OA19256A

OA19256A - Anti-Viral Compounds.

Info

Publication number: OA19256A
Application number: OA1201400136
Authority: OA
Inventors: Kerry W. Fowler; Shawn P. Iadonato; Kristin Bedard; Myra Wang IMANAKA
Original assignee: Kineta, Inc.
Priority date: 2011-09-30
Filing date: 2012-09-27
Publication date: 2020-06-05

Abstract

Disclosed herein are compounds and related compositions for the treatment of viral infection, including RNA viral infection, and compounds that can modulate the RIG-I pathway in vertebrate cells, including compounds that can activate the RIG-I pathway.

Description

The compounds and methods disclosed herein shift the focus of viral drug development away from the targeting of viral proteins to the development of drugs that target and enhance the host’s innate antiviral response. Such compounds and methods are likely to be more effective, less susceptible to the emergence of viral résistance, cause fewer side effects and be effective against a range of different viruses.

The RIG-I pathway is intimately involved in regulating the innate immune response to RNA virus infections. RIG-I agonists are expected to be useful for the treatment of many viruses including, without limitation, HCV, influenza, and West Nile virus. Accordingly, the présent disclosure relates to compounds and methods for treating viral infection, including infection by RNA viruses, wherein the compounds can modulate the RIG-I pathway.

One embodiment of the présent disclosure includes a compound represented by the formula

Formula 1 wherein a dashed line indicates the presence or absence of a bond; R¹ may be R^a, OR² or NR²R³; each R^a may independently be independently H, optionally substituted hydrocarbyl, optionally substituted aryl, optionally substituted heteroaryl; R² and R³ may each independently be R^a, COR^a, or SO2R^a; Y¹, Y², Y³ and Y⁴ may each independently be CR⁴ or N; each R⁴ may independently be R², OR^a, NR²R³. SR^a, SOR^a, SO2R^a, SO2NHR^a NCOR^a, halogen, trihalomethyl, CN, S=O, or nitro; R⁵ may be R^a, COR^a, SO2R^a, or is not présent; V may be CR², CR²R³, C=O, COCR²R³, or C=NR²; and, W and X may each independently be N, NR^a, O, S, CR²R⁴or CR⁴.

Some embodiments of the présent disclosure include a compound represented by the formula (the “KIN1000” compound):

Additional embodiments include a compound represented by the formula

R¹⁶ wherein R¹⁰, R¹³, R¹⁴, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independently R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl; each R^b is independently H or C1.3 hydrocarbyl, and each R^c is independently H or Cv3 alkyl.

Further embodiments of the présent disclosure include a compound represented by the formula (the “KIN1148” compound):

H

Some embodiments of the présent disclosure include a pharmaceutical composition comprising any of the compounds as described herein.

Some embodiments of the présent disclosure include methods of treating or preventing a viral infection in a vertebrate comprising administering to the vertebrate a pharmaceutical composition as described herein. In some embodiments, the viral infection is caused by a virus from one or more of the following families: Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae. In some embodiments, the viral infection is influenza virus, Hepatitis C virus, West Nile virus, SARS-coronavirus, poliovirus, measles virus, Dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, llheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, Kyasanur forest disease virus, respiratory syncytial virus or HIV.

Some embodiments of the methods of the présent disclosure include administering any of the pharmaceutical compositions described herein as an adjuvant for a prophylactic or therapeutic 3 vaccine. In some embodiments, the method includes vaccinating a vertebrate by additionally administering a vaccine against influenza virus, Hepatitis C virus, West Nile virus, SARScoronavirus, poliovirus, measles virus, Dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, llheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, Kyasanur forest disease virus or HIV.

Some embodiments of the présent disclosure include methods of modulating the innate immune response in a eukaryotic cell, comprising administering to the cell any of the compounds as described herein. In some embodiments the cell is in vivo. In other embodiments the cell is in vitro.

Brief Description of the Drawings

Figure 1 shows validation and characterization of compound KIN1000 (“RLU” = relative luciferase units). In Figure 1A, initial “hit” compounds were validated by demonstrating dosedependent induction of the ΙΡΝβ-luciferase (ΙΡΝβ-LUC, left), ISG56-luciferase (ISG56-LUC, center), and the ISG54-luciferase (ISG54-LUC, right) reporter genes. Figure 1B confirms the specificity of Kl N1000, which does not induce the non-specific β-actin promoter (“0.5% DMSO” = vehicle control; “10μΜ KIN1000” = β-actin-luciferase reporter in presence of KIN1000; “10μΜ Compound X” = positive control β-actin induction). In Figure 1C, the MTS assay demonstrated that KIN1000 did not show évident cytotoxicity to human cells treated for 48 hours with the compound. The O.D. value that represents 50% cell mortality is shown by a horizontal line, also demonstrating that the CC50 of KIN1000 is greaterthan 20μΜ.

Figure 2 shows activation of transcription factors by KIN1000. In Figure 2A, HeLa cells treated with increasing amounts of KIN1000 showed dose-dependent increase in IRF-3 translocation to the nucléus, quantified by nuclear intensity minus cytoplasmic intensity (“normalized nuclear intensity”). In Figure 2B, HeLa cells treated with increasing amounts of KIN1000 showed dosedependent increase in NFkB translocation, quantified by nuclear intensity minus cytoplasmic intensity. “SeV” refers to Sendai virus infection, the positive control.

Figure 3 shows anti-viral activity of KIN1000. MRC5 cells treated with increasing amounts of KIN1000 showed dose-dependent decrease in infection by influenza virus.

Figure 4 shows Luminex® (Luminex Corp., Austin TX) quantified levels of cytokine expression induced by KIN1000. Human dendritic cells treated with increasing amounts of KIN1000 showed dose-dependent expression of cytokines including IL-8, MCP-1 (CCL2), and MIP-1a and β (CCL3 and CCL4, respectively).

Figure 5 shows induction of gene expression by KIN1000 and its dérivative compound KIN1148. Figure 5A shows gene expression levels of IFIT2 (left) and OAS1 (right) in HeLa cells over time from 4-24 hours post treatment with 10uM KIN1000 (grey) or KIN1148 (black). Figure 5B shows gene expression levels of IFIT2 in PH5CH8 cells (left) treated with Kl N1000 (solid grey bars) or KIN1148 (solid black bars), and in HeLa cells (right) treated with KIN1000 (grey striped bars) or 4

KIN1148 (black checked bars). In each test group, the three vertical bars represent 5, 10, and 20 μΜ compound (KIN1000 or KIN1148), respectively. Figure 5C shows gene expression levels of IFIT2 (left), OAS1 (center), and MxA (right) in primary HUVEC cells that were treated with 1μΜ Kl N1000 (grey) or 1μΜ KIN1148 (black).

Figure 6 shows antiviral activity of KIN1000 and KIN1148 against respiratory syncytial virus. Figure 6A shows that HeLa cells treated with increasing amount of KIN1000 and KIN1148 showed dose-dependent decrease in infection by RSV. Figure 6B shows that KIN1148 showed antiviral activity against RSV when drug is added up to 24 hours prior to infection.

Figure 7 shows antiviral activity of KIN1148 against Influenza A virus Udorn/72. H292 cells (left) and HEK293 cells (right) treated with 2μΜ (H292) or 10μΜ (HEK293) of KIN1148 showed decrease in infection by virus.

Figure 8 shows antiviral activity of KIN1148 against Dengue virus type 2. Huh 7 cells treated with increasing amounts of KIN1148 showed dose-dependent decrease in infection by virus.

Figure 9 shows antiviral activity of KIN1148 against Hepatitis B virus. HepAD38 cells treated with increasing amounts of KIN1148 showed dose-dependent decrease in supernatant levels of virus. The O.D. value that represents no HBV in the supernatant is shown by a horizontal line labeled “NO HBV CELLS.”

Figure 10 shows IgG antibody production induced by KIN1000 and KIN1148 in vivo. Animais (Lewis female rats, 10-12 weeks old) were vaccinated with suspensions of OVA in PBS, OVA+polyl:C, OVA+KIN1000 or OVA+KIN1148 subcutaneously in the footpad and base of tail (0.025 mL injection volume per site). Animais were boosted identically at 2 and 8 weeks post priming. Animais were bled at the indicated time points, sera was prepared and antibody levels were detected by ELISA. OD450 values for vaccine préparations containing KIN1000 (large checked bars) and KIN1148 (horizontal striped bars) were normalized to values obtained from animais that received OVA in PBS alone as vaccines. Poly l:C (small checked bars) was used as a control adjuvant.

Figure 11 shows cellular response elicited by KIN compound vaccination. Delatyed type hypersensitivity responses elicited 2 weeks after the first boost (4 weeks post prime) were measured. Animais were challenged by injection of 0.02 mL of PBS (left ear pinna) or 0.02 mL of OVA (1 mg/mL) in PBS (right ear pinna) at indicated time point. 24 hours later ear thickness was measured with calipers. The calculated différence between right ear and left ear is shown. “OVA+K1148” (vertical striped bar) = différence in ear thickness in animal injected with vaccine containing KIN1148. Poly l:C (“OVA+pl:C;” horizontal striped bar) was used as a control adjuvant.

Detailed Description

The présent disclosure provides compounds and methods that shift the focus of viral treatments away from the targeting of viral proteins to the development of drugs that target and enhance the host (patient’s) innate antiviral response. Such compounds and methods are likely to be 5 more effective, less susceptible to the emergence of viral résistance, cause fewer side effects and be effective against a range of different viruses.

The RIG-I pathway is intimately involved in regulating the innate immune response to RNA virus infections. RIG-I is a cytosolic pathogen récognition receptor that is essential for triggering immunity to a wide range of RNA viruses. RIG-I is a double-stranded RNA helicase that binds to motifs within the RNA virus genome characterized by homopolymeric stretches of uridine or polymeric U/A motifs. Binding to RNA induces a conformation change that relieves RIG-I signaling repression by an autologous repressor domain, thus allowing RIG-I to signal downstream through its tandem caspase activation and recruitment domains (CARDs). RIG-I signaling is dépendent upon its NTPase activity, but does not require the helicase domain. RIGI signaling is silent in resting cells, and the repressor domain serves as the on-off switch that governs signaling in response to virus infection.

RIG-I signaling is transduced through IPS-1 (also known as Cardif, MAVs, and VISA), an essential adaptor protein that résides in the outer mitochondrial membrane. IPS-1 recruits a macromolecular signaling complex that stimulâtes the downstream activation of IRF-3, a transcription factor that induces the expression of type I IFNs and virus-responsive genes that control infection. Compounds that trigger RIG-I signaling directly or through modulation of RIG-I pathway components, including IRF-3, présent attractive therapeutic applications as antivirals or immune modulators.

A high-throughput screening approach was used to identify compounds that modulate the RIG-I pathway, a key regulator of the cellular innate immune response to RNA virus infection. In particular embodiments, validated RIG-I agonist lead compounds were demonstrated to specifically activate interferon regulatory factor-3 (IRF-3). In additional embodiments they exhibit one or more of the following: they induce the expression of interferon-stimulated genes (ISGs), hâve low cytotoxicity in cell-based assays, are suitable for analog development and SAR studies, hâve drug-like physiochemical properties, and hâve antiviral activity against influenza A virus and/or HCV.

As discussed below, these compounds represent a new class of potential antiviral therapeutics. Although the disclosure is not bound by a spécifie mechanism of action of the compounds in vivo, the compounds are selected for their modulation of the RIG-I pathway. In certain embodiments, the modulation is activation of the RIG-I pathway. Compounds and methods disclosed herein function to, one or more of, decrease viral protein, viral RNA, and infectious virus in cell culture models of HCV and/or influenza virus.

