WO2019233474A1 - Antiviral compounds and methods of use thereof - Google Patents

Antiviral compounds and methods of use thereof Download PDF

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Publication number
WO2019233474A1
WO2019233474A1 PCT/CN2019/090301 CN2019090301W WO2019233474A1 WO 2019233474 A1 WO2019233474 A1 WO 2019233474A1 CN 2019090301 W CN2019090301 W CN 2019090301W WO 2019233474 A1 WO2019233474 A1 WO 2019233474A1
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substituted
viral
forms
pharmaceutically acceptable
compound
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PCT/CN2019/090301
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French (fr)
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Yi Tsun Richard Kao
Fang Yang
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The University Of Hong Kong
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    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • C07C233/66—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by halogen atoms or by nitro or nitroso groups
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    • C07C233/67—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/75—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
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    • C07C233/80—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
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    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
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    • C07C235/24—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
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    • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/64—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen 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
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    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles 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 in position 2
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    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
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    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
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    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans 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 carbon atoms of the hetero ring
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    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring 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 atoms of the carbocyclic ring
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Definitions

  • the disclosed invention is generally in the field of a class of compounds that inhibit viral infection, more specifically inhibitors of viral nucleoprotein, methods of making, and using thereof.
  • Influenza A virus is the major causative agent for mortality and morbidity in pandemic and epidemic flu. It remains as the major threat to public health and global economy.
  • M2 Amantadine, Rimantadine
  • NA Optamivir, Zanamivir
  • IAV has a segmented genome consisting of eight RNA molecules that are individually encapsidated into vRNAP (RNA polymerase) . This represents the minimum sets of proteins for functional viral transcription and replications. It is an attractive strategy to develop antivirals that target nucleoprotein (NP) , as NP is the major structural protein in IAV with multiple functions throughout the virus infection life cycle. NP was previously reported to interact with RNA and oligomerize by self-interaction to form an oligomer to provide the structural role in RNP formation. Furthermore, NP is also required for viral transcription and replication.
  • NP target nucleoprotein
  • NP The major role of NP is to encapsidate RNA molecules into RNP complex and maintain the conformation of the RNA template in order to facilitate the viral transcription, replication, and assembly into the newly formed virions.
  • NP or RNP is also known to translocate between the cytoplasm and nucleus with the help of other viral proteins and host factors.
  • compositions, and methods of use thereof treat, inhibit, reduce the risk of, or prevent viral infections.
  • the present disclosure relates to compounds with antiviral activity, methods for the preparation of such compounds, and their use to inhibit viral nucleoprotein activity, viral transcription, viral replication, and assembly into the newly formed virions.
  • A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring
  • R 2 -R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 10 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 9 ; -CONHR 9 ) , tertiary amide (e.g., -NR 9 COR 9 ; -CONR 9 R 9 ) , secondary carbamate (e.g., -OCONHR 9 ; -NH
  • R 4 of Formula I is the same as R 1 .
  • the compounds Formula II can have the following structures.
  • R 12 -R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 20 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 19 ; -CONHR 19 ) , tertiary amide (e.g., -NR 19 COR 19 ; -CONR 19 R 19 ) , secondary carbamate (e.g., -OCONHR 19 ; -NH
  • R 4 of Formula I can be a secondary amide (-NHCOR 9 )
  • R 9 can be one of the following structures:
  • R 21 -R 29 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 32 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 31 ; -CONHR 3 ) , tertiary amide (e.g., -NR 31 COR 31 ; -CONR 31 R 31 ) , secondary carbamate (e.g., -OCONHR 31 ; -NH
  • the compounds disclosed herein can be in the form of a pharmaceutically acceptable salt or a prodrug thereof.
  • the one or more compounds can be in a pharmaceutical composition including and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical compositions can include one or more of the compounds, and can optionally include one or more pharmaceutically acceptable excipients.
  • the disclosed compounds are present in an effective amount to inhibit viral transcription.
  • the disclosed compounds are present in an effective amount to inhibit replication.
  • the disclosed compounds are present in an effective amount to inhibit viral protein synthesis.
  • the disclosed compounds are present in an effective amount to block vRNP export and/or suppress the virion budding process.
  • Also disclosed are methods of treating an infection including administering one or more of the disclosed compounds or compositions in an amount effective to treat a subject in need thereof.
  • An effective amount of the one or more compounds having the structure of Formula I or II may be an effective amount that is enough of the one or more compounds to treat, inhibit, or prevent a viral infection, such as by influenza A virus, through transcription, replication, protein synthesis, vRNP export, and/or virion budding process.
  • a viral infection such as by influenza A virus, through transcription, replication, protein synthesis, vRNP export, and/or virion budding process.
  • the method includes the one or more compounds are in an amount effective to inhibit an infection comprising: inhibiting viral transcription; inhibiting viral replication; inhibiting viral protein synthesis; blocking vRNP export; or inhibiting virion budding process; or inhibiting viral nucleoprotein activity, or a combination thereof.
  • the infection treated by the methods disclosed herein with the compounds disclosed is a viral infection involving a viral nucleoprotein.
  • the viral infection treat by the methods disclosed herein with the compounds disclosed is influenza A virus.
  • the methods of administering one or more of the compounds by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration are disclosed herein.
  • A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring
  • R 2 -R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 2 The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein R 2 -R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH, -COO - , -COR 10 , -NH 2 CO, -CONH 2 , -CONHR 9 , -CONR 9 R 9 , -NHCOR 9 , -NR 9 COR 9 , -OCONHR 9 ; -NHCOOR 9 , -OCONR 9 R 9 ; -NR 9 COOR 9 , -NHCONHR 9 ; -NR 9 CONHR 9 , -
  • each R 8 -R 10 of R 2 -R 7 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 3 The compound of solution 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is of Formula I and R 4 is the same as R 1 .
  • Solution 4 The compound of solution 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein Formula II is
  • each R 12 -R 17 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 5 The compound of solution 4 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 12 -R 17 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH; -COO - ; -COR 20 ; -NH 2 CO; -CONH 2 ; -CONHR 19 ; -CONR 19 R 19 ; -NHCOR 19 ; -NR 19 COR 19 ; -OCONHR 19 ; -NHCOOR 19 ; -OCONR 19 R 19 ; -NR 19 COOR 19 ; -NHCONHR 19 ; -NR 19 CONHR 19
  • each R 18 -R 20 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 6 The compound of solution 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein when R 4 is -NHCOR 9 , then R 9 is
  • each R 21 -R 29 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 7 The compound of solution 6 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 21 -R 29 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH; -COO - ; -COR 32 ; -NH 2 CO; -CONH 2 ; -CONHR 31 ; -CONR 31 R 31 ; -NHCOR 31 ; -NR 31 COR 31 ; -OCONHR 31 ; -NHCOOR 31 ; -OCONR 31 R 31 ; -NR 31 COOR 31 ; -NHCONHR 31 ; -NR 31 CONHR 31
  • each R 30 -R 32 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  • Solution 8 The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is a compound selected from the group consisting of:
  • Solution 9 The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound has the structure:
  • Solution 10 A pharmaceutical composition comprising one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof and one or more pharmaceutically acceptable carriers.
  • Solution 11 The pharmaceutical composition of solution 10, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof are present in an effective amount to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, or a combination thereof.
  • Solution 12 The pharmaceutical composition of solution 10 or 11 further comprising one or more pharmaceutically acceptable excipients.
  • Solution 13 A method of treating a subject in need thereof, the method comprising administering one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition of any of solutions 10-12 in an amount effective to treat the subject in need thereof.
  • Solution 14 The method of solution 13, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
  • Solution 15 The method of solution 13 or 14, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
  • Solution 16 The method of solution 15, wherein the viral infection is an influenza A viral infection.
  • Solution 17 The method of any one of solutions 13-16, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
  • Solution 18 The method of any one of solutions 13-17, wherein the subject has a viral infection.
  • Solution 19 The method of any one of solutions 13-17, wherein the subject is at risk of developing a viral infection.
  • Solution 20 The method of solution 18 or 19, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
  • Solution 21 Use of one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition of any of solutions 10-12 in manufacture of a medicament for treating a subject in need thereof.
  • Solution 22 Use of solution 21, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
  • Solution 23 Use of solution 21 or 22, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
  • Solution 24 Use of solution 23, wherein the viral infection is an influenza A viral infection.
  • Solution 25 Use of any one of solutions 21-24, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
  • Solution 26 Use of any one of solutions 21-25, wherein the subject has a viral infection.
  • Solution 27 Use of any one of solutions 21-25, wherein the subject is at risk of developing a viral infection.
  • Solution 28 Use of solution 26 or 27, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
  • Solution 29 Compound of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof for use in treating a subject in need thereof.
  • Solution 30 Compound according to claim 29 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
  • Solution 31 Compound according to claim 29 or 30 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
  • Solution 32 Compound according to claim 31 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the viral infection is an influenza A viral infection.
  • Solution 33 Compound according to any one of solutions 29-32 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
  • Solution 34 Compound according to any one of solutions 29-33 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject has a viral infection.
  • Solution 35 Compound according to any one of solutions 29-33 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject is at risk of developing a viral infection.
  • Solution 36 Compound according to solution 34 or 35 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
  • Solution 37 The composition according to any of solutions 10-12 for use in treating a subject in need thereof.
  • Solution 38 The composition according to claim 37, wherein the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
  • Solution 39 The composition according to claim 37 or 38, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
  • Solution 40 The composition according to claim 39, wherein the viral infection is an influenza A viral infection.
  • Solution 41 The composition according to any one of solutions 37-40, wherein the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
  • Solution 42 The composition according to any one of solutions 37-41, wherein the subject has a viral infection.
  • Solution 43 The composition according to any one of solutions 37-41, wherein the subject is at risk of developing a viral infection.
  • Solution 44 The composition according to solution 42 or 43, wherein the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
  • Figure 1 is an EC 50 curve showing the broad spectrum antiviral activities of FA-6005 against A/WSN/1933, A/Hong Kong/HKU38/2004 [H3N2] , A/Houston/21OS/2009 [H1N1] and H7N9 clinical isolate with an EC 50 of 2.82 ⁇ 0.33 ⁇ M, 1.51 ⁇ 0.58 ⁇ M, 6.22 ⁇ 0.07 ⁇ M and 1.37 ⁇ 0.10 ⁇ M, respectively.
  • Figure 2A-B shows antiviral activities of Compound 26 (FA-6005)
  • A Efficacies of FA-6005 in murine influenza A/Puerto Rico/8/1934 [H1N1] infection model.
  • B Zanamivir and FA-6005 reduced the viral load in the lungs.
  • FIG. 3 shows FA-6005 is targeting Influenza A NP. Escape mutant virus and recombinant virus carrying the I41T substitution in influenza A NP confer resistance to high concentrations of FA-6005. Antiviral activities of FA-6005 against A/WSN/33 virus, I41T escape mutant virus or I41T variant virus generated by reverse genetics were determined by PRA.
  • Figure 4A-B shows FA-6005 inhibits virus transcription and replication.
  • A Time-of-addition experiments examining the effect of FA-6005 on various stages of IAV life cycle.
  • B FA-6005 exhibit inhibition of the parental virus NP activity, but not the resistant I41T variant virus NP in a luciferase reporter assay.
  • Figure 5 shows FA-6005 inhibits all kinds of viral RNAs synthesis.
  • Figure 6 shows FA-6005 abolishes viral protein synthesis.
  • DMSO was added as a negative control.
  • Cell lysates were collected at 2, 4, 6, and 8 hpi, and further analyzed by western blotting.
  • This application describes compounds that were screened from a compound library along with its derivatives and identified as influenza virus inhibitors.
  • the target of these compounds was shown to be nucleoprotein, which played multiple roles in Influenza A virus infection cycle.
  • small molecule inhibitors of influenza A viral nucleoprotein and methods of using thereof are disclosed.
  • Analog and “Derivative, ” are used herein interchangeably, and refer to a compound that possesses the same core as a parent compound, but differs from the parent compound in bond order, the absence or presence of one or more atoms and/or groups of atoms, and combinations thereof.
  • the derivative can differ from the parent compound, for example, in one or more substituents present on the core, which may include one or more atoms, functional groups, or substructures.
  • the derivative can also differ from the parent compound in the bond order between atoms within the core.
  • a derivative can be imagined to be formed, at least theoretically, from the parent compound via chemical and/or physical processes.
  • Co-administration includes simultaneous and sequential administration.
  • An appropriate time course for sequential administration may be chosen by the physician, according to such factors as the nature of a patient’s illness, and the patient’s condition.
  • “Pharmaceutically acceptable, ” as used herein, refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.
  • Prodrug refers to a pharmacological substance (drug) that is administered to a subject in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized in the body (in vivo) into a compound having the desired pharmacological activity.
  • therapeutically effective amount refers to an amount of the therapeutic agent that, when incorporated into and/or onto the self-assembled gel composition, produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment.
  • the effective amount may vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, or the severity of the disease or condition.
  • a carboxylic acid is the group–COOH. Unless specified otherwise the term carboxylic acid embraces both the free acid and carboxylate salt.
  • An alkyl is the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C 30 for straight chain, C 3 -C 30 for branched chain) , more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms.
  • the chain has 1-6 carbons.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value.
  • high, ” “higher, ” “increases, ” “elevates, ” or “elevation” refer to increases above basal levels, e.g., as compared to a control.
  • low, ” “lower, ” “reduces, ” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
  • inhibitor means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
  • monitoring refers to any method in the art by which an activity can be measured.
  • providing refers to any means of adding a compound or molecule to something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo.
  • preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • in need of treatment refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case ofhumans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver’s expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.
  • a caregiver e.g. physician, nurse, nurse practitioner, or individual in the case ofhumans; veterinarian in the case of animals, including non-human mammals
  • subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent) , a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans) .
  • arthropod e.g., insects and crustaceans
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • treatment and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • treatment while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention.
  • the effects of treatment can be measured or assessed as described herein and as known in the art
  • a cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject.
  • a “cell” can be a cell from any organism including, but not limited to, a bacterium.
  • the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
  • an effective amount of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount. ” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
  • alkyl includes both “unsubstituted alkyls” and “substituted alkyls, ” the latter of which refers to alkyl moieties having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • lower alkyl refers to an alkyl group having from one to ten carbons, more preferably from one to six carbon atoms, in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls.
  • the alkyl groups may also contain one or more heteroatoms within the carbon backbone. Examples include oxygen, nitrogen, sulfur, and combinations thereof. In certain forms, the alkyl group contains between one and four heteroatoms.
  • Alkenyl and alkynyl refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C 2 -C 30 ) and possible substitution to the alkyl groups described above.
  • Aryl refers to 5-, 6-and 7-membered aromatic rings.
  • the ring may be a carbocyclic, heterocyclic, fused carbocyclic, fused heterocyclic, bicarbocyclic, or biheterocyclic ring system, optionally substituted as described above for alkyl.
  • “Ar, ” as used herein, includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms.
  • Examples include, but are not limited to, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.
  • Those aryl groups having heteroatoms in the ring structure may also be referred to as “heteroaryl, ” “aryl heterocycles, ” or “heteroaromatics.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, --CF 3 , and --CN.
  • substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,
  • Ar also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings” ) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles, or both rings are aromatic.
  • Alkylaryl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group) .
  • Heterocycle or heterocyclic refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, containing carbon and one to four heteroatoms each selected from non-peroxide oxygen, sulfur, and N (Y) wherein Y is absent or is H, O, (C 1-4 ) alkyl, phenyl or benzyl, and optionally containing one or more double or triple bonds, and optionally substituted with one or more substituents.
  • the term “heterocycle” also encompasses substituted and unsubstituted heteroaryl rings.
  • heterocyclic ring examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1, 5, 2-dithiazinyl, dihydrofuro [2, 3-b] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indoli
  • Heteroaryl refers to a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms each selected from non-peroxide oxygen, sulfur, and N (Y) where Y is absent or is H, O, (C 1 -C 8 ) alkyl, phenyl, or benzyl.
  • Non-limiting examples ofheteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide) , thienyl, pyrimidinyl (or its N- oxide) , indolyl, isoquinolyl (or its N-oxide) , quinolyl (or its N-oxide) and the like.
  • heteroaryl can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • heteroaryl examples include, but are not limited to, furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide) , thientyl, pyrimidinyl (or its N-oxide) , indolyl, isoquinolyl (or its N-oxide) , quinolyl (or its N-oxide) , and the like.
  • nitro means-NO 2 .
  • Halogen refers to fluorine, chlorine, bromine, or iodine.
  • substituted refers to all permissible substituents of the compounds described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms, such as oxygen, sulfur, or nitrogen, grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen, may have hydrogen substituents and/or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. “Substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • Numerical ranges disclosed herein disclose individually each possible number in such range, as well as any sub-ranges and combinations of sub-ranges encompassed therein.
  • acarbon range i.e., C 1 -C 10
  • a carbon length range of C 1 -C 10 discloses C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , and C 10 , as well as discloses sub-ranges encompassed within, such as C 2 -C 9 , C 3 -C 8 , C 1 -C 5 , etc.
  • an integer value range of 1-10 discloses the individual values of 1, 2, 3, 4, 5, 6, 7, 8, and 10, as well as sub-ranges encompassed within.
  • a concentration range or weight percent range or volume percent range such as from l%to 2%by weight of the composition, discloses the individual values and fractions thereof, such as 1%, 1.1%, 1.2%, 1.32%, 1.48%etc., as well as sub-ranges encompassed within.
  • R 2 -R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 10 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 9 ; -CONHR 9 ) , tertiary amide (e.g., -NR 9 COR 9 ; -CONR 9 R 9 ) , secondary carbamate (e.g., -OCONHR 9 ; -NH
  • R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
  • R 4 is the same as R 1
  • R 3 or R 6 and R 5 or R 3 are alkoxy, such as methoxy.
  • R 4 is a heteroaryl, such as furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, pyridyl, (or its N-oxide) , thienyl, pyrimidinyl (or its N-oxide) , indolyl, isoquinolyl (or its N-oxide) , or quinolyl (or its N-oxide) .
  • a heteroaryl such as furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, pyridyl, (or its N-oxide) , thienyl, pyrimidin
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is a substituted alkenyl, such as an ethenylbenzene.
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is a substituted heteroaryl, such as a substituted or unsubstituted furanyl, pyridine, or benzothiophenyl.
  • the benzothiphenyl can be further substituted with a halogen, such as Cl.
  • R 2 or R 6 is an alkoxy, such as a methoxy
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is a substituted heteroaryl, such as a substituted or unsubstituted benzofuranyl.
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is a substituted alkoxy, such as a methoxy the methoxy can be substituted with a substituted aryl, such as a phenyl, the phenyl can be further substituted with a halogen, such as Cl, or a nitro at the ortho position or an alkyl, such as a methyl at the para position.
