WO2013074965A1

WO2013074965A1 - Aminoalkyl phenol ether inhibitors of influenza a virus

Info

Publication number: WO2013074965A1
Application number: PCT/US2012/065582
Authority: WO
Inventors: Arnab Basu; Debra M. Mills; John D. Williams; Norton P. Peet; Donald T. Moir; Terry L. Bowlin
Original assignee: Microbiotix Inc
Current assignee: Microbiotix Inc
Priority date: 2011-11-16
Filing date: 2012-11-16
Publication date: 2013-05-23
Anticipated expiration: 2014-05-16

Abstract

The present invention is directed to the discovery of novel nonpeptidic small molecules that function as inhibitors of the influenza virus infection. In particular, the present invention is directed to the discovery of anti-influenza entry inhibitors with an aminoalkyl phenol ether structure that specifically target the influenza virus group 1 hemagglutinin (HA), the influenza virus envelope glycoprotein which mediates influenza virus entry through receptor binding and fusion of the virus with host cells.

Description

AMINOALKYL PHENOL ETHER INHIBITORS OF INFLUENZA A VIRUS

Priority Claim

This application claims priority to U.S. Provisional Application No. 61/560,612 filed November 16, 201 1.

Statement Regarding Federally Sponsored Research

The invention described herein was supported by National Institutes of Health grant no. 1R43A 1072861 -01 A2. Accordingly, the United States Government has certain rights in the invention.

Field of the Invention

The present invention is directed to the discovery of novel nonpeptidic small molecules that function as inhibitors of influenza virus replication. In particular, the present invention is directed to the discovery of influenza viral entry inhibitors having an aminoalkyl phenol ether core structure, which compounds specifically target the influenza viruses group 1 hemagglutinin (HA), the influenza virus envelope glycoprotein, which mediates influenza virus entry through receptor binding and fusion of the virus with host cells.

Background of the Invention

Influenza virus causes annual epidemics and occasional pandemics. The virus undergoes regular antigenic changes brought about by either (a) point mutations in the genes coding for surface envelope glycoproteins hemagglutinin (HA) and/or neuraminidase (NA), or (b) by re- assortment of genes from two or more distinct types of influenza viruses. This constant evolution of the antigenic elements of the virus, referred to as "antigenic shift", increases the overall virulence of influenza virus. Pandemics occur when a new influenza virus emerges which, due to antigenic shift, the human population has no immunity. See, MMWR Morb Mortal Wkly Rep 58(33): 913- 918 (2009); MMWR Morb Mortal Wkly Rep 58(17): 467-470 (2009); Fauci, A. S., Cell 124(4), 665- 670 (2006); Govorkova et al., J Virol, 84(16): 8042-8050 (2010); Korteweg, C., and Gu, J.

Biochem. Cell Biol . 88(4), 575-587 (2010).

Vaccines, currently the primary strategy for protection against influenza infection, are only effective if they match the circulating virus type(s). Since the timing and subtype of the next influenza pandemic cannot be predicted, a "pandemic vaccine" cannot be developed in advance against new emerging strain(s). See, Horimoto, T., and Kawaoka, Y. Curr. Top. Microbiol. Immunol, 333: 165-176 (2009); Kemble, G., and Greenberg, H. Vaccine, 21 (16): 1789-1795 (2003); Tscherne, D. M., and Garcia-Sastre, A. J. Clin. Invest., 121(1 ): 6-13 (201 1).

In 2009, a novel influenza A HIN1 virus (A/HIN1/2009) that is antigenically divergent from the seasonal HlNl, emerged through reassortment, and caused the first influenza pandemic of the 21st century (Horimoto, T., and Kawaoka, Y., Curr. Top. Microbiol. Immunol., 333: 165-176 (2009); Kemble, G., and Greenberg, H., Vaccine, 21(16): 1789-1795 (2003); Tscherne, D. M., and Garcia-Sastre, A., J Clin. Invest., 121(1): 6-13 (201 1)).

Antiviral drugs, therefore, form an important part of a strategy for dealing with a new influenza outbreak. Effective antiviral therapy is an essential component of therapeutic options in the fight against influenza. The strategy described herein for developing new anti-influenza therapeutics is to target the surface protein hemagglutinin (HA), which mediates influenza virus entry through receptor binding and fusion of the virus with host cells (Lamb, R. A., and Krug, R. M. Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. R, and Webster, R. G. Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins (2001)). The focus of the present invention is to isolate, identify, and develop nonpeptidic small molecules that function to inhibit influenza virus entry. Conceptual support for this approach comes from the previous identification of several small molecules that interfere with RSV and MV entry (Ha et al., EMBO J., 21(5): 865-875 (2002); Mochalova et al., Antiviral Res., 23(3-4): 179-190 (1994); Sakagami et al., In Vivo, 6(5): 491-495 (1992); Sidwell et al, Antivir. Chem. Chemother., 10(4): 187-193 (1999)). HA is a class I fusion protein, a class that includes HIV Gpl20 and F protein of paramyxoviruses (RSV and MV) (Cianci et al.,. J. Antimicrob.

Chemother., 55(3): 289-292 (2005); Cianci et al., Antimicrob. Agents Chemother., 48(2): 413-422 (2004); Doyle et al., J Virol, 80(3): 1524-1536 (2006) Plemper et al., Antimicrob. Agents Chemother., 49(9): 3755-3761 (2005); Plemper et al., Proc. Natl. Acad. Sci. USA, 101(15): 5628- 5633 (2004); Sun et al., J. Med. Chem., 49(17): 5080-5092 (2006)).

The Orthomyxoviridae family includes influenza A, B, and C viruses, and Thogoto- and Isavirus (Lamb, R. A., and Krug, R. M., Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. F., and Webster, R. G.

Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins (2001 )). Influenza pandemics in humans are caused by influenza A viruses. Influenza A viruses contain 8 single-stranded, negative- sense viral RNAs (vRNAs) that encode 10-1 1 proteins. Influenza A virus contains two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (see Fig.1). Based on the antigenicity of the HA and NA proteins, 16 different HA subtypes (HI -HI 6) and 9 different NA subtypes (Nl- N9) of influenza A viruses have been identified. Of these, only a limited number of virus subtypes circulate in humans (i.e., H1-H3, and Nl, N2). The protorypic HA is synthesized as a single polypeptide and is subsequently cleaved into HA1 and HA2 subunits. HA cleavage is required for infectivity (Sidwell et al., Antimicrob. Agents Chemother., 40(1 1): 2626-2631 (1996); Sidwell et ah, Antiviral Res., 37(2): 107-120 (1998)) because it generates the hydrophobic N-terminus of HA2, which mediates fusion between the viral envelope and the cell membrane (Lamb, R. A., and Krug, R. M. Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. F., and Webster, R. G. Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins (2001)). Comparisons of the HA sequences indicate that there are two groups of HAs: group 1 contains HI, H2, H5, H6, H8, H9, HI 1, H12, H13, and H16, and group 2 contains H3, H4, H7, H10, H14, and H15. The two groups are divided into four clades: HI, H2, H5, H6, HI 1 and H13; H8, H9 and H12; H3, H4 and H14; and H7, H10 and H15 (Russell et al., Virology, 325(2): 287-296 (2004); Gamblin, S. J., and Skehel, J. J., J. Biol. Chem., 285(37):

28403-28409 (2010); Skehel, J. Biologicals, 37(3): 177-178 (2009); Skehel, J. J., and Wiley, D. C, Annu. Rev. Biochem., 69: 531-569 (2000)). Comparisons of the three-dimensional structures of representative HAs from three of the clades, H3, H5 and H9 (Ha et al, Embo J., 21(5): 865-875 (2002)) suggest that all three have very similar overall structure: The HA1 polypeptide of each of the identical subunits of the trimers mainly forms a membrane-distal domain that contains the receptor-binding and vestigial esterase subdomains; the HA2 polypeptide forms the fusion subdomain, the stem of the trimers, in which three long central a-helices are prominent (Skehel, J. J., and Wiley, D. C, Annu. Rev. Biochem., 69: 531-569 (2000)). However, the rotation of the membrane-distal subdomains relative to the central stem varied between the three HA clades and the variation appeared to be related to differences in the structure and positioning of a loop in HA2 that links a-helix A, HA2 residues 38-58, and a-helix B, HA2 residues 75-127. Additionally, the location and nature of ionisable residues near the "fusion peptide", HA2 residues 1-10, that could be involved in activation of the membrane fusion function of HA at endosomal H, also varied. Both of these features were proposed to be clade-specific and together with analyses of HA sequences were suggested as the basis for a structural classification of HAs.

HA appears to be critical in influenza virus replication and represents a validated target for pharmacological intervention. Several steps in the HA-mediated entry process are attractive targets for new anti-influenza therapeutics:

(1) Attachment of HA to its sialic acid receptors: The receptor-binding site of HA is a pocket located on each subunit at the distal globular part of HA 1 , and binds to cell surface sialic acid residues in a multivalent attachment process. The residues (Y98, W153, H183, E190, L194) forming the pocket are largely conserved among all subtypes of influenza (Skehel, J. J., and Wiley, D. C., Annu. Rev. Biochem., 69: 531-569 (2000)). Therefore, it would be advantageous to develop inhibitors that block binding of the virus to cells by either binding to receptor binding sites or preventing the interaction through some other mechanism. Several high molecular weight polymers, such as polyphenol and lignin, have been reported to inhibit binding of the virus to the cell membrane. Mochalova et al., Antiviral Res., 23(3-4): 179-190 (1994); Sakagami et al., In Vivo, 6(5): 491 -495 (1992); Sidwell et al., Antivir. Chem. Chemother., 10(4): 187-193 (1999); Sidwell et al., Antimicrob. Agents Chemother., 40(1 1): 2626-2631 (1996); Sidwell et al., Antiviral. Res., 37(2): 107-120 (1998); Sidwell et al., Antiviral Res., 6(6): 343-353 (1986); Sidwell et al., Chemotherapy, 40(1): 42-50 (1994).

(2) HA-mediated virus-cell fusion: Influenza virus enters its host cell by receptor-mediated endocytosis, followed by acid-activated membrane fusion in endosomes. The low pH environment in the endosomes is required to trigger the transition of HA from the non-fusogenic to the fusogenic conformation. This confonnational change relocates the fusion peptide segment from the amino- terminus of HA2 to the tip of the molecule. Following this conformational change, the fusion peptides fuse the viral envelope with the endosomal membrane. Inhibition of endosomal H+- ATPase, that blocks the acidification of endosomes, strongly inhibits the replication of influenza virus in MDCK cells (Hernandez et al., Annu. Rev. Cell Dev. Biol, 12: 627-661 (1996)). However, endosomal H+-ATPase activity is not a virus-specific target and interfering with H+-ATPase activity may lead to undesirable toxic side effects;

(3) The fusogenic trimer-of-hairpins structure: HA is a class I fusion protein as are HIV Gpl20 and F protein of paramyxoviruses (Cianci et al., Proc. Natl. Acad. Sci. U SA, 101(42): 15046-15051 (2004); Cianci et al., J. Antimicrob. Chemother., 55(3): 289-292 (2005); Cianci et al., Antimicrob. Agents Chemother., 48(2): 413-422 (2004)). Class I fusion proteins undergo a series of

conformational rearrangements from prefusion to fusion form that leads to the association of C- terminal and N-terminal heptad repeats. The resultant structure, a stable six-helix bundle, promotes the juxtaposition of the viral and cellular envelopes during fusion (Beigel et al., N. Engl. J. Med, 353(13): 1374-1385 (2005); Le et al., Nature, 437(7062): 1 108 (2005); Hayden et al., J. Infect. Dis., 189(3): 440-449 (2004)). This final fusion hairpin structure is sustained by protein-protein interactions. Small molecule entry inhibitors of paramyxoviruses have been identified that interfere with the formation or consolidation of the final fusion hairpin structure (Cianci et al., Proc. Natl. Acad. Sci. USA, 101(42): 15046-15051 (2004); Cianci et al., J. Antimicrob. Chemother., 55(3): 289-292 (2005); Cianci et al., Antimicrob. Agents Chemother., 48(2): 413-422 (2004)). Because a similar structure is present in influenza HA, the hydrophobic pocket structure is recognized as a potential target site for small molecule inhibitors. Moreover, the fact that the receptor binding domain and fusogenic trimer-of-hairpins structure are highly conserved lessens the probability of changes conferring resistance to drugs that target such structures.

Influenza Pandemics Influenza pandemics are caused by "antigenic shift", i.e., the introduction of new HA (or new HA and NA) subtypes into the human population (Lamb, R. A., and Krug, R. M.,

Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. F., and Webster, R. G., Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins(2001)). The lack of prior exposure to the new HA (or HA and NA) subtypes creates a population that is immunologically naive to the "antigenic shift" variants, resulting in extremely high infection rates and rapid spread worldwide. In humans, the historical pandemics in 1918, 1977, and 2009, in 1957, and in 1968 were caused by the H1NI, H2N2, and H3N2 viruses, respectively ((Lamb, R. A., and Krug, R. M., Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. F., and Webster, R. G., Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins(2001)). For the 1918, 1957, and 1968 pandemics, evidence suggests that the HAs were derived from avian viruses, and the Nl and N2 NAs of 1918 and 1957 had a similar derivation. In 1968, the N2 NA of the H3N2 virus was derived from the 1967 H2N2 virus. In 2009, both HI HA and Nl NA appear to have been derived from a porcine source ((Lamb, R. A., and Krug, R. M., Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001); Wright, P. F., and Webster, R. G., Orthomyxoviruses, 4th Ed., Lippincott Williams and Wilkins(2001)). The origin of the 1977 H1N1 virus is unknown. The 1918/1919 'Spanish influenza' is the most devastating infectious disease on record. An estimated 20-50 million people died worldwide and life expectancy in the US was reduced by 10 years (Tscherne, D. M., and Garcia-Sastre, A., J. Clin. Invest, 121(1): 6-13 (2011)).

