2,3-fluorinated glycosides as neuraminidase inhibitors, their method of preparation, pharmaceutical

专利摘要:
2,3-FLUORINATED GLYCOSIDS AS NEURAMINIDASE INHIBITORS AND THEIR USE AS ANTI-VIRALS. The present invention relates to compounds of formula (I) useful for the treatment or prophylaxis of viral infection, particularly viral influenza, which are provided. Pharmaceutical preparations of the same and methods for their preparation are also described. The therapeutic effect is obtained via inhibition of viral neuraminidases, also known as viral sialidases. These neuraminidases are classified under the family of viral enzymes GH34.
公开号:BR112012001061B1
申请号:R112012001061-1
申请日:2010-07-15
公开日:2020-10-27
发明作者:Stephen Withers；Andrew Graham Watts；Jin Hyo Kim；Tom Wennekes
申请人:The University Of British Columbia；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] This application claims the benefit of US Provisional Patent Application Serial No. 61 / 213,786 entitled “NEURAMIDINASE INHIBITORS: COMPOSITIONS and METHOD OF USE FOR THE TREATMENT OF INFLUENZA”, filed on July 15, 2009, which is incorporated here by reference in full. TECHNICAL FIELD
[0002] The present invention relates to therapeutic products, their uses and methods for the treatment or prophylaxis of viral infection. In particular, the invention relates to compounds, compositions, therapies and methods of treatment for viral infections, such as influenza. BACKGROUND
[0003] Infection and invasion by influenza virus requires the intervention of sialic acid residues on the surface of the host cell. Sialic acid and neuraminic acid are used interchangeably. Similarly, sialidase and neuraminidase are used interchangeably. Initial attachment of the virus to the host cell occurs via the binding of the virus to these sialic acids (charged, sugars with 9 carbons) through the hemagglutinin protein of the virus. Once inside the cell, the virus reproduces taking advantage of the host's cellular machinery. However, in order to remain optimally infectious, the virus has developed a neuraminidase that cuts the sialic acid from the surface of the host cell to help the virus escape from the host cell to infect other cells. Failure to cut sialic acid from the surface of the host cell results in retention of the virus through attachment to the host cell.
[0004] The GH33 family of neuraminidases contains all sialidases, except viral enzymes (GH34 family). The GH33 and GH34 families are structurally distinct and in sequence (see Cantarei BL. Etal. (2009); and Henrissat B. and Davies GJ (1997) for foundations on Family classifications). Previous work has shown that 2,3-difluoro-sialic acids are effective inhibitors of GH33 neuraminidases and that GH33 neuraminidases are processed through a covalent intermediate (see, for example, Watts, A. etal. (2003); Amaya, MF etal. (2004); Watts, AG and Withers, SG (2004); Watts, AG et al. (2006); Newstead, S. etal. (2008); Damager, I. etal. (2008); and Buchini, S. etal. (2008)).
[0005] The most likely mechanism for GH34 sialidase (ie, viral sialidases) reported in the literature is one involving an ion pair intermediate (von Itzstein M. (2007)).
[0006] A number of compounds are known to inhibit neuraminidases. Some well-known neuraminidase inhibitors are sialic acid analogs containing alkene (for example, Laninamivir CAS # 203120-17-6; Oseltamivir (Tamiflu) CAS # 204255-11-8; and Zanamivir (Relenza) CAS # 139110-80-8 see also US 5,360,817 and Ikeda et al. Bioorganic and Medicinal Chemistry (2006) 14: 7893-7897). It has been shown that fluorinated sugar derivatives with fluoride (reactive) driving groups are inhibitors of a range of "retention" glycosidases and work via the formation of particularly stable glycosyl enzyme intermediates (for example, Hagiwara et al. (1994); and Buchini et al. (2008)). These reagents have been shown to be very specific with respect to their target enzymes, are highly bio-stable and even capable of crossing the blood-brain barrier. Such inhibitors have their action-based mechanism, making the development of virus resistance difficult, so any mutations in the viral enzyme that reduce inhibition must necessarily reduce the efficiency of the enzyme on the natural substrate, sialic acid and therefore less likely to be tolerated. SUMMARY
[0007] The present invention is based, in part, on the fortuitous discovery that compounds having a covalent intermediate, as described here, modulate neuraminidase. Specifically, compounds identified here show neuraminidase inhibition, which can be useful for the treatment or prophylaxis of viral infection. In particular, the treatment or prophylaxis of influenza.
[0008] The compounds described here can be used for in vivo or in vitro (i.e., non-clinical) research uses to investigate the mechanisms of neuraminidase inhibition. In addition, these compounds can be used individually or as part of a kit for in vitro or in vivo research to investigate neuraminidase inhibition using recombinant proteins, viral strains, cells maintained in culture and / or animal models. Alternatively, the compounds described here can be combined with commercial packaging and / or instructions for use.
[0009] The present invention is also based, in part, on the discovery that the compounds described here can also be used to modulate neuraminidase activity in vitro or in vivo for research and therapeutic uses. The compounds can be used in an effective amount, so that neuraminidase activity can be modulated. Neuraminidase can be viral. Neuraminidase can be an influenza neuraminidase. In particular, the compounds can be used to inhibit neuraminidase activity. Modulatory activity compounds can be used in an in vivo or in vitro model for the study of viral infection. For example, influenza infection. In addition, activity modulatory compounds can be used for the treatment or prophylaxis of viral infection. Viral infection can be influenza.
[00010] Furthermore, the present invention is based, in part, on the assessment that 3-fluoro-sialic acid compounds can be GH34 sialidase inhibitors, as long as the compounds have a sufficient conduction group on carbon 2 (position Z in Formula I), in addition to the appropriate stereochemistry, as described here. Compounds identified here show neuraminidase inhibition, which can be useful for the treatment or prophylaxis of viral infection. In particular, the treatment or prophylaxis of influenza.
[00011] According to one embodiment, compounds having a structure of Formula I are provided:
on what:
[00012] Té C (O) NH2, COOH or COOR1,
[00013] wherein R1 is a linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00014] wherein the optional substituent is selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00015] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00016] Z is F, Cl, Br, or OSO2R2,
[00017] wherein R2 is a linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00018] wherein the optional substituent is selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00019] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00020] A is selected from the group including: H, F, Cl, Br, OH, CN, OR3, NO2, SO2R3, SR3 and COR3,
[00021] where R3 is a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00022] where the optional substituent is selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and [
[00023] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00024] D can be selected from the group including: H, F, Cl, Br, OH, CN, OR4, NO2, SO2R4, SR4 and COR4, as long as A and D are not both H and
[00025] where R4 may be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00026] wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00027] where zero to ten carbons of the optionally substituted alkyl group main chain can be optional and independently substituted with O, N or S;
[00028] collectively, A and D optionally form an oxo group;
[00029] X can be selected from the group including: N3, NH2, NHR5, NHCH3, NHCH2CH3, NHC (NH) NH2, NHC (NH) NHR5, NR5R6, and NHC (NH) N (R5) R6,
[00030] wherein R5 and R6 can independently be CeHs, CH2C6H5 or a C1-6 alkyl group;
[00031] E can be selected from the group including: NH2, NHC (O) CH3, OR7, NHR7 and N (R7) (R8),
[00032] wherein R7 and R8 may independently be a linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00033] wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00034] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00035] Q can be selected from the group including: CH2OH, CH2R9, CH (R9) (R10), C (R9) (R10) (R11),
on what:
[00036] R9, R10 and R11 can be independently CH3 or CH2CH3 and
[00037] each of J and G can be independently selected from the group: H, OH, OAc, OC (O) CH3, F, Cl, Br, NO2, CN, OR12, SO2R12, COR12 and SR12,
[00038] where R12 can be CH3, CH2CH3 or CH2CH2CH3 and
[00039] M can be H, OH, OAc, OC (O) CH3, NH2, F or Cl and
[00040] L can be H, OH, OAc, OC (O) R13 or NH2,
[00041] where R13 may be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-20 alkyl group,
[00042] wherein the optional substituent can be selected from one or more of the group: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00043] where zero to ten carbons of the optionally substituted alkyl group main chain can be optional and independently substituted with O, N or S.
[00044] According to another embodiment, compounds having a structure of formula I are provided:
on what:
[00045] T can be C (O) NH2, COOH or COOR1,
[00046] wherein R1 may be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group,
[00047] wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00048] wherein zero to ten carbons of the optionally substituted alkyl group main chain can be optional and independently substituted with O, N or S;
[00049] Z can be F, Cl, Br, or OSO2R2,
[00050] wherein R2 may be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group,
[00051] wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00052] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00053] A can be F or Cl;
[00054] D can be H;
[00055] X can be selected from the group including: NH2, NHCH3, NHCH2CH3, NHCH2CH2CH3, NHCH2CH2CH2CH3, and NHC (NH) NH2;
[00056] E can be NH2 or NHC (O) CH3;
[00057] Q can be selected from the group including:
on what:
[00058] each of J and G can be independently selected from the group: H, OH, OAc, OC (O) CH3, F, Cl, Br, NO2, CN, OR12, SO2R12, COR12 and SR12,
[00059] where R12 can be CH3, CH2CH3 or CH2CH2CH3 and
[00060] M can be H, OH, OAc, OC (O) CH3, NH2, F or Cl and
[00061] L can be H, OH, OAc, OC (O) R13 or NH2,
[00062] where R13 may be a C1-10 linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-10 alkyl group,
[00063] wherein the optional substituent can be selected from one or more of the group: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00064] where zero to ten carbons of the optionally substituted alkyl group main chain can be optional and independently substituted with O, N or S.
[00065] According to another embodiment, compounds having a structure of formula I are provided:
on what:
[00066] T can be C (O) NH2, COOH or COOR1,
[00067] where R1 may be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group,
[00068] wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2, and
[00069] wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S;
[00070] Z can be F or Cl;
[00071] A can be F or Cl;
[00072] D can be H;
[00073] X can be selected from the group including: NH2, NHCH3, NHCH2CH3, NHCH2CH2CH3, NHCH2CH2CH2CH3, and NHC (NH) NH2;
[00074] E can be NH2 or NHC (O) CH3;
[00075] Q can be selected from the group including:
on what:
[00076] each of J and G can be independently selected from the group: H, OH, OAc, OC (O) CH3, F, Cl, Br, NO2, CN,
[00077] M can be H, OH, OAc; and
[00078] L can be H, OH, OAc.
[00079] According to another embodiment, compounds are provided as described here for modulation of viral neuraminidase activity. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00080] According to another embodiment, compounds are provided as described here for use in preparing a medicament for modulating viral neuraminidase activity. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00081] According to another embodiment, compounds are provided as described here for modulation of viral neuraminidase activity. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00082] According to another embodiment, pharmaceutical compositions are provided which can include one or more compounds as described herein and a pharmaceutically acceptable excipient. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00083] According to another embodiment, compounds or pharmaceutically acceptable salts thereof are provided as described herein for modulation of viral neuraminidase activity. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00084] According to another embodiment, a method of modulating viral neuraminidase activity with one or more compounds described herein or a pharmaceutically acceptable salt thereof is provided. The viral neuraminidase can be a GH34 neuraminidase. Modulation of viral neuraminidase activity can be for the treatment of influenza in an animal. The animal can be a mammal. The animal can be a human being.
[00085] According to another embodiment, a commercial package is provided which may contain one or more compounds described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof. The commercial package may optionally contain instructions for the use of the compounds or pharmaceutically acceptable salt thereof or pharmaceutical composition thereof in the treatment of influenza.
[00086] T can be C (O) NH2, COOH or COOR1, where R1 can be a C1-20 linear, branched or cyclic, saturated or unsaturated alkyl group, optionally substituted, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, T can be C (O) OCH3, C (O) OCH2CH3, C (O) OCH2CH2CH3, C (O) OCH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH2CH2CH2 ) OCH2CH2CH2CH2CH2CH2CH2CH3 OR COOH. Alternatively, T can be C (O) OCH3, C (O) OCH2CH3, C (O) OCH2CH2CH3, C (O) OCH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH3OU COOH.
[00087] A can be selected from the group including: F, Cl, Br, OH, CN, OR3, NO2 and COR3, where R3 can be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group , where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2 and where zero to ten carbons of the group's main chain optionally substituted alkyl can be optionally and independently substituted with O, N or S. Alternatively, A can be selected from the group including: F, Cl, Br, OH, CN, OR3 and NO2, where R3 can be a C1-20 alkyl group linear, branched or cyclic, saturated or unsaturated, optionally substituted, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2 and wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S. Alternatively, A can be selected from the group including: F, Cl, Br, OH, CN and NO2. Alternatively, A can be selected from the group including: F, Cl, Br, OR3 and NO2, where R3 can be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, A can be selected from the group including: F, Cl and OR3, where R3 can be a C1-20 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, A can be F, Cl. Alternatively, A can be F.
[00088] D can be selected from the group including: H, F, Cl, Br, OH, CN, OR4, NO2 and COR4, as long as A and D are not both H and where R4 can be a C1-10 alkyl group linear, branched or cyclic, saturated or unsaturated, optionally substituted, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2 and wherein zero to ten carbons of the optionally substituted alkyl group can be optionally and independently substituted with O, N or S. Alternatively, D can be selected from the group including: H, F, Cl, Br, OH, CN, OR4, NO2 and COR4, provided that A and D are not both H and where R4 can be a C1-10 linear, branched or cyclic, saturated or unsaturated, optionally substituted alkyl group, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, D can be selected from the group including: H, F, Cl, Br, OH, CN and NO2, as long as A and D are not both H. Alternatively, D can be selected from the group including: H, F, Cl, Br, OH and NO2, as long as A and D are not both H. Alternatively, D can be selected from the group including: H, F, Cl, Br and OH, as long as A and D are not both H. Alternatively, D can be selected from the group including: H, F, Cl and Br, as long as A and D are not both H. Alternatively, D can be selected from the group including: H, F and Cl, as long as A and D are not both H. Alternatively, D can be selected from the group including: H, F and Cl, as long as A and D are not both H. Alternatively, D can be F or Cl. Alternatively, D can be H or F, as long as A and D are not both H. Alternatively, D can be F. Alternatively, D can be H, as long as A and D are not both H.
[00089] X can be selected from the group including: NH2, NHR5, NHCH3, NHCH2CH3, NHC (NH) NH2, NHC (NH) NHR5 and NR5R6, where R5 and R6 can be independently CeHs, CH2C6H5 or a C1-8 group alkyl. Alternatively, X can be selected from the group including: NH2, NHR5, NHCH3, NHCH2CH3, NHC (NH) NH2 and NHC (NH) NHR5, where R5 can be C6H5, CH2C6H5 or a C1-8 alkyl group. Alternatively, X can be selected from the group including: NH2, NHR5, NHCH3, NHCH2CH3 and NHC (NH) NH2, where R5 can be CeH5, CH2C6H5 or a C1-8 group. Alternatively, X can be selected from the group including: NH2, NHR5, NHCH3, NHCH2CH3 and NHC (NH) NH2, where R5 can be CeH5, CH2C6H5 or a C1-8 alkyl group. Alternatively, X can be selected from the group including: NH2, NHR5, NHCH3, NHCH2CH3 and NHC (NH) NH2, where R5 can be a C1-8 alkyl group. Alternatively, X can be selected from the group including: NH2, NHCH3 and NHC (NH) NH2. Alternatively, X can be selected from the group including: NH2, NHCH2CH3 and NHC (NH) NH2. Alternatively, X can be NH2 or NHC (NH) NH2.
[00090] E can be selected from the group including: NH2, NHC (O) CH3, OR7, NHR7, where R7 can be independently a linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-10 alkyl group, where The optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2 and where zero to ten carbons of the optionally substituted alkyl group main chain can be optionally and independently substituted with O, N or S. Alternatively and can be selected from the group including: NH2, NHC (O) CH3, OR7 and NHR7, where R7 can be independently a linear, branched or cyclic C1-10 alkyl group , optionally substituted, saturated or unsaturated, wherein the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively and can be selected from the group including: NH2, NHC (O) CH3 and OR7, where R7 can be independently a linear, branched or cyclic, saturated or unsaturated, optionally substituted C1-10 alkyl group, where the optional substituent can be selected from one or more of the group including: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, it can be NH2 or NHC (O) CH3. Alternatively and can be NHC (O) CHs.
[00091] Q can be selected from the group including: CH2R9, CH (R9) (R10), C (R9) (R1O) (R11),
where R9, R10 and R11 can be independently CH3 or CH2CH3 and each urn from J and G can be independently selected from the group: H, OH, OAc, OC (O) CH3, F, Cl, Br, NO2, CN, OR12, SO2R12, COR12 and SR12, where R12 can be CH3, CH2CH3 or CH2CH2CH3 and M can be H, OH, OAc, OC (O) CH3, NH2, F or Cl and L can be H, OH, OAc, OC (O ) R13 or NH2, where R13 can be a C1-20 linear, branched or cyclic, saturated or unsaturated alkyl group, optionally substituted and where the optional substituent can be selected from one or more of the group: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2. Alternatively, Q can be selected from the group including:
each urn of J and G can be independently selected from the group: H, OH, OAc, OC (O) CH3, F, Cl, Br, NO2, CN, M can be H, OH, OAc, OC (O) CH3, NH2, F or Cl and L can be H, OH, OAc, OC (O) R13 or NH2. Alternatively, Q can be selected from the group including:
each of J and G can be independently selected from the group: H, OH, OAc, M can be H, OH or OAc and L can be H, OH or OAc. Alternatively, Q can be:
each of J and G can be independently selected from the group: H, OH, OAc, M can be H, OH or OAc. and L can be H, OH or OAc. Alternatively, Q can be:
each of J and G can be independently selected from the group: OH, OAc, M can be OH or OAc and L can be OH or OAc.
[00092] According to another embodiment, a method of preparing compound 2 is provided: the method including: reaction of a SAN3 compound:
with Selectfluor in the presence of MeNO2 / H2O for at least 4 days to form compound 1:
and reaction of compound 1 with diethylamino sulfur trifluoride (DAST), CH2CL2 at between -30 ° C - 0 ° C.
[00093] The method may also include: mixing compound 2 with NaOMe and MeOH; then, mix with Pd / C, H2 and MeOH; and then mix with LiOH, H2O and MeOH to form compound 4:

