WO2001029020A2

WO2001029020A2 - Neuraminidase inhibitors

Info

Publication number: WO2001029020A2
Application number: PCT/US2000/027090
Authority: WO
Inventors: Kent D. Stewart; Dale J. Kempf; Larry L. Klein; Clarence J. Maring; Vincent S. Stoll
Original assignee: Abbott Laboratories
Priority date: 1999-10-19
Filing date: 2000-10-02
Publication date: 2001-04-26
Also published as: AU2039301A; WO2001029020A9; WO2001029020A3

Abstract

The present invention provides compounds which are neuraminidase inhibitors characterized by a unique three-dimensional conformation and orientation relative to the Sites of Occupation 1-4 of the enzyme. Also provided are pharmaceutical compositions containing said compounds, as well as methods of using said compounds and compositions.

Description

NEURAMINIDASE INHIBITORS

Technical Field of the Invention

The present invention relates to novel compounds, compositions, and methods for inhibiting neuraminidase

Background of the Invention

Many disease-causing microorganisms possess a neuraminidase (also known as sialidase) which is involved in the replication process of the microorganism. In particular, viruses of the orthomyxovirus and paramyxovirus groups possess a neuraminidase. Diseases associated with paramyxoviruses include RSV (respiratory syncytial virus- related diseases) , pneumonia and bronchiolitis (associated with paramyxovirus type 3) and laryngotracheobronchitis (associated with paramyxovirus type 1) . Some of the more important disease-causing microorganisms in man and/or animals which possess a neuraminidase include Vibrio cholerae, Clostridium perfringens, Streptococcus pneumoniae, Arthrobacter sialophilus, influenza virus, parainfluenza virus, mumps virus, Newcastle disease virus, fowl plague virus, equine influenza virus and Sendai virus.

Mortality due to influenza is a serious problem throughout the world. The disease is devastating to man, lower mammals and some birds. Although vaccines containing attenuated influenza virus are available, those vaccines only provide immunological protection toward a few influenza strains and are less effective in otherwise immunologically compromised populations such as the elderly, young children, and in those who suffer from chronic respiratory illness. The productivity loss from absence due to sickness from influenza virus infection has been estimated to be more than $1 billion per year. There are two major strains of influenza virus (designated A and B) . Currently, there are only a few pharmaceutical products approved for treating influenza. These include amantadine and rimantadine, which are active only against the A strain of influenza viruses, and ribavirin, which suffers from dose-limiting toxicity. Mutant virus which is resistant to amantadine and rimantadine emerges quickly during treatment with these agents .

Very recently the first influenza neuraminidase inhibitor, zanamivir, was approved. However, it can only be administered by inhalation. Therefore, there is a continuing need for improved agents for treatment and/or prevention of influenza infection. Neuraminidase is one of two major viral proteins which protrude from the envelope of influenza virus. During the release of progeny virus from infected cells, neuraminidase cleaves terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides on the cell surface. Inhibition of neuraminidase enzymatic activity leads to aggregation of progeny virus at the surface. Such virus is incapable of infecting new cells, and viral replication is therefore retarded or blocked. X-ray crystallographic studies and sequence alignments have shown that the residues which directly contact the sialic acid portion of the substrate are strictly conserved in the neuraminidase from all A and B influenza strains. Thus, a compound which binds to the sialic acid binding region of the neuraminidase active site will block the replication of both the A and B strains of influenza virus. Compounds which are influenza neuraminidase inhibitors will be useful for the prevention of influenza infection and will be useful for the treatment of influenza infection.

The following references disclose neuraminic acid derivatives with the disclosed utility listed after each reference :

L. Von Itzstein, et al . , European Patent Application No. EP539204, published April 28, 1993 (antiviral agent);

T. Honda, et al . , European Patent Application No. EP823428, published February 11, 1998 (sialidase inhibitor; influenza treatment) ;

T. Honda, et al . , International Patent Application No. O98/06712, published February 19, 1998 (sialidase inhibitor; influenza remedy) ;

L. Von Itzstein, et al., International Patent Application No. O95/20583, published August 3, 1995 (viral neuraminidase inhibitor; influenza treatment) ;

P. Smith, International Patent Application No. WO95/18800, published July 13, 1995 (viral neuraminidase inhibitor);

P. Colman, et al . , International Patent Application No. WO92/06691, published April 30, 1992 (viral neuraminidase inhibitor) ;

L. Von Itzstein, et al . , U.S. Patent No. 5,648,379, issued July 15, 1997 (influenza treatment) ;

P. Reece, et al . , International Patent Application No. W097/32214, published September 4, 1997 (bind to influenza virus neuraminidase active site) ; and

P. Reece, et al . , International Patent Application No. W098/21243, published May 23, 1998 (anti-influenza agent) .

The following references disclose sialic acid derivatives with the disclosed utility listed after each reference :

Y. Ohira, et al . , International Patent Application No. O98/11083, published March 19, 1998 (antiviral agent); Y. Ohira, European Patent Application No. EP882721, published December 9, 1998 (antiviral agent) ; and

B. Glanzer, et al . , Helvetica Chimica Acta 74 343-369 (1991) (Vibrio cholerae neuraminidase inhibitor) .

The following references disclose benzene derivatives, cyclohexane derivatives or cyclohexene derivatives with the disclosed utility listed after each reference:

Y. Babu, et al . , U.S. Patent No. 5,602,277, issued February 11, 1997 (neuraminidase inhibitors) ;

M. Luo, et al., U.S. Patent No. 5,453,533, issued September 26, 1995 (influenza neuraminidase inhibitor; influenza treatment) ;

Y. Babu, et al . , International Patent Application No. O96/30329, published October 3, 1996 (neuraminidase inhibitor; viral infection treatment) ;

N. Bischofberger, et al . , U.S. Patent No. 5,763,483, issued June 9, 1998 (neuraminidase inhibitor);

C. Kim, et al . , International Patent Application No. O99/31047, published June 24, 1999 (neuraminidase inhibitor; influenza treatment) ; V. Atigadda, et al . , J. Med. Chem. 42 2332-2343 (1999) (influenza neuraminidase inhibitor) ; and

K. Kent, et al . , International Patent Application No. 98/07685, published February 26, 1998 (intermediates for the preparation of neuraminidase inhibitors) .

