WO2014031571A1 - Treatment of viral infections - Google Patents

Description

TREATMENT OF VIRAL INFECTIONS

[00 i J This invention was made with government support under grants

HHSN26620070010C and IJO i All 074539 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[002] Priority is claimed to provisional application No. 61 /691 ,701 , which was filed in the United States Patent and Trademark Office on August 21 , 2012 and is incorporated herein by reference, in its entirety.

FIELD OF THE INVENTION

[003] The present disclosure is related to compounds that inhibit the replication of negative-sense, single -stranded RNA viruses, such as influenza virus, and the use of those compounds for the treatment or inhibition of viral infection.

BACKGROUND OF THE INVENTION

[004] Influenza virus is a member of the Orthomyxoviridae family. The virus attaches to the ceil surface via sialic acid-containing receptors and enters the cell via pH- dependent endoc tosis. The acidic environment of the endosome triggers fusion of the viral and endosomal membranes, leading to the release of the viral ribonucieoprotein complex (vRNP) into the cytoplasm. The vRNPs - which comprise viral RNA (vRNA), nucleoprotein (NP) and the polymerase proteins FB I , PB2 and PA - then dissociate from the matrix protein (Ml) and enter the nucleus, where vRNA replication and transcription occur. See Palese & Shaw, "Orthomyxoviridae: the Viruses and Their Replication" In: Knipe & Howley (Eds.) Fields Virology, fifth ed., Lippincott Williams and Wilkins, Philadelphia, pp. 1647-1689 (2007). Newly synthesized vRNPs are subsequently exported from the nucleus through the chromosome region maintenance i protein (CRMi)-mediated pathway. Watanabe et ah, Virus Res. (2001 ) 77:31 -42. Virus assembly is mediated by the Ml protein, via interactions with viral membrane proteins [e.g. , hemagglutinin (HA), neuraminidase (MA) and the M2 ion channel] and vRNP complexes. See, AH et al., J. Virol. (2000) 74:8709-8719; Ye et al, J. Virol. (1999) 73 :7467-7473. Virion release from the cell surface is dependent upon neuraminidase activity of MA. Palese & Shaw (2007), supra.

[005] Influenza vims continues to be a significant health and economic burden worldwide. Although drugs are currently available for the treatment of influenza, several strains of the virus have developed resistance to these drags. See, for example, Bright et al, JAMA (2006) 295(8):891 -894; Ison, Curr. Qpiri. Virol. (2011) 1 :563-573. Currently available anti-influenza drugs inhibit viral proteins, such as MA or the M2 ion channel protein. See, for example, Garman & Laver, Curr. Drug Targets (2004) 5:1 19- 136; Pinto and Lamb, Trends Microbiol (1995) 3:271 ; Wharton et al., J. Gen. Virol. (1994) 75(Pt. 4):945- 948. However, the error prone viral polymerase provides the virus with many opportunities to develop resistance.

[006] Konig et al. {Nature (2010) 463:813-817) describe a genome-wide siRMA screen and report that it identified 295 cellular cofactors required for early-stage influenza virus replication. However, Konig et al. are not believed to identify any specific targei(s) for the inhibition or treatment of influenza, and likewise do not identify any drugs or compounds that might be useful for the treatment or inhibition of influenza, international patent publication Mos. WO 2012/018540 and WO 2012/166586 describe compounds that are said to be useful as antiproliferative agents and kinase inhibitors. The compounds are said to be useful for methods of treating cellular proliferative disorders, such as cancer. These publications also mention that the compounds might be useful for treating certain non-cancer proliferative disorders associated with latent viral infections, such as Epstein-Barr virus and/or Hepatitis C. However, the publications to not identify compounds or methods that might be useful for treating virus infections themselves and, in particular, do not describe compounds or methods that might be useful for treating or inhibiting an Orthomyxovindae virus infection, such as influenza virus.

[007] Hence, there is a need to develop new treatments against influenza, as well as other viruses, including a need to develop new drags and compounds that may be useful for such treatments. SUMMARY OF THE INVENTION

[008] The present application is directed to new antiviral agents for the treatment and inhibition of infection with RNA viruses, such as, without limitation influenza viruses, vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV),

[009] Thus, provided herein is a composition comprising a compound having the structure:

or a derivative thereof. For convenience, the foregoing compounds are also identified by the designations ONI 081 10 and ON 123790, respectively, throughout this specification. These and/or similar compounds are described in International Patent Publication Nos.

WO 2012/166586 (Compound 7) and WO 2012/018540 (Compound 15), as well as methods for their- synthesis. However, methods and uses of those compounds according to the present invention are not believed to have been previously disclosed.

[0010] Also provided herein is a method for inhibiting the replication of a negative- sense, single- stranded RNA virus in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising an inhibitor of a kinase selected from the group consisting of CAMKK1 , CAMKK2, CDKl/cyclin A, CDKi/cyclin B, CDK2/cyclin A, CDK4/cyclin Dl , CDK4/cyclin D3, CDK5/p25, CDK5. p35, CDK6/cyclin Dl , CDK6/cyclm D3, CDK cyclin K, CDK9/cyclin Ti, CK2a, CK2a2, DYRK1B, DYRK2, GSK3a, GSK3b, HGK/MAP4K4, MLK3/MAP3K1 1, MUSK, NLK, PIM3, and SGKl . In certain aspects of the method, the composition comprises a compound having the structure:

or a derivative thereof.

[001 1 ] Also provided herein is a method of treating or inhibiting a viral infection in a subject, the method comprising administering to a subject in need thereof an inhibitor of a kinase selected from the group consisting of CAMKKl, CAMKK2, CDKl/cyclin A, CDKl/cyclin B, CDK2/cyclin A, CDK4/cyclin Dl, CDK4/cyciin D3, CDK5/p25, CDK5/p35, CDK6/cyciin Di , CDK6/cyclin D3, CDK9/cyclin K, CDK9/cyciin TL CK2a, CK2a2, DYR.K1B, DYRK2, GSK3a, GSK3b, HGK MAP4K4 , MI.K3/MAP3 1 1 , MUSK, NLK, ΡΪΜ3, and SGK l . in certain aspects, the composition comprises a compound having the structure:

or a derivative thereof.

[0012] In certain aspects of the methods provided herein, the virus to he inhibited is a member of a virus family selected from the group consisting of Orthomyxoviridae, Rhabdoviridae, and Paramyxoviridae. In still other aspects, the virus is selected from the group consisting of Influenza A, Influenza B, Influenza C, Infectious salmon anemia (ISA), Thogoto, vesicular stomatitis Indiana virus (VSV), Sendai vims, and Newcastle disease virus (NDV).

[001 3] These and other aspects of the present disclosure will be apparent to those of ordinary skill in the art in light of the present specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A to IE are bar graphs quantifying cell viability (expressed as percent (%) of DMSO control) following exposure to DMSO or the indicated concentrations (μΜ) of the kinase inhibitors, ON108050 (Fig. l A), ON108080 (Fig. IB), ON1081 10 (Fig. 1 C), ON 123300 (Fig. ID), and ON123790 (Fig. I E) in DMSO. [0015] Figure 2 shows the chemical structure of ONI 081 10.

[001 6] Figures 3A and 3B are bar graphs quantifying viability of uninfected A549

(Fig. 3A) and LA4 (Fig. 3B) ceils and viral titer in A549 (Fig. 3A) or LA4 (Fig. 3B) cells infected with influenza virus (A/WSN/33) at an MOl of 0.01, following incubation of the cells with DMSO or the indicated concentration (μΜ) of ONI 08110 in DMSO. "XT' means the cells were left untreated. R esults are expressed as percent of DMSO control.

[0017] Figure 4 is a bar graph quantifying viral entry into A549 cells, as determined by a luciferase reporter assay, incubated for 2 to 4 hours in the presence of ONI 08110 (3.13 uM), or solvent DMSO, or diphyilm (2.1 uM) as positive control, followed by exposure to pseudoparticies bearing influenza vims HA/NA proteins and encoding the Gaussia luciferase reporter gene.

[001 8] Figure 5 is a bar graph quantifying at 5 and 7 hours post infection (hpi) export of influenza vRNPs into the cytoplasm (expressed as percent nuclear accumulation of vRNPs) of A549 cells infected with influenza virus (A/WSN/33) and treated with ON1081 10 (3.13 μΜ), or solvent DMSO. Asterisks indicate statistical significance as determined using two-way ANOVA (pO.OOl).

[0019] Figures 6A and 6B are bar graphs quantifying viability of uninfected VSV

(Fig. 6 A) and NDV (Fig. 6B) and viral titer of VSV or NDV ceils infected with VSV-GFP at an MOl of 0,0001 (Fig. 6A) or NDV (B i strain) at an MOl of 2 (Fig. 6B), following exposure to DMSO or the indicated concentration (μΜ) of ON 108110 in DMSO. "U" means cells were left untreated. Results are expressed as percent of DMSO control.

[0020] Figures 7-10 are the structures of compounds ONI 08080, ON123300,

ONI 23790, and ON I 08050, respectively,

[0021] Figures 11A-11C depict exemplary synthesis schemes for synthesizing substituted 2-benzylidene-2H-benzo[b][l,4]thiazin-3(4H)-ones, such as ON3088110. The sysnthesis schemes are referred to as Schemes 1-3, respectively, in the examples below.

[0022] Figure 12 depicts an exemplary synthetic scheme for synthesis of the compound ON 10881 10. DETAILED DESCRIPTION OF THE INVENTION

Overview

[0023] Influenza A virus is a member of the Orthomyxoviriciae family of enveloped, negative-stranded RNA viruses. It binds to sialic acid-containing receptors and enters cells via pH-dependent endocytosis. The acidic environment of the endosome triggers fusion of the viral and endosomal membranes, leading to the release of the virai ribonuefeoprotein comple (vRNP) into the cytoplasm. The vRNPs, composed of the viral RNA (vRNA), nucleoprotein (NP) and the polymerase proteins (PB1 , PB2, PA), dissociates from the matrix protein (Ml) and enters the nucleus, where vRNA replication and transcription occur. Newly synthesized vRNPs are exported from the nucleus through the chromosome region maintenance i protein (CRMi)-mediated pathway. Virus assembly is mediated by the Ml protein, which interacts with viral membrane proteins hemagglutinin (HA), neuraminidase (NA), M2 ion channel protein and vRNP complexes. Virion release from the cell surface requires the neuraminidase activity of NA.

[0024] Currently available anti-influenza drugs inhibit virai proteins such as NA or the M2 ion channel protein, however resistance to currently available drugs in becoming an increasing problem. One way to circumvent drug resistance is to develop drugs that target host factors required for virai growth instead of viral proteins.

[0025] A genome-wide siRNA screen was performed and 295 host factors that are required for the replication of influenza virus were identified (Konig et al. (2010) Nature 463, 813-817), A majority of those host factors were kinases. Therefore, it was investigated whether kinase inhibitors can impair the replication of influenza virus.

[0026] As disclosed in Examples 1-6, below, the multikinase inhibitor ONI 08110 impairs the replication of influenza vims at concentrations that are not toxic to human and mouse lung cells. ON1081 10 was found to impair influenza vRNP nuclear export. ON1081 10 was also found to inhibit a number of kinases (listed in Table 2, below) that have not been previously shown to be involved in vRNP export. The kinases listed in Table 2 thus represent targets for the inhibition of influenza A virus infection and/or replication. The compound ONl OSi 10 was also found to inhibit two other negative-stranded RNA viruses, vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). ON I 081 10 and its derivatives are thus therapeutic agents for the treatment of viral infections, such as, for example and without limitation, infections with influenza virus and other negative stranded RNA viruses

Definitions

[0027] As used herein, the term "derivative", e.g., in the context of a derivative of a compound of the present disclosure (e.g., ONI 081 10), includes pharmaceutically acceptable salts, hydrates, solvates, prodrugs, analogs or other derivati ve of ON 1081 10 that maintain one or more of the functions of the compound. Preferably, such derivative maintains the ability to inhibit one or more of viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual.

