WO2012115945A1 - Viral inhibitors - Google Patents

Abstract

The present invention generally relates to compounds to treat viral infections and methods of their use. In particular, compounds of the present invention inhibit the action of NSl protein, thereby mitigating viral infection and, in particular, influenza virus infection. Accordingly, NSl inhibitors and methods of treatment that employ such inhibitors are contemplated by the present invention.

Description

DESCRIPTION VIRAL INHIBITORS

1. Priority Claim

This application claims benefit of priority to U.S. Provisional Application Serial No. 61/444,962, filed February 21, 2011, the entire contents of which are hereby incorporated by reference.

2. Statement of Government Support

This invention was made with government support under grant number NIH R01 GM06715908 Al and AI079110 awarded by the National Institutes of Health (NIH) The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention generally relates to the fields of virology, molecular biology and medicine. More particularly, it concerns the discovery of compounds that inhibit pathways targeted by influenza virus NS 1 protein. 2. Description of Related Art

Influenza viruses cause approximately 36,000 deaths annually in the United States (Simonsen et al., 1997) and -500,000 deaths worldwide per year (Smith et al., 2004). Strains that are extremely pathogenic have been responsible for high numbers of deaths worldwide, such as the 1918 pandemic which led to -30 million deaths around the world (Webster, 1999). Currently, there are only two basic therapeutic approaches available for treating pandemic influenza: vaccination and inhibitors of virus infection or replication. Vaccination, although highly effective against certain strains, is limited by the highly mutable nature of the virus and must be reconstituted annually to address the changing viral ecology. A number of drugs have been developed that inhibit various steps in viral infection and replication, but they have demonstrated only limited efficacy. Thus, the availability of additional therapeutic modalities for the treatment of influenza viral diseases is presently unsatisfactory. Other treatments and routes of influenza virus mitigation are therefore needed. SUMMARY OF THE INVENTION

The present invention generally provides compounds and their use as antiviral agents. More particularly, the inventors have identified small organopharmaceuticals that inhibit the pathway influenced by NSl protein of influenza A virus, a major virulence factor. NSl protein also inhibits interferon (IFN) gene induction and IFN-modulated immune responses. As such, the compounds described herein are also novel inhibitors of viral replication and pathogenesis that act by preventing NSl protein-mediated inhibition of IFN-dependent immune responses to viral infection.

Thus, in accordance with the present invention, there are provided compounds of the formula:

wherein:

n is 2, 3, 4, 5, 6, 7 or 8;

A is -CHRi- -0-, -NRi- or -S-; and

Ri is:

hydrogen, hydroxy or amino; or

alkyl₍c<i2), alkenyl_(C<i₂), alkynyl_(C<i2), aryl_(C≤12), aralkyl_(C<i₂), heteroaryl_(C<i₂), heteroaralkyl₍c<i2), acyl_(C<₁₂), alkoxy_(C<i2), aryloxy_(C<i2), aralkoxy_(C<i2), heteroaryloxy(c<i2), heteroaralkoxy(c≤i₂), acyloxy(c≤i₂), alkylaminO(c<i2), dialkylaminO(c≤i₂), arylaminO(c<i₂), aralkylaminO(c≤i₂), heteroarylamino(c≤i2), heteroaralkylamino(c≤i₂), amido(c≤i₂), or a substituted version of any of these groups;

or a pharmaceutically acceptable salt or tautomer thereof. In particular, n may be 5. In particular, A may be -CHRi- and Ri may be hydrogen. In particular, A may be -0-. In particular, A may be -NRi^~ and Ri may be hydrogen. In particular, A may be -NRi^~ and Ri may be heteroaryl₍c<8), optionally where i is pyrimidyl or methylphenyl. In particular, A may be -NRi^~ and Ri may be aryl₍c<₈₎. In particular, A may be -NRi^~ and Ri may be heteroatom-substituted aryl₍c<₈₎, optionally where Ri may be methoxyphenyl or trifluoromethylphenyl. In particular, A may be -S-. Specifically, the compound may be selected from the group consisting of:

Accordingly, certain methods of the present invention contemplate a method of treating or preventing a viral infection in a patient comprising administering to said patient an effective amount of a compound as shown above. Certain methods of the present invention are drawn to solely treatment of viral infections comprising administering to said patient an effective amount of the compound. The patient may be a mammal, such as a mouse, rabbit, or human.

In any method of the present invention that contemplates a viral infection, the viral infection may be influenza. The viral infection may be caused by, for example, influenza A virus. Other viruses are also contemplated. For example, the structure of NS1 protein of influenza B virus resembles NS1 protein of the influenza A virus; accordingly, influenza B virus may be associated with a viral infection of the present invention. Members of other virus families have been shown to be sensitive to interferon and to inhibit the production of interferon, and may do this through mechanisms similar to influenza A virus involving the same molecular pathways. Thus, compounds that inhibit the influenza A virus NS1- influenced pathway may also block the inhibition of interferon by these other viruses. In this regard, therefore, certain embodiments of the present invention contemplate a viral infection caused by, e.g., a bunyavirus (such as LaCross virus), an arenavirus, or an encephalitis virus {e.g., West Nile virus). Other viruses contemplated by the present invention include rabies or a filovirus {e.g., Ebola virus and Marburg virus). Any one or more of these compounds may optionally be excluded from any generic compound discussed herein, or may optionally be excluded from the class of viral inhibitors.

Compounds of the present invention may be administered to a cell, tissue, organism, or patient in any manner known to those of skill in the art. For example, in certain embodiments, an inhibitor may be administered to a patient via a method selected from the group consisting of an inhaled aerosol, a nasal spray, an oral formulation and an injection. The dosage of an inhibitor may also be administered to a cell, tissue, organism, or patient in any manner known to those of skill in the art. In certain embodiments, the dosage ranges from about 1 mg/kg to about 50 mg/kg, or any range derivable therein. In certain embodiments, the dosage ranges from about 10 to about 40 mg/kg. In certain embodiments, the dosage ranges from about 5 to about 45 mg/kg.

Other methods of the present invention contemplate a method of inhibiting NS1 activity comprising administering to a cell an effective amount of an inhibitor as described herein. The cell may be in vitro or in vivo.

Yet another method of the present invention contemplates a method of inhibiting influenza A virus cytopathic effect in a cell comprising administering to said cell an effective amount of an inhibitor as described herein.

In certain embodiments, a method of reducing the severity or duration of a viral infection in a patient comprising administering to said patient an effective amount of an inhibitor as describe herein is provided. For example, such a method may reduce the severity or duration of viral infection symptoms. The viral infection may be influenza, such as influenza A virus, or any other virus discussed herein. In the case of influenza viruses, such methods may comprise a method of reducing the severity or duration of influenza virus symptoms, such as headache, fever, sore throat, muscle pain, weakness, cough, and/or overall discomfort.

A method of treating or preventing a viral infection in a patient comprising administering to said patient an effective amount of an inhibitor as described herein in combination with another agent is another method contemplated by the present invention. In particular embodiments, only methods of treatment are contemplated. The second agent may be, for example, a neuraminidase inhibitor, such as Relenza™ or Tamiflu™, or an M2 proton channel inhibitor, such as amantadine or rimantadine. Methods employing these types of compounds will typically be employed to treat influenza virus infection. Another method of the present invention contemplates a method of selecting for a compound that inhibits NS l activity comprising:

a) infecting a cell with plasmids expressing luciferase and NS 1 protein, b) contacting the cell with a target compound, and

c) quantifying the luciferase signal;

wherein a decrease in the luciferase signal relative to the signal obtained in the absence of target compound indicates that the target compound is an NS 1 activity inhibitor.

Also encompassed by the present invention are pharmaceutical compositions. Any inhibitor described herein is contemplated as comprised in a pharmaceutical composition. For example, the present invention contemplates pharmaceutical compositions comprising a pharmaceutically acceptable carrier, diluent, and/or excipient and any one or more of the following:

As used herein, an "NS l inhibitor" is an organopharmaceutical (that is, a small organic molecule) that inhibits NSl protein activity but does not affect NS l protein gene expression. In particular, compounds that affect the signaling pathway influenced by NS l are contemplated. Inhibitors typically have a molecular weight of about 500 g/mol or less.

The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification, includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 99%), or more, or any range derivable therein, reduction of activity compared to normal. In a further example, following administering of a NS l protein inhibitor, a patient may experience a reduction in severity or duration of one or more viral infection symptoms, such as influenza symptoms as described herein.

The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a compound of the present invention is administered or delivered to a target cell or are placed in direct juxtaposition with the target cell. The terms "administered" and "delivered" are used interchangeably with "contacted" and "exposed."

As used herein, the term "effective" (e.g., "an effective amount") means adequate to accomplish a desired, expected, or intended result. For example, an "effective amount" may be an amount of a compound sufficient to produce a therapeutic benefit (e.g., effective to reproducibly inhibit decrease, reduce, or otherwise reduce the severity of a viral infection).

"Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a subject or patient (e.g., a mammal, such as a human) having a viral infection may be subjected to a treatment comprising administration of a compound of the present invention.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the onset, frequency, duration, or severity of the signs or symptoms of a disease. For example, a therapeutically effective amount of a compound of the present invention (e.g. , an NS 1 protein inhibitor) may be an amount sufficient to treat or prevent a viral infection.

The term "substantially" and its variations are defined as being largely but not necessarily wholly what is specified as understood by one of ordinary skill in the art, and in one non-limiting embodiment 'substantially' refers to ranges within about 10%, within about 5%, within about 1%, or within about 0.5%. An NS1 protein inhibitor may, for example, be administered to a subject, e.g., a human, suffering from a viral infection until the viral infection has substantially disappeared.

It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.

As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

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

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-C. Identification of small molecules that revert the inhibition of gene expression mediated by the influenza virus NS 1 protein and protect cells from virus-induced cell death. (FIG. 1A) Luciferase expression in HEK 293T cells transfected with NS1 and treated individually with 200,000 synthetic compounds (5 μΜ) was normalized to values for on-plate controls treated with 0.3% DMSO. Values are expressed as Z scores using the mean value and s.d. of the experimental population screened on the same day. Red circle shows compound 1 studied here. (FIG. IB) Inhibition of influenza virus-mediated cell death. The 640 most active compounds were tested at three concentrations for the ability to inhibit the cytopathic effect of A/WSN/1933 influenza virus infection in HBECs. Z scores for compounds assayed at 1.7 μΜ are plotted according to activity. (FIG. 1C) The structure of the most active naphthalimide from the primary screen (1), an inactive analog (2) and a more potent related compound (3) are shown.

FIGS. 2A-E. Compound 3 is less cytotoxic, more stable than 1 and reverts influenza virus-mediated cytotoxicity and mRNA export block. (FIG. 2A) MDCK cells were treated for 30 h with compounds 1, 2 and 3 at the various concentrations depicted, and control cells were treated with the same concentration of DMSO as those in the wells containing compound. Cell viability was determined by measuring cell ATP concentrations. RLU, relative light units. (FIG. 2B) The fraction of compound remaining in cells treated with 1 or 3 as a function of incubation time was determined by mass spectrometry. LN, natural log. (FIG. 2C) MDCK cells were pretreated for 17 h with DMSO or with the indicated concentrations of 3 and subsequently mock infected or infected with A/WSN/1933 virus at m.o.i. 0.001 for 48 h. The indicated concentrations of compound were present during infection. Differential interference contrast (DIC) microscopy imaging was performed in a Zeiss Axiovert 200M. Cell survival was determined by counting live cells. Scale bar, 90 μιη. (FIG. 2D) MDCK cells, mock infected or infected with A/WSN/1933 in the presence or absence of 25 μΜ 3, were fixed and subjected to oligo-dT in situ hybridization to detect poly(A) RNA distribution in the nucleus and cytoplasm. Influenza proteins were detected by immunofluorescence using antibodies specific for influenza proteins. Yellow arrowheads point to cells with mR A export block, whereas white arrowheads point to cells that do not show blockage. Scale bar, 15 μηι. (FIG. 2E) Data from triplicate experiments as depicted in d were quantified and the percentage of infected cells retaining mRNA in the nucleus is shown. Data represent mean values ^s.d.

FIGS. 3. Compound 3 inhibits virus replication but does not induce I FN response.

(FIGS. 3A-C) MDCK cells mock infected or infected at m.o.i. 0.001 with the influenza virus strains shown were left untreated or treated with compounds at the depicted concentrations, and the virus titers of culture supernatants were determined by plaque assay. Strain A/WSN/1933 is in FIG. 3 A, A/Texas/36/91 is in FIG. 3B and A/Brevig/Mission 1/1918 is in c. (FIG. 3D) Intracellular viral protein concentrations were measured by immunoblot analysis with specific antibodies to the indicated proteins. (FIG. 3E) Human A549 cells treated with DMSO or 25 μΜ 3 were mock infected or infected with A/WSN/1933 at m.o.i. 0.001, and after 36 h, RNA was isolated and the expression of the IFN-responsive genes shown was quantified by real-time PCR. (FIG. 3F) MDCK cells mock infected or infected with VSV- GFP (m.o.i. = 0.001) were untreated or treated with the indicated compounds. At 24 h after infection, virus titers were determined in the supernatants. Data represent mean values + s.d.

