WO2016073947A1 - Composés anti-viraux, compositions pharmaceutiques et méthodes d'utilisation de ces derniers - Google Patents

Composés anti-viraux, compositions pharmaceutiques et méthodes d'utilisation de ces derniers Download PDF

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Publication number
WO2016073947A1
WO2016073947A1 PCT/US2015/059611 US2015059611W WO2016073947A1 WO 2016073947 A1 WO2016073947 A1 WO 2016073947A1 US 2015059611 W US2015059611 W US 2015059611W WO 2016073947 A1 WO2016073947 A1 WO 2016073947A1
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virus
compound
lower alkyl
compounds
fused
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PCT/US2015/059611
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English (en)
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Shawn P. Iadonato
Kristin M. Bedard
Kerry W. Fowler
Shari KAISER
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Kineta, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/20—Oxygen atoms
    • C07D215/22—Oxygen atoms attached in position 2 or 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/88—Oxygen atoms
    • C07D239/91—Oxygen atoms with aryl or aralkyl radicals attached in position 2 or 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring

Definitions

  • HCV chronic liver diseases
  • 130 million of these are chronic carriers at risk of developing chronic liver diseases (cirrhosis, carcinoma, and liver failure).
  • HCV is responsible for two thirds of all liver transplants in the developed world.
  • Recent studies show that the death rate from HCV infection is rising due to the increasing age of chronically infected patients.
  • RIG-I signaling is transduced through IPS-1 (also known as Cardif, MAVs, and VISA), an essential adaptor protein that resides in the outer mitochondrial membrane.
  • IPS-1 recruits a macromolecular signaling complex that stimulates the downstream activation of interferon regulatory factor-3 (IRF-3), a transcription factor that induces the expression of type I interferons (IFNs) and virus-responsive genes that control infection.
  • IRF-3 interferon regulatory factor-3
  • IFNs type I interferons
  • FIG. 1 B shows quantitation of HCV viral RNA by real-time quantitative PCR (RT-qPCR) done in Huh7 cells untreated (Untreated), treated with interferon (I FN), or pre-treated with compound 2 (CPD 2) for 18 hours and infected with HCV2a at MOI of 1.0 for 72 hours. Viral RNA was isolated and quantitated in the supernatant of infected cultures.
  • FIG. 1 C shows a similar quantitation of HCV viral RNA by RT-qPCR done in Huh7 cells infected with HCV2a at MOI of 1.0 for 4 hours and then treated with compound 2.
  • 7E shows that compound 7 (20mg/kg) inhibited flu replication in the lung compared to vehicle treatment (V) when administered by intranasal instillation either -24 hours prior (prophylactic; pre) or +24 hours post (therapeutic; post) lethal infection with PR8 flu.
  • Lung tissue was harvested 72 hours after infection and flu RNA was quantitated by PCR.
  • FIGs. 9A, 9B, and 9C show the in vitro antiviral activity of compounds of the disclosure.
  • FIG. 9A shows anti-influenza activity of compounds 35 and 37.
  • FIG. 9B shows antiviral activity against H3N2 influenza virus.
  • the lead compounds shows a >3 log reduction in FLU (H3N2) viral titer, and lead compounds are 850-fold more potent than the library hit compound against influenza.
  • FIG. 9C shows broad spectrum in vitro influenza efficacy of compounds 32, 37, and 43.
  • FIGs. 1 1 A, 1 1 B, and 1 1 C show the broad in vivo antiviral efficacy of compound 7 compared with control (HPBCD) in mice.
  • FIG. 1 1A shows influenza lung titer
  • FIG. 1 1 B shows MHV lung titer
  • FIG. 1 1 C shows DENV plasma titer.
  • FIG. 12 shows that a single prophylactic intranasal treatment with compound 7 protected against lethal (10X LD50) H1 N1 influenza challenge in mice.
  • a and A' are optional linker groups between the core bicyclic ring structure and the substituent R 3 or W, respectively. That is, A and/or A' may each be present or absent depending on the particular embodiment of the compound as shown by the value for s and r, i.e., when s or r is 1 then the respective group A or A' is present and when s or r is 0 then the respective group A or A' is absent.
  • substituents on the ring structures can include a groups wherein s can be 1 , A may be O, S(O), NR d and R 3 can be 3-propynyl; S0 2 CH 3 ; CF 2 H; CF 3 ; CONHCH 3 or alkyl-CONR 4 R 5 or alkyl-NR 4 R 5 where R 4 and R 5 can come together to form a ring such as morpholine, N- methylpiperazine, or pyrrolidine; or alternatively where s can be 0 and R 3 may be SO2CH3, COR 4 , CONR 4 R 5 , N-imidazolinyl or N-maleimido; and wherein r can be 0 and W can be 1 - naphthyl, cyclopentyl, 2-thiazolyl, 2-pyrazinyl, 2-benzoxazolyl, or 4-R 6 -1 -phenyl and R 6 is tert- butyl, Br, OCF3 or -NH
  • the compounds can have a structure:
  • each R 6 can be independently selected from H, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, lower alkyl, haloalkyl, aryl, alkenyl, alkynyl, alkylaryl, arylalkyl, alkoxy, aryloxy, arylalkoxy, alkoxyalkylaryl, alkylamino, arylamino, heteroalkyl, heteroaryl, cycloalkyl, cyclic heteroalkyl, acyl, NH 2 , OH, CN, N0 2 , OCF 3 , CF 3 , Br, CI, F, -NHSO2R 7 , 1 -amidino, 2-amidino, alkylcarbonyl, morpholino, piperidyl, dioxanyl, pyr
  • Other example compounds include wherein s is 1 , A is NR' where R' is H, methyl, or ethyl; R 3 is H, 3-propynyl, SO2CH3, CF 2 H, CF 3 , CON HCH3, C 2 H 4 NR 4 R 5 , or CH 2 CONR 4 R 5 ; where R 4 and R 5 come together to form a morpholino ring, an N-acetyl piperazinyl ring, an N- methanesulfonyl piperazinyl ring, or an N-methyl piperazinyl ring; or s is 0 and R 3 is SO2CH3, COR 4 , CONR 4 R 5 , N-imidazolinyl, or N-maleimido.
  • r is 0 and W is 1-naphthyl, cyclopentyl, 2-thiazolyl, 2-pyrazinyl, 2-benzoxazolyl, or 4-R 6 -1 -phenyl and R 6 is tert-butyl, Br, OCF3, or -NHSO2R 7 , where R 7 is N-piperidyl or phenyl; or r is 1 , and W is phenyl.