[0028] As used herein, the term "infection" means the invasion by, multiplication and/or presence of a virus in a ceil or a subject. In one embodiment, an infection is an "active" infection, i.e., one in which the virus is replicating in a cell or a subject. Such an infection is characterized by the spread of the virus to other cells, tissues, and/or organs, from the cells, tissues, and/or organs initially infected by the virus. An infection may also be a latent infection, i.e., one in which the virus is not replicating. In one embodiment, an infection refers to the pathological state resulting from the presence of the virus in a ceil or a subject, or by the invasion of a cell or subject by the virus.

[0029] As used herein, the term "inhibit" is used to refer to a reduction in viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual. As used herein, the term "inhibitor" refers to a compound that decreases viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual. Viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another ceil, tissue or organ or to another individual can be determined using assays taught herein and/or as known to one of skill in the art.

[0030] The term "subject" or "individual" as used herein refers to an animal having an immune system, preferably a mammal (e.g., rodent, such as mouse). In particular, the term refers to humans.

[0031 ] As used herein, "treating" or "treatment" of a state, disorder or condition includes: ( 1) inhibiting the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0032] As used herein, the term "treating a viral infection" means that at least one symptom of the viral infection is decreased. Symptoms of viral infection (e.g., of influenza, parainfluenza, and infections with other negative-sense, single-stranded RNA virus) are well known to those of skill in the art. Treating a viral infection typically includes inhibiting one or more of viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another ceil, tissue or organ in the individual.

Compounds of the Invention

[0033] Methods of synthesizing substituted 2-benzylidene-2H-benzo[b][l ,4]thiazin-

3(4H)-ones, such as ON10881 10, are described in U.S. application no. 61/490,786, filed May 27, 201 1 and international application PCT/US2012/039544, filed May 25, 2012, which are incorporated herein by reference, and portions of which is reproduced herein as Synthesis Examples A and B. See also International Patent Publication No. WO 2012/166586 which published on December 6, 2012. An exemplary synthetic scheme for synthesis of compound ON 10881 10 is shown in Figure 12. Methods of synthesizing 2-substituted-8-alkyl-7-oxo- 7,8-dihydropyrido[2,3-D] pyrimidine-6-carbonitriles, such as ON123790 are described in international application PCT US201 1/044807, which published as WO 2012/018540 Ai, and which is incorporated herein by reference.

[0034] In certain embodiments, the present disclosure provides a composition comprising a compound having the structure:

or a derivative thereof. This compound is referred to in the present Examples as ONI 081 10.

[0035] Derivatives of ON1081 10 maintain one or more of the functions of

ON1081 10. For example, in one embodiment, a derivative (e.g.,. pharmaceutically acceptable salt, hydrate, solvate, prodrug, or analog) of QN1081 10 maintains the ability to inhibit one or more of the kinases: CAMKK1, CAMKK2, CDKl/cyclin A, CDKl/cyclin B, CDK2/cyclin A, CDK4/cyclin Di, CDK4/cyclm D3, CDK5/p25, CDK5/p35, CDK6/cycIin Di, CDK6/cycIin D3, CDK9/cyclin K, CDK9/cyciin Ti , CK2a, CK2a2, DYRKIB, DYRK2, GSK3a, GSK3b, HGK/MAP4K4, MLK3/MAP3K1 1 , MUSK, NLK, PIM3, and SGKI ,

[0036] In another embodiment, the present disclosure provides a composition comprising a compound having the structure:

or a derivative thereof. This compound is referred to in the present Examples as ON123790.

[0037] In another embodiment, the present disclosure provides an inhibitor of one or more of the following kinases: CAMKK 1 , CAMKK2, CDKl/cyclin A, CDKl/cyclin B, CDK2/eyeim A, CDK4/cyclin DI, CDK4/cyclin D3, CDK5/p25, CDK5/p35, CDK6/cyclin DI, CDK6/cyclin D3, CDK9/cyclm K, CDK9/cyclin TI , CK2a, CK2a2, DYRKI B, DYRK2, GSK3a, GSIOb, HGK/MAP4K4, MLK3/MAP3K1 1, MUSK, NLK, PIM3, and SGKI . Non- limiting examples of kinase inhibitors are provided in Table I , below: Table 1 : Exemplary Kisiase Inhibitors

[0038] Additional compounds (e.g., selected from a compound library) that inhibit the above-disclosed kinases can be identified using methods well known in the art. Further, the screening assays and in vivo testing described below can be used to determine whether the candidate compound has the desired activity, such as the ability to inhibit one or more of viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a vims from a cell, tissue, or organ to another ceil, tissue or organ or to another individual (subject).

Com ou nd Screening Assay;

[0039] The effect of an inhibitor provided herein on viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a vims from a ceil, tissue, or organ to another cell, tissue or organ or to another individual can be determined according to any suitable assay known in the art. Further, the effect of a compound on the replication or other viral activity of any type, subtype or strain of a negative-sense, single-stranded RNA virus may be determined. The negative-sense, single-stranded RNA virus may be a non- segmented or a segmented vir u s. Non- limiting examples of non-segmented, negative-sense, single- stranded RNA viruses include: rhab do viruses (e.g., VSV, rabies, and rabies-related viruses), paramyxoviruses (e.g., NDV, Sendai vims, measles virus, mumps virus, parainfluenza vims, and pneumoviruses such as respiratory syncytial vims (RSV) and metapneumovirus), filoviruses (e.g., Ebola virus and Marburg virus), hepatitis delta virus, and bornavimses. Non-limiting examples of segmented, negative- sense, single-stranded RNA viruses include: orthomyxoviruses (e.g., influenza A vims, iniluenza B virus, influenza C virus, thogoto virus, and infectious salmon anemia vims), bunyaviruses (e.g., bunyamwcra virus, Hantaan vims, Dugbe vims, Rift Vaiiey fever virus, and tomato spotted wilt virus), and arenaviruses (e.g., Lassa virus, Junin virus, Machupo virus, and lymphocytic choriomeningitis virus). In a specific embodiment, the negative-sense, single-stranded RNA virus is an enveloped vims. In another specific embodiment, the negative- sense, single- stranded RNA virus is iniluenza virus (e.g., an influenza A virus, influenza B virus, or influenza C virus). In another embodiment, the negative- sense, single-stranded RNA virus is a parainfluenza virus or a respiratory syncytial virus (RSV). In another embodiment, the virus is NDV or VSV.

[0040] In some embodiments, the effect of a compound on the replication of an attenuated negative-sense, single-stranded RNA virus is determined. In some embodiments, the effect of a compound on the replication of a naturally occurring strain, variant or mutant of a negative-sense, single-stranded RNA vims, a mutagenized negative-sense, single- stranded RNA virus, a reassortant negative-sense, single-stranded RNA virus and/or a genetically engineered negative-sense, single-stranded RNA virus can be assessed. In a specific embodiment, the effect of a compound on the replication of a vaccine strain of a negative-sense, single-stranded RNA virus is determined. [0041] By way of example, a decrease in viral replication can be measured using a high throughput assay, as described below. In one embodiment, a decrease in viral replication can be measured by: (a) contacting a compound with a cell before (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours or more before), concurrently and/or subsequent to (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours or more after) infection; and (b) measuring virus replication. The cells used in the assay should be susceptible to infection by the chosen virus and can be infected at different MOIs. The effect of a compound on virus replication can be assessed by measuring virus replication at different times post-infection. For example, virus replication may be measured 6 hours, 12 hours, 1 6 hours, 24 hours, 48 hours or 72 hours post-infection, using any method known to one of skill in the art can be used measure virus replication. In another embodiment, a decrease in viral replication is assessed by measuring viral liter (as determined, e.g., by plaque formation). In another embodiment, a decrease in viral replication is assessed by measuring the production of viral proteins (as determined, e.g., by Western blot analysis, ELISA or flow cytometry). In another embodiment, a decrease in viral replication is assessed by measuring the production of viral nucleic acids (as determined, e.g., by RT-PCR or Northern blot analysis) using techniques known to one of skill in the art.

[0042] The effect of a compound can also be assessed by measuring viral replication by: (a) contacting a compound with a cell before (e.g., 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours or more before), concurrently and/or subsequent to (e.g., 1 5 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours or more after) infection with a negative-sense, single-stranded R A virus; and (b) measuring virus replication. The ceils can be infected at different MOIs and the effect of a compound on virus replication can be assessed. For example, the MOIs may be 0.001 , 0.005, 0.01 , 0.05, 0.1, 0.5, 1, 2.5, or 5. The effect of different concentrations of a compound on virus replication can also be assessed. The ceils or other substrate that contains cells (e.g., embryonated eggs) used in the assay should be susceptible to infection by the chosen negative-sense, single-stranded RNA virus. The ceils may be primary cells or established cell lines. With respect to influenza virus, for example, the following cells may be used in the assay: chicken cells (e.g., primary chick embryo cells or chick kidney cells), Vero ceils, LA4 cells, MDCK ceils, human respiratory epithelial cells (e.g., A549 cells), calf kidney ceils, and mink lung cells, in one embodiment, the ceils used to assess the effect of a compound on virus replication are selected from the following cells or cell lines: MEF, 293T, Huh 7.5, Detroit, and human tracheobronchial epithelial (HTBE; primary lung cells) cells, in one embodiment, the cell or cell line is biologically relevant to virus infection.

[0043] Virus replication can be measured at different times post-infection. For example, virus replication may be measured 6 hours, 12 hours, 16 hours, 24 hours, 48 hours or 72 hours post-infection. Any method known to one of skill in the art can be used measure virus replication. For example, viral replication may be assessed by measuring viral titer (as determined, e.g., by plaque formation), the production of viral proteins (as determined, e.g., by western blot analysis, ELISA or flow cytometry), or the production of viral nucleic acids (as determined, e.g., by RT-PCR or Northern blot analysis) using techniques known to one of skill in the art, and as discussed below.

[0044] In the assays described above, a compound that decreases the replication of a negative- sense, single-stranded RNA virus is identified if the replication of the virus is decreased in the cell contacted with the compound relative to the replication of the viru s in a cell contacted with a negative control (e.g., PBS or saline).

[0045] In certain embodiments, an inhibitor of viral replication, viral genome replication, or viral protein synthesis is identified if a compound reduces the viral replication, viral genome replication, or viral protein synthesis by at least 1.5 fold, 2 fold, 3 ibid, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 100 fold, 500 fold, or 1000 fold relative to virus replication in the absence of compound or the presence of a negative control. In certain embodiments, an inhibitor of viral replication, viral genome replication, or viral protein synthesis is identified if a compound reduces the virus replication by 1.5 to 3 fold, 2 to 4 fold, 3 to 5 fold, 4 to 8 fold, 6 to 9 fold, 8 to 10 fold, 2 to 10 fold, 5 to 20 fold, 10 to 40 fold, 10 to 50 fold, 25 to 50 fold, 50 to 100 fold, 75 to 100 fold, 100 to 500 fold, 500 to 1000 fold, or 10 to 1000 fold. In a specific embodiment, an inhibitor of viral replication, viral genome replication, or viral protein synthesis is identified if a compound reduces the virus replication by approximately 2 logs or more, approximately 3 logs or more, approximately 4 logs or more, approximately 5 logs or more, or 2. to 10 logs or 2 to 5 logs relative to virus replication in the absence of compound or the presence of a negative control. [0046] In certain embodiments, a compound results in about a 2 fold or more reduction of viral yieid per round of viral replication, in a specific embodiment, a compound results in about a 10 fold or more reduction of viral yield per round of viral replication.

[0047] In certain embodiments, a compound is considered an inhibitor of viral replicatio if it reduces viral replicatio by at least 2 wells of hemagglutinin (HA) in a hemagglutination assay, which equals approximately a 75% reduction in viral titer.

[0048] In certain embodiments, a compound is considered an inhibitor of viral replication if it reduces viral titer by 50% or more, by 55% or more, by 60% or more, by 65% or more, by 70% or more, by 75% or more, by 80% or more, by 85% or more, by 90% or more, or by 95% or more.

[001 ] Standard assays for influenza virus replication have been described, See, e.g.,

Sidwell et ai., Antiviral Research, 2000, 48 :1 -16. These assays can also be adapted for use with other negative-sense, single-stranded RNA viruses. By way of non-limiting example, exemplary assays for measuring effects of a compound on viral functions (e.g., replication, cell entry, etc.) are described in more detail below.