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is a substituted alkyl, such as a methyl the methyl can be substituted with a substituted aryl, such as a phenyl, the phenyl can be further substituted with an alkoxy, such as a methoxy at the meta and para positions.
  • R 3 or R 5 and R 6 or R 2 are alkoxy, such as methoxy or ethoxy
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is an aryl, such as a phenyl.
  • R 5 or R 3 is an alkoxy, such as a methoxy
  • R 2 or R 6 is halogen, such as a Cl
  • R 4 is a secondary amide (-NHCOR 9 )
  • R 9 is an aryl, such as a phenyl.
  • R 4 is a secondary amide (–NHCOR 9 )
  • R 9 is an aryl, such as a substituted or unsubstituted phenyl.
  • the R 9 phenyl is substituted with alkoxy, halogen, nitro, alkyl, and/or a combination thereof.
  • X is N, O, S, or C
  • R 33 -R 39 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 42 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 41 ; -CONHR 41 ) , tertiary amide (e.g., -NR 41 COR 41 ; -CONR 41 R 41 ) , secondary carbamate (e.g., -OCONHR 41 ; -NH
  • R 4 substituted heterocyclic X is N, Y is NR 37 , R 37 is a substituted alkyl, such as a methyl substituted with a substituted phenyl.
  • R 4 substituted heterocyclic for the R 4 substituted heterocyclic, X is N, Y is NR 37 , R 37 is a substituted alkyl, such as a methyl substituted with a substituted phenyl with a halogen, such as F, at the ortho position.
  • R 4 substituted heterocyclic X is N, Y is NR 37 , R 37 is a substituted carbonyl (-COR 42 ) , R 42 is a substituted phenyl with an alkyl, such as methyl at the ortho position.
  • Formula I has the following structure:
  • R 40 -R 47 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 50 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 49 ; -CONHR 49 ) , tertiary amide (e.g., -NR 49 COR 49 ; -CONR 49 R 49 ) , secondary carbamate (e.g., -OCONHR 49 ; -NH
  • R 9 is an aryl, such as a substituted or unsubstituted phenyl.
  • R 9 phenyl is substituted at R 44 or R 46 with a halogen, such as Br or Cl.
  • the R 9 phenyl is substituted at R 43 or R 47 and R 45 with a halogen, such as Cl.
  • the R 9 phenyl is substituted at R 43 or R 47 with an alkoxy, such as a methoxy, and at R 46 or R 44 with a halogen, such as Cl.
  • the R 9 phenyl is substituted at R 46 or R 44 with nitro and at R 43 or R 47 with a halogen, such as Cl. In some forms, the R 9 phenyl is substituted at R 44 or R 46 with nitro and at R 43 or R 47 with an alkyl, such as methyl. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with a halogen, such as Cl, and at R 45 with nitro. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with an alkyl, such as methyl, and at R 42 with an alkyl, such as methyl.
  • the R 9 phenyl is substituted at R 43 and R 47 with an alkoxy, such as a methoxy. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with a halogen, such as F, and at R 45 with a cyano. In some forms, the R 9 phenyl is substituted at R 44 or R 46 with an alkyl, such as methyl, and at R 43 or R 47 with an alkoxy, such as methoxy.
  • R 4 is a secondary amide (–NHCOR 9 )
  • R 9 is an aryl or a heteroaryl, such as:
  • R 21 -R 29 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 32 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 31 ; -CONHR 31 ) , tertiary amide (e.g., -NR 31 COR 31 ; -CONR 31 R 31 ) , secondary carbamate (e.g., -OCONHR 31 ; -NH
  • R 9 Q is O
  • T is O
  • U is CR 25
  • R 26 R 4 and R 5 are hydrogen
  • R 21 , R 22 , and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkyl
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is hydrogen
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkyl
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 and R 26 are hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is halogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is halogen
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is halogen
  • R 21 , R 22 , and R 23 are halogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is hydrogen
  • R 21 , R 22 , or R 23 are halogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is halogen
  • R 21 , R 22 , or R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkoxy
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkoxy
  • R 21 , R 22 , and R 23 are alkoxy.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkoxy
  • R 21 , R 22 , or R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkoxy
  • R 21 , R 22 , or R 23 are halogen.
  • R 9 Q is N
  • T is NR 27
  • R 27 is hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is N
  • T is NR 27
  • R 27 is hydrogen
  • R 21 , R 22 , and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
  • R 9 Q is N
  • T is NR 27
  • R 27 is alkyl
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is N
  • T is NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is N
  • T is NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is N
  • T is NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 a halogen
  • R 9 Q is N
  • T is NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , and R 23 hydrogen.
  • R 9 Q is N
  • T is NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , or R 23 are alkoxy.
  • R 9 Q is N
  • T is NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , or R 23 are alkyl.
  • R 9 Q is N
  • T is NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , or R 23 are halogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 and R 29 are hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 and R 29 are hydrogen
  • R 21 , R 22 , and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is alkyl
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 R 28 orR 29 is hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is alkyl
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is halogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is halogen
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is halogen
  • R 21 , R 22 , and R 23 are halogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 or R 29 is halogen
  • R 21 , R 22 , or R 23 are halogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 orR 29 is alkoxy
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 orR 29 is alkoxy
  • R 21 , R 22 , and R 23 are alkoxy.
  • R 9 Q is O
  • T is O
  • U is CR 25 R 26
  • R 25 or R 26 is alkoxy
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • V and W are CR 28 R 29 , R 28 orR 29 is alkoxy
  • R 21 , R 22 , and R 23 are halogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 are hydrogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 are hydrogen
  • R 21 , R 22 , and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is alkyl
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is halogen
  • R 21 , R 22 , and R 23 are halogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , and R 23 are hydrogen.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , or R 23 are alkoxy.
  • R 9 Q is O
  • T is O
  • U is NR 24
  • R 24 is alkoxy
  • R 21 , R 22 , or R 23 are alkyl.
  • R 9 Q is O
  • T is O
  • V and W are NR 27
  • R 27 is alkoxy
  • R 21 , R 22 , or R 23 are halogen.
  • R 4 is a thiourea (–NHCSNHR 8 )
  • R 8 is a carbonyl (-COR 10 )
  • R 10 is a substituted or unsubstituted alkyl, such as a substituted or unsubstituted methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.
  • the R 10 is isobutyl or tert-butyl.
  • R 4 is a thiourea (–NHCSNHR 8 )
  • R 8 is a carbonyl (-COR 10 )
  • R 10 is an aryl, such as a substituted or unsubstituted phenyl.
  • the R 10 phenyl is substituted with alkoxy, halogen, nitro, alkyl, and/or a combination thereof.
  • R 4 is a thiourea (–NHCSNHR 8 )
  • R 8 is a carbonyl (-COR 10 )
  • R 10 is an heteroaryl, such as a substituted or unsubstituted benzofuranyl.
  • Formula I has the following structure.
  • R 51 -R 55 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 58 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 57 ; -CONHR 57 ) , tertiary amide (e.g., -NR 57 COR 57 ; -CONR 57 R 57 ) , secondary carbamate (e.g., -OCON
  • R 4 is a thiourea (–NHCSNHR 8 )
  • R 8 is a carbonyl (-COR 10 )
  • R 10 is an aryl, such as a substituted or unsubstituted phenyl.
  • R 10 phenyl is substituted at R 51 or R 55 with a halogen, such as Cl.
  • the R 10 phenyl is substituted at R 51 or R 55 and R 53 with a halogen, such as Cl.
  • the R 10 phenyl is substituted at R 51 or R 55 with an alkoxy, such as a methoxy, and at R 54 or R 52 with a halogen, such as Cl. In some forms, the R 10 phenyl is substituted at R 53 with an alkoxy, such as propoxy. In some forms, the R 10 phenyl is substituted at R 54 or R 52 with nitro and at R 55 or R 51 with an alkyl, such as methyl. In some forms, the R 10 phenyl is substituted at R 51 and R 55 with an alkoxy, such as methoxy. In some forms, the R 10 phenyl is substituted at R 52 and R 54 with an alkyl, such as methyl.
  • the R 10 phenyl is substituted at R 53 with an alkyl, such as ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some forms, the R 10 phenyl is not a methyl at R 53 .
  • A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring
  • R 2 -R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 10 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 9 ; -CONHR 9 ) , tertiary amide (e.g., -NR 9 COR 9 ; -CONR 9 R 9 ) , secondary carbamate (e.g., -OCONHR 9 ; -NH
  • R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
  • Formula II has the following structure.
  • R 12 -R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 20 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 19 ; -CONHR 19 ) , tertiary amide (e.g., -NR 19 COR 19 ; -CONR 19 R 19 ) , secondary carbamate (e.g., -OCONHR 19 ; -NH
  • R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
  • Formula II has the following structure.
  • R 12 -R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO - ) , carbonyl (e.g., -COR 20 ) , primary amide (e.g., -NH 2 CO; -CONH 2 ) , secondary amide (e.g., -NHCOR 19 ; -CONHR 19 ) , tertiary amide (e.g., -NR 19 COR 19 ; -CONR 19 R 19 ) , secondary carbamate (e.g., -OCONHR 19 ; -NH
  • R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
  • the compounds of formula I or formula II, or a pharmaceutically acceptable salt or a prodrug thereof is a compound selected from the group consisting of:
  • the compounds described herein may have one or more chiral centers, and thus exist as one or more stereoisomers.
  • Such stereoisomers can exist as a single enantiomer, a mixture of enantiomers, a mixture of diastereomers, or a racemic mixture.
  • stereoisomers refers to compounds made up of the same atoms having the same bond order but having different three-dimensional arrangements of atoms that are not interchangeable. The three-dimensional structures are called configurations.
  • enantiomers refers to two stereoisomers that are non-superimposable mirror images of one another.
  • optical isomer is equivalent to the term “enantiomer. ”
  • diastereomer refers to two stereoisomers which are not mirror images but also not superimposable.
  • racemate, ” “racemic mixture” or “racemic modification” refer to a mixture of equal parts of enantiomers.
  • chiral center refers to a carbon atom to which four different groups are attached. Choice of the appropriate chiral column, eluent, and conditions necessary to effect separation of the pair of enantiomers is well known to one of ordinary skill in the art using standard techniques (see e.g. Jacques, J. et al., “Enantiomers, Racemates, and Resolutions, ” John Wiley and Sons, Inc. 1981) .
  • the compounds can also be a pharmaceutically acceptable salt of any of the compounds described above.
  • Formula I also encompasses pharmaceutically acceptable esters, amides, and salts of such compounds, as will be explained in detail, infra.
  • Such compounds of the formula (I) and their pharmaceutically acceptable esters, amides, and salts are referred to herein as the inventive compounds.
  • Formula I also encompasses pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base.
  • Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like.
  • the reaction is conducted in water, alone or in combination with an inert, water- miscible organic solvent, at a temperature of from about 0°C to about 100°C, such as at room temperature.
  • the molar ratio of compounds of structural formula (I) to base used are chosen to provide the ratio desired for any particular salts.
  • the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt.
  • non-aqueous media including ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams&Wilkins, Baltimore, MD, 2000, p. 704; and “Handbook of Pharmaceutical Salts: Properties, Selection, and Use, ” P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids, such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids.
  • inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids
  • organic acids such as acetic, benzenesul
  • Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids.
  • suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, mesylate, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate) , methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclo
  • the pharmaceutically acceptable salt may include alkali metal salts, including sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
  • Base salts can also be formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts.
  • Organic salts may be made from secondary, tertiary or quaternary amine salts, such as tromethamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) , and procaine.
  • secondary, tertiary or quaternary amine salts such as tromethamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) , and procaine.
  • Basic nitrogen-containing groups may also be quaternized with agents such as lower alkyl (C 1 -C 6 ) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides) , dialkyl sulfates (e.g., dimethyl, diethyl, dibuytl, and diamyl sulfates) , long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides) , arylalkyl halides (e.g., benzyl and phenethyl bromides) , and others.
  • agents such as lower alkyl (C 1 -C 6 ) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides) , dial
  • the compound can also be a pharmaceutically acceptable prodrug of any of the compounds described above.
  • Prodrugs are compounds that, when metabolized in vivo, undergo conversion to compounds having the desired pharmacological activity.
  • Prodrugs can be prepared by replacing appropriate functionalities present in the compounds described above with "pro-moieties" as described, for example, in H. Bundgaar, Design of Prodrugs (1985) .
  • prodrugs examples include ester, ether or amide derivatives of the compounds described above, polyethylene glycol derivatives of the compounds described above, N-acyl amine derivatives, dihydropyridine pyridine derivatives, amino-containing derivatives conjugated to polypeptides, 2-hydroxybenzamide derivatives, carbamate derivatives, N-oxides derivatives that are biologically reduced to the active amines, and N-mannich base derivatives.
  • prodrugs see, for example, Rautio, J. et al. Nature Reviews Drug Discovery. 7: 255-270 (2008) .
  • compositions are provided containing a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt or prodrug thereof, in combination with one or more pharmaceutically acceptable excipients.
  • Representative excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof.
  • Suitable pharmaceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) , and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • the compounds described herein can be formulated with one or more additional active agents, such as anti-infectious agents, analgesic, etc.
  • compositions can also include one or more vitamins, minerals, dietary supplements, nutraceutical agents, such as proteins, carbohydrates, amino acids, fatty acids, antioxidants, and plant or animal extracts, or combinations thereof.
  • nutraceutical agents such as proteins, carbohydrates, amino acids, fatty acids, antioxidants, and plant or animal extracts, or combinations thereof.
  • Suitable vitamins, minerals, nutraceutical agents, and dietary supplements are known in the art, and disclosed, for example, in Roberts et al., (Nutriceuticals: The Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing Foods, American Nutriceutical Association, 2001) .
  • Nutraceutical agents and dietary supplements are also disclosed in Physicians' Desk Referencefor Nutritional Supplements, 1st Ed. (2001) and The Physicians' Desk Referencefor Herbal Medicines, 1st Ed. (2001) .
  • Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.
  • compositions may be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Excipients, including plasticizers, pigments, colorants, stabilizing agents, and glidants, may also be used to form coated compositions for enteral administration. Delayed release dosage compositions may be prepared as described in standard references such as “Pharmaceutical dosage form tablets, ” eds. Liberman et al.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name (Roth Pharma, Westerstadt, Germany) , zein, shellac, and polysaccharides.
  • cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate
  • polyvinyl acetate phthalate acrylic acid polymers and copolymers
  • methacrylic resins that are commercially available under the trade name (Roth Pharma, Westerstadt, Germany) , zein, shellac,
  • Diluents also referred to as “fillers, " are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.
  • Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
  • Binders are used to impart cohesive qualities to a solid dosage composition, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms.
  • Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol) , polyethylene glycol, waxes, natural and synthetic gums, such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers, such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.
  • Lubricants are used to facilitate tablet manufacture.
  • suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
  • Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP ( XL from GAF Chemical Corp) .
  • Stabilizers are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.
  • Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT) ; ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites, such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA) .
  • Oral dosage forms such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release.
  • the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup.
  • the particles can be formed of the drug and a controlled release polymer or matrix.
  • the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.
  • the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids.
  • aqueous medium such as physiological fluids.
  • the matrix swells entrapping the active agents, which are released slowly over time by diffusion and/or degradation of the matrix material.
  • Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.
  • the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coatings or extended release coatings.
  • the coating or coatings may also contain the compounds and/or additional active agents.
  • the extended release compositions are generally prepared as diffusion or osmotic systems, for example, as described in “Remington–The science and practice of pharmacy” (20th ed., Lippincott Williams&Wilkins, Baltimore, MD, 2000) .
  • a diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art.
  • the matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form.
  • the three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds.
  • Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene.
  • Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses, such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and 934, polyethylene oxides and mixtures thereof.
  • Fatty compounds include, but are not limited to, various waxes, such as carnauba wax and glyceryl tristearate, and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.
  • the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly (acrylic acid) , poly (methacrylic acid) , methacrylic acid alkylamine copolymer poly (methyl methacrylate) , poly (methacrylic acid) (anhydride) , polymethacrylate, polyacrylamide, poly (methacrylic acid anhydride) , and glycidyl methacrylate copolymers.
  • acrylic acid and methacrylic acid copolymers including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
  • the acrylic polymer is comprised of one or more ammonio methacrylate copolymers.
  • Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
  • the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename
  • the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames RL30D and RS30D, respectively.
  • RL30D and RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth) acrylic esters being 1: 20 in RL30D and 1: 40 in RS30D.
  • the mean molecular weight is about 150,000.
  • S-100 and L-100 are also preferred.
  • the code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents.
  • RL/RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.
  • RL/RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release composition having a desirable dissolution profile.
  • Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% RL, 50% RL and 50% RS, and 10% RL and 90% RS.
  • acrylic polymers may also be used, such as, for example, L.
  • extended release compositions can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form.
  • the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.
  • the devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units.
  • multiple units include, but are not limited to, multilayer tablets andcapsules containing tablets, beads, or granules.
  • An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.
  • Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation. Their compositions usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient.
  • the usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders.
  • Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar.
  • Powdered cellulose derivatives are also useful.
  • Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose.
  • Natural and synthetic gums including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used.
  • Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders.
  • a lubricant is necessary in a tablet composition to prevent the tablet and punches from sticking in the die.
  • the lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
  • Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method.
  • the congealing method the drug is mixed with a wax material and either spray-congealed or congealed and screened and processed.
  • Delayed release compositions can be created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.
  • the delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material.
  • the drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core” dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule.
  • Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional "enteric" polymers.
  • Enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon.
  • Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename (Rohm Pharma; Westerstadt, Germany) , including L30D-55 and L100-55 (soluble at pH 5.5 and above) , L-100(soluble at pH 6.0 and above) ,
  • the preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.
  • the coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc.
  • a plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. %to 50 wt. %relative to the dry weight of the polymer.
  • typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides.
  • a stabilizing agent is preferably used to stabilize particles in the dispersion.
  • Typical stabilizing agents are nonionic emulsifiers, such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. %to 100 wt. %of the polymer weight in the coating solution.
  • One effective glidant is talc.
  • Other glidants such as magnesium stearate and glycerol monostearates, may also be used.
  • Pigments, such as titanium dioxide may also be used.
  • Small quantities of an anti-foaming agent such as a silicone (e.g., simethicone) , may also be added to the coating composition.
  • the composition can provide pulsatile delivery of the one or more of the compounds disclosed herein.
  • pulsatile is meant that a plurality of drug doses are released at spaced apart intervals of time.
  • release of the initial dose is substantially immediate, i.e., the first drug release "pulse” occurs within about one hour of ingestion.
  • This initial pulse is followed by a first time interval (lag time) during which very little or no drug is released from the dosage form, after which a second dose is then released.
  • a second nearly drug release-free interval between the second and third drug release pulses may be designed.
  • the duration of the nearly drug release-free time interval will vary depending upon the dosage form design e.g., a twice daily dosing profile, a three times daily dosing profile, etc.