C. Kim, et al., International Patent Application No. 098/17647, published April 30, 1998 discloses piperidine derivatives that are useful as neuraminidase inhibitors.

N. Bischofberger, et al . , International Patent Application No. W096/26933, published September 6, 1996 and N.

Bischofberger, et al . , International Patent Application No. 099/14185, published March 25, 1999 disclose various substituted 6-membered ring compounds that are useful as neuraminidase inhibitors .

The following references disclose dihydropyran derivatives that are useful as viral neuraminidase inhibitors :

D. Andrews, et al . , International Patent Application No. WO97/06157, published February 20, 1997 and U.S. Patent No. 5,919,819, issued July 6, 1999; and P. Cherry, et al . , International Patent Application No. 096/36628, published November 21, 1996.

C. Kim, et al . , U.S. Patent No. 5,512,596, issued April 30, 1996 discloses 6-membered aromatic ring derivatives that are useful as neuraminidase inhibitors.

G. Diana, et al., International Patent Application No. O98/03487, published January 29, 1998 discloses substituted pyridazines that are useful for treatment of influenza.

B. Horenstein, et al . , International Patent Application No. O99/06369, published February 11, 1999 discloses piperazine derivatives that are useful as neuraminidase inhibitors .

The following references disclose substituted cyclopentanes that are useful as neuraminidase inhibitors and treatments for influenza:

Y. Babu, et al . , International Patent Application No. 097/47194, published December 18, 1997; and Y. Babu, et al . , International Patent Application No. W099/33781, published July 8, 1999.

L. Czollner, et al . , Helvetica Chimica Acta 73 1338-1358 (1990) discloses pyrrolidine analogs of neuraminic acid that are useful as Vibrio cholerae sialidase inhibitors.

. Brouillette, et al . , International Patent Application No. W099/14191, published March 25, 1999, discloses substituted pyrrolidin-2-one compounds that are useful as neuraminidase inhibitors and treatments for influenza.

The following references disclose siastatin B analogs that are useful as neuraminidase inhibitors:

Y. Nishimura, et al . , Natural Product Letters 1 39-44 (1992) ; and

Y. Nishimura, et al . , Natural Product Letters 1 33-38 (1992) .

C. Penn, UK Patent Application No. GB2292081, published February 14, 1996 discloses the use of a neuraminidase inhibitor in combination with an influenza vaccine.

All of the above references describe neuraminidase inhibitors with polar or charged substituents that bind within regions of the active site of the enzyme, herein denoted as Sites of Occupation 1-3.

Thus, it would be an important contribution to the art to provide compounds which are neuraminidase inhibitors and which also provide novel 3 -dimensional characteristics.

Summary of the Invention

The present invention relates to compounds which are neuraminidase inhibitors .

The instant invention additionally relates to pharmaceutical compositions containing a compound of the invention and the use of said compounds in the inhibition of neuraminidase, as well as the treatment of influenza thereby.

The invention still further relates to a method for selecting a compound which inhibits neuraminidase.

Brief Description of the Drawings

Figure 1 illustrates Sites of Occupation 1 through 4 within the neuraminidase active site. 2.0 Angstrom (small) and 3.0 Angstrom (large) spheres are shown for each of the four sites. The inhibitor 4-Gua-Neu5Ac2en (thick black lines) and protein (thin black lines) are taken from Protein Data Bank entry 1NNC.

Detailed Description of the Invention

The present invention provides compounds which are neuraminidase inhibitors. Said compounds, when bound to the enzyme, are characterized by a unique three-dimensional conformation and orientation relative to the neauraminidase .

A compound of the present invention binds to the ectodomain of influenza neuraminidase and inhibits the catalytic activity of that enzyme. By binding to the ectodomain of neuraminidase, the compounds prevent the catalytic action of this enzyme, and thereby, exhibit antiviral action upon the influenza virus. The potency of prefered compounds of this invention will be at IC50 = 1 micromolar, or less, when measured in standard enzyme assays of substrate hydrolysis, as described hereinbelow. The inhibitor binds to the active site of influenza neuraminidase, which active site is well known in the art (P.M. Colman, J.N. Varghese & W. G. Laver, "Structure of the catalytic and antigenic sites in influenza virus neuraminidase", Nature, 303, 41-44, 1983). When bound to the enzyme active site, the inhibitor possesses substituent groups, of which at least one atom or a centroid is contained within various Sites of Occupation, as defined below. The Site of Occupation volume is defined by locating the center point of a spherical volume together with a defined radius around that center point. The center point of the spherical volume is defined by the point of intersection of vectors of a certain length (loci) emanating from particular alpha carbon atoms of the bound neuraminidase enzyme. One of skill in the art will recognize that any of the alpha carbons of the bound neuraminidase enzyme can used as points of reference. The reference points for location of the center point include the alpha carbons of protein residues within the neuraminidase enzyme. Protein X-ray crystal structures of influenza neuramindase at atomic resolution have shown that while non-active site insertions, deletions, and residue variation can occur between neuraminidase enzymes from different strains and subtypes, the active site residues are conserved and spatially equivalent. Thus, numbering of protein residues and reference to their alpha carbons may refer to different sequence numbering, but refer to identical positions in 3 -dimensional atomic space. A table of equivalent residues for three sequences of influenza neuraminidase, N2/Tokyo67, N9/Tern, and B/Lee is provided in Table 1 hereinbelow. In each case, the 3-dimensional structure has been determined by protein x-ray crystallography, and the coordinates are available from the Protein Data Bank under the accession codes IIVF, INNC, and 1B9S, respectively. Only the residues of influenza neuraminidase from strain B/Lee will be used in the definition of the Sites of Occupation, but it is understood that the corresponding residues as provided below in Table 1 from other neuraminidase enzymes are encompassed by the discussion.

Table 1

Spatially Equivalent Protein Residues of Influenza

Neuraminidases from Different Strains and Subtypes

Definition of the Sites of Occupation

Four separate Sites of Occupation are defined below:

Site 1:

The volume is of spherical shape and has its center point defined by loci selected from the group of loci consisting of 8.9 to 9.9 Ang from the alpha carbon of residue 116, 10.4 to 11.4 Ang from the alpha carbon of residue 292, 9.5-10.5 Ang from the alpha carbon of residue 374, and 7.6-8.6 Ang from the alpha carbon of residue 409.