Viral Titer Assay

[002] In this non-limiting example, a monolayer of the target mammalian cell line is infected with different amounts (e.g., multiplicity of 3 plaque forming units (pfu) or 5 pfu) of virus (e.g., influenza) and subsequently cultured in the presence or absence of various dilutions of compounds (e.g., 0.1 g/mi, 1 . fig/ml, 5 .ug-'ml, or 10 g/'mi). Infected cultures are harvested 48 hours or 72 hours post infection and titered by standard plaque assays known in the art on the appropriate target ceil line (e.g., A549 ceils).

Flow Cytometry Assay

[003] Flow cytometry can be utilized to detect expression of vims antigens in infected target cells cultured in the presence or absence of compounds (see, e.g., McSharry et al, Clinical Microbiology Rev., 1994, 7:576-604). Non-limiting examples of viral antigens that can be detected on cell surfaces by flow cytometry include, but are not limited to HA of influenza; and H and F of measles virus. In other embodiments, intracellular viral antigens or viral nucleic acid can be deiected by flow cytometry with techniques known in the art. Viral Cytopathic Effect (CPE) Assay

[004] CPE is the morphological changes that cultured cells undergo upon being infected by most viruses. These morphological changes can be observed easily in unfixed, unstained cells by microscopy. Forms of CPE, which can vary depending on the virus, include, but are not limited to, rounding of the cells, appearance of inclusion bodies in the nucleus and/or cytoplasm of infected cells, and formation of syncytia, or polykaryocytes (large cytoplasmic masses that contain many nuclei).

[005] The CPE assay can provide a measure of the effect of a compound on virus replication. In a non- limiting example of such an assay, compounds are serially diluted (e.g. 1000, 500, 100, 50, 10, 1 , 0.1 ji mi) and added to 3 wells containing a cell monolayer (preferably mammalian cells at 80-100% confluent) of a 96-weli plate. Within 5 minutes, viruses are added and the plate sealed, incubated at 37 ^°C for the standard time period required to induce near-maximal viral CPE (e.g., approximately 48 to 120 hours, depending on the virus and multiplicity of infection). When assaying a compound for its potential inhibitory activity, CPE is read microscopically after a known positive control drug (an antiviral) is evaluated in parallel with compounds in each test. A non-limiting example of a positive control is ribavirin for influenza, measles, respiratory syncytial, and arainfluenza. The data is expressed as 50% effective concentrations or approximated virus- inhibitory concentration, 50% endpoint (EC50) and cell-inhibitory concentration, 50% endpoint (IC5₀). General selectivity index ("SI") is calculated as the IC5₀ divided by the EC5₀. These values can be calculated using any method known in the art, e.g., the computer software program MacSynergy II by M, N. Prichard, K. R. Asaltine, and C. Shipman, Jr., University of Michigan, Arm Arbor, Mich.

[006] In one embodiment, a compound has an SI of greater than 3, 4, 5, 6, 7, 8, 9,

10, 1 1 , 12, 13, 14, 15, 20, 21 , 22, 23, 24, 25, 30, 35, 39, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1,000, or 10,000. In some embodiments, a compound has an SI of greater than 10. In a specific embodiment, compounds with an SI of greater than 10 are further assessed in other in vitro and in vivo assays described herein or others known in the art to characterize safety and efficacy.

Neutral Red (NR) Dye Uptake Assay

[007] The NR Dye Uptake assay can be used to validate the CPE inhibition assay.

In a non-limiting example of such an assay, the same 96-wcii microplates used for the CPE inhibition assay can be used. Neutral red is added to the medium, and cells not damaged by virus take up a greater amount of dye. The percentage of uptake indicating viable cells is read on a microplate autoreader at dual wavelengths of 405 and 540 nm, with the difference taken to eliminate background, (see McManus et al., Appl. Environment. Microbiol. 31 :35- 38, 1976), An EC50 is determined for samples with infected ceils and contacted with compounds, and an TC50 s determined for samples with uninfected cells contacted with compounds.

[008J For compounds that enhance viral replication, the compound is tested for its ability to increase cell damage caused by virus, as compared to a control wherein the cell is treated with viras alone and a control wherein the cell is treated with the compound alone.

Plaque Assay

[009] In a non-limiting example of a plaque assay, the virus is diluted into various concentrations and added to each well containing a monolayer of the target ceils in triplicate. The plates are then incubated for a period of time to achieve effective infection of the control sample (e.g., 1 hour with shaking every fifteen minutes). After the incubation period, an equal amount of 1 % agarose is added to an equal volume of each compound dilution prepared in 2x concentration. In certain embodiments, final compound concentrations between 0.03 Ltg/ml to 100 .iig/mi can be tested with a final agarose overlay concentration of 0.5%, The drug agarose mixture is applied to each well in 2 ml volume and the plates are incubated for three days, after which the ceils are stained with a 1.5% solution of neutral red. At the end of the 4-6 hour incubation period, the neutral red solution is aspirated, and plaques counted using a stereomicroscope. Alternatively, a final agarose concentration of 0.4% can be used. In other embodiments, the plates are incubated for more than three days with additional overlays being applied on day 4 and on day 8 when appropriate. In another embodiment, the overlay medium is liquid rather than semi-solid.

Hemagglutination Assays

[0010] In a no n- limiting example of a hemagglutination assay, cells are contacted with a compound and are concurrently or subsequently infected with the virus (e.g., at an MOT. of 1) and the virus is incubated under conditions to permit virus replication (e.g., 20-24 hours). The compounds are preferably present throughout the course of infection. Viral replication and release of viral particles is then determined by hemagglutination assays using 0.5% chicken red blood cells, in some embodiments, a compound is considered an inhibitor of viral replication if it reduces viral replication by at least 2 wells of HA, which equals approximately a 75% reduction in viral titer. In specific embodiments, an inhibitor reduces viral titer in this assay by 50% or more, by 55% or more, by 60%) or more, by 65%) or more, by 70% or more, by 75% or more, by 80% or more, by 85% or more, by 90% or more, or by 95% or more.

Cyto toxic ity Assays

[001 1 ] In some embodiments, compounds differentially affect the viability of uninfected cells and ceils infected with virus. The differential effect of a compound on the viability of viraily infected and uninfected cells may be assessed using techniques known to one of skill in the art or described herein. In certain embodiments, compounds are more toxic to cells infected with a virus than uninfected ceils. In specific embodiments, compounds preferentially affect the viability of cells infected with a vims.

[0012] Many assays well -known in the art can be used to assess viability of cells

(infected or uninfected) or cell lines following exposure to a compound and, thus, determine the cytotoxicity of the compound. For example, cell proliferation can be assayed by measuring Bromodeoxyuridine (BrdU) incorporation (see, e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et aL, 1988, J. Immunol. Meth. 107:79), (³H) thymidine incorporation (see, e.g., Chen, J., 1996, Oncogene 13:1395-403; Jeo ng, J., 1995, J. Biol. Chem. 270:18367 73), by direct cell count, or by detecting changes in transcription, translation or activity of known genes such as proto-onco genes (e.g., fos, myc) or ceil cycle markers (Rb, cdc2, cyclin A, D i , D2, D3, E, etc). The levels of such protein and mR A and activity can be determined by any method well known in the art. For example, protein can be quantitated by known immunodiagnostic methods such as EOS A, Western blotting or immunoprecipitation using antibodies, including commercially available antibodies. mRNA can be quantitated using methods that are well known and routine in the art, for example, using northern analysis, RNase protection, or polymerase chain reaction in connection with reverse transcription. Cell viability can be assessed by using trypan-bl e staining or other cell death or viability markers known in the art. In a specific embodiment, the level of cellular ATP is measured to determine ceil viability.

[0013] In specific embodiments, cell viability is measured in three-day and seven-day periods using an assay standard in the art, such as the CellTiter-Glo Assay Kit (Promega), which measures levels of intracellular ATP. A reduction in cellular ATP is indicative of a cytotoxic effect. In another specific embodiment, cell viability can be measured in the neutral red uptake assay. In other embodiments, visual observation for morphological changes may include enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic- very heavy- -80%), PH (partially toxic-heavy— 60%), P (partially toxic-40%), Ps (partially toxic-slight— 20%), or 0 (no toxicity— 0%), conforming to the degree of cytotoxicity seen. A 50% cell inhibitory (c totoxic) concentration (IC50) is determined by regression analysis of these data.

[001 4] in a specific embodiment, the cells used in the cytotoxicity assay are animal cells, including primary cells and cell lines. In some embodiments, the ceils are human cells. In certain embodiments, cytotoxicity is assessed in one or more of the following cell lines: U937, a human monocyte ceil line; primary peripheral blood mononuclear cells (PBMC); Huh7, a human hepatoblastoma cell line; 293T, a human embryonic kidney cell line; and THP- i , monocytic cells. In certain embodiments, cytotoxicity is assessed in one or more of the following cell lines: MDCK, MEF, Huh 7.5, Detroit, or human tracheobronchial epithelial (TiTBE) cells.

[0015] Compounds can be tested for in vivo toxicity in animal models. For example, animal models, described herein and/or others known in the art, used to test the activities of compounds can also be used to determine the in vivo toxicity of these compounds. For example, animals are administered a range of concentrations of compounds. Subsequently, the animals are monitored over time for iethaiity, weight loss or failure to gain weight, and/or levels of serum markers that may be indicative of tissue damage (e.g., creatine phosphokinase level as an indicator of general tissue damage, level of glutamic oxalic acid transaminase or pyruvic acid transaminase as indicators for possible liver damage). These in vivo assays may also be adapted to test the toxicity of various administration mode and/or regimen in addition to dosages.

[001 6] The toxicity and/or efficacy of a compound in accordance with the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED5₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5₀ ED5₀. Generally, although not necessarily, a compound identified in accordance with the present disclosure that exhibits large therapeutic indices is preferred. While a compound identified in accordance with the present disclosure that exhibits toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0017] The data obtained from the ceil culture assays and animal studies can be used in formulating a range of dosage of a compound identified in accordance with the present disclosure for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED5₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in a method disclosed herein, the therapeutically effective dose can be estimated initially from ceil culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in ceil culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high- performance liquid chromatography. Additional information concerning the assays described herein as well as dosage determination is provided below.

Viral Entry Assay

[001 J Inhibition of viral entry into ceils can be determined by any suitable assa known in the art. For example, in the present Examples, viral entry was assayed using a previously described protocol (Konig ei al. (2010) Nature 463, 813-817). In this method, cells (e.g., A549, Vero, or other permissive cell line) are seeded in appropriate tissue culture plates. Subsequently, the cells are treated with a candidate compound approximately 2 to 4 hours prior to addition of pseudoparticles bearing WSN-HA/NA proteins and encoding a reporter gene (e.g., the Gaussia luciferase reporter gene). Cells are subsequently washed after an incubation period (e.g., 1 8 hours) and new medium is added. Reporter activity is subsequently measured using an appropriate detection system.

vRNP localization

[002] Inhibition of viral replication can also be measured by quantifying viral RNP export from the nucleus to the cytoplasm of an infected cell. Any suitable assay know in the art may be used. For example, and without limitation, as described in the present Examples, A549 cells were seeded on coverslips in a 24- ell plate at 7.5 x 10⁴ cells per well. The next day, cells were treated with the test compound or solvent DMSO for 2 hours prior to infection with influenza virus. Cells were washed and incubated with medium containing the compound. At 3, 5 and 7 hours post infection (hpi), cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, stained with a polyclonal rabbit anti-NP serum and Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen). 4'- 6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. The coverslips were mounted on a slide using Prolong gold antifade reagent (Invitrogen), and the cells were viewed under an AxioplahZIE fluorescence microscope.