FIGS. 4. Influenza virus activates the mTORC 1 pathway and naphthalimide requires the mTORCl inhibitor REDD1 for its antiviral activity. (FIG. 4A) A549 cells were untreated or treated with 30 μΜ 3 for the indicated time periods, in the absence or presence of actinomycin D (0.5 μg/ml). REDD1 mRNA levels were quantified by real time PCR. (FIG. 4B) A549 cells were untreated or treated with 30 μΜ 3 (in the absence or presence of 0.5 μg/ml actinomycin D as indicated) for 18 h before infection and during infection. Cell extracts were obtained at 6 h after infection and subjected to immunoblot analysis with the indicated antibodies. Densitometry analysis was performed to determine the ratio of REDD 1 over loading control (Mito-70 kDa) using ImageJ (FIGS. 24A-B). (FIG. 4C) A549 cells were untreated or treated as in FIG. 4B before and during infection. Cell extracts were subjected to immunoblot analysis with depicted antibodies (FIG. 25). (FIG. 4D) Phosphorylation of Akt or S6K was measured by immunoblot analysis in cell extracts of A549 cells infected with influenza virus in the presence or absence of 3. Compound was added prior to and during infection as in FIG. 4B (FIG. 26). (FIGS. 4E-F) REDD1+/+ cells were untreated or treated with 3 and mock-infected or infected at m.o.i 0.01 with A/WSN/1933 for 72 h (e). REDD1- /- MEF cells, untreated or treated with 3, were infected with A/WSN/1933 at m.o.i. 0.001 for 48 h (FIG. 4F). Cell survival was determined by Trypan blue exclusion assay and virus titers were measured by plaque assays. Data represent mean values ± s.d. FIGS. 5. Viruses activate the mTORCl pathway via down-regulation of REDDl expression. Extracts from cells mock infected or infected with influenza virus (FIG. 5A) or VSV-GFP (FIG. 5B) were subjected to immunoblot analysis with depicted antibodies. Densitometry analysis was performed to determine the ratio of REDDl overloading control (Mito-70 kDa) using ImageJ. (FIG. 5C) Wild-type or REDD IV- MEF cells were infected with VSV-GFP at m.o.i. of 0.001 for 24 h. DIC or fluorescent images of VSV-GFP are shown. Scale bar, 50 μιη. (FIG. 5D) Supernatants of cells from FIG. 5C were subjected to plaque assays. Data represent mean values ± s.d.

FIGS. 6A-F. REDDl regulates viral protein expression in an mTORCl -dependent manner. (FIG. 6 A) REDD1+/+ and REDDlV-cells were infected with influenza virus WSN at m.o.i. 2 for 1 h at 22 °C and then shifted to 37 °C. Viral protein levels were monitored over time by immunoblot analysis with the depicted antibodies. (FIG. 6B) Viral protein levels were monitored as in FIG. 6A. (FIG. 6C) WSN-infected REDD1+/+ and REDD IV- cells were treated with 100 nM rapamycin. Rapamycin was added 1 h after infection. NSl levels were monitored over time by immunoblot analysis. (FIG. 6D) U20S cells, untreated or treated with tetracycline to induce REDDl overexpression, were infected as in FIG. 6A but with both influenza virus and VSV. NSl or VSV-M protein levels were monitored by immunoblot analysis. (FIGS. 6E-F) TSC2+/+ and TSC2V- cells were pretreated with 10 μΜ 3. Cells were then infected with influenza virus WSN at m.o.i. 2 for 1 h at 22 °C and then shifted to 37 °C in the absence of compound. After 1 h of infection, 3 was added back. Cell extracts were obtained at 5 h and 6 h after infection in FIG. 6E and 8 h and 9 h after infection in FIG. 6F, then subjected to immunoblot analysis with the indicated antibodies.

FIG. 7. Compound 3 is a more potent inhibitor of influenza virus replication than other 1 analogs. To first measure cell toxicity, MDCK cells were treated for 24 h with 20 μΜ of each compound and ATP levels were measured. Values are normalized to controls treated with DMSO and represent triplicate values that had standard errors less than 10%. From a separate experiment, supernatants of cells infected with A/WSN/1933 influenza virus (m.o.i. 0.001) and treated for 24 h with 20 μΜ of each compound were subjected to hemagglutination (HA) assays (data not shown) and plaque assays (shown in the table), and values were normalized to that of control cultures treated with DMSO. Plaque assays were not performed for compounds that exhibited no differences in HA assays (nd). Compound 8 was not tested for virus inhibition due to its rapid cytotoxicity, nd, not determined. Except for 3, which was synthesized as described above, all the other compounds in this table can be obtained from ChemBridge, ChemDiv, ComGenex, TimTek, and their purities were equal or above 90%.

FIG. 8. Bulk Protein Synthesis is not inhibited by 3. Protein synthesis was measured

35

by pulse labeling MDCK cells with S-methionine, untreated or treated with 50 μΜ of 3.

35

Samples were collected at the indicated time points and S-methionine labeled proteins were measured.

FIG. 9. Influenza virus replication is inhibited by 3 in human carcinomic alveolar basal epithelial cells. A549 cells were untreated or pre-treated with 30 μΜ of 3 overnight. Cells were then infected with A/Texas/36/1991 at m.o.i. 0.001 for 1 h at 22 °C, in the absence of compound. Cells were shifted to 37 °C and 3 was added back to cells that were pre-treated with 3. Virus titers of culture supematants harvested at 48 h post-infection were determined by plaque assay.

FIGS. 10A-B. Interferon Response is not Required for Naphthalimide Antiviral Activity. (FIG. 10A) Vero cells were untreated or treated with 3 (50 μΜ) and ATP levels were measured. No cytotoxicity was observed at this concentration. Vero cells were then infected with A/WSN/1933 at an m.o.i. 0.01 or 0.001 for the indicated time points. Cells were treated with DMSO or 3 (50 μΜ) during infection. Supematants were subjected to hemaglutinin assays (HA) to measure viral titers. HAU, hemagglutination unit. Since Vero cells are interferon-deficient cells and were protected from vims replication by 3, this compound does not act via interferon. (FIG. 10B) STAT1-/- cells were untreated or treated with 3 (40 μΜ) and ATP levels were measured. No cytotoxicity was observed at this concentration. STAT1 -/- cells were then infected with A/WSN/1933 at an m.o.i. 0.01 for 72 h. Cells were untreated or treated with 3 (40 μΜ) in the absence or presence of vims and cell survival was determined by measuring ATP levels. STAT1-/- cells were significantly protected from viral-mediated cell death in the presence of 3.

FIG. 11. Influenza Vims Protein Levels are Down-Regulated by 3 in Cells with Impaired Interferon Response. Vero cells were pre-treated with 3 (50 μΜ) for 2 h and then infected with A/WSN/1933, at m.o.i. 1, for 1 h in the absence of compound. As shown in FIGS. 10A-B, 3 is not cytotoxic at the concentration used here. One hour post-infection, 3 was added back and incubated for various time periods as depicted in the figure. Cell extracts were subjected to immunoblot analysis with antibodies against influenza vims proteins or with an antibody against a mitochondrial protein, used as loading control. FIG. 12. The niTOR Pathway Is Regulated by 3. A549 cells were treated with DMSO or with 30 μΜ 3 for 3 h. RNA was isolated and processed for microarray analysis. The results of triplicate experiments were subjected to Gene Set Enrichment Analysis as described in Methods. Response networks after enrichment analysis of cells treated with 3 versus DMSO alone are shown. Node colors refer to -fold changes - white denoting no change to dark blue indication down-regulation of a three-fold or more, and red depicts up-regulation. Oval shapes refer to enriched genes in the particular gene set, rectangles denote other genes that have been identified to function in the response network by NetworkExpress. Edge colors indicate edge scores after NetworkExpress analysis using average fold changes between connected nodes. Yellow edges indicate high edge score. Edge arrow shapes denote different types of interactions with arrows indicating metabolic reactions, circles identifying phosphorylation, and no arrow shape refers to protein-protein interactions. The diagram displays the MTORPATHWAY response network with 40 nodes and 92 edges calculated with parameters k= 3 and 1= 5.

FIG. 13. Rapamycin treatment reduced the levels of influenza virus NS1 protein.

A549 cells were treated with 100 nM rapamycin for 18 h and then infected with influenza virus A/WSN/1933 for 6 h in the presence of rapamycin. Cell extracts were obtained and immunoblot analysis was performed with anti-NSl antibodies.

FIGS. 14A-B. Compound 3 inhibits the mTORCl pathway independent of virus infection. (FIG. 14A) Human lung cancer H358 cells have chronically activated S6K signaling indicated by S6K p-Thr389, which is inhibited by 3 in the absence of virus, but not by 2. (FIG. 14B) In two additional cancer cell lines with chronically activated S6K, 3 inhibited phosphorylation of S6K on Thr389, but did not inhibit Akt phosphorylation.

FIG. 15. REDDl-/- cells are permissive to influenza virus infection. REDD1+/+ and REDDl-/- cells were infected with A/WSN/1933 at m.o.i. 0.001 for 48 h. Supematants of infected cells were subjected to plaque assays to determine viral titers.

FIG. 16. Cell Survival of REDD1+/+ and REDDl-/- Cells Treated with 3. REDD1+/+ cells were treated with 10 μΜ 3 for 72 h and ATP levels were measured.

FIG. 17. REDDl-/- Cells Express Higher Levels of VSV Proteins than REDD1+/+ Cells. REDD1+/+ and REDDl-/- cells were infected with VSV at m.o.i. 2 for 5 h, 6 h, or 7 h. Cell extracts were subjected to immunoblot analysis with antibodies against VSV proteins.

FIGS. 18A-C. REDDl is Required for Naphthalimide Antiviral Activity Independent of Autophagy. (FIG. 18 A) REDD1+/+ or (FIG. 18B) REDDl-/- MEFs were untreated or pre- treated for 2 h with 3 (10 μΜ) and then infected with VSV at m.o.i. 0.001 for 1 h in the absence of compound. Then, 3 was added back and infection proceeded for 24 h. Supernatants of infected cells were collected and subjected to plaque assays. (FIG. 18C) ATG5-/- cells were infected and processed as in FIG. 18A.

FIG. 19. REDD1-/- cells express the same amount of influenza viral proteins in the absence or presence of autophagy inhibitor. REDD1-/- cells were infected with influenza virus over time in the absence or presence of 50 μΜ chloroquine, which was added 6 h postinfection. Cells lysates were subjected to immunoblot analysis with the depicted antibodies. As positive control, LC3-II levels were monitored and were enhanced in the presence of chloroquine treatment, γ-tubulin was used as loading control.

FIG. 20. REDD1-/- Cells Express Higher Levels of vRNAs than REDD1+/+ Cells.

REDD1+/+ and REDD1-/- cells were infected with WSN at m.o.i. 2 for 1 h at 22 °C and then shifted to 37 °C. Viral RNA (vRNA) levels were monitored by quantitative real time PCR at 3 h and 6 h. Briefly, total RNA was isolated at 3 and 6 h post-infection using Rneasy Mini Kit (Qiagen, Valencia, CA), following manufacture's protocol. RT was carried out using First Strand cDNA Synthesis using the Superscript II RT kit (Invitrogen) and specific primers. Real time PCR was performed using gene-specific primers and normalized to HPRT1 and RPS11.

FIG. 21. Rapamvcin Down-Regulates PB1 Levels. WSN-infected REDD1-/- cells were treated with 100 nM Rapamycin for 6 h. Rapamycin was added one 1 h post-infection.

FIG. 22. Activation of AKT and S6K is not down-regulated by naphthalimide in

WSN-infected REDD1-/- cells. REDD1-/- cells were untreated or treated with 3 and mock infected or infected with influenza virus at m.o.i. 2. Cell extracts were obtained at 8 and 9 h post-infection and subjected to immunoblot analysis with the depicted antibodies.

FIGS. 23A-C. Analog of the naphthalimide 3 more effectively inhibits the highly pathogenic H1N1/1918 influenza virus. (FIG. 23 A) MDCK cells were pre-treated for 17 h with DMSO or with 25 μΜ 3 or 4 and subsequently mock infected or infected with A/WSN/1933 at m.o.i. 0.001 for 48 h. Compounds were present during infection. DIC imaging was performed. (FIG. 23B) Cells were untreated or treated with 25 μΜ of 4 and mock infected or infected with A/WSN/1933 at m.o.i. 1 for 6 h. Cell extracts were subjected to immunoblot analysis with the depicted antibodies. (FIG. 23C) H1N1/1918 virus replication in MDCK cells, in the absence or presence of 3 or 4, was measured by plaque assays.

FIGS. 24A-B. REDD1 is induced by compound 3. (FIG. 24A) Renal carcinoma cells were untreated or treated with 20 μΜ 3 for 6 h. Cell extracts were analyzed by immunoblot analysis with anti-REDDl or anti-Mito-70 kD antibodies. (FIG. 24B) A549 cells were untreated or treated with 30 μΜ 3 (in the absence or presence of 0.5 μ /ηι1 actinomycin D as indicated) for 18 h prior to infection and during infection. Cell extracts were obtained at 6 h post-infection and subjected to immunoblot analysis with anti-REDDl or anti-Mito-70 kD antibodies. * cross-reacting bands and Mito-70 kD serve as loading controls. The REDD1 bands are broad in FIG. 24A because this is a 15-well gel as opposed to FIG. 24B, which is a 10-well gel. In addition, the broadness of REDD 1 bands vary between cells types.

FIG. 25. Influenza virus activated the mTORCl pathway. A549 cells were untreated or treated with 30 μΜ 3 for 18 h prior to infection and during infection. Cell extracts were obtained at 4 h and 7 h post-infection and subjected to immunoblot analysis with the depicted antibodies.

FIG. 26. Compound 3 blocks S6 activation independent of Akt. Compound 3 inhibited phosphorylation of S6K in cells infected with influenza virus prior to a decrease in the levels of phosphorylated AKT. Phosphorylation of Akt or S6K was measured by immunoblot analysis in cell extracts of A549 cells infected with influenza virus in the presence or absence of 3. Compound was added prior and during infection.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Influenza virus nonstructural protein NSl is a major virulence factor for viral pathogenesis that alters multiple host functions. During infection, NSl localizes both in the nucleus and cytoplasm (Li et al., 1998) . The cytoplasmic pool of NSl inhibits interferon (IFN) gene induction by interfering with the cytoplasmic signal transduction pathway mediated by RIG-I (Guo et al., 2007; Mibayashi et al., 2007; Opitz et al., 2007; Pichlmair et al., 2006). NSl also prevents IFN action by sequestering double-stranded RNA and/or targeting the function of downstream antiviral effector proteins, such as PKR and the RNase L pathways (Li et al., 2006; Min and Krug, 2006). In addition, NSl has been shown to activate phosphatidylinositol 3-kinase signaling (PI3K), which is important for promoting viral replication (Hale et al., 2006). A key nuclear function of NSl is to selectively inhibit host mRNA processing and export, thus blocking expression of host antiviral genes but not export of viral RNAs (Nemeroff et al., 1998; Satterly et al., 2007). Genetic studies have shown that disruption of NS 1 functions by mutation results in highly attenuated viruses that can only replicate in immunocompromised hosts (Garcia-Sastre et al., 1998). The present inventors developed an assay for NSl protein function and screened a library of organopharmaceuticals to identify compounds that blocked NSl protein function. Active compounds from the first assay were screened in a second assay for virus growth and those that inhibited virus growth were selected for further study. Since NSl protein is highly conserved across major influenza subtypes and is not a major antigen for generating anti- influenza response, there is no positive selective pressure for natural variants of NSl protein. This suggests that drugs that target NS 1 protein will be broadly effective against influenza subtypes. Indeed, by attenuating the virual infection, the NSl protein inhibitors described herein may prevent disease while simultaneously allowing an immune response to the infecting strain, thereby giving lasting protection to that particular strain of virus.