[00094] According to another embodiment, a method of preparing compound 12 is provided, the method may include mixing with NaOMe and MeOH; then, mix of compost 2:
with AcOH until neutral; then, mix with PMes, H2O and MeOH; and then reaction with compound VII:
in EtsN, MeOH and DMF to produce compound VIII:
then, reaction of compound VIII with LiOH, H2O and THF; and then reaction with Pd / C, H2, H2O and THF.
[00095] According to another embodiment, compounds are provided which can be selected from one or more of the compounds shown in TABLES 2A and 2B. BRIEF DESCRIPTION OF THE DRAWINGS
[00096] Figure 1 shows a mechanism of neuraminidase.
[00097] Figure 2 shows inactivation of neuraminidase by 2,3-difluoro-sialic acid (1).
[00098] Figures 3A and 3B show a time-dependent inactivation of influenza sialidase (subtype N9) by compound 4, indicated by concentration (3A) and re-plotting of the kinetic constants of inactivation of pseudo-first magnitude (K Obs) versus concentration of compound 4 (3B).
[00099] Figures 4A and 4B show the plasma levels of DFSA-4Gu after IV and IN administration.
[000100] Figures 5A, 5B, and 5C show the tracheal levels of DFSA-4Gu after IV and IN administration.
[000101] Figures 6A, 6B, and 6C show lung levels of DFSA-4Gu after IV and IN administration.
[000102] Figure 7 shows lung levels of Zanamivir after IV and IN administration.
[000103] Figure 8 shows a plot of survival of mice infected with Influenza A virus HK1 H2N3 and treated with neuraminidase inhibitors. DETAILED DESCRIPTION
[000104] As used here, the phrase “Cx y alkyl” or “Cx-Cy alkyl” is used as commonly understood by those skilled in the art and often refers to a chemical entity that has a carbon skeleton or main chain of carbon comprising a number of x and y (with all individual integers within the range included, including integers x and y) of carbon atoms. For example, a "C1-10 alkyl" is a chemical entity that has 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atom (s) in its carbon skeleton or main chain.
[000105] As used herein, the term "branched" is used as normally understood by those skilled in the art and often refers to a chemical entity that comprises a skeleton or main chain that divides into more than one contiguous chain. The parts of the skeleton or main chain that split in more than one direction can be linear, cyclic or any combination of them. Non-limiting examples of branched alkyl are tert-butyl and isopropyl.
[000106] As used herein, the term "unbranched" is used as normally understood by those skilled in the art and often refers to a chemical entity that comprises a skeleton or main chain that does not split into more than one contiguous chain. Non-limiting examples of unbranched alkyls are methyl, ethyl, n-propyl and n-butyl.
[000107] As used herein, the term "substituted" is used as commonly understood by those skilled in the art and often refers to a chemical entity that has a chemical group replaced by a different chemical group that contains one or more hetero atoms. Unless otherwise specified, a substituted alkyl is an alkyl in which one or more hydrogen atoms are replaced with one or more atoms that are not hydrogen. For example, chloromethyl is a non-limiting example of a substituted alkyl, more particularly an example of a substituted methyl. Aminoethyl is another non-limiting example of substituted alkyl, more particularly it is substituted ethyl. The functional groups described here can be replaced, for example and without limitation, by 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substituents.
[000108] As used herein, the term "unsubstituted" is used as normally understood by those skilled in the art and often refers to a chemical entity that is a hydrocarbon and / or does not contain a heteroatom. Non-limiting examples of unsubstituted alkyls include methyl, ethyl, tert-butyl and pentyl.
[000109] As used here, the term "saturated", when referring to a chemical entity, is used as normally understood by those skilled in the art and often refers to a chemical entity comprising only single bonds. Non-limiting examples of saturated chemical entities include ethane, ferc-butyl and N + Hs.
[000110] As used herein, the term "halogenated" is used as will normally be understood by those skilled in the art and refers to a chemical portion or entity in which a hydrogen atom is replaced by a halogen atom, such as chlorine , fluorine, iodine or bromine. For example, a fluorinated side chain refers to a side chain in which one or more hydrogen atoms are replaced with one or more fluorine atoms.
[000111] Non-limiting examples of C1-C10 saturated alkyl may include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl, sec-pentyl, t-pentyl, n-hexyl, i-hexyl, 1,2-dimethylpropyl, 2-ethylpropyl, 1-methyl-2-ethylpropyl, 1-ethyl-2-methylpropyl, 1,1, 2-trimethyl I propyl, 1,1,2-triethyl I propyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 2-ethylbutyl, 1,3-dimethylbutyl, 2-methylpentyl, 3-methylpentyl, sec- hexyl, t-hexyl, n-heptyla, i-heptyla, sec-heptyla, t-heptyla, n-octyla, i-octyla, sec-octyla, t-octyla, n-nonila, i-nonila, sec-nonila, t-nonila, n-decila, i-decila, sec-decila and t-decila. Non-limiting examples of C2-C10 alkenyl may include vinyl, allyl, isopropenyl, 1-propene-2-yl, 1-butene-1-yl, 1-butene-2-yl, 1-butene-3-yl, 2- butene-1-yl, 2-butene-2-yl, octenyl and decenyl. Non-limiting examples of C2-C10 alkynyl may include ethyl, propynyl, butynyl, pentynyl, hexynyl, heptinyl, octinyl, noninyl and decynyl. C1-C10 saturated alkyl, C2-C10 alkenyl or C2-C10 alkynyl can be, for example and without limitation, interrupted by one or more heteroatoms which are independently nitrogen, sulfur or oxygen.
[000112] Non-limiting examples of the Ce-Cio aryl group may include phenyl (Ph), pentalenyl, indenyl, naphthyl and azulenyl. The Ce-9 aryl-C1-4 alkyl group can be, for example and without limitation, a C1-4 alkyl group as defined anywhere herein above having a CΘ-9 aryl group as defined elsewhere herein as a substituent . The Ce-8 aryl-C2-4 alkenyl group can be, for example and without limitation, a C2-4 alkenyl as defined any part herein above having a Ce-8 aryl group as defined elsewhere herein as a substituent. The Ce-8 aryl-C2-4 alkynyl group can be, for example and without limitation, a C2-4 alkynyl group as defined anywhere here above having a Ce-8 aryl group as defined anywhere above as a substituent . Non-limiting examples of the non-aromatic heterocyclic group of 4 to 10 elements containing one or more heteroatoms which are independently nitrogen, sulfur or oxygen may include pyrrolidinyl, pyrrolinyl, piperidinyl, piperazinyl, imidazolinyl, pyrazolidinyl, imidazolidinyl, morpholinyl, tetrahydrohydridan, tetrahydropyridine , oxetanil, oxathiolanil, phthalimide and succinimide. Non-limiting examples of the aromatic heterocyclic group of 5 to 10 elements containing one or more heteroatoms which are independently nitrogen, sulfur or oxygen may include pyrrolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, imidazolyl, thiazolyl and oxazolyl.
[000113] The modalities involving the formulas as described here include all possible stereochemical alternatives, including those illustrated or described here.
[000114] In some embodiments, the compounds as described herein or acceptable salts thereof can be used for systemic treatment or prophylaxis of a viral infection. In some embodiments, the compounds as described herein or acceptable salts thereof can be used in the preparation of a medicament or composition for systemic treatment or prophylaxis of a viral infection. In some embodiments, methods of systemic treatment of any of the infections described here are also provided. Some embodiments make use of compositions comprising a compound described herein and a pharmaceutically acceptable excipient or vehicle. In some embodiments, viral infection is caused, at least in part, by an influenza virus. Methods of treating any of the indications described here are also provided. Such methods may include administering a compound as described herein or a composition of a compound as described herein or an effective amount of a compound as described herein or composition of a compound as described herein to an individual who needs them.
[000115] Compounds as described here can be in the free form or in the form of a salt thereof. In some embodiments, the compounds as described herein can be in the form of a pharmaceutically acceptable salt, which are known in the field (Berge et al., J. Pharm. Sci. 1977, 66, 1). Pharmaceutically acceptable salt as used herein includes, for example, salts that have the desired pharmacological activity of the parent compound (salts which retain the biological efficacy and / or properties of the parent compound and which are not biologically and / or otherwise undesirable ). Compounds as described herein having one or more functional salt-forming groups can, for example, be formed as a pharmaceutically acceptable salt. Compounds containing one or more basic functional groups may be capable of forming a pharmaceutically acceptable salt, for example, with a pharmaceutically acceptable organic or inorganic acid. Pharmaceutically acceptable salts can be derived, for example and without limitation, from acetic acid, adipic acid, alginic acid, aspartic acid, ascorbic acid, benzoic acid, benzene sulfonic acid, butyric acid, cinnamic acid, citric acid, camphoric acid, camphor-sulfonic, cyclopentanopropionic acid, diethylacetic acid, digluconic acid, dodecyl-sulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glycerophosphoric acid, glycolic acid, hemi-sulfonic acid, heptanoic acid , hexanoic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, 2-hydroxyethane sulfonic acid, isonicotinic acid, lactic acid, malic acid, maleic acid, malonic acid, mandelic acid, methanesulfonic acid, 2-naphthalene sulfonic acid, naphthalenedi-sulfonic acid, p-toluene-sulfonic acid, nicotinic acid, nitric acid, oxalic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, phosphonic acid rich, picric acid, pyelic acid, pivalic acid, propionic acid, pyruvic acid, salicylic acid, succinic acid, sulfuric acid, sulfamic acid, tartaric acid, thiocyanic acid or undecanoic acid. Compounds containing one or more acid functional groups may be capable of forming pharmaceutically acceptable salts with a pharmaceutically acceptable base, for example and without limitation, inorganic bases based on alkali metals or alkaline earth metals or organic bases, such as primary amine compounds, secondary amine compounds, tertiary amine compounds, quaternary amine compounds, substituted amines, naturally occurring substituted amines, cyclic amines or basic ion exchange resins. Pharmaceutically acceptable salts can be derived, for example and without limitation, from a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, such as ammonium, sodium, potassium, lithium, calcium, magnesium, iron, zinc, copper, manganese or aluminum, ammonia, benzathine, meglumine, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, lysine, arginine hydrabamine, choline, betaine, ethylene diamine, glucosamine, glucamine, methylglucamine, theobromine, purines, piperazine, piperidine, procaine, N-ethylpiperidine, theobromine, tetramethylammonium compounds, tetraethylammonium compounds, pyridine, N, N-dime-tilylaniline N-methylpiperidine, morpholine, N-methylmorpholine, N-ethylmorpholine, dicyclohexylamine, dibenzylamine, N, N-dibenzylphenethylamine, 1-efenamine, N, N'-dibenzylethylenediamine or res polyamine lines. In some embodiments, the compounds as described herein may contain acidic and basic groups and may be in the form of internal salts or zwitterions, for example and without limitation, betaines. Salts, as described here, can be prepared by conventional processes known to those skilled in the field, for example and without limitation, by reacting the free form with an organic acid, an inorganic acid, an organic base or an inorganic base or through anion exchange or cation exchange from other salts. Those skilled in the art will appreciate that the preparation of salts can occur in situ during isolation and / or purification of the compounds or preparation of the salts can occur through separate reaction of an isolated and / or purified compound.
[000116] In some embodiments, compounds and all their different forms (for example, free forms, salts, polymorphs, isomeric forms), as described here, may be in the form of solvent addition, for example, solvates. Solvates contain stoichiometric or non-stoichiometric amounts of a solvent in physical association with the compound or salt thereof. The solvent can be, for example and without limitation, a pharmaceutically acceptable solvent. For example, hydrates are formed when the solvent is water or alcoholates are formed when the solvent is an alcohol.
[000117] In some embodiments, compounds and all their different forms (for example, free forms, salts, solvates, isomeric forms), as described here, may include crystalline and / or amorphous forms, for example, polymorphs, pseudopolymorphs, conformational polymorphs, amorphous forms or a combination thereof. Polymorphs include different crystal conditioning configurations of the same elemental composition as a compound. Polymorphs usually have X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, different stability and / or solubility. Those skilled in the art will appreciate that several factors, including recrystallization solvent, crystallization rate and storage temperature, can cause a single crystal form to dominate.
[000118] In some embodiments, compounds and all the different forms thereof (for example, free forms, salts, solvates, polymorphs), as described here, include isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, individual enantiomers, individual diastereomers, racemates, diastereomeric mixtures and combinations thereof and are not limited by the description of the illustrated formula for convenience.
[000119] In some embodiments, pharmaceutical compositions according to the present invention may comprise a salt of such a compound, preferably a pharmaceutically or physiologically acceptable salt. Pharmaceutical preparations will typically comprise one or more vehicles, excipients or diluents acceptable for the mode of administration of the preparation, whether by administration by injection, inhalation, topical, washing or other modes suitable for the selected treatment. Selected vehicles, excipients or diluents include those known in the field for use in such modes of administration.
[000120] Suitable pharmaceutical compositions can be formulated by means known in the field and their mode of administration and dose determined by the expert. For parenteral administration, a compound can be dissolved in sterile water or saline or a pharmaceutically acceptable carrier for administration of non-water-soluble compounds, such as those used for vitamin K. For enteral administration, the compound can be administered in a tablet, capsule or dissolved in liquid form. The tablet or capsule can be coated enteric or in a sustained release formulation. Many suitable formulations are known, including polymeric or protein microparticles that encapsulate a compound to be released, ointments, pastes, gels, hydrogels or solutions which can be used topically or locally to administer a compound. A sustained release patch or implant can be employed to provide release over an extended period of time. Many techniques known to those skilled in the art are described in Remington: The Science and Practice of Pharmacy by Alfonso Gennaro, 20aed., Lippencott Williams and Wilkins, (2000). Formulations for parenteral administration may contain, for example, excipients, polyalkylene glycols, such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes. Biocompatible, biodegradable, lactose / glycolide copolymer / polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful systems for parenteral delivery for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems and liposomes. Inhalation formulations can contain excipients, for example, lactose or they can be aqueous solutions containing, for example, polyoxyethylene-9 lauryl ether, glycocholate and deoxycholate or they can be oily solutions for administration in the form of nasal drops or as a gel. The formulations can be specifically prepared for intranasal delivery. For example, nasal inhalation.
[000121] Pharmaceutical compounds or compositions according to the present invention or for use in the present invention can be administered by means of a medical device or utensil, such as an implant, graft, prosthesis, stent, etc. Also, implants can be designed which are designed to contain and release such compounds or compositions. An example would be an implant made of a polymeric material adapted to release the compound over a period of time.
[000122] An "effective amount" of a pharmaceutical composition, as described herein, includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to obtain the desired therapeutic result, such as reduced viral load, increased life expectancy or increased life span. A therapeutically effective amount of a compound can vary according to factors such as the condition of the disease, age, sex and weight of the individual and the ability of the compound to stimulate a desired response in the individual. Dosage regimens can be adjusted to provide the optimal therapeutic response. A therapeutically effective amount is also one in which any toxic or harmful effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an effective amount, at the dosages and for periods of time necessary, to obtain the desired prophylactic result, such as less severe or delayed infection or no onset, increased life expectancy, increased lifespan or prevention of progression of infection. Typically, a prophylactic dose is used in individuals before or at an early stage of the disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
[000123] It should be noted that the dosage values may vary with the severity of the condition to be relieved. For any particular individual, specific dosage regimens can be adjusted according to individual needs and the professional judgment of the person who is administering or supervising the administration of the compositions. Dosage ranges shown here are exemplary only and do not limit the dosage ranges that can be selected by doctors. The amount of active compound (s) in the composition can vary according to factors such as the condition of the disease, age, sex and weight of the individual. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. It may be advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.
[000124] In some embodiments, the compounds and all the different forms thereof, as described here, can be used, for example and without limitation, in combination with other treatment methods.
[000125] In general, the compounds described here will be used without causing substantial toxicity. The toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing on cell cultures or experimental animals and determining the therapeutic index, that is, the ratio of LDso (the lethal dose to 50% population) and LD100 (the lethal dose for 100% of the population). In some circumstances, however, as in severe unhealthy conditions, it may be necessary to administer substantial excesses of the compositions. Some compounds described here can be toxic in some concentrations. Titration studies can be used to determine toxic and non-toxic concentrations. Toxicity can be assessed by examining the specificity of a particular compound or composition by cell lines. Animal studies can be used to provide an indication of whether the compound has any effects on other tissues.
[000126] Compounds as described here can be administered to an individual. As used here, an "individual" can be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The individual may be suspected of having or at risk of having an infection, such as a viral infection, or suspected of having or at risk of having a viral infection. In particular, the infection can be mediated by a neuraminidase. Diagnostic methods for viral infection, such as influenza, and clinical design of viral infection, such as influenza, are well known to those skilled in the field.
[000127] TABLE 1. Compounds made and tested for modulatory neuraminidase activity. TABLE 1: 2,3-fluorinated glycosides