Outbreaks of highly pathogenic H5N1 avian influenza viruses

Most avian influenza subtypes cause only mild infections or no illness at all in humans (Beare, A. S., and Webster, R. G., Arch. Virol., 119(1-2): 37-42 (1991)). The H5N1 virus that has emerged in southeast Asia is much more virulent than other avian influenza A viruses, and it has an unusually broad host range, causing lethal disease in many species of wild and domestic birds and in small and large felines, rodents and primates. Avian influenza represents a likely pathogen to cause the next pandemic, according to the World Health Organization (WHO) and Centers for Disease Control (CDC). Although highly pathogenic H5N1 viruses have not yet caused a human pandemic, their continued transmission to humans and high mortality rate in humans have made the development of therapies to these viruses a priority. The first transmission of highly pathogenic H5N1 avian influenza viruses to humans occurred in Hong Kong in 1997 when 6 of the 18 individuals infected succumbed to the infection. Since 2003, highly pathogenic H5N1 avian influenza viruses have become prevalent in southeast Asia and endemic in poultry in some countries in that region (Fauci, A. S., Cell, 124(4): 665-670 (2006)). More than 4200 outbreaks have been reported in Asian, African, and European countries (Beigel et al., N. Engl. J. Med. , 353(13): 1374- 1385 (2005)), resulting in the death or slaughter of more than 100 million poultry. Close contact between humans and poultry in rural areas of these regions likely facilitates virus transmission to humans. 236 human infections with 138 fatalities have been reported in 9 different countries (Beigel et al, N. Engl. J. Med, 353(13): 1374-1385 (2005); Le et al., Nature, 437(7062): 1 108 (2005)). Furthermore, the appearance of H5N1 strains resistant to the NA inhibitor oseltamivir (see Le et al., Nature, 437(7062): 1 108 (2005)) indicates that novel therapeutic treatments are urgently needed.

Options for pandemic control

A desirable way to combat the H5N1 avian influenza virus or any emerging or re-emerging influenza virus in humans is to inhibit or at least reduce the likelihood of interspecies transfer, and this requires a comprehensive, multifaceted approach. Currently, vaccination is the proven effective strategy for protection against influenza infection. However, its efficacy during a pandemic will be limited as a "pandemic vaccine" cannot be developed in advance against new emerging mutant strain(s) (Kemble, G., and Greenberg, H., Vaccine 21(16): 1789-1795 (2003); Hayden et al., J. Infect. Dis., 189(3), 440-449 (2004)). The current inactivated trivalent vaccine does not provide protection against the H5 and H7 avian influenza strains (Kemble, G., and Greenberg, H., Vaccine, 21(16): 1789-1795 (2003); Hayden, F. G., Pediatr. Infect. Dis. J. , 23(1 1 Suppl): S262-269 (2004)). Moreover, at this point we cannot predict whether the currently circulating H5N1 will be the next pandemic strain. In addition, the vaccine production capabilities will be strained during a pandemic by the need to immunize a vast number of individuals worldwide in a short period of time.

Antiviral drugs will be the first line of medical intervention during an influenza pandemic. Currently FDA approved drugs for the treatment of influenza virus infections are the influenza M2 ion channel blockers (amantadine and rimantadine) and the NA inhibitors (oseltamivir and zanamivir). The M2 ion channel blockers, amantadine and rimantadine, exert their antiviral activity by blocking the M2 ion channel, preventing virion uncoating and the release of genome segments into the cytoplasm. The efficacy of these antivirals has been undermined by the evolution of resistant strains. Moreover, historically, the inhibitors have been used almost exclusively to prevent infection or to reduce the duration of uncomplicated seasonal influenza; their benefit in treating severe disease has not been defined.

Influenza NA, a key enzyme in viral replication, spread, and pathogenesis, is considered to be one of the most important targets for combating influenza. The amino acids that make up the active site of NA are highly conserved across the 9 known NA subtypes. Current anti-influenza drugs oseltamivir and zanamivir efficiently block the NA activity of the 2009 pandemic strain and 2004 H5N1 viruses in vitro (Gubareva et al., Lancet, 355(9206): 827-835 (2000); Hayden et al., N. Engl. J. Med., 343(18): 1282-1289 (2000); Mase et al., Virology, 339(1): 101-109 (2005); Mase et al., Virology, 332(1): 167-176 (2005); Ward et al., J. Antimicrob. Chemother. , 55 Suppl 1 : i5-i21 (2005)), indicating their effectiveness in influenza chemotherapy and prophylaxis against H5N1 virus infection. However, during the 2008-2009 season, almost all seasonal influenza A/H1N1 isolates in the US were resistant to oseltamivir (99%, compared to 11 % in 2007-2008). The pandemic 2009 influenza A/H1N1 virus and the HPAI influenza H5N1 strains have been generally susceptible to NA inhibitors. However, oseltamivir-resistant strains have been isolated from patients infected with both these strains (Skehel, J. J., and Wiley, D. C, Annu. Rev. Biochem., 69: 531-569 (2000); Cianci et al., J. Antimicrob. Chemother., 55(3): 289-292 (2005); Cianci et al., Antimicrob. Agents Chemother.; 48(2): 413-422 (2004); Le et al., Nature, 437(7062): 1 108 (2005); Chen et al., J. Infect. Dis., 203(6): 838-846 (201 1); Moss et al., J. Antimicrob. Chemother., 65(6): 1086-1093 (2010); van der Vries et al., N. Engl. J. Med., 363(14): 1381-1382 (2010); van der Vries et al., N. Engl. J. Med., 359(10): 1074-1076 (2008); Sambhara, S., and Poland, G. A., Annu. Rev. Med., 61 : 187-198 (2010); Shinde et al., N. Engl. J. Med., 360(25): 2616-2625 (2009)). Therefore, it is unclear whether the NA inhibitors will be sufficient for use alone in future influenza pandemics, and broad-spectrum therapeutics against influenza virus infections are critically needed to address the problem of influenza pandemics, a major threat to the public health globally.

Interfering with viral entry is a novel and attractive therapeutic strategy to control virus infection. Proof of principle of this approach has come from the peptidic HIV inhibitor enfuvirtide. The present invention relates to the development of a nonpeptidic small molecule to inhibit influenza virus entry. Multiple routes of administration are conceivable for these inhibitor molecules, and highly cost-effective production strategies can be easily achieved. Combination therapies using multiple drugs that have different mechanisms of antiviral activity can be employed for synergistic antiviral effects and to prevent the emergence of resistant strains. The nonpeptidic small molecule inhibitors described herein act synergistically with both the NA inhibitors and M2 ion channel blockers.

The discovery and development of inhibitors of influenza infection, as outlined herein, will provide life-saving therapy worldwide and will prove invaluable in dealing with a potentially catastrophic influenza pandemic. Interfering with virus entry as described herein is a novel and attractive therapeutic strategy to control virus infection.

Summary of the Invention

The present invention is directed to the discovery of novel nonpeptidic small molecule inhibitors against influenza virus. In a preferred embodiment, the compounds described herein are effective inhibitors against infection by the influenza A virus. The novel inhibitors described herein are suitable for the treatment and/or prevention of influenza virus pathogenesis in humans and other mammalian and avian species.

Specifically the invention is related to the identification and characterization of nonpeptidic small molecule inhibitors comprising an aminoalkyl phenol ether core structure that prevent entry of the virus into a host cell. Even more particularly, the invention is related to the identification of nonpeptidic small molecule inhibitors for preventing the entry of influenza A virus into a host cell. More particularly, the invention is directed to the identification of nonpeptidic small molecule inhibitors for preventing the entry of influenza A virus with group 1 HA, into a host cell. In a preferred embodiment, the inhibitors of the present invention will target, i.e., be specific for, the group 1 hemagglutinin (HA) envelope glycoprotein that mediates viral entry into the cell.

However, it will be appreciated that, by following the procedures described herein, nonpeptidic small molecule inhibitors for any influenza virus, virus subtype, or viral target can be identified.

In a preferred embodiment, the present invention is directed to an influenza virus inhibitor comprising the compound of Formula I

Formula I wherein:

Ar is an aryl or heteroaryl group;

L is a linker which may be a direct bond or a divalent linear or branched alkyl, alkenyl, or alkynyl chain, optionally containing from 1-4 heteroatoms and optionally substituted by one or more halo, hydroxy, alkoxy, alkoxycarbonyl, or alkylthio groups; and

1 and R² are selected independently from a monovalent alkyl, alkenyl, or alkynyl group that may be unsubstituted or substituted with one or more substituents selected from halo, amino, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, acyl, carboxy, alkoxycarbonyl, amino, alkylamino, acyiamino, amido, sulfonamido, mercapto, alkylthio, arylthio, thioacyl, alkylsulfonyl, or aminosulfonyl, or R¹ and R² together may be covalently linked to form a cyclic structure of between 5-8 ring atoms, including the nitrogen atom to which R¹ and R² are attached;

and pharmaceutically acceptable salts thereof.

The aminoalkyl phenol ether compounds described herein are useful as anti-influenza agents and may be used to treat influenza viral infections. Accordingly, an individual infected with or exposed to influenza virus, for example, Influenza A virus of avian or swine origin, may be treated by administering to the individual in need thereof, an effective amount of a compound according to Formula I.

The present invention is also related to the use of one or more or a combination of the compounds disclosed herein to treat viral influenza infection and, especially, use of one or more or a combination of the above compounds to treat Influenza A infection. In particular, use of one or more or a combination of the above compounds for the treatment of Influenza A of avian or swine origin is advantageously carried out by following the teachings herein. In a particular embodiment, the viral inhibitory compounds described herein are useful for targeting the group 1 hemagglutinin (HA) envelope glycoprotein that mediates viral entry into the cell.

The present invention also provides pharmaceutical compositions containing one or more of the influenza inhibitor compounds disclosed herein and a pharmaceutically acceptable carrier or excipient. The use of one or more of the influenza inhibitor compounds in the preparation of a medicament for treating the viral infection is disclosed.

An influenza inhibitor compound or combination of inhibitor compounds described herein may also be used as a supporting or adjunctive therapy for the treatment of influenza infection in an individual (human or other animal).

In yet another embodiment, a composition comprising an influenza inhibitor or a combination of influenza inhibitors described herein may also comprise a second agent (second active ingredient, second active agent) that possesses a desired therapeutic or prophylactic activity other than or in addition to the influenza virus inhibition effected by the influenza inhibitors) described herein. Such a second active agent includes, but is not limited to, an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic agent (e.g., a nonsteroidal antiinflammatory drug (NSAID), acetaminophen, an opioid, a COX-2 inhibitor), an immunostimulatory agent (e.g., a cytokine), a hormone (natural or synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations of such second agents.

In a preferred embodiment, the small molecules of the present invention are formulated into a pharmaceutically acceptable carrier and are applied by an injection, including, without limitation, intradermal, transdermal, intramuscular, intraperitoneal and intravenous. According to another embodiment of the invention, the administration is oral and the inhibitor may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule, which simplifies oral application. The production of these forms of administration is within the general knowledge of a practitioner in this field. However, multiple routes of administration are envisioned for the molecules of the present invention, and highly cost-effective production strategies can be easily achieved. Compositions comprising an influenza inhibitor described herein may be formulated for administration to an individual (human or other animal) by any of a variety of additional routes including, but not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, parenteral, intraperitoneal, sublingual (under the tongue), buccal (cheek), oral (for swallowing), topical (epidermis), transdermal (absorption through skin and lower dermal layers to underlying vasculature), nasal (nasal mucosa), intrapulmonary (lungs), intrauterine, vaginal, intracervical, rectal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrarenal, nasojejunal, and intraduodenal.

To identify inhibitors that prevent entry of the influenza virus into host cells according to the present invention, a pseudotype virus expressing HA (H5 subtype) was developed as a model to mimic HA-mediated entry of the live virus into a cell. The pseudotype virus provides a means for safely replicating the viral entry mechanism and identifying inhibitors thereof, which inhibitors are then tested against viral infection under strict regulatory conditions that are not required for initial screenings with the pseudotype viruses.

Therefore, in another aspect, the present invention is directed to a high throughput system (HTS) assay for rapidly screening potential small molecule inhibitors of influenza virus using the pseudotype viruses described herein.

It is an object of the present invention to target envelope glycoprotein hemagglutinin (HA), which mediates influenza virus entry through receptor binding and fusion with host cells.

Therefore, in another aspect, the present application is directed to the discovery of anti- influenza entry inhibitors and to develop entry therapeutics that are active against seasonal influenza, in particular, H5N1, 2009 H1N1 swine flu pandemic strain, and other potential pandemic influenza viruses.

Brief Description of the Drawings

Figure 1 is a schematic diagram of the influenza A virus.

Figure 2 shows the effect of neuraminidase (NA) treatment (0-40 units/ml) on the infectivity of HIV/HA pseudotype virus in 293T producer cells. Cells treated 26 hr post- transfection with NA (0-40 units/ml) displayed higher levels of infectivity as compared to controls.

Figure 3 shows a comparison of the ability of HIV/HA and HIV/HA(USDA) pseudotype viruses to infect A549 cells with or without pretreatment with trypsin. Treatment with trypsin did not have any effect on HIV/HA entry since HA(H5) contains a ubiquitous protease cleavage site and is cleaved naturally in the infection process. Pretreatment with trypsin enhanced entry of HIV/HA(USDA). HA(H5) of the USDA strain does not have the ubiquitous protease cleavage site and requires additional enzymatic treatment for full infectivity. The HA(H5) of the USDA can be cleaved only by protease from cells of the upper respiratory tract. Since we are using human kidney cells, HI HA(USDA) infection requires trypsin treatment.