  • the nearly drug release-free interval has a duration of approximately 3 hours to 14 hours between the first and second dose.
  • the nearly drug release-free interval has a duration of approximately 2 hours to 8 hours between each of the three doses.
  • the pulsatile release profile is achieved with dosage forms that are closed and preferably sealed capsules housing at least two drug-containing "dosage units" wherein each dosage unit within the capsule provides a different drug release profile.
  • Control of the delayed release dosage unit (s) is accomplished by a controlled release polymer coating on the dosage unit, or by incorporation of the active agent in a controlled release polymer matrix.
  • Each dosage unit may comprise a compressed or molded tablet, wherein each tablet within the capsule provides a different drug release profile. For dosage forms mimicking a twice a day dosing profile, a first tablet releases drug substantially immediately following ingestion of the dosage form, while a second tablet releases drug approximately 3 hours to less than 14 hours following ingestion of the dosage form.
  • a first tablet releases drug substantially immediately following ingestion of the dosage form
  • a second tablet releases drug approximately 3 hours to less than 10 hours following ingestion of the dosage form
  • the third tablet releases drug at least 5 hours to approximately 18 hours following ingestion of the dosage form. It is possible that the dosage form includes more than three tablets. While the dosage form will not generally include more than a third tablet, dosage forms housing more than three tablets can be utilized.
  • each dosage unit in the capsule may comprise a plurality of drug-containing beads, granules or particles.
  • drug-containing beads refer to beads made with drug and one or more excipients or polymers.
  • Drug-containing beads can be produced by applying drug to an inert support, e.g., inert sugar beads coated with drug or by creating a "core” comprising both drug and one or more excipients.
  • drug-containing "granules” and “particles” comprise drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, granules and particles do not contain an inert support.
  • Granules generally comprise drug particles and require further processing. Generally, particles are smaller than granules, and are not further processed. Although beads, granules and particles may be formulated to provide immediate release, beads and granules are generally employed to provide delayed release.
  • parenteral administration means administration by any method other than through the digestive tract or non-invasive topical or regional routes.
  • parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, by injection, and by infusion.
  • compositions can be prepared as aqueous compositions using techniques is known in the art.
  • such compositions can be prepared as injectable compositions, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable compositions for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • emulsions such as water-in-oil (w/o) emulsions, oil-
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) , oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. ) , and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. )
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl- ⁇ -alanine, sodium N-lauryl- ⁇ -iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the composition can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the composition may also contain an antioxidant to prevent degradation of the active agent (s) .
  • composition is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water-soluble polymers are often used in compositions for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
  • parenteral compositions described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.
  • the compounds, and optionally one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release.
  • the composition contains two or more drugs
  • the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile) or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc. ) .
  • the compounds and/or one or more additional active agents can be incorporated into polymeric microparticles that provide controlled release of the drug (s) .
  • Release of the drug (s) is controlled by diffusion of the drug (s) out of the microparticles and/or degradation of the polymeric particles by hydrolysis and/or enzymatic degradation.
  • Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.
  • Polymers that are slowly soluble and form a gel in an aqueous environment may also be suitable as materials for drug containing microparticles.
  • Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides) , polyhydroxy acids, such as polylactide (PLA) , polyglycolide (PGA) , poly (lactide-co-glycolide) (PLGA) , poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.
  • PLA polylactide
  • PGA polyglycolide
  • P4HB poly-4-hydroxybutyrate
  • the drug (s) can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and/or mechanical erosion.
  • slowly soluble in water refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof.
  • Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol) , fatty acids and derivatives, including, but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and tri-glycerides) , and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes.
  • waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax.
  • a wax-like material is defined as any material that is normally solid at room temperature and has a melting point of from about 30 to 300°C.
  • rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above.
  • rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch) , cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose) , alginic acid, lactose and talc.
  • a pharmaceutically acceptable surfactant for example, lecithin may be added to facilitate the degradation of such microparticles.
  • Proteins that are water insoluble can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof that are water soluble can be formulated with drug into microparticles and subsequently cross- linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.
  • Encapsulation or incorporation of drug into carrier materials to produce drug containing microparticles can be achieved through known pharmaceutical composition techniques.
  • the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof.
  • Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion.
  • wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools.
  • the molten wax-drug mixture can be extruded and spheronized to form pellets or beads.
  • Detailed descriptions of these processes can be found in “Remington-The science and practice of pharmacy, ” 20th Edition, Jennaro et al., (Phila, Lippencott, Williams, and Wilkens, 2000) .
  • a solvent evaporation technique to produce drug containing microparticles.
  • drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.
  • drug in a particulate form is homogeneously dispersed in a water-insoluble or slowly water-soluble material.
  • the drug powder itself may be milled to generate fine particles prior to composition.
  • the process of jet milling known in the pharmaceutical art, can be used for this purpose.
  • drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture.
  • a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.
  • the particles can also be coated with one or more modified release coatings.
  • Solid esters of fatty acids which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles.
  • Zein is an example of a naturally water-insoluble protein. It can be coated onto drug containing microparticles or drug particles by spray coating or by wet granulation techniques.
  • some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of non-soluble networks.
  • Many methods of cross-linking proteins initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical cross-linking agents.
  • cross-linking agents examples include aldehydes (gluteraldehyde and formaldehyde) , epoxy compounds, carbodiimides, and genipin.
  • aldehydes gluteraldehyde and formaldehyde
  • epoxy compounds carbodiimides
  • genipin examples include aldehydes (gluteraldehyde and formaldehyde) , epoxy compounds, carbodiimides, and genipin.
  • oxidized and native sugars have been used to cross-link gelatin (Cortesi, R., et al., Biomaterials 19 (1998) 1641-1649) .
  • Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products.
  • cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.
  • a water soluble protein can be spray coated onto the microparticles and subsequently cross-linked by the one of the methods described above.
  • drug containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently cross-linked.
  • suitable proteins for this purpose include gelatin, albumin, casein, and gluten.
  • Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations that cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions.
  • Active agents can be formulated for depot injection.
  • the active agent is formulated with one or more pharmaceutically acceptable carriers that provide for the gradual release of active agent over a period ofhours or days after injection.
  • the depot composition can be administered by any suitable means; however, the depot composition is typically administered via subcutaneous or intramuscular injection.
  • depot compositions contain one or more biodegradable polymeric or oligomeric carriers.
  • Suitable polymeric carriers include, but are not limited to poly (lactic acid) (PLA) , poly (lactic-co-glycolic acid) (PLGA) , poly (lactic acid) -polyethyleneglycol (PLA-PEG) block copolymers, polyanhydrides, poly (ester anhydrides) , ppolyglycolide (PGA) , poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4- hydroxybutyrate (P4HB) , polycaprolactone, cellulose, hydroxypropyl methylcellulose, ethylcellulose, as well as blends, derivatives, copolymers, and combinations thereof.
  • the carrier and active agent can be formulated as a solution, an emulsion, or suspension.
  • One or more compounds, and optionally one or more additional active agents, can also be incorporated into polymeric or oligomeric microparticles, nanoparticles, or combinations thereof.
  • the composition is fluid and designed to solidify or gel (i.e., forming a hydrogel or organogel) upon injection. This can result from a change in solubility of the composition upon injection, or for example, by injecting a pre-polymer mixed with an initiator and/or crosslinking agent.
  • the polymer matrix, polymer solution, or polymeric particles entrap the active agent at the injection site.
  • the active agent is released, either by diffusion of the agent out of the matrix and/or dissipation of the matrix as it is absorbed.
  • the release rate of the active agent from the injection site can be controlled by varying, for example, the chemical composition, molecular weight, crosslink density, and/or concentration of the polymeric carrier. Examples of such systems include those described in U.S. Patent Nos. 4,938,763, 5,480,656 and 6,113,943.
  • Depot composition can also be prepared by using other rate-controlling excipients, including hydrophobic materials, including acceptable oils (e.g., peanut oil, corn oil, sesame oil, cottonseed oil, etc. ) and phospholipids, ion-exchange resins, and sparingly soluble carriers.
  • acceptable oils e.g., peanut oil, corn oil, sesame oil, cottonseed oil, etc.
  • phospholipids e.g., phospholipids, ion-exchange resins, and sparingly soluble carriers.
  • the depot composition can further contain a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) , oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. ) , and combinations thereof.
  • a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) , oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. ) , and combinations thereof.
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. )
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or
  • Solutions and dispersions of the compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl- ⁇ -alanine, sodium N-lauryl- ⁇ -iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
  • the composition can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the composition may also contain an antioxidant to prevent degradation of the active agent (s) .
  • composition is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water-soluble polymers are often used in compositions for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
  • Implantation of a slow-release or sustained-release system such that a constant level of dosage is maintained is also contemplated herein.
  • the active agent (s) provided herein can be dispersed in a solid matrix optionally coated with an outer rate-controlling membrane.
  • the compound diffuses from the solid matrix (and optionally through the outer membrane) sustained, rate-controlled release.
  • the solid matrix and membrane may be formed from any suitable material known in the art including, but not limited to, polymers, bioerodible polymers, and hydrogels.
  • the respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream.
  • the respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli.
  • the upper and lower airways are called the conducting airways.
  • the terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung, where the exchange of gases occurs.
  • the alveolar surface area is the largest in the respiratory system and is where drug absorption occurs.
  • the alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. Effective delivery of therapeutic agents via pulmonary routes requires that the active agent be formulated so as to reach the alveoli.
  • compositions can be divided into dry powder compositions and liquid compositions. Both dry powder and liquid compositions can be used to form aerosol compositions.
  • aerosol refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.
  • compositions, and methods of manufacture are described by Caryalho, et al., J Aerosol Med Pulm Drug Deliv. 2011 Apr; 24 (2) : 61-80. Epub 2011 Mar 16, for delivery of chemotherapeutic drugs to the lungs.
  • Dry powder compositions are finely divided solid compositions containing one or more active agents which are suitable for pulmonary administration.
  • the one or more active agents can be incorporated in crystalline or amorphous form.
  • Dry powder compositions can be administered via pulmonary inhalation to a patient without the benefit of any carrier, other than air or a suitable propellant.
  • the dry powder compositions include one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier may include a bulking agent, such as carbohydrates (including monosaccharides, polysaccharides, and cyclodextrins) , polypeptides, amino acids, and combinations thereof.
  • a bulking agent such as carbohydrates (including monosaccharides, polysaccharides, and cyclodextrins) , polypeptides, amino acids, and combinations thereof.
  • Suitable bulking agents include fructose, galactose, glucose, lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol, palatinite, raffinose, stachyose, sucrose, trehalose, xylitol, hydrates thereof, and combinations thereof.
  • the pharmaceutical carrier may include a lipid or surfactant.
  • Natural surfactants such as dipalmitoylphosphatidylcholine (DPPC) , are the most preferred. This is commercially available for treatment of respiratory distress syndrome in premature infants.
  • Synthetic and animal derived pulmonary surfactants include:
  • Exosurf-a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents Pumactant (Artificial Lung Expanding Compound or ALEC) -a mixture of DPPC and PG KL-4-composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B. Venticute-DPPC, PG, palmitic acid and recombinant SP-C
  • the pharmaceutical carrier may also include one or more stabilizing agents or dispersing agents.
  • the pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate.
  • the pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride.
  • Dry powder compositions are typically prepared by blending one or more active agents with a pharmaceutical carrier.
  • additional active agents may be incorporated into the mixture.
  • the mixture is then formed into particles suitable for pulmonary administration using techniques known in the art, such as lyophilization, spray drying, agglomeration, spray coating, extrusion processes, hot melt particle formation, phase separation particle formation (spontaneous emulsion particle formation, solvent evaporation particle formation, and solvent removal particle formation) , coacervation, low temperature casting, grinding, milling (e.g., air-attrition milling (jet milling) , ball milling) , high pressure homogenization, and/or supercritical fluid crystallization.
  • milling e.g., air-attrition milling (jet milling) , ball milling
  • An appropriate method of particle formation can be selected based on the desired particle size, particle size distribution, and particle morphology.
  • the method of particle formation is selected so as to produce a population of particles with the desired particle size, particle size distribution for pulmonary administration.
  • the method of particle formation can produce a population of particles from which a population of particles with the desired particle size, particle size distribution for pulmonary administration is isolated, for example by sieving. It is known in the art that particle morphology affects the depth of penetration of a particle into the lung as well as uptake of the drug particles. As discussed above, drug particles should reach the alveoli to maximize therapeutic efficacy.
  • dry powder compositions is processed into particles having the appropriate mass median aerodynamic diameter (MMAD) , tap density, and surface roughness to achieve delivery of the one or more active agents to the deep lung.
  • MMAD mass median aerodynamic diameter
  • Particles having a mass median aerodynamic diameter (MMAD) of greater than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed.
  • Particles having diameters of about 3 to about 5 microns are small enough to reach the upper-to mid-pulmonary region (conducting airways) , but may be too large to reach the alveoli.
  • Smaller particles, (i.e., about 0.5 to about 3 microns) are capable of efficiently reaching the alveolar region.
  • Particles having diameters smaller than about 0.5 microns can also be deposited in the alveolar region by sedimentation, although very small particles may be exhaled.
  • the precise particle size range effective to achieve delivery to the alveolar region will depend on several factors, including the tap density of particles being delivered. Generally speaking, as tap density decreases, the MMAD of particles capable of efficiently reaching the alveolar region of the lungs increases. Therefore, in cases of particles with low tap densities, particles having diameters of about 3 to about 5 microns, about 5 to about 7 microns, or about 7 to about 9.5 microns can be efficiently delivered to the lungs.
  • the preferred aerodyanamic diameter for maximum deposition within the lungs can be calculated. See, for example, U.S. Patent No. 7,052,678 to Vanbever, et al.
  • the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 10 microns, more preferably between about 0.5 microns to about 7 microns, most preferably between about 0.5 to about 5 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 3 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 3 to about 5 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 5 to about 7 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 7 to about 9.5 microns.
  • At least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of less than about 10 microns, more preferably less than about 7 microns, most preferably about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have an aerodynamic diameter of greater than about 0.1 microns.
  • At least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns and less than about 10 microns, more preferably greater than about 0.5 microns and less than about 7 microns, most preferably greater than about 0.5 microns and less than about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns and less than about 3 microns.
  • At least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 3 microns and less than about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 5 microns and less than about 7 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 7 microns and less than about 9.5 microns.
  • the particles have a tap density of less than about 0.4 g/cm 3 , more preferably less than about 0.25 g/cm 3 , most preferably less than about 0.1 g/cm 3 .
  • Features which can contribute to low tap density include irregular surface texture and porous structure.
  • the particles are spherical or ovoid in shape.
  • the particles can have a smooth or rough surface texture.
  • the particles may also be coated with a polymer or other suitable material to control release of one or more active agents in the lungs.
  • Dry powder compositions can be administered as dry powder using suitable methods known in the art.
  • the dry powder compositions can be suspended in the liquid composition s described below, and administered to the lung using methods known in the art for the delivery of liquid compositions.
  • Liquid compositions contain one or more compounds dissolved or suspended in a liquid pharmaceutical carrier.
  • Suitable liquid carriers include, but are not limited to distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate buffered saline (PBS) , Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.
  • PBS phosphate buffered saline
  • Ringer's solution Ringer's solution
  • isotonic sodium chloride or any other aqueous solution acceptable for administration to an animal or human.
  • liquid compositions are isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, more preferably from about pH 6.0 to pH 7.0.
  • the liquid pharmaceutical carrier can include one or more physiologically compatible buffers, such as a phosphate buffers.
  • physiologically compatible buffers such as a phosphate buffers.
  • Liquid compositions may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin.
  • Liquid compositions may also include one or more preservatives, such as ethyl or n-propylp-hydroxybenzoate.
  • the liquid compositions may contain one or more solvents that are low toxicity organic (i.e., nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to readily aerosolize the composition. Any such solvent included in the liquid composition should not detrimentally react with the one or more active agents present in the liquid composition. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as a freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid composition as desired to increase the volatility and/or alter the aerosolizing behavior of the solution or suspension.
  • solvents that are low toxicity organic (i.e., nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahyd
  • Liquid compositions may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art.
  • minor amounts means no excipients are present that might adversely affect uptake of the one or more active agents in the lungs.
  • Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art.
  • aerosol as used herein refers to any preparation of a fine mist of solid or liquid particles suspended in a gas.
  • the gas may be a propellant; however, this is not required. Aerosols may be produced using a number of standard techniques, including as ultrasonication or high pressure treatment.
  • a dry powder or liquid compositions as described above is formulated into aerosol compositions using one or more propellants.
  • propellants include air, hydrocarbons, such as pentane, isopentane, butane, isobutane, propane and ethane, carbon dioxide, chlorofluorocarbons, fluorocarbons, and combinations thereof.
  • Suitable fluorocarbons include 1-6 hydrogen containing fluorocarbons, such as CHF 2 CHF 2 , CF 3 CH 2 F, CH 2 F 2 CH 3 , and CF 3 CHFCF 3 as well as fluorinated ethers, such as CF 3 -O-CF 3 , CF 2 H-O-CHF 2 , and CF 3 -CF 2 -O-CF 2 -CH 3 .
  • Suitable fluorocarbons also include perfluorocarbons, such as 1-4 carbon perfluorocarbons including CF 3 CF 3 , CF 3 CF 2 CF 3 , and CF 3 CF 2 CF 2 CF 3 .
  • the propellants include, but not limited to, one or more hydrofluoroalkanes (HFA) .
  • HFA propellants include but are not limited to, 1, 1, 1, 2, 3, 3-heptafluoro-n-propane (HFA 227) , 1, 1, 1, 2-tetrafluoroethane (HFA 134) 1, 1, 1, 2, 253, 3, 3-heptafluoropropane (Propellant 227) , or any mixture of these propellants.
  • the one or more propellants have sufficient vapor pressure to render them effective as propellants.
  • the one or more propellants are selected so that the density of the mixture is matched to the density of the particles in the aerosol composition in order to minimize settling or creaming of the particles in the aerosol composition.
  • the propellant is preferably present in an amount sufficient to propel a plurality of the selected doses of the aerosol composition from an aerosol canister.
  • a device is used to administer the compositions to the lungs.
  • Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.
  • Inhalation can occur through the nose and/or the mouth of the patient.
  • Administration can occur by self-administration of the composition while inhaling, or by administration of the composition via a respirator to a patient on a respirator.
  • the dry powder compositions described above can be administered to the lungs of a patient using a dry powder inhaler (DPI) .
  • DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which can then be inhaled by the patient.
  • the dose to be administered is stored in the form of a non-pressurized dry powder and, on actuation of the inhaler, the particles of the powder are inhaled by the subject.
  • a compressed gas i.e., propellant
  • the DPI may be breath actuated, meaning that an aerosol is created in precise response to inspiration.
  • dry powder inhalers administer a dose of less than a few tens of milligrams per inhalation to avoid provocation of cough. DPIs function via a variety of mechanical means to administer compositions to the lungs.