[000128] TABLE 2. Compounds having neuraminidase modulatory activity. TABLE 2A: 2,3-fluorinated glycosides with neuraminidase modulating activity

[000129] TABLE 2B. Compounds produced and expected to have neuraminidase modulatory activity. TABLE 2B: 2,3-fluorinated glycoside compounds


[000130] The compounds described here can also be used in assays and for research purposes.
[000131] Compounds for use in the present methods can be synthesized using the methods described here.
[000132] Various alternative modalities and examples are described here. These modalities and examples are illustrative and should not be construed as limiting the scope of the invention.
[000133] Previous work published by Hagiwara et al (1994) reported 3-fluoro-sialic acids as only modest sialidase inhibitors. Specifically, they report two compounds, one with an OH group on carbon 2 (Z position in Formula I). However, the OH group is not a sufficient driving group to allow retention of a covalent intermediate. Consequently, the Hagiwara OH compound is etal. (in C2 equivalent to Z in Formula I) showed minimal inhibition. In addition, the other compound tested by Hagiwara et al., Which has fluorine (a sufficient driving group) in C2 (equivalent to Z in Formula I), does not have the correct stereochemistry. Consequently, any appreciation of these requirements was absent in Hagiwara et al. GENERAL METHODOLOGIES Synthesis
[000134] General methodologies for the preparation of compounds of formula I are described in the exemplary non-limiting schemes below.

[000135] Selectfluor (3.5 eq.) Was added to a solution of SAN3 (1 eq.) In MeNO2 / water (3/1 ~ 4/1) and the solution stirred for 3 days or more to end the reaction in room temperature (SAN3 Synthesis - Chandler, M. et al. Journal of the Chemical Society-Perkin Transactions 1, 1995; 1173-1180). The reaction mixture was dissipated with saturated NaHCOs and extracted with EtOAc. The presence of the compound (1) can be easily confirmed via TLC. The axial compound-F (1) has a lower Rf value than the starting material and the equatorial-F compound and any other stereoisomers (anomeric hydroxy isomers) will have higher Rf values than the starting material. Fluoride in C3 was detected at ~ 204 ppm during a 19F NMR experiment and stereochemistry was attributed based on the coupling constants in 1H- and 19F-NMR. The coupling constants JH3 / F3 (47.9 Hz) and JF3 / H4 (31.0 Hz) are indicative of an axial configuration of the fluorine atoms in C3, respectively. Table 3: Examination for C3 fluorination with Selectfluor.

[000136] The reaction can be monitored for termination by means of UV over TLC, because only the initiation material is detected under short UV. The reaction is considered complete when the active compound disappears by UV.

[000137] DAST (1.1 eq.) Was added dropwise to a suspended solution of compound 1 (1 eq.) In DCM -40 ° C and the solution stirred vigorously for 30 min. After the mixture became clear, the reaction mixture was dissipated with saturated NaHCOs and extracted with DCM and EtOAc to provide compound 2. If the reaction mixture does not become clear after 20 min, the reaction mixture is placed in a bath at -10 ° C for the last 10 min.
[000138] Compound 2 was hydrogenated with Pd / C in MeOH overnight at room temperature, the catalyst was then filtered and 6M NaOMe was added to the reaction mixture. The mixture was then acidified with IR120 (H +, strong) to remove Na +. The resin was filtered and the filtrate was evaporated and chromatographed (EtOAc / MeOH / water = 15/2/1). Hydrogenation was monitored by staining with nihydrin solution. Compounds 3 and 4 were isolated.

[000139] To a solution of compound 3 in MeCN / water (5/1) was added acetaldehyde (2 eq) at room temperature. After 30 min, 4 eq. of NaCNBHs were added and the reaction mixture stirred for 10 min at the same temperature. The reaction mixture was dissipated with 5% citric acid and chromatographed with EtOAc / acetone (9/1).
[000140] The monoacetylated amine (6) was deprotected with 6M NaOMe in wet MeOH to provide compound 7.

[000141] Compound 4 was re-esterified with HCI catalysis (2M HCI in Et2O) in dry EtOH at room temperature. The reaction mixture was left to stand overnight and then evaporated. 2 eq. of HCI were usually added. The ethyl ester can be separated by chromatography on silica gel.

[000142] The difluorinated compound 2 was deprotected with 6M NaOMe in moist MeOH at room temperature and compound 8 was easily purified by silica gel chromatography with 90% yields after acidification with IR120 (H + strong).

[000143] Azide 2 was hydrogenated with Pd / C to provide compound 3, then NHs (g) was bubbled to remove acetate and provide amide 4 in good yield.

[000144] For the preparation of compound 11, amine 3 was acetylated with AC2O in pyridine and O-acetylates and methyl ester were selectively removed with NaOMe. The 11 / V-acetylated compound was purified and obtained in a yield of 70% (three steps in total).

[000145] Guanylated compound 12 was prepared with 3,5-dimethylpyrazole-1-carboxamidine and EtsN in MeOH at 60 ° C for 3 weeks. The reaction mixture was dissipated after three weeks and 50% of the starting material 4 was successfully recovered. The 4-guanylated compound 12 was isolated in 80% yield and 0 ethyl ester 13 in 80% yield after re-esterification with catalytic HCl in EtOH at room temperature for 5 hours.
[000146] Compounds of formula I can be prepared using the chemical methodologies described in the exemplary non-limiting scheme below.

[000147] Synthesis of 4-amino derivatives of 2,3-difluoro-sialic acid. Reagents and conditions are as follows: Ref 1 is Chandler etal. (1995) J Chem Soc. Perk. Trans 1, 1173-1180; (a) 4 eq. Selectfluor, MeNO2 / water (3/1), rt .; (b) DAST, DCM, -40 ° C. (c) NaOMe, wet MeOH, rt .; (d) Pd / C, H2, MeOH, rt .; (e) EtOH, HCI cat., rt .; (f) Acetaldehyde for 4, Benzaldehyde for 5, NaCNBHs, acetone, rt. and (g) 3,5-dimethylpyrazole-1-carboxamidine nitrate, MeOH, 60 ° C.
[000148] Compounds of formula I having a C4 amine can alternatively be prepared using the chemical methodologies described in the exemplary non-limiting scheme below.

[000149] Alternatively, compounds of formula I having a C4 guanidine, can be prepared by means of the chemical methodologies described in the exemplary non-limiting scheme below.

[000150] Alternatively, compounds of formula I can be prepared using the chemical methodologies described in the exemplary non-limiting scheme below.

[000151] Alternatively, compounds of formula I having modifications in C1 can be prepared using the chemical methodologies described in the exemplary non-limiting scheme below.

[000152] It will be appreciated by those skilled in the art that variations in the length of the alkyl chain can be obtained by replacing 1-octanol (C8 - having 8 carbons) with an alternative alcohol. For example, 1-octanol in the above scheme can be replaced by an alternative primary alcohol 0 which can, for example, be selected from one or more of the following: Propan-1-ol (C3); Butanol (C4); 1-Pentanol (C5); 1-Hexanol (C6); 1-Heptanol (C7); 1-Nonanol (C9); 1-Decanol (C10); Undecanol (C11); Dodecanol (C12); 1- Tetradecanol (014); cetyl alcohol (016); stearyl alcohol (018); and arachidyl alcohol (C20). Similarly, it will be appreciated that an alternative substrate for this reaction can be chosen. For example, instead of DFSA-4NH2 (compound 4), compound 12 (DFSA-4Gu) or compound 7, etc. can be used.
[000153] Alternatively, compounds of formula I having modifications in C1 can be prepared using the chemical methodologies described in the exemplary non-limiting scheme below. For example, a 5-acetamido-4-guanyl-2,3,4,5, -tetradeoxy-3-fluoro-D-erythro-β-L-mano-non-2-ulopyran hydrochloric acid salt ethyl silonate is shown below, which also adds an ethyl group at C1 (R1). It will be appreciated by those skilled in the art that variations in the produced salt can be obtained by substituting for an alternative acid and that the length of the C1 alkyl group can be adjusted by substituting for an alternative alcohol, as presented above.

[000154] Alternatively, compounds of formula I having modifications to the C6 ring can be prepared using the chemical methodologies described in the exemplary non-limiting scheme below.