  • W a can be Br, aryl, CF 3 , lower alkyl, cycloalkyl, heterocycloalkyl, CHF 2 , C(CH 3 ) 3 , NHS0 2 W b ;
  • W b can be phenyl, cycloalkyl, heterocycloalkyl, or lower alkyl;
  • W c can be lower alkyl.
  • R 1 can be H, lower alkyl or OR c , where R c is H or lower alkyl and R 3 can be lower alkyl, phenyl, phenol, OR d , NR d , OR d R e , or NR d R e .
  • in can have a structure
  • R 10 and R 11 can each independently be H, lower alkyl, an alkoxy group, an alkylamino group, or a hydroxyl group. In some cases, R 10 and R 11 can be fused to form one or more substituted cyclic groups or one or more unsubstituted cyclic groups.
  • the one or more substituted cyclic groups, the one or more unsubstituted cyclic groups, or both can include at least one ring structure including at least four members, at least one ring structure including at least five members, at least one ring structure including at least six members, or at least one ring structure including at least seven members.
  • R 10 and R 11 can be fused to form a substituted morpholino ring.
  • R 10 and R 11 can be fused to form a substituted diazine ring.
  • R 10 and R 11 can be fused to form an unsubstituted diazine ring.
  • R 10 and R 11 can be fused to form a substituted or unsubstituted piperazinyl ring.
  • R 10 and R 11 can be fused to form a two ringed structure with a first ring including four members having at least one nitrogen atom and a second ring including four members having at least one oxygen atom.
  • compounds can also have a structure:
  • compounds can have the following structures:
  • A can be a five-membered heterocyclic ring including at least one N atom.
  • R 3 can be (CH2) q NR c R d , where q is 1 , 2, or 3, and R c and R d are each independently H, lower alkyl, or OR a with R a being H or lower alkyl.
  • alkyl refers to a functional group including a straight-chain, branched-chain, or cyclic hydrocarbon (cycloalkyl) containing from 1 to 20 carbon atoms linked exclusively by single bonds.
  • “Lower alkyl” refers to a functional group containing from 1 to 20 carbon atoms. An alkyl group may be optionally substituted as defined herein.
  • substituted alkyls, alkenyls, and alkynyls refer to alkyls, alkenyls, and alkynyls substituted with one to five substituents from the group 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-
  • Alkylene refers to a saturated aliphatic group derived from a straight or branched chain saturated hydrocarbon attached at two or more positions, such as methylene (-CH 2 -). Unless otherwise specified, the term “alkyl” may include “alkylene” groups.
  • heteroaryl refers to a functional group including 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.
  • hydroxy refers to the functional group hydroxyl (-OH).
  • Compounds can also be provided as alternate solid forms, such as polymorphs, solvates, 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.
  • Compounds also include pharmaceutically acceptable salts of the compounds.
  • Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid.
  • inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
  • Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, ⁇ -hydroxybutyric, malonic, galactic, and galacturonic acid.
  • Pharmaceutically acceptable acidic/anionic salts also include, the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate,
  • Suitable pharmaceutically acceptable base addition salts include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N, N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine. All of these salts can be prepared by conventional means from the corresponding compound represented by the disclosed compounds by treating, for example, the disclosed compounds with the appropriate acid or base.
  • Pharmaceutically acceptable basic/cationic salts also include, the diethanolamine, ammonium, ethanolamine, piperazine and triethanolamine salts.
  • a prodrug includes a compound which is converted to a therapeutically active compound after administration, such as by hydrolysis of an ester group or some other biologically labile group.
  • the pharmaceutical composition including a compound of the disclosure can be formulated in a variety of forms depending upon the particular indication being treated and will be apparent to one of ordinary skill in the art. Formulating pharmaceutical compositions including one or more compounds of the disclosure can employ straightforward medicinal chemistry processes.
  • the pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional adjuvants, such as buffering agents, preservatives, isotonicifiers, stabilizers, wetting agents, emulsifiers, etc.
  • Preservatives can be added to pharmaceutical compositions to retard microbial growth, and are typically added in amounts of 0.2%-1 % (w/v).
  • Suitable preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
  • Additional miscellaneous excipients can include chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, and vitamin E) and cosolvents.
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine, and vitamin E
  • cosolvents e.g., ascorbic acid, methionine, and vitamin E
  • the pharmaceutical compositions can be made up in a solid form (including granules, powders, or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the compounds can be admixed with adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium 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.
  • adjuvants such as lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alg
  • the pharmaceutical composition can be in solid or liquid form, e.g., in the form of a capsule, tablet, powder, granule, suspension, emulsion, or solution.
  • Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules.
  • the compound can be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms can also include, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms can also include buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • the pharmaceutical compositions can take the form of tablets or lozenges formulated in conventional manners.
  • Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
  • Such pharmaceutical compositions can also include adjuvants, such as wetting, sweetening, flavoring, and perfuming agents.
  • the pharmaceutical compositions can be formulated for parenteral administration by injection, e.g. by bolus injection, or infusion.
  • Formulations for injection can be presented in unit dosage form, e.g. in glass ampoule or multi-dose containers, e.g. glass vials.
  • the pharmaceutical compositions for injection can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as antioxidants, buffers, non-ionic detergents, dispersants, isotonicifiers, suspending agents, stabilizers, preservatives, dispersing agents and/or other miscellaneous additives.
  • Parenteral formulations to be used for in vivo administration generally are sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
  • the compounds 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 colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • coascervation techniques for example hydroxymethylcellulose, gelatin or poly-(methylmethacylate) microcapsules
  • colloidal drug delivery systems for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • macroemulsions for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions disclosed herein can be used to treat a viral infection in a subject; wherein the viral infection is caused by a virus from one the following families: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Picobirnaviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae
  • the pharmaceutical compositions can be used to treat a viral infection caused by one or more of Alfuy virus, Banzi virus, bovine diarrhea virus, Chikungunya virus, Dengue virus (DNV), Encephalomyocarditis virus (EMCV), hepatitis B virus (HBV), HCV, human cytomegalovirus (hCMV), HIV, llheus virus, influenza virus (including avian and swine isolates), Japanese encephalitis virus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, louping-ill virus, measles virus, MERS-coronavirus (MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus, parainfluenza virus, poliovirus, Powassan virus, respiratory syncytial virus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louis encephalitis virus, tick-borne ence
  • the actual dose amount administered to a particular subject can be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of viral infection, previous or concurrent therapeutic interventions, idiopathy of the subject, and route of administration.
  • compositions can be administered intravenously to a subject for treatment of viral infections in a clinically safe and effective manner, including one or more separate administrations of the composition.