In one embodiment, the disclosure herein relates to a class of compounds of represented by the following formula:

Formula 1

wherein a dashed line indicates the presence or absence of a bond; R¹ may be R^a, OR² or NR²R³; each R^a may independently be independently H, optionally substituted hydrocarbyl, optionally substituted aryl, optionally substituted heteroaryl; R² and R³ may each independently be R^a, COR^a, or SO2R^a; Y¹, Y², Y³ and Y⁴ may each independently be CR⁴ or N; each R⁴ may independently be R², OR^a, NR²R³ SR^a, SOR^a, SO2R^a, SO2NHR^a NCOR^a, halogen, trihalomethyl, CN, S=O, or nitro; R⁵ may be R^a, COR^a, SO2R^a, or is not présent; V may be CR², CR²R³, C=O, COCR²R³, or C=NR²; and, W and X may each independently be N, NR^a, O, S, CR²R⁴or CR⁴.

With respect to Formula 1, Y¹ may be CR⁴ or N. In some embodiments, Y¹ is CR⁴.

With respect to Formula 1, Y² may be CR⁴ or N. In some embodiments, Y² is CR⁴.

In some embodiments, Y¹ and Y² are both CR⁴, and togetherform an additional heterocyclic ring optionally substituted by R¹⁸. In some embodiments, V may be:

With respect to Formula 1, Y³ may be CR⁴ or N. In some embodiments, Y³ is CR⁴.

With respect to Formula 1, Y⁴ may be CR⁴ or N. In some embodiments, Y⁴ is CR⁴.

In some embodiments, Y¹, Y², Y³ and Y⁴ are CR⁴. In some embodiments, Y¹, Y², Y³ and Y⁴ are

CH. In some embodiments, Y¹ and Y² are

Some embodiments include compounds represented by any of Formulas 2-12.

Formula 7

Formula 3

R¹⁶

Formula 9

Formula 6

With respect to any relevant structural feature herein, each R^a can independently be H; optionally substituted hydrocarbyl, such as C-|.12 or C^e hydrocarbyl; optionally substituted aryl, such as optionally substituted C6-i2 aryl, including optionally substituted phenyl; optionally substituted heteroaryl, including optionally substituted C2.-|2 heteroaryl, such as optionally substituted pyridinyl, optionally substituted furyl, optionally substituted thienyl, etc. In some embodiments, each R^a can independently be H, or C-m2 alkyl, including: linear or branched alkyl having the formula C_aH_a+1, or cycloalkyl having the formula CaH^, wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, such as linear or branched alkyl of the formula: CH₃, C₂H₅, C₃H₇, C₄H₉, CsHn, C₆H₁₃, C₇H₁₅, C₈H_17i C₉H₁₉, C_1oH₂₁, etc., or cycloalkyl of the formula: C₃H₅, C₄H₇, C₅H₉, CeHn, C₇H₁₃, C₈H_15i C₉Hi₇, Ci₉Hi₉, etc.

With respect to R^a, in some embodiments, the aryl group is substituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid, nitro or acylamino.

With respect to R^a, in some embodiments, the heteroaryl group is single or fused. In some embodiments, the single heteroaryl group is imidazole. In some embodiments, the fused heteroaryl group is benzimidazole. In some embodiments, the heteroaryl group is substituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid, nitro or acylamino. In some embodiments, the alkyl group is branched, cyclic or polycyclic.

With respect to R^a, a hydrocarbyl may be alkyl, alkenyl, or alkynyl. In some embodiments, the alkyl group is substituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, heteroaryl, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid, nitro, or acylamino. In some embodiments, the heteroaryl group is single or fused. In some embodiments, the single heteroaryl group is imidazole. In some embodiments, the fused heteroaryl group is benzimidazole. In some embodiments, the alkenyl group is branched, cyclic or polycyclic. In some embodiments, the alkenyl group is substituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, heteroaryl, alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid, nitro, or acylamino.

With respect to any relevant structural feature herein, R^b may be H, or C-1.3 hydrocarbyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, CH=CH₂, CH₂CH=CH₂, C^CH, CH₂C^CH, etc.

With respect to any relevant structural feature herein, R^c may be H, or C-|.₃ alkyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, etc. In some embodiments, R^c is H.

With respect to any relevant formula or structural depiction herein, such as Formula 1, Formula 2, Formula 3, or Formula 4, R¹ is R^a, OR² or NR²R³. In some embodiments, R¹ is optionally substituted phenyl. In some embodiments, R¹ is unsubstituted phenyl. In some embodiments, R¹is optionally substituted naphthyl. In some embodiments, R¹ is unsubstituted naphthyl.

In some embodiments, R¹ is

With respect to any relevant structural feature herein, R² may be R^a, COR^a, or SO₂R^a. In some embodiments, R² may be H, methyl, ethyl, a propyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl, cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomer thereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In some embodiments, R²is H.

With respect to any relevant structural feature herein, R³ may be R^a, COR^a, or SO₂R^a. In some embodiments, R³ may be H, methyl, ethyl, a propyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl, cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomer thereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In some embodiments, R³ is H.

With respect to any relevant structural feature herein, each R⁴ may independently be R², OR^a, COR^a, CO2R^a, OCOR^a, CONR²R³ NR²R³ NR^bCOR^a, SR^a, SOR^a, SO2R^a, SO2NR^aR^b, NCOR^a, halogen, trihalomethyl, CN, S=O, nitro, or C2.₅ heteroaryl. In some embodiments, R⁴ is H.

Generally R⁵ and R^s-R³², may be H or any substituent, such as a substituent having from 0 to 6 carbon atoms and from 0 to 5 heteroatoms independently selected from: O, N, S, F, Cl, Br, and I, and/or having a molecular weight of 15 g/mol to 300 g/mol. Any of R⁵ and R⁸-R³² may comprise: a) 1 or more alkyl moieties optionally substituted with, or optionally connected by, b) 1 or more functional groups, such as C=C, C=C, CO, CO₂, CON, NCO₂, OH, SH, O, S, N, N=C, F, Cl, Br, I, CN, NO₂, CO₂H, NH₂, etc.; or may be a substituent having no alkyl portion, such as F, Cl, Br, I, NO₂, CN, NH₂, OH, COH, CO₂H, etc.

With respect to any relevant structural feature herein, In some embodiments, R⁵ may be R^a, COR^a, SO2R^a, or may not be présent. Some non-limiting examples of R⁵ may include H or C-|_3 alkyl, such as CH₃, C₂H₅, C₇, cyclopropyl, etc. In some embodiments, R⁵ may be CH₃ In some embodiments, R⁵ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R⁸ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R⁸ is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C^CH, or NO₂. In some embodiments, R⁸ is H.

morpholino

With respect to any relevant formula or structural depiction above, some non-limiting examples of R⁹ may include R^b, COR^b, CO2R^b, CONR^bR°, NR^bCOR^c, SO2NR^bR^c, CF3, CN, or C2.5 heterocyclyl. In some embodiments, R⁹ is H, CH3 CH₂CH₃, SO₂NH₂, or CH₂C^CH. In some embodiments, R⁹ is H, CH3, CH2CH3, CH2CH2CH3, CH2CH=CH2, or ΟΗ2ΟξΟΗ. In some embodiments, R⁹ is CH2C^CH. In some embodiments, R⁹ is H.

SO₂NR^bR^c

NR^bCOR^c

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁰ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R¹⁰ is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹⁰ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹¹ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R¹¹ is H, CH3] CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹¹ is H, Cl or Br. In some embodiments, R¹¹ is Cl. In some embodiments, R¹¹ is Br. In some embodiments, R¹¹ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹² may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R¹² is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹² is H, Cl, or SO2NH2. In some embodiments, R¹² is H. In some embodiments, R¹² is Cl. In some embodiments, R¹² is SO2NH₂. In some embodiments, R¹² is H.

^v°>

In some embodiments, R¹¹ and R¹² are together: / θ

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹³ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R¹³ is H, CH3] CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C^CH, or NO₂. In some embodiments, R¹³ is H or Cl. In some embodiments, R¹³ is H. In some embodiments, R¹³ is Cl.In some embodiments, R¹¹ and R¹³ are Cl.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁴ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, CF3, CN, NO₂, F, Cl, Br, or I. In some embodiments, R¹⁴ is H, CH₃. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, CH₂C=CH, or NO₂. In some embodiments, R¹⁴ is H.

In some embodiments, R¹⁰ and R¹⁴ are H. In some embodiments, R¹⁰, R¹², and R¹⁴ are H. In some embodiments, R¹⁰, R¹³, and R¹⁴ are H. In some embodiments, R¹⁰, R¹¹, R¹³, and R¹⁴ are H. In some embodiments, R¹⁰, R¹¹, R¹², and R¹⁴ are H. In some embodiments, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁶ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br,or C2.5 heterocyclyl. In some embodiments, R¹⁶ is H, CH3 CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C=CH, or NO₂. In some embodiments, R¹⁶ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁷ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2_5 heterocyclyl. In some embodiments, R¹⁷ is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹⁷ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁸ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R¹⁸ is H, CH3, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹⁸ is H or CH3. In some embodiments, R¹⁸ is H. In some embodiments, R¹⁸ is CH3. In some embodiments, R¹⁸ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R¹⁹ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, or C2.5 heterocyclyl. In some embodiments, R¹⁹ is H, CH3 CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R¹⁹ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁰ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²⁰ is H, CH3 CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂CeCH, or NO₂. In some embodiments, R²⁰ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²¹ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²¹ is H, CH3,

Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)_2i SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R²¹ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²² may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²² is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments, R²² is H.

In some embodiments, R¹⁹, R²⁰, R²¹, and R²² are H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²³ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²³ is H, CH3> CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂CeCH, or NO₂. In some embodiments, R²³ is H or SO₂NH₂. In some embodiments, R²³ is H. In some embodiments, R²³is SO2NH2.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁴ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²⁴ is H, CH3 CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments, be R²⁴ may H.

In some embodiments, R²³ and R²⁴ are H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁵ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, or C2.5 heterocyclyl. In some embodiments, R²⁵ is H, CH3iCH₂CH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C^CH, or NO₂. In some embodiments, R²⁵ is CH₃ or H. In some embodiments, R²⁵ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁶ may include R^b, CF3, CN, or NO2. In some embodiments, R²⁶ is H, CH3. or CH₂CH₃. In some embodiments, R²⁶ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁷ may include R^b, OR^b, SR^b, COR^b, CO2R^b, CONR^bR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²⁷ is H, CH3, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, SO₂NH₂, ΟΗ₂ΟξΟΗ, or NO₂. In some embodiments, R²⁷ is H, (CH2)3CH3, CH2CH2OCH3, CH2CH2N(CH3)2, CH2CH2-morpholino, or CH2CH2SCH3. In some embodiments, R²⁷ is H. In some embodiments, R²⁷ is (CH2)₃CH₃. In some embodiments, R²⁷ is CH2CH2OCH3. In some embodiments, R²⁷ is CH2CH₂N(CH₃)₂. In some embodiments, R²⁷ is CH2CH2-morpholino. In some embodiments, R²⁷ is CH2CH₂SCH₃.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁸ may include R^b, OR^b, SR^b, COR^b, CO₂R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c,

SO₂NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²⁸ is H, CH3i CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R²⁸ is H, CH2CH3, OCH3, N(CH3)2, morpholino, or SCH3. In some embodiments, R²⁸ is H. In some embodiments, R²⁸ is CH2CH₃. In some embodiments, R²⁸ is OCH3. In some embodiments, R²⁸ is CN(CH3)₂. In some embodiments, R²⁸ is morpholino. In some embodiments, R²⁸ is SCH₃.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R²⁹ may include R^b, OR^b, SR^b, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R²⁹ is H, CH3i or CH₂CH₃. In some embodiments, R²⁹ is H.