A. Influenza

Influenza viruses have been a major cause of mortality and morbidity in man throughout recorded history. Epidemics occur at regular intervals which vary widely in severity but which always cause significant mortality and morbidity, most frequently in the elderly population. The cause of influenza epidemics was first attributed to a virus by R. E. Shops, who showed that influenza epidemics could be transmitted with filtered mucus. Influenza viruses are currently divided into three types: A, B, and C, based upon differences in internal antigenic proteins. Only influenza A viruses are further classified by subtype on the basis of the two main surface glycoproteins hemagglutinin and neuraminidase. Influenza A viruses can infect birds and mammals and a reservoir of virus is maintained in non-human species that cannot be eliminated. It is by crossing species into the human population that new influenza A virus subtypes cause human pandemics. Influenza A subtypes and B viruses are further classified by strains.

New strains of influenza caused by antigenic drift appear at regular frequency, usually annually, and begin a cycle of infection which typically travels around the globe. Approximately every year, at least one minor change occurs in either the hemagglutinin or neuraminidase antigens (or both), but that change is sufficient to render those persons who had a previous strain susceptible to the new strain. As influenza is caused by a variety of species and strains of viruses, in any given year some strains can die out while others create epidemics while yet another strain can cause a pandemic. Little is known about how individual epidemics are initiated. Non-limiting exemplary strains include A/Wisconsin/67/2005 (H3N2)-like virus (A/Wisconsin/67/2005 or A/Hiroshima/52/2005 strains), A/New Caledonia/20/99 (H1N1), B/Malaysia/2506/2004-like virus (B/Malaysia/2506/2004 or B/Ohio/1/2005 strains), and A/Solomon Islands/3/2006 (H1N1)- like virus.

An influenza infection produces an acute set of symptoms including headache, fever, sore throat, muscle pain, weakness, cough, and/or overall discomfort. In severe cases or situations involving pre-existing pulmonary or cardiovascular disease, hospitalization is required. Pneumonia due to direct viral infection or due to secondary bacterial or viral invasion is the most frequent complication. For a review on the clinical aspects of influenza virus infection (Douglas, 1990).

B. Influenza A Virus

The genome of the influenza A virus consists of 8 negative single-strand R A segments that encode 10 genes necessary for viral replication and virulence (Knipe and Howley, 2001). Influenza viruses are unusual among negative strand RNA viruses in that they replicate in the nucleus of the cell unlike others that display a predominantly or exclusively cytoplasmic life cycle. This feature has led to the evolution of additional complexities in the influenza virus life cycle and in its interaction with its host's cellular machinery. These complexities present potential vulnerabilities that might be exploited for therapeutic benefit.

Similar to most viral infections, replication of influenza virus in vertebrate cells is recognized by elements of the innate immune system, triggering a signal transduction pathway leading to type I IFN production and response. If fully functional, the type I IFN pathway would produce a potent antiviral state through induction of a large battery of antiviral effector proteins that preclude further viral replication. However, like many evolutionarily successful viruses, influenza virus has evolved mechanisms for inhibiting this innate response (Guo et al., 2006; Levy and Garcia-Sastre, 2001; Li et al., 2006; Mibayashi et al., 2007; Min and Krug, 2006; Opitz et al., 2007; Pichlmair et al., 2006), mainly through functions of the NS1 protein, described below (Garcia-Sastre et al., 1998). Negative strand RNA viruses induce innate immunity by two cellular pathways, a cytoplasmic recognition pathway that operates in most cell types, and a transmembrane pathway that operates predominantly in dendritic and monocytic cells. Both pathways can trigger type I IFN gene transcription through activation of latent transcription factors of the IRF and NF-κΒ families. However, genetic evidence suggests that the cytoplasmic pathway predominates for protection against influenza viral infections and that the transmembrane pathway may in fact exacerbate infection (Guillot et al., 2005; Le Goffic et al., 2006; Le Goffic et al., 2007). The cytoplasmic signaling pathway operates in the primary targets for respiratory viral infections, bronchial and pulmonary epithelial cells and alveolar macrophages. This signal transduction pathway is triggered by recognition of viral RNA or ribonucleoprotein particles (RNP) by the cytoplasmic RNA helicase RIG-I (Guo et al., 2006; Mibayashi et al., 2006; Opitz et al., 2007; Pichlmair et al., 2006) leading to activation of the downstream adaptor and effector proteins, MAVS (also known as IPS-1, VISA, or Cardif), TBK-1 (also known as T2K or NAK), ΙΚΚ-ε (also known as IK -i), IRF3, and IRF7 (Akira et al, 2006; Kawai and Akira, 2006). Activated IRF3, in conjunction with activated NF-kB and AP-1 transcription factors, is essential for induction of IFN-β gene expression, while activated IRF7 mediates most IFN-a gene expression (Akira et al., 2006; Kawai and Akira, 2006; Marie et al., 1998). Because IRF7 and many other components of the signaling pathway are expressed at low levels in epithelial cells until induced in response to an initial IFN stimulation, IFN-a gene expression is highly dependent on positive feedback through the IFN response pathway (Marie et al., 1998; Taniguchi and Takaoka, 2001). Compounds of the present invention may trigger IFN-β and/or IFN-a gene expression (see Enniga, 2002).

C. Interferons

As noted above, NS1 protein inhibits interferon (IFN) gene induction and IFN- modulated immune responses. As such, the NS1 inhibitors described herein are also novel inhibitors of viral replication and pathogenesis that act by preventing NS 1 protein-mediated inhibition of IFN-dependent immune responses to viral infection.

Interferons are important cytokines characterized by antiviral, antiproliferative and immunomodulatory activities. Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface, thereby preventing viral replication within the cells. There are several groups of interferons (IFN), including a (formerly ai), Ω (formerly a₂), β, γ and τ. Mature human interferons are between 165 and 172 amino acids in length. In humans IFN-a and IFN-Ω are encoded by multiple, closely related non-allelic genes. Additionally, there are pseudo-genes of IFN-a and IFN-Ω. By contrast, IFN-β and IFN-γ are encoded by unique genes.

The interferons can also be grouped into two types. IFN-γ is the sole type II interferon; all others are type I interferons. Type I and type II interferons differ in gene structure (type II interferon genes have three exons; type I, one), chromosome location (in humans, type II is located on chromosome- 12; the type I interferon genes are linked and on chromosome-9), and the types of tissues where they are produced (type I interferons are synthesized ubiquitously, type II by lymphocytes). Type I interferons competitively inhibit each other's binding to cellular receptors, while type II interferon has a distinct receptor (reviewed by Sen and Lengyel, 1992).

IFN-a has become most widely used for therapeutic purposes. Among the interferons of human origin, the IFN-as are divided into several subtypes, which are either encoded by different gene loci or alleles of those. The function of each subtype is still not clear, and the molecular or cellular targets of their antiviral and antineoplastic activities is thus not fully investigated. Human IFN-as are encoded by a multigene family consisting of about 20 genes; each gene encodes a single subtype of the human IFN-a. Human IFN-a polypeptides are produced by a number of human cell lines and human leukocyte cells after exposure to viruses or double-stranded RNA, or in transformed leukocyte cell lines (e.g., lymphoblastoid lines). IFN-as interact with cell- surface receptors and induce the expression, primarily at the transcriptional level, of a broad but specific set of cellular genes. Several IFN-a-induced gene products have been used as markers for the biological activity of interferons. These include, for instance, ISG15, ISG54, IRF1, GBP, and IP10.

Human IFN-β is a regulatory polypeptide with a molecular weight of 22 kDa consisting of 166 amino acid residues. It can be produced by most cells in the body, in particular fibroblasts, in response to viral infection or exposure to other biologies. It binds to a multimeric cell surface receptor, and productive receptor binding results in a cascade of intracellular events leading to the expression of IFN-β inducible genes which, in turn, produces effects which can be classified as antiviral, antiproliferative, or immunomodulatory.

D. NS1 protein

The nonstructural NS1 proteins of pathogenic strains of influenza virus are major virulence factors for viral pathogenesis. NS1 protein inhibits host gene expression and signal transduction required to mount innate and adaptive immune responses. In infected cells, NS1 protein is localized in the nucleus and the cytoplasm. The nuclear pool of NS1 protein inhibits mRNA processing and nuclear export of mRNAs, preventing proper expression of antiviral genes, while the cytoplasmic pool inhibits signal transduction pathways necessary for antiviral gene induction and effector proteins necessary for antiviral defense. Genetic studies have shown that abrogation of NS1 protein functions by mutation results in highly attenuated viruses that can only replicate in immunocompromised hosts (Garcia-Sastre et al., 1998; Krug et al, 2003). For example, in animals or cells deficient in type I IFN responses, influenza viral mutants lacking NS1 protein replicate at near wild type levels and cause diseases similar to wild type viruses (Garcia-Sastre et al, 1998). These observations define NS 1 protein as an essential element of viral virulence and suggest that its importance for viral pathogenesis is to selectively debilitate innate immunity.

Inhibition of either the primary activation or the secondary viral response pathways in cells described above in section A severely impairs innate immune responses by blocking IFN protection, and it is in this manner that NS1 protein promotes virulence. NS1 protein inhibits IRF3 and NF-κΒ activation and therefore IFN gene induction by interfering with the cytoplasmic signal transduction pathway (Donelan et al, 2004; Talon et al, 2000) through inhibiting the function of RIG-I (Guo et al, 2006; Mibayashi et al, 2006; Opitz et al, 2007; Pichlmair et al, 2006). NS1 protein also prevents IFN action by sequestering double-stranded RNA and/or targeting the function of downstream antiviral effector proteins, such as PKR and the RNase L pathway (Li et al, 2006; Min and Krug, 2006).

NS1 protein is a 230-amino acid protein that contains two major domains and forms a homodimer (Knipe and Howley, 2001). The amino terminal region of NS1 protein (residues 1-73) encompasses an RNA-binding domain that is able to interact non-specifically with dsRNA (Knipe and Howley, 2001). Structural and biochemical studies have shown that arginine-38 (R38) is required for binding dsRNA. This interaction is of low affinity compared to other RNA binding proteins; nevertheless, recent studies of mutant influenza viruses with impaired dsRNA binding ability have demonstrated that this function contributes to virulence. Mutations of NS1 protein that abrogate dsRNA binding resulted in attenuated viruses that grow to lower titers, induced increased IFN production, and failed to effectively block antiviral effector functions (Donelan et al, 2003; Min and Krug, 2006). However, abrogation of RNA binding attenuates virulence less than complete loss of the NS 1 protein. Thus, additional sequences of the NS1 protein are also critical for virulence. A region within the amino terminal domain of NS1 protein, from amino acids 19 to 38, is required for NS1 protein-mediated inhibition of mRNA nuclear export (Qian et al, 1994), which, as mentioned below, is a key nuclear function of NS 1 protein that inhibits expression of host antiviral genes. In fact, the present inventors have recently shown that the amino terminal domain of NS1 protein is involved in its interaction with the mRNA export machinery, namely the NXFl-pl5 heterodimer, Rael and E1B-AP5 (Satterly et al, 2007), which are mRNA export factors known to form a complex and to mediate nuclear exit of mRNAs (Bachi et al, 2000; Blevins et al, 2003; Satterly et al, 2007). The carboxyl terminal domain of NSl protein, amino acids 134 to 161, is also required for the inhibitory effect of NSl protein on mRNA nuclear export (Qian et al, 1994). The carboxy terminus of NSl protein is also termed the effector domain and is the region that binds the human 30 kD subunit of the cleavage and polyadenylation specificity factor (CPSF) and the poly(A)-binding protein II (PABII), which are involved in binding the AAUAAA polyadenylation signal and in the elongation of the poly(A) chain, respectively (Chen et al, 1999; Nemeroff et al, 1998). The interaction of NSl protein with these proteins inhibits 3 'end processing of host mRNAs and contributes to nuclear retention of host mRNAs. A mutant influenza virus that expresses an NSl protein with a mutated CPSF binding site is highly attenuated and cells infected with this virus produce high levels of IFN- β mRNA (Noah et al, 2003; Twu et al, 2006). These effects are also likely caused by changes in interactions between the mutant NSl protein and additional host proteins directly involved in nuclear export of mRNAs, as the inventors demonstrate herein. mRNA processing and export are connected - some proteins remain bound to mRNAs throughout these processes and others are exchanged with factors specific for each step. In fact, combinatorial assembly of complexes that share some common factors are being revealed as mechanisms to generate specific functions and/or redundancy (Rochette-Egly, 2005).

The present inventors have found that NS 1 protein binds cellular factors involved in nuclear export of bulk mRNAs, while viral mRNAs exit the nucleus via a distinct pathway. This inhibition of mRNA export can be reverted by increased expression of the mRNA export factors targeted by NSl protein. In contrast, cells from mice that express low levels of specific mRNA export factors are highly permissive to influenza virus replication and pathogenesis. Similarly, the cytoplasmic pool of NSl protein inhibits IFN gene induction by interfering with the signal transduction pathway triggered by viral infection. In the absence of this NSl protein-imposed block, viral replication is highly attenuated and pathogenesis is reduced, except in mice with mutations in elements normally targeted by NSl protein. Compounds of the present invention may inhibit only nuclear and/or both nuclear and cytoplasmic NSl protein functions.