[000155] It will be appreciated by those skilled in the art that variations in the length of the alkyl chain can be obtained by replacing the trimethyl ortho-octyrate (C8 - having 8 carbons) or trimethyl ortho-butyrate (C4 - having 4 carbons) with a derivative of alternative ortho ester. For example, trimethyl orthooctirate or trimethyl orthobutyrate in the above schemes can be replaced with an alternative ortho ester derivative which can, for example, be selected from one or more of the following: trimethyl orthoacetate (C2); trimethyl orthopropionate (C3); trimethyl orthopentionate (C5); trimethyl orthohexanate (C6); trimethyl orthoheptanate (C7); trimethyl orthonate (C9); trimethyl orthodecanate (C10). Similarly, it will be appreciated that an alternative substrate for this reaction can be chosen. Description (2R, 3R) -4-Azido-4-deoxy-3-fluoro-7,8,9-tri-O-acetyl-sialic acid methyl ester (1)
[000156] ESI-MS m / z 515.1 (+ Na); 19F-NMR (CFCh, 282 MHz) δ - 204.7 (dd, J 47.9 and 31.0 Hz); 1H NMR (300 MHz) δ 5.32 (1H, m); 5.14 (1H, m); 5.00 (1H, dd, J31.4 and 1.5 Hz), 4.86 (1H, dd, J 4.5 and 1.9 Hz), 4.25 (1H, m), 4.15 ( 1H, m), 4.04 (1H, dd, J 12.5 and 8.5 Hz), 3.83 (1H, m), 3.77 (3H, OMe), 2.02 (12H, 4s, 4 Ac). (2R, 3R) -4-Azido-2,4-dideoxy-2,3-difluoro-7,8,9-tri-O-acetyl-sialic acid methyl ester (2)
[000157] ESI-MS m / z = 517.0 (+ Na); 19F-NMR (CFCh, 282 MHz) δ - 123.2 (1F, t, 8.46 Hz), -217.2 (1F, m); 1H-NMR (300MHz) δ 7.23 (1H, d, NH), 5.97 (1H, m), 5.37 (1H, m), 5.24 (1H, m), 5.10 (1H , dd, J49.4 and 7.3 Hz), 4.65 (1H, dt, 28.1 and 9.4 Hz), 4.51 (1H, m), 4.27 (2H, m), 3 , 88 (3H, d, J7.0 Hz, OMe), 3.39 (1H, m), 2.10 (12H, m, 4Ac), 13C NMR (100MHz) δ 21.6, 21.7, 21 , 8, 24.5, 49.1, 54.7, 57.8 (dd), 62.7, 68.5, 69.6, 71.6 (d), 78.2, 87.5 (dd ), 105.5 (dd), 165.0 (dd), 170.6, 171.4, 172.2, 172.5. (2R, 3R) -4-Azido-2,4-dideoxy-2,3-difluoro-sialic acid (8)
[000158] ESI-MS m / z = 353.2 (-H); F-NMR (CFCh, 282 MHz) δ -122.3 (1F, d, J 11.3 Hz), -216.4 (1F, ddd, J 50.1, 29.2 and 11.3 Hz); 1H-NMR (300MHz) δ 5.30 (1H, dm, J 50.2 Hz), 4.34 (1H, t, J 10.8 Hz), 4.07 (1H, dd, J 29.1 and 11.2 Hz), 3.90 (1H, d, J 10.5 Hz), 3.74 (2H, m), 3.50 (3H, m), 1.95 (3H, s, Ac), 13C NMR (100MHz) δ 23.0, 45.9 (d), 61.5 (dd), 63.9, 68.7, 71.1, 74.1 (d), 88.0 (dd), 106.5 (dd), 169.2 (dd), 175.8. (2R, 3R) -4-Amino-2,4-dideoxy-2,3-difluoro-sialylamide (9)
[000159] ESI-MS m / z = 350.1 (+ Na); 19F-NMR (CFCh, 282 MHz) δ-121.1 (1F, d, J8.46 Hz), -219.4 (1F, m); 1H-NMR (300MHz) δ 5.10 (1H, dm, J49.3 Hz), 4.20 (1H, t, J 10.8 Hz), 3.96 (1H, d J 10.6 Hz), 3.73 (2H, m), 3.50 (3H, m), 1.95 (3H, d, J 1.2 Hz, Ac), 13C NMR (100MHz) δ 22.2, 46.1, 51 , 5 (dd), 63.2, 67.5, 70.1, 74.1, 87.0 (dd) 105.0 (dd), 168.2 (dd), 175.2. (2R, 3R) -4- (N-Acetyl) amino-2,4-dideoxy-2,3-difluoro-sialic acid (11)
[000160] ESI-MS m / z = 415.1 (+ 2 Na); 19F-NMR (CFCh, 282 MHz) δ -121.8 (1F, d, J 11.9 Hz), -214.4 (1F, m); 1H-NMR (300MHz) δ 5.05 (1H, d, J 50.5 Hz), 4.50 (1H, m), 4.18 (1H, m), 4.05 (1H, d J 10, 4Hz), 3.82 (1H, d, J 10.3 Hz), 3.75 (2H m), 3.48 (2H, m), 1.89 (3H, s, Ac), 1.84 ( 3H, s, Ac), 13C NMR (100MHz) δ 21.6, 21.8 (d), 45.2, 50.8 (m), 63.1, 68.0, 70.5, 70.6 , 73.3 (d), 73.5, 169.5 (dd), 174.2, 174.8. (2R, 3R) -4- (N-Ethyl) amino-2,4-dideoxy-2,3-difluoro-sialic acid (7)
[000161] ESI-MS m / z = 355.3 (-H); 19F-NMR (CFCh, 282 MHz) δ - 121.8 (1F, d, J 11.5 Hz), -216.4 (1F, m); 1H-NMR (300MHz) δ 5.30 (1H, dm, J 50.2 Hz), 4.29 (1H, t, J 10.7 Hz), 3.82 (1H, d J 10.6 Hz) , 3.73 (2H, m), 3.52 (3H, m), 3.00 (2H, m), 1.95 (3H, s, Ac), 1.10 (3H, t, J 7, 3 Hz), 13C NMR (100MHz) δ 12.4, 23.2, 41.3, 45.2, 58.0 (dd), 63.9, 64.2 (d), 68.7, 71, 2, 74.2 (d), 85.5 (dd), 107.2 (dd), 170.0 (dd), 176.1. (2R, 3R) -4-Amino-2,4-dideoxy-2,3-difluoro-sialic acid (4)
[000162] ESI-MS m / z = 351.2 (+ Na); 19F-NMR (CFCh, 282 MHz) δ - 122.0 (1F, d, J 11.3 Hz), -217.4 (1F, m); 1H-NMR (400MHz) δ 5.10 (1H, ddt, J 50.4, 10.0 and 5.2 Hz), 4.16 (1H, m), 3.73 (3H m), 3.45 (3H, m), 1.91 (3H, dd, J 5.1 and 1.4 Hz, Ac), 13C NMR (100MHz) δ 23,1,46,8, 53,1 (dd), 63, 9, 64.1.68.5, 71.4, 74.4, 89.8 (dd), 108.5 (dd), 170.4 (dd), 176.0. (2R, 3R) -4-Ethyl-2,4-dideoxy-2,3-difluoro-sialylate (5)
[000163] ESI-MS m / z = 379.1 (+ Na); 19F-NMR (CFCh, 282 MHz) δ - 122.3 (1F, d, J 5.6 Hz), -219.0 (1F, m); 1H-NMR (400MHz) δ 5.10 (1H, ddt, J 49.2, 10.0 and 2.8 Hz), 4.45 (2H, m), 4.14 (1H, m), 4, 05 (1H, d J 10.4Hz), 3.88 (3H m), 3.66 (3H, m), 2.10 (3H, d, J 1.6 Hz, NAc), 1.30 (3H , dt, J 70.4 and 7.2 Hz), 13C NMR (100MHz) δ 13.4, 17.1, 22.4, 44.6 (dd), 52.4 (dd), 57.7, 63.2, 65.0, 67.7 (d), 70.1 (d), 73.3 (d), 86.7 (dm), 105.1 (tm), 167.0 (dm), 175.4 (d). (2R, 3R) -4-Guanyl-2,4-dideoxy-2,3-difluoro-sialic acid (12)
[000164] ESI-MS m / z = 370.3 (-H); 19F-NMR (CFCh, 282 MHz) δ - 121.3 (1F, d, J 14.4 Hz), -214.7 (1F, m); 1H-NMR (300MHz) δ 5.00 (1H, dm, J 50.2 Hz), 4.19 (1H, t, J 8.9 Hz), 3.81 (2H, m), 3.52 ( 3H, m), 3.10 (1H, q, J 7.3 Hz), 1.90 (3H, s, Ac), 1.12 (4H, m), 13C NMR (100MHz) δ 22.3, 43.5, 46.5, 55.1, 63.9, 68.9, 71.5, 74.2, 89.5 (dd), 107.0 (dd), 161.5, 170.1 ( dd), 175.8. (2R, 3R) -4-Guanyl-2,4-dideoxy-2,3-difluoro-sialylate (13)
[000165] ESI-MS m / z = 421.4 (+ Na); 19F-NMR (CFCh, 282 MHz) δ - 122.5 (1F, d, J 12.0 Hz), -216.0 (1F, m); 1H-NMR (300MHz) δ 5.16 (1H, dm, J 49.5 Hz), 4.32 (2H, m), 4.18 (2H, m), 4.03 (1H, d, J 9 Hz), 3.70 (2H, m), 3.50 (3H, m), 3.09 (2H, q, J 7.1 Hz), 1.90 (3H, s, Ac), 1.23 (3H, t, J 6.2 Hz).
5-Acetamido-4-amino-2,3,4,5 fluoride hydrochloric acid salt, - tetrade0xi-3-fluoro-D-erythro-β-L-mano-non-2-ulopyran-octyl silonate
[000166] ESI-MS m / z = 463.3 (M + Na); 1H-NMR (400 MHz, CH3OD) δ 5.37 (1H, ap dt, J49.92, 5.04), 4.50 - 4.44 (1H, m), 4.38 - 4.27 (2H , m), 4.21 - 4.11 (2H, m), 3.80 - 3.77 (1H, m), 3.75 (1H, dd, J 6.91, 2.30), 3, 69 - 3.66 (1H, m), 3.60 - 3.55 (1H, m), 2.04 (3H, s), 1.80 - 1.73 (2H, m), 1.42 - 1.28 (10, m), 0.91 (3H, t, J 7.00).
5-Acetamido-7,8,9-tri-Q-acetyl-4-amino-2,3,4,5-tetrade0xi-3-fluoro-D-erythro-β-L-mano fluoride hydrochloric acid salt -non-2-ulopyran-ethyl silonate
[000167] ESI-MS m / z = 483.3 (M + H); 1H-NMR (400 MHz, CDCl3) δ 6.99 (1H, s), 5.70 - 5.23 (3H, m), 5.02 (1H, s), 4.61 - 4.08 (6H , m), 2.28 -1.23 (15H, m).
Ethyl 5-acetamido-4-amino-213,4,5, -tetrade0xi-3-fluoro-D-erythro-β-L-mano-non-2-ulopyranosilonate
[000168] ESI-MS m / z = 379.1 (M + Na); 1H-NMR (400 MHz, D2O) δ 5.18 (1H, after d, J 49.34), 4.38 (2H, q, J 7.16), 4.20-4.14 (1H, m), 4.01 (1H, d, J 10.51), 3.87-3.76 (2H, m), 3, 61 (1H, dd, J 11.95, 5.86), 3.55 (1H, d, J9.29), 3.39 (1H, dd, J 30.00, 10.96), 2.03 (3H, s), 1.32 (3H, t, J7.16).
5-Acetamido-4-amino-9-butyryl-2,314,51-tetrade0xi-3-fluoro-D-erythro-β-L-mano-non-2-ulopyrano-silonate hydrochloric acid salt
[000169] ESI-MS m / z = 421.3 (M + Na); 1H-NMR (400 MHz, MeOD) δ 5.34 (1H, ap d, J51.62), 4.47 (1H, appt, J10.66), 4.36 (1H, d, J10.20), 4.16 (1H, dd, J 11.42, 6.24), 4.09 - 3.97 (3H, m), 3.55 (1H, d, J 9.14), 2.34 (2H , t, J 7.31), 2.03 (3H, s), 1.70-1.61 (2H, m), 0.96 (3H, t, J 7.31).
5-Acetamido-4-Amino-9-Octanoyl- 213,4,51-tetrade0xi-3-fluoro-D-erythro-β-L-mano-non-2-ulopyrano-silonate fluoride hydrochloric acid salt
[000170] ESI-MS m / z = 453.3 (MH); 1H-NMR (400 MHz, MeOD) δ 5.35 (1H, ap d, J45.84), 4.58 -4.28 (2H, m), 4.23 - 3.88 (4H, m), 3.63 - 3.50 (1H, m), 2.48-2.26 (2H, m), 2.15-1.94 (3H, m), 1.72-1.54 (2H, m ), 1.51 - 1.14 (8H, m), 1.03-0.88 (3H, m).
Hydrochloric acid salt of ethyl 5-acetamido-4-chanyl-2,3,4,5, -tetradeoxy-3-fluoro-D-erythro-β-L-mano-non-2-ulopyrane-silonate
[000171] ESI-MS m / z = 421.4 (M + Na); 1H-NMR (300MHz, D2O) δ5.16 (1H, after d, J49.5 Hz), 4.32 (2H, m), 4.18 (2H, m), 4.03 (1H, d, J9 Hz), 3.70 (2H, m), 3.50 (3H, m), 3.09 (2H, q, J 7.1 Hz), 1.90 (3H, s), 1.23 (3H , t, J 6.2 Hz). Enzyme Kinetics
[000172] All experiments were performed in 20 mM I CaCh 50 mM Tris buffer, pH 7.6 containing 0.1% BSA. The cuvettes had a path length of 1 cm and were used in a Cary 4000 or Cary 300 UV / visible light spectrophotometer connected to a circulating water bath. The data were analyzed using the GraFit 4.0 program (Erithacus Software). Time-dependent inactivations were performed by pre-incubating the enzyme at 30 ° C in the presence of various concentrations of inactivator. Residual enzymatic activity was determined at appropriate time intervals by adding an aliquot of the inactivation mixture to a test solution containing 0.5 mM 4-trifluoromethylumbelliferyl acid (CF3MUSA). The kinetic parameters were determined by measuring the initial linear increase in absorbance at 385 nm. The initial rates at each time point were plotted as a function of time to obtain time-dependent exponential decline curves, from which K Obs could be obtained for each concentration of inactivator using the equation: (rate) t = ( rate) t = oe (ki obst) + compensation