Site 2: The volume is of spherical shape and has its center point defined by loci selected from the group of loci consisting of 6.2 to 7.2 Ang from the alpha carbon of residue 117, 3.7-4.7 Ang from the alpha carbon of residue 149, 5.0-6.0 Ang from the alpha carbon of residue 177, and 7.5 to 8.5 Ang from the alpha carbon of residue 226.

Site 3;

The volume is of spherical shape and has its center point defined by loci selected from the group of loci consisting of 6.4 to 7.4 Ang from the alpha carbon of residue 150, 6.6-7.6 Ang from the alpha carbon of residue 177, 6.1-7.1 Ang from the alpha carbon of residue 221, and 4.8 to 5.8 Ang from the alpha carbon of residue 223.

Site 4;

The volume is of spherical shape and has its center point defined by loci selected from the group of loci consisting of 5.7-6.7 Ang from the alpha carbon of residue 149, 8.8-9.8 Ang from the alpha carbon of residue 226, 9.2- 10.2 Ang from the alpha carbon of residue 245, and 8.9-9.9 Ang from the alpha carbon of residue 409.

A substituent is considered to be within a Site of Occupation if any atom of that substituent is within the Site of Occupation. An atom is within the Site of Occupation if the coordinates for the center of that atom are within the volume defined for a particular Site of Occupation. Definition of the Volume Defined at each Site of Occupation

The volume of space defined at each Site of Occupation is defined by spheres of a given radius emanating from the loci given above. It is understood that spheres of lower radius claim smaller volumes, and thus in turn, encompass fewer molecules. Most preferred are radii of 4.0 Angstroms or less, prefered are radii of 2.0 or 3.0 Angstoms or less, less prefered are radii of 1.0 Angstrom or less. An atom of a substituent is within this volume if the coordinates for the center of that atom fall within the volume defined for a particular Site of Occupation. In the case of Site of Occupation 4, a substituent/ring system is within this volume if the centroid of the ring atoms is found within the volume. A centroid of a ring is defined as the geometric or arithmetic centerpoint of the coordinates of the atoms that comprise the ring. Methods for determining if atomic coordinates of a atom of a substituent falls within a Site of Occupation

Means for determining the spatial orientation of a compound of this invention are well known in the art. One preferred method for determination of the spatial orientation of a neuraminidase inhibitor is by the technique of X-ray crystallography. The process of determining the structures of protein/inhibitor complexes using the X-ray technique is well known (see T. L. Blundel and L. N. Johnson, Protein Crystallography, Academic Press, (1976) and Methods in Enzymology, volumes 114 and 115, H. W. Wyckoff et al . , eds . , Academic Press (1985)). This technique can employ, for instance, a highly purified preparation of neuraminidase in a buffered solution (typically at a pH of between about 4.5 and about 8.0) . The complex is allowed to crystallize in the presence of a precipitation agent (such as ammonium sulfate) under conditions which yield single crystals of the complex.

Specific conditions for crystallizing neuraminidase with various inhibitors have been well documented (see, for example P.M. Colman, Protein Science, 3, 1687-1696 (1994)). Alternatively, crystals of un-inhibited neuraminidase can be grown and inhibitors soaked into the crystal lattice and the structure determined. Typical soaking conditions employ, for instance, placing a crystal of uninhibited neuraminidase into a buffered solution at pH 7.0. An amount of the compound of interest is dissolved in a cosolvent, for example dimethyl sulfoxide, and an aliquot of this inhibitor solution added to the liquid containing the crystal. After sitting for a time not shorter than 15 minutes nor longer than 30 days, the crystal is removed from the liquid and applied to a x-ray diffraction device. Application of a concentrated X-ray beam (from rotating anode X-ray generator or synchrotron) to an appropriately prepared and mounted crystal will yield a diffraction pattern from the reflected X-ray beam. Detection of the diffracted rays may be carried out by using a multiwire area detector (such as that manufactured by Siemmens Analytical X-Ray Instruments, Inc. (Madison, WI) ) or an R- axis II image plate system from Rigaku Corporation, The Woodlands, TX) . Refinement of the X-ray diffraction data using computer software such as X-PLOR (A.T. Brunger, X- PLOR, Version 3.1: Yale University Press: New Haven, CT, 1992, distributed by Molecular Simulations, Inc.)^' will yield a three dimensional structure. In general, the above technique will yield a structure which may be refined to about 2 to 3 A with an R-value of about 0.25 or less. As the skilled artisan can appreciate, these values are adequate to determine the interactions between neuraminidase and a given compound such that it will be clear if the features described herein are present. Visualization of the complex of neuraminidase and an inhibitor may be carried out using computer software such as Insightll (Biosym/Molecular Simulations, Inc., San Diego) or Quanta (Molecular Simulations, Inc., Burlington MA) , and distances and spherical volumes may be generated using tools available within those programs. An example of the use of X-ray crystallography in determining the spatial orientation of a neuraminidase inhibitor in B/Lee influenza neuraminidase protein is found in Janakiraman, M. N. et al., Biochemistry, 33, 8172-8179 (1994).

A second means for determining spatial orientation is the technique of Nuclear Magnetic Resonance (NMR) Spectroscopy . The process of determining the structures of protein/inhibitor complexes using the NMR technique is well known (see K. Wuthrich, NMR of Proteins and Nucleic Acids, John Wiley, (1986)) . This technique can employ, for instance, a preparation of neuraminidase complexed with an inhibitor of interest in a buffered solution (typically at a pH of between about 3.0 and about 8.0) . Either single or multi-dimensional techniques may be applied. Advantageously, the enzyme and/or the inhibitor may be enriched with stable isotopes such as ¹³C, ¹⁵N, or ²H to more easily determine the binding conformation and proximity. In general, the NMR technique will yield a structure which has no distance violation greater than about 0.3 A, and an RMS deviation between the family of structures generated from the average structure of about 0.6 A. As the skilled artisan can appreciate, these values are adequate to determine the interactions between neuraminidase and a given compound such that it will be clear if the features described herein are present. Visualization of the complex of neuraminidase and an inhibitor may be carried out using computer software such as Insightll (Biosym/Molecular Simulations, Inc., San Diego) or Quanta (Molecular Simulations, Inc., Burlington MA) , and distances and spherical volumes may be generated using tools available within those programs.