  • a doctor blade or shutter slides across the dry powder composition contained in a reservoir, culling the composition into a flowpath whereby the patient can inhale the powder in a single breath.
  • the dry powder composition is packaged in a preformed dosage form, such as a blister, tabule, tablet, or gelcap, which is pierced, crushed, or otherwise unsealed to release the dry powder composition into a flowpath for subsequent inhalation.
  • a preformed dosage form such as a blister, tabule, tablet, or gelcap, which is pierced, crushed, or otherwise unsealed to release the dry powder composition into a flowpath for subsequent inhalation.
  • Still others DPIs release the dry powder composition into a chamber or capsule and use mechanical or electrical agitators to keep the dry powder composition suspended in the air until the patient inhales.
  • Dry powder compositions may be packaged in various forms, such as a loose powder, cake, or pressed shape for insertion in to the reservoir of a DPI.
  • suitable DPIs for the administration of the compositions described above include the inhaler (Astrazeneca, Wilmington, Del. ) , the inhaler (Innovata, Ruddington, Nottingham, UK) , the inhaler (Glaxo, Greenford, Middlesex, UK) , the (Orion, Expoo, FI) , the inhaler (Pfizer, New York, N. Y. ) , the inhaler (Microdose, Monmouth Junction, N.J. ) , and the inhaler (Dura, San Diego, Calif. ) .
  • liquid compositions described above can be administered to the lungs of a patient using a pressurized metered dose inhaler (pMDI) .
  • pMDI pressurized metered dose inhaler
  • Pressurized Metered Dose Inhalers generally include at least two components: acanister in which the liquid composition is held under pressure in combination with one or more propellants, and a receptacle used to hold and actuate the canister.
  • the canister may contain a single or multiple doses of the composition.
  • the canister may include a valve, typically a metering valve, from which the contents of the canister may be discharged. Aerosolized drug is dispensed from the pMDI by applying a force on the canister to push it into the receptacle, thereby opening the valve and causing the drug particles to be conveyed from the valve through the receptacle outlet.
  • the liquid composition atomized, forming an aerosol.
  • pMDIs typically employ one or more propellants to pressurize the contents of the canister and to propel the liquid composition out of the receptacle outlet, forming an aerosol.
  • Any suitable propellants including those discussed above, may be utilized.
  • the propellant may take a variety of forms.
  • the propellant may be a compressed gas or a liquefied gas.
  • Chlorofluorocarbons (CFC) were once commonly used as liquid propellants, but have now been banned. They have been replaced by the now widely accepted hydrofluororalkane (HFA) propellants.
  • pMDIs are available from a number of suppliers, including 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura.
  • the patient administers an aerosolized composition by manually discharging the aerosolized composition from the pMDI in coordination with inspiration. In this way, the aerosolized composition is entrained within the inspiratory air flow and conveyed to the lungs.
  • a breath-actuated trigger such as that included in the inhaler (MAP Pharmaceuticals, Mountain View, Calif. ) may be employed that simultaneously discharges a dose of the composition upon sensing inhalation.
  • MAP Pharmaceuticals Mountain View, Calif.
  • These devices, which discharge the aerosol composition when the user begins to inhale, are known as breath-actuated pressurized metered dose inhalers (baMDIs) .
  • Nebulizers are liquid aerosol generators that convert the liquid composition described able, usually aqueous-based compositions, into mists or clouds of small droplets, preferably having diameters less than 5 microns mass median aerodynamic diameter, which can be inhaled into the lower respiratory tract. This process is called atomization.
  • the droplets carry the one or more active agents into the nose, upper airways or deep lungs when the aerosol cloud is inhaled.
  • Any type of nebulizer may be used to administer the composition to a patient, including, but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.
  • Pneumatic (jet) nebulizers use a pressurized gas supply as a driving force for atomization of the liquid composition. Compressed gas is delivered through a nozzle or jet to create a low pressure field which entrains a surrounding liquid composition and shears it into a thin film or filaments. The film or filaments are unstable and break up into small droplets that are carried by the compressed gas flow into the inspiratory breath. Baffles inserted into the droplet plume screen out the larger droplets and return them to the bulk liquid reservoir. Examples of pneumatic nebulizers include, but are not limited to, PARI LC PARI LC Devilbiss and Boehringer Ingelheim
  • Electromechanical nebulizers use electrically generated mechanical force to atomize liquid compositions.
  • the electromechanical driving force can be applied, for example, by vibrating the liquid composition at ultrasonic frequencies, or by forcing the bulk liquid through small holes in a thin film.
  • the forces generate thin liquid films or filament streams which break up into small droplets to form a slow moving aerosol stream which can be entrained in an inspiratory flow.
  • the electromechanical nebulizer is an ultrasonic nebulizer, in which the liquid compositions is coupled to a vibrator oscillating at frequencies in the ultrasonic range.
  • the coupling is achieved by placing the liquid in direct contact with the vibrator (such as a plate or ring in a holding cup) , or by placing large droplets on a solid vibrating projector (a horn) .
  • the vibrations generate circular standing films which break up into droplets at their edges to atomize the liquid composition.
  • Examples of ultrasonic nebulizers include Drive Medical Beetle Octive Tech and John Bunn
  • the electromechanical nebulizer is a mesh nebulizer, in which the liquid compositions is driven through a mesh or membrane with small holes ranging from 2 to 8 microns in diameter, to generate thin filaments which break up into small droplets.
  • the liquid composition is forced through the mesh by applying pressure with a solenoid piston driver (for example, the nebulizer) , or by sandwiching the liquid between a piezoelectrically vibrated plate and the mesh, which results in a oscillatory pumping action (for example or nebulizer) .
  • the mesh vibrates back and forth through a standing column of the liquid to pump it through the holes.
  • nebulizers include the AeroNeb AeroNeb PARI Omron and Aradigm
  • EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions.
  • Examples of EHD aerosol devices are known in the art. See, for example, U.S. Patent No. 4,765,539 to Noakes et al. and U.S. Patent No. 4,962,885 to Coffee, R.A.
  • the electrochemical properties of the composition may be important parameters to optimize when delivering the liquid composition to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art.
  • compositions containing one or more of the compounds described herein can be administered to treat viral infections.
  • the method for treating the viral infection by inhibiting viral nucleoprotein activity with an effective amount of one or more of the compound disclosed herein.
  • a method for inhibiting viral infection including inhibiting viral transcription, replication, protein synthesis, blocking vRNP export, and/or inhibiting virion budding process with an effective amount of one or more of the compound disclosed herein. The method of treating and inhibiting a viral infection where the virus is influenza A virus.
  • the dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician based on the clinical condition of the subject involved.
  • the dose, schedule of doses and route of administration can be varied.
  • one or more of the compounds are in an amount effective to inhibit viral transcription. In some forms, one or more of the compounds are in an amount effective to inhibit viral replication. In some forms, one or more of the compounds are in an amount effective to inhibit viral protein synthesis. In some forms, one or more of the compounds are in an amount effective to suppress the virion budding process. In some forms, one or more of the compounds are in an amount effective to inhibit viral nucleoprotein activity. In some forms, one or more of the compounds are in an amount effective to block vRNP export.
  • the precise dosage administered to a patient will depend on many factors, including the physical characteristics of the patient (e.g., weight) , the degree of severity of the disease or disorder to be treated, and the presence or absence of other complicating diseases or disorders and can be readily determined by the prescribing physician.
  • the compound (s) is administered at a dosage equivalent to an oral dosage of between about 0.005 mg and about 500 mg per kg of body weight per day, more preferably between about 0.05 mg and about 100 mg per kg of body weight per day, most preferably between about 0.1 mg and about 10 mg per kg of body weight per day.
  • the dosage can be effective to produce a concentration of one or more of the compounds within the range of 0.5 ⁇ M to 1 ⁇ M, 0.5 ⁇ M to 2 ⁇ M, 0.5 ⁇ M to 3 ⁇ M, 0.5 ⁇ M to 4 ⁇ M, 0.5 ⁇ M to 5 ⁇ M, 0.5 ⁇ M to 10 ⁇ M, 0.5 ⁇ M to 20 ⁇ M, 0.5 ⁇ M to 30 ⁇ M, 0.5 ⁇ M to 40 ⁇ M, 0.5 ⁇ M to 50 ⁇ M, 0.5 ⁇ M to 60 ⁇ M, 0.5 ⁇ M to 70 ⁇ M, 0.5 ⁇ M to 80 ⁇ M, 0.5 ⁇ M to 90 ⁇ M, 0.5 ⁇ M to 100 ⁇ M, 0.5 ⁇ M to 130 ⁇ M, 0.5 ⁇ M to 150 ⁇ M, 0.5 ⁇ M to 180 ⁇ M, 0.5 ⁇ M to 200 ⁇ M, 0.5 ⁇ M to 300 ⁇ M, 0.5 ⁇ M to 400 ⁇ M, 0.5 ⁇ M to 500 ⁇ M, 0.5 ⁇ M to 600 ⁇ M, 0.5 ⁇ M to 700 ⁇ M, 0.5 ⁇ M to 800 ⁇ M, 0.5 ⁇ M to
  • the dosage can be effective to produce a concentration of one or more compounds of 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 11 ⁇ M, 12 ⁇ M, 13 ⁇ M, 14 ⁇ M, 15 ⁇ M, 16 ⁇ M, 17 ⁇ M, 18 ⁇ M, 19 ⁇ M, 20 ⁇ M, 21 ⁇ M, 22 ⁇ M, 23 ⁇ M, 24 ⁇ M, 25 ⁇ M, 26 ⁇ M, 27 ⁇ M, 28 ⁇ M, 29 ⁇ M, 30 ⁇ M, 31 ⁇ M, 32 ⁇ M, 33 ⁇ M, 34 ⁇ M, 35 ⁇ M, 36 ⁇ M, 37 ⁇ M, 38 ⁇ M, 39 ⁇ M, 40 ⁇ M, 41 ⁇ M, 42 ⁇ M, 43 ⁇ M, 44 ⁇ M, 45 ⁇ M, 46 ⁇ M, 47 ⁇ M, 48 ⁇ M, 49 ⁇ M, 50 ⁇ M, 60 ⁇ M, 70 ⁇ M, 80 ⁇ M, 90 ⁇ M, 100 ⁇ M, 110 ⁇ M, 120 ⁇ M, 130 ⁇ M, 140 ⁇ M
  • the dosage can be effective to produce a concentration of one or more of the compounds within the range of 1 ⁇ M to 5 ⁇ M, 1 ⁇ M to 10 ⁇ M, 1 ⁇ M to 50 ⁇ M, 5 ⁇ M to 10 ⁇ M, 5 ⁇ M to 50 ⁇ M, 10 ⁇ M to 50 ⁇ M, 20 ⁇ M to 50 ⁇ M, 30 ⁇ M to 50 ⁇ M, 40 ⁇ M to 50 ⁇ M, 10 ⁇ M to 20 ⁇ M, 10 ⁇ M to 30 ⁇ M, 10 ⁇ M to 40 ⁇ M, 20 ⁇ M to 30 ⁇ M, 20 ⁇ M to 40 ⁇ M, or 30 ⁇ M to 40 ⁇ M in a body fluid of the subject.
  • the efficacy of administration of a particular dose of the compounds or compositions according to the methods described herein can be determined by evaluating the particular aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of influenza A virus or other diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field.
  • a subject for example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed) , (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.
  • the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
  • any of the compounds having the formula I can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • the compounds described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of compositions of the compounds described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • a compound or pharmaceutical composition described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • a compound or pharmaceutical composition described herein can be administered as an ophthalmic solution and/or ointment to the surface of the eye.
  • a compound or pharmaceutical composition can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. Parenteral administration, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's , or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.
  • Pharmaceutical compositions may be administered, for example, in a single dosage, as a continuous dosage, one or more times daily, or less frequently, such as once a week.
  • the pharmaceutical compositions can be administered once a day or more than once a day, such as twice a day, three times a day, four times a day or more. In certain forms, the compositions are administered orally, once daily or less.
  • the pharmaceutical compositions are administered in an effective amount and for an effective period of time to elicit the desired therapeutic benefit.
  • the pharmaceutical composition is administered for a period of at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer.
  • compositions may also be administered prophylactically, e.g., to patients or subjects who are at risk for infection.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, sex, weight and general condition of the subject, extent of the disease in the subject, route of administration, whether other drugs are included in the regimen, and the like. Thus, it is not possible to specify an exact dosages for every composition. However, an appropriate dosage can be determined by one of ordinary skill in the art using only routine experimentation. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • the compounds disclosed herein can be co-administered with one or more additional therapeutic, prophylactic, or diagnostic agents.
  • Co-administration includes administration within the same dosage form or within different dosage forms.
  • the dosage forms can be administered simultaneously (e.g., at the same time or essentially at the same time) or sequentially. “Essentially at the same time” as used herein generally means within ten minutes, preferably within five minutes, more preferably within two minutes, most preferably within in one minute.
  • Dosage forms administered sequentially can be administered within several hours of each other, e.g., with ten hours, nine hours, eight hours, seven hours, six hours, five hours, four hours, three hours, two hours, one hour, 30 minutes, 20 minutes, or 15 minutes.
  • MDCK Madin-Darby canine kidney
  • H1N1 Madin-Darby canine kidney
  • H3N2 Madin-Darby canine kidney
  • Influenza A virus subtypes A/WSN/33 [H1N1] , A/PR/8/34 [H1N1] were propagated in MDCK cell culture in either plain MEM supplemented with 0.2%FBS.
  • Other virus strains, including A/Hong Kong/415742/2009 [H1N1] , A/California/NHRC0007/2005 [H3N2] , and clinical isolate [H7N9] were propagated in either MEM or DMEM without FBS. All experiments involving live clinical isolate [H7N9] followed the standard operating procedures of the approved Biosafety Level 3 facility.
  • a Preliminary High throughput screening of the structurally diverse small molecule library was performed to identify an influenza A/WSN/1933 [H1N1] inhibitor. After the initial screening a total of 82 analogues of compound 82 were ordered for further testing. The analogues were tested against an influenza A/WSN/1933 [H1N1] and influenza A/California/NHRC0007/2005 [H3N2] .
  • PRA Plaque Reduction Assay
  • the PRA assay was performed in triplicate by seeding confluent MDCK cells in 24-well tissue culture plates (TPP, Switzerland) using MEM (Thermo Fisher Scientific, USA) in 10%FBS, prior to conducting the assay. After 16 to 24 h, cells were infected with either 50 plaque forming unit (P) of influenza virus for 24 well plates with or without the addition of serial diluted compounds. Infected cells were incubate at 37°C with 5%CO 2 for 1.5 h before removing unbound viral particles by aspiration.
  • P plaque forming unit
  • CellTiterGlo kit (Promega) was used to test the cell viability of selected compounds by detection of ATP levels as a function of cell viability according to the manufacturer’s instructions.
  • the assay was performed in triplicate by seeding MDCK cells at 20,000 cells/well in 100 ⁇ L MEM supplemented with 10%FBS in 96-well cell culture plates one day prior to conducting the assay. After 24 h, cells were washed once with 1 ⁇ PBS and replenished with MEM before addition of 2X serially diluted compounds mixed in MEM with 1%FBS, starting from 1 mM. The cells were then incubated at 37°C with 5%CO2 for 24 h.
  • mice were then subjected to intranasal inoculation with 30 LD 50 (6000 PFU) of the influenza A/PR/8/34 H1N1 virus in Zanamivir, FA-6005 or DMSO.
  • the infected mice were then treated with two doses per day of i.p. zanamivir, FA-6005 or DMSO solution for 7 days.
  • the mice were monitored for illness and survival rates for 14 days until death.
  • Five mice from each group were sacrificed 6 th day post-infection to determine viral titre and pathological changes in the lungs by plaque assay (half lung) .
  • Preliminary High throughput screening of the structurally diverse small molecule library identified Compound 82 to be an influenza A/WSN/1933 [H1N1] inhibitor.
  • a total of 81 analogues to compound 82 were ordered for further testing.
  • the analogues were tested against an influenza A/WSN/1933 [H1N1] , influenza A/California/NHRC0007/2005 [H3N2] and influenza A/Houston/21O2/2009 [H1N1 swine] results are shown in Table 1.
  • Compound 26 (FA-6005) was finalized for more in depth studies. Compound 26 showed desirable protection against an influenza A/WSN/1933 [H1N1] during the analogue screen and exhibited a low micromolar EC 50 against other strains of influenza A, including influenza A/Houston/21O2/2009 [H1N1 swine] , influenza A/Hong Kong/HKU38/2004 [H3N2] , influenza A/Puerto Rico/8/1934 [H1N1] , A/WSN/1933 [H1N1] , and influenza A/Hong Kong/clinical isolate [H7N9] ( Figure 1) .
  • Compound 26 was also tested for the CC 50 (Cytotoxic concentration 50%) and estimated to be around 200 ⁇ m in cell viability assay, giving this compound a good selective index. Since, Compound 26 (F-6005) exhibits broad spectrum antiviral activities against different strains of influenza, we speculate Compound 26 would be advantageous to inhibit possible new outbreaks of influenza.
  • Compound 26 (FA-6005) in vivo potency was evaluated in murine animal experiments. 5-7 weeks old female BALB/C mice were infected with influenza A/Puerto Rico/8/1934 [H1N1] , divided into three groups and subjected to different treatments twice for 7 days after infection. Survival rate was monitored, groups treated with existing neuraminidase inhibitor drug Zanamivir were 100%rescued, groups with DMSO treatment succumbed to infections starting from day 8 after exposed to the virus, and groups treated with Compound 26 showed 80%survival after the virus challenge. Compound 26 (FA-6005) also demonstrated a delay in time of death (Figure 2A) .
  • Table 1 Shows normalized viral protection of MDCK cells for each analog at 50 ⁇ M and 10 ⁇ M concentration against influenza A/WSN/1933 [H1N1] , and influenza A/California/NHRC0007/2005 [H3N2] .
  • IAV resistant to FA-6005 was generated by serial passages of A/WSN/1933 [H1N1] in MDCK cells in the presence of increasing concentration of FA-6005.
  • the A/WSN/1933 [H1N1] was also passaged in MDCK cells without the addition of FA-6005 as control.
  • the lower concentration series were first passage with 5 X the A/WSN/1933 [H1N1] initial EC 50 , the higher concentration being 10 X the initial EC 50 .
  • the desired FA-6005 resistant clones were purified by plaque isolation on MDCK cell monolayers in the presence of compound.
  • Viral RNA were purified from FA-6005 escape mutants by viral RNA extraction, complementary DNA (cDNA) of all 8 segments were obtained by reverse transcriptions using Superscript III reverse transcriptase (Invitrogen) . Genomic sequencing was conducted to identify any mutations.
  • Recombinant influenza virus was generated by pHW2000 eight-plasmid system by reverse genetics.