Animal Model Studies

[003] Compounds and compositions are preferably assayed in vivo for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vivo assays can be used to determine whether it is preferable to administer a compound and/or another therapeutic agent. For example, to assess the use of a compound to inhibit a viral infection, the compound can be administered before the animal is infected with the virus. Alternatively, or in addition, a compound can be administered to the animal at the same time that the animal is infected with the virus. To assess the use of a compound to treat or manage a viral infection, in one embodiment, the compound is administered after a viral infection in the animal. In another embodiment, a compound is administered to the animal at the same time that the animal is infected with the virus to treat and/or manage the viral infection. In a specific embodiment, the compound is administered to the animal more than one time,

[004] Compounds can be tested for antiviral activity against virus in animal models systems including, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, goats, sheep, dogs, rabbits, guinea pigs, etc. In a specific embodiment of the present disclosure, compounds are tested in a mouse model system. Such model systems are widely used and well-known to the skilled artisan. Compounds can also be tested for replication enhancing activity toward virus replication in animal models systems including, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, goats, sheep, dogs, rabbits, guinea pigs, etc. In a specific embodiment of the present disclosure, compounds are tested in a mouse model system. Such model systems are widely used and well-known to the skilled artisan.

[005] Typically, animals are infected with virus and concurrently or subsequently treated with a compound or placebo. Alternatively, animals are treated with a compound or placebo and subsequently infection with virus. Samples obtained from these animals (e.g., serum, urine, sputum, semen, saliva, plasma, or tissue sample) can be tested for viral replication via well known methods in the art, e.g., those that measure altered yirai titers (as determined, e.g., by plaque formation), the production of viral proteins (as determined, e.g., by Western blot, ELTSA, or flow cytometry analysis) or the production of viral nucleic acids (as determined, e.g., by RT-PCR or northern blot analysis). For quantitation of virus in tissue samples, tissue samples are homogenized in phosphate-buffered saline (PBS), and diiutions of clarified homogenates are adsorbed for 1 hour at 37 ^°C onto monolayers of cells (e.g., Vero, A549 or MDCK ceils). In other assays, histopathologic evaluations are performed after infection, preferably evaluations of the organ(s) the virus is known to target for infection. Vims immunohistochemistry can be performed using a viral-specific monoclonal antibody.

[006] The effect of a compound on the virulence of a virus can also be determined using in vivo assays in which the titer of the virus in an infected subject administered a compound, the length of survival of an infected subject administered a compound, the immune response in an infected subject administered a compound, the number, duration and/or severity of the symptoms in an infected subject administered a compound, and/or the time period before onset of one or more symptoms in an infected subject administered a compound is assessed. Techniques known to one of skill in the art can be used to measure such effects.

Influenza Virus Animal Models

[007] Animal models, such as ferret, mouse, guinea pig, and chicken, developed for use to test antiviral agents against influenza virus have been described, see, e.g., Sidwell et al, Antiviral Res., 2000, 48: 1-16; Lowen A. C. et ai. PMAS., 2006, 103: 9988-92: and McCauley et al., Antiviral Res., 1995, 27: 179-186. For mouse models of influenza, non- limiting examples of parameters that can be used to assay antiviral activity of compounds administered to the influenza-infected mice include pneumonia-associated death, serum al - acid glycoprotein increase, animal weight, lung virus assayed by hemagglutinin, lung virus assayed by plaque assays, and hist op atho logical change in the lung. Statistical analysis is carried out to calculate significance (e.g., a P value of 0.05 or less).

[008] Nasal turbinates and trachea may be examined for epithelial changes and subepithelial inflammation. The lungs may be examined for bronchioiar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. The alveoli are also evaluated for inflammatory changes. The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelial ceils with ciliated apical borders and basal pseudostratified nuclei; minimal inflammation); 1 + (epithelial layer columnar and even in outline with only slightly increased proliferation; cilia still visible on many cells); 2+ (prominent changes in the epithelial layer ranging from attenuation to marked proliferation; cells disorganized and layer outline irregular at the luminal border); 3+ (epithelial layer markedly disrupted and disorganized with necrotic cells visible in the lumen; some bronchioles attenuated and others in marked reactive proliferation).

[009] The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal: Lined by medium to tall columnar epithelial cells with ciliated apical border, nuclei basal and pseudostratified. Cytoplasm evident between apical border and nucleus. Occasional small focus with squamous ceils); 1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffuse squamous metaplasia of much of the epithelial layer, cilia may be evident focally); 2.5+ (diffuse squamous metaplasia with very few cilia evident).

[001 0] Virus immunohistochemistry is performed using a viral-specific monoclonal antibody (e.g. NP-, N- or UN-specific monoclonal antibodies). Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (few infected cells); 1+ (few infected cells, as widely separated individual cells); 1.5+ (few infected cells, as widely separated singles and in small clusters); 2+ (moderate numbers of infected cells, usually affecting clusters of adjacent ceils in portions of the epithelial layer lining bronchioles, or in small sub lobular foci in alveoli); 3+ (numerous infected cells, affecting most of the epithelial layer in bronchioles, or widespread in large sublobular foci in alveoli).

Assays in Humans

[001 i] In one embodiment, candidate compounds that modulate replication of a negative- sense, single-stranded RNA virus are assessed in human subjects suffering from such an infection with such a virus, in accordance with this embodiment, a candidate compound or a control compound is administered to the human subject, and the effect of a test compound on viral replication is determined by, e.g., analyzing the level of the virus or viral nucleic acids in a biological sample (e.g., serum or plasma), A candidate compound that alters the virus replication can be identified by comparing the level of virus replication in a subject or group of subjects treated with a control compound to that in a subject or group of subjects treated with a candidate compound. Alternatively, alterations in viral replication can be identified by comparing the le vel of the virus replication in a subject or group of subjects before and after the administration of a candidate compound. Techniques known to those of skill in the art can be used to obtain the biological sample and analyze the mRNA or protein expression.

[0012] In another embodiment, the effect of a candidate compound on the severity of one or more symptoms associated with a negative-sense, single- stranded RNA vims is assessed in a subject having such a virus infection. In accordance with this embodiment, a candidate compound or a control compound is administered to a human subject suffering from a negative-sense, single-stranded RNA virus infection and the effect of a candidate compound on one or more symptoms of the virus infection is determined . A candidate compound that reduces one or more symptoms can be identified by comparing the subjects treated with a control compound to the subjects treated with the test compound. Techniques known to physicians familiar with infectious diseases can be used to determine whether a candidate compound reduces one or more symptoms associated with the infectious disease.

Compositions

[0013] Any compound described herein may optionally be in the form of a composition comprising the compound and a carrier, excipient or diluent. In certain embodiments provided herein, compositions (including pharmaceutical compositions) comprise a compound and a pharmaceutic ally acceptable carrier, excipient, or diluent.

[0014] in other embodiments, provided herein are pharmaceutical compositions comprising an effective amount of a compound and a pharmaceutically acceptable carrier, excipient, or diluent. In a specific embodiment, the pharmaceutical compositions comprise an inhibitor of a negative-sense, single-stranded RNA virus (e.g., the compound ONI 081 10). The pharmaceutical compositions are suitable for veterinary and/or human administration.

[0015] The pharmaceutical compositions provided herein ca be i any form that allows for the composition to be administered to a subject, preferably a human.

[0016] In a specific embodiment and in this context, the term "pharmaceutically acceptable carrier, excipient or diluent" means a carrier, excipient or diluent approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a specific carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0017] Typical compositions and dosage forms comprise one or more excipients.

Suitable excipients are well-known to those skilled in the art of pharmacy, and non limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, cthanoi and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0018] Lactose free compositions can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (U8P)8P(XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Specific lactose free dosage forms comprise a compound, macrocrystalline cellulose, pre gelatinized starch, and magnesium stearate.

[0019] Further provided herein are anhydrous pharmaceutical compositions and dosage forms comprising one or more compounds, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long term storage in order to determine characteristics such as shelf life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drag Stability: Principles & Practice, 2d. Ed., Marcel Dekker, MY, N.Y., 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations. [0020] Anhydrous compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Compositions and dosage forms that comprise lactose and at least one compound that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

[0021] An anhydrous composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0022] Further provided herein are compositions and dosage forms that comprise one or more agents that reduce the rate by which a compound will decompose. Such agents, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[0023] The compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a compound preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a specific embodiment, the compositions or single unit dosage forms are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject,

[0024] Compositions provided herein are formulated to be compatible with the intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In a specific embodiment, a composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as iignocaine to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non aqueous liquid suspensions, oil in water emulsions, or a water in oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0025] The composition, shape, and type of dosage forms of the present disclosure will typically vary depending on their use.

[0026] Generally, the ingredients of compositions provided herein are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0027] Pharmaceutical compositions provided herein that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed,, Mack Publishing, Easton Pa. (1990).

[0028] Typical oral dosage forms provided herein are prepared by combining a compound in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

[0029] Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed, if desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0030] Examples of excipients that can be used in oral dosage forms provided herein include, but are not limiied to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), micro crystalline cellulose, and mixtures thereof.

[0031 ] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms provided herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions provided herein is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form. [0032] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL PH 101 , AVICEL PH 103 AVICEL RC 581 , AVICEL PH 105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC 581. Suitable anhydrous or low moisture excipients or atiditives include AVICEL PH 103 and Starch 1 500 LM.

[0033] Disintegrants are used in the compositions provided herein to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much dismtegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of dismtegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms provided herein. The amount of dismtegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of dismtegrant, specifically from about 1 to about 5 weight percent of disintegrant.

[0034] Disintegrants that can be used in pharmaceutical compositions and dosage forms provided herein include, but are not limited to, agar, aiginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.

[0035] Lubricants that can be used in pharmaceutical compositions and dosage forms provided herein include, but are not limited to, calcium stearate, magnesium stcaratc, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROS1L 200, manufactured by W .R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Piano, Tex.), CAB O SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated. [0036] A compound can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat Nos. 3,845,770; 3,916,899; 3,536,809; 3,598, 123; and 4,008,719, 5,674,533, 5,059,595, 5,591 ,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566. Such dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled release formulations known to those of ordinary skill in the an, including those described herein, can be readily selected for use with the active ingredients of the present disclosure. The present disclosure thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled release.

[0037] All controlled release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non controlled counterparts, ideally, the use of an optimally designed controlled release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0038] Most controlled release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or agents,

[0039] Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' Datural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0040] Suitable vehicles that can be used to provide parenteral dosage forms pro vided herein are well known to those skilled in the art. Examples include, but are not limited to: Water for injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl and benzyl benzoate,

[0041 ] Agents that increase the solubility of one or more of the compounds provided herein can also be incorporated into the parenteral dosage fonns provided herein.

[0042] Transdermal, topical, and mucosal dosage forms provided herein include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. ( 1980 & 1990); and Introduction to Pharmaceutical Dosage Fonns, 4th ed., Lea & Febiger, Philadelphia ( 1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.

[0043] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms provided herein are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, et anol, ethylene glycol, propylene glycol, butane 1,3 dioi, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments. which are noil toxic and pharmaceutically acceptable. Moisturizers or humect ants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990).

[0044] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with a compound. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone: various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0045] The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more compounds. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Agents such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or iipophilicity of one or more compounds so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration enhancing agent. Different salts, hydrates or solvates of the compounds can be used to further adjust the properties of the resulting composition,

[0046] in certain specific embodiments, the compositions are in oral, injectable, or transdermal dosage forms. In one specific embodiment, the compositions are in oral dosage forms. In another specific embodiment, the compositions are in the form of injectable dosage forms. In another specific embodiment, the compositions are in the form of transdermal dosage forms.

Dosages

[0047] The amount of an inhibitor, or the amount of a composition comprising an inhibitor, that will be effective in the inhibition, treatment and/or management of a negative- sense, single-stranded RNA virus infection, and in the inhibition, treatment and/or management of a viral infection (e.g., influenza infection) can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend, e.g., on the route of administration and the seriousness of the infection, and should be decided according to the judgment of the practitioner and each patient's or subject's circumstances.