Figure 4 shows the effect of bafilomycin A (0-150 nM) which blocks acidification of endosomes and NH4CI (0-25 nM), a weak base, on the infectivity of HIV/HA pseudotype virus on A549 cells. Pretreatment of A549 cells with bafilomycin prevented HIV/HA infection of the cells indicating entry of HIV/HA into the cell is dependent on a low pH environment.

Figure 5 shows the susceptibility to infection of a number of target cell lines to the HIV/HA pseudotype virus. The human lung cell lines A549 and NCI/H661 were highly susceptible to infection by HIV/HA. These two cell lines were used for screening the compound libraries for small molecule inhibitors that prevent entiy of the influenza virus.

Figure 6 shows the development of an HA binding assay to susceptible cells to elucidate the mode of HA/inhibitor interactions. Figure 6A shows the four different regions of H5 1 HA1 that were fused with human IgG Fc to generate fusion proteins. Figure 6B shows one microgram of each purified fusion protein run on SDS-PAGE and stained with Coomassie blue. Figure 6C shows the binding of the fusion proteins (1 μg-20 g) to 293T cells as measured by flow cytometry.

Constructs #1 and #4 exhibited good binding to 293T cells. Figure 6D shows the binding of the fusion proteins to susceptible 293T and A549 cells and resistant Jurkat (human T cell line) cells. As expected, construct #4 showed significant binding to the 293T and A549 cells but not to the Jurkat cells. Therefore these results indicate that a sensitive HA binding assay has been developed

Figure 7 is a diagrammatic representation of the process for producing the HIV/HA pseudotype virus and infection of cells with the virus.

Figure 8 is a workflow diagram for advancing small molecule screening "hits" from the initial screening stage with HIV/HA to the stage of validated HA-specific influenza inhibitor.

Figure 9 shows the structure of isolated compound MBX2329, which was used as a preliminary SAR scaffold to study analogous small molecule inhibitor compounds identified according to the present invention as described in Example 9.

Figure 30 displays the antiviral activity of MBX2329 against HIV/HA (H5) pseudotype virus and cell culture grown virus in vitro. Panel A: Comparison of antiviral activity and cell toxicity of compound MBX2329 against HIV/HA (H5). HIV/HA(H5) was incubated with A549 cells at a MOI for 3 h in the presence or absence of compound MBX2329 in a dose dependent manner. The diamonds represent anti-HIV/HA activity while the squares represent cytotoxicity. Panels B and C: The inhibitory effect of compound MBX2329 against influenza HlNl strain A/WS/33 (HlNl) [panel B] and 2009 pandemic HlNl swine flu strain A/California/I0/2009/H1N1 (swine flu)] [panel C]. Figure 1 1 displays a time-of-addition study of compound MBX2329 inhibition during virus entry. A single cycle time of addition experiment was done with HIV/HA(H5) to determine the stage of influenza virus entry blocked by compound MBX2329. A549 cells were infected with 100 μΐ of p24-norrnalized HIV/HA(H5). Compound MBX2329 was added 2 hours before infection (-2h), during adsorption (Oh), and for 1 hour, 2 hours, 3 hours, 5 hours, and 24 hours after infection (+lh, +2h, +3h, +5h, and +24h, respectively). Infected monolayers were washed with PBS and incubated for 72 hours. Inhibition of HIV/HA(H5) pseudotype infection was detected as a reduced luciferase signal.

Figure 12 reveals that MBX2329 does not inhibit HA-mediated influenza virus binding to the cell surface. Influenza A virus has the ability to adsorb onto chicken RBCs, resulting in hemagglutination. Inhibition of agglutination of blood cells was used to investigate whether the inhibitors blocked viral attachment. For this assay, viruses were 2-fold serially diluted in a 96-well plate, and an equal volume of 0.5% chicken red blood cells (cRBCs) were added in the presence (20 μΜ) of the inhibitors. The plates were kept at 4°C for 60 min., and agglutination was determined visually. The cRBCs sedimented and formed red buttons in negative control wells (no virus), whereas positive control wells (virus only) had an opaque appearance with no sedimentation at high virus concentrations due to hemagglutination (left side of plate, Fig. 12). MBX2329 did not block HA hemagglutination activity, indicating that the compounds do not inhibit viral attachment.

Figure 13 displays the synergistic inhibition of influenza A/California 10/2009 by

MBX2329 and oseltamivir.

Definitions

In order that the invention may be more clearly understood, the following abbreviations and terms are used as defined below.

Abbreviations for various substituents (side groups, radicals) of organic molecules are those commonly used in organic chemistry. Such abbreviations may include "shorthand" forms of such substituents. For example, "Ac" is an abbreviation for an acetyl group, "Ar" is an abbreviation for an aryl (including heteroaryl) group, and "halo" or "halogen" indicates a halogen radical (e.g., F, CI, Br, I). "Me" and "Et" are abbreviations used to indicate methyl (CH₃-) and ethyl (CH₃CH₂-) groups, respectively; and "OMe" (or "MeO") and "OEt" (or "EtO") indicate methoxy (CH₃0-) and ethoxy (CH₃CH₂O-), respectively. Hydrogen atoms are not always shown in organic molecular structures or may be only selectively shown in some structures, as the presence and location of hydrogen atoms in organic molecular structures are understood and known by persons skilled in the art. Likewise, carbon atoms are not always specifically abbreviated with "C", as the presence and location of carbon atoms, e.g., between or at the end of bonds, in structural diagrams are known and understood by persons skilled in the art. Minutes are commonly abbreviated as "min."; hours are commonly abbreviated as "hr." or "h".

A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step. It is also understood that an element or step "selected from the group consisting of refers to one or more of the elements or steps in the list that follows, including combinations of any two or more of the listed elements or steps.

The term "halo" or "halogen" as used herein means fluorine, chlorine, bromine, or iodine. The term "alkyl" is intended to mean a straight or branched chain monovalent or divalent aliphatic hydrocarbon radical of 1-12, preferably 1-4, carbon atoms, such as methyl (Me), ethyl (Et), propyl (Pr), isopropyl (z^'Pr), butyl (Bu), isobutyl ^'Bu), sec-butyl (sBu), teri-butyl (/Bu), and the like, which may be unsubstituted, or substituted at any carbon atom by replacement of one or more hydrogen atoms with by a suitable substituent, as found herein.

The term "haloalkyl" is intended to mean an alkyl moiety that is substituted with one or more identical or different halogen atoms, e.g., -CH₂CI, -CF₃, -CH₂CF₃, -CH₂CC1₃, and the like.

The term "alkenyl" is intended to mean a straight-chain or branched hydrocarbon radical having 2-8 carbon atoms and at least one double bond, e.g., ethenyl, 3-buten-l-yl, 3-hexen-l-yl, cyclopent-l-en-3-yl, and the like, which may be unsubstituted, or substituted by one or more suitable substituents found herein. The term "cycloalkenyl" refers to cyclic hydrocarbon radicals of 2- 12 carbon atoms having at least one double bond.

The term "alkynyl" is intended to mean a straight-chain or branched hydrocarbon radical having 2-8 carbon atoms and at least one triple bond, e.g., ethynyl, 3-butyn-l-yl, 2-butyn- 1 -yl, 3- 65582

pentyn-l-yl, and the like, which may be unsubstituted, or substituted by one or more suitable substituents found herein.

The term "cycloalkyl" is intended to mean a non-aromatic monovalent or divalent

monocyclic or polycyclic radical having 3-12 carbon atoms, e.g., cyclopentyl, cyclohexyl,

decalinyl, and the like, which may be unsubstituted or substituted at any carbon atom by one or more of the suitable substituents found herein, and to which may be fused one or more aryl groups, heteroaryi groups, or heterocycloaikyi groups, which themselves may be unsubstituted or

substituted by one or more suitable substituents found herein.

The term "heterocycloaikyi" is intended to mean a non-aromatic monovalent or divalent, monocyclic or polycyclic radical having 2-12 carbon atoms, and 1-5 heteroatoms selected from nitrogen, oxygen, or sulfur, e.g., pyrrolodinyl, tetrahydropyranyl, morpholinyl, piperazinyl, oxiranyl, hexahydroazepinyl (azepanyl), and the like, unsubstituted, or substituted by one or more of the suitable substituents found herein, and to which may be fused one or more aryl groups, heteroaryi groups, cycloalkyl or heterocycloaikyi groups, which themselves may be unsubstituted or substituted by one or more suitable substituents found herein.

The term "aryl" is intended to mean an aromatic monovalent or divalent, monocyclic or polycyclic radical comprising between 6 and 18 carbon ring members, e.g., phenyl, biphenyl, naphthyl, phenanthryl, and the like, which may be substituted by one or more suitable substituents found herein, and to which may be fused one or more heteroaryi groups or heterocycloaikyi groups, which themselves may be unsubstituted or substituted by one or more suitable substituents found herein.

The term "heteroaryi" is intended to mean an aromatic monovalent or divalent, monocyclic or polycyclic radical comprising between 3 and 18 carbon ring members and at least 1 heteroatom selected from nitrogen, oxygen, or sulfur, e.g., pyridyl, pyrazinyl, pyridizinyl, pyrimidinyl, furanyl, thienyl, azapinyl, triazolyl, quinolinyl, imidazolinyl, benzimidazolinyl, indolyl, and the like, which may be substituted by one or more of the suitable substituents found herein, and to which may be fused one or more aryl, heteroaryi groups or heterocycloaikyi groups, which themselves may be unsubstituted or substituted by one or more suitable substituents found herein.

The term "hydroxyl" is intended to mean the radical -OH.

The term "alkoxy" is intended to mean the radical -OR where R is an alkyl or cycloalkyl group.

The term "aryloxy" is intended to mean the radical -OAr where Ar is an aryl grpup. The term

"heteroaryloxy" is intended to mean the radical -O(HAr) where HAr is a heteroaryi group. The term "acyl" is intended to mean a -C(0)R radical where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl, e.g. acetyl, benzoyl, and the like.

The term "carboxy" is intended to mean the radical -C(0)OH.

The term "alkoxycarbonyl" is intended to mean a -C(0)OR radical where R is alkyl, alkenyl, alkynyl, or cycloalkyl.

The term "aryloxycarbonyl" is intended to mean a -C(0)OR radical where R is aryl or heteroaryl.

The term "amino" is intended to mean the radical -NH₂.

The term "alkylamino" is intended to mean the radical -NRR' where R, and R' are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

The term "acylamino" is intended to mean the radical -NHC(0)R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl, e.g., acetylamino, benzoylamino, and the like.

The term "amido" in intended to mean the radical -C(0)NRR' where R and R' are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or

heterocycloalkyl.

The term "sulfonylamino" is intended to mean the radical -NHS0₂R where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

The term "amidino" is intended to mean the radical -C(NR)NR'R", where R, R', and R" are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and wherein R, R', and R" may form heterocycloalkyl rings, e.g., carboxamido, imidazolinyl,

tetrahydropyrimidinyl.

The term "guanidine" is intended to mean the radical -NHC(NR)NR'R", where R, R', and R" are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and wherein R, R', and R" may form heterocycloalkyl rings.

The term "mercapto" is intended to mean the radical -SH.

The term "alkylthio" is intended to mean the radical -SR where R is an alkyl or cycloalkyl group.

The term "arylthio" is intended to mean the radical -SAr where Ar is an aryl group.

The term "hydroxamate" is intended to mean the radical -C(0)NHOR where R is an alkyl or cycloalkyl group.

The term "thioacyl" is intended to mean a -C(S)R radical where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

The term "alkylsulfonyl" is intended to mean the radical -S0₂R where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. The term "aminosulfonyl" is intended to mean the radical -S0₂NRR' where R and R' are, independently, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or

heterocycloalkyl.

In the context of therapeutic use of the influenza inhibitor compounds described herein, the terms "treatment", "to treat", or "treating" will refer to any use of the influenza inhibitor compounds calculated or intended to arrest or inhibit the virulence or viral entry of influenza into host cells. Thus, treating an individual may be carried out after any diagnosis indicating possible viral infection, i.e., whether an infection by a particular influenza strain has been confirmed or whether the possibility of infection is only suspected, for example, after an individual's exposure to the virus or to another animal or individual infected by the virus. Furthermore, where an individual is administered an "effective amount" of a compound of this invention, it is meant .that a sufficient quantity of the active compound is administered to inhibit or preferably arrest the infection of the treated individual by influenza virus. Determination of effective amounts for a given dosage form and given mode of administration may be readily determined by practitioners in this field.

It is further contemplated that the compounds of the present invention will be routinely combined with other active ingredients such as antibiotics, antibodies, antiviral agents, anticancer agents, analgesics (e.g., a non-steroidal anti-inflammatory drug (NSAID), acetaminophen, opioids, COX-2 inhibitors), immunostimulatory agents (e.g., cytokines or a synthetic immunostimulatory organic molecules), hormones (natural, synthetic, or semi-synthetic), central nervous system (CNS) stimulants, antiemetic agents, anti-histamines, erythropoietin, agents that activate complement, sedatives, muscle relaxants, anesthetic agents, anticonvulsive agents, antidepressants, antipsychotic agents, and combinations thereof.

The meaning of other terms will be understood by the context as understood by the skilled practitioner in the art, including the fields of organic chemistry, pharmacology, and virology.

Unless otherwise indicated, it is understood that description of the use of an inhibitor compound in a composition or method also encompasses the embodiment wherein a combination of two or more such inhibitor compounds are employed as the source of influenza inhibitory activity in a composition or method of the invention.

Pharmaceutical compositions according to the invention comprise an influenza inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof, as the "active ingredient" and a pharmaceutically acceptable carrier (or "vehicle"), which may be a liquid, solid, or semi-solid compound. By "pharmaceutically acceptable" is meant that a compound or composition is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect the influenza inhibitor or any other component that may be present in a composition in such a way that would compromise the desired therapeutic and/or preventative benefit to a patient. Pharmaceutically acceptable carriers useful in the invention include those that are known in the art of preparation of pharmaceutical compositions and include, without limitation, water, physiological pH buffers, physiologically compatible salt solutions (e.g., phosphate buffered saline), and isotonic solutions. Pharmaceutical compositions of the invention may also comprise one or more excipients, i.e., compounds or compositions that contribute or enhance a desirable property in a composition other than the active ingredient.