In some embodiments, R⁸ and R²⁹ are H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R³⁰ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, or I. In some embodiments, R³⁰ is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂CeCH, or NO₂. In some embodiments, R³⁰ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R³¹ may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R³¹ is H, CH3] CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R³¹ is H.

With respect to any relevant formula or structural depiction above, some non-limiting examples of R³² may include R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl. In some embodiments, R³² is H, CH3. CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CeCH, or NO₂. In some embodiments, R³² is H or NO2. In some embodiments, R³² is H. In some embodiments, R³² is NO2.

In some embodiments, R³⁰, R³¹, and R³² are H. In some embodiments, R³¹ and R³² are H.

NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂CsCH, or NO₂. In some embodiments, R³³ is H.

In another embodiment disclosed herein the compound is (the “KIN1000” compound)

Unless otherwise indicated, any reference to a compound herein by structure, formula, name or any other means, includes pharmaceutically acceptable salts, such as sodium, potassium, and ammonium salts; prodrugs, such as ester prodrugs; alternate solid forms, such as polymorphs, solvatés, hydrates, etc.; tautomers; or, any other Chemical species that may rapidly convert to a compound described herein under conditions in which the compounds are used as described herein.

Unless stereochemistry is unambiguously depicted, any structure, formula or name for a compound can refer to any stereoisomer or any mixture of stereoisomers of the compound.

As used herein, the term “functional group” refers to an atom or a group of atoms that hâve similar Chemical properties whenever they occur in different compounds, and as such the functional group defines the characteristic physical and Chemical properties of families of organic compounds.

Unless otherwise indicated, when any compound or Chemical structural feature (collectively referred to herein as a “compound”), such as for example alkyl, aryl, etc., is referred to as being “optionally substituted,” that compound can hâve no substituents (in which case it is “unsubstituted”), or it can include one or more substituents (in which case it is “substituted”). The term “substituent” has the ordinary meaning known to one of ordinary skill in the art. In some embodiments, the substituent may be an ordinary organic moiety known in the art, which can hâve a molecular weight (e.g., the sum of the atomic masses of the atoms of the substituent) of 15 g/mol to 50 g/mol, 15 g/mol to 100 g/mol, 15 g/mol to 150 g/mol,15 g/mol to 200 g/mol, 15 g/mol to 300 g/mol, or 15 g/mol to 500 g/mol. In some embodiments, the substituent comprises: 0-30, 0-20, 0-10, or 0-5 carbon (C) atoms; and/or 0-30, 0-20, 0-10, or 0-5 heteroatoms including N, O, S, Si, F, Cl, Br, or I; provided that the substituent comprises at least one atom including C, N, O, S, Si, F, Cl, Br, or I in a substituted compound. Examples of substituents include, but are not limited to, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, hydroxy, alkoxy, aryloxy, acyl, acyloxy, alkylcarboxylate, thiol, alkylthio, cyano, halo, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxyl, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, etc. For convenience, the term “molecular weight” is used with respect to a moiety or part of a molécule to indicate the sum of the atomic masses of the atoms in the moiety or part of a molécule, even though it may not be a complété molécule.

As used herein, the term “hydrocarbyl” has the broadest meaning generally understood in the art, and can include a moiety composed of carbon and hydrogen. Some examples can include alkyl, alkenyl, alkynyl, aryl, etc., and combinations thereof, and can be linear, branched, cyclic, or a combination thereof. Hydrocarbyl can be bonded to any other number of moieties (for example, can be bonded to one other group, such as -CH₃, -CH=CH₂, etc.; two other groups, such as -phenyl-, -C^C-, etc.; or any number of other groups) that the structure can bear, and in some embodiments, can contain from one to thirty-five carbon atoms. Examples of hydrocarbyl groups include but are not limited to C-ι alkyl, C₂ alkyl, C₂ alkenyl, C₂ alkynyl, C₃ alkyl, C₃alkenyl, C₃ alkynyl, C₄ alkyl, C₄ alkenyl, C₄ alkynyl, C₅ alkyl, C₅ alkenyl, C₅ alkynyl, C₆ alkyl, C₆alkenyl, C₆ alkynyl, phenyl, etc.

As used herein the term “alkyl” has the broadest meaning generally understood in the art, and can include a moiety composed of carbon and hydrogen containing no double or triple bonds and not having any cyclic structure. Alkyl can be linear alkyl, branched alkyl, cycloalkyl, or a combination thereof, and in some embodiments, can contain from one to thirty-five carbon atoms. In some embodiments, alkyl can include Ci.₁₀ linear alkyl, such as methyl (-CH₃), ethyl (CH₂CH₃), n-propyl (-CH₂CH₂CH₃), n-butyl (-CH₂CH₂CH₂CH₃), n-pentyl (-CH₂CH₂CH₂CH₂CH₃), n-hexyl (-CH₂CH₂CH₂CH₂CH₂CH₃), etc.; C₃._w branched alkyl, such as C₃H₇ (e.g. iso-propyl), C₄H₉ (e.g., branched butyl isomers), C₅Hn (e.g., branched pentyl isomers), C₆H₁₃ (e.g., branched hexyl isomers), C₇H₁₅ (e.g., branched heptyl isomers), etc.; C₃.₁₀ cycloalkyl, such as C₃H₅ (e.g. cyclopropyl), C₄H₇ (e.g., cyclobutyl isomers such as cyclobutyl, methylcyclopropyl, etc.), C5H9 (e.g., cyclopentyl isomers such as cyclopentyl, methylcyclobutyl, dimethylcyclopropyl, etc.) CeHn (e.g., cyclohexyl isomers), C₇H₁₃ (e.g., cycloheptyl isomers), etc.; and the like.

The terms “alkyl,” “alkenyl” and “alkynyl” refer to substituted and unsubstituted alkyls, alkenyls and alkynyls, respectively. An alkyl group can be optionally substituted as defined herein.

Substituted alkyls, alkenyls and alkynyls refers to alkyls, alkenyls and alkynyls substituted with one to five substituents including H, lower alkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy, arylalkoxy, alkoxyalkylaryl, alkylamino, arylamino, NH₂, OH, CN, NO₂, OCF₃, CF₃, F, 1-amidine, 2-amidine, alkylcarbonyl, morpholinyl, piperidinyl, dioxanyl, pyranyl, heteroaryl, furanyl, thiophenyl, tetrazolo, thiazolyl, isothiazolyl, imidazolyl, thiadiazolyl, thiadiazole S-oxide, thiadiazole S,S-dioxide, pyrazolo, oxazolyl, isoxazolyl, pyridinyl, pyrimidinyl, quinolinyl, isoquinolinyl, SR, SOR, SO₂R, CO₂R, COR, CONR’R”, CSNR’R” and SO_nNR’R”.

As used herein, either alone or in combination, the term alkynyl refers to a functional group comprising a straight-chain or branched-chain hydrocarbon containing from 2 to 20 carbon atoms and having one or more carbon-carbon triple bonds and not having any cyclic structure. An alkynyl group may be optionally substituted as defined herein. Examples of alkynyl groups include, without limitation, ethynyl, propynyl, hydroxypropynyl, butynyl, butyn-1-yl, butyn-2-yl, 3methylbutyn-1-yl, pentynyl, pentyn-1-yl, hexynyl, hexyn-2-yl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, nonadecynyl, eicosynyl, and the like.

The term alkylene as used herein, alone or in combination, refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene (-CH₂-). Unless otherwise specified, the term alkyl may include alkylene groups.

As used herein, either alone or in combination, the term alkylcarbonyl or alkanoyl refers to a functional group comprising an alkyl group attached to the parent molecular moiety through a carbonyl group. Examples of alkylcarbonyl groups include, without limitation, methylcarbonyl, ethylcarbonyl, and the like.

As used herein, either alone or in combination, the term heteroalkyl refers to a functional group comprising a straight-chain or branched-chain hydrocarbon containing from 1 to 20 atoms linked exclusively by single bonds, where at least one atom in the chain is a carbon and at least one atom in the chain is O, S, N, or any combination thereof. The heteroalkyl group can be fully saturated or contain from 1 to 3 degrees of unsaturation. The non-carbon atoms can be at any interior position of the heteroalkyl group, and up to two non-carbon atoms may be consecutive, such as, e.g., -CH2-NH-OCH3. In addition, the non-carbon atoms may optionally be oxidized and the nitrogen may optionally be quaternized.

As used herein, either alone or in combination, the term “alkyloxy” or alkoxy refers to a functional group comprising an alkyl ether group. Examples of alkoxys include, without limitation, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tertbutoxy, and the like.

As used herein, either alone or in combination, the term hydroxy refers to the functional group hydroxyl (-OH).

As used herein, either alone or in combination, the term carboxyl or carboxy refers to the functional group -C(=O)OH or the corresponding carboxylate anion -C(=O)O-, Examples include, without limitation, formic acid, acetic acid, oxalic acid, benzoic acid. An O-carboxyl group refers to a carboxyl group having the general formula RCOO, wherein R is an organic moiety or group. A C-carboxyl group refers to a carboxyl group having the general formula COOR, wherein R is an organic moiety or group.

As used herein, either alone or in combination, the term oxo refers to the functional group =0. As used herein, the term “carbocyclic” has the broadest meaning generally understood in the art, and includes a ring or ring System wherein the ring atoms are ail carbon. Examples include, but are not limited to, phenyl, naphthyl, anthracenyl, cycloalkyl, cycloalkenyl, cycloalkynyl, etc., and combinations thereof.

As used herein, the term “heterocyclic” has the broadest meaning generally understood in the art, and includes a ring or ring System wherein at least one of the ring atoms is not carbon, such as N, O, S, etc. Examples include, but are not limited to, heteroaryl, cycloheteroalkyl, cycloheteroalkenyl, cycloheteroalkynyl, etc., and combinations thereof.

As used herein, either alone or in combination, the term “cycloalkyl,” “carbocyclicalkyl” and “carbocyclealkyl” refers to a functional group comprising a substituted or unsubstituted nonaromatic hydrocarbon with a non-conjugated cyclic molecular ring structure of 3 to 12 carbon atoms linked exclusively with carbon-carbon single bonds in the carbon ring structure. A cycloalkyl group can be monocyclic, bicyclic or polycyclic, and may optionally include one to three additional ring structures, such as, e.g., an aryl, a heteroaryl, a cycloalkenyl, a heterocycloalkyl, or a heterocycloalkenyl.

As used herein, either alone or in combination, the term lower cycloalkyl refers to a functional group comprising a monocyclic substituted or unsubstituted non-aromatic hydrocarbon with a non-conjugated cyclic molecular ring structure of 3 to 6 carbon atoms linked exclusively with carbon-carbon single bonds in the carbon ring structure. Examples of lower cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

As used herein the term “aryl” has the broadest meaning generally understood in the art, and can include an aromatic ring or aromatic ring System. An aryl group can be monocyclic, bicyclic or polycyclic, and may optionally include one to three additional ring structures; such as, for example, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl. The term aryl includes, without limitation, phenyl (benzenyl), thiophenyl, indolyl, naphthyl, tolyl, xylyl, anthracenyl, phenanthryl, azulenyl, biphenyl, naphthalenyl, 1-methylnaphthalenyl, acenaphthenyl, acenaphthylenyl, anthracenyl, fluorenyl, phenalenyl, phenanthrenyl, benzo[a]anthracenyl, benzo[c]phenanthrenyl, chrysenyl, fluoranthenyl, pyrenyl, tetracenyl (naphthacenyl), triphenylenyl, anthanthrenyl, benzopyrenyl, benzo[a]pyrenyl, benzo[e]fluoranthenyl, benzo[ghi]perylenyl, benzo[j]fluoranthenyl, benzo[k]fluoranthenyl, corannulenyl, coronenyl, dicoronylenyl, helicenyl, heptacenyl, hexacenyl, ovalenyl, pentacenyl, picenyl, perylenyl, tetraphenylenyl, etc.