[0048] In some embodiments, the dosage of an inhibitor is determined by extrapolating from the no observed adverse effective level (NOAEL), as determined in animal studies. This extrapolated dosage is useful in determining the maximum recommended starting dose for human clinical trials. For instance, the NOAELs can be extrapolated to determine human equivalent dosages (HED). Typically, TIED is extrapolated from a non-human animal dosage based on the doses that are normalized to body surface area (i.6,, mg/m²). In specific embodiments, the NOAELs are determined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs, primates, primates (monkeys, marmosets, squirrel monkeys, baboons), micropigs or minipigs. For a discussion on the use of NOAELs and their extrapolation to determine human equivalent doses, See Guidance for Industry Estimating the Maximum Safe Starting Dose in initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER), Pharmacology and Toxicology, July 2005. in one embodiment, an inhibitor or composition thereof is administered at a dose that is lower than the human equivalent dosage (HED) of the NOAEL over a period of 1 week, 2 weeks, 3 weeks, i month, 2 months, three months, four months, six months, nine months, 1 year, 2 years, 3 years, 4 years or more.

[0049] In certain embodiments, a dosage regime for a human subject can be extrapolated from animal model studies using the dose at which 10% of the animals die (LDio). In general the starting dose of a Phase I clinical trial is based on preclinical testing. A standard measure of toxicity of a drug in preclinical testing is the percentage of animals that die because of treatment. It is well within the skill of the art to correlate the LDio in an animal study with the maximal-tolerated dose (MTD) in humans, adjusted for body surface area, as a basis to extrapolate a starting human dose. In some embodiments, the interrelationship of dosages for one animal model can be converted for use in another animal, including humans, using conversion factors (based on milligrams per meter squared of body surface) as described, e.g., in Freireich et al, Cancer Chemother. Rep., 1966, 50:219-244. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. In certain embodiments, the adjustment for body surface area includes host factors such as, for example, surface area, weight, metabolism, tissue distribution, absorption rate, and excretion rate. In addition, the route of administration, excipient usage, and the specific disease or virus to target, are also factors to consider.

[0050] In one embodiment, the standard conservative starting dose is about 1/10 the murine LD_{J O}, although it may be even lower if other species (i.e., dogs) were more sensitive to the inhibitor. In other embodiments, the standard conservative starting dose is about 1/100, 1/95, 1 /90, 1/85, 1/80, 1 /75, 1/70, 1/65, 1 /60, 1/55, 1/50, 1 45, 1/40, 1 /35, 1 /30, 1/25, 1/20, 1/15, 2/10, 3/10, 4/10, or 5/10 of the murine LDj₀. In other embodiments, a starting dose amount of an inhibitor in a human is lower tha the dose extrapolated from animal model studies. In another embodiment, a starting dose amount of an inhibitor in a human is higher than the dose extrapolated from animal model studies, it is well within the skill of the art to start doses of the active composition at relatively low levels, and increase or decrease the dosage as necessary to achieve the desired effect with minimal toxicity.

[0051 ] Exemplary doses of inhibitors or compositions include milligram or microgram amounts per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 5 micrograms per kilogram to about 100 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). In specific embodiments, a daily dose is at least 50 mg, 75 mg, 100 mg, 150 mg, 250 mg, 500 mg, 750 mg, or at least 1 g.

[0052] In another embodiment, the dosage is a unit dose of 5 mg, preferably 10 mg,

50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg or more. In another embodiment, the dosage is a unit dose that ranges from about 5 mg to about 100 mg, about 100 mg to about 200 mg, about 150 mg to about 300 mg, about 150 mg to about 400 mg, 250 mg to about 500 mg, about 500 mg to about 800 mg, about 500 mg to about 1000 mg, or about 5 mg to about 1000 mg.

[0053] In certain embodiments, suitable dosage ranges for oral administration are about 0.001 milligram to about 500 milligrams of a compound, per kilogram body weight per day. In specific embodiments, the oral dose is about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0,1 milligram to about 75 milligrams per kilogram body weight per day or about 0.5 milligram to 5 milligrams per kilogram body weight per day. The dosage amounts described herein refer to total amounts administered; that is, if more than one compound is administered, then, in some embodiments, the dosages correspond to the total amount administered, in a specific embodiment, a compound disclosed is about 10% to about 95% by weight of an oral compositions provided herein.

[0054] Suitable dosage ranges for intravenous (i.v.) administration are about 0.01 milligram to about 100 milligrams per kilogram body weight per day, about 0.1 milligram to about 35 milligrams per kilogram body weight per day, and about 1 milligram to about 10 milligrams per kilogram body weight per day.

[0055] In some embodiments, suitable dosage ranges for intranasal administration are about 0.01 pg/kg body weight per day to about 1 mg/kg body weight per day.

[0056] Suppositories generally contain about 0.01 milligram to about 50 milligrams of a compound of the present disclosure per kilogram body weight per day and comprise active ingredient in the range of about 0.5% to about 10% by weight,

[0057] Recommended dosages for intradermal, intramuscular, intraperitoneal, subcutaneous, epidural, sublingual, intracerebral, intravaginal, transdermal administration or administration by inhalation are in the range of about 0.001 milligram to about 500 milligrams per kilogram of body weight per day. Suitable doses for topical administration include doses that are in the range of about 0.001 milligram to about 50 milligrams, depending on the area of administration. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.

[0058] in another embodiment, a subject is administered one or more doses of a prophylactically or therapeutically effective amount of an inhibitor or a composition, wherein the prophylactically or therapeutically effective amount is not the same for each dose.

[0059] In other embodiments, a subject is administered an inhibitor or a composition in an amount effective to inhibit viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another ceil, tissue or organ or to another individual, by at least 20% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. in certain embodiments, a subject is administered an inhibitor or a composition in an amount effective inhibit viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual, by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or other known to one of skill in the art.

[0060] In other embodiments, a subject is administered an inhibitor or a composition in an amount effective to inhibit viral replication by 1 log, 1.5 logs, 2 logs, 2.5 logs, 3 logs, 3.5 logs, 4 logs, 5 logs or more relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0061] In certain embodiments, a dose of an inhibitor or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, two, three or four doses of an inhibitor or a composition is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks. In some embodiments, a dose(s) of an inhibitor or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, or 21 days, in certain embodiments, a dose of an inhibitor or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more,

[0062] The dosages of prophylactic or therapeutic agents which have been or are currently used for the inhibition, treatment and/or management of a negative-sense, single- stranded R A virus infection can be determined using references available to a clinician such as, e.g., the Physicians' Desk Reference (61 ^st ed. 2007). In a specific embodiment, dosages lower than those which have been or are currently being used to inhibit, treat and/or manage the infection are utilized in combination with one or more inhibitors or compositions. For inhibitors which have been approved for uses other than inhibition, treatment or management of viral infections, safe ranges of doses can be readily determined using references available to clinicians, such as e.g., the Physician's Desk Reference (61^st ed. 2007).

[0063] The above-described administration schedules are provided for illustrative purposes only and should not be considered limiting. A person of ordinary skill in the art will readily understand that ail doses are within the scope of the present disclosure. More generally, dose and frequency will depend in part on recession of pathological signs and clinical and subclinical symptoms of a disease condition or disorder contemplated for treatment with the present compounds.

Ther ap eu tic Methods o f Us e

[0064] in certain embodiments, methods for inhibiting replication of a negative-sense, single-stranded RNA virus, utilizing an inhibitor described herein, are provided. Also provided are methods for inhibiting replication of negative-sense, single-stranded RNA viruses in a subject, comprising administering an inhibitor of viral replication to the subject. In other embodiments, methods for inhibiting viral genome replication, viral protein synthesis, viral titer, and/or the spread of a virus from a ceil, tissue, or organ to another ceil, tissue or organ or to another individual, wherein the virus is a negative-sense, single-stranded RNA virus, are provided. In a specific embodiment, methods for treating a viral infection (e.g. with a negative-sense, single- stranded RNA virus (e.g., influenzavirus, NDV, Sendai, etc.) are provided, in other embodiments, methods for inhibiting a viral infection in a subject are provided. Thus, such methods may be carried out either in vitro (e.g., in a cell line) and/or in vivo (e.g., in a subject, e.g., an avian or mammalian subject), and can comprise administering an inhibitor of a function (e.g., viral replication, decreasing kinetics of viral replication, viral protein synthesis, viral infeciivity, etc.) of a negative-sense, single-stranded RNA virus, as described herein.

[0065] In a specific embodiment, the inhibitor is administered to a subject infected with the virus (e.g., a virus of the family Or thorny xoviridae (Influenzavirus), Rhabdovirida (VSV), or Paramyxoviridae (Newcastle disease virus (NDV) or Sendai virus). The inhibitor may also be administered to a subject prior to viral infection (i.e., prophyiacticaily), in order to inhibit a viral infection (e.g., influenzavirus infection). In certain embodiments, the inhibitor is an inhibitor of one or more the kinases: CAMKK1 , CAMKK2, CDKl/cyciin A, CDKl/cyelin B, CD 2/cyciin A, CDK4/cyclin Dl , CDK4/cyclin D3, CDK5/p25, CDK5 p35, CDK6/cyclin Dl , CDK6/cyclm D3, CDK cyclin K, CDK9/cyclin Tl, CK2a, CK2a2, DYRKiB, DYRK2, GSK3a, GSK3b, HGK/M AP4K4 , MLK3 /MAP3 K 1 1 , MUSK, NLK, PIM3, and SGK1. In a specific embodiment, the inhibitor has the structure shown in Figure 2 (the compound referred to herein as"ON1081 10"). In another embodiment, the inhibitor is a pharmaceutically acceptable salt, hydrate, solvate, prodrug, analog or other derivative of ON 1081 10. In another embodiment, the inhibitor has the structure shown in Figure 9 (the compound referred to herein as"ON123790"). in another embodiment, the inhibitor is a pharmaceutically acceptable salt, hydrate, solvate, prodrug, analog or other derivative of ON 123790.

[0066] Typically, when administered to a subject, e.g., for the treatment of an infection with a negative- sense, single-stranded RNA virus, a compound or composition provided herein inhibits one or more of viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual, by at least 2.0% to 25%, preferably at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In certain embodiments, a subject is administered a composition (e.g., comprising an inhibitor) provided herein in an amount effective to inhibit viral genome replication, viral protein synthesis, viral titer, viral replication, and/or the spread of a virus from a cell, tissue, or organ to another cell, tissue or organ or to another individual, by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 1 0 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or other known to one of skill in the art

[0067] When administered to a subject, preferably a compound or composition of the present disclosure is administrated in an "effective" or "therapeutically effective" amount. Within the context of the present disclosure, the terms "therapeutically effective" and "effective amount," used interchangeably, in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s). In certain embodiments, an "effective amount" in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a viral infection or a symptom associated therewith; (ii) reduce the duration of a viral infection or a symptom associated therewith; (iii) inhibit the progression of a viral infection or a symptom associated therewith; (iv) cause regression of a viral infection or a symptom associated therewith; (v) inhibit the development or onset of a viral infection or a symptom associated therewith; (vi) inhibit the recurrence of a viral infection or a symptom associated therewith; (vii) reduce or inhibit the spread of a virus from one cell to another ceil, one tissue to another tissue, or one organ to another organ; (ix) inhibit or reduce the spread of a virus from one subject to another subject; (x) reduce organ failure associated with a viral infection; (xi) reduce hospitalization of a subject; (xii) reduce hospitalization length; (xiii) increase the survival of a subject with a viral infection; (xiv) eliminate a virus infection; (xv) inhibit virus replication; (xvi) inhibit the entry of a virus into a host cell(s); (xviii) inhibit replication of the viral genome; (xix) inhibit synthesis of viral proteins; (xx) inhibit assembly of viral particles; (xxi) inhibit release of viral particles from a host celi(s); (xxii) reduce viral titer; and/or (xxiii) enhance or improve the prophylactic or therapeutic effeci(s) of another therapy.

[0068] Further, in certain embodiments, a compound or composition of the invention may be administered in combination with another therapy (e.g., another antiviral therapy or immune enhancing agent or therapy). As used herein, the term "in combination," in the context of the administration of two or more therapies to a subject, refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies are administered to a subject with a viral infection. A first therapy (e.g., a first prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject with a viral infection.