E. Chemical Definitions

When used in the context of a chemical group, "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =0; "halo" means independently -F, -CI, -Br or -I; "amino" means -NH₂ (see below for definitions of groups containing the term amino, e.g., alkylamino); "hydroxyamino" means -NHOH; "nitro" means -N0₂; imino means =NH (see below for definitions of groups containing the term imino, e.g., alkylimino); "thio" means =S.

The symbol "-" means a single bond, "=" means a double bond. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed.

For the groups and classes below, the following parenthetical subscripts further define the group/class as follows: "(Cn)" defines the exact number (n) of carbon atoms in the group/class. "(C≤n)" defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group in question, e.g. , it is understood that the minimum number of carbon atoms in the group "alkenyl₍c<8)" or the class "alkene(c<8)" is two. For example, "alkoxy(c≤io)" designates those alkoxy groups having from 1 to 10 carbon atoms (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g. , 3 to 10 carbon atoms). (Cn-n') defines both the minimum (n) and maximum number (η') of carbon atoms in the group. Similarly, "alkyl₍c_2-10)" designates those alkyl groups having from 2 to 10 carbon atoms (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g. , 3 to 10 carbon atoms)).

The term "alkyl" when used without the "substituted" modifier refers to a non- aromatic monovalent group with a saturated carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH₃ (Me), -CH₂CH (Et), -CH₂CH₂CH₃ (n-Pr), -CH(CH₃)₂ (iso-Pr), -CH(CH₂)₂ (cyclopropyl), -CH₂CH₂CH₂CH₃ (n- Bu), -CH(CH₃)CH₂CH₃ (sec-butyl), -CH₂CH(CH₃)₂ (wo-butyl), -C(CH₃)₃ (ieri-butyl), -CH₂C(CH₃)₃ (neo-pentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl groups. The term "substituted alkyl" refers to a non-aromatic monovalent group with a saturated carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The following groups are non-limiting examples of substituted alkyl groups: -CH₂OH, -CH₂C1, -CH₂Br, -CH₂SH, -CF₃, -CH₂CN, -CH₂C(0)H, -CH₂C(0)OH, -CH₂C(0)OCH₃, -CH₂C(0)NH₂, -CH₂C(0)NHCH₃, -CH₂C(0)CH₃, -CH₂OCH₃, -CH₂OCH₂CF₃, -CH₂OC(0)CH₃, -CH₂NH₂, -CH₂NHCH₃, -CH₂N(CH₃)₂, -CH₂CH₂C1, -CH₂CH₂OH, -CH₂CF₃, -CH₂CH₂OC(0)CH₃, -CH₂CH₂NHC0₂C(CH₃)₃, and -CH₂Si(CH₃)₃.

The term "alkanediyl" when used without the "substituted" modifier refers to a nonaromatic divalent group, wherein the alkanediyl group is attached with two σ-bonds, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH₂- (methylene), -CH₂CH₂-, -CH₂C(CH )₂CH₂-,

-CH₂C

are non- limiting examples of alkanediyl groups. The term

"substituted alkanediyl" refers to a non-aromatic monovalent group, wherein the alkynediyl group is attached with two σ-bonds, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched, cyclo, cyclic or acyclic structure, no carbon-carbon double or triple bonds, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The following groups are non- limiting examples of substituted alkanediyl groups: -CH(F)-, -CF₂- -CH(Cl)-, -CH(OH)-, -CH(OCH₃)- and -CH₂CH(C1)-.

The term "alkane" when used without the "substituted" modifier refers to a nonaromatic hydrocarbon consisting only of saturated carbon atoms and hydrogen and having a linear or branched, cyclo, cyclic or acyclic structure. Thus, as used herein cycloalkane is a subset of alkane. The compounds CH₄ (methane), CH₃CH₃ (ethane), CH₃CH₂CH₃ (propane), (CH₂)₃ (cyclopropane), CH₃CH₂CH₂CH₃ (n-butane), and CH₃CH(CH₃)CH₃ (isobutane), are non-limiting examples of alkanes. A "substituted alkane" differs from an alkane in that it also comprises at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The following compounds are non- limiting examples of substituted alkanes: CH₃OH, CH₃C1, nitromethane, CF₄, CH₃OCH₃ and CH₃CH₂NH₂.

The term "alkenyl" when used without the "substituted" modifier refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non- limiting examples of alkenyl groups include: -CH=CH₂ (vinyl), -CH=CHCH , -CH=CHCH₂CH₃, -CH₂CH=CH₂ (allyl), -CH₂CH=CHCH₃, and -CH=CH-C₆H₅. The term "substituted alkenyl" refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, a linear or branched, cyclo, cyclic or acyclic structure, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The groups, -CH=CHF, -CH=CHC1 and -CH=CHBr, are non-limiting examples of substituted alkenyl groups.

The term "alkenediyl" when used without the "substituted" modifier refers to a nonaromatic divalent group, wherein the alkenediyl group is attached with two σ-bonds, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups, -CH=CH-,

CH=C(CH₃)CH₂- -CH=CHCH₂- and

are non-limiting examples of alkenediyl groups. The term "substituted alkenediyl" refers to a non-aromatic divalent group, wherein the alkenediyl group is attached with two σ-bonds, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The following groups are non-limiting examples of substituted alkenediyl groups: -CF=CH-, -C(OH)=CH- and -CH₂CH=C(C1)-.

The term "alkene" when used without the "substituted" modifier refers to a nonaromatic hydrocarbon having at least one carbon-carbon double bond and a linear or branched, cyclo, cyclic or acyclic structure. Thus, as used herein, cycloalkene is a subset of alkene. The compounds C₂H₄ (ethylene), CH₃CH=CH₂ (propene) and cylcohexene are non- limiting examples of alkenes. A "substituted alkene" differs from an alkene in that it also comprises at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S.

The term "alkynyl" when used without the "substituted" modifier refers to a monovalent group with a nonaromatic carbon atom as the point of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups, -C≡CH, -C≡CCH₃, -C≡CCeH₅ and -CH₂C≡CCH₃, are non-limiting examples of alkynyl groups. The term "substituted alkynyl" refers to a monovalent group with a nonaromatic carbon atom as the point of attachment and at least one carbon-carbon triple bond, a linear or branched, cyclo, cyclic or acyclic structure, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The group, -C≡CSi(CH₃)3, is a non-limiting example of a substituted alkynyl group. The term "alkynediyl" when used without the "substituted" modifier refers to a non- aromatic divalent group, wherein the alkynediyl group is attached with two σ-bonds, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups, -C≡C^~, -C≡CCH₂-, and -C≡CCH(CH3)- are non-limiting examples of alkynediyl groups. The term "substituted alkynediyl" refers to a non-aromatic divalent group, wherein the alkynediyl group is attached with two σ-bonds, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one carbon-carbon triple bond, and at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The groups -C≡CCFH- and -C≡CHCH(C1)- are non-limiting examples of substituted alkynediyl groups.

The term "alkyne" when used without the "substituted" modifier refers to a non- aromatic hydrocarbon having at least one carbon-carbon triple bond and a linear or branched, cyclo, cyclic or acyclic structure. Thus, as used herein, cycloalkene is a subset of alkene. The compounds C₂H₂ (acetylene), CH3C≡CH (propene) and cylcooctyne are non- limiting examples of alkenes. A "substituted alkene" differs from an alkene in that it also comprises at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S.

The term "aryl" when used without the "substituted" modifier refers to a monovalent group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C₆H₄CH₂CH₃ (ethylphenyl), -C₆H₄CH₂CH₂CH₃ (propylphenyl), -C₆H₄CH(CH₃)₂, -C₆H₄CH(CH₂)₂, -C₆H₃(CH₃)CH₂CH₃ (methylethylphenyl), -C₆H₄CH=CH₂ (vinylphenyl), -C₆H₄CH=CHCH₃, -C₆H₄C≡CH, -C₆H₄C≡CCH₃, naphthyl, and the monovalent group derived from biphenyl. The term "substituted aryl" refers to a monovalent group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group further has at least one atom independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. Non- limiting examples of substituted aryl groups include the groups: -C₆H₄F, -C₆H₄C1, -C₆H₄Br, -C₆H₄I, -C₆H₄OH, -C₆H₄OCH₃, -C₆H₄OCH₂CH₃, -C₆H₄OC(0)CH₃, -C₆H₄NH₂, -C₆H₄NHCH₃, -C₆H₄N(CH₃)2, -C₆H₄CH₂OH, -C₆H₄CH₂OC(0)CH₃, -C₆H₄CH₂NH₂, -C₆H₄CF₃, -C₆H₄CN, -C₆H₄CHO, -C₆H₄CHO, -C₆H₄C(0)CH₃, -C₆H₄C(0)C₆H₅, -C₆H₄C0₂H, -C₆H₄C0₂CH₃, -C₆H₄CONH₂, -C₆H₄CONHCH₃, and -C₆H₄CON(CH₃)₂.

The term "aralkyl" when used without the "substituted" modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples of aralkyls are: phenylmethyl (benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and 2,3-dihydro- indenyl, provided that indenyl and 2,3-dihydro-indenyl are only examples of aralkyl in so far as the point of attachment in each case is one of the saturated carbon atoms. When the term "aralkyl" is used with the "substituted" modifier, either one or both the alkanediyl and the aryl is substituted. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)- methyl, 2-oxo-2-phenyl-ethyl (phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl, chromanyl where the point of attachment is one of the saturated carbon atoms, and tetrahydroquinolinyl where the point of attachment is one of the saturated atoms.

The term "heteroaryl" when used without the "substituted" modifier refers to a monovalent group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the monovalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples of aryl groups include acridinyl, furanyl, imidazoimidazolyl, imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl, indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl, pyrroloimidazolyl, chromenyl (where the point of attachment is one of the aromatic atoms), and chromanyl (where the point of attachment is one of the aromatic atoms). The term "substituted heteroaryl" refers to a monovalent group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of an aromatic ring structure wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the monovalent group further has at least one atom independently selected from the group consisting of non-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, CI, Br, I, Si, and P.

The term "heteroaralkyl" when used without the "substituted" modifier refers to the monovalent group -alkanediyl-fieteroaryl, in which the terms alkanediyl and heteroaryl are each used in a manner consistent with the definitions provided above. Non-limiting examples of aralkyls are: pyridylmethyl, and thienylmethyl. When the term "heteroaralkyl" is used with the "substituted" modifier, either one or both the alkanediyl and the heteroaryl is substituted.

The term "acyl" when used without the "substituted" modifier refers to a monovalent group with a carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclo, cyclic or acyclic structure, further having no additional atoms that are not carbon or hydrogen, beyond the oxygen atom of the carbonyl group. The groups, -CHO, -C(0)CH₃ (acetyl, Ac), -C(0)CH₂CH₃, -C(0)CH₂CH₂CH₃, -C(0)CH(CH₃)₂, -C(0)CH(CH₂)₂, -C(0)C₆H₅, -C(0)C₆H₄CH₃, -C(0)C₆H₄CH₂CH₃, -COC₆H₃(CH₃)₂, and -C(0)CH₂CeH₅, are non-limiting examples of acyl groups. The term "acyl" therefore encompasses, but is not limited to groups sometimes referred to as "alkyl carbonyl" and "aryl carbonyl" groups. The term "substituted acyl" refers to a monovalent group with a carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclo, cyclic or acyclic structure, further having at least one atom, in addition to the oxygen of the carbonyl group, independently selected from the group consisting of N, O, F, CI, Br, I, Si, P, and S. The groups, -C(0)CH₂CF₃, -C0₂H (carboxyl), -C0₂CH₃ (methylcarboxyl), -C0₂CH₂CH₃, -C0₂CH₂CH₂CH₃, -C0₂C₆H₅, -C0₂CH(CH₃)₂, -C0₂CH(CH₂)₂, -C(0)NH₂ (carbamoyl), -C(0)NHCH₃, -C(0)NHCH₂CH₃, -CONHCH(CH₃)₂, -CONHCH(CH₂)₂, -CON(CH₃)₂, -CONHCH₂CF₃, -CO-pyridyl, -CC-imidazoyl, and -C(0)N₃, are non- limiting examples of substituted acyl groups. The term "substituted acyl" encompasses, but is not limited to, "heteroaryl carbonyl" groups.

The term "alkoxy" when used without the "substituted" modifier refers to the group -OR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkoxy groups include: -OCH₃, -OCH₂CH₃, -OCH₂CH₂CH₃, -OCH(CH₃)₂, -OCH(CH₂)₂, -O-cyclopentyl, and -O-cyclohexyl. The term "substituted alkoxy" refers to the group -OR, in which R is a substituted alkyl, as that term is defined above. For example, -OCH₂CF₃ is a substituted alkoxy group.

The term "alcohol" when used without the "substituted" modifier corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. Alcohols have a linear or branched, cyclo, cyclic or acyclic structure. The compounds methanol, ethanol and cyclohexanol are non-limiting examples of alcohols. A "substituted alkane" differs from an alcohol in that it also comprises at least one atom independently selected from the group consisting of N, F, CI, Br, I, Si, P, and S. Similarly, the terms "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "hetero aralkoxy" and "acyloxy", when used without the "substituted" modifier, refers to groups, defined as -OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl, respectively, as those terms are defined above. When any of the terms alkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is modified by "substituted," it refers to the group -OR, in which R is substituted alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.

The term "alkylamino" when used without the "substituted" modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkylamino groups include: -NHCH₃, -NHCH₂CH₃, -NHCH₂CH₂CH₃, -NHCH(CH₃)₂, -NHCH(CH₂)₂, -NHCH₂CH₂CH₂CH₃, -NHCH(CH₃)CH₂CH₃, -NHCH₂CH(CH₃)₂, -NHC(CH₃)₃, -NH-cyclopentyl, and -NH-cyclohexyl. The term "substituted alkylamino" refers to the group -NHR, in which R is a substituted alkyl, as that term is defined above. For example, -NHCH₂CF is a substituted alkylamino group.