A third means for determining spatial orientation is the technique of Molecular Modeling. This process of creating theoretical models of protein-inhibitor complexes is well known (see G. L. Seibel and P. A. Kollman, "Molecular Mechanics and the Modeling of Drug Structures, Ch. 18.2 in Comprehensive Medicinal Chemistry, C. Hansch, Ed., Pergammon Press, (1990) and T.J. Perun and C. L. Propst, Eds. Computer-Aided Drug Design, Marcel Dekker, Inc. (1989)). Computer software such as Insightll

(Biosym/Molecular Simulations, Inc., San Diego) or Quanta (Molecular Simulations, Inc., Burlington MA) is used to construct a possible 3D arrangement of inhibitor and protein. Typically, coordinates for the protein are derived from prior X-ray or NMR structures taken from the Protein Data Base (Brookhaven National Laboratories, New York) . Coordinates for the inhibitor are adopted which uses standard bond lengths and angles derived from structures of organic compounds (for example, data found within the Cambridge Crystallographic Database (University Chemical Laboratory, Cambridge, United Kingdom) . The candidate inhibitor is aligned in 3 -dimensional space with other, related inhibitors whose bound conformations have previously been determined by either x-ray crystallography or NMR spectroscopy. Both Van der Waals volume and electrostatic potentials are used to direct the alignment process. Typically the protein and inhibitor molecules are allowed to achieve conformations that are lower in energy from the initial starting geometry by energy minimization in which a force field is used to mathematically search for the lowest energy conformation. Suitable force fields such as AMBER (S. J. Weiner et al, Journal of Computational Chemistry, 7, 230-252 (1986) or CVFF (J. R. Maple et al, Journal of Computational Chemistry, 15, 162-182 (1994)) are available within the software listed above. Typically, energy minimization of the inhibitor is initially carried out with the enzyme atoms held fixed in space followed by more extensive energy minimization of the entire protein/inhibitor complex. Conformational searching

(torsion driver, rotor library, or dynamics/Monte Carlo methods are available within the software listed above) is carried out to explore additional or alternative binding modes of the inhibitor within the active site of neuraminidase. Water molecules may be added during this analysis to more closely simulate the aqueous environment. Usually, only one or a small number of possible inhibitor conformations remain after this entire modeling process. The accuracy of these theoretical models is frequently comparable to the accuracy of structures determined by x- ray or NMR methods, particularly when the candidate inhibitor compound is closely related to the previously studied inhibitor compounds. An example of the use of molecular modeling methods in determining the spatial orientation of a neuraminidase inhibitor when bound to influenza neurminidase protein is found in M. von Itzstein et al.. Nature, 363, 418-423, 1993. Visualization of the complex of neuraminidase and an inhibitor may be carried out using computer software such as Insightll (Biosym/Molecular Simulations, Inc., San Diego) or Quanta (Molecular Simulations, Inc., Burlington MA), and distances and spherical volumes may be generated using tools available within those programs.

The binding mode of a compound of formula I

whose synthesis is disclosed in von Itzstein, M. et al, Nature, 363, 418-423 (1993), and whose binding mode is disclosed in Varghese, J. N. et al . , Protein Science, 4, 1081-1087 (1995) . Is also known in the art as . A compound of formula I is known in the art as 4-Guanidino-Neu5Ac2en.

A compound of formula I is further represented in Figure 1, wherein the binding of said compound to the active site of the neuraminidase enzyme is depicted. As shown in Figure 1, the compound of formula I possesses s carboxylate substituent within Site of Occupation 1, a guanidine substituent within Site of Occupation 2, and amide group within Site of Occupation 3, and the centroid of a 6-membered non-aromatic ring within Site of Occupation

The compounds of the present invention can be used in the form of salts derived from inorganic or organic acids. These salts include but are not limited to the following: acetate, trifluoroacetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide , hydroiodide, 2-hydroxy-ethanesulfonate (isethionate) , lactate, maleate, methanesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, p- toluenesulfonate and undecanoate . Also, basic nitrogen- containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil- soluble or dispersible products are thereby obtained.

Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, lithium, calcium or magnesium or with ammonium or N(R**)4+ salts (where R** is loweralkyl) .

In addition, salts of the compounds of this invention with one of the naturally occurring amino acids are also contemplated.

Preferred salts of the compounds of the invention include hydrochloride, methanesulfonate, sulfonate, phosphonate and isethionate.

The compounds of this invention may have a substituent which is an acid group (for example, -C02H, -S03H, -S02H, -P03H2, -P02H) . Compounds of this invention having a substituent which is an ester of such an acidic group are also encompassed by this invention. Such esters may serve as prodrugs. The prodrugs of this invention are metabolized in vivo to provide the above-mentioned acidic substituent of the parental compound. Prodrugs may also serve to increase the solubility of these substances and/or absorption from the gastrointestinal tract. These prodrugs may also serve to increase solubility for intravenous administration of the compounds. Prodrugs may also serve to increase the hydrophobicity of the compounds . Prodrugs may also serve to increase the oral bioavailability of the compounds by increasing absorption and/or decreasing first- pass metabolism. Prodrugs may also serve to increase tissue penetration of the compounds, thereby leading to increased activity in infected tissues and/or reduced rate of clearance.

Such esters contemplated by this invention include:

alkyl esters, especially loweralkyl esters, including, but not limited to, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl esters and the like;

alkoxyalkyl esters, especially, loweralkoxyloweralkyl esters, including, but not limited to, methoxymethyl, 1- ethoxyethyl, 2-methoxyethyl, isopropoxymethyl, t- butoxymethyl esters and the like; alkoxyalkoxyalkyl esters, especially, alkoxyalkoxy- substituted loweralkyl esters, including, but not limited to, 2-methoxyethoxymethyl esters and the like;

aryloxyalkyl esters, especially, aryloxy-substituted loweralkyl esters, including, but not limited to, phenoxymethyl esters and the like, wherein the aryl group is unsubstituted or substituted as previously defined herein;

haloalkoxyalkyl esters, especially, haloalkoxy- substituted loweralkyl esters, including, but not limited to, 2 , 2 , 2-trichloroethoxymethyl esters and the like;