Use of inhibitors in Cell Culture and as Disinfectants

[0069] The present disclosure provides for the use of inhibitors as ingredients in cell culture- elated products in which it is desirable to have antiviral activity. In one embodiment, at least one inhibitor is added to cell culture media. In certain embodiments, inhibitors that prove too toxic or are not used in subjects are added to cell culture-related products, such as media. The present disclosure also provides for the use of inhibitors as ingredients in disinfectants and soaps. Thus, for example, the compounds disclosed in the present Examples (ONI 08110, OM 108080, ONI 081 10, ON 12.3300 and ONI 23790) may also be used in such disinfectants, soaps, and/or cell culture-related products.

[0070] In accordance with the present disclosure, there may be employed conventional molecular biology, microbiology, recombinant DNA, immunology, cell biology and other related techniques within the skill of the art. See, e.g., Sambrook et al., (2.001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N. Y.; Ausubel et al., eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc. : Hoboken, .J.; Bonifacino et al., eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, .J.; Coico et al., eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al., eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.: Hoboken, N.J.; Hames et al., eds.

( 1999) Protein Expression: A Practical Approach. Oxford University Press: Oxford; Freshney

(2000) Culture of Animal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; among others. The Current Protocols listed above are updated several times every year.

[0071 ] The present disclosure is further described by way of the follo ing particular examples. However, the use of such examples is illustrative only and is not intended to limit the scope or meaning of this disclosure or of any exemplified term. Nor is the disclosure limited to any particular preferred embodiments) described herein. indeed, many modifications and variations of the disclosure will be apparent to those skilled in the art upon reading this specification, and such "equivalents" can be made without departing from the disclosure in spirit or scope. The disclosure is therefore limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

EXAMPLES

[0072] The present disciosure is described further below in working examples which are intended to further describe the disclosure without limiting the scope therein.

[0073] In the Examples 1-6 below, the following materials and methods were used.

Cells

[0074] A549 (human lung adenocarcinoma ceil line) and LA4 (mouse lung adenoma cell line) cells were maintained in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cell viability assay

[0075] A549 or LA4 cells were seeded in 96-well plates at 8,000 cells per well one day prior to treatment with two- fold serial dilutions of the compound of interest, or solvent dimethyl sulfoxide (DMSO). The amount of DMSO was kept the same for ail dilutions of the compound. At 24 hours post-treatment, ceil viability was measured using the CellTiter-Glo assay (Promega) according to the manufacturer's instructions.

Viral infection.

[0076] A549 or LA4 cells were seeded in 24 -well plates at I x 10^s cells per well one day prior to treatment with two-fold serial dilutions of the compound of interest, or the solvent DMSO. Two to four hours post-treatment, cells were washed with phosphate buffered saline (PBS) and infected with influenza virus (A/WSN/33) ("WSN virus") at an MOl of 0.0 i , or vesicular stomatitis virus (VSV-GFP) at an MOl of 0.0001 , or Newcastle disease virus (NDV-B 1 strain) at an MOl of 2. After virus inoculation, medium containing the compound of interest was added back to the ceils. At 2.4 hours post-infection (hpi), the amount of virus in the supematants was determined using either a hemagglutination (HA) or a plaque assay.

Hemagglutination assay

[0077] Hemagglutination assay was performed by making two-fold serial dilutions of the supernatant with PBS in a 96-well V-bottom plate, followed by addition of 0.5% chicken red blood cell (RBC) suspension. The plate was shaken for 30 seconds, incubated for 45 minutes at room temperature and observed for hemagglutination. The number of HA wells was counted for each sample.

Plaque Assay

[0078] Plaque assay for NDV-B 1 was performed using DF1 cells (chicken embryo fibroblast cell line). Cells were fixed, permeabilized and incubated with an antiserum raised against the virus for 1 hour at room temperature. After 3 washes with 0.1 % PBS-Tween 20, an HRP-iinked anti-rabbit IgG (GE Healthcare NA9340V) was added and incubated for 1 hour at room temperature. After 3 washes with 0.1 % PBS-Tween 20, True Blue Peroxidase substrate was added and the number of plaques was counted. Entry assay

[0079] Viral entry was assayed using a previously described protocol (Konig et a!.

(2010) Nature 463, 813-817). Briefly, A549 cells were seeded in 96-well plates at 3 x 10⁴ cells per well. The next day, cells were treated with ONI 08110, or diphyliin, or solvent DMSO in medium supplemented with 4 με/^'ηιΐ poly rene for 2 to 4 hours prior to addition of pseudoparticles bearing WSN-HA/NA proteins and encoding the Gaussia luciterase reporter gene. Cells were washed four times after 18 hours and new medium was added. Twenty- four hours after addition of medium, Gaussia luciferase activity was measured using the Renilla luciferase assay system (Promega).

vRNP localization

[0080] A549 cells were seeded on coverslips in a 24-well plate at 7.5 x 10⁴ cells per well The next day, cells were treated with ON1081 10 (3.13 μΜ) or solvent DMSO for 2 hours prior to infection with influenza virus (A/WSN/33) ("WSN virus") at an MOI of 3. Cells were washed and incubated with medium containing the compound. At 3, 5 and 7 hpi, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS, stained with a polyclonal rabbit anti-NP serum and Alexa Fluor 488 donkey anti- rabbit IgG (Invitrogen). 4 '-6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. The coverslips were mounted on a slide using Prolong gold aniifade reagent (Invitrogen). Cells were viewed under an Axioplan2IE fluorescence microscope.

Example 1: Screening of kinase inhibitors

for activity against influenza A virus

[0081 ] This example describes the identification and characterization of compounds that inhibit replication of influenza A virus.

[0082] Five kinase inhibitors (ON1081 10, ON108080, ON123300, ON123790, and

ONI 08050) were selected Irom a compound library and tested for their ability to inhibit influenza A virus. The structures of ONI 08110, ONI 08080, ON123300, ON123790, and ON 108050 are shown in Figures 2 and 7-10, respectively. A549 ceils were treated with twofold serial dilutions of each compound in order to determine whether test concentrations were toxic to cells. The amount of solvent DMSO was kept the same for ail dilutions of each compound. At 2.4 hours post-treatment, cell viability was measured using the CellTiter-Glo assay, which measures the amount of AT in cells. The results were expressed relative to the DM80 control (Fig. 1). ON 108050 was toxic to the ceils (55% viability at the lowest concentratio tested) so this compound was not tested further. ONI 08080, ON1081 10, ON 123300 and ON123790 had varying effects on cell viability, as shown in Figures IB to E.

[0083] To determine whether any of those compounds inhibits replication of influenza virus (A/WSN/33), A549 ceils were treated with each compound at concentrations that yielded at least 70% viability relative to the DMSO control. Two (2) to four (4) hours post-treatment, ceils were infected with the viras at a multiplicity of infection (MOl) of 0.01 to allow multicycle replication, Supernatants were harvested 24 hours post-infection (hpi) and a hemagglutination (HA) assay was performed. The ability of the compounds to inhibit the virus was expressed as the reduction in the number of HA wells relative to the DMSO control, as summarized in Table 2, below:

Table 2: Activity of kinase inhibitors against influenza A virus

[0084] ONI 08080 and ON123300 did not inhibit the virus at any of the

concentrations that were tested. ON I 081 10 reduced the number of HA wells by 5 at a concentration of 3, 13 to 25 μΜ, ON 123790 reduced the number of HA wells by 2 at a concentration of 3.13 μΜ and by 5 at a concentration of 6,25 μΜ, Taken together with the results in Figure 1, this indicates that ON108110 and ON 123790 inhibit influenza virus at concentrations that are not toxic to cells.

[0085] This example demonstrates the chemical structure of ONI 081 10 and its kinase inhibition profile.

[0086] The chemical structure of ON 1081 10 is shown in Figure 2. To determine the kinases that ON 1081 10 inhibits, the compound was sent to Reaction Biology Corporation (Malvern, PA) for kinase inhibition profiling. Among 300 kinases that were tested, ON 1081 10 inhibited 25 kinases significantly (with IC₅₀ of less than 100 nM), as summarized in Table 3, below:

Table 3: Kinase inhibition profile of ONI 08110

Exemplar}' GenBank Accession No, or Publication^)

Kinase ICso (nM)

Describing Kinase

ML 3/MAP3K11 NP 002410 87.4

MUSK NP 005583 56.14

NLK NP 057315 35.61

P1M3 NP 001001852 1.92

SGK1 NP 005618 82.29

Example 3: ONlOSilO inhibits replication

of influenza A virus

[0087] This example demonstrates that ON l OS i lO reduces influenza A virus replication in a dose-dependent manner at concentrations that are not toxic to cells.

[0088] To quantify the degree to which ON 108 U 0 inhibits replication of influenza virus, A549 cells were treated with two-fold serial dilutions of ONI 081 10 (0.78 to 6.25 μΜ) for 2-4 hours prior to infection with WSN virus at an MOI of 0.01. The amount of virus present in the supernatant at 24 hpi was measured using a plaque assay. ON108110 inhibited viral replication in a dose-dependent manner at concentrations that are not toxic to ceils (Fig. 3 A). At the highest concentration tested (6.25 μΜ), the ceil viability was 81 % relative to the DMSO control, and the amount of virus in the supernatant was 0.007% relative to the DMSO control.

[0089] To determine whether ON 1081 10 inhibits the replication of influenza virus in a species-specific manner, the compound was tested in a mouse lung adenoma ceil line (LA4). Ceils were treated with two-fold serial dilutions of ON I 081 10 (0.78 to 25 μΜ) and infected with WSN virus in the same manner. Figure 3B shows that ONl OSi lO inhibited viral replication in a dose-dependent manner at concentrations that are not toxic to LA4 cells. At the highest concentration tested (25 μΜ), the ceil viability was 87% relative to the DMSO control, and the amount of virus in the supernatant was 0.05% relative to the DMSO control. Taken together with the results in Fig. 3 A, these results indicate that ONI 081 10 inhibits viral replication in both A549 and LA4 cells, and was most effective in inhibiting viral replication in A549 cells. Example 4 ; Effect of ONI 08110 on influenza A virus entry

[0090] This example demonstrates that ON 1081 10 does not inhibit viral entry.

[0091] Next, it was determined whether ON 1081 10 inhibits entry of influenza virus.

A549 cells were treated with ONl OS l l O at 3.13 μΜ, a concentration that yielded good cell viability (90% relative to the DMSO control) and good viral inhibition ( 1 1 % relative to the DMSO control). Two (2) to four (4) hours post-treatment, cells were incubated with pseudoparticles bearing WSN-HA/NA proteins and encoding the Gaussia lueiferase reporter gene. Diphyllin, a compound previously shown to inhibit influenza virus entry was used as a positive control. After 18 hours, ceils were washed and medium was added. A lueiferase assay was performed 24 hours after addition of medium. Figure 4 shows that diphyllin reduced viral entry to 8% relative to the DMSO control, whereas ONlOS l lO did not inhibit viral entry.

Example 5; Effect of ON108110 on export of

vRNPs into cytoplasm

[0092] This example demonstrates that ONl OSl lO impairs the export of influenza viral RNPs (vRNPs) into the cytoplasm of A549 cells.

[0093] To determine whether ON1081 10 inhibits vRNP trafficking, A549 cells were treated with ONlOS l l O at 3.13 μΜ for 2 hours prior to infection with WSN virus at an MOI of 3. Cells were washed several times and medium containing the compound was added back to the cells. At 5 and 7 hpi, cells were fixed, permeabilized and stained with a polyclonal rabbit anti-NP serum, Alexa Fluor 488 donkey anti-rabbit IgG (In vitro gen), and DAPl, and then viewed under an Axioplan2TE fluorescence microscope. At 5 hpi, there were no differences in the vRNP localization between the DMSO- and ON 1081 10- treated cells. The percentages of nuclear accumulation for DMSO- and ONI 081 10-treated cells were 63 ± 8 % and 72 ± 2 %, respectively (Fig. 5). At 7 hpi, the vRNP localization was predominantly cytoplasmic for DMSO-treated cells. In contrast, the vRNP localization was predominantly nuclear for ON 1081 10-treated ceils. The percentages of nuclear accumulation for DMSO- and ON 1081 10-treated cells were 1 ± 1 % and 86 ± 2 %, respectively (Fig. 5), These results strongly indicate that ON I 081 10 impairs the export of influenza vRNPs into the cytoplasm of A549 cells. Example 6 : Effect of ONI 08110 on VSV and NDV re lic tion

[0094] This example demonstrates that ON108110 reduces the replication of vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV) in a dose-dependent manner at concentrations that are not toxic to cells.

