycol and ethyl alcohol.

In formulating a pharmaceutical preparation of the present invention for oral administration, a solid carrier would typically be mixed with a lubricant, such as magnesium stearate, calcium stearate or a polyethylene glycol wax, and then compressed into tablet form. In keeping with common practice, tablets can be coated with a concentrated sugar solution which may contain components like gum arabic, gelatine, talcum and titanium dioxide. Alternatively, tablets can be coated with a lacquer dissolved in a readily volatile organic solvent.

A pharmaceutical composition within the present invention contains a virus-inhibiting amount of an active agent as described above. The optimum dosage of the active compound will vary with the particular case, and can be determined routinely in the clinical context, which may be prophylactic or therapeutic. 'Prophylactic' treatment is to be understood to mean treatment intended to prevent or retard second-cycle infection as defined below, thus preventing the establishment of the complete clinical manifestations of the disease caused by that virus. 'Therapeutic' treatment is to be understood to mean treatment intended to alleviate the symptoms and severity of infection which is already

established, by disrupting release of virus particles and thus preventing further cycles of viral replication.

Generally, the amount of active agent present in a

pharmaceutical composition of the present invention should be sufficient to inhibit at least second-cycle infection by orthomyxovirus or paramyxovirus in an animal. That is, an initial viral infection of a cell culminates in the assembly and budding of virus particles at the cell-membrane surface, which would be followed in the normal course by release of the particles and infection thereby ("second-cycle

infection") of other cells. A suitable amount of active agent to include in a pharmaceutical composition of the present invention would thus retard at least this second cycle of infection by virus, it is thought by inhibiting the action of neuraminidase that results in release of virus particles from the membrane surface.

For administration by inhalation, the daily dosage as employed for treatment, according to the present

invention, of an adult human of approximately 70 kg body weight will range from lmg to 1000 mg, preferably between 5 mg and 500 mg, and may take the form of single or multiple doses, e.g., one to six times a day. For oral

administration, the daily dosage (again, for treatment of a 70 kg adult) will typically range from about 1 mg to 5 g, preferably between 5 mg and 2 g, and may be given, for example, in single to four doses per day. It will therefore be convenient for a pharmaceutical composition of the present invention to contain active (antiviral) agent at a

concentration in the range of 0.000001 to 100 mg/ml.

Other objects, features and advantages of the present invention will become apparent from the preceding detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Claims

What Is Claimed Is:

1. A pharmacologically active composition comprising:

(i) a virus-inhibiting amount of a compound that binds the active site of influenza virus neuraminidase and that displays in vivo activity against orthomyxovirus or paramyxovirus; and

(ii) a pharmaceutically-acceptable carrier for said compound.

2. A pharmacologically active composition according to Claim 1, wherein said carrier is suitable for intranasal administration.

3. A pharmacologically active composition according to Claim 2, wherein (a) said compound is micronised and (b) said carrier comprises a propellant suitable for pressurized aerosol administration.

4. A pharmacologically active composition according to Claim 3, wherein said carrier further comprises a fatty acid, a surface-active agent or a detergent.

5. A pharmacologically active composition according to Claim 2, wherein said compound and carrier form a solution or a suspension of said compound in said carrier, said solution or suspension being suitable for administration directly to nasal mucosa.

6. A pharmacologically active composition according to Claim 1, wherein said carrier is sterile water that is substantially pyrogen-free.

7. A pharmacologically active composition according to Claim 1, wherein said compound displays in vivo activity against a virus selected from the group consisting of

influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus and Sendai virus.

8. A pharmacologically active composition according to Claim 7, wherein said virus is an influenza virus.

9. A pharmacologically active composition according to Claim 1, wherein said compound possesses a K_i value, with respect to said active site, of less than 10^-7 M.

10. A pharmacologically active composition according to Claim 9, wherein said K_i value is less than about 0.5 x 10^-8 M.

11. A pharmacologically active composition according to Claim 1, wherein said compound is a carbocyclic or

heterocyclic compound comprised of a 5-, 6- or 7-membered ring carrying a substituent selected from a carboxylate moiety and an analogue thereof, said ring and said

substituent being positioned in the same plane.

12. A pharmacologically active composition according to Claim 11, wherein said compound is represented by the

structural formula

wherein

A denotes 0,

R¹ denotes COOH, P(O)(OH)₂, NO₂, SOOH, SO₃H, tetrazol, CH₂CHO, CHO, CH(CHO)₂ or, where R¹ is COOH,

P(O)(OH)₂, SOOH or SO₃H, an ethyl, methyl or pivaloyl ester thereof,

R⁴ denotes NHC-R⁷, where R⁷ is an unsubstituted or halogen-substituted linear or cyclic alkyl group of 1 to 6 carbon atoms, or SR⁶, OR⁶, COOH or alkyl/aryl ester thereof, NO₂, C(R⁶)₃, CH₂COOH or alkyl/aryl ester thereof, CH₂NO₂ or CH₂NHR⁷, and

R⁵ denotes CH₂YR⁶, CHYR⁶CH₂YR⁶ or CHYR⁶CHYR⁶CH₂YR⁶ where Y is O, S or H, and successive Y moieties in an R⁵ group are the same or different.