  • 0.05 mg/kg to 5.0 mg/kg can be administered to a subject per day in one or more doses (e.g., doses of 0.05 mg/kg once-daily (QD), 0.10 mg/kg QD, 0.50 mg/kg QD, 1 .0 mg/kg QD, 1 .5 mg/kg QD, 2.0 mg/kg QD, 2.5 mg/kg QD, 3.0 mg/kg QD, 0.75 mg/kg twice-daily (BID), 1.5 mg/kg BID or 2.0 mg/kg BID).
  • QD 0.05 mg/kg once-daily
  • BID 0.10 mg/kg QD
  • 0.50 mg/kg QD 0.50 mg/kg QD
  • 1 .0 mg/kg QD 1 .5 mg/kg QD
  • 2.0 mg/kg QD 2.0 mg/kg QD
  • the total daily dose of a compound can be 0.05 mg/kg to 3.0 mg/kg administered intravenously to a subject one to three times a day, including administration of total daily doses of 0.05-3.0, 0.1 -3.0, 0.5-3.0, 1.0-3.0, 1 .5-3.0, 2.0-3.0, 2.5-3.0, and 0.5-3.0 mg/kg/day of compounds of Table 1 using 60-minute QD, BID, or three times daily (TID) intravenous infusion dosing.
  • TID three times daily
  • HCV drugs for potential administration in combination or conjunction with the pharmaceutical compositions disclosed herein include ACH-1625 (Achillion); Glycosylated interferon (Alios Biopharma); ANA598, ANA773 (Anadys Pharm); ATI-0810 (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, IDX- 375 (Idenix); IMO-2125 (Idera Pharmaceuticals); INX-189 (Inhibitex); ITCA-638 (Intarcia Therapeutics); ITMN-191/RG7227 (Interm
  • W is aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, heteroalkyl, substituted heteroalkyl, heterocycloalkyl, substituted heterocycloalkyl, arylalkyl, or heteroaryl alkyl;
  • Embodiment 5 A compound of any one of embodiments 1-4, wherein W has a structure selected from:
  • each R 8 is independently selected from H, alkyl, haloalkyl, cycloalkyi, aryl, alkenyl, alkynyl, alkylaryl, arylalkyl, alkoxyalkylaryl, heteroalkyl, heteroaryl, cyclic heteroalkyl, acyl, CF3, alkylcarbonyl, tetrazolo, thiazole, isothiazolo, imidazolo, thiadiazole, thiadiazole S-oxide, thiadiazole S,S-dioxide, pyrazolo, oxazole, isoxazole, pyridinyl, pyrimidinyl, quinoline, isoquinoline, CO2R 4 , COR 4 , CONR 4 R 5 , SO2CH3, or two adjacent R 8 groups can come together to form a fused 5- or 6-membered cycloalkyi ring, heterocycloalkyi ring, methylene di
  • p and t are each independently 0, 1 , 2, 3, 4, or 5, provided that p + t ⁇ 5;
  • q is 1 , 2, 3, or 4.
  • Embodiment 6 A compound of embodiment 5, wherein R 6 is H, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, CI, Br, CF 3 , OCF 3 , or -NHSO2R 7 , where R 7 is lower alkyl, cycloalkyi, heterocycloalkyi, aryl, or heteroaryl.
  • Embodiment 9 A compound of any one of embodiments 6-8, wherein r is 0 and W is 4- (OR 8 )-1 -phenyl and (OR 8 ) is trifluoromethoxy, butanyloxy, cyclopropylmethoxy, dimethylpropoxy, trifluoroethoxy, difluoromethoxy, oxanylmethoxy, oxanylmethoxy, or dimethylbutoxy.
  • Embodiment 10 A compound of any one of embodiments 6-9, wherein s is 1 , A is O or NR' where R' is H or lower alkyl, and R 3 is H, 3-propynyl, SO2CH3, CF 2 H, CF 3 , CONHCH3, or CH2CONR 4 R 5 ; where R 4 and R 5 come together to form a morpholino ring, an N-acetyl piperazinyl ring, an N-methanesulfonyl piperazinyl ring, or an N-methyl piperazinyl ring; or s is 0 and R 3 is SO2CH3, COR 4 , CONR 4 R 5 , N-imidazolinyl, or N-maleimido.
  • Embodiment 12 A compound having a structure
  • R 1 is H, lower alkyl, or OR a ;
  • R 10 and R 11 are each independently H, lower alkyl, an alkoxy group, or an alkylamino group; optionally R 10 and R 11 can be fused to form one or more substituted cyclic groups or one or more unsubstituted cyclic groups;
  • n 1 , 2, 3, or 4;
  • p can be 0, 1 , 2, 3, 4, or 5.
  • R 1 and R 2 are each independently H, R 6 is Br, CF 3 , OCF 3 , or C(CH 3 ) 3 , m is 2 or 3, and R 10 and R 11 are fused to form a substituted or unsubstituted six-membered heterocyclic ring structure including at least one nitrogen atom and at least one oxygen atom.
  • Embodiment 17 A compound of embodiment 12, wherein R 1 and R 2 are each independently H, R 6 is Br, CF 3 , OCF 3 , or C(CH 3 ) 3 , m is 2 or 3, and R 10 and R 11 are fused to form a two ring structure with a first ring including four members having at least one nitrogen atom and a second ring including four members having at least one oxygen atom.
  • Embodiment 18 The compound of any one of embodiments 12-17 having a structure:
  • n 0, 1 , 2, or 3;
  • R 6 is H, Br, CI, F, phenyl, CF 3 , lower alkyi, heteroaryl, cycloalkyi, OW a , C(CH 3 ) 3 , OCH 2 W a , or OCH 2 W b , NHS0 2 W b or NW c S0 2 W c ;
  • W c is lower alkyi
  • R c is H or lower alkyi
  • R h is alkynyl
  • Embodiment 30 The compound of embodiment 26, wherein Z 1 and X 2 are N, R 1 is H, R 3 is CH 3 , and R 6 is OCH 2 CF 3 .
  • Embodiment 31 The compound of any one of embodiments 26-30 having a structure:
  • Embodiment 38 A compound of embodiment 19 for use in modulating an innate immune response in a eukaryotic cell, the use comprising administering the compound to the eukaryotic cell.
  • Embodiment 41 A compound for use according to embodiment 40 wherein the compound has a structure as shown in embodiment 31.