[0095] Above, it is shown that ONI 081 10 inhibits the replication of influenza A virus by impairing vRNP nuclear export. Since ONI 081 10 inhibits a variety of host kinases, it was next determined whether the compound could also inhibit other negative stranded RNA viruses (e.g. VSV and NDV) that rely on host machinery (e.g. host kinases) for replication.

[0096] A549 ceils were treated with two-fold serial dilutions (0.78 to 12.5 μΜ) of

ON1081 10 for 2 to 4 hours prior to infection with VSV-GFP at an MO I of 0.0001. Supernatants were harvested 24 hpi and plaque assay was performed. Figure 6A shows that ONI 081 10 inhibits the replication of VSV in a dose-dependent manner at concentrations that are not toxic to cells.

[0097] To determine whether ON 1081 10 inhibits the replication of NDV, A549 cells were treated with two-fold serial dilutions (0.78 to 25 μΜ) of ON1081 10 for 2 to 4 hours prior to infection with NDV (Bl strain) at an MOI of 2. The amount of virus in the supernatant at 24 hpi was measured using a plaque assay. As shown in Figure 6B, ON1081 10 inhibited the replication of NDV in a dose-dependent manner at concentrations that were not toxic to cells.

[0098] Unlike influenza vRNAs, VSV and NDV vRNAs are replicated and transcribed in the cytoplasm. Viral RNPs of these two viruses are not exported from the nucleus. Thus, this suggests that ON1081 10 inhibits VSV and NDV in a manner distinct from that of influenza virus.

PropSietie Example 1.· In vivo properties of ON108110 and its analogs

[0099] ON 1081 10 and derivatives (e.g., analogs) of ONI 081 10 having pharmacokinetic properties optimized for in vivo administration are tested in an in vivo animal model of viral infection. Animals (e.g., mice) are infected with influenza A virus or another negative-sense, single-stranded RNA virus (e.g., VSV, Sendai virus, NDV, avian flu virus) and treated with a test compound (e.g., ON1081 10 or an analog thereof) or with a control, to determine whether treatment with the test compound treats the viral infection. In another set of experiments, the animals are first treated with a composition containing ON1081 10 or an analog thereof, and subsequently exposed to virus. The ability of the vaccine to confer protection to viral infection is assessed.

Syn thesis Example A :

Synthesis of 2-Ben¾yjidene~2H-Benz ?¾j H,41thiazia-3(4H)-ones

[00100] Exemplary starting materials and methods for synthesizing substituted 2- benzylidene-2H-benzo[b][l ,4]thiazin-3(4H)~ones, such as ONI 088110, are described in international Patent Publication No, WO 2012/166586 (the entirety of which is incorporated herein, by reference), portions of which are reproduced in this Example. In particular, this example describes methods that may be used to synthesize compounds of Formula I, below:

Formula I

[00101] wherein

[00102] n is 0, 1 or 2;

[00103] R¹ is selected fro the group consisting of-H, -(CrCejaikyi, -(C2-C,s)alkenyi,

-(C2-C6)alkynyl, optionaliy substituted aryi, optionaliy substituted heteroaryl, optionally substituted aryl-(Ci-C6)alkyJ, optionally substituted heterQaryl-(C_}-C₆)alkyl,

C₆)alkyi, -C(=0)(C₂-C6)alkenyl, -C(=0)-optionally substituted aryi, ·(/·; OHC/i l - s · optionally substituted aryi, and -C(=0)(CH₂)_p-optionally substituted heteroaryl;

[00104] R Β and R⁴ are independently selected from the group consisting of -H, halogen, -CN, -NRⁱ⁰R^{! !} , -OH, -OR¹³, -(C_;-C₆)aikoxy, -N<¼, -(d-C^alkyl, -(C_r C₆)pertluoroalkyI, -iCi-C«)perfiuoroalkoxy, -C(=0)R.¹⁵ _s -C(=O)0R¹⁵, -OC(=0)R¹², -

OCiOiOR¹ -C(==0)NR¹⁷R¹⁸, -S(Ci-C₆)alkyl, -S(-===0)R^1J, -S(- 3)₂R",

-GS(===0)₂Rⁱ³, ^■■S(===0)_qR¹⁵ _> -OS(===0)qR¹⁵, -SH3)₂NR^{! 7}R^{i S}, -S(==0)NR^{i 7}R^] \ optionaliy substituted aryi, optionally substituted aryl-(C t-Q alky optionally substituted heteroaryl, optionally substituted heteroaryl-iCj -C-6)alkyl, optionally substituted (C2-C )heterocyclyl, optionaliy substituted (C₂-C¾)heterocyclyl-(Ci -Q alkyl, ^»NH(CI-i₂)_mQ ===0)OR¹⁴, - C(=NR¹⁴)^'NR^{i 4}2, -C(=N-OR.¹⁴)^'NRⁱ 2 , -P(=G)(GRⁱ⁴)_2s and -OP(=0)(ORⁱ )₂; [00105] Ar is optionally substituted heteroaryl, optionally substitu ted (Cio-Ct- aryl, or

[00106] wherein

[00107] R^s, R⁶, R'₅ R^s, and R⁹ are independently selected from the group consisting of

-H, -OH, -OR¹³, -NO2, halogen, -CN, NRⁱ⁰R^!1, -(CH2)_mNR¹⁰Rⁿ, -()(ΠΗ···ΝΚ"^!Κ". -(Q- C₆)alkyi, -(CH₂)_raO(C_f-C6)atkyl, -(Ci-C₆)alkoxy, -(CrCelperfluoroalkyl, -(C_f- C₆)perfluoroalkyoxy, -SH, -S(Ci-C₆)alkyl, -SR^B, -S(=0)R¹⁵, -8(=0)₂R^!5, -C(=0)R¹⁵, - C(=0)OR¹⁵, -C(=0)NRⁱ⁷R¹⁸, -OC(=0)R¹⁶, -OC(=0)OR¹², -OU 0;XR^: R^1". heterocyclyl, optionally substituted heteroaryl, -NH(CH₂)mC(<>)OR¹⁴, -OS( OI K¹". -C(== R¹ )NR¹⁴ ₂, - C(===N-0R¹⁴)NRⁱ⁴ ₂, -P(=0)(OR¹⁴)¾ and -OP(=0)(OR^l4)₂;

[00108] each R'° and R¹¹ is independently selected from the group consisting of -H, -

(CrC₆)alkyi, -(Cj-C₆)alkyoxy, ·(/·; () R^!'. -C(=0)NR¹⁷R¹⁸, -C(=0)OR¹², -C(=0)OR¹², ^■■ C(=NR¹ )NR^,7R' R^IJ, optionally substituted aryl, optionally substituted heteroaryl, and - C(=NR^I'^,)Rⁱ⁵; or R¹⁰ and Rⁿ, together with the nitrogen to which they are bound, form an optionally substituted (C^-Csjheterocycle;

[00109] each Rⁱ is independently selected from the group consisting of -(Ci-C6)alkyl, and optionally substituted aryl;

[00110] each R^1"' is independently selected from the group consisting of optionally substituted aryl and -(CH₂)_mR¹⁶;

[00111] each R¹⁴ is independently selected from the group consisting of -H and -(Ci-

C)alkyl; or two occurrences of R¹⁴ bound to the same nitrogen form a (C₂-C6)taeterocycle, together with the nitrogen atom to which they are bound;

[00112] each R¹⁵ is independently selected from the group consisting of -H, -{€^■■-

Celaikyl, optionally substituted aryl, and NR^!" ₂; [001 13] each R^!o is independently selected from the group consisting of ---(CrCeWkyi, NR^{! 4} ₂, and Ar';

[001 14] each R^! and R¹⁸ is independently selected from the group consisting of -H, -

(Cj -C alkyl, -(Ci-C6)alkyoxy, R'³, optionally substituted aryl, and optionally substituted heteroaryl; or R^{l ?} and R¹⁸, together with the nitrogen to which they are bound, form an optionally substituted (C₂-C5)heierocycle;

[001 15] m is independently at each occurrence 1 , 2, 3 , 4 or 5;

[001 16] p is independently at each occurrence 0, 1 , 2, or 3;

[001 17] q is independently at each occurrence 0, 1 or 2;

[001 18] each optionally substituted aryl, optionally substituted (Cio-Ci₄)aryl, optionally substituted heteroaryl, optionally substituted aryl-(Ci-C½)a!kyl, optionally substituted heteroaryi-i Cj -C^alkyl, optionally substituted (C2-C¾)heterocyclyl, optionally substituted (C2-C¼)heterocyclyl-(Ci-C6)a1kyl, and optionally substituted (Ci-C$ )heterocycle is optionally substituted with one or more sobstituents independently selected from the group consisting of halogen, -CN, -NR.'⁴?.,

C₆ialkyl, -NR^l4C(===0)0(C₃-C₆)alkyl, -NR¹⁴C(==0)NR¹⁴ ₂, -NR^l C(===NRⁱ⁴)NR⁵ ₂, - NH(CH₂)_J,_iC(===0)OR¹⁴ ₅ -OH, -N0₂, -{C_rC₆)aikyl -(CH_?)_r,P(CrC₆)alkyl, -(Ci-Q alkoxy, ^■■ (C₂-G;)aikenyl, -(C₂-C¾alkynyl₅ ~SR ^{i 4}, -S(=0)R¹⁵, -Si=G)₂R^{i 5}, -NR¹⁴Si=G)₂R^{i 5}, -(C₃- CftjperfluoroalkyL -(Ci-C6)perfluoroalkoxy, -Ci=0)R¹⁴, -C(=0)OR¹⁴, -C(-0)NR^w ₂, - OC(===0)R¹⁴, -0C(=O)NR"^"¾ -OC(===0)0(C] -C₆)alkyi, -PC===0^')(OR¹ )¾ -0P(O)(0R^l4)₂, heterocyclyl, and heteroaryl;

[00119] Ar ' is a radical according to Formla H:

Formula H

[00120] R'⁹, R~°, R²¹, R", and R^2j are independently selected from the group consisting of -H, -OH, -N0₂, halogen, -CN, NRⁱ⁰Rⁿ, -(CH₂)_mNR^SQRⁿ, -O(CH₂)_mNR¹⁰R¹ - (Ci-C₆)alkyl, -(CH₂)_mO(d -CsJalkyl, -(C_t-C₆)alkoxy, -(Ci-C₆)perfluoroalkyl, -(C_r C₆)perfluoroalkyoxy, -SH, -8Rⁱ², -S(=0)R^f5, -S(=0)₂R¹⁵, -C(=0)R¹⁵, -C(=0)OR¹⁵, - C(=0)NR¹⁷R¹⁸, -0C(O)R^!6, -OC(=0)OR¹², -OC(=0)NR^!7R^!8, heterocyclyl, optionally substituted heteroaryl, -NH(CH₂)_mC(=0)OR¹⁴, -OS(=0)₂R¹⁶, -C(-NR^M)NR^!4 _¾ -C(-N- OR^l4)NR ⁴ ₂, -P(=0)(OR¹⁴)₂, and -OP(=0)(OR^w)₂;

[00121] provided that:

(i) at least one of R², R³, or R is other than hydrogen;

(ii) when none of R², R³ and R⁴ are -OR¹³, -N R¹³, -SR¹³, -S(=0)R^!3, or -S(=0)₂R¹³, and Ar is

then at least one of R° and R⁸ is -NO? and at least R' is other than hydrogen or halogen; and

(iii) when Ar is optionally substituted heteroaryl and none of R^{^}, R^J, or R⁴ are - oR^! \ -NHR¹³, -Si 0)R ^: . or S( ()) R ^s \ then R¹ is other than hydrogen.

[00122] There are provided processes for preparing compounds according to

Formula I, intermediates that are useful in the preparation of such compounds, and processes for preparing such intermediates.

[00123] The compounds of Formula I can be prepared by condensing an appropriately substituted aldehyde according to Formula ΙΠ

Formula Ml

[00124] wherein Ar is defined elsewhere herein, with a compound according to

Formula IV:

Formula IV

[00125] wherein R¹, R², R³, R⁴, and n are as defined elsewhere herein.