13. A pharmacologically active composition according to Claim 12, wherein said compound is DANA or FANA.

14. A pharmacologically active composition according to Claim 1, wherein said compound is a heterocylic compound comprising a heterooxygen and an anomeric carbon carrying substituents that are non-mutarotatable.

15. A pharmacologically active composition according to Claim 14, wherein said compound is a C9-carbohydrate.

16. A pharmacologically active composition according to Claim 1, wherein said compound is represented by the

structural formula

wherein

A denotes 0,

R denotes hydrogen, CN, CH-NHR⁶, CH₂OR⁶, CH₂F,

R¹ denotes COOH, P(O)(OH)₂, NO₂, SOOH, SO₃H, tetrazole, CH₂CHO, CHO, CH(CHO)₂ or, where R¹ is COOH,

P(O)(OH)₂, SOOH or SO₃H, an ethyl, methyl or pivaloyl ester thereof, R² denotes H, OR⁶, F, Cl, Br, CN, NHR⁶, SR⁶ or CH₂X, wherein X is NHR⁶, halogen or OR⁶ and

R⁶ is hydrogen; an acyl group having 1 to 4 carbon atoms; a linear or cyclic alkyl group having 1 to 6 carbon atoms, or a halogen-substituted analogue thereof; or an unsubstituted aryl group or an aryl substituted by a halogen, an allyl group, an OH group, an N0₂ group, an NH₂ group or a COOH group,

R⁵ denotes CH₂YR⁶, CHYR⁶CH₂YR⁶ or

CHYR⁶CHYR⁶CH₂YR⁶ where Y is O, S or H, and successive Y

moieties in an R⁵ group are the same or different.

17. A pharmacologically active composition according to Claim 16, wherein said compound is selected from the group consisting of 2-deoxy-N-acetyl-α-D-neuraminic acid, methyl N- acetyl-4,7,8,9-tetra-O-acetyl-2-deoxy-2α-allylthio- neuraminate, sodium N-acetyl-2-deoxy-2α-allylthioneuraminate, methyl N-acetyl-4,7,8,9-tetra-O-acetyl-2-deoxy-2α-fluoro-D- neuraminate and sodium N-acetyl-2-deoxy-2α-fluoro-D- neuraminate.

18. A compound that binds the active site of influenza virus neuraminidase and that displays in vivo activity against orthomyxovirus or paramyxovirus, wherein said

compound is not one selected from the group consisting of the compounds set out in Table 1.

19. A compound according to Claim 18, wherein said compound binds said active site with a K_i value of less than 10^-7 M.

20. A compound according to Claim 19, wherein said K_i value is less than about 0.5 x 10^-8 M.

21. A compound according to Claim 18, wherein said compound is a carbocyclic or heterocyclic compound comprised of a 5-, 6- or 7-membered ring carrying a substituent

selected from a carboxylate moiety and an analogue thereof, said ring and said substituent being positioned in the same plane.

22. A compound according to Claim 21, wherein said compound is represented by the structural formula:

wherein

A denotes O,

P(O)(OH)₂, SOOH or SO₃H, an ethyl, methyl or pivaloyl ester thereof,

O

'

R⁵ denotes CH₂YR⁶, CHYR⁶CH₂YR⁶ or CHYR⁶CHYR⁶CH₂YR⁶ where Y is O, S or H, and successive Y moieties in an R⁵ group are the same or different,

subject to the provisos that

(i) when R³ or R³' is OR⁶ or hydrogen, then said compound cannot have both

(a) an R² that is hydrogen and

(b) an R⁴ that is NH-acyl,

and

(ii) R⁶ represents a covalent bond when Y is hydrogen.

23. A compound according to Claim 18, wherein said compound is a heterocyclic compound comprising a heterooxygen and an anomeric carbon carrying substituents that are non- mutarotatable.

24. A compound according to Claim 23, wherein said compound is a C9-carbohydrate.

25. A compound according to Claim 18, wherein said compound is represented by the structural formula

wherein

A denotes O,

R denotes hydrogen, CN, CH-NHR⁶, CH₂OR⁶, CH₂F,

R¹ denotes COOH, P(O)(OH)₂, NO₂, SOOH, SO₃H,

tetrazole, CH₂CHO, CHO, CH(CHO)₂ or, where R¹ is COOH,H P(O)(OH)₂, SOOH or SO₃H, an ethyl, methyl or pivaloyl ester thereof,

R⁵ denotes CH₂YR⁶, CHYR⁶CH₂YR⁶ or

(i) when R³ or R³' is OR⁶ or hydrogen, then said compound cannot have both

(a) an R² that is hydrogen and

(b) an R⁴ that is NH-acyl,

substituted cyclohexyl and

(iii) R⁶ represents a covalent bond when Y is hydrogen.