Additionally, as used herein, either alone or in combination, the term “aryl,” hydrocarbyl aryl or “aryl hydrocarbon” can refer to a functional group comprising a substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic molecular ring structure of 3 to 12 carbon atoms. Substituted aryl refers to aryls substituted with one to five substituents including H, lower alkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy, arylalkoxy, alkoxyalkylaryl, alkylamino, arylamino, NH₂, OH, CN, NO₂, OCF₃, CF₃, Br, Cl, F, 1-amidino, 2-amidino, alkylcarbonyl, morpholino, piperidinyl, dioxanyl, pyranyl, heteroaryl, furanyl, thiophenyl, tetrazolo, thiazole, isothiazolo, imidazolo, thiadiazole, thiadiazole S-oxide, thiadiazole S,S-dioxide, pyrazolo, oxazole, isoxazole, pyridinyl, pyrimidinyl, quinoline, isoquinoline, SR, SOR, SO₂R, CO₂R, COR, CONR’R”, CSNR’R”, SO_nNR’R”, etc.

As used herein, either alone or in combination, the term “lower aryl” refers to a functional group comprising a substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic molecular ring structure of 3 to 6 carbon atoms. Examples of lower aryl groups include, without limitation, phenyl and naphthyl.

As used herein, either alone or in combination, the term heteroaryl refers to a functional group comprising a substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic molecular ring structure of 3 to 12 atoms, where at least one atom in the ring structure is a carbon and at least one atom in the ring structure is O, S, N, or any combination thereof. A heteroaryl group can be monocyclic, bicyclic or polycyclic, and may optionally include one to three additional ring structures, such as, e.g., an aryl, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, or a heterocycloalkenyl. Examples of heteroaryl groups include, without limitation, acridinyl, benzidolyl, benzimidazolyl, benzisoxazolyl, benzodioxinyl, dihydrobenzodioxinyl, benzodioxolyl, 1,3-benzodioxolyl, benzofuryl, benzoisoxazolyl, benzopyranyl, benzothiophenyl, benzo[c]thiophenyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, carbazolyl, chromonyl, cinnolinyl, dihydrocinnolinyl, coumarinyl, dibenzofuranyl, furopyridinyl, furyl, indolizinyl, indolyl, dihydroindolyl, imidazolyl, indazolyl, isobenzofuryl, isoindolyl, isoindolinyl, dihydroisoindolyl, isoquinolyl, dihydroisoquinolinyl, isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl, phenanthrolinyl, phenanthridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrrolinyl, pyrrolyl, pyrrolopyridinyl, quinolyl, quinoxalinyl, quinazolinyl, tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl, thiophenyl, thiazolyl, thiadiazolyl, thienopyridinyl, thienyl, thiophenyl, triazolyl, xanthenyl, and the like.

As used herein, either alone or in combination, the term lower heteroaryl refers to a functional group comprising a monocyclic or bicyclic, substituted or unsubstituted aromatic hydrocarbon with a conjugated cyclic molecular ring structure of 3 to 6 atoms, where at least one atom in the ring structure is a carbon and at least one atom in the ring structure is O, S, N, or any combination thereof.

The phenyl structure associated with some of the embodiments described herein is depicted below, This structure can be unsubstituted, as shown below, or can be substituted such that a substituent can independently be in any position normally occupied by a hydrogen atom when the structure is unsubstituted. Unless a point of attachment is indicated by -|, attachment may occur at any position normally occupied by a hydrogen atom.

Phenyl

Each R_a can independently be H; optionally substituted hydrocarbyl; optionally substituted aryl, such as optionally substituted phenyl or optionally substituted aryl; optionally substituted heteroaryl, such as optionally substituted pyridinyl, optionally substituted furyl, optionally substituted thienyl, etc. In some embodiments, each R_a can independently be H, or Ci.₁₂ alkyl, including: linear or branched alkyl having the formula C_aH_a+1, or cycloalkyl having the formula CaHa.!, wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, such as linear or branched alkyl of the formula: CH₃, C₂H₅, C₃H₇, C₄H₉, CsHn, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉, Ci₀H₂₁, etc., or cycloalkyl of the formula: C₃H₅, C₄H₇, C₅H₉, CeHn, C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

The term “treat” includes one or more of the diagnosis, cure, mitigation, vaccination, augmentation of a therapy or prévention of disease in man or other animais

As used herein, the term “vertebrate” includes ail living vertebrates such as, without limitation, mammals, humans, birds, dogs, cats, livestock, farm animais, free-range herds, etc.

Many RNA viruses share biochemical, regulatory, and signaling pathways. These viruses include but are not limited to influenza virus (including avian and swine isolâtes), respiratory syncytial virus, Hepatitis C virus, West Nile virus, SARS-coronavirus, poliovirus, measles virus, Dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, llheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, and the Kyasanur forest disease virus. The compounds and methods disclosed herein can be used to treat these viruses.

Relevant taxonomie families of RNA viruses include, without limitation, Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Paramyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, and Tymoviridae. The compounds and methods disclosed herein can be used to treat viruses within these families of viruses as part of a pharmaceutically acceptable drug formulation. Other relevant virus families include, without limitation, Hepadnaviridae, Herpesviridae, and Papillomaviridae.

Particular embodiments provide for pharmaceutical compositions comprising the compounds, alone or in combination with an antigen, for the purpose of treating and/or preventing disease in an animal including a vertebrate animal. As such, in some embodiments the pharmaceutical compositions can be used as vaccines.

The disclosure provides for the use of the compounds as adjuvants.

The compounds and methods disclosed herein can be additive or synergistic with other thérapies currently in development or use. For example, ribavirin and interferon-α provide an effective treatment for HCV infection when used in combination. Their efficacy in combination can exceed the efficacy of either drug product when used alone. The compositions of the disclosure can be administered alone or in combination or conjunction with interferon, ribavirin and/or a variety of small molécules that are being developed against both viral targets (viral proteases, viral polymerase, assembly of viral réplication complexes) and host targets (host proteases required for viral Processing, host kinases required for phosphorylation of viral targets such as NS5A, and inhibitors of host factors required to efficiently utilize the viral internai ribosome entry site, or IRES).

The compounds and methods disclosed herein could be used in combination or conjunction with, without limitation, adamantane inhibitors, neuraminidase inhibitors, alpha interferons, nonnucleoside or nucleoside polymerase inhibitors, NS5A inhibitors, antihistamines, protease inhibitors, helicase inhibitors, P7 inhibitors, entry inhibitors, IRES inhibitors, immune stimulators, HCV réplication inhibitors, cyclophilin A inhibitors, A₃ adenosine agonists, and microRNA suppressors.

Cytokines that could be administered in combination or conjunction with the compounds and methods disclosed herein include, without limitation, IL-2, IL-12, IL-23, IL-27, or IFN-γ. New HCV drugs that are or will be available for potential administration in combination or conjunction with the compounds and methods disclosed herein include, without limitation, ACH-1625 (Achillion); Glycosylated interferon (Alios Biopharma); ANA598, ANA773 (Anadys Pharm); ATI0810 (Arisyn Therapeutics); AVL-181 (Avila Therapeutics); LOCTERON® (Biolex); CTS-1027 (Conatus); SD-101 (Dynavax Technologies); Clemizole (Eiger Biopharmaceuticals); GS-9190 (Gilead Sciences); GI-5005 (Globallmmune BioPharma); Resiquimod / R-848 (Graceway Pharmaceuticals); Albinterferon alpha-2b (Human Genome Sciences); IDX-184, IDX-320, IDX375 (Idenix); IMO-2125 (Idera Pharmaceuticals); INX-189 (Inhibitex); ITCA-638 (Intarcia Therapeutics); ITMN-191/RG7227 (Intermune); ITX-5061, ITX-4520 (iTherx Pharmaceuticals); MB11362 (Metabasis Therapeutics); Bavituximab (Peregrine Pharmaceuticals); PSI-7977, RG7128, PSI-938 (Pharmasset); PHX1766 (Phenomix); Nitazoxanide / ALINIA® (Romark Laboratories); SP-30 (Samaritan Pharmaceuticals); SCV-07 (SciClone); SCY-635 (Scynexis); TT-033 (Tacere Therapeutics); Viramidine/taribavirin (Valeant Pharmaceuticals); Telaprevir, VCH-759, VCH-916, VCH-222, VX-500, VX-813 (Vertex Pharmaceuticals); and PEG-INF Lambda (Zymogenetics).

New influenza and West Nile virus drugs that are or will be available for potential administration in combination or conjunction with the compounds and methods disclosed herein include, without limitation, neuraminidase inhibitors (Peramivir, Laninamivir); triple therapy neuraminidase inhibitors ribavirin, amantadine (ADS-8902); polymerase inhibitors (Favipiravir); reverse transcriptase inhibitor (ANX-201); inhaled chitosan (ANX-211); entry / binding inhibitors (Binding Site Mimetic, FLUCIDE™ (NanoViricides, West Haven, Connecticut); entry inhibitor (FLUDASE® (NexBio, San Diego, California); fusion inhibitor, (MGAWN1 for West Nile); host cell inhibitors (lantibiotics); cleavage of RNA genome (RNAi, RNAse L); immune stimulators (Interferon, Alferon-LDO; Neurokininl agonist, Homspera, Interferon Alferon N for West Nile); and TG21.

Other drugs for treatment of influenza and/or hepatitis that are available for potential administration in combination or conjunction with the compounds and methods disclosed herein include, without limitation:

Table 1. Hepatitis and influenza drugs

Branded Name	Generic Name	Approved Indications
PEGASYS® (Genentech, South San Francisco, California)	PEGinterferon alfa-2a	Hepatitis C, Hepatitis B
PEGINTRON® (Merck,	PEGinterferon alfa-2b	Hepatitis C

Whitehouse Station, New Jersey)
COPEGUS® (Roche Pharmaceuticals, Nutley, New Jersey)	Ribavirin	Hepatitis C
REBETOL® (Schering Plough, Kenilworth, New Jersey)	Ribavirin	Hepatitis C
—	Ribavirin	Hepatitis C
TAMIFLU® (Roche Pharmaceuticals, Nutley, New Jersey)	Oseltamivir	Influenza A, B, C
RELENZA® (GlaxoSmithKIine, London, UK)	Zanamivir	Influenza A, B, C
—	Amantadine	Influenza A
—	Rimantadine	Influenza A

These agents can be incorporated as part of the same pharmaceutical composition or can be administered separately from the compounds of the disclosure, either concurrently or in accordance with another treatment schedule.

The compounds and methods disclosed herein can be additive or synergistic with other compounds and methods to enable vaccine development. By virtue of their antiviral and immune enhancing properties, the compounds can be used to affect a prophylactic or therapeutic vaccination. The compounds need not be administered simultaneously or in combination with other vaccine components to be effective. The vaccine applications of the compounds are not limited to the prévention or treatment of virus infection but can encompass ail therapeutic and prophylactic vaccine applications due to the general nature of the immune response elicited by the compounds.