The term "dialkylamino" when used without the "substituted" modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl having two or more saturated carbon atoms, at least two of which are attached to the nitrogen atom. Non-limiting examples of dialkylamino groups include: -NHC(CH₃)₃, -N(CH₃)CH₂CH₃, -N(CH₂CH₃)₂, N-pyrrolidinyl, and N- piperidinyl. The term "substituted dialkylamino" refers to the group -NRR', in which R and R' can be the same or different substituted alkyl groups, one of R or R' is an alkyl and the other is a substituted alkyl, or R and R' can be taken together to represent a substituted alkanediyl with two or more saturated carbon atoms, at least two of which are attached to the nitrogen atom.

The terms "alkoxyamino", "alkenylamino", "alkynylamino", "arylamino",

"aralkylamino", "heteroarylamino", "heteroaralkylamino", and "alkylsulfonylamino" when used without the "substituted" modifier, refers to groups, defined as -NHR, in which R is alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl, respectively, as those terms are defined above. A non-limiting example of an arylamino group is -ΜΚ₆Η₅. When any of the terms alkoxyamino, alkenylamino, alkynylamino, arylamino, aralkylamino, heteroarylamino, heteroaralkylamino and alkylsulfonylamino is modified by "substituted," it refers to the group -NHR, in which R is substituted alkoxy, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl, respectively. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, in which R is acyl, as that term is defined above. A non-limiting example of an acylamino group is -NHC(0)CH₃. When the term amido is used with the "substituted" modifier, it refers to groups, defined as -NHR, in which R is substituted acyl, as that term is defined above. The groups -NHC(0)OCH₃ and -NHC(0)NHCH₃ are non- limiting examples of substituted amido groups.

The term "alkylimino" when used without the "substituted" modifier refers to the group =NR, wherein the alkylimino group is attached with one σ-bond and one π-bond, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkylimino groups include: =NCH , =NCH₂CH and =N-cyclohexyl. The term "substituted alkylimino" refers to the group =NR, wherein the alkylimino group is attached with one σ-bond and one π-bond, in which R is a substituted alkyl, as that term is defined above. For example, =NCH₂CF is a substituted alkylimino group.

The term "alkylthio" when used without the "substituted" modifier refers to the group -SR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkylthio groups include: -SCH₃, -SCH₂CH₃, -SCH₂CH₂CH₃, -SCH(CH₃)₂, -SCH(CH₂)₂, -S-cyclopentyl, and -S-cyclohexyl. The term "substituted alkylthio" refers to the group -SR, in which R is a substituted alkyl, as that term is defined above. For example, -SCH₂CF₃ is a substituted alkylthio group.

In addition, atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ¹³C and ¹⁴C. Similarly, it is contemplated that one or more carbon atom(s) of a compound of the present invention may be replaced by a silicon atom(s). Furthermore, it is contemplated that one or more oxygen atom(s) of a compound of the present invention may be replaced by a sulfur or selenium atom(s).

Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to the atom.

As used herein, a "chiral auxiliary" refers to a removable chiral group that is capable of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with such compounds, and many are commercially available. The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

The term "effective," as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.

As used herein, the term "IC₅o" refers to an inhibitory dose which is 50% of the maximum response obtained.

An "isomer" of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.

As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non- limiting examples of human subjects are adults, juveniles, infants and fetuses.

"Pharmaceutically acceptable" means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.

"Pharmaceutically acceptable salts" means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, /?-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, /?-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002).

As used herein, "predominantly one enantiomer" means that a compound contains at least about 85% of one enantiomer, or more preferably at least about 90% of one enantiomer, or even more preferably at least about 95% of one enantiomer, or most preferably at least about 99% of one enantiomer. Similarly, the phrase "substantially free from other optical isomers" means that the composition contains at most about 15% of another enantiomer or diastereomer, more preferably at most about 10% of another enantiomer or diastereomer, even more preferably at most about 5% of another enantiomer or diastereomer, and most preferably at most about 1% of another enantiomer or diastereomer.

"Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

"Prodrug" means a compound that is convertible in vivo metabolically into an inhibitor according to the present invention. The prodrug itself may or may not also have activity with respect to a given target protein. For example, a compound comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, phosphates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis- -hydroxynaphthoate, gentisates, isethionates, di-/?-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, /?-toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids, and the like. Similarly, a compound comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

A "repeat unit" is the simplest structural entity of certain materials, for example, frameworks and/or polymers, whether organic, inorganic or metal-organic. In the case of a polymer chain, repeat units are linked together successively along the chain, like the beads of a necklace. For example, in polyethylene, -[- ¾ ¾-]_η-, the repeat unit is -CH2CH2-. The subscript "n" denotes the degree of polymerisation, that is, the number of repeat units linked together. When the value for "n" is left undefined, it simply designates repetition of the formula within the brackets as well as the polymeric nature of the material. The concept of a repeat unit applies equally to where the connectivity between the repeat units extends three dimensionally, such as in metal organic frameworks, cross-linked polymers, thermosetting polymers, etc.

The term "saturated" when referring to an atom means that the atom is connected to other atoms only by means of single bonds.

A "stereoisomer" or "optical isomer" is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers.

The invention contemplates that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures.

"Substituent convertible to hydrogen in vivo" means any group that is convertible to a hydrogen atom by enzymological or chemical means including, but not limited to, hydrolysis and hydrogenolysis. Examples include hydrolyzable groups, such as acyl groups, groups having an oxycarbonyl group, amino acid residues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl, diphenylphosphinyl, and the like. Examples of acyl groups include formyl, acetyl, trifluoroacetyl, and the like. Examples of groups having an oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl (-C(0)OC(CH₃)₃), benzyloxycarbonyl, /?-methoxybenzyloxycarbonyl, vinyloxycarbonyl, β-(/?- toluenesulfonyl)ethoxycarbonyl, and the like. Suitable amino acid residues include, but are not limited to, residues of Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), He (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn (ornithine) and β- Ala. Examples of suitable amino acid residues also include amino acid residues that are protected with a protecting group. Examples of suitable protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and /?-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (-C(0)OC(CH₃)₃), and the like. Suitable peptide residues include peptide residues comprising two to five amino acid residues. The residues of these amino acids or peptides can be present in stereochemical configurations of the D-form, the L- form or mixtures thereof. In addition, the amino acid or peptide residue may have an asymmetric carbon atom. Examples of suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom. Examples of suitable amino acid protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and /?-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (-C(0)OC(CH₃) ), and the like. Other examples of substituents "convertible to hydrogen in vivo" include reductively eliminable hydrogenolyzable groups. Examples of suitable reductively eliminable hydrogenolyzable groups include, but are not limited to, arylsulfonyl groups (such as o-toluenesulfonyl); methyl groups substituted with phenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such as β,β,β-trichloroethoxycarbonyl and β-iodoethoxycarbonyl). "Effective amount," "Therapeutically effective amount" or "pharmaceutically effective amount" means that amount which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

"Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.

The above definitions supersede any conflicting definition in any of the reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.

Compounds of the present invention may contain one or more asymmetric centers and thus can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In certain embodiments, a single diastereomer is present. All possible stereoisomers of the compounds of the present invention are contemplated as being within the scope of the present invention. However, in certain aspects, particular diastereomers are contemplated. The chiral centers of the compounds of the present invention can have the S- or the ^-configuration, as defined by the IUPAC 1974 Recommendations. In certain aspects, certain compounds of the present invention may comprise S- or ^-configurations at particular carbon centers.

Compounds of the present invention may be purchased, such as SigmaAldrich

(Milwaukee, WI); Chemical Diversity Laboratories (San Diego, CA); and ChemBridge Corp. (San Diego, CA). Compounds may also be ordered from companies that prepare customized organic compounds (e.g., AsisChem, Inc., SynChem, Inc.), or prepared using synthetic organic techniques. For example, the scheme below depicts one means of synthetically accessing certain NS 1 inhibitors of the present invention

over 40 commer

analogs available

Other synthetic techniques to prepare compounds of the present invention as well as derivatives are well-known to those of skill in the art. For example, Smith and March, 2001 discuss a wide variety of synthetic transformations, reaction conditions, and possible pitfalls relating thereto. Methods discussed therein may be adapted to prepare compounds of the present invention from commercially available starting materials.

Solvent choices for preparing compounds of the present invention will be known to one of ordinary skill in the art. Solvent choices may depend, for example, on which one(s) will facilitate the solubilizing of all the reagents or, for example, which one(s) will best facilitate the desired reaction (particularly when the mechanism of the reaction is known). Solvents may include, for example, polar solvents and non-polar solvents. Solvents choices include, but are not limited to, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride and acetonitrile. More than one solvent may be chosen for any particular reaction or purification procedure. Water may also be admixed into any solvent choice. Further, water, such as distilled water, may constitute the reaction medium instead of a solvent.

Persons of ordinary skill in the art will be familiar with methods of purifying compounds of the present invention. One of ordinary skill in the art will understand that compounds of the present invention can generally be purified at any step, including the purification of intermediates as well as purification of the final products. In preferred embodiments, purification is performed via silica gel column chromatography or HPLC.

In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof.

F. Pharmaceutical Formulations and Routes for Administration

Pharmaceutical compositions of the present invention comprise an effective amount of one or more candidate substances (e.g., an NS 1 protein inhibitor) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The candidate substance may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intraarterially, intraperitoneally, intracranially, intrapleurally, intratracheally, intranasally (e.g., via a nasal spray), topically, subcutaneously, intravesicularlly, mucosally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 1990).

In particular embodiments, the composition is administered to a subject using a drug delivery device. Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of an NS 1 protein inhibitor.

The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.

In certain embodiments, it may be desirable to provide a continuous supply of a pharmaceutical composition to the patient. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an NS1 protein inhibitor. In other embodiments, the NS1 protein inhibitor may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens {e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. The NS1 protein inhibitor may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. Other bases and salts are described herein.

In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. It may be preferable to include isotonic agents, such as, for example, sugars, sodium chloride, or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in certain embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

In certain embodiments the candidate substance is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. In certain embodiments, carriers for oral administration comprise inert diluents (e.g., glucose, lactose, or mannitol), assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

In certain embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent (e.g., water) first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

G. Combination Therapy

In order to enhance or increase the effectiveness of an NS1 inhibitor of the present invention, the NS1 inhibitor may be combined with another therapy, such as another agent that combats viral infection. For example, NS1 inhibitors of the present invention may be provided in a combined amount with an effective amount another agent to reduce or block viral replication in infected cells. It is contemplated that this type of combination therapy may be used in vitro or in vivo.

These processes may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit. This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.

The compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously {i.e., within less than about a minute) as the candidate substance. In other aspects, one or more agents may be administered about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 1 1 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 , about 2, about 3, about 4, about 5, about 6, about 7 or about 8 weeks or more, and any range derivable therein, prior to and/or after administering the candidate substance.

Various combination regimens of the agents may be employed. Non-limiting examples of such combinations are shown below, wherein an NS 1 protein inhibitor is "A" and a second agent is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

In a non-limiting examples, an agent that combats viral infections may be an influenza virus neuraminidase inhibitor (e.g., Relenza™ or Tamiflu™). Another non-limiting example of an agent that combats viral infections is an M2 proton channel inhibitor (e.g., amantadine or rimantadine).

H. Examples

The following examples are included to demonstrate certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 - MATERIALS AND METHODS

Compound screen. The UT Southwestern Compound Library is composed of 200,000 synthetic-drug-like compounds arrayed in DMSO in 384-well plates. HEK 293T cells were transfected with an approximately 10: 1 ratio of plasmid pCMV-Luc encoding luciferase and pCAGGS-NSl encoding NS1 using Lipofectamine 2000 (Invitrogen). Cells were transfected with the luciferase plasmid alone as a positive control. After 16 h, cells were dispensed at 5,000 cells per well in 384-well plates. After 1 h, compounds from the library were added to a final concentration of 5 μΜ in 1% (v/v) DMSO in a one compound per well format. Experimental samples were limited to columns 3 to 22, with controls treated with 1% DMSO in the first and last two columns of wells. Wells in the first column of each plate contained cells transfected with the luciferase plasmid alone; all other wells received cells transfected with both plasmids. Plates were incubated for 22 h at 37 °C in 5% C0₂, then cooled to 22 °C, incubated with Bright-Glo luciferase substrate (Promega) for 4 min, then luminescence was recorded. Experiments producing plates with standard (Z) scores lower than 0.45 were repeated. Experimental values were normalized to the mean of the luciferase-only control on the same plate. Compounds were ranked by Z score, and the 640 compounds with the most positive Z scores were selected and retested in the assay at concentrations of 15 μΜ, 5 μΜ and 1.7 μΜ. These compounds were also tested for the ability to prevent cell death of immortalized human bronchial epithelial cells (HBECs) that had been infected with A/WS/33 influenza virus by measuring cell ATP levels with ATP-lite (PerkinElmer). A table describing this screen can be found in Table 1.

Table 1 - Small Molecule Screening Data

were transfected with the luciferase plasmid alone as a positive control. After 16 hours, cells were dispensed at 5000 cell/well in 384 well plates. After one hour, compounds from the library were added to a final concentration of 5 μΜ in 1% DMSO in a one compound/one well format. Experimental samples were limited to columns 3 to 22, with controls treated with 1% DMSO in the first and last two columns of wells. Wells in the first column of each plate contained cells transfected with the luciferase plasmid alone; all other wells received cells transfected with both plasmids. Plates were incubated 22 h at 37°C in 5% C02, then cooled to room temperature and incubated with Bright-Glo luciferase substrate (Promega) for 4 min and luminescence was recorded.

Additional comments Care was taken each day to transfect subconfluent cells that had been cultured for 16-18 hours.