alkoxycarbonylalkyl esters, especially, loweralkoxycarbonyl-substituted loweralkyl esters, including, but not limited to, methoxycarbonylmethyl esters and the like;

cyanoalkyl esters, especially, cyano-substituted loweralkyl esters, including, but not limited to, cyanomethyl, 2-cyanoethyl esters and the like;

thioalkoxymethyl esters, especially, lowerthioalkoxy- substituted methyl esters, including, but not limited to, methylthiomethyl, ethylthiomethyl esters and the like; alkylsulfonylalkyl esters, especially, loweralkylsulfonyl-substituted loweralkyl esters, including, but not limited to, 2-methanesulfonylethyl esters and the like;

arylsulfonylalkyl esters, especially, arylsulfonyl- substituted loweralkyl esters, including, but not limited to, 2-benzenesulfonylethyl and 2-toluenesulfonylethyl esters and the like;

acyloxyalkyl esters, especially, loweralkylacyloxy- substituted loweralkyl esters, including, but not limited to, formyloxymethyl, acetoxymethyl, pivaloyloxymethyl , acetoxyethyl, pivaloyloxyethyl esters and the like;

cycloalkylcarbonyloxyalkyl esters including cyclopentanecarbonyloxymethyl , cyclohexanecarbonyloxymethyl, cyclopentanecarbonyloxyethyl, cyclohexanecarbonyloxyethyl esters and the like;

arylcarbonyloxyalkyl esters including, but not limited to, benzoyloxymethyl esters and the like;

(alkoxycarbonyloxy) alkyl esters, especially, (loweralkoxycarbonyloxy) -substituted loweralkyl esters, including, but not limited to, methoxycarbonyloxymethyl. ethoxycarbonyloxymethyl . 1- (methoxycarbonyloxy) ethyl, 2- (ethoxycarbonyloxy) ethyl esters and the like;

(cycloalkyloxycarbonyloxy) alkyl esters, especially, (cycloalkyloxycarbonyloxy) -substituted loweralkyl esters, including, but not limited to, cyclohexyloxycarbonyloxymethyl , cyclopentyloxycarbonyloxyethyl , cyclohexyloxycarbonyloxypropyl esters and the like;

oxodioxolenylmethyl esters including, but not limited to, (5-phenyl-2-oxo-l, 3-dioxolen-4-yl) methyl, [5- (4- methylphenyl) -2-oxo-l, 3-dioxolen-4-yl] methyl, [5- (4- methoxyphenyl) -2-oxo-l , 3-dioxolen-4-yl] methyl, [5- (4- fluorophenyl) -2-oxo-l, 3-dioxolen-4-yl] methyl, [5- (4- chlorophenyl) -2-oxo-l, 3 -dioxolen-4-yl] methyl, (2-oxo-l, 3- dioxolen-4-yl) methyl, (5-methyl-2-oxo-l , 3-dioxolen-4- yl) methyl, (5-ethyl-2-oxo-l , 3-dioxolen-4-yl) methyl, (5-^' propyl-2-oxo-l, 3-dioxolen-4-yl) methyl , (5-isopropyl-2-oxo- 1, 3-dioxolen-4-yl) methyl, (5-butyl-2-oxo-l, 3-dioxolen-4- yl) methyl esters and the like;

phthalidyl esters wherein the phenyl ring of the phthalidyl group is unsubstituted or substituted as defined previously herein, including, but not limited to, phthalidyl, dimethylphthalidyl, dimethoxyphthalidyl esters and the like;

aryl esters including, but not limited to, phenyl, naphthyl, indanyl esters and the like;

arylalkyl esters, especially, aryl-substitued loweralkyl esters, including, but not limited to, benzyl, phenethyl, 3-phenylpropyl, naphthylmethyl esters and the like, wherein the aryl part of the arylalkyl group is unsubstituted or substituted as previously defined herein;

dialkylaminoalkyl esters, especially dialkylamino- substituted loweralkyl esters, including, but not limited to, 2- (N,N-dimethylamino) ethyl, 2- (N,N-diethylamino) ethyl ester and the like

(heterocyclic) alkyl esters, especially, heterocyclic- substituted loweralkyl esters wherein the heterocycle is a nitrogen-containing heterocycle, including, but not limited to, (heterocyclic) methyl esters and the like, wherein the heterocyclic part of the (heterocyclic) alkyl group is unsubstituted or substituted as previously defined herein; and carboxyalkyl esters, especially, carboxy-substituted loweralkyl esters, including, but not limited to carboxymethyl esters and the like;

and the like.

Preferred prodrug esters of acid-containing compounds of the the instant invention are loweralkyl esters, including, but not limited to, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl esters, 3- pentyl esters, cycloalkyl esters, cycloalkylalkyl esters and benzyl esters wherein the phenyl ring is unsubstituted or substituted as previously defined herein.

Methods for the preparation of prodrug esters of compounds of the instant invention are well-known in the art and include:

reacting the acid with the corresponding halide (for example, chloride or acyl chloride) and a base (for example, triethylamine, DBU, N,N-dimethylaminopyridine and the like) in an inert solvent (for example, DMF, acetonitrile, N-methylpyrrolidone and the like) ;

reacting an activated derivative of the acid (for example, an acid chloride, sulfonyl chloride, monochlorophosphonate and the like) with the corresponding alcohol or alkoxide salt; and the like.

Other examples of prodrugs of the present invention include amides derived from the substituent which is an acid group.

Such amides contemplated by this invention include:

simple amides, such as -C(0)NH2 and the like;

alkylamino amides, especially, loweralkylamino amides, including, but not limited to, methylamino, ethylamino, n-propylamino, isopropylamino amides and the like ;

cylcoalkylamino amides, including, but not limited to, cylopropylamino, cylcobutylamino, cyclopentylamino, cyclohexylamino amides and the like;

acylamino amides, including, but not limited to acetylamino, propionylamino, butanoylamino amides and the like;

cylcoalkylcarbonylamino amides, including, but not limited to, cyclopropylcarbonylamino, cyclobutylcarbonylamino amides and the like; alkoxycarbonylalkylamino amides, including, but not limited to, ethoxycarbonylmethylamino, t- butyloxycarbonylmethylamino and the like;

aminoacylamino amides, including, but not limited to, aminoacetylamino amides and the like;

dialkylaminoacylamino amides, including, but not limited to, dimethylaminoacetylamino, diethylaminoacetylamino amides and the like;

(heterocyclic) acylamino amides, including, but not limited to, piperidin-1-ylacetylamino amides and the like;

amides derived from single naturally occuring L-amino acids (or from acid-protected L-amino acids, for example, esters of such amino acids and the like) or from dipeptides comprising two naturally occuring L-amino acids wherein each of the two amino acids is the same or is different (or from acid-protected dipeptides, for example, esters of such dipeptides and the like) ;

and the like.