As is understood by one of ordinary skill in the art, vaccines can be against viruses, bacterial infections, cancers, etc. and can include one or more of, without limitation, a live attenuated vaccine (LAIV), an inactivated vaccine (IIV; killed virus vaccine), a subunit (split vaccine); a subvirion vaccine; a purified protein vaccine; or a DNA vaccine. Appropriate adjuvants include one or more of, without limitation, water/oil émulsions, non-ionic copolymer adjuvants, e.g., CRL 1005 (Optivax™; Vaxcel Inc., Norcross, Ga.), aluminum phosphate, aluminum hydroxide, aqueous suspensions of aluminum and magnésium hydroxides, bacterial endotoxins, polynucleotides, polyelectrolytes, lipophilie adjuvants and synthetic muramyl dipeptide (norMDP) analogs such as N-acetyl-nor-muranyl-L-alanyl-D-isoglutamine, N-acetyl-muranyl-(6-Ostearoyl)-L-alanyl-D-isoglutamine or N-Glycol-muranyl-LalphaAbu-D-isoglutamine (Ciba-Geigy

Ltd.).

The pharmaceutical composition comprising a compound of the disclosure can be formulated in a variety of forms; e.g., as a liquid, gel, lyophilized, or as a compressed solid. The preferred form will dépend upon the particular indication being treated and discernible by one of ordinary skill in the art. In one embodiment, the disclosed RIG-I agonists include formulations for oral delivery that can be small-molecule drugs that employ straightforward médicinal chemistry processes.

The administration of the formulations of the présent disclosure can be performed in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intrathecally, vaginally, rectally, intraocularly, or in any other acceptable manner. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques known in the art, such as pumps (e.g., subcutaneous osmotic pumps) or implantation. In some instances the formulations can be directly applied as a solution or spray.

An example of a pharmaceutical composition is a solution designed for parentéral administration. Although in many cases pharmaceutical solution formulations are provided in liquid form, appropriate for immédiate use, such parentéral formulations can also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those of ordinary skill in the art that lyophilized préparations are generally more stable than their liquid counterparts. Such lyophilized préparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as, without limitation, stérile water for injection or stérile physiological saline solution.

Parenterals can be prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the compound having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art (ail of which are termed excipients), for example buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic détergents, antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximates physiological conditions. They are typically présent at a concentration ranging from 2 mM to 50 mM. Suitable buffering agents for use with the présent disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additional possibilities are phosphate buffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives can be added to retard microbial growth, and are typically added in amounts of 0.2%-1% (w/v). Suitable preservatives for use with the présent disclosure include, without limitation, phénol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., benzalkonium chloride, bromide or iodide), hexaméthonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers can be added to ensure isotonicity of liquid compositions and include, without limitation, polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can be présent in an amount between 0.1% and 25% by weight, typically 1% to 5%, taking into account the relative amounts of the other ingrédients.

Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent dénaturation or adhérence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human sérum albumin, bovine sérum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically présent in the range of from 0.1 to 10,000 parts by weight based on the active compound weight.

Additional miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, méthionine, vitamin E) and cosolvents.

The active ingrédient can also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules, in colloïdal drug delivery Systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 21^st Ed., published by Lippincott Williams & Wilkins, A Wolters Kluwer Company, 2005, the teachings of which are incorporated by reference herein.

Parentéral formulations to be used for in vivo administration generally are stérile. This is readily accomplished, for example, by filtration through stérile filtration membranes.

Suitable examples of sustained-release préparations include semi-permeable matrices of solid hydrophobie polymers containing the compound or composition, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylenevinyl acetate, degradable lactic acid-glycolic acid copolymers such as the PROLEASE® technology (Alkermes, Cambridge, Massachusetts) or LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate; Abbott Laboratories, Abbott Park, Illinois), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molécules for long periods such as up to or over 100 days, certain hydrogels release compounds for shorter time periods.

Oral administration of the compounds and compositions is one intended practice of the disclosure. For oral administration, the pharmaceutical composition can be in solid or liquid form, e.g., in the form of a capsule, tablet, powder, granule, suspension, émulsion or solution. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingrédient. A suitable daily dose for a human or other vertebrate can vary widely depending on the condition of the patient and other factors, but can be determined by persons of ordinary skill in the art using routine methods.

In solid dosage forms, the active compound can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances, e.g., lubricating agents such as magnésium stéarate. In the case of capsules, tablets and pills, the dosage forms can also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

The compounds or compositions can be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnésium stéarate, magnésium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, they can be dissolved in saline, water, polyethylene glycol, propylene glycol, éthanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are known in the pharmaceutical art. The carrier or diluent can include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials known in the art.

The présent disclosure further includes the use and application of the compounds, compositions and methods herein in vitro in a number of applications including but not limited to developing thérapies and vaccines against viral infections, research in modulation of the innate immune response in eukaryotic cells, etc. The compounds, compositions and methods of the présent disclosure can also be used in animal models. The results of such in vitro and animal in vivo uses of the compounds, compositions and methods of the présent disclosure can, for example, inform their in vivo use in humans, or they can be valuable independent of any human therapeutic or prophylactic use.

Examples

The Examples below describe the antiviral and pharmacological properties of the disclosed compounds. The Examples are included to demonstrate particular embodiments of the disclosure. It should be appreciated by those of ordinary skill in the art that the techniques disclosed in the Examples represent techniques and compositions discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of ordinary skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments which are disclosed and still obtain a like or similar resuit without departing from the spirit and scope of the disclosure. For example, the Examples below provide in vitro methods for testing the compounds of the disclosure. Other in vitro virus infection models include but are not limited to flaviviruses such as bovine diarrheal virus, West Nile Virus, and GBV-C virus, other RNA viruses such as respiratory syncytial virus, and the HCV replicon Systems. Furthermore, any appropriate cultured cell competent for viral réplication can be utilized in the antiviral assays.

Example 1. Biological activity of Kl N1000

Luciferase assay to identify active compounds. Cultured human cells that were stably transfected with a luciferase reporter gene coupled with a RIG-I signaling pathway responsive promoter (ΙΡΝβ, ISG56, or ISG54 promoter) were seeded and allowed to grow overnight. The compound “KIN1000” was then added and cells were grown in the presence of KIN1000 for 1820 hours. Steady-GIo luciferase substrate (Promega) was added and luminescence was read on a luminometer (Berthold).

Figure 1A shows that KIN1000 as described herein was validated by demonstrating dosedependent induction of the luciferase reporter gene coupled to the promoters for ΙΡΝβ (ΊΡΝβLUC,” left), ISG56 (“ISG56-LUC,” center), and ISG54 (“ISG54-LUC,” right). Additionally, KIN1000 did not induce a nonspecific promoter (β-actin-LUC, Figure 1B).

MTS assay to détermine cytotoxicity. Cultured human HeLa cells were treated with increasing amounts of compound or équivalent amounts of DMSO diluted in media for 48 hours to see their effect on cell viability. The proportion of viable cells was calculated using a cell viability assay that measures conversion of a tétrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner sait; or MTS] to a colored formazan compound in live cells.

The conversion of MTS to formazan was detected in a 96-well microtiter plate reader, and the resulting optical densities plotted directly to estimate cell viability. Cell Titer One (Promega) was the one-step reagent used, as manufacturées protocol suggested, and cells were incubated for three hours in the presence of reagent before optical density (O.D.) reading was done. Compounds were diluted to final concentrations of 0, 1, 5, 10, and 20 μΜ in media containing 0.5% DMSO. Négative control wells contained no compound, and positive control for cytotoxicity was examined using 10% DMSO. Each Kl N1000 concentration and control was done in triplicate wells. Kl N1000 showed no cytotoxicity to multiple cell types (MTS assay, Figure 1C).

Immunofluorescent cytochemistry assay to détermine IRF-3 activation and translocation to the nucléus. The induction of ISG expression mediated by RIG-I is conferred by phosphorylation, dimerization, and nuclear translocation of the IRF-3 transcription factor. Cultured human HeLa cells were treated with increasing amounts of compound or équivalent amounts of DMSO diluted in media for 20 hours. Positive control wells were infected with 100 HA/mL Sendai virus for an équivalent time period. IRF-3 was detected using polyclonal rabbit sérum spécifie to IRF-3 and a secondary antibody conjugated to DyLight® (Pierce Biotechnology, Inc., Rockford, IL) 488. KIN1000 shows a dose dépendent increase in nuclearcytoplasmic différence for IRF-3 (Figure 2A).

Immunofluorescent cytochemistry assay to détermine NFkB activation. The innate immune response dépendent on RIG-I also activâtes the NFkB transcription factor and thus increases nuclear levels. Cultured human HeLa cells were treated with increasing amounts of compound or équivalent amounts of DMSO diluted in media for 20 hours. Positive control wells were infected with 100 HA/mL Sendai virus for an équivalent time period. NFkB was detected using monoclonal mouse antibody spécifie to the p65 subunit of NFkB and a secondary antibody conjugated to DyLight 488.

Quantification of immunofluorescent assays. 96-well plates containing cultured human cells treated with compound and stained for either IRF-3 or NFkB were scanned and quantified using the ArrayScan® instrument and software (Cellomics). Activation of transcription factor was evidenced by increased nuclear intensity normalized for cytoplasmic intensity, or nuclearcytoplasmic différence. ΚΙΝ1000 shows a dose dépendent increase in nuclear-cytoplasmic différence for NFkB (Figure 2B).

Other compounds as described herein likewise can be evaluated by the methods described in this example, and other cell types can also be used.

Example 2. Antmral activity of KIN1000 against influenza WSN strain

MRC5 cells were treated with increasing amounts of Kl N1000 12-24 hours prior to infection by influenza virus. The number of infected cells 24 hours after introduction of virus was then quantified by an immunofluorescent assay of viral protein in cells. The KIN1000 compound disclosed herein demonstrated efficient activity against influenza virus strain WSN. Figure 3 shows that MRC5 cells treated with increasing amounts of KIN1000 showed a dose-dependent decrease in infection by influenza virus.

Example 3. Ex vivo immune stimulatory activity of KIN1000

The activity of KIN1000 in primary immune cells was assayed to détermine whether KIN1000 stimulâtes immune responses. Cultured human primary dendritic cells were treated with 0, 1, or 10 μΜ of Kl N1000 for 24 hours. Supernatant from treated wells was isolated and tested for levels of cytokine protein. Cytokines were detected using spécifie antibodies conjugated to magnetic beads and a secondary antibody that reacts with Streptavidin/Phycoerythrin to produce a fluorescent signal. The bound beads were detected and quantified using the Magpix® (LuminexCorp.) instrument, although similar techniques as are known in the art may be used to measure fluorescent protein production, such as for example an ELISA.

KIN1000 was shown to induce expression of the chemokines IL-8, MCP-1, MIP-1a and ΜΙΡ-1β by dendritic cells (Figure 4).

Other cells from which cytokine sécrétion can be measured include, for example but without limitation, human peripheral blood mononuclear cells, human macrophages, mouse macrophages, mouse splénocytes, rat thymocytes, and rat splénocytes.

Example 4. antiviral activity and pharmacological properties using structure-activity RELATIONSHIP (SAR) STUDIES

This Example describes optimization of compounds for antiviral action. First, a small analog dérivative set is used to define a structural class. The active analogs that are identified in this first stage are then used to define a subset of structural classes of interest for further optimization (Stage 2).