  • Embodiment 43 A method of embodiment 42 wherein the viral infection is caused by a virus from one or more of the following families: Arenaviridae, Arterivirus, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Closteroviridae, Comoviridae, Coronaviridae, Cystoviridae, Flaviviridae, Flexiviridae, Hepadnaviridae, Hepevirus, Herpesviridae, Leviviridae, Luteoviridae, Mesoniviridae, Mononegavirales, Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Picobirnaviridae, Picobirnavirus, Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Ronivirida
  • Embodiment 44 A method of embodiment 42, wherein the viral infection is caused by one or more of influenza virus, Alfuy virus, Banzi virus, bovine diarrhea virus, Chikungunya virus, Dengue virus (DNV), hepatitis B virus (HBV), hepatitis C virus (HCV), human cytomegalovirus (hCMV), human immunodeficiency virus (HIV), llheus virus, influenza virus (including avian and swine isolates), Japanese encephalitis virus, Kokobera virus, Kunjin virus, Kyasanur forest disease virus, louping-ill virus, measles virus, MERS-coronavirus (MERS), metapneumovirus, any of the Mosaic Viruses, Murray Valley virus, parainfluenza virus, poliovirus, Powassan virus, respiratory syncytial virus (RSV), Rocio virus, SARS-coronavirus (SARS), St. Louis encephalitis virus, tick-borne ence
  • Embodiment 45 A method of embodiment 42, wherein the pharmaceutical composition is administered as an adjuvant for a prophylactic or therapeutic vaccine.
  • Embodiment 47 A method of modulating the innate immune response in a eukaryotic cell, comprising administering to the cell a compound having a structure
  • Embodiment 49 A method of embodiment 47, wherein the cell is in vitro.
  • Embodiment 50 A method of treating a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition thereby treating the viral infection caused by Ebola virus in the subject, wherein said pharmaceutical composition comprises at least one of the compounds having a structure .
  • Chromenones may be prepared by a wide variety of methods reviewed in publications including T.A. Geissman The Chemistry of Flavonoid Compounds, MacMillan, New York, 1962; P.M. Dewick Isoflavonoids. In The Flavonoids: Advances in Research, J.B. Harborne and T. J. Mabry, Eds. Chapman & Hall, New York, 1982; E. Wong The Isoflavonoids. In The Flavonoids, J.B. Harborne, T.J. Mabry, and Helga Mabry, Eds., Academic Press, New York San Francisco, 1975; Paul M. Dewick Isoflavonoids. In The Flavonoids: Advances in research since 1986, J.B.
  • Step 1 Synthesis of the intermediate 1 -(2,4-dihydroxyphenyl)-2-[4- (trifluoromethoxy)phenyl]ethanone.
  • [4-(Trifluoromethoxy)phenyl]acetic acid (5.0 g)and resorcinol (2.5 g) were dissolved in 70 mL BF3 etherate and the mixture was heated at 80 °C for 4 hours. The mixture was cooled and poured into 300 mL water and stirred for 3 hours. The organic layer was separated, diluted with 100 mL ether and stirred with an aqueous solution of 10 g sodium carbonate in 200 ml water for 3 hours. The organic layer was separated and the solvent was removed under reduced pressure to yield 6.0 g of oil which was purified by silica gel chromatography to yield 3.1 g (44%) of the product.
  • Step 2 Synthesis of the intermediate 7-hydroxy-3-[4-(trifluoromethoxy)phenyl]-4H- chromen-4-one.
  • the ketone (2.1 g) from step 1 was dissolved in 20 mL DMF and 19 mL of BF3 etherate was added with cooling followed by addition of 5 mL methanesulfonyl chloride. The mixture was heated at 90 °C for 2 h then poured into 300 mL water and stirred vigorously. The resulting solid was filtered off, washed with water and dried to afford crude product which was recrystallized from hot ethanol to yield 1.6 g (74%) of the phenolic chromenone.
  • Step3 Synthesis of 7-[2-(4-acetylpiperazin-1-yl)ethoxy]-3-[4-(trifluoromethoxy)phenyl]- 4H-chromen-4-one.
  • the phenol from step 2 (1 .7 g) and 1-[4-(2-hydroxyethyl)piperazin-1 - yl]ethanone (1.1 g) were dissolved in 20 mL THF and cooled to 0 °C.
  • DIAD diisopropyl azodicarboxylate
  • the library hit compounds 1 and 2 were tested for antiviral activity in vitro.
  • Huh7 cells were seeded in 96-well plates at a density of 2-5x103 cells/well. Cells were grown for 16 hours and compounds that were diluted to 5, 10, 20, or 50uM in media containing 0.5% dimethyl sulfoxide (DMSO) were added to each well. Cells were incubated for 18 - 24 hours and then infected with 750 pfu HCV2a strain. Diluted virus was added directly to the well and compound was not removed. Infected cells were grown for 24 - 72 hours post compound treatment and then fixed. Cells were fixed with 4% paraformaldehyde and stained for HCV protein.
  • DMSO dimethyl sulfoxide
  • FIGs. 1 A, 1 B, and 1 C show the antiviral activity of the compounds 1 and 2 against HCV.
  • FIG. 1A shows an HCV focus-forming assay done in Huh7 cells pre-treated with compound 1 for 24 hours and infected with HCV2a at a multiplicity of infection (MOI) of 0.5 for 48 hours. HCV proteins were detected by immunofluorescent staining with viral-specific serum and foci were normalized to negative control cells that were not drug treated (equal to 1 ).
  • MOI multiplicity of infection
  • FIG. 1 B shows quantitation of HCV viral RNA by real-time quantitative PCR (RT-qPCR) done in Huh7 cells untreated (Untreated), treated with interferon (I FN), or pre-treated with compound 2 (CPD 2) for 18 hours and infected with HCV2a at MOI of 1.0 for 72 hours. Viral RNA was isolated and quantitated in the supernatant of infected cultures.
  • FIG. 1 C shows a similar quantitation of HCV viral RNA by RT-qPCR done in Huh7 cells infected with HCV2a at MOI of 1.0 for 4 hours and then treated with compound 2.
  • Huh7 cells were grown under normal growth conditions and treated with the indicated amount of compound 2 in media containing 0.5% DMSO. The cells were grown in the presence of compound for 5 hours and then infected with 250 pfu Murine EMCV obtained from ATCC #VR-129B. Infected cells were grown for an additional 18 hours and then cell viability was measured using an MTS assay. Negative control cells were treated with buffer alone containing 0.5% DMSO. Interferon treatment was used as a positive control for virus inhibition and was added similar to compound treatments at a final concentration of 10 lU/mL Interferon-a: Intron A, from Schering-Plough.
  • EMCV encephalomyocarditis virus
  • MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay
  • Antiviral activity of compound 2 against RSV was measured by immunofluorescent based focus-forming assay.
  • Cultured human HeLa cells were seeded in 6-well tissue-culture plates at a density of 4x10 5 cells per well and grown for 24 hours. Cells were infected with RSV A2 Long strain (ATCC VR-26) at a MOI of 0.1 for 2 hours and then removed.