Methods for preparation of prodrug amides of compounds of the invention are well-known in the art and include reacting the acid with the appropriate amine in the presence of an amide bond or peptide bond-forming coupling reagent or reacting an activated derivative of the acid with the appropriate amine and the like.

Other examples of prodrugs of the present invention include esters of hydroxyl-substituted compounds of the invention which have been acylated with a blocked or unblocked amino acid residue, a phosphate function, a hemisuccinate residue, an acyl residue of the formula R100C(O)- or R100C(S)- wherein R100 is hydrogen, lower alkyl, haloalkyl, alkoxy, thioalkoxy, alkoxyalkyl, thioalkoxyalkyl or haloalkoxy, or an acyl residue of the formula Ra-C(Rb) (Rd)-C(O)- or Ra-C(Rb) (Rd)-C(S)- wherein Rb and Rd are independently selected from hydrogen or lower alkyl and Ra is -N(Re) (Rf) , -ORe or -SRe wherein Re and Rf are independently selected from hydrogen, lower alkyl and haloalkyl, or an amino-acyl residue having the formula R101NH(CH2)2NHCH2C(O) - or R101NH (CH2 ) 20CH2C (O) - wherein R101 is hydrogen, lower alkyl, (aryl) alkyl, (cycloalkyl) alkyl, acyl, benzoyl or an -amino acyl group. The amino acid esters of particular interest are of glycine and lysine; however, other amino acid residues can also be used, including any of the naturally occuring amino acids and also including those wherein the amino acyl group is -C(O) CH2NR102R103 wherein R102 and R103 are independently selected from hydrogen and lower alkyl, or the group -NR102 R103, where R102 and R103, taken together, forms a nitrogen containing heterocyclic ring.

Other prodrugs include a hydroxyl-substituted compound of the invention whgrein the hydroxyl group is functionalized with a substituent of the formula

-CH(R104)OC(O)R105 or -CH (R104 ) OC (S) R105 wherein R105 is lower alkyl, haloalkyl, alkoxy, thioalkoxy or haloalkoxy and R104 is hydrogen, lower alkyl, haloalkyl, alkoxycarbonyl, aminocarbonyl , alkylaminocarbonyl or dialkylaminocarbonyl . Such prodrugs can be prepared according to the procedure of Schreiber (Tetrahedron Lett. 1983, 24, 2363) by ozonolysis of the corresponding methallyl ether in methanol followed by treatment with acetic anhydride.

The preparation of esters of hydroxyl-substituted compounds of the invention is carried out by reacting a hydroxyl-substituted compound of the instant invention with an activated amino acyl, phosphoryl, hemisuccinyl or acyl derivative.

Prodrugs of hydroxyl-substituted-compounds of the invention can also be prepared by alkylation of the hydroxyl substituted compound of the invention with (halo) alkyl esters, transacetalization with bis- (alkanoyl) acetals or condensation of the hydroxyl group with an activated aldehyde followed by acylation of the intermediate hemiacetal .

In preparing prodrugs it often is necessary to protect other reactive functional groups, in order to prevent unwanted side reactions. After protection of the reactive groups the desired group can be functionalized. The resulting functionalized product is then deprotected, to remove the protecting groups that were added to prevent unwanted side reactions. This will provide the desired prodrug. Suitable reaction conditions for preparing protecting groups are well known in the art. One source for reaction conditions is found in T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley & Sons, New York (1991),

This invention also encompasses compounds which are esters or prodrugs and which are also salts. For example, a compound of the invention can be an ester of a carboxylic acid and also an acid addition salt of an amine or nitrogen-containing substituent in the same compound. Preferred compounds of the instant invention have the following characteristics:

1. The compound will possess a carboxylate, sulfonate, sulfinate, phosphate, or phosphinate substituent within Site of Occupation 1.

2. The compound will possess a substituent with at least two sp2 carbon centers within Site of Occupation

2. A list of examples of such substituents is given in Table 2.

3. The compound will possess an amide or sulfonamide group within Site of Occupation 3

4. The compound will possess a 4, 5, 6, 7, or 8- membered non-aromatic ring system, whose centroid lies within Site of Occupation 4.

The volume of each of the Sites of Occupation have radius 4.0 Angstroms

Most preferred compounds of the instant invention are those which have the following characteristics:

1. The compound will possess a carboxylate substituent within Site of Occupation 1. 2. The compound will possess a substituent with at least two sp2 carbon centers within Site of Occupation

2. A list of examples of such substituents is given in Table 2.

3. The compound will possess an amide group within Site of Occupation 3

4. The compound will possess a 5- or 6-membered ring system, whose centroid lies within Site of Occupation 4.

The volume of each of the Sites of Occupation have radius 4.0 Angstroms .

Table 2 provided hereinbelow illustrates substituents containing at least two sp2 carbon centers that bind within the Site of Occupation 2. The point of attachment of the substituent to the remainder of the inhibitor molecule is designated by wavy underline. All other points of attachment on these substituents are included within the scope of the invention. Table 2

10 11 12

13 14 15 16

17 18 19 20

21 22 23 24

25 26 27 28

29 30 31 32

33 34 35 36

37 38 39 40

41 42 43 44

45 46 47 48

_/N. ,N. HN ^{^} N

V — M N

49 50 51 52

53 54 55 56

57 58 59 60

61 62 63 64

65 66 67 68

69 70 71 72

73 74 75 76

77 78 79 80

81 82 83 84

85 86 87 88

Cl. CH₃ H₃C pi H₃C CH₃ Cl pH₃

89 90 91 92

93 94 95 96

97 98 99 100

101 102 103 104

105 106 107 108

H,C CF, F,C CH, F,C CH,

109 110 111 112

113 114 115 116

117 118 119 120

121 122 123 124

125 126 127 128

F₃C Cl F,C Cl F₃C pi F₃C CF₃

^■3^ Cl X

129 130 131 132

Cl

133 134 135 136

137 138 139 140

141 142 143 144

145 146 147 148

149 150 151 152

^H3C CF₃ H₃C Cl Cl CF₃ F,C Cl

Cl F,C H,C H,C

153 154 155 156

157 158 159 160

161 162 163 164

165 166 167 168

169 The compounds of the instant invention exclude those substituents of formula II, III, IV, V, and VI, which lie within Site of Occupation 2 as provided below:

IV

Compounds of the instant invention still further exclude the compounds of formula VII, VIII, VXI, X, and XI as shown hereinbelow (whose synthesis is disclosed in Compound of Kok, G. B., and von Itzstein, M. , J. Chem. Soc . Perkin Trans. I, 905-908 (1998)), wherein any of the alcoholic -OH groups are esterified or not, or whether the carboxylic acid group is esterified or not:

The ability of the compounds of the present invention to inhibit neuraminidase in vitro can be determined according to the methods described hereinbelow. Neuraminidase Inhibition Assay:

Influenza virus A/Nl/PR/8/34 was grown in the allantoic cavity of fertilized eggs and purified by sucrose density gradient centrifugation (Laver, W. G. (1969) in "Fundamental Techniques in Virology" (K. Habel and N. P. Salzman, eds . ) pp. 92-86, Academic Press, New York). Influenza virus A/N2/Tokyo/3/67 was obtained from the tissue culture supernatents of virus grown on MDCK cells. Neuraminidase from B/Memphis/3/89 virus was prepared by digestion of the virus with TPCK-trypsin followed by centrifugation and then purification of the neuraminidase catalytic fragment using sucrose density gradient centrifugation and dialysis as described previously (Air, G. M., Laver, W. G. , Luo, M. , Stray, S. J. , Legrone, G., and Webster, R. G. (1990) Virology 177, 578-587). The neuraminidase inhibition assays used the neuraminidase enzymatic activity associated with the A/Nl/PR/8/34 or A/N2/Tokyo/3/67 whole virus, or the B/Memphis/3/89 catalytic head fragment. The whole virus or catalytic fragment was diluted appropriately with 20 mM N- ethylmorpholine, 10 mM calcium choride, pH 7.5 buffer on the day of the experiment. Neuraminidase inhibition assays were conducted in 20 mM N-ethylmorpholine, 10 mM calcium choride, pH 7.5 buffer with 5% DMSO. Reaction mixtures included neuraminidase, inhibitor (test compound) and 20-30

μM 4-methylumbelliferyl sialic acid substrate in a total volume of 200 μh and were contained in white 96-well U- shaped plates. Typically, five to eight concentrations of inhibitor were used for each Ki value measurement . The reactions were initiated by the addition of enzyme and allowed to proceed for 30-60 minutes at room temperature. The fluorescence for each well of the plate was measured once each minute during the reaction period by a Fluoroskan II plate reader (ICN Biomedical) equipped with excitation and emission filters of 355 +/- 35 nm and 460 +/- 25 nm, respectively. The plate reader was under the control of DeltaSoft II software (Biometallics) and a Macintosh computer. If the compound exhibited linear reaction velocities during the reaction period, then the reaction velocities for the dose-response study were fit to equation 1 using a nonlinear regression program (Kaleidagraph) to determine the overall Ki value (Segel, I. H. (1975) in Enzyme Kinetics, pp. 105-106, Wiley- Interscience, New York) .

( 1 - Vi/Vo) = [ I ] / { [ I ] + Ki ( l + [S] /Km) } eqn 1 In equation 1, Vi and Vo represent inhibited and uninhibited reaction velocities, respectively, and Km = 16 - 40 μM depending on the neuraminidase strain tested. For those compounds exhibiting slow-binding inhibition (Morrison, J. F. (1982) Trends Biochem. Sci . 7, 102- 105) , a second experiment was performed in a manner identical to the first except that neuraminidase and inhibitor were preincubated in the absence of substrate for 2 hours at room temperature prior to initiating the reactions with substrate. Data analysis for the resulting linear velocities was conducted as described above.

Equation 2 was used to measure Ki values in the sub- nanomolar range (Morrison, J. F. And Stone, S. R. (1985) Comments Mol . Cell Biophys . 2, 347-368) .

V = A{sqrt{(Ki' + It -Et) ^Λ2 + 4Ki'Et} - (Ki' + It - Et)] eqn. 2

In equation 2, V = velocity; A = αkcat [S] /2 (Km + [S] ) ; a is a factor to convert fluorescence units to molar concentrations; Ki' = Ki (1 + [S] /Km) ; It = total inhibitor concentration and Et = total active concentration of neuraminidase. The compounds of the invention inhibit influenza A neuraminidase and influenza B neuraminidase with Ki values between about 0.1 nanomolar and about 500 micromolar. Preferred compounds of the invention invention inhibit influenza A neuraminidase and influenza B neuraminidase with Ki values between about 0.1 nanomolar and about 3.5 micromolar.

The ability of the compounds of the invention to inhibit plaque formation in cell culture can be determined by the method described below.

Cell Culture Plague Formation Inhibition Assay- Cell Cultures: MDCK cells obtained from the American Type Culture Collection were grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (GibcoBRL) supplemented with 10% fetal calf serum (JRH Biosciences) , 40 mM HEPES buffer (GibcoBRL) and antibiotics (GibcoBRL) . Cells were routinely cultured in flasks or roller bottles at 37°C and 5% C02. At confluence cells were reduced to a density of 500,000 cells in a ml using trypsin/EDTA (GibcoBRL) treatment of the monolayer followed by cell centrifugation, resuspension, and dilution into growth media. Cells were planted at a volume to surface area ratio of 1 ml over 1 cm2 of growth surface .

Plaque Assay Protocol: On MDCK cell confluent 6 well plates growth media was removed and the cells were overlaid with 1.5 ml of assay media (DMEM with 1% fetal calf serum, 40 mM HEPES buffer and antibiotics) containing pre-mixed virus (influenza A/Tokyo/3/67 [H2N2] ) (40 -100 plaque forming units) and 2x concentration test compound. The plates were placed on a rocker and incubated for 2 hours at room temperature. During the virus adsorption period agar overlay media was prepared. In a microwave oven 2X agarose (final concentration of 0.6% agarose) in overlay media (DMEM with 40 mM HEPES buffer) was melted and then placed in a 48°C water bath for temperature equilibration. After the virus adsorption period was completed 1.5 ml agar over media was added and mixed with the 1.5 ml virus and test compound containing media per well.