Detailed Description of the Invention

The present invention is directed to the discovery of novel nonpeptidic small molecule inhibitors against influenza virus. In a preferred embodiment, the compounds comprise a family of compounds sharing an aminoalkyl phenol ether scaffold of Formula I. In another preferred embodiment, the compounds are effective inliibitors against infection by the influenza A virus. In a particularly preferred embodiment, the influenza inhibitors described herein are specific for the group 1 hemagglutinin (HA) envelope glycoprotein that mediates viral entry into the cell. The influenza inhibitors described herein are suitable for the treatment and/or prevention of influenza virus in humans and other mammalian and avian species.

In a particularly preferred embodiment, the present invention is related to the identification and characterization of novel nonpeptidic small molecule inhibitors compounds comprising an aminoalkyl phenol ether scaffold, which compounds are effective to prevent entry of the influenza virus into a host cell. The novel compounds described herein are suitable for the treatment or prevention of influenza. Even more particularly, the invention is related to the identification of nonpeptidic small molecule inhibitors for preventing the entry of influenza A virus into a host cell. Even more particularly, the present invention is related to the identification of nonpeptidic small molecule inhibitor for preventing the entry of influenza A virus with group 1 HA, into a host cell. In a preferred embodiment, the inhibitors of the present invention will target, i.e., be specific for, the group 1 hemagglutinin (HA) envelope glycoprotein that mediates viral entry into the host cell. However, it will be appreciated that, by following the procedures described herein, nonpeptidic small molecule inhibitors for any influenza virus, virus subtype, or viral target can be identified.

Also contemplated herein is the use of one or more, or a combination, of the compounds disclosed herein to treat viral influenza infection. Especially, use of one or more or a combination of the compounds have an aminoalkyl phenol ether scaffold for treating Influenza A. In particular, use of one or more or a combination of the above compounds for the treatment of Influenza A of avian or swine origin is advantageously carried out by following the teachings herein.

In a preferred embodiment, the novel compounds of the present invention will be administered as an orally active therapeutic, since that is the most convenient and rapid method of 2 065582

administration to a large exposed population in case of pandemic. However, the inhibitors described herein will also be suitable for IV administration, because it is envisioned that in case of a natural outbreak the infected patients may require IV administration. Therefore, the inhibitors described herein will provide an effective, safe, and easy therapeutic option for any newly emerged pandemic strain(s).

Multiple routes of administration are envisioned for the compounds of the present

invention, and highly cost-effective production strategies can be easily achieved. Compositions comprising an influenza inhibitor described herein may be formulated for administration to an individual (human or other animal) by any of a variety of routes including, but not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, parenteral, intraperitoneal, sublingual

(under the tongue), buccal (cheek), oral (for swallowing), topical (epidermis), transdermal

(absorption through skin and lower dermal layers to underlying vasculature), nasal (nasal mucosa), intrapulmonary (lungs), intrauterine, vaginal, intracervical, rectal, intraretinal, intraspinal,

intrasynovial, intrathoracic, intrarenal, nasojejunal, and intraduodenal.

Formula 1 wherein:

Ar is an aryl or heteroaryl group;

R¹ and R² are selected independently from a monovalent alkyl, alkenyl, or alkynyl group that may be unsubstituted or substituted with one or more substituents selected from halo, amino, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, acyl, carboxy, alkoxycarbonyl, amino, alkylamino, acylamino, amido, sulfonamido, mercapto, alkylthio, arylthio, thioacyl, alkylsulfonyl, or aminosulfonyl, or R¹ and R² together may be covalently linked to form a cyclic structure of between 5-8 ring atoms, including the nitrogen atom to which R¹ and R² are attached;

or a pharmaceutically acceptable salt thereof.

Particular embodiments of the compound of Formula I include the following:

HCI

21

22







Particularly preferred compounds of the present invention include the following:

HCI

The present invention also provides pharmaceutical compositions containing one or more of the influenza inhibitor compounds disclosed herein and a pharmaceutically acceptable carrier or excipient. The use of one or more of the influenza inhibitor compounds in the preparation of a medicament for combating the viral infection is also contemplated.

Compositions comprising an influenza inhibitory compounds described herein may be formulated for administration to an individual (human or other animal) by any of a variety of routes including, but not limited to, oral (for swallowing), intravenous, intramuscular, subcutaneous, intraarterial, parenteral, intraperitoneal, sublingual (under the tongue), buccal (cheek), , topical (epidermis), transdermal (absorption through skin and lower dermal layers to underlying vasculature), nasal (nasal mucosa), intrapulmonary (lungs), intrauterine, vaginal, intracervical, rectal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrarenal, nasojejunal, and

intraduodenal.

Pharmaceutical compositions according to the invention comprise a novel influenza inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof, as the "active ingredient" and a pharmaceutically acceptable carrier (or "vehicle"), which may be a liquid, solid, or semi-solid compound. Pharmaceutically acceptable carriers useful in the invention include those that are known in the art of preparation of pharmaceutical compositions and include, without limitation, water, physiological pH buffers, physiologically compatible salt solutions (e.g., phosphate buffered saline), and isotonic solutions. Pharmaceutical compositions of the invention 12 065582

may also comprise one or more excipients, i.e., compounds or compositions that contribute or enhance a desirable property in a composition other than the active ingredient.

Various aspects of formulating pharmaceutical compositions, including examples of various excipients, dosages, dosage forms, modes of administration, and the like are known to those skilled in the art of pharmaceutical compositions and also available in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), Remington: The Science and Practice of Pharmacy, Volumes 1 & 2, 19th edition, Alfonso R. Gennaro, ed., (Mack Publishing Co., Easton, PA 1995), or other standard texts on preparation of pharmaceutical compositions.

Pharmaceutical compositions may be in any of a variety of dosage forms particularly suited for an intended mode of administration. Such dosage forms, include, but are not limited to, aqueous solutions, suspensions, syrups, elixirs, tablets, lozenges, pills, capsules, powders, films, suppositories, and powders, including inhalable formulations. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or a multiple of a dose that is calculated to produce effective inhibition of influenza.

Pharmaceutical compositions described herein may be packaged in a variety of ways appropriate to the dosage form and mode of administration. These include but are not limited to vials, bottles, cans, packets, ampoules, cartons, flexible containers, inhalers, and nebulizers. Such compositions may be packaged for single or multiple administrations from the same container. Kits may be provided comprising a composition, preferably as a dry powder or lyophilized form, comprising an influenza inhibitor and preferably an appropriate diluent, which is combined with the dry or lyophilized composition shortly before administration. Pharmaceutical compositions comprising the novel influenza inhibitory compounds described herein may also be packaged in single use pre-filled syringes or in cartridges for auto-injectors and needleless jet injectors. Multi- use packaging may require the addition of antimicrobial agents such as phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalconium chloride, and benzethonium chloride, at concentrations that will prevent the growth of bacteria, fungi, and the like, but that are non-toxic when administered to a patient.

Consistent with good manufacturing practices, which are in current use in the

pharmaceutical industry and which are well known to the skilled practitioner, all components contacting or comprising a pharmaceutical composition must be sterile and periodically tested for sterility in accordance with industry norms. Methods for sterilization include ultrafiltration, autoclaving, dry and wet heating, exposure to gases such as ethylene oxide, exposure to liquids, such as oxidizing agents, including sodium hypochlorite (bleach), exposure to high energy electromagnetic radiation (e.g., ultraviolet light, x-rays, gamma rays, ionizing radiation). Choice of method of sterilization will be made by the skilled practitioner with the goal of effecting the most efficient sterilization that does not significantly alter a desired biological function of the influenza inhibitor or other component of the composition.

In yet another embodiment, a composition comprising an influenza inhibitor or a combination of influenza inhibitors described herein may also comprise a second agent (second active ingredient, second active agent) that possesses a desired therapeutic or prophylactic activity other than that of the influenza inhibition. Such a second active agent includes, but is not limited to, an antibiotic, an antibody, an antiviral agent, an anticancer agent, an analgesic agent (e.g., a nonsteroidal anti-inflammatory drug (NSAID), acetaminophen, an opioid, a COX-2 inhibitor), an immunostimulatory agent (e.g., a cytokine), a hormone (natural or synthetic), a central nervous system (CNS) stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

Construction of Pseudotype Virus

To identify novel inhibitory compounds that prevent entry of the influenza virus into host cells, a pseudotype virus expressing HA (H5 subtype) was developed as a model to mimic HA- mediated entry of the live virus into a cell. The pseudotype virus provides a means for safely replicating the viral entry mechanism and identifying inhibitors thereof, which inhibitors are then tested against viral infection under strict regulatory conditions that are not required for initial screenings with the pseudotype viruses.

To construct the pseudotype virus, cDNA encoding the HA gene of the highly pathogenic H5N1 influenza virus (Quinghai strain) was cloned into the pcDNA3 mammalian expression vector under the control of the CMV promoter (Rumschlag-Booms et al., J. Antivir. Antiretrovir., 7(3): 8-10 (2011).

To generate an HIV derived influenza pseudotype expressing HA (HIV/HA), the HIV-1 proviral genome containing a luciferase reporter gene (pNL4.3.Luc.R-E-) was co-transfected with the H5 HA (pcDNA3-HA) into 293T cells. The HIV/HA contains a luciferase gene reporter gene. Upon entry into a target cell, the viral RNA is reverse transcribed, actively imported into the nucleus, and stably integrated into the genome. Luciferase activity in the transduced cells provides a measure of virus infectivity (Basu et al., J. Virol. , 85(7): 3106-3119 (2011); Manicassamy et a!., J. Virol, 79(8): 4793-4805 (2005); Wang, et al., Virol. Sin., 26(3): 156-170 (201 1)). The luciferase assay is highly sensitive and is suitable for a high throughput screening (HTS) 96-well plate format. In addition, the use of non-replicating HIV derived influenza pseudotypes make HTS feasible without the need for stringent biohazard conditions required for handling pathogenic avian influenza strains. This is a key advantage of HIV/HA for developing an HTS assay to measure virus attachment and entry.

Therefore, the present application also addresses the optimization of several important parameters related to the discovery of small molecule inhibitors according to the present invention including: (a) generation of HIV/HA, (b) target cells to be used, (c) titration of HIV/HA, and (d) controls to maximize infectivity and signal to background (S/B) ratio (> 100/1).

Following generation, the HIV/HA pseudotype virus was collected and assessed for infectivity following standard protocols. A particular advantage of the pseudotype virus is it provides a means to safely and reliably mimic the entry of the "live" virus into a host cell while eliminating the dangers of working directly with the live virus. Potential inhibitors identified in this screening process can later be tested for the ability to directly prevent entry of the live virus into a host cell, however these later tests will be conducted under strict regulatory conditions which are not required for working with the pseudotype virus. The "primary hits" of inhibitors from the pseudotype virus assays may then be evaluated against a H5N1 strain in an enhanced BSL3 laboratory. This secondary screening will rapidly identify those compounds that are active against infectious viruses.

Another advantage of conducting the initial screening with the pseudotype virus as opposed to the live infectious virus is its focus on detecting entry inhibitors. By contrast, working with the live virus will lead to the identification of compounds that will not only inhibit viral entry, but also viral replication and egress.

The initial screen was performed with a compound library of > 140,000 compounds for entry inhibition, thus even a small increase in the number of primary hits (due to the presence of replication and assembly inhibitors) could have resulted in the necessity for additional secondary testing of thousands of compounds. In contrast, initial screening with the pseudotype virus as described herein reduced the number of primary hits because it identified only putative entry inhibitors of H5 influenza virus. Therefore, the method of primary screening with pseudotype virus reduced both the number of compounds handled and the screens needed.

Following the procedures outlined below, approximately 140,000 discrete compounds were screened and 141 primary hits identified. The Z' factor for the HTS was 0.5±0.2. Primary hits were counter-screened with pseudotype virus expressing an unrelated glycoprotein (VSV-G) and infectious H1N1 virus for their specificity. Compounds were evaluated for their potency and cytotoxicity with resynthesized compounds. Only 36 of the primary hits specifically inhibited the HA-mediated entry process. The final hit rate from the HTS was 0.09%. All of the 36 hit compounds exhibited IC 0 values of <25μΜ. Structurally, the HA inhibitors can be represented as clusters of >2 members each and singletons.

Therefore, by following the procedures described herein, a HTS assay has been developed using HIV/HA(H5) to screen for HA (H5) inhibitors. A total of 36 HA(H5) specific inhibitors have been identified with 1C₉₀ < 25μΜ and CC₅₀ > 25μΜ, and all 36 compounds inhibited cell culture · grown influenza virus (H1N1)(P 8). Particular compounds are shown in Tables 3 and 4 below.

A preferred compound of this invention has the structure shown in Figure 9.

In another embodiment, the novel small molecule influenza inhibitory compounds of the present invention are formulated into a pharmaceutically acceptable carrier and are applied by an injection, including, without limitation, intradermal, transdermal, intramuscular, intraperitoneal and intravenous. According to another embodiment of the invention, the administration is oral and the inhibitor may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule, which simplifies oral application. The production of these forms of administration is within the general knowledge of practitioners in this field.

Compositions and Methods

Influenza inhibitor compounds as described herein may be synthesized using established chemistries as shown below.