  • Compound dilutions were prepared in 0.5% DMSO and used to treat cells at final concentrations of compound ranging from 0.001 to 10 ⁇ per well. Vehicle control wells contained 0.5% DMSO and were used to compare to compound-treated cells. RSV infections after compound treatment were allowed to proceed for 48 hours.
  • Virus supernatants were then harvested and used to infect new monolayers of HeLa cells seeded in 96-well tissue-culture plates at a density of 8x10 3 cells per well. The newly infected cells were incubated overnight (18 - 24 hours) and used to measure the level of infectious virus in the original supernatants by immunofluorescent staining of viral protein. The cells were fixed with ice-cold 1 :1 methanol and acetone solution and stained for RSV F protein. Primary mouse anti-RSV monoclonal antibody (EMD Millipore) was used at a 1 :2000 dilution.
  • EMD Millipore Primary mouse anti-RSV monoclonal antibody
  • FIG. 2A shows cell viability following infection with RSV A2 and treatment with compound 2.
  • FIG. 2B shows compound 2 treatment decreased RSV viral RNA 48 hours post infection.
  • Antiviral activity against influenza virus in vitro was measured for selected compounds.
  • Cultured human 293 cells were seeded in 6-well tissue-culture plates at a density of 3x10 5 cells per well for the flu focus-forming assay and grown for 24 hours.
  • Cells were infected with influenza virus A/Udorn/72 H3N2 strain at a MOI of 0.1 for 2 hours and then removed.
  • Compound dilutions were prepared in 0.5% DMSO and used to treat cells at final concentrations of compound ranging from 0.001 to 10 ⁇ per well.
  • Vehicle control wells contained 0.5% DMSO and were used to compare to compound-treated cells. Replication was then allowed to proceed for 24 hours.
  • Virus supernatants were then harvested and used to infect new monolayers of permissive MDCK cells that were seeded 24 hours prior in 96-well tissue-culture plates at a density of 1 .5x10 4 cells per well.
  • the newly infected cells were incubated overnight (18 - 24 hours) and used to measure the level of infectious virus in the original supernatants by immunofluorescent staining of viral protein.
  • the cells were fixed with ice-cold 1 :1 methanol and acetone solution and stained for influenza nucleoprotein (NP).
  • Primary mouse anti-NP monoclonal antibody (Chemicon) was used at a 1 :3000 dilution.
  • FIGs. 3A, 3B, and 3C show results from the influenza focus-forming assay. Decrease in foci is graphed as percent inhibition of viral infection by compound.
  • FIG. 3A shows that compound 2 showed dose-dependent decrease in viral infection of 293 cells; compounds 3, 5, 6, 7, 8, 1 1 , 16, and 14 improved on this antiviral activity as shown by decreased viral titer.
  • FIG. 3B shows that compounds 19, 21 , 22, 23, 26, 28, 7, 29, 30, and 12 showed dose-dependent decrease in viral infection of 293 cells.
  • FIG. 3C shows determined I C50 values of exemplary compounds in the influenza antiviral assay.
  • Antiviral activity against DNV in vitro was measured for selected compounds.
  • Cultured human Huh7 cells were seeded in 6-well tissue-culture plates at a density of 4x10 5 cells per well for the DNV focus-forming assay and grown for 24 hours. Cells were infected with DNV type 2 strain at a MOI of 0.1 for 2 hours and then removed. Compound dilutions were prepared in 0.5% DMSO and used to treat cells at final concentrations of compound ranging from 0.001 to 10 ⁇ per well. Vehicle control wells contained 0.5% DMSO and were used to compare to compound- treated cells. Replication was then allowed to proceed for 48 hours.
  • FIG. 4A shows a dose-dependent decrease in viral protein in cells infected with DNV and treated with increasing amounts of compound 2.
  • the results of the DNV focus-forming assay for antiviral activity are shown in FIG. 4B.
  • the decrease in foci is graphed as percent inhibition of viral infection by compound.
  • the compounds 2 black dashed line
  • 3, 7, 19, 22, 23, and 26 showed dose-dependent decreases in viral infection of Huh7 cells.
  • I C50 values (in M) are shown.
  • HFF human foreskin fibroblasts
  • ATCC hCMV AD169 strain
  • Compound dilutions were prepared in 0.5% DMSO and used to treat cells at final concentrations of compound ranging from 0.001 to 10 ⁇ per well.
  • Vehicle control wells contained 0.5% DMSO and were used to compare to compound-treated cells. Replication was then allowed to proceed for 48 - 96 hours.
  • Virus supernatants were harvested at 48, 72, and 96 hours and used to infect new monolayers of HFFs that have been seeded 24 hours prior in 96-well tissue-culture plates at a density of 3x10 4 cells per well.
  • the newly infected cells were incubated for 24 hours and used to measure the level of infectious virus in the original supernatants by immunofluorescent staining of viral protein.
  • the cells were fixed with ice-cold 1 :1 methanol and acetone solution and stained for hCMV IE1 protein similarly to previously described methods for the other in vitro virus systems.
  • FIGs. 5A and 5B show the antiviral activity of selected compounds against human cytomegalovirus (hCMV).
  • FIG. 5A shows dose-dependent decrease in hCMV as measured by foci (FFU/mL) in samples treated with compounds 26, 28, 29, and 30.
  • FIG. 5B shows dose-dependent decrease in hCMV as measured by foci (FFU/mL) in samples treated with compounds 7, 3, 22, 19, and 23.
  • RIG-I signaling pathway activation by compound 7 was measured by assaying activation of IRF-3 dependent signaling. This was done by measuring IRF-3 dependent gene expression by RT-qPCR in cells treated with compound. Cultured human cells were treated with 0.001 - 10 ⁇ of compound 7 or DMSO vehicle control and incubated for up to 24 hours. Cells are harvested at time points from 4 - 24 hours after treatment. RNA isolation, reverse transcription, and qPCR were performed using well known techniques. 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).
  • AACt relative expression analysis
  • FIG. 6 shows interferon regulatory factor-3 (IRF-3) responsive gene expression induced by compound 7 in 293 cells.
  • DMSO treatment D was used as negative control.
  • Influenza infection F was used as a positive control for induction of gene expression.
  • Antiviral activity of compound 7 was measured using a mouse influenza model. Virus infection was achieved with non-surgical instillation of influenza virus strains A/Puerto Rico/8/1934 (PR8). Compound 7 was administered daily by intranasal administration of 10 mg/kg in 10% hydroxypropyl-3-cyclodextrin (HPBCD) or vehicle-only control over the entire course of infection. Animals were evaluated for study endpoints including daily clinical observations, mortality, body weight, and body temperature. Virus titer was measured in lung tissue.