Stage 2, dérivative expansion. Stage 2 focuses on creating structural diversity and evaluating core variants. Structural dérivatives are tested for biological activity in the IRF-3 translocation assay, antiviral activity, and cytotoxicity in one or more cell lines or peripheral blood mononuclear cells. Optimized molécules that show improved efficacy and low cytotoxicity are further characterized by additional measures of in vitro toxicology and absorption, distribution, metabolism, and élimination (ADME). Their mechanism of action and breadth of antiviral activity are also studied.

Chemical design in SAR studies. To design analog structures, the drug-like properties, metabolic lability, and toxic potential of the lead compounds are analyzed. Drug-like properties, as measured by Lipinski’s Rules, and related physiochemical properties are primary indicators of bioavailability. Structural features that suggest metabolic and toxicological liabilities may indicate limited stability, reduced half-life, reactive intermediates, or idiosyncratic toxicity and will therefore be removed. A 5- to 10-compound analog set is constructed to remove or alter chemically reactive or metabolically susceptible structural features, thereby developing a preliminary SAR.

Compounds are tested for in vitro antiviral activity against HCV 2A, respiratory syncytial virus, dengue virus type 2, and influenza A virus strains. Viral protein and RNA levels are assessed following drug treatment using the assays described herein.

Following several itérative rounds of SAR, compounds are selected for characterization of their in vitro toxicological and ADME properties and for further mechanistic study. The SAR studies are designed to provide lead compounds with picomolar to nanomolar potency, which is adéquate to support preclinical development.

In vitro pharmacology. In vitro pharmacology studies are performed to measure performance of the most promising analogs in one or more assays of intestinal permeability, metabolic stability and toxicity. Key in vitro characterization studies can include plasma protein binding; sérum, plasma, and whole-blood stability in human and model organisme; intestinal permeability; intrinsic clearance; human Ether-à-go-go (hERG) channel inhibition; and genotoxicity.

For each analog, an HPLC- and/or HPLC-mass spectrometry-based analytical method is used to evaluate drug and métabolite concentrations in various test Systems. Although the spécifie analytical method is optimized for each molécule, reverse-phase chromatography can be used alone or in combination with quadrupole mass spectrometry to characterize the identity and purity of several of the lead molécules. Initially, drug stability over time in increasing concentrations of sérum, plasma, and whole blood from mammalian species (such as mouse, cynomolgus macaque, and human) is evaluated by HPLC, and a half-life is determined.

Prominent métabolites characterized by mass spectrometry. Human plasma protein binding are evaluated by partition analysis using equilibrium dialysis. For intestinal permeability modeling, apical-to-basolateral flux is assessed in the human épithélial cell line TC7. Hepatic clearance is estimated for a subset of the most promising analogs by measuring the rate of disappearance of the parent compound during incubation in human liver microsomes. As above, spécifie métabolites can be isolated and characterized.

In vitro toxicology. In vitro toxicology studies are performed to evaluate the potential cardiac and genetic toxicity of lead analogs. Automated patch-clamp is used to assess the impact of each compound on hERG channel currents in a recombinant Chinese hamster ovary (CHO) cell line transgenically expressing the human Kv11.1 gene. Concentrations up to the lesser of 30 times the maximum sérum concentration or the limit of solubility of each compound are evaluated in order to détermine an IC50 for the molécule on the hERG channel. A subset of compounds is evaluated over a range of concentrations for their ability to induce mutation reversion in Salmonella typhimurium strains TA98 and TA100 or to promote micronucleus formation in CHO cells in culture.

EXAMPLE 5. ACTIVATION OF GENE EXPRESSION BY KIN1000 AND DERIVATIVE COMPOUNDS

Gene expression in HeLa cells. Cultured human cells were treated with 20μΜ, 10μΜ, 5μΜ of compound or a DMSO control and incubated for up to 24 hours. Cells were harvested and RNA was isolated using the QIAshredder columns and RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Reverse transcription was performed and the cDNA template was used for quantitative real-time PCR. PCR reactions were performed using commercially available, validated TaqMan gene expression assays (Applied Biosystems/Life Technologies) according to manufacturer instructions. Gene expression levels were measured using a relative expression analysis (AACt).

Gene expression in PH5CH8 cells. Cultured human cells were treated with 10uM, 5uM, 1uM or a DMSO control and incubated for up to 24 hours. Cells were harvested and RNA was isolated using the QIAshredder columns and RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Reverse transcription was performed and the cDNA template was used for quantitative real-time PCR. PCR reactions were performed using commercially available, validated TaqMan gene expression assays (Applied Biosystems/Life Technologies) according to manufacturer instructions. Gene expression levels were measured using a relative expression analysis (AACt).

Gene expression in HUVEC primary cells. Cells were thawed and seeded in 6-well plates at 2.4x10⁴ cells per well and allowed to grow to 80% confluence, typically 5 days in culture with fresh media replaced every 48 hours. Compound was added at 10μΜ, 1μΜ or a DMSO control and incubated for up to 24 hours. Cells were harvested and RNA was isolated using the QIAshredder columns and RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Reverse transcription was performed and the cDNA template was used for quantitative real-time PCR. PCR reactions were performed using commercially available, validated TaqMan gene expression assays (Applied Biosystems/Life Technologies) according to manufacturer instructions. Gene expression levels were measured using a relative expression analysis (AACt).

Figure 5 shows induction of gene expression by KIN1000 and its dérivative compound KIN1148. Figure 5A shows gene expression levels of IFIT2 (left) and OAS1 (right) in HeLa cells over time from 4-24 hours post treatment with 10μΜ KIN1000 (grey) or 10μΜ KIN1148 (black). Figure 5B shows gene expression levels of IFIT2 in PH5CH8 cells (left) treated with KIN1000 (solid grey bars) or KIN1148 (solid black bars), and in HeLa cells (right) treated with KIN1000 (grey striped bars) or KIN1148 (black checked bars). In each test group, the three vertical bars represent 5, 10, and 20 μΜ compound (KIN1000 or KIN1148), respectively. Figure 5C shows gene expression levels of IFIT2 (left), OAS1 (center), and MxA (right) in primary HUVEC cells that were treated with 1μΜ KIN1000 (grey) or 1μΜ KIN1148 (black). The différence in axis scaling demonstrates that compounds are more active in a primary cell type. These data demonstrate that compounds are active in cells by inducing responsive gene expression.

Gene expression can be similarly assayed in cell types that include, without limitation: primary blood mononuclear cells, human macrophages, THP-1 cells, Huh 7 cells, A549 cells, MRC5 cells, rat splénocytes, rat thymocytes, mouse macrophages, mouse splénocytes, and mouse thymocytes. Expression of other genes of interest can be assayed as described herein.

Example 6. Antiviral activity of KIN1000 against various viruses

Antiviral action in cell culture infection models. To further characterize the breadth of antiviral activity of optimized molécules, cell culture infection models are used to analyze different viruses, including but not limited to different strains of influenza virus, HCV, Dengue virus, RSV, and West Nile virus (WNV), an emerging public health concern. The studies include treating cells with compound 2-24 hours prior to infection or treating cells up to 8 hours after infection. Virus production and cellular ISG expression are assessed over a time course to analyze antiviral effects of représentative compounds from lead structural classes. ΙΡΝβ treatment is used as a positive control.

Virus production is measured by focus-forming or plaque assay. In parallel experiments, viral RNA and cellular ISG expression are measured by qPCR and immunoblot analyses. These experiments are designed to validate compound signaling actions during virus infection, and assess compound actions to direct innate immune antiviral programs against various strains of viruses and in the setting of virus countermeasures. Detailed dose-response analyses of each compound are conducted in each virus infection System to détermine the effective dose that suppresses virus production by 50% (IC50) and 90% (IC90) as compared with control cells for both the pre-treatment and post-treatment infection models.

Table 2. Virus Systems and study design for antiviral analysis of lead compounds

Virus	Virus Strain	Study Design
HCV	H77 (génotype 1a) JFH1 (génotype 2a)	Assays Plaque or focus forming assays (infectious virus) qPCR (RNA levels) Immunoblot and ELISA (protein levels) Study Design Compound treatment of cells preand post-infection Détermine EC₅₀ and EC_go Inhibition of viral life cycle
FLU	High pathogenicity in mice A/PR/8/34 (H1N1 mouse-adapted virus) A/WSN/33 (H1N1 mouse-adapted neurovirulent virus) Low pathogenicity in mice A/Texas/36/91 (H1N1 circulating virus) A/Udorn/72 (H3N2)
WNV	TX02 (lineage 1) MAD78 (lineage 2)

EXAMPLE 7. Activity of KIN1000 and dérivative compounds against respiratory

SYNCYTIAL VIRUS.

HeLa cells were seeded the previous day in 6-well plates at 4x10⁵ cells per well. The next day, the media was replaced with RSV in media without FBS at an MOI of 0.1. Virus binding occurred at 37°C for 2 hours. After 2 hours the cells were washed with warm complété media and replaced with media containing drug at varying concentrations of 10μΜ, 5μΜ, 1μΜ or a DMSO control. Cells were placed in a 37°C incubatorfor 48 hours.

For virus détection and titration, HeLa cells were seeded in 96-well plates at 8x10³ cells per well 24hrs prior to collecting virus supernatant. After the 48 hour incubation period, the virus supernatant from the infected plate was harvested and used to infect these cells at a 1/10 final dilution. Cells were placed in a 37°C incubator for 24 hours.

hours after infection, cells were washed twice with PBS and fixed with methanol/acetone solution. After fixing the cells were washed twice with PBS and replaced with blocking buffer (10% horse sérum, 1g/mL BSA and 0.1% Triton-100X in PBS) for 1 hour. The blocking buffer is replaced with binding buffer containing a 1/2000 dilution of primary antibody for 2 hours at room température. The primary antibody was a mouse monoclonal antibody against RSV. The cells were washed twice with PBS and replaced with binding buffer containing 1/3000 dilution of the Alexa Fluor-488 goat anti-mouse secondary antibody and a Hoechst nuclear stain for 1 hour at room température. The cells were washed twice with PBS and PBS is added to ail wells. The 96-well plate is sealed and fluorescence activity associated with virus infectivity was determined by immunofluorescent assay using the Array Scan instrument (Thermo-Fischer).

Figure 6 shows experiments performed using the protocol of the Example, demonstrating the antiviral activity of KIN1000 and KIN1148 against respiratory syncytial virus. Figure 6A shows that HeLa cells treated with increasing amount of KIN1000 and KIN1148 showed dosedependent decrease in infection by RSV. Figure 6B shows that KIN1148 showed antiviral activity against RSV when drug is added up to 24 hours prior to infection.

Treatment with compounds prior to infection. In variations of this method, the compounds are added at varying time points prior to infection with virus. Virus détection and titration is conducted as described.

Analog testing and SAR studies. Antiviral activity against RSV was used as a criterion to measure activity of structural dérivatives of KIN1000. Table 3 shows select structural dérivatives of KIN1000 that demonstrated antiviral activity against RSV. Compared to KIN1000 parent compound, these analogs showed varying levels of antiviral activity against RSV. +++ = greater than 70% inhibition of infection, ++ = greater than 50% inhibition, + = greater than 30% inhibition, - = less than 30% inhibition.