  • the eight plasmids contain the cDNA of the 8 segments of the influenza A/WSN/1933 [H1N1] genome: pHW2000-PB1, pHW2000-PB2, HW2000-PA, pHW2000-HA, pHW2000-NP, pHW2000-NA, pHW2000-M and pHW2000-NS. Drug resistant mutations were introduced to the parental plasmid using site-directed mutagenesis.
  • the array of plasmids were transfected into co-cultured293T and MDCK cells with TransLT-Oligo Transfection Reagent (Mirus) according to manufacturer's manual. The infectious particles from the supernatants were harvested at 72 hours post-transfection, and the recombinant virus titer was determined by plaque assay.
  • HEK293T cells were seeded in 96-wells plate at 2x10 4 Cells per well one day prior assay.
  • Components of the RNP complex consist of pHW2000-NP, plus pHW2000-PA, pHW2000-PBI, and pHW2000-PB2, or corresponding mutant plasmids were combined with a luciferase reporter plasmid, pHY-Luci.
  • the plasmids were co-transfected into cultured HEK293T cells using TransLT-Oligo Transfection Reagent (Mirus) according to manufacturer's manual. Plasmid phRL-TK (promega) expressing renilla luciferase, was co-transfected as internal control for data normalization.
  • MDCK Cells infected with influenza A/WSN/1933 [H1N1] at MOI 10 in fresh MEM supplemented with 0.2%FBS and 1 ⁇ g/mL TPCK-treated trypsin with 20 ⁇ M FA-6005 or DMSO were collected at 2, 4, 6, and 8 h post-infection. Unbounded viruses were removed and cells were washed once with PBS and lysed. The lysate were centrifuged and supernatants were collected, normalized to same amount and subjected to SDS-PAGE. The protein bands were transferred into nitrocellulose membrane, blocked with 10%w/v skim milk in PBST.
  • Membranes were incubated with appropriate dilutions of primary antibodies of ⁇ -H1N1 and GADPH in a blocking solution overnight, then washed with PBST three times before incubated with recommended dilution of conjugated secondary antibodies in a blocking solution. The membrane was washed three times with TBST, dried and imaged with Odyssey Imagine system (LI-COR) .
  • LI-COR Odyssey Imagine system
  • cells were harvested followed by extraction of viral RNAs using the RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. Standard curve of vRNA was generated for quantification of viral RNA.
  • Viral gene segments in the pHW2000 vector were amplified with T7 promoter and T7 terminator, and linearized PCR products were purified by using PureLink PCR Purification Kit (Thermo Fisher Scientific) .
  • RNA standard was synthesized from purified PCR product templates using the RiboMAX Large Scale RNA Production System-T7 kit (Promega Co. ) according to the manufacturer's manual.
  • DNA templates in RNA transcript were digested by DNase I (New England Biolab) at 37°C for IS min followed by a purification of the RNA transcript by using the RNeasy Mini Kit (QIAGEN) .
  • Concentrations of the RNA transcripts and extracted RNA were determined using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc. ) , molecular copies of the synthetic RNA were then calculated.
  • Reverse transcription was performed in the presence of saturated trehalose using PrimeScript RT-PCR Kit (Clontech) according to manufacturer's manual.
  • Real-time PCR qPCR was performed using a FastStart Universal SYBR Green Master (Roche Life Science) and monitored with ABI VIA7 (Thermo Fisher Scientific Inc. ) .
  • FISH Fluorescence in situ hybridization
  • IF Immunofluorescence microscopy
  • the slides preparation for simultaneous FISH and IF were carried out according to the manufacturer's instruction.
  • the slides were imaged with Leica DMIL inverted microscope with DC300F digital imaging system (Leica Microsystems) or LSM710 confocal microscopy (Carl Zeiss AG) .
  • MEM infection medium
  • l ⁇ g/ml TPCK-treated trypsin l ⁇ g/ml TPCK-treated trypsin
  • M1 protein For the detection of M1 protein, 2 h after the infection, the infected cells were fixed and permeabilized with 0.1%of Triton-X 100 in PBS and further incubated with M1 monoclonal antibody (Abcam) for 1 h. Cells were washed with PBS then incubated with secondary anti-mouse IgG-AF488. Nuclei were stained with ProLong Gold anti-fade reagent with DAPI (Thermo Fisher Scientific) . RNP import was detected at 3 h post infection; the infected cells were stained with NP monoclonal antibodies in PBS and further incubated with anti-mouse IgG-AF488 secondary antibody.
  • IgG mouse immunoglobulin G
  • the bounded magnetic beads were wash with washing buffer containing 50 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 0.5%NP40, and 10%glycerol.
  • Bounded proteins were eluted by boiling in SDS-PAGE sample buffer. The proteins were subjected to SDS-PAGE and Western blot.
  • the monolayer was washed with phosphate buffer, cells were scrapped into phosphate buffer and harvested by centrifugation. The supernatant was replaced with fresh phosphate buffer and processed by Electron Microscope Unit at HKU. Images were acquired by FEI Philips CM100 transmission electron microscope equipped with a Deben AMT digital camera and an EDAX Genesis XM4 EDX system.
  • Compound 82 and Compound 26 appear to inhibit influenza A virus by a mode of action different from other known antiviral drugs. Raising escape mutants against a drug is a rapid way to decipher its interacting partner, particularly in virus. Similar strategies were employed with gradual increasing of Compound 26 (FA-6005) concentration to generate resistant mutant virus from influenza A/WSN/1933 [H1N1] . The mutants had their genomes sequenced and mutation re-introduced into infectious particles by reverse genetics and tested against Compound 26 (FA-6005) to confirm the mutation sites. The substitution of isoleucine to threonine found on nucleoprotein amino acid41 was identified to be a highly resistant mutant against Compound 26 (FA-6005) in plaque reduction assays ( Figure 3) .
  • NP-Compound interaction was modeled with AutoDock Vina, using the published crystal structures of influenza A/WSN/1933 [H1N1] NP protein and Compound 26 (FA-6005) .
  • the docking model reveals that Compound 26 (FA-6005) could access into a pocket, which is in close proximity to residue 41, and mediate a hydrogen bond interaction with the isoleucine residue ( Figure 4) .
  • the threonine substitution might lead to abolishment of the interaction between Compound 26 (FA-6005) and the NP protein, resulting in the resistance of the compound.
  • NP protein is the most abundantly expressed protein during influenza infection, and serves multiple functions throughout the viral life cycle. Ranging from nuclear import of vRNPs, replication, transcription, to nuclear export of newly synthesized viral genome. Given that NP was likely the molecular target of Compound 26 (FA-6005) , it was speculated that Compound 26 (FA-6005) would exert inhibitory effect on the virus replication and trafficking. To differentiate the effect of the compound on the viral infection cycle, a time of addition experiments was conducted. Compounds were added at different time points, before and after exposure to virus. A strong inhibitory effect was observed from-1 to 6 h post infection, indicating that Compound 26 (FA-6005) , interferes with the early to late stage of the influenza virus ( Figure 5a) .
  • NP protein Apart from viral transcription and replication processes, NP protein also participates in the RNP translocation between cytoplasm and nucleus. FISH combined immunofluorescence imaging was used to visualize the migration of viral nucleoprotein as well as the vRNA and mRNA of segment 5 (containing NP) under the influence of Compound 26 (FA-6005) . The result indicated that the nuclear export process of vRNPs was inhibited at 8 h post-infection with the addition of Compound 26 (FA-6005) when compared to controls. Similar experiments with the NP 141 T mutants was not affected by Compound 26 (FA-6005) , the nuclear export process of the mutant virus was not inhibited.
  • Compound 26 (FA-6005) in inhibitory function with NP and its interactive site with the 41 isoleucine residue.
  • the addition of Compound 26 (FA-6005) will result in impaired virion budding process ( Figure 9) with the generation of defective virions.
  • This defective virion budding process can be correlated with the impede vRNP cytoplasmic trafficking, resulting in the decrease in titre output in TIA assay in the late stage of the viral infection.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about, ” it will be understood that the particular value forms another, specifically contemplated form that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.

Abstract

The present disclosure relates to compounds with antiviral activity, methods for the preparation ofsuch compounds, and their use to inhibit viral nucleoprotein activity, viral transcription, replication, and assembly into the newly formed virions.

Description

ANTIVIRAL COMPOUNDS AND METHODS OF USE THEREOF FIELD OF THE INVENTION
The disclosed invention is generally in the field of a class of compounds that inhibit viral infection, more specifically inhibitors of viral nucleoprotein, methods of making, and using thereof.
BACKGROUND OF THE INVENTION
Influenza A virus (IAV) , is the major causative agent for mortality and morbidity in pandemic and epidemic flu. It remains as the major threat to public health and global economy. The emergence of antiviral resistance strains to currently available drugs targeting M2 (Amantadine, Rimantadine) or NA (Oseltamivir, Zanamivir) were reported. Therefore, there is demand for new antiviral drugs to treat, combat IAV transmission, and prevent the future IAV pandemics.
IAV has a segmented genome consisting of eight RNA molecules that are individually encapsidated into vRNAP (RNA polymerase) . This represents the minimum sets of proteins for functional viral transcription and replications. It is an attractive strategy to develop antivirals that target nucleoprotein (NP) , as NP is the major structural protein in IAV with multiple functions throughout the virus infection life cycle. NP was previously reported to interact with RNA and oligomerize by self-interaction to form an oligomer to provide the structural role in RNP formation. Furthermore, NP is also required for viral transcription and replication. The major role of NP is to encapsidate RNA molecules into RNP complex and maintain the conformation of the RNA template in order to facilitate the viral transcription, replication, and assembly into the newly formed virions. NP or RNP is also known to translocate between the cytoplasm and nucleus with the help of other viral proteins and host factors.
There is a need for drugs and treatments that are effective against viral infections, such as influenza infections. There is an especial need for drugs and treatments that can be effective against a broad range of different strains of influenza A.
Therefore, it is an object of the present invention to provide a new class of compounds that treat, inhibit, reduce the risk of, or prevent viral infections.
It is a further object of the present invention to provide compositions, and methods of use thereof, treat, inhibit, reduce the risk of, or prevent viral infections.
It is also an object of the present invention to provide new inhibitors of NP and methods of using thereof.
BRIEF SUMMARY OF THE INVENTION
The present disclosure relates to compounds with antiviral activity, methods for the preparation of such compounds, and their use to inhibit viral nucleoprotein activity, viral transcription, viral replication, and assembly into the newly formed virions.
The compounds disclosed herein having the following formulas.
Figure PCTCN2019090301-appb-000001
wherein
A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring;
R 1 is
Figure PCTCN2019090301-appb-000002
wherein
G is independently C=O or SO 2;
R 2-R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 10) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 9; -CONHR 9) , tertiary amide (e.g., -NR 9COR 9; -CONR 9R 9) , secondary carbamate (e.g., -OCONHR 9; -NHCOOR 9) , tertiary carbamate (e.g., -OCONR 9R 9; -NR 9COOR 9) , urea (e.g., -NHCONHR 9; -NR 9CONHR 9, -NHCONR 9R 9, -NR 9CONR 9R 9) , thiourea (e.g., -NHCSNHR 8) , carbinol (e.g., -CH 2OH; -CHR 9OH, -CR 9R 9OH) , ester (e.g., -COOR 9) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 9) , tertiary amine (e.g., -NR 9R 9) , thioether (e.g., -SR 9) , sulfinyl  group (e.g., -SOR 9) , and sulfonyl group (e.g., -SOOR 9) , wherein R 8-R 10 are defined the same as R 2-R 7.
In some forms, R 4 of Formula I is the same as R 1.
In some forms, the compounds Formula II can have the following structures.
Figure PCTCN2019090301-appb-000003
E is independently O, S, N, NR 15, CR 16R 17, or C=O;
D and J are independently O, S, N, NR 15, CR 16R 17, or C=O;
L and M are independently O, S, N, NR 15, CR 16R 17, or C=O;
R 11 is
Figure PCTCN2019090301-appb-000004
wherein
G is independently C=O or SO 2;
R 12-R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 20) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 19; -CONHR 19) , tertiary amide (e.g., -NR 19COR 19; -CONR 19R 19) , secondary carbamate (e.g., -OCONHR 19; -NHCOOR 19) , tertiary carbamate (e.g., -OCONR 19R 19; -NR 19COOR 19) , urea (e.g., -NHCONHR 19; -NR 19CONHR 19, -NHCONR 19R 19, -NR 19CONR 19R 19) , thiourea (e.g., -NHCSNHR 18) , carbinol (e.g., -CH 2OH; -CHR 19OH, -CR 19R 19OH) , ester (e.g., -COOR 19) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 19) , tertiary amine (e.g., -NR 19R 19) ,  thioether (e.g., -SR 19) , sulfinyl group (e.g., -SOR 19) , and sulfonyl group (e.g., -SOOR 19) , wherein R 18-R 20 are defined the same as R 12-R 17.
In some forms, R 4 of Formula I can be a secondary amide (-NHCOR 9) , and R 9 can be one of the following structures:
Figure PCTCN2019090301-appb-000005
wherein
U is independently O, S, N, NR 24, CR 25R 26, or C=O;
Q and T are independently O, S, N, NR 27, CR 28R 29, or C=O;
V and W are independently O, S, N, NR 27, CR 28R 29, or C=O;
R 21-R 29 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 32) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 31; -CONHR 3) , tertiary amide (e.g., -NR 31COR 31; -CONR 31R 31) , secondary carbamate (e.g., -OCONHR 31; -NHCOOR 31) , tertiary carbamate (e.g., -OCONR 31R 31; -NR 31COOR 31) , urea (e.g., -NHCONHR 31; -NR 31CONHR 31, -NHCONR 31R 31, -NR 31CONR 31R 31) , thiourea (e.g., -NHCSNHR 30) , carbinol (e.g., -CH 2OH; -CHR 31OH, -CR 31R 31OH) , ester (e.g., -COOR 31) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 31) , tertiary amine (e.g., -NR 31R 31) , thioether (e.g., -SR 31) , sulfinyl group (e.g., -SOR 31) , and sulfonyl group (e.g., -SOOR 31) , wherein R 30-R 32 are defined the same as R 21-R 29.
The compounds disclosed herein can be in the form of a pharmaceutically acceptable salt or a prodrug thereof.
In one form, the one or more compounds can be in a pharmaceutical composition including and one or more pharmaceutically acceptable carriers.
In another form the pharmaceutical compositions can include one or more of the compounds, and can optionally include one or more pharmaceutically acceptable excipients. In some forms, the disclosed compounds are present in an effective amount to inhibit viral transcription. In some forms, the disclosed compounds are present in an effective amount to inhibit replication. In some forms, the disclosed compounds are present in an effective amount to inhibit viral protein synthesis. In some forms, the disclosed compounds are present in an effective amount to block vRNP export and/or suppress the virion budding process.
Also disclosed are methods of treating an infection including administering one or more of the disclosed compounds or compositions in an amount effective to treat a subject in need thereof.
An effective amount of the one or more compounds having the structure of Formula I or II, in this context, may be an effective amount that is enough of the one or more compounds to treat, inhibit, or prevent a viral infection, such as by influenza A virus, through transcription, replication, protein synthesis, vRNP export, and/or virion budding process.
In some forms, the method includes the one or more compounds are in an amount effective to inhibit an infection comprising: inhibiting viral transcription; inhibiting viral replication; inhibiting viral protein synthesis; blocking vRNP export; or inhibiting virion budding process; or inhibiting viral nucleoprotein activity, or a combination thereof.
In some forms, the infection treated by the methods disclosed herein with the compounds disclosed is a viral infection involving a viral nucleoprotein.
In some forms, the viral infection treat by the methods disclosed herein with the compounds disclosed is influenza A virus.
In some forms, the methods of administering one or more of the compounds by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration are disclosed herein.
The present provides the following technical solutions:
Solution 1. A compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof or a prodrug thereof:
Figure PCTCN2019090301-appb-000006
wherein
A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring;
R 1 is
Figure PCTCN2019090301-appb-000007
wherein
G is independently C=O or SO 2;
R 2-R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 2. The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein R 2-R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH, -COO -, -COR 10, -NH 2CO, -CONH 2, -CONHR 9, -CONR 9R 9, -NHCOR 9, -NR 9COR 9, -OCONHR 9; -NHCOOR 9, -OCONR 9R 9; -NR 9COOR 9, -NHCONHR 9; -NR 9CONHR 9, -NHCONR 9R 9, -NR 9CONR 9R 9, -NHCSNHR 8, -CH 2OH; -CHR 9OH, -CR 9R 9OH, -COOR 9, -SH, -NH 2, -NHR 9, -NR 9R 9, -SR 9, -SOR 9, and -SOOR 9;
wherein each R 8-R 10 of R 2-R 7 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 3. The compound of  solution  1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is of Formula I and R 4 is the same as R 1.
Solution 4. The compound of  solution  1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein Formula II is
Figure PCTCN2019090301-appb-000008
E is independently O, S, N, NR 15, CR 16R 17, or C=O;
D and J are independently O, S, N, NR 15, CR 16R 17, or C=O;
L and M are independently O, S, N, NR 15, CR 16R 17, or C=O;
R 11 is
Figure PCTCN2019090301-appb-000009
wherein G is independently C=O or SO 2;
each R 12-R 17 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano;  formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 5. The compound of solution 4 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 12-R 17 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH; -COO -; -COR 20; -NH 2CO; -CONH 2; -CONHR 19; -CONR 19R 19; -NHCOR 19; -NR 19COR 19; -OCONHR 19; -NHCOOR 19; -OCONR 19R 19; -NR 19COOR 19; -NHCONHR 19; -NR 19CONHR 19; -NHCONR 19R 19; -NR 19CONR 19R 19; -NHCSNHR 18; -CH 2OH; -CHR 19OH; -CR 19R 19OH; -COOR 19; -SH; -NH 2; -NHR 19; -NR 19R 19; -SR 19; -SOR 19; and -SOOR 19,
wherein each R 18-R 20 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 6. The compound of  solution  1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein when R 4 is -NHCOR 9, then R 9 is
Figure PCTCN2019090301-appb-000010
wherein
U is independently O, S, N, NR 24, CR 25R 26, or C=O;
Q and T are independently O, S, N, NR 27, CR 28R 29, or C=O;
V and W are independently O, S, N, NR 27, CR 28R 29, or C=O;
wherein each R 21-R 29 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 7. The compound of solution 6 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 21-R 29 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH; -COO -; -COR 32; -NH 2CO; -CONH 2; -CONHR 31; -CONR 31R 31; -NHCOR 31; -NR 31COR 31; -OCONHR 31; -NHCOOR 31; -OCONR 31R 31; -NR 31COOR 31; -NHCONHR 31; -NR 31CONHR 31; -NHCONR 31R 31; -NR 31CONR 31R 31; -NHCSNHR 30; -CH 2OH; -CHR 31OH; -CR 31R 31OH; -COOR 31; -SH;-NH 2; -NHR 31; -NR 31R 31; -SR 31; -SOR 31; and -SOOR 31,
wherein each R 30-R 32 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
Solution 8. The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is a compound selected from the group consisting of:
Figure PCTCN2019090301-appb-000011
Figure PCTCN2019090301-appb-000012
Figure PCTCN2019090301-appb-000013
Figure PCTCN2019090301-appb-000014
Figure PCTCN2019090301-appb-000015
Figure PCTCN2019090301-appb-000016
Figure PCTCN2019090301-appb-000017
Figure PCTCN2019090301-appb-000018
Figure PCTCN2019090301-appb-000019
Figure PCTCN2019090301-appb-000020
Figure PCTCN2019090301-appb-000021
Figure PCTCN2019090301-appb-000022
Figure PCTCN2019090301-appb-000023
Figure PCTCN2019090301-appb-000024
Figure PCTCN2019090301-appb-000025
Figure PCTCN2019090301-appb-000026
Figure PCTCN2019090301-appb-000027
Figure PCTCN2019090301-appb-000028
Figure PCTCN2019090301-appb-000029
Solution 9. The compound of solution 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound has the structure:
Figure PCTCN2019090301-appb-000030
Solution 10. A pharmaceutical composition comprising one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof and one or more pharmaceutically acceptable carriers.