[000173] The compensation was used because the rates do not decline to zero. The inactivation rate constant (K) and the reversible dissociation constant for the inactivator (Kd) were determined by plotting K Obs versus concentration of inactivator to the equation: ki obs = ki [l] / (Kd + [l]).
[000174] In the case where [I] «Kd, a second magnitude rate constant (ki / Kd) was determined by adapting the data to the equation: ki obs = kj [l] / Kd -
[000175] Time-dependent reactivations were performed by applying the inactivated enzyme solution (50 pl) to an Amicon ™ 10 K filter (Millipore ™) to remove the excess inactivator. The filter was washed 5 times with 150 µl of buffer at 4 ° C. The enzymatic activity was tested at intervals of time by adding an aliquot of eluted enzyme to a test solution containing 0.5 mM CF3MUSA. First rate constants for reactivation at each acceptor concentration (kr obs) were determined by directly adapting the activity versus time data to a first magnitude equation. Reactivation experiments were attempted, but no significant enzymatic activity could be detected over time, suggesting that hydrolysis of the sialyl enzyme intermediate is very slow. Ki was determined by adapting the data to the equation: Ki = Kd (/ Chid // Ci). Cell-based assay of influenza antiviral activity
[000176] Compounds were tested for antiviral activity using a cell-based assay, which consists of making 2-fold serial dilutions of antiviral compounds (from 1: 2 to 1: 4096 in MegaVir medium in sufficient volume for the number of tested viruses - 60 uL per virus), to which 100 infectious units of the specific influenza virus are added and the preparations are transferred to MDCK cell monolayers on a microtiter slide. The test was performed on a 96-well microtiter slide. The slide is monitored for the development of cytopathic effects of influenza from days 3 to 5 post-infection. Antiviral activity is determined by inhibiting the development of cytopathic effects. The greatest dilution of the compound in which the monolayers are intact is taken as the endpoint. Zanamivir was used as a positive control. Dilution of Preparations: 1. In row A, on a transparent 96-well microtiter slide, prepare 2-fold serial dilutions of antiviral compounds, from 1: 2 to 1: 4096 in MegaVir medium in sufficient volume for the number of viruses tested (60 uL per virus). 2. Transfer 55 μl of the 2-fold dilution series to a clean row on the 96-well microtiter slide. 3. At 55 μl of the dilution series, add 55 μl of the diluted influenza virus (at 100 TCID50 per 25 μl). Also add viruses to the positive control wells. 4. To the 110 μL mixture, add 55 μL of 4X trypsin treated with TPCK. Add trypsin also to the positive and negative control wells. Mix well. 5. Also prepare 2-fold serial dilutions from 1: 2 to 1: 256 for the inoculation virus in MegaVir medium for titration again. Slide inoculation: 6. In a 96-well microtiter slide containing confluent monolayers of MDCK cells in ~ 200 μL of MegaVir medium, transfer 75 μL of the mixture to 2 respective rows as duplicates. 7. Transfer 50 μl of the positive control and 25 μl of the negative controls to the respective wells. 8. Also transfer 25 uL of the new virus titration in duplicates. 9. Therefore, in each well: a. Samples: 25 μl of compounds + 25 μl of virus + 25 μl of trypsin b. Positive control: 25 μL of virus + 25 μL of trypsin (without compounds) c. Negative control: 25 µL trypsin (without compounds or viruses) d. New titration: 25 uL of virus 10. The slides are incubated at 37 ° C in a CO2 incubator for 3 days, then observed for cytopathic effects on day 3 and day 5. In vivo Pharmacokinetic Profile (PK) Study Dose Administration
[000177] Intravenous Injections (IV) - The mice were injected with the volume required to administer the prescribed dose (mg / kg) to the animals based on the individual weight of the mouse using a 28G needle. The injection volume was 200 pL / 20 g of mouse. The mice were quickly held during IV injections for approximately 1 minute. Vein dilation was obtained by keeping the animals under a heating lamp for a period of between 1-2 minutes.
[000178] Intranasal Administration (IN) - The mice were anesthetized with isoflurane at 2% and 2 L / 02 / min until the absence of a flexion reflex by pain. The animals were kept upright and, using a micropipette, 10 pL of the compounds were instilled in each nostril of the animals per 15 g of body weight. The mice were quickly held during the instillation procedure for approximately 20 seconds and the release rate was adjusted to allow the mice to inhale the compounds without blistering. The mice were returned to the anesthetic chamber in an inverted position for a further 2 minutes or until their breathing returned to normal. Pharmacokinetic Sampling
[000179] The mice were individually weighed and evenly distributed in groups according to weight. The mice (n = 20 / group and n = 4 / time point) were injected with the test articles, as described in the dose administration section.
[000180] Blood collection: Blood was collected at the time points indicated in the study cluster table. For blood collection, the mice were sacrificed through CO2 inhalation and blood was collected through cardiac puncture. At the last breath, the mice were removed from the inhalation chamber and approximately 500-700 piL of blood was collected by cardiac puncture with a 25G needle and placed in a Microtainer tube with appropriate EDTA. Each tube was inverted several times to ensure uniform mixing of blood and EDTA to prevent clotting. Blood samples were stored on ice until all samples were collected for a particular time point and then further processed to generate plasma.
[000181] Plasma Preparation: Plasma was prepared by centrifuging the samples at 2500 rpm for 15 minutes at 4 ° C (rpm based on a Beckman GH 3.8A rotor, 250 xg average RCF), then pipetted and placed in labeled bottles on ice and then frozen at - 80 ° C. The samples were transported on dry ice.
[000182] Tissue Collections: After blood collection, the tissue of the trachea and lung were collected. Briefly, the ventral side of the neck was cut to expose the thyroid and sternotiroidal muscle. The muscles were gently cut to expose the larynx and trachea, a hemostat was used to close the trachea (close to the larynx) which was then cut just posterior to the larynx and the entire trachea, the bronchial tree was removed with the lungs trapped . The trachea was separated from the lungs. Fabric was not rinsed in saline. Each tissue sample was transferred to an individual labeled bottle on ice and then frozen at -80 ° C. The samples were transported on dry ice. Animal Observation
[000183] The mice were continuously monitored for acute signs of toxicity during the first two hours after administration of the test compound. For groups of mice at the last point of time (7 days), the mice were monitored 2X a day before sacrifice and tissue collection. Body weights of individual mice were measured every Monday, Wednesday and Friday during the course of the study. Data collect
[000184] The actual time of blood collection (period of the day), body weights and behavioral parameters, as described in Experimental Design: Sampling for Pharmacokinetics.
[000185] The following records were collected: • Manual distribution by body weight • Individual body weights • Observations • Comments • Real time of blood and tissue collection • Identity of tissue samples • Reasons / findings related to any premature animal sacrifice Animal Observations
[000186] Drug-Induced Stress Assessment - All animals were observed post-administration and at least once a day, more if deemed necessary, during pre-treatment and treatment periods for mortality and morbidity. In particular, signs of illness included loss of body weight, changes in appetite and behavioral changes, such as altered gait, lethargy and manifestations of stress with the naked eye. When signs of severe illness were observed, the animals were sacrificed (asphyxiation with CO2) and an autopsy was performed to assess other signs of toxicity. The following organs were examined: liver, gallbladder, spleen, lung, kidney, heart, intestine, lymph nodes and bladder. Any other unusual findings were also noted.
[000187] Dying animals were sacrificed for humanitarian reasons and the decision to sacrifice was at the discretion of the technician who cared for the animal and the study director. These findings were recorded as raw data and the time of death log was calculated the next day. Influenza model in mice in vivo Animals
[000188] A model of influenza with mice was used for this study. Female mice aged 6-7 weeks (Balb / C - Mus musculus) were provided with HK1 influenza virus adapted to 1250 pfu per mouse. Stimulus doses were 3x LDso determined in in vivo titration studies. The animals were housed in level 2 isolation. Activity Sequence in the Study