  • HPBCD hydroxypropyl-3-cyclodextrin
  • Antiviral activity of compound 7 was measured using a mouse DNV model. Virus infection is achieved using intraperitoneal injection of DNV type 2 strain. Compound 7 was administered daily by IP injection of 10 mg/kg or vehicle-only control over the entire course of infection. Animals were evaluated for study endpoints including daily clinical observations, mortality, body weight, and body temperature. Virus RNA was measured in serum.
  • FIGs. 7A, 7B, 7C, 7D, and 7E show in vivo broad spectrum antiviral activity and bioavailability of compound 7.
  • Compound 7 (10mg/kg in 10% HPBCD; 7) intranasal treatment reduces replication and titer of influenza (shown in FIG. 7A) and mouse hepatitis virus (MHV) (FIG. 7B) in the lung compared to vehicle treatment (V).
  • FIG. 7C shows level of compound 7 in plasma over time when dosed at 10mg/kg via intraperitoneal injection or intravenous injection.
  • FIG. 7D shows that compound 7 inhibited DNV as measured in serum compared to vehicle treatment (V) when dosed IP 10 mg/kg/day.
  • 7E shows that compound 7 (20mg/kg) inhibited flu replication in the lung compared to vehicle treatment (V) when administered by intranasal instillation either -24 hours prior (prophylactic; pre) or +24 hours post (therapeutic; post) lethal infection with PR8 flu.
  • Lung tissue was harvested 72 hours after infection and flu RNA was quantitated by PCR.
  • Example 1 Antiviral activity and pharmacological properties using Quantitative Structure-Activity Relationship (QSAR) studies
  • This Example describes analog compound design using QSAR approach of the compounds described herein for antiviral action.
  • the QSAR studies are designed to provide lead compounds with picomolar to nanomolar potency. Optimization of the compounds focuses on creating structural diversity and evaluating core variants and group modifications. Analogs are tested for antiviral activity against several viruses including the virus assay models described herein. Furthermore, analogs are tested for cytotoxicity in one or more cell lines or peripheral blood mononuclear cells. Optimized compounds that show improved efficacy and low cytotoxicity are further characterized by additional measures of in vitro and in vivo toxicology and absorption, distribution, metabolism, and elimination (ADME). Their mechanism of action and breadth of antiviral activity are also studied.
  • ADME in vitro and in vivo toxicology and absorption, distribution, metabolism, and elimination
  • Embodiments of compounds disclosed herein can be described as chromenone compounds.
  • Phenyl chromenones known as isoflavones are best known as natural products isolated from the Leguminosae (legume) family and are usually polyhydroxylated and pharmacologically active as phytoestrogenics and antioxidants.
  • the most recognizable member of this class is genistein, which has been reported to have anticancer activities and to induce thymic and immune changes in mammals. It is relevant that a preliminary screen of a Natural Cancer Institute (NCI) natural product library revealed genistein as a validated hit for interferon- stimulated gene (ISG) induction. This correlation demonstrates the potential for broad flexibility in functional group modifications and analog design while retaining biological activity.
  • NCI Natural Cancer Institute
  • a (high-performance liquid chromatography) HPLC- and/or HPLC- mass spectrometry-based analytical method is used to evaluate compound and metabolite concentrations in various test systems.
  • 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 compounds.
  • compound stability over time in increasing concentrations of serum, plasma, and whole blood from mammalian species (such as mouse, cynomolgus macaque, and human) will be evaluated by HPLC, and a half-life will be determined.
  • prominent metabolites are characterized by mass spectrometry.
  • RIG-I signaling pathway activation by compounds One example of an assay to measure RIG-I pathway activation is the measurement of downstream gene expression by RT- qPCR in cells treated with compound.
  • the transcription factor IRF-3 is activated through RIG-I signaling and the increased expression of IRF-3 dependent genes indicate activation of the RIG-I pathway.
  • Other genes that are associated with the host innate immune antiviral response are also measured as indicators of compound activity.
  • Gene expression can be similarly assayed in cell types that include: primary blood mononuclear cells, human macrophages, THP-1 cells, Huh7 cells, A549 cells, MRC5 cells, rat splenocytes, rat thymocytes, mouse macrophages, mouse splenocytes, and mouse thymocytes. Expression of other genes of interest can be assayed as described herein. In addition, gene expression can be assayed in the presence of virus in order to determine compound activity in the context of active viral infection.
  • Innate immune response induction by compounds can be assayed in primary immune cells to determine whether compound treatment stimulates immune response pathways.
  • One example is to assay cytokine expression in cultured human primary blood cells such as dendritic cells. Cells are seeded in tissue culture dishes and treated with compound ranging from 0.001 - 10 ⁇ of compound.
  • supernatants from treated wells are isolated 24 - 48 hours after compound treatment and tested for levels of cytokine protein.
  • Cytokines are detected using specific antibodies conjugated to magnetic beads and a secondary antibody that reacts with Streptavidin/Phycoerythrin to produce a fluorescent signal.
  • the bound beads are detected and quantified using the MAGPIX® (Luminex Corp.) instrument, although similar techniques as are known in the art may be used to measure fluorescent protein production, such as for example an ELISA.
  • Other cells from which cytokine secretion can be measured include, for example human peripheral blood mononuclear cells, human macrophages, mouse macrophages, mouse splenocytes, rat thymocytes, and rat splenocytes.
  • Cytotoxicity is evaluated using standard in vitro assays including MTS assay and caspase assay. Protocols to perform these assays are known to those skilled in the art and there are several commercially available kits to measure assay readout, such as a colorimetric based assay to measure conversion of MTS to formazan (Cell Titer One, Promega) and a sandwich ELISA based assay to measure levels of activated caspase-3 (PATHSCAN® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190, Cell Signaling Technology, Inc., Danvers, MA).
  • Cultured human cells are treated with increasing amounts of compound from 0 up to at least 50 ⁇ or equivalent amounts of DMSO diluted in media to evaluate their effect on cell viability.
  • Cultured human cell lines that are used in this assay include Huh7, PH5CH8, A549, or HeLa cells.
  • In vitro pharmacology and toxicology This description of toxicological assays is exemplary. In vitro studies are performed to measure performance of the most promising analogs in one or more assays of intestinal permeability, metabolic stability, and toxicity. These studies can include plasma protein binding; serum, plasma, and whole-blood stability in human and model organisms; intestinal permeability; intrinsic clearance; human Ether-a-go-go (hERG) channel inhibition to test potential cardiac toxicity; and genotoxicity using for example a reversion mutation assay (Ames test) and/or a micronucleus formation assay. Human plasma protein binding will be evaluated by partition analysis using equilibrium dialysis.