Solution 11. The pharmaceutical composition of solution 10, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof are present in an effective amount to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, or a combination thereof.
Solution 12. The pharmaceutical composition of  solution  10 or 11 further comprising one or more pharmaceutically acceptable excipients.
Solution 13. A method of treating a subject in need thereof, the method comprising administering one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition of any of solutions 10-12 in an amount effective to treat the subject in need thereof.
Solution 14. The method of solution 13, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
Solution 15. The method of  solution  13 or 14, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
Solution 16. The method of solution 15, wherein the viral infection is an influenza A viral infection.
Solution 17. The method of any one of solutions 13-16, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
Solution 18. The method of any one of solutions 13-17, wherein the subject has a viral infection.
Solution 19. The method of any one of solutions 13-17, wherein the subject is at risk of developing a viral infection.
Solution 20. The method of solution 18 or 19, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
Solution 21. Use of one or more compounds of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition of any of solutions 10-12 in manufacture of a medicament for treating a subject in need thereof.
Solution 22. Use of solution 21, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
Solution 23. Use of solution 21 or 22, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
Solution 24. Use of solution 23, wherein the viral infection is an influenza A viral infection.
Solution 25. Use of any one of solutions 21-24, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
Solution 26. Use of any one of solutions 21-25, wherein the subject has a viral infection.
Solution 27. Use of any one of solutions 21-25, wherein the subject is at risk of developing a viral infection.
Solution 28. Use of solution 26 or 27, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
Solution 29. Compound of any one of solutions 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof for use in treating a subject in need thereof.
Solution 30. Compound according to claim 29 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
Solution 31. Compound according to claim 29 or 30 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
Solution 32. Compound according to claim 31 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the viral infection is an influenza A viral infection.
Solution 33. Compound according to any one of solutions 29-32 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
Solution 34. Compound according to any one of solutions 29-33 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject has a viral infection.
Solution 35. Compound according to any one of solutions 29-33 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the subject is at risk of developing a viral infection.
Solution 36. Compound according to solution 34 or 35 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound or a pharmaceutically acceptable salt thereof or a prodrug thereof inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
Solution 37. The composition according to any of solutions 10-12 for use in treating a subject in need thereof.
Solution 38. The composition according to claim 37, wherein the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
Solution 39. The composition according to claim 37 or 38, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
Solution 40. The composition according to claim 39, wherein the viral infection is an influenza A viral infection.
Solution 41. The composition according to any one of solutions 37-40, wherein the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
Solution 42. The composition according to any one of solutions 37-41, wherein the subject has a viral infection.
Solution 43. The composition according to any one of solutions 37-41, wherein the subject is at risk of developing a viral infection.
Solution 44. The composition according to solution 42 or 43, wherein the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an EC 50 curve showing the broad spectrum antiviral activities of FA-6005 against A/WSN/1933, A/Hong Kong/HKU38/2004 [H3N2] , A/Houston/21OS/2009 [H1N1] and H7N9 clinical isolate with an EC 50 of 2.82±0.33μM, 1.51±0.58μM, 6.22±0.07μM and 1.37±0.10μM, respectively.
Figure 2A-B shows antiviral activities of Compound 26 (FA-6005) (A) Efficacies of FA-6005 in murine influenza A/Puerto Rico/8/1934 [H1N1] infection model. (B) Zanamivir and FA-6005 reduced the viral load in the lungs.
Figure 3 shows FA-6005 is targeting Influenza A NP. Escape mutant virus and recombinant virus carrying the I41T substitution in influenza A NP confer resistance to high concentrations of FA-6005. Antiviral activities of FA-6005 against A/WSN/33 virus, I41T escape mutant virus or I41T variant virus generated by reverse genetics were determined by PRA.
Figure 4A-B shows FA-6005 inhibits virus transcription and replication. (A) Time-of-addition experiments examining the effect of FA-6005 on various stages of IAV life cycle. (B) FA-6005 exhibit inhibition of the parental virus NP activity, but not the resistant I41T variant virus NP in a luciferase reporter assay.
Figure 5 shows FA-6005 inhibits all kinds of viral RNAs synthesis. Total cellular RNA was collected at 2, 4, 6, and 8 hpi from MDCK cells infected with A/WSN/33 virus NP or I41T variant NP virus at MOI=10.
Figure 6 shows FA-6005 abolishes viral protein synthesis. MDCK cells were infected with IAV at MOI=10 in the presence of 20μM FA-6005. DMSO was added as a negative control. Cell lysates were collected at 2, 4, 6, and 8 hpi, and further analyzed by western blotting.
DETAILED DESCRIPTION OF THE INVENTION
This application describes compounds that were screened from a compound library along with its derivatives and identified as influenza virus inhibitors. The target of these compounds was shown to be nucleoprotein, which played multiple roles in Influenza A virus infection cycle.
In particular, small molecule inhibitors of influenza A viral nucleoprotein and methods of using thereof are disclosed.
A. Definitions
“Analog” and “Derivative, ” are used herein interchangeably, and refer to a compound that possesses the same core as a parent compound, but differs from the parent compound in bond order, the absence or presence of one or more atoms and/or groups of atoms, and combinations thereof.  The derivative can differ from the parent compound, for example, in one or more substituents present on the core, which may include one or more atoms, functional groups, or substructures. The derivative can also differ from the parent compound in the bond order between atoms within the core. In general, a derivative can be imagined to be formed, at least theoretically, from the parent compound via chemical and/or physical processes.
“Co-administration, ” as used herein, includes simultaneous and sequential administration. An appropriate time course for sequential administration may be chosen by the physician, according to such factors as the nature of a patient’s illness, and the patient’s condition.
“Pharmaceutically acceptable, ” as used herein, refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.
“Prodrug, ” as used herein, refers to a pharmacological substance (drug) that is administered to a subject in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized in the body (in vivo) into a compound having the desired pharmacological activity.
The term "therapeutically effective amount" refers to an amount of the therapeutic agent that, when incorporated into and/or onto the self-assembled gel composition, produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, or the severity of the disease or condition.
A carboxylic acid is the group–COOH. Unless specified otherwise the term carboxylic acid embraces both the free acid and carboxylate salt.
An alkyl is the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1-C 30 for straight chain, C 3-C 30 for branched chain) , more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. In some forms, the chain has 1-6 carbons. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The  ranges provided above are inclusive of all values between the minimum value and the maximum value.
The terms “high, ” “higher, ” “increases, ” “elevates, ” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low, ” “lower, ” “reduces, ” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
The term “monitoring” as used herein refers to any method in the art by which an activity can be measured.
The term “providing” as used herein refers to any means of adding a compound or molecule to something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo.
The term “preventing” as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case ofhumans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver’s expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.
As used herein, “subject” includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity. The subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent) , a fish, a bird or a reptile or an amphibian. The subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans) . The term does not denote a particular age  or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.
By “treatment” and "treating" is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitiative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
A cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject. A “cell” can be a cell from any organism including, but not limited to, a bacterium.
In one aspect, the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
By the term “effective amount” of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount. ” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
The term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls, ” the latter of which refers to alkyl moieties having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl) , thiocarbonyl (such as a thioester, a thioacetate, or a thioformate) , alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, “lower alkyl” refers to an alkyl group having from one to ten carbons, more preferably from one to six carbon atoms, in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls.
The alkyl groups may also contain one or more heteroatoms within the carbon backbone. Examples include oxygen, nitrogen, sulfur, and combinations thereof. In certain forms, the alkyl group contains between one and four heteroatoms.
Alkenyl and alkynyl refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C 2-C 30) and possible substitution to the alkyl groups described above.
Aryl refers to 5-, 6-and 7-membered aromatic rings. The ring may be a carbocyclic, heterocyclic, fused carbocyclic, fused heterocyclic, bicarbocyclic, or biheterocyclic ring system, optionally substituted as described above for alkyl. Broadly defined, “Ar, ” as used herein, includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms. Examples include, but are not limited to, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Those aryl groups having heteroatoms in the ring structure may also be referred to as “heteroaryl, ” “aryl heterocycles, ” or “heteroaromatics. ” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, --CF 3, and --CN. The term “Ar” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings” ) wherein at least one of the rings is aromatic, e.g.,  the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles, or both rings are aromatic.
Alkylaryl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group) .
Heterocycle or heterocyclic refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, containing carbon and one to four heteroatoms each selected from non-peroxide oxygen, sulfur, and N (Y) wherein Y is absent or is H, O, (C 1-4) alkyl, phenyl or benzyl, and optionally containing one or more double or triple bonds, and optionally substituted with one or more substituents. The term “heterocycle” also encompasses substituted and unsubstituted heteroaryl rings. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H, 6H-1, 5, 2-dithiazinyl, dihydrofuro [2, 3-b] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1, 2, 5-thiadiazinyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, 1, 3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl.
Heteroaryl refers to a monocyclic aromatic ring containing five or six ring atoms containing carbon and 1, 2, 3, or 4 heteroatoms each selected from non-peroxide oxygen, sulfur, and N (Y) where Y is absent or is H, O, (C 1-C 8) alkyl, phenyl, or benzyl. Non-limiting examples ofheteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide) , thienyl, pyrimidinyl (or its N- oxide) , indolyl, isoquinolyl (or its N-oxide) , quinolyl (or its N-oxide) and the like. The term "heteroaryl" can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto. Examples ofheteroaryl include, but are not limited to, furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide) , thientyl, pyrimidinyl (or its N-oxide) , indolyl, isoquinolyl (or its N-oxide) , quinolyl (or its N-oxide) , and the like.
As used herein, the term "nitro" means-NO 2. Halogen refers to fluorine, chlorine, bromine, or iodine.
The term “substituted” refers to all permissible substituents of the compounds described herein. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms, such as oxygen, sulfur, or nitrogen, grouping in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C 3-C 20 cyclic, substituted C 3-C 20 cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, and polypeptide groups.
Heteroatoms, such as nitrogen, may have hydrogen substituents and/or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. “Substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
Numerical ranges disclosed herein disclose individually each possible number in such range, as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example, acarbon range (i.e., C 1-C 10) is intended to disclose individually every possible carbon value and/or  sub-range encompassed within. For example, a carbon length range of C 1-C 10 discloses C 1, C 2, C 3, C 4, C 5, C 6, C 7, C 8, C 9, and C 10, as well as discloses sub-ranges encompassed within, such as C 2-C 9, C 3-C 8, C 1-C 5, etc. Similarly, an integer value range of 1-10 discloses the individual values of 1, 2, 3, 4, 5, 6, 7, 8, and 10, as well as sub-ranges encompassed within. Further, a concentration range or weight percent range or volume percent range, such as from l%to 2%by weight of the composition, discloses the individual values and fractions thereof, such as 1%, 1.1%, 1.2%, 1.32%, 1.48%etc., as well as sub-ranges encompassed within.
B. Compounds
Compounds having Formula I and methods of using are described herein.
Figure PCTCN2019090301-appb-000031
wherein
R 1 is
Figure PCTCN2019090301-appb-000032
wherein
G is independently C=O, or SO 2;
R 2-R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 10) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 9; -CONHR 9) , tertiary amide (e.g., -NR 9COR 9; -CONR 9R 9) , secondary carbamate (e.g., -OCONHR 9; -NHCOOR 9) , tertiary carbamate (e.g., -OCONR 9R 9; -NR 9COOR 9) , urea (e.g., -NHCONHR 9; -NR 9CONHR 9, -NHCONR 9R 9, -NR 9CONR 9R 9) , thiourea (e.g., -NHCSNHR 8) ,  carbinol (e.g., -CH 2OH; -CHR 9OH, -CR 9R 9OH) , ester (e.g., -COOR 9) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 9) , tertiary amine (e.g., -NR 9R 9) , thioether (e.g., -SR 9) , sulfinyl group (e.g., -SOR 9) , and sulfonyl group (e.g., -SOOR 9) , wherein R 8-R 10 are defined the same as R 1-R 7.
In some forms, for R 1, G is C=O of Formula I.
In some forms, for R 1, G is C=O and R 7 is hydrogen or lower alkyl, such as methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl, at the ortho, meta, or para position. In other forms, R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position of Formula I, and R 4 is the same as R 1.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is the same as R 1, and R 3 or R 6 and R 5 or R 3 are alkoxy, such as methoxy.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, and R 2 or R 6 and R 5 or R 3 are alkoxy, such as methoxy.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 3 or R 5 and R 6 or R 2 are halogen, such as Cl, and R 4 is nitro.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 2 or R 6 is a halogen, such as Cl, or an alkoxy, such as methoxy, and R 4 is nitro.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, and R 4 is a heteroaryl, such as furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, pyridyl, (or its N-oxide) , thienyl, pyrimidinyl (or its N-oxide) , indolyl, isoquinolyl (or its N-oxide) , or quinolyl (or its N-oxide) .
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, and R 4 is a heterocyclic, such as pyrrolidinyl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, and R 4 is not a tetrazole.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a primary amine.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a primary amine, and R 2 or R 6 is hydroxy.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (-NHCOR 9) , R 9 is an alkyl, such as a methyl, ethyl, or propyl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted alkenyl, such as an ethenylbenzene.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted heteroaryl, such as a substituted or unsubstituted furanyl, pyridine, or benzothiophenyl. The benzothiphenyl can be further substituted with a halogen, such as Cl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 2 or R 6 is an alkoxy, such as a methoxy, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted heteroaryl, such as a substituted or unsubstituted benzofuranyl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 3 or R 5 is an alkoxy, such as a methoxy, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted aryl, such as a substituted or unsubstituted furan.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted alkoxy, such as a methoxy the methoxy can be substituted with a substituted aryl, such as a phenyl, the phenyl can be further substituted with a halogen, such as Cl, or a nitro at the ortho position or an alkyl, such as a methyl at the para position.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (-NHCOR 9) , R 9 is a substituted alkyl, such as a methyl the methyl can be substituted with a substituted aryl, such as a phenyl, the phenyl can be further substituted with an alkoxy, such as a methoxy at the meta and para positions.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 3 or R 5 and R 6 or R 2 are alkoxy, such as methoxy or ethoxy, R 4 is a secondary amide (-NHCOR 9) , R 9 is an aryl, such as a phenyl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 5 or R 3 is an alkoxy, such as a methoxy, R 2 or R 6 is halogen, such as a Cl, R 4 is a secondary amide (-NHCOR 9) , R 9 is an aryl, such as a phenyl.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (–NHCOR 9) , R 9 is an aryl, such as a substituted or unsubstituted phenyl. In some forms, the R 9 phenyl is substituted with alkoxy, halogen, nitro, alkyl, and/or a combination thereof.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 3 or R 5 is halogen, such as Cl, and R 4 is a substituted or unsubstituted heterocyclic, such as pyrrolidinyl. In some forms, R 4 is a substituted or unsubstituted heterocyclic, such as:
Figure PCTCN2019090301-appb-000033
X is N, O, S, or C;
Y is N, O, SO 2, C=O, NR 37, or CR 38R 39;
R 33-R 39 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 42) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 41; -CONHR 41) , tertiary amide (e.g., -NR 41COR 41; -CONR 41R 41) , secondary carbamate (e.g., -OCONHR 41; -NHCOOR 41) , tertiary carbamate (e.g., -OCONR 41R 41; -NR 41COOR 41) , urea (e.g., -NHCONHR 41; -NR 41CONHR 41, -NHCONR 41R 41, -NR 41CONR 41R 41) , thiourea (e.g., -NHCSNHR 40) , carbinol (e.g., -CH 2OH; -CHR 41OH, -CR 41R 41OH) , ester (e.g., -COOR 41) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 41) , tertiary amine (e.g., -NR 41R 41) , thioether (e.g., -SR 41) , sulfinyl group (e.g., -SOR 41) , and sulfonyl group (e.g., -SOOR 41) , wherein R 40-R 42 is defined the same as R 33-R 39.
In some forms, for the R 4 substituted heterocyclic, X is N, Y is NR 37, R 37 is a substituted alkyl, such as a methyl substituted with a substituted phenyl.
In some forms, for the R 4 substituted heterocyclic, X is N, Y is NR 37, R 37 is a substituted alkyl, such as a methyl substituted with a substituted phenyl with a halogen, such as F, at the ortho position.
In some forms, for the R 4 substituted heterocyclic, X is N, Y is NR 37, R 37 is a substituted carbonyl (-COR 42) , R 42 is a substituted phenyl with an alkyl, such as methyl at the ortho position.