Administration of Intranasal Dose
[000189] The mice were anesthetized with isoflurane at 2% and 2 L / 02 / min until the absence of a flexion reflex by pain. The animals were kept upright and, using a micropipette, 10 µL of the compounds were instilled in each nostril of the animals. The mice were quickly held during the instillation procedure for approximately 20 seconds and the release rate was adjusted to allow the mice to inhale the compounds without blistering. The mice were returned to the anesthetic chamber in an inverted position for a further 2 minutes or until their breathing returned to normal. Intranasal Virus Inoculation
[000190] The mice were anesthetized with isoflurane at 2% and 2 L / 02 / min until the absence of a flexion reflex by pain. The animals were kept upright and, using a micropipette, 10 µl of a viral preparation containing 3,000 pfu of influenza A virus, A / HK / 1/68 (H3N2), was instilled in each nostril (total inoculum of 1,250 pfu per animal). The virus was prepared as a suspension in serum-free DMEM. The mice were quickly held during the instillation procedure for approximately 20 seconds and the release rate was adjusted to allow the mice to inhale the compounds without blistering. The mice were returned to the anesthetic chamber in an inverted position for a further 2 minutes or until their breathing returned to normal. Data collect
[000191] The following records were collected: • Manual distribution by body weight • Individual body weights • Observations • Comments • Reasons / findings related to any premature animal sacrifice Assessment of Drug-Induced Stress or Illness
[000192] All animals were observed post-administration, twice daily during treatment periods and once daily for mortality and morbidity. Signs of illness included loss of body weight, change in appetite, difficulty breathing and behavioral changes such as altered gait / posture, lethargy and manifestations of stress with the naked eye. When signs of severe illness (determined by weight loss> 15%), the animals reached the endpointe were sacrificed (asphyxiation with CO2).
[000193] Dying animals were sacrificed for humanitarian reasons and the decision to sacrifice was at the discretion of the technician who cared for the animal and the study director. These findings were recorded as raw data and the log of time of death was calculated the next day. EXAMPLES
[000194] Other modalities are described with reference to the following non-limiting examples. EXAMPLE 1: Assays with the viral sialidase enzyme
[000195] Incubation of influenza sialidase with different concentrations of the various compounds resulted in time-dependent decreases in enzyme activity, as expected for mechanism-based inhibitors and as shown for compound 4 in figures 3A and B. Figures 3A and 3B show the time-dependent inactivation of influenza sialidase (subtype N9) by compound 4. The enzyme was incubated with the indicated concentrations of compound 4 and aliquots assayed with 0.5 mM CF3MUSA. Inactivation by compound 4 at the indicated concentration (3A) and re-plotting inactivation kinetic constants of pseudo-first magnitude (ki obs) versus concentration of compound 4 (3B).
[000196] All 3-fluoro-sialyl fluorides showed excellent inactivation profiles against influenza sialidase at 30 ° C, except compound 8 and the half-time for total sialidase inactivation was estimated to be approximately 10 min for all derivatives amine (compounds 4, 7, 9, 11 and 12) (summary in Table 4). Interestingly, 4-amino compound 4 and 4-guanylated compound 12 showed good inactivation values (Aj / Kd = 25 min'1mM'1 for compound 4 and Áj / Kd = 24 min'1mM'1 for compound 12 ). In addition, compounds 4 and 12 showed slow reactivation in buffer at the same temperature, with the half-time for total reactivation of inactivated sialidase being determined as 4.8 h for compound 4 and 6.7 h for compound 12 Consequently, the 3-fluoro-sialyl enzyme intermediate very stably blocked the active site in influenza sialiasis and its effective Ks were in the nanomolar range (93 nM for compound 4 and 70 nM for compound 12).
[000197] Inactivation parameters for 2,3-difluoro-sialic acid, (23DFSA) and the compound of 4-azide 8 could not be measured at 30 ° C due to the rapid hydrolysis of the sialyl enzyme intermediate. Thus, the kinetic values for these two inhibitors were measured at 4 ° C and the estimated inactivation numbers were provided. The Aj / Kd value of compound 23DFSA was determined to be 196 min ^ mM'1 and the compound of 4-azide 8 and the compound of 4-amine 4 showed kinetic values 26 times lower at 4 ° C (Âj / Kd = 7 , 5 min'1mM'1 for compound 8 and 7.3 min ^mM'1 for compound 4). Although the 4-amine derivatives showed lower kinetic values of inactivation, these compounds proved to be much better inhibitors than the 4-hydroxylated derivative (23DFSA) due to their very slow reactivation.TABLE 4: Kinetic parameters for the sialidase reaction of influenza with 3-fluoro-sialilaa fluorides
a All experiments were carried out in 20 mM TRIS buffer / 50 mM CaCh, pH 7.6 containing 0.1% BSA at 30 ° C. b The kinetic values were collected at 4 ° C. ND = not determined. EXAMPLE 2: Assays with the human sialidase enzyme
[000198] Incubation of different concentrations of each compound with human sialidase did not result in inactivation of human sialidase, even at very high concentrations (10 mM) of compound, demonstrating the specificity of the compounds by influenza sialidases. EXAMPLE 3: Cytoprotection assays with influenza A strains
[000199] Candidate inhibitors (compounds 4, 5, 7-9 and 11 -13) have been tested against Zanamivir and / or 2,3-DFSA for their cytopathic effect (CPE) against two strains of influenza A. It is important note that although the present cytoprotection assay is a good qualitative indicator of antiviral activity, the results are often variable (as noted by others, Tisdale M. (2000)) and care must be taken when performing a quantitative analysis. TABLE 5. The activity of compounds against influenza A / Brisbane / 10/2007 (H3N2)
TABLE 6. The activity of compounds against influenza A / Denver / 1/57 (H1N1)
TABLE 7. The activity of compounds 4 and 12 against a variety of influenza A viruses and an Oseltamivir-resistant strain (OsR)