Synthesis Scheme 1 :

= 3-CI, 4-CI, 5-CI, 6-CI

3-OMe, 4-OMe, 5-OMe, 6-OMe

General Procedure A:

Mossy zinc (2.48 g) was added to a solution of mercury (II) chloride (0.25 g) in water (20 mL) and concentrated aqueous HC1 (120 μΐ_^). The solution was decanted, and the Zn-Hg amalgam was added to a suspension of toluene (2 mL), water (1.5 mL), and the substituted acetophenone (1.0 g) in concentrated aqueous HC1 (3.5 mL). The resulting mixture was heated to reflux for 24 h, and additional concentrated aqueous HC1 (4 mL) was added after 12 h. The mixture was then cooled and extracted with EtOAc (100 mL). The organic extract was dried over MgS04, filtered, and evaporated to yield a residue. The residue was subjected to flash chromatography on silica gel (40 g) with 0-30% EtOAc/hexane. Product-containing fractions were pooled and evaporated to provide the desired ethylphenol.

2-Ethyl-3-methoxyphenol

2-Hydroxy-6-methoxyacetophenone (1.0 g, 6.0 mmol) was reduced according to General Procedure A to provide 1.03 g (112%) of 2-ethyl-3-methoxyphenol as a clear oil: ^!H-NMR (CDCI₃) δ 6.98 (t, 1H), 6.43 (dd, 1H), 3.78 (s, 3H), 2.66 (q, 2H), 1.12 (t, 3H).

2-Ethyi-4-methoxyphenoI

2-Hydroxy-5-methoxyacetophenone (1.0 g, 6.0 mmol) was reduced according to General Procedure A to provide 0.57 g (62%) of 2-ethyl-3-methoxyphenol as a clear oil: 'H-NMR (CDC1₃) δ 6.72-6.58 (m, 3H), 4.94 (s, br, 1H), 3.76 (s, 3H), 2.60 (q, 2H), 1.19 (t, 3H).

2-Ethyl-5-methoxyphenol

2-Hydroxy-4-methoxyacetophenone (1.0 g, 6.0 mmol) was reduced accoiding to General Procedure A to provide 0.98 g (107%) of 2-ethyl-5-methoxyphenol as a clear oil: Ή-ΝΜΕ. (CDC1₃) δ 7.00 (d, 1H), 6.43 (dd, 1H), 6.37 (d, 1H), 5.99 (s, 1H), 3.70 (s, 3H), 2.56 (q, 2H), 1.18 (t, 3H).

2~Ethyl-6-methoxyphenol

2-Hydroxy-3-methoxyacetophenone (1.0 g, 6.0 mmol) was reduced according to General Procedure A to provide 0.54 g (59%) 2-ethyl-6-methoxyphenol as a clear oil: Ή-NM (CDC¾) δ 6.81-6.69 (m, 3H), 5.71 (s, 1H), 3.85 (s, 3H), 2.67 (q, 2H), 1.22 (t, 3H).

-Chloro-2-ethylpheiioI 6-Chloro-2-hydroxyacetophenone (1.0 g, 5.9 mmol) was reduced according to General Procedure A to provide 0.87 g (94%) 3-chloro-2-ethylphenol as a clear oil: Ή-NMR (CDC1₃) δ 6.97-6.90 (m, 2H), 6.67 (q, 1H), 6.08 (s, br, 1H), 4.14 (q, 2H), 1.16 (t, 3H).

4- Chloro-2-ethyIphenoI

5- Chloro-2-hydroxyacetophenone (1.0 g, 5.9 mmol) was reduced according to General Procedure A to provide 0.41 g (45%) of 3-chloro-2-ethylphenol as a clear oil: Ή-NMR (CDC1₃) δ 7.10 (d, 1H), 7.00 (dd, 1H), 6.67 (d, 1H), 4.98 (s, 1H), 2.59 (q, 2H), 1.21 (t, 3H).

5-ChIoro-2-ethyIphenoI

4-ChIoro-2-hydroxyacetophenone (1.0 g, 5.9 mmol) was reduced according to General Procedure A to provide 0.83 g (90%) 5-chloro-2-ethylphenol as a clear oil: ¾-NMR (CDCI₃) 57.01 (d, 1H), 6.83 (d, 1H), 6.79 (m, 1H), 6.68 (s, br, 1H), 2.58 (q, 2H), 1.18 (t,3H).

6-Chloro-2-ethylphenoI

3-Chloro-2-hydroxyacetophenone (1.0 g, 5.9 mmol) was reduced according to General Procedure A to provide 0.81 g (88%) of 6-chloro-2-ethylphenol as a clear oil: ^-NMR (CDC1₃) δ 7.14 (dd, 1H), 7.03 (d, 1H), 6.79 (dd, 1H), 5.60 (s, 1H), 2.67 (q, 2H), 1.23 (t, 3H).

Synthesis Scheme 2:

1) /"CI

(R)₂N

General Procedure B:

To a solution of substituted phenol in acetone (15 mL) were added potassium carbonate (4.0 eq) and l-(2-chloroethyl)azepane hydrochloride (1.5 eq). The suspension was heated to 100° C for 48 h, then cooled and filtered to remove residual solids. The solids were rinsed with acetone (10 mL), and the combined filtrate was evaporated and subjected to flash chromatography on silica gel (40 g) with 0-10% MeOH/CHCl₃. Product-containing fractions were pooled and evaporated to yield a residue that was acidified with concentrated aqueous HCI, evaporated repeatedly with

EtOH/hexane, triturated with Et₂0, then filtered and dried to provide the desired

(phenoxyethyl)azepane as the hydrochloride salt.

•HCI

1- [2-(2-MethyIphenoxy)ethyI]azepane hydrochloride (MBX 2643A)

2- Methylphenol (182 mg, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 250 mg (64%) of 1 -[2-(2-methylphenoxy)ethyl] azepane hydrochloride as an off-white solid: R_f 0.32 (10% MeOH/CHCl₃); mp 137-140° C; ¾- NMR (CDC1₃) δ 12.42 (s, br, 1H), 7.15 (d, 2H), 6.94-6.86 (m, 2H), 4.58 (s, br, 2H), 3.68 (s, br, 2H), 3.52 (s, br, 2H), 3.20 (s, 2H), 2.20 (s, 5H), 1.89 (s, 4H), 1.68 (s, 2H); m/z expected 233.2 found 234.2 (M+H)⁺.

•HCI

1- [2-(2-Ethylphenoxy)ethylJazepane hydrochloride (MBX 2329 A)

2- Ethylphenol (0.61 g, 5.0 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 0.75 g (53%) of l-[2-(2-methylphenoxy)ethyl]azepane hydrochloride as an off-white solid: R_f 0.35 (10% MeOH/CHCI₃); mp 122-123° C; ^!H-NMR (DMSO-i/₆) δ 11.03 (s, br, 1H) 7.21-7.16 (m, 2H), 6.98-6.90 (m, 2H), 4.42 (t, 2H), 3.56-3.43 (m, 4H), 3.31-3.20 (m, 2H), 2.58 (q, 2H), 1.88-1.84 (m, 4H), 1.72-1.55 (m, 4H), 1.16 (t, 3H).

•HCI

l-[2-(3-Ethylphenoxy)ethyl]azepane hydrochloride (MBX 2656A)

3-Ethylphenol (205 mg, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 196 mg (63%) of l-[2-(3-ethylphenoxy)ethyl]azepane hydrochloride as a white powder: R_f 0.28 (10% MeOH/CHC¾); mp 96-100°C; 'H-NMR (CDC1₃) δ 12.51 (s, br, IH), 7.21 (t, 1H), 6.86 (d, 1H), 6.74 (m, 2H), 4.55 (s, 2H), 3.63 (s, 2H), 3.46 (s, 2H), 3.15 (m, 2H), 2.62 (q, 2H), 2.20 (mt, 2H), 1.88 (m, 4H), 1.67 (s, 2H), 1.20 (t, 3H); m/z expected 247.2, found 248.2 (M+H)⁺. 2012/065582

•HCI

l-[2-(4-Ethylphenoxy)ethyl]azepane hydrochloride (MBX 2655A)

4-Ethylphenol (205 rag, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 200 mg (85%) of l-[2-(4-ethylphenoxy)ethyl]azepane hydrochloride as a white powder: R_f 0.33 (10% MeOH/CHCl₃); mp 141-149°C; Ή-NMR (CDC1₃) δ 12.51 (s, br, IH), 7.7.12 (d, 2H), 6.92 (d, 2H), 4.52 (s, 2H), 3.62 (m, 2H), 3.46 (m, 2H), 3.15 (q, 2H), 2.59 (m, 2H), 2.17 (t, 2H), 1.91-1.85 (m, 4H), 1.69 (m, 2H), 1.20 (t, 3H); m/z expected 247.2, found 248.2 (M+H)⁺.

1- [2-(2-Ch!orophenoxy)ethyIJazepane hydrochloride (MBX 2646A)

2- Chlorophenol (216 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 268 mg (86%) of 1 -[2-(2-chlorophenoxy)ethyl] azepane hydrochloride as an off-white powder: R_f 0.23 (10% MeOH/CHCl₃); mp 107-1 10°C; ¾- NMR (CDC1₃) δ 12.49 (s, br, IH), 7.36 (d, IH), 7.23 (d, IH), 6.96 (t, 2H), 4.63 (s, br, 2H), 3.67 (s, br, 2H), 3.54 (s, 2H), 3.26 (m, 2H), 2.19 (m, 2H), 1.87 (s, 4H), 1.69 (s, 2H); m z expected 253.1 found 254.2 ( +H)⁺.

•HCI

l-[2-(3-Chlorophenoxy) ethyljazepane hydrochloride (MBX 2640A)

3-Chlorophenol (216 mg, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 283 mg (88%) of l-[2-(3-chlorophenoxy)

ethyl]azepane hydrochloride as a white powder: R_f 0.27 (10% MeOH/CHCl₃); mp 140-145°C; Ή- NMR (CDC1₃) δ 12.58 (s, br, IH), 7.23 (t, IH), 6.99 (d, IH), 6.91 (s, IH), 6.81 (d, Hi), 4.57 (s, br, 2H), 3.63 (s, br, 2H), 3.47 (s, 2H), 3.13 (s, 2H), 2.18 (s, 2H), 1.88 (s, 4H), 1.69 (s, 2H); m/z

expected 253.1, found 254.2 (M+H)⁺.

l-[2-(4-Chlorophenoxy)ethyl]azepane hydrochloride (MBX 2647A) U 2012/065582

4-Chlorophenol (216 mg, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 287 mg (73%) of l-[2-(4-chlorophenoxy)ethyl] azepane hydrochloride as an off-white powder: R_f 0.27 (10% MeOH/CHCl₃); mp 167-169° C; Ή- NMR (CDC1₃) δ 12.60 (s, br, IH), 7.25 (d, 2H), 6.84 (d, 2H), 4.55 (s, br, 2H), 3.63 (m, 2H), 3.46 (s, br, 2H), 3.13 (pent, 2H), 2.17 (t, 2H), 1.88 (t, 4H), 1.69 (t, 2H); m/z expected 253.1 found 254.2 (M+H)⁺.

1- {2-[2-(TrifluoromethyI)phenoxy]ethyl}azepane hydrochloride (MBX 2651A)

2- (Trifluoromethyl)phenol (272 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 1 14 mg (62%) of 1 -{2-[2- (trifluoromethyl)phenoxy] ethyl} azepane hydrochloride as an beige powder: R_f 0.27 (10%

MeOH CHCl₃); mp 119-133° C; 'H-NMR (CDC1₃) δ 12.54 (s, br, IH), 7.59-7.50 (m, 2H), 7.11- 7.04 (m, 2H), 4.70 (s, 2H), 3.65 (m, 2H), 3.51 (s, 2H), 3.21-3.11 (m, 2H), 2.18 (m 2H), 1.87 (t, 4H), 1.69 (d, 2H); m/z expected 287.1, found 288.2 (M+H)⁺.

l-{2-[3-(TrifluoromethyI)phenoxy]ethyl}azepane hydrochloride (MBX 2641A)

3-(Trifluoromethyl)phenoI (209 mg, 1.3 mmol) was treated with l-(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 271 mg (70%) of l-{2-[3- (trifluoromethyl)phenoxyJethyl}azepane hydrochloride as a beige powder: R_f 0.31 (10%

MeOH CHCl₃); mp 1 10-125°C; ¾-NMR (CDC1₃) δ 12.65 (s, IH), 7.43 (t, IH), 7.27 (t, IH), 7.11 (d, 2H), 4.62 (t, 2H), 3.69-3.62 (m, 2H), 3.52-3.47 (m, 2H), 3.15 (q, 2H), 2.18 (q, 2H), 1.89 (m, 4H), 1.73-1.68 (m, 2H); m z expected 287.1, found 288.3 (M+H)⁺.

l-{2-[4-(Trifluoromethyl)phenoxy]ethyl}azepane hydrochloride (MBX 2652A)

4-(Trifluoromethyl)phenol (272 mg, 1.7 mmol) was treated with 1 -(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 202 mg (88%) of 1 -{2-[4- (trifluoromethyl)phenoxy]ethyl}azepane hydrochloride as a white powder: R_f 0.38 (10%

MeOH CHCls); mp 161-163°C; 'H-NMR (CDC1₃) δ 12.65 (s, br, IH), 7.57 (d, 2H), 7.00 (d, 2H), 4.64 (t, 2H), 3.68-3.61 (m, 2H), 3.49 (t, 2H), 3.14 (q, 2H), 2.18 (m, 2H), 1.89-1.76 (m, 4H), 1.73- 1.67 (m, 2H); m/z expected 287.1 found 288.2 (M+H)⁺.

l-[2-(3-Methoxyphenoxy)ethyl]azepane hydrochloride (MBX 2661A)

3-Methoxyphenol (156 mg, 1.3 mmol) was treated with I -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 135 mg (60%) of l-[2-(3-methoxyphenoxy)ethyl] azepane hydrochloride as a white powder: R_f 0.28 (10% MeOH/CHCl₃); mp 96-100°C; Ή-NMR (CDC1₃) δ 12.30 (s, br, IH), 7.20 (t, IH), 6.58-6.46 (m, 3H), 4.54 (s, br, 2H), 3.79 (s, 3H), 3.64 (s, br, 2H), 3.49 (s, br, 2H), 3.15 (s, br, 2H), 2.17 (s, br, 2H), 1.88 (m, 4H), 1.67 (m, 2H); m/z expected 249.2, found 250.2 (M+H)⁺.

l-[2-(4-Methoxyphenoxy)ethyl]azepane hydrochloride (MBX 2642A)

4-Methoxyphenol (156 mg, 1.3 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 57 mg (66%) of l-[2-(4-methoxyphenoxy)ethyl] azepane hydrochloride as a beige powder: R_f 0.27 (10% MeOH/CHCl₃); mp 136-140° C; Ή-NMR (CDCI₃) 5 12.41 (s, br, IH), 6.84 (s, 4H), 4.49 (s, 2H), 3.77 (s, 3H), 3.65 (t, 2H), 3.45 (s, 2H), 3.14 (m, 2H), 2.18 (q, 2H), 1.87 (t, 4H), 1.68 (d, 2H); m z expected 249.2, found 250.2 (M+H)⁺.