Library Library size 200,000

Library composition Commercially available chemical compounds

Source ChemDiv, ChemBridge, Comgenex,

TimTek

Additional comments Compounds in the library represent all the desirable chemical diversity available from the first three companies as of 2004-2005. All compounds passed 48 selective filters and chemical diversity was calculated with CheD software (TimTek).

Screen Format 384 well plates

Concentration(s) tested 5 μΜ

Plate controls Column 1 positive control, cells transfected with luciferase alone and treated with final concentration of 1% DMSO. Columns 2, 23 and 24, negative controls transfected with both plasmids and treated with a final concentration of 1% DMSO.

Reagent/compound dispensing Biomek FX for compounds. Titertek system Multidrop for other reagents

Detection instrument and Perkin Elmer envision and software software

Assay validation/QC Z' factors were calculated for each plate using the means and standard deviations of the on plate positive and negative controls. Plates with Z' <0.45 were repeated.

Correction factors

Normalization For each plate experimental values were normalized to the mean of the positive control.

Additional comments Post-HTS Hit criteria Hits were defined as wells in which analysis luciferase expression exceeded 3.5

standard deviations of the negative controls on the same plate and that had not been previously identified in other screens at UTSW. The top 640 compounds were selected for analysis.

Hit rate .0032

Additional assay(s) The primary assay was repeated in

triplicate at 15, 5 and 1.67 μΜ concentrations. All 640 compounds were tested for cytotoxicity on HBEC and MDCK cells, using Celtiter Glo (Promega).

All 640 compounds were tested for the ability to inhibit influenza virus cytopathic effects as measured by Celtiter Glo.

Confirmation of hit purity and After resupply from commercial sources or structure in-house synthesis, purity and identity of

compounds was determined by LC/MS with an Agilent 1100 series instrument.

Additional comments 72 compounds were selected as compounds

of interest after additional assays were completed. Most of these fell into 8 structure classes and representatives of these 8 were purchased for additional testing.

Compound half-life. Compound half-lives were measured in HBECs by LC/MS/MS. Metabolic stability half-life was determined by substrate depletion48. Cell survival and cytotoxicity measurements. MEFs, HBECs or MDCK cells were seeded in white-walled 96- well plates at a density of 3 x 10 cells per well, 16 h before compound addition. Compounds dissolved in sterile DMSO (Sigma) at a concentration of 25 mM were diluted to 100 μΜ in OptiMEM I (Invitrogen) in triplicates. The 100-μΜ starting dilutions were serially diluted in two-fold steps to a final concentration of 0.2 μΜ. Control experiments, performed in the absence of compound, had the same final concentration of DMSO as compound-treated samples. At the time points depicted in the figures (24 h, 48 h and 72 h), cells were lysed, and ATP levels were measured by luminescence using the Cell Titer-Glo kit (Promega), following manufacturer instructions. In parallel, cells were also counted at the beginning and at the end of each experiment, and cell survival was quantified by Trypan blue exclusion assay.

Influenza virus replication. MDCK cells were infected with various strains of influenza virus depicted in the figures at an m.o.i. of 0.001 p.f.u. per cell for 1 h. Next, cells were washed with PBS and overlaid with OptiMEM containing two-fold compound dilutions ranging from 100 μΜ to 0.8 μΜ. Samples containing only the same volume of DMSO as the compounds were included. At 30 h after infection, culture medium was collected, and cell debris was removed by centrifugation at l,000g for 10 min and frozen at -80 °C. Viral titers were determined by plaque assay. The experiments conducted with the H1N1/1918 strain were performed in a high-containment (BSL3++) facility. For experiments performed with U20S cells, cells were plated in 12-well plates in DMEM containing 10% (v/v) FBS and incubated overnight. Cells were then incubated in medium containing tetracycline (1 μg/ml) for 2 h to induce REDD1 overexpression. Cells were washed with PBS and infected with A/WSN/1933 or VSV at m.o.i. 2 for 1 h. Tetracycline was again added 1 h after infection, and cell lysates were prepared at various time points after infection, as indicated in FIGS. 6A- F.

VSV replication assay. VSV replication: MDCK cells seeded in 35 mm-diameter dishes were infected with VSV-GFP at m.o.i. 0.001 p.f.u. per cell. At 24 h after infection, supematants were clarified and used for titration on Vero cells. Four-fold serial dilutions of virus containing supematants were made in PBS containing serum and antibiotics. Fifty microliters of each dilution were mixed with an equal volume of complete growth medium containing 8,000 Vero cells and incubated at 37 °C for 48 h in 96-well plates. Cells were fixed in 4% (v/v) paraformaldehyde. The number of wells with GFP expression were counted by fluorescence microscopy and subsequently used to calculate relative vims titers. Infection of U20S cells with VSV was performed in the same manner as influenza vims infection described above. In situ hybridization. mR A distribution in MDCK cells infected with influenza vims in the presence or absence of compounds was performed as previously describedl8. Influenza proteins were detected with mouse influenza-specific antibody (Biodesign International) and FITC-labeled mouse-specific antibody (Invitrogen).

Phospho-S6K analysis. Cells were starved for 18 h and then mock infected or infected as described in the legend of FIGS 5A-E. Five percent semm was added to induce S6K phosphorylation in control lanes. H358 and H1993 cells were treated with 10 μΜ 3, and LnCap cells were treated with 30 μΜ.

All data presented here are representative of at least three independent experiments. In the line graphs or histograms, data represent mean values + s.d.

Reagents. Human A549, 293T, LnCap, and MDCK cells were obtained from the

American Type Culture Collection and cultured in DMEM (Invitrogen) containing 10% fetal bovine semm and 1% penicillin (Invitrogen). Female ICR/CD-I mouse hepatocytes, InVitroGRO HI and HT Medium, and Celsis Torpedo Antibiotic Mix were purchased from Celsis/In Vitro Technologies (Baltimore, MD). The immortalized human bronchial epithelial cells (HBEC) (Ramirez et al., 2004) were obtained originally from John Minna (UT Southwestern) and were cultured in keratinocyte serum- free medium (SFM, Invitrogen). H358 and H1993 cells were also obtained from John Minna. REDDl cells are immortalized mouse embryo fibroblasts according to a 3T3 protocol (Vega-Rubin-de-Celis et al, 2010). The following antibodies were used for immunoblot analysis: anti-p70 S6 Kinase rabbit polyclonal (Cell Signaling, MA), anti-P-p70 S6 Kinase (T389) (108D2) rabbit monoclonal (Cell Signaling), anti-NXFl/TAP (Proteintech) and anti-P-Akt (S473) (D9E) rabbit monoclonal antibodies (Cell signaling); a-tubulin and γ-tubulin monoclonal antibody (Sigma); goat polyclonal anti-PBl antibodies (Santa Cruz Biotechnology). phospho-4E-BPl (Thr37/46) (236B4) Rabbit mAb (Cell Signaling Technology); REDDl (DDIT4) rabbit antibody (Novus Biologicals); β-actin (SIGMA); LC3 rabbit antibody (Novus Biologicals). Monoclonal antibody against Complex II subunit 70 kD (Mito-70 kD) was obtained from Mitoscience. Anti-VSV M protein antibody was generated against recombinant full-length protein. Anti-VSV proteins antibodies were a gift from G. Barber.

Plasmids. pCMVLuc expressing luciferase and pGAGGS-NSl expressing NS1 were used in the compound screen and validation assays. The UT Southwestern Compound Library is composed of 200,000 synthetic drug-like compounds purchased from commercial sources and arrayed in DMSO in 384 well plates.

Methods. Representative synthetic procedure for the synthesis of naphthalimide analogs Step 1 : Synthesis of 6-(6-chloro-l,3-dioxo-lH-benzo[de]isoquinolin-2(3H)- yl)hexanoic acid A solution of 4-chloro-l,8-naphthalic anhyrdie (10.0 g, 43 mmol) and 6- aminocaproic acid (5.6 g, 43 mmol) in N-methylpyrrolidone (100 mL) was heated to 110 °C in a 250 mL round bottom flask under an atmosphere of nitrogen. The reaction was monitored by LC/MS for product formation. After 90 minutes at 110 °C, the reaction was complete consumption of both starting materials. The reaction was cooled to room temperature and then diluted with ethyl acetate (250 mL) and a dilute aqueous NaCl solution (120 mL). The resulting layers were separated and the aqueous layer back-extracted with EtOAc (2 x 100 mL). The combined organic layers were then washed with water (10 x 100 mL) followed by a final brine wash (50 mL) and the dried over anhydrous Na₂S0₄. Concentration gave a crude dark solid (9.6 g) which was purified using normal phase silica gel chromatography (50% ethyl acetate in hexanes) to provide the desired naphthalimide as a light yellow amorphous solid (7.7 g, 52% yield). 1H NMR (CDC1₃) 8.65 (d, 1H), 8.60 (d, 1H), 8.52 (d, 1H) 7.9-7.8 (m, ²H), 4.2 (t, ²H), 2.39 (t, ²H), 1.76 (m, ⁴H), 1.5 (m, ²H). MS (ESI) 346 (M+H), 368 (M+Na). Step 2: Synthesis of analog 3 [6-(l,3-dioxo-6-(piperidin-l-yl)-lH- benzo[de]isoquinolin-2(3H)-yl)hexanoic acid] A solution of 6-(6-chloro-l,3-dioxo-lH- benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (74 mg, 0.22 mmol, from Step 1) in piperidine (2.0 mL) was heated to 80 °C in a sealed scintillation vial. The reaction was monitored by LC/MS for product formation. After 105 min, the reaction was complete by evidence of complete consumption of the starting material. The reaction was cooled and diluted with EtOAc (20 mL) and then washed with water (~10 x 5 mL) until the aqueous washings were clear. The combined aqueous layers were then acidified with 3N HC1 to a pH = 4.0 and then extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (5 mL) and dried over Na₂S0₄. The crude orange solid obtained after concentration was purified by recrystallization from EtOH to provide 3 as a light orange crystalline solid (40 mg, 46% yield). 1H NMR (CDC1₃) 8.58 (d, 1H), 8.49 (d, 1H), 8.38 (d, 1H), 7.53 (t, 1H), 7.18 (d, 1H), 4.20 (t, ²H), 3.21 (br m, ⁴H), 2.35, (t, ²H), 1.80 (m, ⁴H), 1.75 (m, ⁷H), 1.49 (m, ²H). MS (ESI) 395 (M+H), 417 (M+Na). ¹³C NMR (500 MHz, DMSO) of 3: δ 175.1 1, 164.17, 163.63, 157.31, 132.88, 131.21, 131.17, 129.76, 126.45, 126.06, 123.12, 115.65, 115.54, 54.63, 34.17, 28.00, 26.73, 26.40, 24.91, 24.54 Purity: 93% at 254 nm ¹³C NMR (500 MHz, DMSO) of 4: δ 175.12, 164.16, 163.64, 156.09, 149.45, 132.83, 131.32, 131.13, 130.16, 129.72, 128.75, 126.77, 125.98, 123.21, 116.56, 116.42, 115.81, 53.26, 49.56, 34.17, 28.00, 26.73, 24.91, 20.78. Purity: 91% at 254 nm,

RNA isolation and real-time RT-PCR. A549 cells were seeded in 35-mm-diameter dishes and infected with A/WSN/1933 virus at an m.o.i. of 0.001 pfu/cell. After one hour, cells were washed and compound-containing medium was overlaid onto the monolayers. At 36 h p.i., the medium was harvested to confirm compound activity by HA assay. From the same wells, total RNA was isolated using TRIzol reagent (Invitrogen) as recommended by the manufacturer. Isolated RNA was treated with 2 U of Turbo DNase (Ambion) at 37 °C for 30 min to remove potential genomic DNA contamination. Reverse transcription and real-time RT-PCR was performed by the Mount Sinai Microarray, PCR and Bioinformatics Shared Research Facility as described previously. In brief, cDNA was synthesized using Affiniscript RT (Stratagene) in combination with oligo dT18 primers (Integrated DNA Technologies) for cDNA synthesis of cellular genes or an influenza virus NP specific primer for cDNA synthesis of viral NP RNA. Real-time PCR was performed using Platinum Taq polymerase (Invitrogen) and SYBR Green I (Molecular Probes) using an ABI 7900HT real-time PCR machine. Primer nucleotide sequences are available upon request. The results of quantification were normalized to the amount of a-tubulin, β-actin and ribosomal protein S 11 mR A in the same sample. Each PCR was performed in triplicate, and median values and standard deviations were calculated. The amount of R A was determined with respect to standardized samples and expressed in relative units. A similar assay was performed to measure REDD1 mRNA levels in the absence or presence of actinomycin D (0.5 μ§/ι 1), as described in the legend of FIG. 5 A.