In some forms, Formula I has the following structure:
Figure PCTCN2019090301-appb-000034
wherein
R 9 is
Figure PCTCN2019090301-appb-000035
R 40-R 47 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 50) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 49; -CONHR 49) , tertiary amide (e.g., -NR 49COR 49; -CONR 49R 49) , secondary carbamate (e.g., -OCONHR 49; -NHCOOR 49) , tertiary carbamate (e.g., -OCONR 49R 49; -NR 49COOR 49) , urea (e.g., -NHCONHR 49; -NR 49CONHR 49, -NHCONR 49R 49, -NR 49CONR 49R 49) , thiourea (e.g., -NHCSNHR 48) , carbinol (e.g., -CH 2OH; -CHR 49OH, -CR 49R 49OH) , ester (e.g., -COOR 49) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 49) , tertiary amine (e.g., -NR 49R 49) , thioether (e.g., -SR 49) , sulfinyl group (e.g., -SOR 49) , and sulfonyl group (e.g., -SOOR 49) , wherein R 48-R 50 is defined the same as R 2-R 6.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (–NHCOR 9) , R 9 is an aryl, such as a substituted or unsubstituted phenyl. R 9 phenyl is substituted at R 44 or R 46 with a halogen, such as Br or Cl. In some forms, the R 9 phenyl is substituted at R 43 or R 47and R 45 with a halogen, such as Cl. In some forms, the R 9 phenyl is  substituted at R 43 or R 47 with an alkoxy, such as a methoxy, and at R 46 or R 44 with a halogen, such as Cl. In some forms, the R 9 phenyl is substituted at R 46 or R 44 with nitro and at R 43 or R 47 with a halogen, such as Cl. In some forms, the R 9 phenyl is substituted at R 44 or R 46 with nitro and at R 43 or R 47 with an alkyl, such as methyl. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with a halogen, such as Cl, and at R 45 with nitro. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with an alkyl, such as methyl, and at R 42 with an alkyl, such as methyl. In some forms, the R 9 phenyl is substituted at R 43 and R 47 with an alkoxy, such as a methoxy. In some forms, the R 9 phenyl is substituted at R 43 or R 47 with a halogen, such as F, and at R 45 with a cyano. In some forms, the R 9 phenyl is substituted at R 44 or R 46 with an alkyl, such as methyl, and at R 43 or R 47 with an alkoxy, such as methoxy.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a secondary amide (–NHCOR 9) , R 9 is an aryl or a heteroaryl, such as:
Figure PCTCN2019090301-appb-000036
wherein
U is independently O, S, N, NR 24, CR 25R 26, or C=O;
Q and T are independently O, S, N, NR 27, CR 28R 29, or C=O;
V and W are independently O, S, N, NR 27, CR 28R 29, or C=O;
R 21-R 29 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 32) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 31; -CONHR 31) , tertiary amide (e.g., -NR 31COR 31; -CONR 31R 31) , secondary carbamate (e.g., -OCONHR 31; -NHCOOR 31) , tertiary carbamate (e.g., -OCONR 31R 31; -NR 31COOR 31) , urea (e.g., -NHCONHR 31; -NR 31CONHR 31, -NHCONR 31R 31, -NR 31CONR 31R 31) , thiourea (e.g., -NHCSNHR 30) , carbinol (e.g., -CH 2OH; -CHR 31OH, -CR 31R 31OH) , ester (e.g., -COOR 31) ,  thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 31) , tertiary amine (e.g., -NR 31R 31) , thioether (e.g., -SR 31) , sulfinyl group (e.g., -SOR 31) , and sulfonyl group (e.g., -SOOR 31) , wherein R 30-R 32 are defined the same as R 21-R 29.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 4 and R 5 are hydrogen, and R 21, R 22, and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkyl, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is hydrogen, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkyl, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 and R 26 are hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is halogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is halogen, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is halogen, and R 21, R 22, and R 23 are halogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is hydrogen, and R 21, R 22, or R 23 are halogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is halogen, and R 21, R 22, or R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkoxy, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkoxy, and R 21, R 22, and R 23 are alkoxy.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkoxy, and R 21, R 22, or R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkoxy, and R 21, R 22, or R 23 are halogen.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is hydrogen, and R 21, R 22, and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is alkyl, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is halogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is halogen, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is halogen, and R 21, R 22, and R 23 a halogen.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is alkoxy, and R 21, R 22, and R 23 hydrogen.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is alkoxy, and R 21, R 22, or R 23 are alkoxy.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is alkoxy, and R 21, R 22, or R 23 are alkyl.
In some forms, for R 9 Q is N, T is NR 27, U is C=O, R 27 is alkoxy, and R 21, R 22, or R 23 are halogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 and R 29 are hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 and R 29 are hydrogen, and R 21, R 22, and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is alkyl, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29 R 28 orR 29 is hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is alkyl, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is halogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is halogen, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is halogen, and R 21, R 22, and R 23 are halogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 or R 29 is halogen, R 21, R 22, or R 23 are halogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 orR 29 is alkoxy, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 orR 29 is alkoxy, and R 21, R 22, and R 23 are alkoxy.
In some forms, for R 9 Q is O, T is O, U is CR 25R 26, R 25 or R 26 is alkoxy, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, V and W are CR 28R 29, R 28 orR 29 is alkoxy, and R 21, R 22, and R 23 are halogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 are hydrogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 are hydrogen, and R 21, R 22, and R 23 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is alkyl, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is halogen, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is halogen, and R 21, R 22, and R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is halogen, and R 21, R 22, and R 23 are halogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is alkoxy, and R 21, R 22, and R 23 are hydrogen.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is alkoxy, and R 21, R 22, or R 23 are alkoxy.
In some forms, for R 9 Q is O, T is O, U is NR 24, R 24 is alkoxy, and R 21, R 22, or R 23 are alkyl.
In some forms, for R 9 Q is O, T is O, V and W are NR 27, R 27 is alkoxy, and R 21, R 22, or R 23 are halogen.
In some other forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a thiourea (–NHCSNHR 8) , R 8 is a carbonyl (-COR 10) , R 10 is a substituted or unsubstituted alkyl, such as a substituted or unsubstituted methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some forms, the R 10 is isobutyl or tert-butyl.
In some other forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a thiourea (–NHCSNHR 8) , R 8 is a carbonyl (-COR 10) , R 10 is an aryl, such as a substituted or unsubstituted phenyl. In some forms, the R 10 phenyl is substituted with alkoxy, halogen, nitro, alkyl, and/or a combination thereof.
In some other forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a thiourea (–NHCSNHR 8) , R 8 is a carbonyl (-COR 10) , R 10 is an heteroaryl, such as a substituted or unsubstituted benzofuranyl.
In some forms, Formula I has the following structure.
Figure PCTCN2019090301-appb-000037
wherein
R 10 is
Figure PCTCN2019090301-appb-000038
R 51-R 55 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 58) , primary amide  (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 57; -CONHR 57) , tertiary amide (e.g., -NR 57COR 57; -CONR 57R 57) , secondary carbamate (e.g., -OCONHR 57; -NHCOOR 57) , tertiary carbamate (e.g., -OCONR 57R 57; -NR 57COOR 57) , urea (e.g., -NHCONHR 56; -NR 57CONHR 57, -NHCONR 57R 57, -NR 57CONR 57R 57) , thiourea (e.g., -NHCSNHR 57) , carbinol (e.g., -CH 2OH; -CHR 57OH, -CR 57R 57OH) , ester (e.g., -COOR 57) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 57) , tertiary amine (e.g., -NR 57R 57) , thioether (e.g., -SR 57) , sulfinyl group (e.g., -SOR 57) , and sulfonyl group (e.g., -SOOR 57) , wherein R 56-R 58 are defined the same as R 51-R 55.
In some other forms, for R 1, G is C=O and R 7 is tert-butyl at the para position, R 4 is a thiourea (–NHCSNHR 8) , R 8 is a carbonyl (-COR 10) , R 10 is an aryl, such as a substituted or unsubstituted phenyl. R 10 phenyl is substituted at R 51 or R 55 with a halogen, such as Cl. In some forms, the R 10 phenyl is substituted at R 51 or R 55and R 53 with a halogen, such as Cl. In some forms, the R 10 phenyl is substituted at R 51 or R 55 with an alkoxy, such as a methoxy, and at R 54 or R 52 with a halogen, such as Cl. In some forms, the R 10 phenyl is substituted at R 53 with an alkoxy, such as propoxy. In some forms, the R 10 phenyl is substituted at R 54 or R 52 with nitro and at R 55 or R 51 with an alkyl, such as methyl. In some forms, the R 10 phenyl is substituted at R 51 and R 55 with an alkoxy, such as methoxy. In some forms, the R 10 phenyl is substituted at R 52 and R 54 with an alkyl, such as methyl. In some forms, the R 10 phenyl is substituted at R 53 with an alkyl, such as ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl. In some forms, the R 10 phenyl is not a methyl at R 53.
Compounds having Formula II and methods of using are described herein.
Figure PCTCN2019090301-appb-000039
wherein
R 1 is
Figure PCTCN2019090301-appb-000040
A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring;
G is independently C=O, or SO 2;
R 2-R 7 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 10) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 9; -CONHR 9) , tertiary amide (e.g., -NR 9COR 9; -CONR 9R 9) , secondary carbamate (e.g., -OCONHR 9; -NHCOOR 9) , tertiary carbamate (e.g., -OCONR 9R 9; -NR 9COOR 9) , urea (e.g., -NHCONHR 9; -NR 9CONHR 9, -NHCONR 9R 9, -NR 9CONR 9R 9) , thiourea (e.g., -NHCSNHR 8) , carbinol (e.g., -CH 2OH; -CHR 9OH, -CR 9R 9OH) , ester (e.g., -COOR 9) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 9) , tertiary amine (e.g., -NR 9R 9) , thioether (e.g., -SR 9) , sulfinyl group (e.g., -SOR 9) , and sulfonyl group (e.g., -SOOR 9) , wherein R 8-R 10 are defined the same as R 1-R 7.
In some forms, for R 1, G is C=O of Formula II.
In some forms, for R 1, G is C=O and R 7 is hydrogen or lower alkyl, such as methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl, at the ortho, meta, or para position. In other forms, R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
In some forms, for R 1, G is C=O and R 7 is tert-butyl at the para position of Formula II.
In some forms, Formula II has the following structure.
Figure PCTCN2019090301-appb-000041
E is independently O, S, N, NR 15, CR 16R 17, or C=O;
D and J are independently O, S, N, NR 15, CR 16R 17, or C=O;
R 11 is
Figure PCTCN2019090301-appb-000042
wherein
G is independently C=O or SO 2;
R 12-R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 20) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 19; -CONHR 19) , tertiary amide (e.g., -NR 19COR 19; -CONR 19R 19) , secondary carbamate (e.g., -OCONHR 19; -NHCOOR 19) , tertiary carbamate (e.g., -OCONR 19R 19; -NR 19COOR 19) , urea (e.g., -NHCONHR 19; -NR 19CONHR 19, -NHCONR 19R 19, -NR 19CONR 19R 19) , thiourea (e.g., -NHCSNHR 18) , carbinol (e.g., -CH 2OH; -CHR 19OH, -CR 19R 19OH) , ester (e.g., -COOR 19) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 19) , tertiary amine (e.g., -NR 19R 19) ,  thioether (e.g., -SR 19) , sulfinyl group (e.g., -SOR 19) , and sulfonyl group (e.g., -SOOR 19) , wherein R 18-R 20 are defined the same as R 12-R 17.
In some forms, for R 11, G is C=O of Formula IIa.
In some forms, for R 11, G is C=O and R 7 is hydrogen or lower alkyl, such as methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl, at the ortho, meta, or para position. In other forms, R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position of Formula IIa.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 and R 17 are hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 and R 17 are hydrogen, and R 12, R 13, and R 14 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkyl, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkyl, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is hydrogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are alkoxy.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, E is CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is hydrogen, and R 12, R 13, and R 14 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is alkyl, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is hydrogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is halogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is alkoxy, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is alkoxy, and R 12, R 13, or R 14 are alkoxy.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is alkoxy, and R 12, R 13, or R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is N, J is NR 15, E is C=O, R 15 is alkoxy, and R 12, R 13, or R 14 are halogen.
In some forms, Formula II has the following structure.
Figure PCTCN2019090301-appb-000043
E is independently O, S, N, NR 15, CR 16R 17, or C=O;
D and J are independently O, S, N, NR 15, CR 16R 17, or C=O;
L and M are independently O, S, N, NR 15, CR 16R 17, or C=O;
R 11 is
Figure PCTCN2019090301-appb-000044
wherein
G is independently C=O or SO 2;
R 12-R 17 is independently absent or selected from hydrogen, halogen, nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxy, cyano, formyl, acyl, carboxylic acid (-COOH) , carboxylate (-COO -) , carbonyl (e.g., -COR 20) , primary amide (e.g., -NH 2CO; -CONH 2) , secondary amide (e.g., -NHCOR 19; -CONHR 19) , tertiary amide (e.g., -NR 19COR 19; -CONR 19R 19) , secondary carbamate (e.g., -OCONHR 19; -NHCOOR 19) , tertiary carbamate (e.g., -OCONR 19R 19; -NR 19COOR 19) , urea (e.g., -NHCONHR 19; -NR 19CONHR 19, -NHCONR 19R 19, -NR 19CONR 19R 19) , thiourea (e.g., -NHCSNHR 18) , carbinol (e.g., -CH 2OH; -CHR 19OH, -CR 19R 19OH) , ester (e.g., -COOR 19) , thiol (-SH) , primary amine (-NH 2) , secondary amine (e.g., -NHR 19) , tertiary amine (e.g., -NR 19R 19) , thioether (e.g., -SR 19) , sulfinyl group (e.g., -SOR 19) , and sulfonyl group (e.g., -SOOR 19) , wherein R 18-R 20 are defined the same as R 12-R 17.
In some forms, for R 1, G is C=O of Formula IIb.
In some forms, for R 11, G is C=O and R 7 is hydrogen or lower alkyl, such as methyl, ethyl, n-propyl, or isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl, at the ortho, meta, or para position. In other forms, R 7 is substituted with a lower alkyl, such as tert-butyl at the para position.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position of Formula IIb.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 and R 17 are hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 and R 17 are hydrogen, and R 12, R 13, and R 14 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkyl, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkyl, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is hydrogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are alkoxy.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are CR 16R 17, R 16 or R 17 is alkoxy, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 are hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 are hydrogen, and R 12, R 13, and R 14 are alkyl, such as methyl, ethyl, propyl, butyl, isobutyl, or tert-butyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is alkyl, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 are hydrogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is halogen, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is halogen, and R 12, R 13, and R 14 are halogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is halogen, and R 12, R 13, and R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is alkoxy, and R 12, R 13, and R 14 are hydrogen.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is alkoxy, and R 12, R 13, and R 14 are alkoxy.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is alkoxy, and R 12, R 13, or R 14 are alkyl.
In some forms, for R 11, G is C=O and R 7 is tert-butyl at the para position, D is O, J is O, L and M are NR 15, R 15 is alkoxy, and R 12, R 13, or R 14 are halogen.
or the pharmaceutically acceptable salt or ester thereof.
In another form, the compounds of formula I or formula II, or a pharmaceutically acceptable salt or a prodrug thereof, is a compound selected from the group consisting of:
Figure PCTCN2019090301-appb-000045
Figure PCTCN2019090301-appb-000046
Figure PCTCN2019090301-appb-000047
Figure PCTCN2019090301-appb-000048
Figure PCTCN2019090301-appb-000049
Figure PCTCN2019090301-appb-000050
Figure PCTCN2019090301-appb-000051
Figure PCTCN2019090301-appb-000052
Figure PCTCN2019090301-appb-000053
Figure PCTCN2019090301-appb-000054
Figure PCTCN2019090301-appb-000055
Figure PCTCN2019090301-appb-000056
Figure PCTCN2019090301-appb-000057
Figure PCTCN2019090301-appb-000058
Figure PCTCN2019090301-appb-000059
Figure PCTCN2019090301-appb-000060
Figure PCTCN2019090301-appb-000061
Figure PCTCN2019090301-appb-000062
Figure PCTCN2019090301-appb-000063
Figure PCTCN2019090301-appb-000064
Figure PCTCN2019090301-appb-000065
Figure PCTCN2019090301-appb-000066
Figure PCTCN2019090301-appb-000067
The compounds described herein may have one or more chiral centers, and thus exist as one or more stereoisomers. Such stereoisomers can exist as a single enantiomer, a mixture of enantiomers, a mixture of diastereomers, or a racemic mixture.
As used herein, the term “stereoisomers” refers to compounds made up of the same atoms having the same bond order but having different three-dimensional arrangements of atoms that are not interchangeable. The three-dimensional structures are called configurations. As used herein, the term “enantiomers” refers to two stereoisomers that are non-superimposable mirror images of one another. As used herein, the term “optical isomer” is equivalent to the term “enantiomer. ” As used herein the term “diastereomer” refers to two stereoisomers which are not mirror images but also not superimposable. The terms “racemate, ” “racemic mixture” or “racemic modification” refer to a mixture of equal parts of enantiomers. The term “chiral center” refers to a carbon atom to which four different groups are attached. Choice of the appropriate chiral column, eluent, and conditions necessary to effect separation of the pair of enantiomers is well known to one of ordinary skill in the art using standard techniques (see e.g. Jacques, J. et al., “Enantiomers, Racemates, and Resolutions, ” John Wiley and Sons, Inc. 1981) .
The compounds can also be a pharmaceutically acceptable salt of any of the compounds described above. In some cases, it may be desirable to prepare the salt of a compound described above due to one or more of the salt's advantageous physical properties, such as enhanced stability or a desirable solubility or dissolution profile.
Also described herein are pharmaceutically acceptable nontoxic ester, amide, and salt derivatives of those compounds of formula (I) containing a carboxylic acid moiety.
Formula I also encompasses pharmaceutically acceptable esters, amides, and salts of such compounds, as will be explained in detail, infra.
Such compounds of the formula (I) and their pharmaceutically acceptable esters, amides, and salts are referred to herein as the inventive compounds.
Formula I also encompasses pharmaceutically acceptable salts. Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. In one aspect, the reaction is conducted in water, alone or in combination with an inert, water- miscible organic solvent, at a temperature of from about 0℃ to about 100℃, such as at room temperature. The molar ratio of compounds of structural formula (I) to base used are chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of pharmaceutically acceptable base to yield a neutral salt.
Generally, non-aqueous media including ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams&Wilkins, Baltimore, MD, 2000, p. 704; and “Handbook of Pharmaceutical Salts: Properties, Selection, and Use, ” P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.
Suitable pharmaceutically acceptable acid addition salts include those derived from inorganic acids, such as hydrochloric, hydrobromic, hydrofluoric, boric, fluoroboric, phosphoric, metaphosphoric, nitric, carbonic, sulfonic, and sulfuric acids, and organic acids, such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic, lactobionic, maleic, malic, methanesulfonic, trifluoromethanesulfonic, succinic, toluenesulfonic, tartaric, and trifluoroacetic acids.
Suitable organic acids generally include, for example, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids. Specific examples of suitable organic acids include acetate, trifluoroacetate, formate, propionate, succinate, glycolate, gluconate, digluconate, lactate, malate, tartaric acid, citrate, ascorbate, glucuronate, maleate, fumarate, pyruvate, aspartate, glutamate, benzoate, anthranilic acid, mesylate, stearate, salicylate, p-hydroxybenzoate, phenylacetate, mandelate, embonate (pamoate) , methanesulfonate, ethanesulfonate, benzenesulfonate, pantothenate, toluenesulfonate, 2-hydroxyethanesulfonate, sufanilate, cyclohexylaminosulfonate, algenic acid, β-hydroxybutyric acid, galactarate, galacturonate, adipate, alginate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, dodecylsulfate, glycoheptanoate, glycerophosphate, heptanoate, hexanoate, nicotinate, 2-naphthalesulfonate, oxalate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, thiocyanate, tosylate, and undecanoate.
In some cases, the pharmaceutically acceptable salt may include alkali metal salts, including sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. Base salts can also be formed from bases which form non-toxic salts, including aluminum, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, meglumine, olamine, tromethamine and zinc salts.