1- {2-[2-(2-Propyl)phenoxy]ethyl}azepane hydrochloride (MBX 2644A)

2- (2-Propyl)phenol (229 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 120 mg (76%) of l-{2-[2-(2-propyl)phenoxy]ethyl} azepane hydrochloride as a beige powder: R_f 0.40 (10% MeOH/CHCl₃); mp 151-169° C; 'H-NMR (CDCI₃) δ 12.62 (s, br, IH), 7.25-7.14 (d, IH), 7.17 (t, IH), 6.99 (t, IH), 6.87 (d, IH), 4.57 (s, 2H), 3.69 (m, 2H), 3.51 (s, 2H), 3.27-3.18 (m, 3H), 2.1 (q, 2H), 1.90 (t, 4H), 1.76-1.67 (m, 2H), 1.20 (d, 6H); m/z expected 261.2, found 262.2 (M+H)⁺.

l-{2-[2-(l-Propyl)phenoxy]ethyl}azepane hydrochloride (MBX 2645A) 2-(l-Propyl)phenol (229 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 20 mg (18%) of l-{2-[2-(l-propyl)phenoxy]ethyl} azepane hydrochloride as a beige powder: R_f 0.31 (10% MeOH/CHCl₃); mp 1 15-1 19° C; 'H-NMR (CDCI₃) δ 12.58 (s, br, IH), 7.16 (t, 2Η), 6.95 (d, 2Η), 6.87 (t, 2H), 4.56 (s, 2H), 3.66 (s, 2Η), 3.51 (s, 2H), 3.19 (s, 2H), 2.55 (t, 2H), 1.91 (s, 4Η), 1.67-1.54 (m, 4H), 0.94 (t, 3H); m/z expected 261.2, found 262.2 (M+H)⁺.

l-[2-(2,6-DiethyIphenoxy)ethyl]azepane hydrochloride (MBX 2662A)

2,6-Diethylphenol (252 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 216 mg (41%) of l-[2-(2,6-diethylphenoxy)ethyl] azepane hydrochloride as a white powder: R_f 0.29 (10% MeOH/CHCl₃); mp 150-154° C; 'H-NMR (CDCfe): δ 12.63 (s, br, IH), 7.06 (s, 3H), 4.34 (s, br, 2H), 3.74 (s, br, 2H), 3.48 (s, 2H), 3.25 (s, br, 2H), 2.68 (d, 2H), 2.63 (d, 2H), 2.25 (s, br, 2H), 1.94 (s, br, 4H), 1.68 (m, 2H), 1.24 (t, 6H); m/z expected 275.2, found 276.1 (M+H)⁺.

1- [2-(2-Ethyl-6-methylphenoxy)ethyl]azepane hydrochloride (MBX 26S3A)

2- Ethyl-6-methylphenol (229 mg, 1.7 mmol) was treated with l-(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 86 mg (17%) of l-[2-(2-ethyl-6- methylphenoxy)ethyl]azepane hydrochloride as a white powder: R_f 0.35 (10% MeOH/CHCI₃); mp 158-159°C; Ή-NMR (CDC1₃) δ 12.60 (s, br, IH), 7.05-6.99 (m, 3H), 4.35 (t, 2H), 3.76 (d, 2H), 3.48 (t, 2H), 3.29-3.19 (m, 2H), 2.64 (q, 2H), 2.31 (s, 3H), 2.27-2.21 (m, 2H), 1.95-1.89 (m, 4H), 1.70 (d, 2H), 1.23 (t, 3H); m/z expected 261.2, found 262.2 (M+H)⁺.

1- {2-(2-Ethyl-3-methoxyphenoxy)ethyl}azepane hydrochloride (MBX 2691A)

2- Ethyl-3-methoxyphenol (730 mg, 4.8 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 71 mg (5%) of l-{2-(2-ethyl-3- methoxyphenoxy)ethyl} azepane hydrochloride as a white powder: R_f 0.22 (10% MeOH/CHCl₃); mp 152-183°C; Ή-NMR (CDC1₃) δ 12.48 (s, br, IH), 7.12 (t, IH), 6.55 (dd, 2H), 4.54 (s, 2H), 3.82 65582

(s, 3H), 3.65 (m, 2H), 3.49 (m, 2H), 3.16 (d, 2H), 2.61 (q, 2H), 2.17 (m, 2H), 1.88 (m, 4H), 1.66 (t, 2H), 1.05 (t, 3H); m/z expected 277.2, found 278.2 (M+H)⁺.

1- {2-(2-EthyI-4-methoxyphenox )ethyl}azepane hydrochloride (MBX 2690A)

2- Ethyl-4-methoxyphenol (384 mg, 2.5 mmol) was treated with 1 -(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 200 mg (26%) of l-{2-(2-ethyl-4- methoxyphenoxy)ethyl}azepane hydrochloride as a white powder: R_f 0.24 (10% MeOH/CHCl₃);

mp 108-114°C; Ή-NMR (CDC1₃) δ 12.32 (s, 1H), 7.03 (t,lH), 6.79 (t, 2H), 4.42 (d, 2H), 3.87 (s, 3H), 3.72 (s, 2H), 3.48 (s, 2H), 3.34-3.27 (m, 2H), 2.62 (q, 2H), 2.24 (q, 2H), 1.93 (s, 4H), 1.71 (m, 2H), 1.19 (t, 3H); m/z expected 277.2, found 278.2 (M+H)⁺.

1- {2-(2-EthyI-5-methoxyphenoxy)ethyl}azepane hydrochloride (MBX 2695A)

2- Ethyl-5-methoxyphenol (950 mg, 6.2 mmol) was treated with l-(2-chloroethyi)azepane

hydrochloride according to General Procedure B to provide 475 mg (24%) of l-{2-(2-ethyl-5- methoxyphenoxy)ethyl}azepane hydrochloride as a white powder: R_f 0.24 (10% MeOH/CHCl₃);

mp 157-160°C; 'H-NMR (CDC1₃) δ 12.60 (s, br, 1H), 7.05 (d, 1H), 6.49 (dd, 1H), 6.44 (d, 1H), 4.53 (s, 2H), 3.78 (s, 3H), 3.6 (m, 2H), 3.49 (s, 2H), 3.66-3.12 (m, 2H), 2.52 (q, 2H), 2.25-2.17 (m, 2H), 1.92-1.86 (m, 4H), 1.69 (d, 2H), 1.15 (t, 3H); m/z expected 277.2, found 278.3 (M+H)⁺.

1- [2-(2-Ethyl-6-methoxyphcnoxy)ethyI]azepane hydrochloride (MBX 2710A)

2- Ethyl-6-methoxyphenol (540 mg, 3.6 mmol) was treated with l-(2-chloroethyl)azepane

hydrochloride according to General Procedure B to provide 148 mg (13%) of l-[2-(2-ethyl-6- methoxyphenoxy)ethyl]azepane hydrochloride as a white powder: R_f 0.34 (10% MeOH/CHCl₃);

mp -148° C; 'H-NMR (CDC1₃) δ 12.15 (s, br, IH), 7.03 (t, 1H), 6.78 (t, 2H), 4.41(m, 2Η), 3.87 (s, 3Η), 3.73 (m, 2Η), 3.49 (m, 2Η), 3.33 (t,2H), 2.62 (q, 2H), 2.23 (m, 2H), 1.89 (m, 4Η), 1.68 (m, 2Η), 1.19 (t, 3H); m/z expected 277.2, found 278.4 (M+H)⁺.

l-(2-(3-ChIoro-2-ethylphenox ) ethyl)azepane hydrochloride (MBX 2712A)

3-Chloro-2-ethylphenol (865 mg, 5.5 mmol) was treated with 1 -(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 400 mg (23%) of l-(2-(3-chloro~2- ethylphenoxy) ethyl)azepane hydrochloride as a white powder: R_f 0.56 (J 0% MeOH/CHCl₃); mp >168° C subl.; 'H-NMR (CDC1₃) δ 12.56 (s, br, IH), 7.12-7.00 (m, 2H), 6.79 (d, IH), 459 (t, 2H), 3.71-3.64 (m, 2H), 3.52 (q, 2H), 3.17 (pent, 2H), 2.79 (q, 2H), 2.19 (q, 2H), 1.95-1.86 (m, 4H), 1.71-1.69 (m, 2H), 1.10 (t, 3H); m/z expected 281.2, found 282.4 (M+H)⁺.

l-{2-(4-Chloro-2-ethylphenoxy)ethyl}azepane hydrochloride (MBX 2693A)

4-Chloro-2-ethylphenol (780 mg, 5.0 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 345 mg (22%) of l-{2-(4-chloro-2- ethylphenoxy)ethyl}azepane hydrochloride as a white powder: R_f 0.24 (10% MeOH/CHCl₃); mp 157-160°C; Ή-NMR (CDC1₃) δ 12.64 (s, br, 1H),7.13 (d, 2H), 6.79 (t, IH), 4.56 (t, 2H), 3.65 (t, 2H), 3.49 (d, 2H), 3.15 (pent, 2H), 2.56 (q, 2H), 2.18 (m, 2H), 1.89 (q, 4H), 1.74-1.67 (m, 2H), 1.17 (t, 3H); m/z expected 281.2, found 282.3 (M+H)⁺.

l-(2-(5-ChIoro-2-ethylphenoxy)ethyI)azepane hydrochloride (MBX 2692A)

5-Chloro-2-ethylphenol (360 mg, 2.3 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 1 12 mg (15%) of 1 -(2-(5-chloro-2- ethylphenoxy)ethyl)azepane hydrochloride as a white powder: R_f 0.24 (10% MeOH/CHCl₃); mp 149-181°C; 'H-NMR (CDC1₃) δ 12.71 (s, br, IH), 7.08 (d, IH), 6.94 (dd, IH), 6.84 (d, IH), 4.55 (t, 2H), 3.66 (t, 2H), 3.50 (d, 2H), 3.15 (pent, 2H), 2.55 (q, 2H), 2.2 (q, 2H), 1.89 (sep, 4H), 1.67 (m, 2H), 1.16 (t, 3H); m/z expected 281.2, found 282.3 (M+H)⁺.

l-(2-(6-Chioro-2-ethylphenoxy)ethyl)azepane hydrochloride (MBX 2716A)

6-Chloro-2-ethylphenol (800 mg, 5.1 mmol) was treated with l-(2-chloroethyl)azepane hydrochloride according to General Procedure B to provide 345 mg (21%) of l-(2-(6-chloro-2- ethylphenoxy)ethyl)azepane hydrochloride as a white powder: R_f 0.41 (10% MeOH/CHCl₃); mp 113-142°C; 'H-NMR (CDC1₃) δ 12.47 (s, br, IH), 7.22 (d, IH), 7.12 (d, IH), 7.04 (t, IH), 4.46 (s, 2H), 3.71 (m, 2H), 3.54 (m, 2H), 3.29 (pent, 2H), 2.69 (q, 2H), 2.25 (m, 2H), 1.92 (m, 3H), 1.70 (m, 3H), 1.23 (t, 3H); m/z expected 281.2, found 282.3 (M+H)⁺.

1- [2-(2-Ethylphenoxy)ethyl]piperidine hydrochloride (MBX 2648A)

2- Ethylphenol (221 mg, 1.8 mmol) was treated with 1 -(2-chloroethyl)piperidine hydrochloride according to General Procedure B to provide 148 mg (74%) of l-[2-(2-ethylphenoxy)ethyl] piperidine hydrochloride as an off-white powder: R_f 0.35 (10% MeOH/CHCl₃); mp 132-133° C; Ή- NMR (CDC1₃) δ 12.62 (s, br, IH), 7.20-702 (m, 2H), 6.95 (t, IH), 6.85 (d, IH), 4.56 (s, 2H), 3.66 (d, 2H), 3.44 (d, 2H), 2.83 (pent, 2H), 2.30 (q, 2H), 1.88 (q, 2H), 1.90-1.86 (m, 3H),1.48 (t, I H), 1.18 (t, 3H); m/z expected 233.2 found 234.2 (M+H)⁺.

•HCI

4-[2-(2-Ethy.lphenoxy)ethyl]morpholine hydrochloride (MBX 2649 A)

2-Ethylphenol (220 mg, 1.8 mmol) was treated with 1 -(2-chloroethyl)morpholine hydrochloride according to General Procedure B to provide 180 mg (62%) of 4-[2-(2-ethylphenoxy)ethyl] morpholine hydrochloride as an off-white powder: R_f 0.54 (10% MeOH/CHCl₃); mp 127-131 ° C; 'H-NMR (CDCI₃) δ 13.64 (s, br, IH), 7.20-7.16 (m, 2H), 6.97 (t, IH), 6.85 (d, IH), 4.64 (t, 2H), 4.0 (d, 2H), 4.26 (t, 2H), 3.61 (d, 2H), 3.48 (m, 2H), 3.10 (m, 2H), 2.60 (q, 2H), 1.89 (t, 3H); m/z expected 235.2, found 236.1 (M+H)⁺.