Multiple experiments were some strains. performed on different days with A: Single measurement; B: Average of two measurements; c: Average of six measurements
[000200] TABLE 7 shows the concentration of antiviral (pM) at which a confluent monolayer of MDCK cells was protected over a period of 5 days against cytopathic viral infection. EXAMPLE 4: Cytoprotection assays with Influenza B strains
[000201] Candidate inhibitors have been tested against Zanamivir for its cytopathic effect (CytoPathic Effect - CPE) against two strains of influenza B. It is important to note that, although the present cytoprotection assay is a good qualitative indicator of antiviral activity, Results are often variable (as noted by others, Tisdale M. (2000)) and care should be taken when performing a quantitative analysis. TABLE 8. The activity of influenza B / HongKong / 5/72 compounds
TABLE 9. The activity of compounds against influenza B / Florida / 04/06
EXAMPLE 5: In vivo pharmacokinetic profile (PK) study
[000202] The purpose of the study was to evaluate the pharmacokinetic profile of a new fluoro-sialic compound (DFSA-4Gu) compared to Zanamivir via the intranasal route and to compare the pharmacokinetic profile of DFSA-4Gu when dosed via the intranasal and injection in Balb / C mice (Mus musculus). TABLE 10. Study parameters


[000203] A PK study was conducted to evaluate DFSA-4Gu in mice, administered via the IN and IV pathways and to evaluate Zanamivir administered via the IN pathway, all at 1 mg / kg in a single dose. Tissue levels and plasma levels for DFSA-4Gu were obtained and lung tissue levels for Zanamivir were obtained. Levels declined with apparent first magnitude kinetics in all tissues through both pathways. Comparison of DFSA-4Gu and Zanamivir via the IN route showed that comparable exposure to the compound in the lung was observed with both agents. Calculated PK Parameters
[000204] PK parameters calculated for DFSA-4Gu and Zanamivir after intranasal dosing with a single dose of 1 mg / kg. TABLE 11 A. Calculated PK values

[000205] PK parameters calculated for DFSA-4Gu after intravenous dosing with a single dose of 1 mg / kg. TABLE 11B. Calculated PK values