Gene Expression Analysis. 5 x 10⁵ A549 cells, in DMEM media (Gibco) supplemented with 10% FBS (Atlas) and 1% Penicillin (Gibco), were seeded overnight in 6- well plates. Compound 3, at 30 μΜ, or DMSO (0.3%) was added for 3 h, and RNA was isolated using the RNeasy Mini Kit (Invitrogen) following the manufacturer's instructions. cDNA was synthesized, labeled, and hybridized to an Illumina HumanRef-8 BeadChip 22K. After baseline correction and normalization of the expression data from the Illumina bead array, the inventors further filtered the expression profile by omitting entries with a p-value of 0.05 or greater. Fold-changes between test and reference-sets have been calculated. For genes with multiple oligonucleotide probes, fold-changes have been calculated prior to the calculation of averages. Only test and reference pairs for each probe were used when both p- values were at or below the 0.05 cutoff value. The inventors then subjected the post- processed gene expression data to Gene Set Enrichment Analysis (GSEA50) using the -fold changes as ranks within the Prerank algorithm of the GSEA software (world-wide-web at broadinstitute.org/gsea) with default parameters and 2000 permutations. The curated C2/CP "canonical pathways" set from the Molecular Signature Database at the Broad Institute has been used as reference gene sets for GSEA, consisting of sets of genes known to function in 639 pathways. Two enriched gene sets, corresponding to different branches of the same pathway were selected for further response network analysis:

1. MTORPATHWAY: 23 genes (20 genes enriched) on the mammalian target of rapamycin (mTOR) that senses mitogenic factors and nutrients, including ATP, and induces cell proliferation, from BioCarta (world-wide-web at biocarta.com/pathfiles/h_MTORPATHWAY.asp)

2. IGF 1 MTORPATHWAY: 20 genes (16 genes enriched) on the growth factor IGF-1 that activates AKT, Gsk3-beta, and mTOR to promote muscle hypertrophy, from BioCarta (world-wide-web at biocarta.com/pathfiles/h_IGFlMTORPATHWAY.asp). For each pathway, the set of enriched genes was used as seed-nodes for further network analysis. NetworkExpress, discussed below, was used to calculate response networks representing the particular response of enriched genes embedded in the constructed human biochemical network omitting interaction data from iHOP. Human biochemical network. To construct a hybrid Homo sapiens interaction and reaction network, protein-protein interactions with directional signal transduction and metabolic reactions were combined. Interaction information from IntAct (Kerrien et al.,

2007) , NetworKin (Linding et al., 2007), the Human Protein Reference Database (HPRD (Keshava et al, 2009) and from Palsson's group (H. sapiens Reconl@ (Pawson and Linding,

2008) yielded a network of -40,000 nodes (genes, proteins and small chemicals) as well as -200,000 interactions (gene-protein, protein-protein) and reactions (chemical, protein- phosphorylation, etc.). In addition, curated information on the influenza virus life-cycle and on host-interactions with influenza factors from the Reactome database (http://www.reactome.org; http://www.reactome.org) were included. The inventors have also integrated the human biochemical network above with a larger literature based network available from iHOP (Hoffmann and Valencia, 2004) with 45,041 nodes and 438,567 interactions, which are about 2/3s of 650,000 interactions predicted by Stumpf et al. (2008). As a third reference network, the Homo sapiens protein interaction network was downloaded from the BioGRID database version 2.0.39 (Stark et al., 2006), which was generated from literature curation of protein interaction data. The data set was filtered to include only direct and physical interactions between human proteins. All loops and duplicate edges were removed. However, duplicate edges from different data sources and different property (e.g. , an interaction identified as generic protein-protein interaction in one data-set and predicted as phosphorylation of a protein by a kinase in another data-set) were kept to emphasize the importance/validity of such interactions.

Network Analysis Tool. The inventors have previously developed a computational method to identify response networks in large biological networks based on expression data (Cabusora et al., 2005; Mawuenyega et al., 2005). This method and the corresponding computer program NetworkExpress are based on superimposing expression values upon the large network, identifying k-shortest paths (Eppstein, 1998; Hershberger et al., 2003) between seed-nodes, scoring the sub-network spanned by the set of k-shortest paths that are shorter than a pre-defined maximum weighted length 1, and finding the best scored subnetwork by optimization techniques. The inventors have a variety of scoring functions available, from simple arithmetic or geometric means to different types of correlation functions for time-series correlations, optionally between same time-points or timeforward/backward. The best-scored sub-network refers to the response network of the system under the specific environmental condition measured by the corresponding expression experiment. NetworkExpress also performs a statistical analysis to validate the significance of the identified sub-graph by comparison to randomly sampled sub-networks using a Monte Carlo approach.

Protein Synthesis. MDCK cells were seeded into 35 mm dishes. Fifty μΜ of 3 or 8 was added for 6, 24, and 48 hrs. Cells were pulsed with 100 μθ 35S-Met for 20 min, harvested, lyzed, and centrifuged at 14,000 x g for 10 min. Supernatant (25μ1) was then blotted on Whatman filter paper (approximately 1 square inch) and allowed to dry. The filters were soaked individually in ice cold 10% TCA for 30 min. Filters were then washed for 5 min with 1 : 1 ethyl ether/ethanol, and again in ethyl ether alone for 5 min. The filters were air- dried and radiation was measured in a scintillation counter.

EXAMPLE 2 - RESULTS

Na hthalimides antagonize NSl and influenza virus. The inventors exploited the potent ability of NSl to inhibit gene expression by blocking mRNA processing and export (Nemeroff et ah, 1998; Satterly et ah, 2007) as the basis for a high-throughput assay that measured the effect of NSl on luciferase expression. Luciferase activity was reduced -95% in cells transfected with plasmids encoding NSl compared to cells expressing luciferase alone, as we previously reported (Satterly et ah, 2007). The inventors screened 200,000 compounds at 5-μΜ concentrations with this assay and then counterscreened for the ability of these small molecules to suppress cytotoxicity caused by influenza virus infection (FIGS. 1 A- B). Among the most active compounds was 4-[N-4-nitro-(l,8-naphthalimide)]-butanoic acid, compound 1 (FIG. 1C). The inventors obtained compounds structurally related to 1 and identified some that had no antiviral activity (2) or more potent activity (3) (FIG. 1C; FIG. 7). Compound 3 was much less cytotoxic (FIG. 2A) and had a much longer half-life than the original compound (1) (FIG. 2B). Compound 3 also did not alter bulk protein synthesis (FIG. 8). To investigate the effect of 3 on virus-mediated cytotoxicity, the inventors infected MDCK cells with influenza A/WSN/1933 virus at a multiplicity of infection (m.o.i.) of 0.001 for 48 h in the presence or absence of 3. Widespread cytopathic effects were observed in MDCK cells in the absence of 3 after 48 h of infection, but 3 largely prevented this effect (FIG. 2C). Because this compound was derived from a screen in which the inventors observed a reversal of NSl -mediated inhibition of gene expression, the inventors expected that a significant reversal of the mRNA-export block induced by influenza virus would occur in the presence of active compound. Indeed, in cell populations infected with influenza virus in the presence of 3, there was a decrease in the number of cells that retained poly(A) RNA in the nucleus compared to the number of infected cells not treated with 3 (FIGS. 2D-E). A subpopulation of infected cells still presented mRNA export block in the presence of 3; thus, it is possible that these cells are at different phases of the cell cycle, which is known to regulate mRNA export (Chakraborty et al., 2008). Thus, 3 partially antagonizes the mRNA export block in virus-infected cells.

Naphthalimide inhibits virus replication. The effect of 3 on virus replication was then assessed using various strains of influenza virus: A/WSN/1933, A/Texas/ 1991 and the highly virulent A/Brevig/Mission/1/1918 strain that killed ~30 million people (Tumpey et al., 2005) (FIGS. 3A-C). Noncytotoxic concentrations of 3 reduced viral titers by 10³ to 10⁶ between 24 to 36 h after infection, depending on the influenza virus strain. The ratio of the concentration causing half-maximum cytotoxicity (CC50) to half-maximum inhibitory concentration (IC₅₀) for 3 was 31 (FIGS. 2A and 3A). Similar results were also observed in human A549 cells (FIG. 9). As shown in FIG. 3D, intracellular influenza virus proteins were also down-regulated in the presence of 3. Thus, 3 decreased viral protein levels, contributing to the reduction of virus replication.

The antiviral effect of 3 was not mediated by IFN. The mRNA levels of IFN-β and IFN effectors were measured by quantitative PCR and microarray analysis, and the results revealed that 3 did not induce IFN production or an IFN-mediated response (FIG. 3E). Furthermore, cells that had impaired IFN response, Vera cells and StatlV-cells, were protected by 3 from influenza virus replication and cell death, respectively (FIGS. 10A-B). Compound 3 also antagonized expression of large amounts of influenza virus proteins in Vera cells (FIG. 11). Thus, 3 partially antagonized the block of mRNA export in virus- infected cells, but this effect did not result in the production of IFN. However, the partial relief of mRNA-export inhibition by 3 was probably a consequence of low NS1 concentrations, which resulted in the expression of host mRNAs that encode antiviral factors. To investigate whether 3 antagonized NS1 directly or promoted host antiviral functions regulated by NS 1 that could also affect replication of other viruses, we infected cells with VSV at 0.001 plaque-forming units (p.f.u.) per cell in the absence or presence of 2 or 3. Compound 3 inhibited VSV replication (FIG. 3F). Thus, 3 targets host cell functions that confer an antiviral state against diverse viruses.

Antiviral activity of naphthalimide requires REDD1. Because 3 targeted the host, we analyzed host pathways by comparing gene expression profiles in human A549 cells in the presence or absence of compound using gene set enrichment analysis (FIG. 12). In cells treated with 3, the mTORCl pathway had one of the highest enrichment scores. REDD1, an inhibitor of the mTORCl pathway (Brugarolas et al., 2004; Ma and Blenis, 2009), was upregulated at the mRNA level (FIG. 4A). The induction of REDD1 mRNA by 3 was abolished in the presence of the transcription inhibitor actinomycin D (FIG. 4A). In addition, REDDl mRNA decayed over time in the absence or presence of 3 and actinomycin D (FIG. 4A). Thus, these results indicate that induction of REDDl mRNA by 3 occurs at the transcriptional level. REDDl protein levels increased approximately six- to eight-fold in the presence of 3 alone or in the presence of both 3 and influenza virus infection (FIG. 4B). Again, this induction of REDDl protein by 3 was abolished in the presence of actinomycin D (FIG. 4B). The inventors found that influenza virus greatly increased the degree of phosphorylation on S6 kinase (p70-S6K) at Thr389 (FIG. 4C), a site phosphorylated by mTORCl, and this effect was greatly reduced in A549 cells treated with 3 (FIG. 4C). That the total amount of S6K protein did not change in the presence of 3 (FIG. 4C) demonstrates that the effect of this small molecule occurred at the level of phosphorylation of S6K at Thr389. The mTORCl inhibitor rapamycin also reduced the amount of influenza virus NS1 protein (FIG. 13).

To investigate whether 3 prevented S6K activation independently of influenza virus, the inventors tested the effect of 3 in H358 non-small cell lung cancer cells, which have chronically active S6K. Cells were treated with 3 and inactive 2, and 3, but not 2, reduced the activation of S6K in H358 cells (FIG. 14A). In two other cancer cell lines with chronically active AKT, H1993 and LnCAP, 3 also reduced the activation of S6K (FIG. 14B). However, 3 did not inhibit phosphorylation of a major active site, Ser473, on AKT (FIG. 4D), which is a target of mTORC2 (24,25). In A549 cells infected with influenza virus for 7 h, 3 blocked S6K activation and had no effect on AKT phosphorylation (FIG. 4D). At 22 h after infection, 3 did not alter phosphorylation at AKT Thr308 but reduced phosphorylation at AKT Ser473; however, this reduction is probably an indirect effect of 3 on the inhibition of viral replication rather than a direct effect of 3 on AKT. Thus, 3 acts in parallel to or downstream of AKT.

To determine whether REDDl was required for the antiviral activity of 3, we tested the antiviral effect of 3 in infected REDD1+/+ or REDD1V- mouse embryonic fibroblasts. Influenza virus-mediated cell death and replication were inhibited by 3 in REDD1+/+ cells infected at m.o.i. 0.01 for 72 h (FIG. 4E); the inventors infected the cells at 72 h because, at this point, enough cell death had occurred so that the inventors could determine protection by 3. Infected REDDIV- cells treated in the same conditions as REDD1+/+ cells were completely dead by 24 h in the presence or absence of compound; therefore, the inventors infected REDDl^- cells with influenza virus at m.o.i. 0.001 for 48 h, in the absence or presence of 3. Even with this low m.o.i. and short infection time, 3 did not protect REDD IV- cells from virus-mediated cell death or virus replication (FIG. 4F). In addition, REDD IV- cells infected at m.o.i. 0.001 for 48 h produced approximately as many viral particles as REDD1+/+ cells infected at m.o.i. 0.01 for 72 h (FIGS. 4E-F). When REDD IV- cells were infected with influenza virus at m.o.i. 0.001, they produced ~200-fold more virus than REDD1+/+ cells infected in the same conditions (FIG. 15). This effect was also observed in VSV-infected REDD1+/+ and REDDl-/- cells (described further below). Treatment of both REDD1+/+ and REDDIV- cells with 3 alone did not cause cytotoxicity (FIG. 16). Thus, REDDl knockout cells were more permissive to influenza virus replication than wild-type cells. As 3 did not inhibit virus replication in the absence of REDDl (FIG. 4F), REDDl is required for the antiviral activity of 3.

REDDl is a host defense factor. These data indicate that REDDl is an important host factor required for antiviral response, raising the possibility that viruses regulate REDDl expression. During influenza virus and VSV infections, REDDl expression initially increased but was then downregulated (FIGS. 5A-B), resulting in activation of S6K (FIG. 4C). The initial upregulation of REDDl probably represented a host antiviral response, which was then inhibited by the virus, resulting in activation of mTORCl . Consistent with REDDl 's involvement in a general host-cell antiviral response, REDDIV- cells were also highly permissive to VSV replication compared to wild-type cells (FIGS. 5C-D), resulting in higher levels of intracellular VSV proteins in REDDl-/- cells than in REDD1+/+ cells (FIG. 17). That 3 did not inhibit VSV replication in the absence of REDDl, as it did in REDD1+/+ cells, shows once again that REDDl is required for its antiviral activity (FIGS. 18A-C).

By preventing viruses from activating mTORCl, REDDl might affect two biological functions potentially important for virus replication: autophagy or protein translation. By preventing activation of mTORCl, enhanced REDDl expression may increase autophagy (Rubinsztein et ah, 2007). However, compound 3 protected ATG5V- cells, which lack an autophagic response, against VSV replication (FIGS. 18A-C). In addition, treatment of cells with chloroquine, an autophagy inhibitor, did not affect the amount of viral protein in REDDIV- cells (FIG. 19). Together, these results indicate that autophagy was not the mechanism involved in 3-mediated inhibition of viral protein expression. Thus, the requirement for activating mTORCl for efficient virus replication is likely to be translation.