N-[2-(2-Ethylphenoxy)ethyl]diethyIamine hydrochloride (MBX 2650A)

2-Ethylphenol (236 mg, 1.9 mmol) was treated with N-(2-chloroethyl)diethylamine hydrochloride according to General Procedure B to provide 21 1 mg (77%) of N-[2-(2-ethylphenoxy)ethyl] diethylamine hydrochloride as a white powder: R_f 0.35 (10% MeOH/CHCl₃); mp 154-159°C; Ή- NMR (CDCI₃) δ 13.64 (s, br, 12.65 (s, br, IH), 7.16 (d, 2H), 6.96 (t, IH), 6.86 (d, IH), 4.54 (s, 2H), 3.50 (s, 2H), 3.29 (s, br, 4H), 2.59 (q, 2H), 1.49 (t, 6H), 1.18 (t, 3H); m z expected 221.2 found 222.1 (M+H)⁺.

l-[2-(2-Ethylphenoxy)ethyl]pyrrolidine hydrochloride (MBX 2654A) 2-Ethylphenol (239 mg, 2.0 mmol) was treated with l -(2-chloroethyl)pyrrolidine hydrochloride according to General Procedure B to provide 180 mg (36%) of 1 -[2-(2-ethylphenoxy)ethyl] pyrrolidine hydrochloride as a white powder: R_f 0.33 (10% MeOH/CHCl₃); mp 162-168°C;Ή- NMR (CDC1₃) δ 12.89 (s, br, 1H), 7.17 (d, 2H), 6.96 (t, 1H), 6.86 (d, 1H), 4.55 (s, 2H), 3.89 (s, 2H), 3.53 (s, 2H), 3.01 (s, 2H), 2.61 (q, 2H), 2.26 (s, 2H), 2.12 (s, 2H), 1.19 (t, 3H); m/z expected 219.2, found 220.1 (Μ+Η)⁺·

EXAMPLES

Example 1. Establishment of HIV/HA pseudotyping system for H5N1 avian influenza entry.

Influenza Virus HA used in this study. The cDNA encoding the HA (H5) gene of the highly pathogenic H5N1 influenza virus, was kindly provided by Dr. George Gao, Institute of Microbiology, Chinese Academy of Sciences, China (Gao et al., Virol. J., 6: 39 (2009);

Rumschlag-Booms et al., Virol. J., 6: 76 (2009)). This HA gene was originally isolated from an influenza A H5N1 virus present in a dead migratory bird (goose) in Qinghai Province, China. The HIV/HA pseudotype virus described herein contains a luciferase gene as reporter gene. Upon entering into a target cell, the viral RNA is reverse transcribed, actively imported into the nucleus, and stably integrated into the genome. Luciferase activity in the transduced cells provides a measure of virus infectivity (Basu et al., J. Virol, 85(7): 3106-31 19 (2011); Manicassamy et al., J. Virol, 79(8): 4793-4805 (2005); Rumschlag-Booms et al., Virol. 1, 6: 16 (2009)). The luciferase assay is highly sensitive and is suitable for a 96-well plate format. In addition, the use of non- replicating HIV derived influenza pseudotypes make HTS feasible without the need for stringent biohazard conditions required for handling pathogenic avian influenza strains. Both are key advantages of HIV/HA for developing an HTS assay to measure virus attachment and entry.

Several important parameters were optimized in order to isolate the small molecules described herein including: (a) generation of HIV/HA, (b) target cells to be used, (c) titration of HIV/HA, and (d) controls, to maximize infectivity and signal to background (S/B) ratio (>100/1).

To construct the pseudotype virus described herein, the envelope-defective proviral genome pNL4.3.LucR-E- (Basu et al., J. Virol, 85(7): 3106-31 19 (2011); Manicassamy et al., J. Virol, 79(8): 4793-4805 (2005); Rumschlag-Booms et al., Virol. J., 6: 76 (2009)) containing a luciferase reporter gene was used as the HIV-I expression vector. In the pNL4.3 uc.R-E- vector, the firefly luciferase gene is inserted into the pNL4-3 nef gene. Two frameshifts (5' Env and Vpr aa 26) render this clone Env- and Vpr- making it competent for only a single round of replication. The vector was licensed from Dr. Ned Landeau from the Salk Institute. To construct the pseudotype virus described herein, the cDNA encoding the HA gene was cloned into the mammalian expression vector pcDNA3 (Invitrogen) under the control of a CMV promoter. To 12 065582

generate an HIV-derived influenza pseudotype expressing HA [HIV/HA], pNL4.3.Luc.R-E- was co-transfected with the H5 HA(pcDNA3-HA) into 293T cells as previously described (Basu et al., J. Wro/., 85(7): 3106-31 19 (201 l); Manicassam et aI., . Virol, 79(8): 4793-4805 (2005); Wang et al., Virol. Sin., 26(3): 156-170 (201 1)). The supernatants containing the pseudotype viruses were collected at 48 h post-transfection, combined, filtered through a 0.45^m-pore-size filter and assessed for infectivity following standard protocols (Basu et al., J. Virol., 85(7): 3106-31 19

(201 1); anicassamy et al., J. Virol., 79(8): 4793-4805 (2005); Rumschlag-Booms et al., Virol. J., 6: 76 (2009)).

Plasmids encoding VSV-G (HIV/VSV-G) and an empty vector were also cotransfected with pNL4.3.Luc.R-E- to generate a control pseudotype viruses. The p24 content of pseudotype viruses was measured using a commercially available kit (Beckman Coulter, CA) for direct comparison of their respective infectivities. 293 T (human), HeLa (human), QT6 (quail), and DF-1 (chicken) cells were infected with p24 normalized HIV/HA or control virions, and the luciferase activities of the cells were determined 48 hours post-challenge (Fig. 3). As expected, all of the four cell lines infected with the VSV-G pseudotype HIV virions displayed high levels of luciferase activity (6.6 to 7.2 logs of RLUs), while the empty vector-infected cells displayed lower levels of luciferase activity (2.8 to 3.1 logs of RLUs). The cells infected by the HIV/HA virions expressed luciferase activity approximately 100-fold higher than background, showing that all of these cells, of both human and avian origin, can be infected by the HIV/HA viruses.

These results demonstrated a functional assay to study the entry mechanism for H5N1 infection. It is important to emphasize that this functional assay greatly improved the safety of the assay as opposed to using a live H5N1 virus for studies of entry mechanism and for screening of entry inhibitors. This functional assay was used to identify potential small molecule inhibitors that prevent the entry of influenza virus into host cells

As described below, by employing this assay, it was demonstrated that human lung cell lines A549 and NCI H661 are highly susceptible to HIV/HA transduction and these cells were used as the target cells in HTS assay to identify small molecule inhibitory compounds that prevent entry of the HIV/HA into these host cells. In addition, it is important to have an adequate number of target cells/well so that the infected cells will give a good luciferase signal-to-noise background ratio (>100/1).

Example 2. Neuraminidase (NA) treatment of the producer cells enhances infectivity of the pseudovirions.

To optimize the HIV/ΉΑ pseudotyping system, neuraminidase (NA) treatment of the 293T producer cells was used to enhance infection of the HIV-based pseudotype viruses. 293 T cells 65582

were co-transfected with pNL4.3.Luc+ R-E- and HA plasmids. Post-transfection (26 h), the transfected 293T cells were treated with NA (New England Biolabs) at concentrations of 0, 5, 10, 20, 40 units/ml. The viral supernatants were diluted 5-fold and used to challenge the target cells. The luciferase activities of the infected cells were determined 48 hours post-infection, and the results are shown in Fig. 2.

Results show that HIV/HA collected from the NA -treated cells displayed at least 10-fold higher luciferase activity than non-treated cells at 5 units/ml NA. Treatment of the producer cells with higher concentrations of NA (10-40 units/ml) further increased the luciferase signals in the target cells, albeit not greatly (5.62 to 5.73 logs). HIV/VSVG and empty vector transfected pseudotype virus were used as controls. These results are consistent with NA treatment facilitating viral release from the producer cells.

In addition, the high signal/background ratio indicates the successful establishment of an efficient HIV/HA pseudotype virus that will be extensively used for the proposed experiments after further HTS optimization.

Therefore, HIV/HA pseudotypes were generated by cotransfection of 293 T cells (3 X 10⁶ cells in a 100mm dish, -70% confluency) with pcDNA3-HA (12 μg) and pNL4.3.Luc.R-E- (^g) using lipofectamine 2000 following standard protocol (see Fig. 7). For a control, an HIV derived VSV-G pseudotype (HIV/VSV-G) was generated by cotransfection of a plasmid carrying VSV-G, (pcDNA3-VSV-G) and pNL4.3.Luc.R-E- in 293T cells following the same protocol, to establish HA target specificity of the compounds. The HIV/HA or HIV/VSV-G pseudotypes are replication- defective, and the pseudotypes will be evaluated for only one round of the infection process. Virus infectivity is measured from the luciferase activity of the transduced cells. Background activity is determined from luciferase activity of cells infected with supernatants of empty pcDNA3 vector and pNL4.3.Luc.R-E- transfected cells.

Example 3. HIV/HA pseudotype virus does not require trypsin treatment.

As stated previously, HA cleavage is required for infectivity (Sidwell et al., Antiviral Res,, 37(2): 107-120 (1998)), because it generates the hydrophobic N-terminus of HA2, which mediates fusion between the viral envelope and the cell membrane (Basu et al., J. Virol. , 85(7): 3106-31 19 (2011); Manicassamy et al, J. Virol., 79(8): 4793-4805 (2005); Rumschlag-Booms et al., Virol. J, 6: 76 (2009)). The effect of trypsin treatment on the infectivity of HIV/HA derived from H5N1 was investigated. For comparison, another HIV/HA pseudotype virus expressing HA of a low pathogenic avian H5N2 isolate (CK/Michoacan/ 28159-530/95) was generated. The HA of this strain does not have a cleavage site that can be cleaved by any ubiquitous protease and requires

TPC -treated trypsin treatment for its cleavage. This was kindly provided by Dr. David L. Suarez, 12 065582

USDA (Lee et al., Avian Pathol., 33(3): 288-297 (2004); Lee et al., Vaccine, 22(23-24): 3175-3181 (2004); Lee et al., J. Virol., 78(15): 8372-8381 (2004)).

The HIV/HA of this low pathogenic avian isolate is designated as HIV/HA(USDA) to differentiate it from the experimental HIV/HA. HIV/HA or HIV/HA (USDA) pseudotype viruses were either treated with trypsin (50 μg/ml) for 30 min at 37°C or no trypsin treatment prior to challenging the target 293T cells. Trypsin treatment did not enhance (or inhibit) HIV/HA mediated viral entry (Fig. 3). In contrast, infection of the trypsin-treated HIV/HA(USDA) was greatly enhanced compared to that with no trypsin treatment.

Example 4. HIV/HA pseudotype virus is sensitive to lysosomotropic compounds during entry.

To test the pH-dependence of HA-mediated viral entry, a predetermined titer of HIV/HA was used to infect 293 T cells in the presence of bafilomycin A and ammonium chloride (NH CI).

Bafilomycin A is a highly selective inhibitor of endosomal H+-ATPases which blocks the

acidification of endosomes, raising the endosomal pH (Bowman et al., Proc. Natl. Acad. Sci. U SA, 85(21): 7972-7976 (1988); Drose et al., Biochemistry, 32(15): 3902-3906 (1993); Marsh, M., and Helenius, A., Virus Res., 36: 107-151 (1989); Marsh, M., and Helenius, A. Cell. 124(4): 729- 740 (2006)). Likewise, H₄C1 is a weak base and raises the endosomal pH ((Bowman et al., Proc. Natl. Acad. Sci. USA, 85(21): 7972-7976 (1988); Drose et al., Biochemistry, 32(15): 3902-3906 (1993); Marsh, M., and Helenius, A., Adv. Virus Res., 36: 107-151 (1989); Marsh, M., and

Helenius, A. Cell. 124(4): 729-740 (2006)). As shown in Fig. 4, panel A, treatment of 293T cells with bafilomycin A efficiently inhibited the infectivity of HIV/HA pseudotype virus between 50- 150 nM. Similarly, exposure of cells to the NH₄C1 [5-25 mM] during the first 3 h of virus

adsorption significantly reduced the virus infection compared to an untreated control (Fig. 4, panel B). Therefore, the results indicate that HIV/HA entry is dependent on low-pH-induced alterations in HA.

Example 5. Human lung cells display maximum infectivity to HIV/HA pseudotype virus.

To characterize and compare the host tropism, infectivity of HIV/HA was assessed on a panel of target cell lines by plaque assay following previously described methods. Human lung cell lines (A549, NCI-H661 and HAPEC), and rat lung cell line (L2) were used as the target cells for infection. The Lecl (Chinese hamster ovary) cell line that is resistant to influenza virus infection was also used. Cells were infected with HIV/HA and HIV/HA(USDA) pseudotype viruses

following previously described protocols. HIV/VSVG and empty vector transfected pseudotype virus were used as positive and negative controls respectively. The human lung cell lines A549 and NCI-H661 were highly susceptible to infection by HIV/HA pseudotype virus as compared to the human lung cell line HAPEC (Fig. 5). HIV/HA (USDA) pseudotype virus, treated with trypsin, was also very infectious to these cells. However, untreated HI HA(USDA) pseudotype virus was not infectious (data not shown). The rat lung cell (L2) was not very susceptible to infection by both HIV/HA and HIWHA(USDA), while Lecl cells were highly resistant to both pseudotypes (Fig. 5). These results indicate that HIV/HA and HIV/HA(USDA) display a preferred entry tropism to human lung cells. The human lung cell lines, A549 and NCI-H661, were used for screening compound libraries for influenza entry inhibitors after further optimization.

These results suggest that HA in HIV/HA pseudotyped virus retains the functional properties of HA present in infectious influenza virus.