[000206] The apparent half-life for the compounds was longer after intranasal dosing than after intravenous dosing. As well as the exposure, expressed as AUC for DFSA-4Gu, it was increased in the trachea and lung by means of intranasal dosage, in relation to the intravenous dosage with proportions of AUC (IN / IV) for the lung = 9.3 and for the trachea = 17.0.
[000207] AUC in the lung after intranasal dosing of Zanamivir and DFSA-4Gu were similar, AUC (Lung) (Zanamivir / DFSA-4Gu) = 1.07. The apparent half-life of the two agents in the lung after intranasal dosing was also similar. EXAMPLE 6: In vivo murine model of influenza infection
[000208] The in vivo efficacy of Zanamivir, DFSA-4Gu and DFSA-4NH2 was tested in a mouse influenza (HK1) model. Compounds were compared with Zanamavir, so the animals received a single intranasal dose of the drug before infection and follow-up doses were provided twice daily for a total of six days. Mortality due to infection was configured as a 15% weight loss, after which the animals were sacrificed. Animal survival was significantly prolonged in groups that had received a dose of 1 mg / kg of DFSA-4GU and Zanamavir, when compared to the untreated control group. Similarly, treatment with DFSA-4NH2 slightly prolonged survival when compared to the untreated control group. However, drug treatments did not prevent mortality, since all treated animals eventually reached the endpoint due to influenza infection. While these results are promising, the use of a different viral strain or an increased dose of drugs can alter survival.TABLE 12. Experimental groups and treatments
t The original protocol required N = 3 for each healthy control group (Groups 6 and 7 in Table 6). In carrying out the study, N = 5 was used in these groups, increasing the total number of animals to 60. TABLE 13. Animal survival rates on days 3, 4, 5, 6, 7 and 8 post-infection. TABLE 13 shows the number of animals left in each group. N = 10 for all groups, except groups 6 and 7 (n = 5). Day 3 pm Day 4 am Day 4 pm Day 5 am Day 5 pm Day 6 am Day 6 pm Day 7 am Day 7 pm Day 8 am Day 8 pm
TABLE 14. Average% weight loss per group on day 4 (am) post-infection. This time point was selected because many animals were about to reach the endpoint, but most groups still had 10 animals. 1
P-value determined by the Student T-test in which each group was individually compared with the mean of the untreated group # 7. A p-value below 0.05 was considered statistically significant.
[000209] With respect to the above results and potential change in the protocol, the use of a different viral strain, a different endpoint or an increased dose of drugs may be justified. For example, another viral strain adapted to mice, such as A / PR / 8/34, can produce different results. Also, a 15% weight loss endpoint can be very low, so animals can still recover after losing 20% or more. For example, Bantia et al. (2001) reported the recovery of animals treated with Zanamavir after a weight loss of more than 20 percent. Similarly, a larger dose of drug may be required to obtain complete protection. For example, in Levena et al. (2001), the authors required doses of Zanamavir at 10 and 50 mg / kg / day before they observed a protective effect.
[000210] Fluorinated compounds of the class described in the present invention are inhibitors of a number of glycosidases and specific with respect to their target enzymes. These compounds have a mechanism based on their inhibitory action. They bind to the enzyme as well as the normal substrate and undergo the first stage of catalysis (intermediate formation) similar to the natural substrate, but then undergo the second stage (renewal via hydrolysis) only very slowly. Importantly, inhibition based on this mechanism would make resistance formation by viruses more difficult. Since the inhibitors are based on the mechanism, any mutations in the viral enzyme that reduce inhibition must necessarily reduce the efficiency of the enzyme on the natural substrate. FIGURE 2 shows an example of neuraminidase inhibition by 3-fluoro-sialyl fluoride (1,23DFSA) (note: the number of sialic acid is different from that of natural sugars due to anomeric carboxylate).
[000211] Fluoro-sials fundamentally differ from Zanamivir and Oseltamivir in two main forms. Zanamivir and Oseltamivir are reversible binding inhibitors that interact with the enzymatic active site very tightly due to their flattened cyclic conformation. Its mode of attachment probably mimics the transition-shaped conformation of sugar during hydrolysis. Therefore, they are transitional state mimics. Fluoro-sialics, in contrast, do not contain double bond, thus adopting a regular chain conformation. They react with the enzyme as if they were substrates and form a covalent bond with the active site nucleophile and hydrolyze the products only very slowly. They derive their very high effectiveness from the lasting nature of the formed intermediate.
[000212] Because of this completely different mode of action, it was not evident whether incorporation of an amine or guanidine substituent would increase its effectiveness, since the sugar ring has a very different conformation in the two cases: the amine / guanidine would be presented in a different way. very different ways in the two cases, so they probably interact very differently. Even more importantly, the main efficacy of fluoro-sialics derives from the formation of a relatively long-lived covalent intermediate. It was not clear whether the incorporation of 4-amine / guanidine in place of 4-hydroxyl would decrease the hydrolysis of this intermediate (deglycosylation) much more than the formation of the intermediate (glycosylation) showed, with the result of a much longer-lived intermediate thus a more effective inhibitor.
[000213] Although various embodiments of the invention are disclosed here, many adaptations and modifications can be made within the scope of the invention according to the common general knowledge of those skilled in the field. Such modifications include the replacement of known equivalents for any aspect of the invention in order to obtain the same result in substantially the same way. Number ranges are inclusive of the numbers that define the range. The word "comprising" is used here as an open-ended term, substantially equivalent to the phrase "including, but not limited to" and the word "comprises" has a corresponding meaning. As used here, the singular forms "one", "one", "o" and "a" include references in the plural, unless the context clearly directs otherwise. So, for example, reference to "one thing" includes more than one such thing. Citing references here is not an admission that such references are prior art to the present invention. BIBLIOGRAPHY
[000214] Amaya, MF, Watts, A., Damager, I., Wehenkel, A., Nguyen, T., Buschiazzo, A., Paris, G., Frasch, AC, Withers, SG and Alzari, PM "Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase ”(2004) Structure, 12, 775-784.
[000215] Bantia S, Parker CD, Ananth SL, Horn LL eries K, Chand P, Kotian PL, Dehghani A, El-Kattan Y, Lin T, Hutchison TL, Montgomery JA, Kellog DL, Babu YS. “Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir.” Antimicrob Agents Chemother. (2001) 45 (4): 1162-7.
[000216] Buchini, S., Buschiazzo, A., Withers, S. G. "Towards a New Generation of Specific Trypanosoma cruzi Trans-sialidase Inhibitors" (2008) Angew. Chemie 47, 2700-2703.
[000217] I will sing BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) “The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics”. Nucleic Acids Res 37: D233-238.
[000218] Damager, I., Buchini, S., Amaya, ML, Buschiazzo, A., Alzari, P., Frasch, ACWatts, A. Withers, SG '' Kinetic and Mechanistic Analysis of Trypanosoma cruzi Trans-sialidase Reveals a Classical Ping-Pong Mechanism with Acid / Base Catalysis ”(2008) Biochemistry, 47, 3507-3512.
[000219] Hagiwara et al. "Inhibition of bacterial and viral sialidases by 3-fluoro-N-acetylneuraminic acid" Carbohydrate Research (1994) 263: 167- 172; and Buchini etal. Agnew. Chem. Int. Ed. (2008) 47: 2700-2703.
[000220] Henrissat B, Davies GJ (1997) "Structural and sequence-based classification of glycoside hydrolases". Curr. Op. Struct. Biol. 7: 637-644.
[000221] Ikeda, K .; Kitani ,. S.; Sato, K. Suzuki, T .; Hosokawa, C. Suzuki, Y. Tanaka, K .; Sato, M. “2b, 3b-difluoro acid derivatives structurally modified at the C-4 position: synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1” Carbohydrate Res. 2004, 339, 1367.
[000222] von Itzstein M "The war against influenza: discovery and development of sialidase inhibitors" Nature Reviews Drug Discovery (2007) 6 (12): 967-974.
[000223] Leneva IA, Goloubeva O, Fenton RJ, Tisdale M, Webster RG. "Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals." Antimicrob Agents Chemother. (2001) 45 (4): 1216-24.
[000224] Newstead, S., Potter, JA, Wilson, JC, Xu, G., Chien, C.- H., Watts, A., Withers, SG and Tailor, GL "The structure of Clostridium perfringens Nani sialidase and its catalytic intermediates "(2008) J. Biol. Chem. 283, 9080-9088.
[000225] Tisdale M. “Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors.” (2000) Rev Med Virol. 10 (1): 45-55.
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权利要求:
Claims (35)
[0001]
1. Compound, characterized by the fact that it presents the formula I:
[0002]
2. Compound according to claim 1, characterized by the fact that it presents the formula I:
[0003]
3. A compound according to claim 1, characterized in that Té COOH or COOR1, R1 is a C1-20 linear, branched or cyclic, saturated or unsaturated alkyl group, optionally substituted, in which the optional substituent is selected from one or more of the group consisting of: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2.
[0004]
Compound according to claim 1 or 3, characterized in that T is C (O) OCH3, C (O) OCH2CH3, C (O) OCH2CH2CH3, C (O) OCH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH2CH2CH3, C (O) OCH2CH2CH2CH2CH2CH2CH2CH3 OR COOH.
[0005]
A compound according to any one of claims 1, 3 and 4, characterized by the fact that A is selected from the group consisting of: F, Cl, Br, OH, CN and NO2.
[0006]
A compound according to any one of claims 1 and 3 to 5, characterized by the fact that A is selected from the group consisting of: F, Cl and OR3, R3 is a C1-20 linear, branched or cyclic, saturated alkyl group or unsaturated, optionally substituted, wherein the optional substituent is selected from one or more of the group consisting of: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2.
[0007]
A compound according to any one of claims 1 and 3 to 6, characterized by the fact that A is F or Cl.
[0008]
A compound according to any one of claims 1 to 7, characterized in that A is F.
[0009]
9. A compound according to any one of claims 1 and 3 to 8, characterized by the fact that D is selected from the group consisting of: H, F, Cl, Br, OH, CN and NO2, provided that A and D are not both H.
[0010]
A compound according to any one of claims 1 and 3 to 9, characterized by the fact that D is selected from the group consisting of: H, F and Cl, provided that A and D are not both H.
[0011]
A compound according to any one of claims 1 and 3 to 10, characterized by the fact that D is F or Cl.
[0012]
12. A compound according to any one of claims 1 and 3 to 11, characterized by the fact that D is H, provided that A and D are not both H.
[0013]
A compound according to any one of claims 1 and 3 to 12, characterized in that X is selected from the group consisting of: NH2, NHR5, NHCH3, NHCH2CH3, NHC (NH) NH2, NHC (NH) NHR5, NHC (NR6) NR5 and NR5R6, where R5 and R6 are, independently, CeHs, CH2C6H5 or a C1-8 alkyl group.
[0014]
Compound according to any one of claims 1 and 3 to 13, characterized in that X is selected from the group consisting of: NH2, NHCH3, NHCH2CH3 and NHC (NH) NH2.
[0015]
A compound according to any one of claims 1 and 3 to 14, characterized in that X is NH2 or NHC (NH) NH2.
[0016]
16. A compound according to any one of claims 1 and 3 to 15, characterized by the fact that E is selected from the group consisting of: NH2, NHC (O) CH3, OR7, NHR7, R7 is independently the C1-10 alkyl group linear, branched or cyclic, saturated or unsaturated, optionally substituted, the optional substituent is selected from one or more of the group consisting of: oxo, OH, F, Cl, Br, I, NH2, CN, SH, SO3H and NO2 and zero to ten carbons of the main chain of the optionally substituted alkyl group are optional and independently substituted with O, N or S.
[0017]
A compound according to any one of claims 1 and 3 to 16, characterized in that E is NH2 or NHC (O) CH3.
[0018]
18. A compound according to any one of claims 1 and 3 to 17, characterized by the fact that E is NHC (O) CH3.
[0019]
19. A compound according to any one of claims 1 and 3 to 18, characterized by the fact that Q is selected from the group consisting of CH2R9, CH (R9) (R10), C (R9) (R10) (R11),
[0020]
20. A compound according to any one of claims 1 and 3 to 19, characterized by the fact that Q is selected from the group consisting of:
[0021]
21. The compound according to any one of claims 1 and 3 to 20, characterized by the fact that Q is selected from the group consisting of:
[0022]
22. A compound according to any one of claims 1 and 3 to 21, characterized by the fact that Q is:
[0023]
23. The compound according to any one of claims 1 and 3 to 22, characterized by the fact that Q is:
[0024]
24. A compound according to any one of claims 1 to 23, characterized in that it is for modulating viral neuraminidase activity.
[0025]
25. The compound according to claim 24, characterized in that the viral neuraminidase is a GH34 neuraminidase.
[0026]
26. The compound according to claim 24 or 25, characterized by the fact that modulation of viral neuraminidase activity is for the treatment of influenza in a human.
[0027]
27. Pharmaceutical composition, characterized in that it comprises a compound as defined in any of the compounds as defined in any one of claims 1 to 23, and a pharmaceutically acceptable excipient.
[0028]
28. Pharmaceutical composition according to claim 27, characterized in that it is for modulating viral neuraminidase activity.
[0029]
29. Pharmaceutical composition according to claim 28, characterized by the fact that the viral neuraminidase is a GH34 neuraminidase.
[0030]
Pharmaceutical composition according to claim 28 or 29, characterized in that the modulation of viral neuraminidase activity is for the treatment of influenza in a human.
[0031]
31. Commercial packaging, characterized by the fact that it comprises one or more compounds as defined in any one of claims 1 to 23, or a pharmaceutically acceptable salt thereof.
[0032]
32. Method of preparing compound 2:
[0033]
33. The method of claim 32, further comprising: mixing compound 2 with NaOMe and MeOH; then, mix with Pd / C, H2 and MeOH; then, mix with LiOH, H2O and MeOH to form compound 4:
[0034]
34. Method of preparing compound 12:
[0035]
35. Composite, characterized by the fact that it is selected from one or more of the following:

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法律状态:
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2020-10-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 27/10/2020, OBSERVADAS AS CONDICOES LEGAIS. |

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优先权:

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申请号 | 申请日 | 专利标题

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US21378609P| true| 2009-07-15|2009-07-15|

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US61/213,786|2009-07-15|

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PCT/CA2010/001063|WO2011006237A1|2009-07-15|2010-07-15|2,3-fluorinated glycosides as neuraminidase inhibitors and their use as anti-virals|

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