TABLE 3

R2

I

KIN	R3	RI		10 uM	5uM	1 uM
1000			H	++	+	-
1014			H	+	-	-
1034	O ω		H	-	+	-
1069	Cl CI^^V	H	H	++	+	-
1072		H	H	++	-	-
1075				+	-	-
1148		H	H	+++	++	-
1169	H \ ^hk . 1		H	+++	++	-
1170	H J N O^J		H	++	-	-
1203	/T”\ z y	H	H	+++	-	-

EXAMPLE 8. Activity of KIN1000 and dérivative compounds against influenza A/Udorn/72 virus

Influenza A/Udorn/72 infection of H292 cells. ,2x10⁶ H292 cells in RPMI1640+10%FCS were treated with 2μΜ KIN1148 in a final concentration of 0.5% DMSO for 6 hours. Compoundcontaining media was aspirated and replaced with 1X MEM containing A/Udorn/72 at an MOI of 0.1 and placed at 37°C in a CO² incubator. Two hours post infection, virus-containing media was aspirated and replaced with 1X MEM containing 1ug/mL TPCK-treated Trypsin, 2μΜ KIN1148, 0.5% DMSO. Cells were placed in 37°C CO₂ incubator for 18 hours. After 20 hours post-infection, virus supernatants were collected and titred on MDCK cells.

Influenza A/Udorn/72 infection of HEK293 cells. 5x10⁵ HEK293 cells were infected with A/Udorn/72 at an MOI of 0.2 in 1X MEM. After 2 hours post-infection, virus-containing media was aspirated and replaced with 1X MEM containing 1pg/mL TPCK-treated Trypsin, 10μΜ KIN1148, 0.5% DMSO. Cells were returned to 37°C, CO₂ incubator for 18 hours. After 20 hours post-infection, virus supernatants were collected and titred on MDCK cells.

Titre in MDCK cells. 10pL of infected supernatant was added to 2x10⁶ MDCK cells in the presence of 2pg/mL TPCK-trypsin and placed in a 37°C CO₂ incubator. After 8 hours, supernatant was removed and cells were fixed and stained with FITC-conjugated antibody spécifie for Influenza NP protein. Number of foci was quantitated using the ArrayScan instrument and software (Cellomics).

Figure 7 shows antiviral activity of KIN1148 against Influenza A virus Udorn/72. H292 cells (left) and HEK293 cells (right) treated with 2uM (H292) or 10uM (HEK293) of KIN1148 showed decrease in infection by virus.

EXAMPLE 9. ACTIVITY OF KIN1000 AND DERIVATIVE COMPOUNDS AGAINST DENGUE VIRUS

Huh 7 cells were seeded the previous day in 6-well plates with 4x10⁵ cells per well. The next day, the media was replaced with Dengue virus type 2 in media without FBS at an MOI of 0.25. Virus binding occurred at 4°C for 1 hour. After 1 hour the cells were washed with warm complété media and replaced with media containing KIN1148 at varying concentrations of 10uM, 5uM, 1uM or a DMSO control. Cells were placed in a 37°C incubator for 48 hours.

Titre in Vero cells. Vero cells were seeded in 96-well plates at 8x10³ cells per well 24hrs prior to collecting virus supernatant. After 48hrs, the virus supernatant was harvested and used to infect Vero cells at a 1/100 final dilution.

hrs after infection, Vero cells were washed 2x with PBS and fixed with methanol/acetone for 15mins. After fixing the cells were wash 2x with PBS and replaced with blocking buffer for 30-45 mins. The blocking buffer was replaced with binding buffer containing a 1/2000 dilution of primary monoclonal antibody targeting the Envelope protein for 2hrs. After 2hrs, the cells were washed 2x with PBS and replaced with binding buffer containing 1/3000 dilution of the Alexa Fluor-488 goat anti-mouse secondary antibody and a Hoechst nuclear stain for 45mins. After 45 mins cells were washed 2x with PBS and PBS was added to ail the well. The 96-well plate was sealed and fluorescence activity associated with virus infectivity was determined by IF using the ArrayScan instrument and software (Cellomics).

Figure 8 shows the results of experiments performed using the protocol of this Example, demonstrating the antiviral activity of KIN1148 against Dengue virus type 2. Huh 7 cells treated with increasing amounts of KIN1148 showed dose-dependent decrease in infection by virus.

EXAMPLE 10. Activity of KIN1000 and dérivative compounds against Hepatitis B virus HepAD38 cells (Hep 2 cells expressing a regulated HBV genome) were grown for 72 hours in the presence of compound (concentrations 1-10 μΜ in 0.5% DMSO media). HepAD38 cells that do not express HBV were used as a négative control. Following 72 hours of treatment 100 pl of media was used in an ELISA to measure HBV surface antigen. The amount of HBV surface antigen produced by the cells was measured in the supernatants by ELISA commercially available HBV sAg ELISA from Creative Diagnostics, NJ.

Figure 9 shows the results of experiments performed using the protocol of this Example, demonstrating the antiviral activity of ΚΙΝ1148 against Hepatitis B virus. HepAD38 cells treated with increasing amounts of ΚΙΝ1148 showed dose-dependent decrease in supernatant levels of virus.

Example 11. In vivo pharmacokinetic, toxicological, and antiviral properties of OPTIMIZED DRUG LEADS IN RELEVANT PRECLINICAL ANIMAL MODELS

Preclinical pharmacokinetic and tolerability profiling. The in vivo pharmacokinetic (PK) profile and tolerability/toxicity of Kl N1000 and related compounds are evaluated in order to conduct further characterization of their antiviral activity in animal models of influenza virus and WNV infection. Mouse is the chosen test species for these studies since it is the most commonly used rodent model of WNV and influenza.

A reverse-phase, HPLC-MS/MS détection method is used for measuring the concentration of each compound in mouse plasma. Prior to PK profiling, an initial oral and intravenous formulation for each compound is developed using a limited formulation component screen that is largely focused on maximizing aqueous solubility and stability over a small number of storage conditions. Any of the analytical methods as are known in the art can be used to measure formulation performance. A formulation is developed for each compound following a three tiered strategy:

Tier 1 : pH (pH 3 to 9), buffer, and osmolality adjustment

Tier 2: addition of éthanol (<10%), propylene glycol (<40%), or polyethylene glycol (PEG) 300 or 400 (<60%) co-solvents to enhance solubility

Tier 3: addition of A/-/V-dimethylacetamide (DMA, <30%), /V-methyl-2-pyrrolidone (NMP, <20%), and/or dimethyl sulfoxide (DMSO, <20%) co-solvents or the cyclodextrins (<40%) as needed to further improve solubility.

For compounds that demonstrate adéquate performance in in vitro antiviral, mechanistic, ADME, and toxicology studies, a preliminary mouse PK study is performed. See Table 3. Each compound is administered as a single dose to animais by oral gavage (<10 ml/kg) or i.v. bolus injection (<5 ml/kg) after an overnight fast. Multiple animais are dosed for each dosing group such that 3 animais can be sampled at each time point. Blood samples are collected by retroorbital sinus prior to dosing and at 5, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hours postdosing. Drug concentrations are measured according to the previously developed bioanalytical method. Pharmacokinetic parameters are evaluated using the WinNonlin software.

Table 4

Study	Experimental design	Route of administration	Outcomes
Mouse PK	Single dose pharmacokinetic study	IV and Oral	Oral bioavailability, C_max, t/₂, Cl, V_d, AUCq-24,0- CO
Mouse tolerability	Phase 1: ascending dose tolerability and MTD détermination; Phase 2: placebo controlled 7-day toxicity at MTD	Oral	MTD, acute toxicity, hematology, sérum chemistry, gross pathology

Based upon performance in exploratory PK studies, compounds are further evaluated for preliminary tolerability and toxicity in mice prior to their characterization in antiviral models. Tolerability studies are performed in two stages: an initial dose escalation stage (up to 5 doses, each separated by a 5-day washout period) to détermine the maximum tolerable dose (MTD, Phase 1), followed by seven daily administrations of the MTD to evaluate acute toxicity (Stage 2). See Table 3. Ail doses are administered by oral gavage. In an exemplary experiment, five animais of each sex are placed on-study in stage 1 and 15 animais per sex per dosing group in Stage 2. Study endpoints include a détermination of the MTD, physical examination, clinical observations, hematology, sérum chemistry and animal bodyweights. Gross pathology is performed on ail animais whether found dead, euthanized in extremis, or at the intended conclusion of the experiment. The toxicology studies are primarily exploratory in nature and intended to identify early toxicological endpoints, and drive sélection of lead candidates for antiviral animal models.

Table 5. In vivo studies of compound actions against WNV and influenza virus_________________

Experiment	Analysis	Goal	Exemplary No. of Mice*
Effective compound dose détermination	Viral burden analysis in sérum	Define in vivo EC₅o and EC₉₀	238
Viral pathogenesis study 1 : EC₅₀ and EC₉₀T reatment	Time to moribund State, clinical scoring for pathologie signs of infection	Define compound action toward limiting viral pathogenesis	739

Viral pathogenesis study 2: EC50 and EC90 treatment and time course analysis	Viral burden analysis in sérum and various target organs	Define compound action toward limiting virus réplication and spread	1056
Viral pathogenesis study 3: (neuroinvasion model) EC50 and EC90 treatment	Time to moribund State, clinical scoring for pathologie signs of infection	Define compound action toward limiting viral pathogenesis in the CNS	370

*Numbers reflect an average of at least two itérations of each experiment

Evaluation of antiviral properties and immune protection using mouse infection models. Optimized compounds are selected based on compound pharmacokinetic, antiviral, and innate immune actions for further évaluation in preclinical mouse models of infection. See Table 4. Innate immune actions of the compounds are measured, and their ability to protect mice from WNV and influenza virus challenge is assessed. For the WNV infection model, subcutaneous footpad infection of wild-type C57BI/6 mice with the virulent lineage 1 strain of WNV (WNV-TX) are performed. Non-surgical trachéal instillation is performed for influenza virus strains A/PR/8/34, A/WSN/33, and A/Udorn/72.

The influenza virus strains used for certain experiments are of two different subtypes (H1N1 and H3N2) and exhibit varying pathogenic properties and clinical présentations in C57BI/6 mice. Mice are monitored for morbidity and mortality over a range of challenge doses (such as, 10 to 1,000 pfu of virus) either alone or in combination with compound treatment beginning 12 hours before or 24 hours after infection and continuing daily subject to the determined plasma half-life of the drug. Compound dose-response analysis and infection time course studies are conducted to evaluate compound efficacy to: 1) limit sérum viral load, 2) limit virus réplication and spread in target organs, and 3) protect against viral pathogenesis.

For WNV, in addition to sérum, viral burden is assessed in lymph nodes, spleen, and brain; for influenza virus, viral burden is assessed in heart, lung, kidney, liver, and brain. Incorporated in the design of these experiments is the détermination of an effective dose for 50% and 90% suppression of sérum viral load (ED50 and ED90) by each compound after a standard challenge of 100 pfu of WNV-TX or 1,000 pfu of influenza virus. Sérum viral loads are determined by qPCR of viral RNA at 24-hour intervals following compound treatment. The compound actions are tested at the ED50 and ED90 toward limiting WNV pathogenesis in the cérébral nervous System using a WNV neuroinvasion model of infection.

Mice are monitored for morbidity and mortality after standard intracranial challenge of 1 pfu of WNV-MAD, either alone or in combination with compound treatment beginning 24 hours after infection.

Example 12. Antiviral activity of KIN1000 and dérivative compounds in vivo

Evaluation of antiviral properties of Kl N1000 and dérivative compounds using mouse infection models. Up to 5 of the most promising compounds will be selected for further évaluation in preclinical mouse models of infection with Influenza and/or respiratory syncytial virus. Table 6 lists the nonclinical studies to measure antiviral efficacy of Kl N1000 and dérivative compounds.