[00126] The aldehyde according to Formula 111 may he purchased from a commercial supplier such as Sigma Aidrich, or the aldehyde of Formula ΪΠ may be prepared using procedures known in the art. The benzothiazinone of Formula IV can be prepared in at least two ways. For example, in one embodiment, a benzothiazinone of Formula IV can be prepared according to Scheme 1 (depicted in Figure 1 LA), wherein R², R* and R⁴ are defined previously herein.

[0012.7] According to Scheme 1 (Figure 11 A), an appropriately substituted nitro, fluorobenzene derivative A, is reacted with thioglycolic acid (R=Ff) or an ester of thioglycolic acid such as methyl or ethyl thioglycolate (R^:::alkyl) to give thioether B. The nitro group in tbioether B can then be reduced. In particular embodiments, the reduction conditions employ Zn and acetic acid, in an alternative embodiment, the reduction protocol employs sodium dithionite (sodium hydrosulfiie). Other nitro group reduction protocols may also be suitable. The resulting amine then undergoes in situ cyclization to give compound C, which is a compound according to Formula TV,

[00128] Optionally the amide functionality in compound C can be alkylated or acylated via deprotonation of compound with appropriate base, examples of which include, but are not limited to, K^COs H, potassium bis(trimethylsiiyl)amide (KHMDS), sodium bis(trimethylsilyl)amide ( aHMDS) and lithium letramethylpiperazine (Li^'TMP). The resulting anion can then be treated with a reactive electrophile, such as, but not limited to, an alkyl or alkenyl halide (e.g., CH₃I, CH₂=CHCH₂Br, etc.), or equivalents thereof. Alternatively, the anion can be treated with a reactive acyl species, such as, but not limited to, an acyl chloride (e.g., acetyl chloride). The resultant product is a compound of Formula IV wherein R^! is other than hydrogen. [00129] In other embodiments, the amide functionality in compound C can be arylated or beterarylated by reacting compound C with Cul, K₃PO₄, an appropriately functionaiized aryl or heteroaryl iodide, and trans- 1 ,2-cyclohexanediamine (10 mol% based on the quivalents of aryl iodide) all in an appropriate solvent, examples of which include, but are not limited to acetonitrile, toluene, dioxane and 1 ,2-dimethoxyethane. Typically the reaction is warmed, and in certain embodiments, warmed to reflux. The reaction is typically carried out under an inert atmosphere. Upon completion, the reaction can be filtered through a silica gel or celite pad to remove solid impurities. The pad can be washed with additional reaction solvent to ensure that ail product has been removed from the filter pad. The solvent of the resultant filtrate can then be evaporated under reduced pressure and the resultant residue purified by flash chromatography. As above, the resultant product is a compound of Formula IV wherein R¹ is other than hydrogen.

[00130] A compound according to Formula TV can also be prepared by fxinctionalizing the commercially available compound 2H- 1 ,4-benzothiazin- 3 (4H)-one . For example, in Scheme 2, depicted in Figure 1 I B, the compound 2H-1 ,4-benzothiazin-3(4H)-one is reacted with chlorosulfonic acid to give 3-oxo-3,4-dihydro-2H-benzo[6][l,4]thiazine-7-sulfonyl chloride (compound "D"). Compound D can then be treated with sodium sulfite (Ν¾8(¾) and sodium hydrogen phosphate (Na₂HP0₄) to give an intermediate (compound "E"), This intermediate can then be reacted with an appropriately substituted benzyl group to give compound F, wherein R^*9, R²⁰, R^{2 i}, R²² and R^"3 are as defined previously herein and X is leaving group such as a haiide, tosylate, mesylate, beiizensulionate, mefhansulfonate or other equivalent group suitable for use in 5^2 type chemistry. Compound F is a compound of Formula IV.

[00131] Optionally, the amide functionality in compound F can be alkylated or acylated via deprotonation of compound F with an appropriate base, examples of which include, but are not limited to, K₇CO₃, Natl, potassium bis(trimethylsilyl)amide (KilMDS), sodium bis(trimethyisilyl)amide (NaHMDS), and lithium tetramethylpiperazine (LiTMP). The resulting anion can then be treated with a reactive electrophiie, such as but not limited to, an alkyl or alkenyl haiide (e.g., CH3I, CH=CHC¾Br, etc.), or equivalents thereof. Alternatively, the anion can be treated with a reactive acyl species, such as, but not limited to, an acyl chloride {e.g., acetyl chloride). The resultant product is a compound of Formula TV whereinR¹ is other than hydrogen. [00132] In other embodiments, the amide functionality in compound F can be arylated or heteroarylated by reacting compound F with Cul, K₃PO₄, an appropriately functionalized aryl or heteroaryl iodide, and trans- 1 ,2-eyelohexanediamine (10 mol% based on the equivalents of aryl iodide) ail in appropriate solvent, examples of which include, but are not limited to, acetonitrile, toluene, dioxane, and 1 ,2^■ dime thoxyethane. Typically the reaction is carried out under an inert atmosphere. Upon completion, the reaction can be filtered through a silica gel or celite pad to remove solid impurities. The pad can be washed with additional reaction solvent to ensure that all product has been removed from the filter pad. The solvent of the resultant filtrate can then be evaporated under reduced pressure and the resultant residue purified by flash chromatography. As above, the resultant product is a compound of Formula IV wherein R¹ is other than hydrogen.

[00133] The aldehyde of Formula HI can then be condensed with the compounds of Formula IV to give a compound of Formula Ϊ. in certain embodiments, the condensation reaction takes place in the presence of a suitable base and an anhydride, wherein the anhydride acts as the solvent for the reaction, in other embodiments, the reaction can be run in a solvent other than anhydride, examples of which include, but are not limited to toluene and acetic acid. In particular embodiments, the base is triethylamine (Et^N) and the anhydride is acetic anhydride (Ac₂0). Other suitable bases, and in particular amine bases, can be substituted for 1¾Ν. Examples of other suitable bases include, but are not limited to, di-isopropyiethyl amine. Likewise, other suitable anhydrides can be used. Examples include, but are not limited to, propionoic anhydride and butyric anhydride. The resulting mixture can then be wanned and, in certain embodiments, heated to reflux to give a compound of Formula I. An exemplary reaction scheme (Scheme 3) is provided in Figure i lC.

[00134] In certain embodiments, the above-described reaction may result in a compound of Formula I whereinR¹ is hydrogen. The nitrogen bound to R¹ can be further functionalized via selective aryiation, heteroaryiation, aikylation or acylation according to the procedures described elsewhere herein for compounds C and F.

[001 35] Compounds of the invention can be prepared and used as their benzothiazinone- 1 -oxides, i.e., compounds of Formula I wherein n is 1. These compounds can be prepared by oxidizing a compound of Formula I (or appropriately functionalized intermediate useful for the preparation of a compound of Formula I) with an oxidizing agent such as meta-chloroperoxybenzoic acid (MCPBA) in a solvent such as, but not limited to, diehioromethane. After appropriate basic aqueous workup, the oxidized compound of Formula 1 can typically be isolated in high yield and purity after recrystallization or flash chro atography.

[00136] Although compounds of Formula 1 are generally produced as their Z isomers, the compounds of the invention can be converted to their E isomeric forms via a photo- induced isomerization (either sunlight or UV7VIS radiation) according to the procedure described in Kamila et ai, "Synthesis of (Z)-hetarylmethyIene and (Z)-substituted benzylidene derivatives of 4Hbenzo[i,4]thiazine-3-thiones and their subsequent conversion to benzothiopyrano-[3,2-i][L4]benzothiazines" AR IVOC 2006 (ii) 1- 14, the entirety of which is hereby incorporated by reference.

General Pro^

Scheme 3

[00137] A compound according to Formula IV (1 mmol), an aldehyde of Formula III (1 mmol), triethyiamine (4 mmol), and acetic anhydride (10 mi) were rcfiuxed under nitrogen atmosphere for two hours. The reaction mixture was cooled to room temperature. Solvent was removed under reduced pressure and the resulting residue was purified by column chromatography to give a compound of Formula I. In certain instances, a solid precipitate formed upon completion and/or cooling the reaction. In either case, the precipitate was collected via filtration. Subsequently, the filtered solid was washed with an appropriate solvent and dried under vacuum to give a compound of Formula I.

[00138] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00139] It is further to be understood that all values are approximate, and are provided for description.

[00140] Patents, patent appiications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for ail purposes.

Claims

What is claimed is:

i . A method for inhibiting the replication of a negative-sense, single-stranded RNA viras in a subject, the method comprising administering to a subject in need thereof an effecti ve amount of a composition comprising an inhibitor of a kinase selected from the group consisting of CAMK L CAMKK2, CDKl/cycJin A, CDKl/cyclm B, CDK2/cyciin A, CDK4/cyclin Dl , CDK4/c clin D3, CDK5/p25, CDK5/p35, CDK6/cyclin D 1 , CDK6/cyciin D3, CDK9/cyciin K, CD 9/cyclin Tl, CK2a, CK2a2, DYRK1B, DYRK2, GSK3a, GSK3b, HGK/ AP4K4, MLK3/MAP3K1 1 , MUSK, NLK, PIM3, and SGK1.

2. The method claim 1 , wherein the composition comprises a compound having the structure:

3. The method of claim 1 , wherein the virus is a member of a virus family selected from the group consisting of Orihomyxovbidae, Rnabdoviridae, and

Pa ratnyxo vi ridae . 4 The method of claim 3, wherein the virus is selected from the group consisting of Influenza A, Influenza B, infl'aenza C, Infectious salmon anemia (ISA), Thogoto, vesicular stomatitis Indiana vims (VSV), Sendai virus, and Newcastle disease virus (NDV).

A method of treating a viral infection in a subject, the method comprising administering to a subject in need thereof an inhibitor of a kinase selected from the group consisting of CAMKKl , CAMKK2, CDKl/cyclin A, CDKl/cyclin B, CDK2/cyclin A, CDK4/cyclin Dl , CDK4/cyclin D3, CDK5/p25, CDK5/p35, CDK6/cyclin D 1 , CDK6/cyciin D3, CDK9/cyclin K, CDK9/cyclm Tl, CK2a, CK2a2, DYR 1B, DYRK2, GSK3a, GSK3b, HGK/MAP4K4, MLK3/MAP3K 1 1 . MUSK, NLK, PIM3, and SGK1.

6. The method of claim 5, wherein the composition comprises a compound having the structure:

or a derivative thereof.

7. The method of claim 5, wherein the virus is a member of a virus family selected from the group consisting of Orthomyxoviridae, Rhabdoviridae, and

Paramyxoviridae .

8. The method of claim 7, wherein the virus is selected from the group consisting of Influenza A, Influenza B, Influenza C, infectious salmon anemia (ISA), Thogoio, vesicular stomatitis Indiana virus (VSV). Sendai virus, and Newcastle disease virus (NDV).

9. A method of inhibiting a viral infection in a subject, the method comprising administering to a subject in need thereof an inhibitor of a kinase selected from the group consisting of CAMKKi, CAMKK2, CDKl/cyclin A, CDKl/cyclin B, CDK2/cyclin A, CDK4/cyclin Dl, CDK4/cyclin D3, CDK5/p25, CD 5/p35, CD 6/cycli Dl, CDK6/cyclin D3, CDK9/cyclin K, CDK9/cyclin Tl , CK2a, CK2a2, DYRK1 B, DYRK2, GSK3a, GSK3b, HGK/MAP4K4, MLK3/MAP3K1 1, MUSK, NLK, PIM3, and SGKl .

10. The method of claim 9, wherein the composition comprises a compound having the structure:

or a derivative thereof. i i. The method of claim 9, wherein the virus is a member of a virus family selected from the group consisting of Orthomyxovitidae, Rhabdoviridae, and

Pammyxo viridae.

12. The method of claim 1 1 , wherein the virus is selected from the group consisting of Influenza A, Influenza B, Influenza C, Infectious salmon anemia (ISA), Thogoto, vesicular stomatitis Indiana virus (VSV), Sendai virus, and Newcastle disease virus (NOV).