To determine whether the enhanced viral infection in REDDIV- cells was due to a general increase in translation or an effect on specific viral proteins, the inventors measured the expression of several influenza virus proteins as a function of time after infection of both REDD1 wild-type and knockout cells. The inventors subjected lysates from REDD1+/+ and REDDlV-cells infected with influenza virus to immunoblot analysis with antibodies against various influenza virus proteins. REDD IV- cells produced large amounts of influenza virus proteins 2-3 h earlier than REDD1+/+ cells (FIGS. 6A-B). The enhanced expression of viral proteins led to increased viral RNA levels (FIG. 20), and the inventors observed similar results upon VSV infection (FIG. 17). To determine whether the effect on viral proteins in REDDl-/- cells was due to the activity of mTORCl in translation, the inventors treated REDD IV- cells with rapamycin. In fact, the down-regulation of viral protein caused by rapamycin treatment in both REDD1+/+ and REDD IV- cells (FIG. 6C) (FIG. 21) indicated that induction of high viral protein levels in REDD IV- cells occurs via activation of mTORCl . Furthermore, in cells conditionally expressing large amounts of REDDl, the concentration of viral protein was reduced, consistent with the function of REDDl as a host defense factor (FIG. 6D).

REDDl prevents the inactivation of the TSC1-TSC2 complex by AKT1 and, thus, blocks activation of the mTORCl pathway (Brugarolas et al., 2004; Vega-Rubin-de-Celis et al., 2010). In TSC2-knockout cells, 3 did not induce downregulation of viral protein expression, as opposed to wild-type cells in which viral protein levels were inhibited by 3 (FIGS. 6E-F). In addition, activation of S6K in REDD IV— infected cells was not inhibited by 3 (FIG. 22), indicating that 3 does not act directly on S6K. Thus, 3 requires TSC2 for downregulating viral protein expression (FIGS. 6E-F). Altogether, these findings show that the antiviral activity of 3 occurs by repressing the activity of mTORCl in a TSC1-TSC2- dependent manner. It is also possible that 3 may act on other pathways. The inventors also designed an analog of 3, termed 4, which has similar antiviral properties to 3. Compound 4 prevented virus replication by inducing REDD 1 but was a more potent inhibitor of the highly pathogenic H1N1/1918 influenza virus strain (FIGS. 23A-C) (Tumpey et al, 2005 ). Altogether, these findings reveal REDDl as a new host antiviral factor and show that the antiviral activity of 3 requires REDD 1.

EXAMPLE 3 - DISCUSSION

There are essentially two approaches for identifying unique host processes that are involved in antiviral functions and can be exploited therapeutically. One is to learn as much as possible about the host mechanisms required by the virus and then test the effects of inhibiting them. The other is to take an unbiased approach and screen for chemical inhibitors of virus functions or host genes required by the virus. Taking the chemical-genetics version of the second approach, the inventors conducted a screen for compounds that antagonized the inhibition of gene expression by NS 1 and identified napthalimides that inhibited replication of influenza viruses and VSV. These compounds functioned by increasing expression of REDD1, a major negative regulator of the mTORCl pathway, and in cells lacking REDD1 the compound lost its antiviral activity.

Many viruses activate AKT by stimulating PI3K (Buchkovich et al, 2008; Cooray, 2004). The direct binding of NSl protein of influenza virus to PI3K results in activation of AKT (Ehrhardt et al, 2007; Hale et al, 2006; Shin et al, 2007; Zhirnow and Klenk, 2007). This has been interpreted either as a means to inhibit apoptosis and prevent the cell from dying prematurely during infection or as a necessary step in promoting virus replication. A recent genome-wide siRNA screen implicated mTORCl in influenza virus replication (Konig et al, 2010), suggesting that activation of that pathway might be one of the functions of elevated AKT1 signaling. These results imply that a major consequence of AKT signaling for influenza virus replication is activation of the mTORCl effector S6K through phosphorylation, as the antiviral napthalimides we identified inhibited phosphorylation of S6K by mTORCl . The inventors showed that the protein upregulated by our napthalimides, the mTORCl inhibitor REDDl, is a new host defense factor. Its production was at first induced by influenza virus or VSV but was then successfully suppressed by the virus. REDDl suppression by viruses promoted virus replication, as REDDl -knockout cells were highly permissive to virus replication.

REDDl is induced by various environmental conditions, including cell confluency, glucocorticoid treatment, hypoxia and other stress-response pathways such as endoplasmic reticulum (ER) stress (Brugarolas, 2010). Both ER stress and hypoxia-inducible factors (HIFs) have a role in immunity and infection (Todd et al., 2008; Zinkernagel et al., 2007). ER stress has been shown to promote plasma-cell development, and the absence of key components in this pathway results in sensitization to viral infection (Todd et al., 2008). Mouse embryonic fibroblasts deficient in the ER protein kinase PERK, which is activated by accumulated unfolded proteins in the ER, are more permissive to VSV replication than wild- type cells (Krishnamoorthy et al, 2008). Upregulation of REDDl in response to ER stress (Jin et al, 2009; Whitney et al, 2009) occurs via the transcription factor ATF4 (Whitney et al, 2009). HIF activation by the hypoxia mimetic cobalt chloride promotes cellular resistance to VSV infection, whereas HIF inhibition by RNA interference or a small molecule antagonist has shown increased sensitivity to viral infection, as measured by enhanced VSV cytotoxicity and replication (Zinkernagel et al., 2007); however, the mechanism is not known. During hypoxia, REDD1 has been shown to be a direct target of the HIF-l transcription factor (Brugarolas et al., 2004), which induces REDD1 expression. Thus, activating a stress response pathway or promoting the expression of a stress-response protein may to a certain extent induce resistance to pathogens and decrease host cytotoxicity. However, the coordination of a stress response to promote cellular resistance without marked damage to the host upon pathogen invasion remains to be further investigated.

The inventors showed that induction of REDD 1 by small molecules is an efficient strategy for interfering with the functions of the mTORCl pathway that are required by viruses. The effect of napthalimide on influenza virus was a sharp attenuation of the production of virus proteins early in infection. The inventors found no effect of the napthalimide on global protein synthesis and no induction of an IFN response. In addition, in cells lacking REDD1, in which expression of influenza virus proteins is enhanced, rapamycin inhibited expression of influenza virus proteins at a concentration that is known not to alter bulk protein synthesis. This indicates selective translational regulation, which has been documented in a number of conditions, including the general amino acid control response (Costa-Mattioli et al., 2009) and other types of processes such as survival or proliferation (Shahbazian et al., 2010). In addition, during nuclear mRNA processing and export, specific sequences within either the untranslated regions or the coding region can dictate the differential binding of RNA-binding proteins (specifically, heterogeneous ribonucleoprotein particles), which will in turn regulate processing and export of specific subsets of mRNAs to result in differential expression (Farny et al., 2008; Keene, 2007). This raises the possibility that the inhibition of the mTORCl pathway may alter translation in a way that is unfavorable to the initiation of specific viral mRNA expression, relative to that of host mRNA. In cells infected at a low m.o.i., the first viral messages must compete with the far larger volume of host messages for access to ribosomes. In this respect, the early viral messages would encounter the same problems as a hostcell message with low abundance, such as mRNAs encoding certain transcription factors. However, at the earliest phases of infection, viruses are largely dependent upon normal host processes, and these processes are the most likely to be the useful therapeutic targets.

Although many viruses can be controlled by vaccination, there is still an important need for antiviral drugs. For viruses that can infect other animals, such as influenza, vaccination will never lead to full eradication. Other human viruses, such as smallpox or measles, can potentially be eradicated by global immunization, but, once the incidence of such diseases becomes very low, global vaccination is inevitably discontinued, leaving the human population vulnerable to reemergence of the virus. The long lead times required to produce sufficient vaccine to protect the human population means that the appearance of a new or reoccurring highly infectious virus can lead to a pandemic of the disease before the vaccine is available, which means that antiviral drugs can be a key boon at such a time. However, antiviral drugs that target viral proteins have the disadvantage that resistance to the drug will arise because of the high rates of mutation inherent in viruses and the large numbers of progeny that they produce. A strategy targeting host processes that are essential for virus replication, such as the one discussed here, avoids this problem, although it is limited by the possibility of toxic side effects. Thus, combinations of non-cytotoxic small molecules that target both viral and host proteins are desirable. Recently, influenza A nucleoprotein was identified as an antiviral target (Kao et al., 2010), and a small molecule that triggered its aggregation and prevented its import into the nucleus protected against influenza virus replication45. In addition, a chemical compound that inhibited host pyrimidine biosynthesis has been recently shown to reduce influenza virus replication (Hoffmann et al., 2011).

In sum, the strategy of chemically inducing host antiviral activities that target host pathways without causing considerable short-term toxic effects will probably have a major impact on antiviral therapy. One such strategy was identified here with the induction of the mTORCl inhibitor REDD1 by naphthalimides. Furthermore, small molecules that inhibit the mTORCl pathway in different ways have the potential for anticancer therapy, as the mTORCl pathway is a major regulator of cell proliferation and cancer (Choo and Blenis, 2009).

All of the methods and apparatuses disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and apparatuses and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A compound of the formula:

wherein:

n is 2, 3, 4, 5, 6, 7 or 8;

A is -CHRi- -0-, -NRi- or -S-; and

Ri is:

hydrogen, hydroxy or amino; or

alkyl₍c<i2), alkenyl_(C<i₂), alkynyl_(C<i₂), aryl_(C<₁₂), aralkyl_(C<i₂₎, hetero- aryl(c≤i2), heteroaralkyl_(C<i₂), acyl_(C<i2), alkoxy_(C≤i2), aryloxy_(C<i2), aralkoxy_(C<i2), heteroaryloxy_(C<i2), heteroaralkoxy(c<i2), acyloxy(c≤i₂), alkylaminO(c<i₂), dialkylamino(c≤i2), arylamino(c<i2), aralkylamino(c≤i2), heteroarylamino(c<i2), heteroaralkylaminO(c<i2), amido(c≤i2), or a substituted version of any of these groups;

or a pharmaceutically acceptable salt or tautomer thereof.

The compound of claim 1 , wherein n is 5.

The compound of claim 1 , wherein A is -CHRi^~.

The compound of claim 3, wherein Ri is hydrogen.

The compound of claim 1 , wherein A is -0-.

The compound of claim 1 , wherein A is -NRi^~.

7. The compound of claim 6, wherein Ri is hydrogen.

8. The compound of claim 6, wherein Ri is heteroaryl(c<8).

9. The compound of claim 8, wherein Ri is pyrimidyl.

10. The compound of claim 6, wherein Ri is aryl_(C<8).

1 1. The compound of claim 8, wherein Ri is methylphenyl.

12. The compound of claim 6, wherein Ri is heteroatom-substituted aryl_(C<8).

13. The compound of claim 12, wherein Ri is methoxyphenyl or trifluoromethylphenyl.

14. The compound of claim 1 , wherein A is -S-.

15. The compound of claim 1 , selected from the group consisting of:

A method of treating or preventing a viral infection in a patient comprising administering to said patient an effective amount of compound having the formula:

wherein:

n is 2, 3, 4, 5, 6, 7 or 8;

A is -CHRi- -0-, -NRi- or -S-; and

Ri is:

hydrogen, hydroxy or amino; or

alkyl₍c<i2), alkenyl_(C<i₂), alkynyl_(C<i2), aryl_(C≤12), aralkyl_(C<i₂), hetero- aryl(c≤i2), heteroaralkyl_(C<i₂), acyl_(C<₁₂), alkoxy_(C<i2), aryloxy₍c<i2), aralkoxy₍c<i₂), heteroaryloxy₍c<i₂), heteroaralkoxy(c<i2), acyloxy(c≤i₂), alkylaminO(c≤i₂), dialkylamino(c≤i2), arylamino(c≤i₂), aralkylamino(c≤i₂), heteroarylamino(c≤i2), heteroaralkylamino(c≤i₂), amido(c≤i₂), or a substituted version of any of these groups;

or a pharmaceutically acceptable salt or tautomer thereof.

17. The compound of claim 1 , wherein n is 5.

18. The method of claim 16,wherein A is -CHR]-.

19. The method of claim 18, wherein Ri is hydrogen.

20. The method of claim 16, wherein A is -0-.

21. The method of claim 16,wherein A is -NRi^~.

22. The method of claim 21 , wherein Ri is hydrogen.

23. The method of claim 21 , wherein Ri is heteroaryl(c<8).

24. The method of claim 23, wherein Ri is pyrimidyl.

25. The method of claim 21 , wherein Ri is aryl(c<8).

26. The method of claim 23, wherein Ri is methylphenyl.

27. The method of claim 21 , wherein Ri is heteroatom-substituted aryl₍c<8).

28. The method of claim 27, wherein Ri is methoxyphenyl or trifluoromethylphenyl.

29. The method of claim 16, wherein A is -S-.

30. The method of claim 16, wherein the compound is selected from the group consisting of:

31. The method of claim 16, wherein the viral infection is caused by the influenza virus, a bunyavirus, an arenavirus, an encephalitis virus, rabies or a filo virus.

32. The method of claim 16, wherein the viral infection is influenza A.

33. The method of claim 16, wherein the viral infection is influenza B.

34. The method of claim 16, wherein the method of administration is selected from the group consisting of an inhaled aerosol, nasal spray, suppository, oral formulation and injection.

35. The method of claim 16, wherein the dose of the compound that is administered is about 1 mg/kg to about 50 mg/kg.

36. A method of inhibiting an NS1 pathway in a cell comprising contacting said cell with an effective amount of a compound of claim 1.

37. The method of claim 36, wherein the cell is in vitro.

38. The method of claim 36, wherein the cell is in vivo.

39. A method of inhibiting influenza A virus cytopathic effect in a cell comprising administering to said cell an effective amount of a compound of claim 1.

40. A method of reducing the severity or duration of a viral infection in a patient comprising administering to said patient an effective amount of a compound of claim 1.

41. The method of claim 40, wherein the viral infection is influenza.

42. The method of claim 40, wherein the viral infection is caused by the influenza A virus.

43. The method of claim 40, wherein the viral infection is caused by the influenza B virus.

44. A method of treating or preventing a viral infection in a patient comprising administering to said patient an effective amount of a compound of claim 1 in combination with a neuraminidase inhibitor or an M2 proton channel inhibitor.

45. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, and/or excipient and a compound of claim 1.