26. A compound according to Claim 25, wherein said compound is methyl N-acetyl-4,7,8,9-tetra-O-acetyl-2-deoxy- 2α-allylthioneuraminate or sodium N-acetyl-2-deoxy-2α- allylthioneuraminate.

27. A method of preventing or ameliorating the symptoms of an orthomyxovirus or paramyxovirus infection, comprising the step of administering to an animal a pharmacologically active composition comprising:

(i) a virus-inhibiting amount of a compound that binds the active site of influenza virus neuraminidase and that displays anti-orthomyxovirus or paramyxovirus activity; and

(ii) a pharmaceutically acceptable carrier for said compound.

28. A method according to Claim 27, wherein the virus is selected from the group consisting of influenza virus, parainfluenza virus, Sendai virus and mumps virus, and the animal is a human.

29. A method according to Claim 27, wherein the virus is Newcastle disease virus or fowl plague virus, and the animal is a bird.

30. A method according to Claim 27, wherein the substance is administered orally, intranasally, buccally, or sublingually.

31. A method according to Claim 27, wherein the substance is administered intranasally.

32. A method of synthesis of a compound according to general formula I, as defined in Claim 25, comprising the steps of:

providing an alkyl N-acetyl neuraminate, reacting said alkyl N-acetyl neuraminate with an alcohol in the presence of an acid catalyst to yield the corresponding ester,

acylating and halogenating the ester by reaction with an acyl halide,

deacylating and de-esterifying the resulting compound under hydrolytic conditions, and

recovering the compound of general formula I.

33. A method of synthesis of a desired compound of general formula I, as defined in Claim 25, which comprises the steps of:

(a) treating a glycal of formula (i)

with hydrogen chloride and then with sodium thiophenoxide to form a thioglycoside of formula (ii)

(b) oxidizing the thioglycoside with

metachloroperoxybenzoic acid to form a sulphone of formula (iii)

and thereafter either (c) reacting the sulphone with lithium diisopropyl amide and then with dimethylcarbonate to form alpha and beta C-1 substituted sugars of formula (iv)

and

(c') isolating the C-1 substituted sugar,

or

(d) reacting the sulphone with lithium diisopropyl amide in the presence of lithium naphthalenide and a compound containing a COOH, P(O)(OH)₂ or SOOH group

and

(d') isolating the desired compound of formula I.

34. A method according to Claim 33, wherein in step (e) the compound containing the P(O)(OH)_H group is diethyl chlorophosphate.

35. A glycosyl halide of general formula III

wherein R may be F, Cl or Br, when R² is not H, F, Cl or Br;

if R³, R³' is OR⁶ or H then R⁴ is NH-acyl; and

A, R¹, R², R³, R³', R⁴, R⁵ and R⁶ are as defined in Claim 25.

36. A method of synthesis of a compound of general formula I, as defined in Claim 25, which comprises the step of reacting a glycosyl halide of general formula III

wherein R may be F, Cl or Br when R² is not H, F,

Cl or Br;

if R³, R³' is OR⁶ or H then R⁴ is NH-acyl; and

A, R¹, R², R³, R³', R⁴, R⁵ and R⁶ are as defined in

Claim 25, with a nucleophile which is a group which can be converted to a desired functional group R¹, and recovering said compound of general formula III.

37. An improved method of synthesis of glycosyl halides of general formula III as defined in Claim 35, comprising the step of treating the corresponding neuraminic acid analogue with excess acetyl halide at room temperature under a nitrogen atmosphere until no starting material is observable by thin layer chromatography, and recovering the desired glycosyl halide.

38. A compound according to Claim 18, synthesised using an intermediate compound selected from the group consisting of 2,3-didehydro-α-D-neuraminic acid; 3,4,6-tri-0-acetyl-2- deoxy-β-L-arabinohexepyranosyl thiophenoxide; 4-0-benzyl-3,6- bis(t-butylmethylsilyloxy)-2-deoxy-β-L-arabino-hexepyranosyl thiophenoxide; 4-0-benzyl-3,6-bis(t-butylmethylsilyloxy)-2- deoxy-β-L-arabinohexepyranosyl phenylsulphone; α-carboxy- methyl-β-phenylsulphonyl-4-0-benzyl-3,6-bis (t-butyldimethyl- silyloxy)-2-deoxy-L-arabinohexepyranose; methyl-4-O-benzyl-

3,6-bis(t-butyldimethylsilyloxy)-2-deoxy-α-L- arabinohexepyranosyl-carboxylate, and methyl-N-acetyl- 4,7,8,9-tetra-O-acetyl-2-β-chloro-2-β-deoxy-D-neuraminate.