  • apical-to-basolateral flux is assessed in a human epithelial cell line such as Caco-2 or 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. Specific metabolites may be isolated and characterized.
  • the compounds disclosed herein have efficient activity against several viruses in vitro. To further characterize the breadth of antiviral activity of optimized compounds, cell culture infection models are used to analyze different viruses as well as different strains of the same virus (Table 4). Assays to measure the antiviral activity of compounds against several of these viruses is described herein.
  • the studies include treating cells with compound 2-24 hours prior to infection and/or treating cells 2-8 hours after infection.
  • Compound is administered at different concentrations ranging from 0.001-1 ⁇ .
  • Positive control treatments used include interferon, ribavirin, oseltamivir, or other known treatment to inhibit the infection of the specific virus.
  • Virus production and cellular ISG expression are assessed over a time course to analyze antiviral activity of each compound (Table 4). Virus production is measured by focus-forming or plaque assay.
  • Antiviral activity against influenza virus in vitro is measured by immunofluorescent based focus-forming assay.
  • Influenza A virus strains that are used in this assay include A/Udorn/72 H3N2 strain and A California/04/09 H1 N1 strain. Experimental conditions are as or substantially similar to those described in Example 6.
  • Antiviral activity against hCMV in vitro is measured by immunofluorescent based focus- forming assay. Experimental conditions are as or substantially similar to those described in Example 7.
  • 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 determine 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.
  • Example 14 In vivo pharmacokinetic and toxicological profiles of optimized compounds in preclinical animal models
  • PK pharmacokinetic
  • tolerability profiling The in vivo PK profile and tolerability/toxicity of optimized compounds are evaluated in order to conduct further characterization of their antiviral activity in animal models of virus infection.
  • Mouse and rat are the chosen test species for these studies because there are several established virus models in the mouse and models of PK, toxicology, and immunology in the rat.
  • Reverse-phase, HPLC-MS/MS detection methods are used to detect and quantify the concentration of each compound in biological samples including plasma and target tissue samples.
  • PK profiling an initial oral and injectable pharmaceutical composition for each compound is developed using a limited pharmaceutical composition component screen that is largely focused on maximizing aqueous solubility and stability over a small number of storage conditions.
  • Existing analytical methods known in the art are used to measure pharmaceutical composition performance.
  • a pharmaceutical composition 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 ethanol ( ⁇ 10%), propylene glycol ( ⁇ 40%), or polyethylene glycol (PEG) 300 or 400 ( ⁇ 60%) co-solvents to enhance solubility
  • Tier 3 addition of N-N-dimethylacetamide (DMA, ⁇ 30%), N-methyl-2-pyrrolidone (NMP, ⁇ 20%), and/or dimethyl sulfoxide (DMSO, ⁇ 20%) co- solvents or the cyclodextrins ( ⁇ 40%) as needed to further improve solubility.
  • DMA N-N-dimethylacetamide
  • NMP N-methyl-2-pyrrolidone
  • DMSO dimethyl sulfoxide
  • Tolerability studies are performed in two stages: an initial dose escalation stage that includes ascending doses up to 5 doses, each separated by a 5-day washout period, to determine the maximum tolerable dose (MTD; Stage 1 ); this is followed by seven daily administrations of the MTD to evaluate acute toxicity (Stage 2).
  • MTD maximum tolerable dose
  • Stage 2 seven daily administrations of the MTD to evaluate acute toxicity
  • Stage 2 seven daily administrations of the MTD to evaluate acute toxicity
  • all doses are administered by oral gavage.
  • five animals of each sex are placed on-study in stage 1 and 15 animals per sex per dosing group in Stage 2.
  • Study endpoints include a determination of the MTD, examination for acute toxicity, physical examination, clinical observations, hematology, serum chemistry, and animal bodyweights. Gross pathology is performed on all animals 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 selection of lead compounds for antiviral animal models.
  • Example 15 In vivo antiviral properties of selected compounds in preclinical animal models.
  • influenza virus strains in these experiments include at least two different subtypes (for example, H1 N1 and H3N2) and exhibit varying pathogenic properties and clinical presentations 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 up to 24 hours before or up to 24 hours after infection and continuing daily subject to the determined plasma half-life of the compound.
  • challenge doses such as, 10 to 1 ,000 pfu of virus
  • Compound dose- response analysis and infection time course studies are conducted to evaluate compound efficacy to: 1 ) limit serum viral load; 2) limit virus replication and spread in target organs; and 3) protect against viral pathogenesis.
  • WNV in addition to serum, viral burden is assessed in lymph nodes, spleen, and brain; for influenza virus, viral burden is assessed in heart, lung, kidney, liver, and brain.
  • ED50 and ED90 serum viral load
  • Serum 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 cerebral 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 17 Broad anti-viral activity of selected compounds.
  • FIG. 8 shows the in vitro antiviral activity of compounds 30, 31 , 32, 34, and 37 against RSV.
  • FIGs. 9A-C show the in vitro antiviral activity of selected compounds of the disclosure.
  • FIG. 9A shows anti-influenza activity of compounds 35 and 37.
  • FIG. 9B shows antiviral activity against H3N2 influenza virus. The compounds showed a >3 log reduction in FLU (H3N2) viral titer, and compounds showed 850-fold improvement in potency over the library hit compound against influenza.
  • FIG. 9C shows broad spectrum in vitro influenza efficacy of compounds 32, 37 and 43.
  • FIGs. 1 1A-C show the broad in vivo antiviral efficacy of compound 7 compared with control (HPBCD) in mice.
  • FIG. 1 1A shows influenza lung titer
  • FIG. 1 1 B shows MHV lung titer
  • FIG. 1 1 C shows DENV plasma titer.
  • FIG. 12 shows that a single prophylactic intranasal treatment with compound 7 protected against lethal (10X LD50) H1 N1 influenza challenge in mice.
  • FIG. 13 shows the in vitro activity of selected compounds of the disclosure against EBOV at 5 ⁇ .
  • the lead compounds 32, 42, 44, and 45 showed greater than a 2 log reduction in EBOV titer in vitro.
  • FIG. 15 shows the potent broad spectrum antiviral activity of selected compounds of the disclosure against influenza (specifically FLU A (strain A/Udorn/72); FLU B (strain B/Mass/2/2012)); RSV (strain A2); HCoV (strain OC43); DENV-2 (strain NGC); DENV-4 (strain H241 ); EBOV (strain Zaire); NiV (strain Malaysia); and LASV (strain Josiah); and WNV (strain Texas02).