Example 6. Compound library screening to identify small molecule inhibitors of influenza virus

As outlined above, pseudotype viruses were produced by co-transfecting 12 μg of construct containing appropriate virus envelope glycoprotein with 12 μg pNL4-3-Luc-R-E- HIV vector into 293 T cells (90% confluent) in 10 cm plates with Lipofectamine 2000 (Invitrogen) according to the supplier's protocol. Cell culture grown influenza H1N1(PR8) viruses were propagated and titrated in MDCK cells over 3 days at 37°C in the presence of ^g/ml tosylsulfonyl phenylalanyl- chloromethylketone (TPCK)-treated trypsin (Sigma-Aldrich) following standard protocol (Lamb, R. A., and Krug, R. M., Orthomyxoviridae: the viruses and their replication., 4th edition Ed., Lippincott Williams and Wilkins(2001)). The chemical libraries screened represent broad and well-balanced collections of over 152,500 compounds. They were purchased from Chembridge (San Diego, CA) and Timtec (Newark, DE), diluted in 96-well master plates at 2.5 mM in dimethyl sulfoxide (DMSO), and stored at -20°C. Compounds were selected in the molecular weight range of 200-500 Da. They have favorable cLogP values (calculated logarithm of n-octanol/water partition coefficient), and encompass over 200 chemotypes.

High throughput screening of combinatorial chemical libraries using pseudotype virus was performed in 96-well plates. Low passage A549 cell monolayers were infected with ΙΟΟμΙ of pseudotype virus containing δμ^ιηΐ polybrene in the presence of 25μΜ (final concentration) test compounds. After 5 h, the inoculum was removed, fresh media was added and the plates were incubated for 72 h at 37°C and 5% C0₂. Infection was quantified using the Britelite PlusTM assay system (Perkin Elmer) in a Wallac EnVision 2102 Multilabel Reader (Perkin Elmer, MA). The percent inhibition was calculated as: 100 x [Relative Luciferase Unit (RLU) in the presence of compound - RLU of negative control / RLU of positive control (without any inhibitor) - RLU of negative control].

AlphaScreen SureFire GAPDH Assay Kit (Perkin Elmer) was used to test cell viability by measuring endogenous cellular GAPDH in cell lysates according to manufacturer's protocol. Results

Approximately 140,000 discrete compounds were screened, and 141 primary hits were identified. The Z' factor for the HTS was 0.5±0.2. Primary hits were counter-screened with pseudotype virus expressing an unrelated glycoprotein (VSV-G) and infectious H1N1 virus for their specificity. They were evaluated for their potency and cytotoxicity with resynthesized compounds. Only 36 of the primary hits specifically inhibited the HA mediated entry process. The final hit rate from the HTS was 0.09%. All the 36 hit compounds exhibited IC₉₀ values of <25μΜ. Structurally, the HA inhibitors can be represented as clusters of >2 members each and singletons. MBX2329 was selected for these primary hits.

The HA inhibitors identified herein included multiple clusters of chemically related structures, as well as singletons. Compound MBX2329, having an aminoalkyl phenol ether scaffold, demonstrated an IC₅o of 0.3 - 10 μΜ and, based on this result further structure activity relationship (SAR) studies with a group of aminoalkyl phenol ethers analogs were conducted. (See Table 1.). The activity of compound MBX2329 was further investigated against other laboratory adapted, pandemic, and drug-resistant influenza A strains. All three compounds were found to be active against the 2009 pandemic influenza A/H1N1 strain (A/California/10/2009; Dr. Mark Prichard's laboratory, UAB) (IC₅₀<0.6 μΜ) and oseltamivir-resistant H1N1

(A/Florida 21/2008/H1N1 H275Y) (Retrovirox, Inc., San Diego, CA)(IC₅₀= 0.3 - 5.8 μΜ)(Τ8Με 3). MBX2329 was also found to be active (IC₅₀ <1.2 μΜ) against an additional H1N1 strain

(A/Washington/ 10/2008), but completely inactive (IC₅₀ >100 μΜ) against an H3N2 influenza strain (A/Texas/ 12/2007) (Nguyen et al., Antimicrob. Agents Chemother., 54(9): 3671 -3677 (2010); Nguyen et al., PLoS One 5(2); e9332 (2010); Nguyen et ah, Antimicrob. Agents Chemother., 53(10): 41 15-4126 (2009)). Importantly, MBX2329 exhibited potent anti-influenza activity and selectivity in four independent laboratories. The anti-influenza strain specificity using an additional pseudotype virus exhibiting the influenza HA H7 subtype was also investigated. MBX2329, was inactive against the H7 pseudotype virus (IC₉₀ >100

1). The 16 subtypes of HA are divided into 2 groups. Both HI and H5 HAs belong to group 1 while H3 and H7 belong to group 2. Therefore, the preliminary data suggest that MBX2329 acts in a highly specific manner, inhibiting only influenza viruses with group 1 HA (HI and H5 subtype) and not influenza viruses with group 2 HA (H3 and H7 subtype).

In addition, as shown in Table 1 , the inhibitors did not prevent the infeclivity of VSV or Lassa pseudotype virus (HIV/LASV). Both LASV and VSV have type 1 membrane proteins similar to that of influenza virus and enter cells by a receptor-mediated endosomal pathway (Radoshitzky et al., Nature, 446 (7131): 92-96 (2007); Radoshitzky et al., Proc. Natl. Acad. Sci. USA, 105(7): 2664-2669 (2008)). Therefore, the results further suggest that MBX2329 does not modulate host factors important for virus replication. The two HA groups have similar architecture but differ from each other in having a group specific pocket at the interface of the HA monomers near the HA2 fusion domain. This region is near the conserved HA domain at the junction of HAl and HA2 that are recognized by the neutralizing antibodies. Since MBX2329 so far has been observed to only inhibit influenza viruses with group 1 HA, it may be hypothesized that they bind to this group specific pocket and inhibit conformational changes of HA to its fusogenic form.

Example 7. MBX2329 inhibits early during infection

HA has multiple functions at both early and late stages of virus infection. To probe the molecular mechanism underlying the antiviral activity of BX2329, a time of addition experiment was performed with HIV/HA(H5) (Fig. 1 1) to determine the stage of influenza virus entry that is blocked by the compounds. Compounds were added lh before adsorption (-lh), during adsorption (Oh) and lh, 2h, 3h, 5h and 24 h post adsorption at IC₉₀ concentrations. Controls consisted of DMSO (no inhibition) and bafilomycin, an endosomal pH modulator that inhibits HA processing in the endosome and inhibits viral entry. MBX2329 inhibited HIV/HA(H5) pseudotype virus infection (>75% inhibition) when added at -2h and Oh, suggesting that the compounds do not act as receptor antagonists. These preliminary findings are consistent with action by MBX2329 acting early during the infection process, possibly causing interference with the HA-mediated virus-cell membrane fusion process.

Example 8: Effect of MBX2329 on hemagglutination activity

Influenza A virus has the ability to adsorb onto chicken RBCs, resulting in

hemagglutination. Inhibition of agglutination of blood cells was used to investigate whether the inhibitors blocked viral attachment. For this assay, viruses were 2-fold serially diluted in a 96-well plate, and an equal volume of 0.5% chicken red blood cells (cRBCs) were added in the presence of the inhibitors (20 μΜ). The plates were kept at 4°C for 60 min., and agglutination was determined visually. The cRBCs sedimented and formed red buttons in negative control wells (no virus), whereas positive control wells (virus only) had an opaque appearance with no sedimentation at high virus concentrations due to hemagglutination (left side of plate, Fig. 12). As shown in Fig. 12, MBX2329 did not block HA hemagglutination activity, indicating that the compound does not inhibit viral attachment. Therefore, MBX2329 inhibits HA-mediated influenza virus entry apparently by blocking fusion, but not binding.

Example 9. Synergy Studies of MBX2329 with Oseltamivir and Amantadine U 2012/065582

MBX2329 inhibited the replication of HINl strains of the virus, but not H3N2 or B strains of the virus. MBX2329 was evaluated against HINl strains A/California/10/2009 and characterized its antiviral activity in combination with other known inhibitors of influenza virus infection. The efficacy of BX2329 and combinations of compounds were evaluated by a neutral red assay using known methods. This neutral red uptake assay was also used to assess cytotoxicity of the

compounds. Briefly, MDCK cells were seeded at a density of 8 ^χ 10⁴ cells/well in 96-well plates in Minimal Essential Medium with Earle's Balanced Salt Solutions (MEM/EBSS) and supplemented with 5% FBS and 50 μg/ml gentamicin. After 24 h incubation (37°C and 5% C0₂), the medium was removed and the wells washed once with 200 μΐ/well MEM/EBSS. For the testing of

individual test compounds, stocks of each compound were diluted in MEM/EBSS and added to the plates containing cell monolayers in a volume of 100 μΐ per well. For the combination assays, 100 μΐ of MEM/EBSS was added to each well and the dilutions of test compounds were performed using the BioMek 2000 liquid handling system. After the dilution matrix was completed, the cells were infected with influenza virus diluted in MEM/EBSS containing 50 μg/ml gentamicin, 2 μg/ml EDTA and 20 U/ml TPCK-Trypsin or wells received medium alone for the cytotoxicity

determination as well as for cell control readings. All plates were incubated for a period of 3 days, after which the viability of the cell monolayer was assessed by adding a Neutral Red solution during an additional 2 h incubation period. The stain was then aspirated, the monolayers washed once with PBS and an extraction buffer was added to each well for 30 minutes. The optical density values were obtained by reading the plates at 540nm on a BioTek plate reader. Experimental data were interpolated to calculate compound concentrations that reduced viral replication by 50% (EC₅₀ values) or cell viability by 50% (CC₅₀ values). Synergy data were evaluated by standard methods.

MBX2329 in combination with oseltamivir resulted in marked synergistic inhibition of influenza virus infection (Table 2). The large volumes of synergy produced by the combination were both reproducible and were statistically significant as indicated by the values at the 95% confidence level. The synergy calculated at each combination of compound concentrations was also plotted to show concentrations where the synergy was observed (Fig. 13). The plots also show that the synergy was observed over a wide range of concentrations.

Table 1 : Selectivity of representative hits against different influenza virus subtypes'

Table 2. Combined efficacy of MBX2329 with oseltamivir or amantadine against A/California/ 10/2009

Values represent the average volume of synergy for two independent experiments with the standard deviation volumes shown.

⁰ Volumes shown represent a minimal estimate of synergy at the 95% confidence level.

12 065582

Table 3- Pseudotype activity data and cytotoxicity for selected aminoaikyl phenol

ethers

- Pseudotype activity data and cytotoxicity for selected aminoalkyi phenol ethers

60 52.1

86966

61.2

The contents of all cited publications are hereby expressly incorporated by reference in their entirety. The practice of the present invention will employ conventional techniques of immunology, molecular biology and cell biology which are well known in the art.

Additional embodiments of the invention may be produced by following the examples and descriptions above. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. The scope of the invention is indicated by the appended claims.

Claims

We Claim:

An influenza virus inhibitor compound of Formula I

R¹

I

Cv . N .

Formula I wherein:

Ar is an aryl or heteroaryl group,

L is a linker which may be a direct bond or a divalent linear or branched alkyl, alkenyl, or alkynyl chain containing from 1 -4 heteroatoms and may be optionally substituted by one or more groups selected from halo, hydroxy, alkoxy, alkoxycarbonyl, and alkylthio groups; and,

R¹ and R² are independently a monovalent alkyl, alkenyl, or alkynyl group that may be unsubstituted or substituted with one or more substituents selected from halo, amino, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, acyl, carboxy, alkoxycarbonyl, amino, alkylamino, acylamino, amido, sulfonamido, mercapto, alkylthio, arylthio, thioacyl, alkylsulfonyl, or aminosulfonyl, or R¹ and R² together may be covalently linked to form a cyclic structure of 5-8 atoms, including the nitrogen atom to which they are attached;

or pharmaceutically acceptable salts thereof.

2. The compound according to Claim 1 selected from the group consisting of:

HCI

66

68

69

70

71

72

3. The compound according to Claim 1 selected from the group consisting of:

HCI

and

HCI

4. The compound according to Claim 1, wherein said influenza virus is Influenza A.

5. The compound according to Claim 4, wherein said Influenza A is selected from avian and swine influenza.

6. A pharmaceutical composition comprising one or more influenza inhibitor compounds according to any one of Claims 1-3 and a pharmaceutically acceptable carrier or excipient.

7. A method of treating an individual for influenza infection comprising administering a compound according to any one of Claims 1-3.

8. A method of treating an individual for influenza infection comprising administering a pharmaceutical composition according to Claims 6.

9. A method for treating an individual infected with or exposed to influenza virus comprising administering to said individual an effective amount to inhibit entry of the virus into host cells of a compound according to any one of Claims 1-3 or a pharmaceutical composition according to Claim 6.

10. The method according to Claim 7, 8, or 9, wherein said individual is human.

1 1. The method according to Claim 10, wherein said virus is Influenza A.

12. The method according to Claim 1 1, wherein said Influenza A virus is of avian or swine origin.

13. The method according to Claim 10, further comprising administering an additional active ingredient selected from the group consisting of an antibiotic, an antibody, an antiviral agent, an analgesic, an immunostimulatory agent, a natural, synthetic or semisynthetic hormone, a central nervous system stimulant, an antiemetic agent, an anti-histamine, an erythropoietin, a complement stimulating agent, a sedative, a muscle relaxant agent, an anesthetic agent, an anticonvulsive agent, an antidepressant, an antipsychotic agent, and combinations thereof.

14. Use of a compound according to any one of Claims 1-3, or a composition according to Claim 6, for the treatment of influenza viral infection.

15. The use according to Claim 14, wherein said influenza is Influenza A.

16. Use of a compound according to any one of Claims 1 -3 for the manufacture of a medicament for treating influenza viral infection.