Cultures were incubated at 35°C for the period required for plaque development, usually several days.

Plaques were fixed with 3.7% formalin in PBS for 20 minutes followed by removal of the agar overlay and staining with 0.1% crystal violet in distilled water for 15 minutes. Plaques were counted and EC 50 concentration determined from multiple concentrations of the tested compound using regression analysis.

Viral Stocks: Stocks were prepared in MDCK confluent roller bottles incubated at 37 °C in DMEM supplemented with 1% FCS, 40mM HEPES buffer, and antibiotics. Bottles were inoculated with a multiplicity of infection of approximately 0.1 plaque forming unit for each cell. Roller bottles were harvested after the cytopathic effect of the virus was observed to be complete. Stocks were prepared from the supernatant resulting from the low speed centrifugation of the media and cell lysate . Stocks were titered and stored at -80 °C.

Compounds of the invention provided plaque formation inhibition for influenza virus A/N2/Tokyo in MDCK cells with EC50 values between about 100 micromolar and about 1 nanomolar. Preferred compounds of the invention provided plaque formation inhibition for influenza virus A/N2/Tokyo in MDCK cells with EC50 values between about 1 micromolar and about 1 nanomolar.

The compounds of the invention can be tested for in vivo antiviral activity using the method described below. In Vivo Antiviral Efficacy Method

Female BALB/c mice were placed under anesthesia (sevoflurane) and inoculated intranasally (IN) with 0.1 ml of influenza A VR-95 (Puerto Rico PR8-34) at 10-2 (diluted from frozen stock) . This viral concentration consistently produced disease in mice within 5 days of inoculation. Animals were treated 4h. pre-infection and 4h. post- infection, andperiodically thereafter, with one of the following therapies: no treatment; test compound (100, 25, 6.25, 1.39 mg/kg/day BID, PO) ; or vehicle (sterile water BID, PO) . A group of ten animals (designated as control) was inoculated with 0.9% saline. Percent survival was determined. On day five, lungs were harvested, weighed and assigned scores of 0, 1, 2, 3 or 4 based on percentage consolidation (0; 10-20; 25-50; 50-75; 75-100%, respectively) . In addition, each lung pair was image analyzed to determine objective lung consolidation percentages .

Claims

WHAT IS CLAIMED IS:

1. A compound, which when bound to influenza neuraminidase and found to inhibit said enzyme, possesses a substituent with at least two sp2 carbon centers within Site of Occupation 2 and at least 2 of the following 3 substituents located in the specified Site of Occupations:

a. a carboxylate, sulfonate, sulfinate, phosphate, or phosphinate substituent within Site of Occupation 1,

b. an amide or sulfonamide group within Site of Occupation 3, or

c. a 4, 5, 6, 7, or 8-membered non-aromatic ring system, whose centroid lies within Site of Occupation 4;

with the proviso that compounds are excluded wherein

(i) the substituent with at least two sp2 carbon centers within Site of Occupation 2 is one of the following substituents when connected to the ring system occupying Site of Occupation 4 through the point of attachment indicated

III IV V

and

(ii) the compound is selected from the following five compounds :

2. A compound of Claim 1 with substituents located within Sites of Occupation with radius equal to 3.0 Angstroms or less.

3. A compound of Claim 1 with substituents located within Sites of Occupation with radius equal to 2.0 Angstroms or less .

4. A compound of Claim 1, which when bound to influenza neuraminidase and found to inhibit said enzyme possesses an IC50 less than or equal to 1 micromolar.

5. A compound of Claim 4 with substituents located within Sites of Occupation with radius equal to 3.0 Angstroms or less.

6. A compound of Claim 4 with substituents located within Sites of Occupation with radius equal to 2.0 Angstroms or less .

7. A compound of Claim 4 which, when bound to influenza neuraminidase and found to inhibit said enzyme with a potency of 1 micromolar or below (IC50 value) , a. possesses a carboxylate substituent within Site of Occupation 1, b. possesses a substituent with at least two sp2 carbon centers within Site of Occupation 2 c. an amide group within Site of Occupation 3, and d. possesses a 5 or 6-membered ring system, whose centroid lies within Site of Occupation 4.

8. A compound of Claim 7 with substituents located within Sites of Occupation with radius equal to 3.0 Angstroms or less.

9. A compound of Claim 7 with substituents located within Sites of Occupation with radius equal to 2.0

Angstroms or less.

10. A compound of Claim 7 which, when bound to influenza neuraminidase and found to inhibit said enzyme with a potency of 1 micromolar or below (IC50 value) , a. possesses a carboxylate substituent within Site of Occupation 1, b. possesses a substituent with at least two sp2 carbon centers within Site of Occupation 2 c. an amide group within Site of Occupation 3, and d. possesses a 5-membered ring system, whose centroid lies within Site of Occupation 4.

11. A compound of Claim 10 with substituents located within Sites of Occupation with radius equal to 3.0 Angstroms or less.

12. A compound of Claim 10 with substituents located within Sites of Occupation with radius equal to 2.0 Angstroms or less.

13. A compound of Claim 7 which, when bound to influenza neuraminidase and found to inhibit said enzyme with a potency of 1 micromolar or below (IC50 value) , a. possesses a carboxylate substituent within Site of Occupation 1, b. possesses a substituent with at least two sp2 carbon centers within Site of Occupation 2 c. an amide group within Site of Occupation 3, and d. possesses a 6-membered ring system, whose centroid lies within Site of Occupation 4.

14. A compound of Claim 13 with substituents located within Sites of Occupation with radius equal to 3.0 Angstroms or less.

15. A compound of Claim 13 with substituents located within Sites of Occupation with radius equal to 2.0 Angstroms or less .

16. A pharmaceutical composition for inhibiting neuaminidase comprising a pharmaceutical carrier and a therapeutically effective amount of the compound of Claim 1

17. A method for inhibiting neuraminidase in a mammal in need thereof comprising administering to said mammal a compound of Claim 1 and a phamaceutically acceptable carrier.