  • the left columns of the table show EC50 values of the compounds calculated in various cell types; the right three columns show the log decrease in viral titer of EBPV, NiV, and LASV in HUVEC cells.
  • FIG. 17A shows the in vitro inhibition of influenza replication by compounds 2, 3, 7, 26, and 38.
  • FIG. 17B shows the basic structure of chemicals studied by functional antiviral activity. SAR studies were directed by functional antiviral activity. The Markush structure represents the sampled chemical space. Each analog was screened for antiviral activity against a panel of RNA viruses to direct the SAR study.
  • the data in FIG. 17A shows the increased potency of compounds generated as listed, resulting in over 3,000-fold increase in potency in Influenza relative to compound 2.
  • FIG. 18A shows the influenza lung titer in mice treated once daily intranasally (IN) for three days with HPBCD, compound 37 (pre) and compound 37 (+24).
  • FIG. 18B shows the coronavirus lung titer in mice treated once daily IN for three days with HPBCD, and compound 37.
  • FIG. 18C shows the Dengue plasma titer in mice treated once daily intraperitoneally (IP) for three days with HPBCD and compound 7. The results show a reduction of FLU-H1 N1 and MHV- coronavirus in the lung at day 3, and inhibition of DENV-2 in the blood at day 3.
  • FIG. 19 shows that delayed treatment up to 48 hours post-infection provided protection from a lethal influenze challenge in mice.
  • the four treatment groups were HPBCD-pre; compound 37-pre; HPBCD +48, and compound 37 +48.
  • a material effect would cause a statistically significant reduction in a disclosed compound's or pharmaceutical composition's ability to treat a viral infection in a subject; reduce viral protein in a subject or assay; reduce viral RNA in a subject or assay or reduce virus in a cell culture.

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Abstract

La présente invention concerne des composés et des compositions pharmaceutiques, et les utilisations de ces derniers pour le traitement d'infections virales, y compris d'infections à virus à ARN. Les composés, compositions pharmaceutiques et méthodes de l'invention peuvent concerner des composés médicamenteux potentiels de type chroménone qui modulent l'immunité innée à travers l'axe RLR-IRF3. Ces composés activent la signalisation immunitaire innée en aval de nombreuses contre-mesures virales et constituent un complément unique aux composés antiviraux classiques en développement ou sur le marché. Ces composés présentent une activité in vitro à large spectre contre divers virus à ARN, y compris les agents pathogènes respiratoires de la grippe, le virus respiratoire syncytial (VRS) et le coronavirus humain (hCoV), avec des valeurs de CE50 dans la tranche nanomolaire basse. Une puissante activité in vitro est également démontrée contre des virus systémiques et des virus émergents, dont les virus de la dengue et Ebola.
PCT/US2015/059611 2014-11-07 2015-11-06 Composés anti-viraux, compositions pharmaceutiques et méthodes d'utilisation de ces derniers WO2016073947A1 (fr)

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CN109810063A (zh) * 2017-11-21 2019-05-28 中国科学院上海药物研究所 一种新型抗流感病毒“孪药”、其制备方法及用途
CN110950828A (zh) * 2019-11-05 2020-04-03 中国人民解放军第二军医大学 一种黄芩素或其衍生物、制备方法与应用
WO2020085952A1 (fr) * 2018-10-23 2020-04-30 Общество С Ограниченной Ответственностью "Валента-Интеллект" Forme pharmaceutique pour le traiement de la grippe et de maladies respiratoires aiguës
CN112694463A (zh) * 2020-12-16 2021-04-23 中国医学科学院医药生物技术研究所 一种异戊烯基色原酮类化合物在制备抗冠状病毒药物中的用途
WO2021207176A1 (fr) * 2020-04-06 2021-10-14 Humanetics Corporation Traitement par génistéine d'une lésion pulmonaire inflammatoire
WO2023280746A1 (fr) * 2021-07-06 2023-01-12 Centre National De La Recherche Scientifique Composé du type 7a,8,9,10,11,11a-hexahydro-1h,7h-pyrano[2,3-c]xanthéne, son procédé de préparation, ses intermédiaires et ses applications thérapeutiques
WO2023143397A1 (fr) * 2022-01-28 2023-08-03 中国科学院上海药物研究所 Composé aryl-pyrone substitué par (alkyl)(amino)alcoxy cyclique et son utilisation
WO2024006287A3 (fr) * 2022-06-27 2024-02-15 University Of Kansas Composés antiviraux utiles contre le sars-cov-2

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109810063A (zh) * 2017-11-21 2019-05-28 中国科学院上海药物研究所 一种新型抗流感病毒“孪药”、其制备方法及用途
WO2020085952A1 (fr) * 2018-10-23 2020-04-30 Общество С Ограниченной Ответственностью "Валента-Интеллект" Forme pharmaceutique pour le traiement de la grippe et de maladies respiratoires aiguës
CN110950828A (zh) * 2019-11-05 2020-04-03 中国人民解放军第二军医大学 一种黄芩素或其衍生物、制备方法与应用
CN110950828B (zh) * 2019-11-05 2021-08-17 中国人民解放军第二军医大学 一种黄芩素或其衍生物、制备方法与应用
WO2021207176A1 (fr) * 2020-04-06 2021-10-14 Humanetics Corporation Traitement par génistéine d'une lésion pulmonaire inflammatoire
CN112694463A (zh) * 2020-12-16 2021-04-23 中国医学科学院医药生物技术研究所 一种异戊烯基色原酮类化合物在制备抗冠状病毒药物中的用途
CN112694463B (zh) * 2020-12-16 2022-06-07 中国医学科学院医药生物技术研究所 一种异戊烯基色原酮类化合物在制备抗冠状病毒药物中的用途
WO2023280746A1 (fr) * 2021-07-06 2023-01-12 Centre National De La Recherche Scientifique Composé du type 7a,8,9,10,11,11a-hexahydro-1h,7h-pyrano[2,3-c]xanthéne, son procédé de préparation, ses intermédiaires et ses applications thérapeutiques
FR3125047A1 (fr) * 2021-07-06 2023-01-13 Centre National De La Recherche Scientifique Composé de type 7a,8,9,10,11,11a-hexahydro-1H,7H-pyrano[2,3-c]xanthene, leur procédé de préparation, leurs intermédiaires et leurs applications thérapeutiques.
WO2023143397A1 (fr) * 2022-01-28 2023-08-03 中国科学院上海药物研究所 Composé aryl-pyrone substitué par (alkyl)(amino)alcoxy cyclique et son utilisation
WO2024006287A3 (fr) * 2022-06-27 2024-02-15 University Of Kansas Composés antiviraux utiles contre le sars-cov-2

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