Table 6. Nonclinical studies to measure drug concentration and antiviral efficacy in vivo

Study	Experimental design	Route Admi.	No. Cpds.	No. Animais	Outcomes
Mouse dosing	Drug measured in blood at 3 dose levels; 2, 8, 24 hours post treatment	Oral/IP	<5	120	Drug concentration in blood; HPLC reverse phase
Mouse Influenza Model	Trachéal instillation of A/WSN/33 or A/Udorn/72; drug treatment at 2 doses w/ placebo	Oral/IP	<5	480	Mortality, viral titer in serum/target organs, body temp., bodyweight, clin, obs., cytokine levels
Mouse RSV Model	Trachéal instillation of RSV A2 Long strain; drug treatment at 2 doses w/ placebo control	Oral/IP	<5	240	Mortality; bodyweight; target organ viral titer; innate immune gene expression; markers of inflammation

Mouse influenza model. We will perform non-surgical trachéal instillation of influenza virus strains A/WSN/33 and A/Udorn/72. These influenza virus strains are two different subtypes (H1N1 and H3N2) and exhibit varying pathogenic properties and clinical présentations in C57BI/6 mice. Lead dérivatives of the KIN1000 family of compounds will be administered daily by oral gavage or IP administration over the entire course of infection (typically 2 weeks) at 2 dose levels plus a placebo control group. Five animais per sex and per group will be evaluated for endpoints, including but not limited to daily clinical observations, mortality, body weight, and body température. Three animais per sex will be used to measure virus titer in sérum, heart, lung, kidney, liver, and brain. Cytokine expression at various time points during infection in compound-treated versus control animais will be assayed.

Mouse RSV model. We will perform non-surgical trachéal instillation of respiratory syncytial virus A2 long strain. BALB-c mice will be infected at a dose of RSV A2 virus that does not cause cytopathic effects followed by daily oral or IP administration of compound at 2 dose levels or a placebo control for up to 21 days. Mice will be monitored as described above, including inspection for morbidity and mortality, viral titer in sérum and blood, cytokine sécrétion, increased immune cell populations, and innate immune gene expression.

Example 13. Adjuvant activity of KIN1000 and dérivative compounds in vivo

To characterize the breadth of adjuvant activity of Kl N1000 and related compounds, in vivo animal models of vaccination and vaccination plus protection are used. The studies include priming animais including but not limited to rats and mice with compound alone or in combination with an antigen and then assessing the adjuvant effect.

Adjuvant effect is measured by assays for modified, enhanced immune humoral and cellular responses. Humoral responses are assessed over time at discrète times post vaccination and/or boosting by collecting blood for sera and determining relative concentrations of antibody classes (IgM, IgG, IgA or IgE) and/or isotypes including lgG1, lgG2a, lgG2b, lgG2c, lgG3 for IgG antibodies. Moreover, affinity and avidity of the generated antibodies is also determined. In instances in which the vaccine préparation includes a combination of compound and antigen, the neutralizing activity of the generated antibodies is also determined.

Cellular mediated immune responses induced by the compounds are measured by established methods in the field including ex vivo stimulation of peripheral blood mononuclear cells, lymph nodes, splénocytes or other secondary lymphoid organs with the antigen and measurement of cytokine or chemokine production in the supernatant at several times thereafter. Cytokines measured include Th1 type of cytokines including but not limited to IFN gamma and TNF alpha, Th2 type cytokines including but not limited to IL-4, IL-10, IL-5 and IL-13 and Th17 cytokines including but not limited to IL-17, IL-21 and IL-23. Chemokines elicited by the compounds are also measured including but not limited to RANTES, IP-10, MIP1a, MIP1b, and IL-8. T cell antigen spécifie production of cytokines can also be measured by intracellular cytokine staining with fluorescently labeled spécifie antibodies and flow cytometry or by ELISPOT. Both CD4+ ad CD8+ T cell populations are studied.

Measurement of adjuvant activity at the cellular level is also determined by immunophenotyping of surface markers of activation by flow cytometry. CD8 T cell antigen-specific responses are also evaluated by intracellular cytokine staining of perforin, cell surface marker expression or prolifération assays including thymidine incorporation.

These experiments are designed to validate compound adjuvant activity in different combinations of prime-boost schemes and assess how the effects of Kl N1000 or related compounds on the innate immune antiviral programs shape the adaptive immune responses mounted to the antigen in the vaccine préparations.

Detailed immune response analyses of each compound as described above are conducted with each selected antigen to détermine the immune correlates for that particular antigen(s) and compound formulation. These results guide the protection studies in which animais vaccinated and boosted with combinations of select optimized compounds and desired antigen(s) formulations from select infectious agents are later challenged with doses of infectious agent that are known to resuit in disease or death of the animal. Protection afforded by vaccination is typically measured by monitoring of clinical symptoms and survival.

A proof of concept experiment was performed. LEWIS female rats at 10-12 weeks of âge were primed with suspensions of antigen (ovalbumin, 0.2 mg/Kg) and KIN1000 (1 mg/Kg) or KIN1148 (1 mg/mL) in phosphate saline buffer (PBS) on day zéro. Control animais received ovalbumin (OVA, InvivoGen Inc.) in PBS or OVA with poly l:C (0.1 mg/Kg, InvivoGen Inc.). Animais were boosted at weeks 2, and 8. Vaccines were delivered subcutaneously in the footpad and base of the tail for priming and on the footpad and flank for the boosts (0.025 mL/site). Blood samples were collected by tail bleed and processed to sérum at 0, 1, 2, 4, 6, and 9 weeks post priming. Titers of OVA spécifie antibodies were determined by ELISA using anti IgM, anti IgG and anti IgG isotype spécifie antibodies. Figure 10 shows IgG antibody levels relative to OVA alone vaccinated Controls in OVA+KIN1000 and OVA+KIN1148 vaccinated animais.

Measurement of cell-mediated adjuvant activity is also determined by determining delayed type hypersensitivity (DTH) to an antigen. In the same proof of concept experiment, cellular responses were evaluated by determining the delayed type hypersensitivity reaction to challenge with OVA 2 weeks after the first boost. Animais were sedated with isoflurane and injected with PBS or OVA (0.02 mL of 1m/gmL solution of OVA in PBS) in the pinna of the left and right ears, respectively. 24 hours later the différence in ear thickness was calculated. Figure 11 shows measured différence between right ear and left ear thickness.

Unless otherwise indicated, ail numbers expressing quantities of ingrédients, properties such as molecular weight, reaction conditions, and so forth used in the spécification and daims are to be understood as being modified in ail instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the spécification and attached daims are approximations that can vary depending upon the desired properties sought to be obtained by the présent disclosure. At the very least, and not as an attempt to limit the application of the doctrine of équivalents to the scope of the daims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the spécifie examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard déviation found in their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following daims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring 41 individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the spécification as if it were individually recited herein. Ail methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ail examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the spécification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

Groupings of alternative éléments or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other éléments found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or délétion occurs, the spécification is deemed to contain the group as modified thus fulfilling the written description of ail Markush groups used in the appended daims.

Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes ail modifications and équivalents of the subject matter recited in the daims appended hereto as permitted by applicable law. Moreover, any combination of the above-described éléments in ail possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Spécifie embodiments disclosed herein may be further limited in the daims using consisting of or and consisting essentially of language. When used in the daims, whether as filed or added per amendment, the transition term “consisting of’ excludes any element, step, or ingrédient not specified in the daims. The transition term “consisting essentially of” limits the scope of a daim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

In closing, it is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the présent disclosure. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the présent disclosure may be utilized in accordance with the teachings herein. Accordingly, the présent disclosure is not limited to that precisely as shown and described.

Claims

1. A compound represented by the formula

wherein a dashed line indicates the presence or absence of a bond;

R¹ is R^a, OR² or NR²R³, optionally substituted naphthyl or substituted phenyl;

each R^a is independently H, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted hydrocarbyl;

R² and R³ are each independently R^a, COR^a, or SO₂R^a;

Y¹, Y², Y³ and Y⁴ are each independently CR⁴ or N, and optionally wherein Y¹ and Y² are both CR⁴ and together form an additional heterocyclic ring optionally substituted by R⁴;

each R⁴ is independently R², OR^a, NR²R³ SR^a, SOR^a, SO2R^a, SO2NHR^a, NCOR^a, halogen, trihalomethyl, CN, S=O, or nitro;

R⁵ is R^a, COR^a, SO2R^a, optionally H or C4_₃ alkyl, or is not présent;

V is C=O, CR², CR²R³, COCR²R³, or C=NR²; and,

W and X are each independently S, O, N, NR^a, CR²R⁴or CR⁴.

2. A compound of claim 1, further represented by a formula

wherein R¹⁰, R¹³, R¹⁴, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² are independently R^b, OR^b, SR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CONR^bR^c, NR^bCOR^c, SO2NR^bR^c, CF3, CN, NO2, F, Cl, Br, I, or C2.5 heterocyclyl; each R^b is independently H or Cv3 hydrocarbyl, and each R^c is independently

H or C_v3 alkyl.

3. A compound of claim 1, wherein Y³ is CR⁴, wherein R⁴ is R^b, OR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CF3, CN, NO₂, F, Cl, Br, or I, wherein R^b and R^c are independently H or C^s alkyl, and/or wherein Y⁴ is CR⁴, wherein R⁴ is R^b, OR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CF3, CN, NO₂, F, Cl, Br, or I, wherein R^b and R^c are independently H or C-|.₃ alkyl.

4. A compound of claim 1, further represented by the formula

wherein R₁₀, Rn, R₁₂, R13, Ru, Rw, R17, and Ri₈ are independently R^b, OR^b, COR^b, CO2R^b, OCOR^b, NR^bR^c, CF3, CN, NO₂, F, Cl, Br, or I, wherein R^b and R^c are independently H or C1.3 5 alkyl; and, R⁵ is H or C_v3 alkyl.

5 optionally, a vaccine against influenza virus, Hepatitis C virus, West Nile virus, SARScoronavirus, poliovirus, measles virus, Dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, St.

Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, llheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, Kyasanur forest disease virus, respiratory syncytial virus or human 10 immunodeficiency virus (HIV).

5. A compound of claim 4, wherein R¹⁸ is CH₃; and/or, wherein R¹³ is Br.

7. A pharmaceutical composition comprising a compound of any one of daims 1-6.

8. A compound of any one of daims 1-6, or a pharmaceutical composition of claim 7, for use in therapy.

5

9. A compound of any one of daims 1-6, or a pharmaceutical composition of claim 7, for the use in treating or preventing a viral infection in a vertebrate, the use comprising administering to the vertebrate a compound of any one of daims 1-6, or a pharmaceutical composition of claim 7;

optionally, wherein the viral infection is caused by a virus from one or more of the 10 following families: Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Flaviviridae,

Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, 15 Tymoviridae, Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae;

optionally, wherein the viral infection is influenza virus, Hepatitis C virus, West Nile virus, SARS-coronavirus, poliovirus, measles virus, Dengue virus, yellow fever virus, tick-borne encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley virus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus, llheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus, Kyasanur forest disease virus, respiratory syncytial virus or human immunodeficiency virus (HIV).

10. A compound of any one of daims 1-6, or a pharmaceutical composition of claim 7, for use as an adjuvant for a prophylactic or therapeutic vaccine;

11. A compound of any one of daims 1-6 for use in modulating the innate immune response in a eukaryotic cell, the use comprising administering to the cell a compound of any one of daims 1-6, optionally wherein the cell is in vivo or in vitro.