Organic salts may be made from secondary, tertiary or quaternary amine salts, such as tromethamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) , and procaine. Basic nitrogen-containing groups may also be quaternized with agents such as lower alkyl (C 1-C 6) halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides) , dialkyl sulfates (e.g., dimethyl, diethyl, dibuytl, and diamyl sulfates) , long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides) , arylalkyl halides (e.g., benzyl and phenethyl bromides) , and others.
The compound can also be a pharmaceutically acceptable prodrug of any of the compounds described above. Prodrugs are compounds that, when metabolized in vivo, undergo conversion to compounds having the desired pharmacological activity. Prodrugs can be prepared by replacing appropriate functionalities present in the compounds described above with "pro-moieties" as described, for example, in H. Bundgaar, Design of Prodrugs (1985) . Examples of prodrugs include ester, ether or amide derivatives of the compounds described above, polyethylene glycol derivatives of the compounds described above, N-acyl amine derivatives, dihydropyridine pyridine derivatives, amino-containing derivatives conjugated to polypeptides, 2-hydroxybenzamide derivatives, carbamate derivatives, N-oxides derivatives that are biologically reduced to the active amines, and N-mannich base derivatives. For further discussion of prodrugs, see, for example, Rautio, J. et al. Nature Reviews Drug Discovery. 7: 255-270 (2008) .
C. Pharmaceutical Compositions
Pharmaceutical Compositions are provided containing a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt or prodrug thereof, in combination with one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) , and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
1. Additional Therapeutics
The compounds described herein can be formulated with one or more additional active agents, such as anti-infectious agents, analgesic, etc.
Pharmaceutical compositions can also include one or more vitamins, minerals, dietary supplements, nutraceutical agents, such as proteins, carbohydrates, amino acids, fatty acids,  antioxidants, and plant or animal extracts, or combinations thereof. Suitable vitamins, minerals, nutraceutical agents, and dietary supplements are known in the art, and disclosed, for example, in Roberts et al., (Nutriceuticals: The Complete Encyclopedia of Supplements, Herbs, Vitamins, and Healing Foods, American Nutriceutical Association, 2001) . Nutraceutical agents and dietary supplements are also disclosed in Physicians' Desk Referencefor Nutritional Supplements, 1st Ed. (2001) and The Physicians' Desk Referencefor Herbal Medicines, 1st Ed. (2001) .
2. Enteral Compositions
Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.
Compositions may be prepared using one or more pharmaceutically acceptable excipients, including diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof. Excipients, including plasticizers, pigments, colorants, stabilizing agents, and glidants, may also be used to form coated compositions for enteral administration. Delayed release dosage compositions may be prepared as described in standard references such as “Pharmaceutical dosage form tablets, ” eds. Liberman et al. (New York, Marcel Dekker, Inc., 1989) , “Remington–The science and practice of pharmacy, ” 20th ed., Lippincott Williams&Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drug delivery systems, ” 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995) . These references provide information on excipients, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.
Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name
Figure PCTCN2019090301-appb-000068
 (Roth Pharma, Westerstadt, Germany) , zein, shellac, and polysaccharides.
Diluents, also referred to as "fillers, " are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium  chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
Binders are used to impart cohesive qualities to a solid dosage composition, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol) , polyethylene glycol, waxes, natural and synthetic gums, such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers, such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid and polyvinylpyrrolidone.
Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross-linked PVP (
Figure PCTCN2019090301-appb-000069
XL from GAF Chemical Corp) .
Stabilizers are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, antioxidants, butylated hydroxytoluene (BHT) ; ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites, such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA) .
a. Controlled release compositions
Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release. For example, the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup. The particles can be formed of the drug and a controlled release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.
In another form, the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids. In the case of gels, the matrix swells entrapping the active agents, which are released slowly over time by diffusion and/or degradation of the matrix material. Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.
In still another form, the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coatings or extended release coatings. The coating or coatings may also contain the compounds and/or additional active agents.
Extended release compositions
The extended release compositions are generally prepared as diffusion or osmotic systems, for example, as described in “Remington–The science and practice of pharmacy” (20th ed., Lippincott Williams&Wilkins, Baltimore, MD, 2000) . A diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses, such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and
Figure PCTCN2019090301-appb-000070
934, polyethylene oxides and mixtures thereof. Fatty compounds include, but are not limited to, various waxes, such as carnauba wax and glyceryl tristearate, and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.
In certain preferred forms, the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly (acrylic acid) , poly (methacrylic acid) , methacrylic acid alkylamine copolymer poly (methyl methacrylate) , poly (methacrylic acid) (anhydride) , polymethacrylate, polyacrylamide, poly (methacrylic acid anhydride) , and glycidyl methacrylate copolymers.
In certain preferred forms, the acrylic polymer is comprised of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups. In one preferred form, the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename
Figure PCTCN2019090301-appb-000071
In further preferred forms, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames
Figure PCTCN2019090301-appb-000072
RL30D and
Figure PCTCN2019090301-appb-000073
RS30D, respectively. 
Figure PCTCN2019090301-appb-000074
RL30D and
Figure PCTCN2019090301-appb-000075
RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth) acrylic esters being 1: 20 in
Figure PCTCN2019090301-appb-000076
RL30D and 1: 40 in
Figure PCTCN2019090301-appb-000077
RS30D. The mean molecular weight is about 150,000. 
Figure PCTCN2019090301-appb-000078
S-100 and
Figure PCTCN2019090301-appb-000079
L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents. 
Figure PCTCN2019090301-appb-000080
RL/RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.
The polymers described above, such as
Figure PCTCN2019090301-appb-000081
RL/RS, may be mixed together in any desired ratio in order to ultimately obtain a sustained-release composition having a desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100%
Figure PCTCN2019090301-appb-000082
RL, 50%
Figure PCTCN2019090301-appb-000083
RL and 50%
Figure PCTCN2019090301-appb-000084
RS, and 10%
Figure PCTCN2019090301-appb-000085
RL and 90%
Figure PCTCN2019090301-appb-000086
RS. One skilled in the art will recognize that other acrylic polymers may also be used, such as, for example, 
Figure PCTCN2019090301-appb-000087
L.
Alternatively, extended release compositions can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.
The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets andcapsules containing tablets, beads, or granules. An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.
Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation. Their compositions usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet composition to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.
Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax material and either spray-congealed or congealed and screened and processed.
Delayed release compositions
Delayed release compositions can be created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.
The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename
Figure PCTCN2019090301-appb-000088
 (Rohm Pharma; Westerstadt, Germany) , including
Figure PCTCN2019090301-appb-000089
L30D-55 and L100-55 (soluble at pH 5.5 and above) , 
Figure PCTCN2019090301-appb-000090
L-100(soluble at pH 6.0 and above) , 
Figure PCTCN2019090301-appb-000091
S (soluble at pH 7.0 and above, as a result of a higher degree of esterification) , and
Figure PCTCN2019090301-appb-000092
NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability) ; vinyl polymers and copolymers, such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers, such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.
The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials,  method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.
The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. %to 50 wt. %relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers, such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. %to 100 wt. %of the polymer weight in the coating solution. One effective glidant is talc. Other glidants, such as magnesium stearate and glycerol monostearates, may also be used. Pigments, such as titanium dioxide, may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone) , may also be added to the coating composition.
Pulsatile Release
The composition can provide pulsatile delivery of the one or more of the compounds disclosed herein. By "pulsatile" is meant that a plurality of drug doses are released at spaced apart intervals of time. Generally, upon ingestion of the dosage form, release of the initial dose is substantially immediate, i.e., the first drug release "pulse" occurs within about one hour of ingestion. This initial pulse is followed by a first time interval (lag time) during which very little or no drug is released from the dosage form, after which a second dose is then released. Similarly, a second nearly drug release-free interval between the second and third drug release pulses may be designed. The duration of the nearly drug release-free time interval will vary depending upon the dosage form design e.g., a twice daily dosing profile, a three times daily dosing profile, etc. For dosage forms providing a twice daily dosage profile, the nearly drug release-free interval has a duration of approximately 3 hours to 14 hours between the first and second dose. For dosage forms providing a three times daily profile, the nearly drug release-free interval has a duration of approximately 2 hours to 8 hours between each of the three doses.
In one form, the pulsatile release profile is achieved with dosage forms that are closed and preferably sealed capsules housing at least two drug-containing "dosage units" wherein each dosage unit within the capsule provides a different drug release profile. Control of the delayed release dosage unit (s) is accomplished by a controlled release polymer coating on the dosage unit, or by  incorporation of the active agent in a controlled release polymer matrix. Each dosage unit may comprise a compressed or molded tablet, wherein each tablet within the capsule provides a different drug release profile. For dosage forms mimicking a twice a day dosing profile, a first tablet releases drug substantially immediately following ingestion of the dosage form, while a second tablet releases drug approximately 3 hours to less than 14 hours following ingestion of the dosage form. For dosage forms mimicking a three times daily dosing profile, a first tablet releases drug substantially immediately following ingestion of the dosage form, a second tablet releases drug approximately 3 hours to less than 10 hours following ingestion of the dosage form, and the third tablet releases drug at least 5 hours to approximately 18 hours following ingestion of the dosage form. It is possible that the dosage form includes more than three tablets. While the dosage form will not generally include more than a third tablet, dosage forms housing more than three tablets can be utilized.
Alternatively, each dosage unit in the capsule may comprise a plurality of drug-containing beads, granules or particles. As is known in the art, drug-containing "beads" refer to beads made with drug and one or more excipients or polymers. Drug-containing beads can be produced by applying drug to an inert support, e.g., inert sugar beads coated with drug or by creating a "core" comprising both drug and one or more excipients. As is also known, drug-containing "granules" and "particles" comprise drug particles that may or may not include one or more additional excipients or polymers. In contrast to drug-containing beads, granules and particles do not contain an inert support. Granules generally comprise drug particles and require further processing. Generally, particles are smaller than granules, and are not further processed. Although beads, granules and particles may be formulated to provide immediate release, beads and granules are generally employed to provide delayed release.
3. Parenteral Compositions
The compounds described herein can be formulated for parenteral administration. “Parenteral administration, ” as used herein, means administration by any method other than through the digestive tract or non-invasive topical or regional routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, by injection, and by infusion.
Parenteral compositions can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable compositions, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions  upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) , oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. ) , and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 
Figure PCTCN2019090301-appb-000093
401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
The composition can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The composition may also contain an antioxidant to prevent degradation of the active agent (s) .
The composition is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
Water-soluble polymers are often used in compositions for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
a. Controlled release compositions
The parenteral compositions described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.
Nano-and microparticles
For parenteral administration, the compounds, and optionally one or more additional active agents, can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release. In forms wherein the composition contains two or more drugs, the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile) or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc. ) .
For example, the compounds and/or one or more additional active agents can be incorporated into polymeric microparticles that provide controlled release of the drug (s) . Release of the drug (s) is controlled by diffusion of the drug (s) out of the microparticles and/or degradation of the polymeric particles by hydrolysis and/or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.
Polymers that are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides) , polyhydroxy acids, such as polylactide (PLA) , polyglycolide (PGA) , poly (lactide-co-glycolide) (PLGA) , poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. Alternatively, the drug (s) can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and/or mechanical erosion. As used herein, the term “slowly soluble in water” refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol) , fatty acids and derivatives, including, but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and tri-glycerides) , and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name
Figure PCTCN2019090301-appb-000094
stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any material that is normally solid at room temperature and has a melting point of from about 30 to 300℃.
In some cases, it may be desirable to alter the rate of water penetration into the microparticles. To this end, rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch) , cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose) , alginic acid, lactose and talc. Additionally, a pharmaceutically acceptable surfactant (for example, lecithin) may be added to facilitate the degradation of such microparticles. Proteins that are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof that are water soluble can be formulated with drug into microparticles and subsequently cross- linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.
Encapsulation or incorporation of drug into carrier materials to produce drug containing microparticles can be achieved through known pharmaceutical composition techniques. In the case of composition in fats, waxes or wax-like materials, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion. In a preferred process, wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. Detailed descriptions of these processes can be found in “Remington-The science and practice of pharmacy, ” 20th Edition, Jennaro et al., (Phila, Lippencott, Williams, and Wilkens, 2000) .
For some carrier materials it may be desirable to use a solvent evaporation technique to produce drug containing microparticles. In this case drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.
In some forms, drug in a particulate form is homogeneously dispersed in a water-insoluble or slowly water-soluble material. To minimize the size of the drug particles within the composition, the drug powder itself may be milled to generate fine particles prior to composition. The process of jet milling, known in the pharmaceutical art, can be used for this purpose. In some forms, drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture. In this case a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.
The particles can also be coated with one or more modified release coatings. Solid esters of fatty acids, which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto drug containing microparticles or drug particles by spray coating or by wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of non-soluble networks. Many methods of  cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde) , epoxy compounds, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized and native sugars have been used to cross-link gelatin (Cortesi, R., et al., Biomaterials 19 (1998) 1641-1649) . Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.
To produce a coating layer of cross-linked protein surrounding drug containing microparticles or drug particles, a water soluble protein can be spray coated onto the microparticles and subsequently cross-linked by the one of the methods described above. Alternatively, drug containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently cross-linked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.
Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations that cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions.
Depot compositions
Active agents can be formulated for depot injection. In a depot injection, the active agent is formulated with one or more pharmaceutically acceptable carriers that provide for the gradual release of active agent over a period ofhours or days after injection. The depot composition can be administered by any suitable means; however, the depot composition is typically administered via subcutaneous or intramuscular injection.
A variety of carriers may be incorporated into the depot composition to provide for the controlled release of the active agent. In some cases, depot compositions contain one or more biodegradable polymeric or oligomeric carriers. Suitable polymeric carriers include, but are not limited to poly (lactic acid) (PLA) , poly (lactic-co-glycolic acid) (PLGA) , poly (lactic acid) -polyethyleneglycol (PLA-PEG) block copolymers, polyanhydrides, poly (ester anhydrides) , ppolyglycolide (PGA) , poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4- hydroxybutyrate (P4HB) , polycaprolactone, cellulose, hydroxypropyl methylcellulose, ethylcellulose, as well as blends, derivatives, copolymers, and combinations thereof.
In depot compositions containing a polymeric or oligomeric carrier, the carrier and active agent can be formulated as a solution, an emulsion, or suspension. One or more compounds, and optionally one or more additional active agents, can also be incorporated into polymeric or oligomeric microparticles, nanoparticles, or combinations thereof.
In some cases, the composition is fluid and designed to solidify or gel (i.e., forming a hydrogel or organogel) upon injection. This can result from a change in solubility of the composition upon injection, or for example, by injecting a pre-polymer mixed with an initiator and/or crosslinking agent. The polymer matrix, polymer solution, or polymeric particles entrap the active agent at the injection site. As the polymeric carrier is gradually degraded, the active agent is released, either by diffusion of the agent out of the matrix and/or dissipation of the matrix as it is absorbed. The release rate of the active agent from the injection site can be controlled by varying, for example, the chemical composition, molecular weight, crosslink density, and/or concentration of the polymeric carrier. Examples of such systems include those described in U.S. Patent Nos. 4,938,763, 5,480,656 and 6,113,943.
Depot composition can also be prepared by using other rate-controlling excipients, including hydrophobic materials, including acceptable oils (e.g., peanut oil, corn oil, sesame oil, cottonseed oil, etc. ) and phospholipids, ion-exchange resins, and sparingly soluble carriers.
The depot composition can further contain a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) , oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc. ) , and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Solutions and dispersions of the compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combination thereof.
Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates, such as sodium dodecylbenzene sulfonate; dialkyl  sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis- (2-ethylthioxyl) -sulfosuccinate; and alkyl sulfates, such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, 
Figure PCTCN2019090301-appb-000095
401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
The composition can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The composition may also contain an antioxidant to prevent degradation of the active agent (s) .
The composition is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
Water-soluble polymers are often used in compositions for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art.
Implants
Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained is also contemplated herein. In such cases, the active agent (s) provided herein can be dispersed in a solid matrix optionally coated with an outer rate-controlling membrane. The compound diffuses from the solid matrix (and optionally through the outer membrane) sustained, rate-controlled release. The solid matrix and membrane may be formed from any suitable material known in the art including, but not limited to, polymers, bioerodible polymers, and hydrogels.
4. Pulmonary compositions
The compounds described herein can be formulated for parenteral administration. Pharmaceutical compositions and methods for the pulmonary administration are known in the art.
The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung, where the exchange of gases occurs.
The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. Effective delivery of therapeutic agents via pulmonary routes requires that the active agent be formulated so as to reach the alveoli.
In the case of pulmonary administration, compositions can be divided into dry powder compositions and liquid compositions. Both dry powder and liquid compositions can be used to form aerosol compositions. The term aerosol as used herein refers to any preparation of a fine mist of particles, which can be in solution or a suspension, whether or not it is produced using a propellant.
Useful compositions, and methods of manufacture, are described by Caryalho, et al., J Aerosol Med Pulm Drug Deliv. 2011 Apr; 24 (2) : 61-80. Epub 2011 Mar 16, for delivery of chemotherapeutic drugs to the lungs.
a. Dry Powder Compositions
Dry powder compositions are finely divided solid compositions containing one or more active agents which are suitable for pulmonary administration. In dry powder compositions, the one or more active agents can be incorporated in crystalline or amorphous form.
Dry powder compositions can be administered via pulmonary inhalation to a patient without the benefit of any carrier, other than air or a suitable propellant. Preferably, however, the dry powder compositions include one or more pharmaceutically acceptable carriers.
The pharmaceutical carrier may include a bulking agent, such as carbohydrates (including monosaccharides, polysaccharides, and cyclodextrins) , polypeptides, amino acids, and combinations thereof. Suitable bulking agents include fructose, galactose, glucose, lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol, palatinite, raffinose, stachyose, sucrose, trehalose, xylitol, hydrates thereof, and combinations thereof.
The pharmaceutical carrier may include a lipid or surfactant. Natural surfactants, such as dipalmitoylphosphatidylcholine (DPPC) , are the most preferred. This is commercially available for treatment of respiratory distress syndrome in premature infants. Synthetic and animal derived pulmonary surfactants include:
Synthetic Pulmonary Surfactants
Exosurf-a mixture of DPPC with hexadecanol and tyloxapol added as spreading agents Pumactant (Artificial Lung Expanding Compound or ALEC) -a mixture of DPPC and PG KL-4-composed of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21 amino acid synthetic peptide that mimics the structural characteristics of SP-B. Venticute-DPPC, PG, palmitic acid and recombinant SP-C
Animal derived surfactants
Alveofact-extracted from cow lung lavage fluid
Curosurf-extracted from material derived from minced pig lung
Infasurf-extracted from calf lung lavage fluid
Survanta-extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin Exosurf, Curosurf, Infasurf, and Survanta are the surfactants currently FDA approved for use in the U.S.
The pharmaceutical carrier may also include one or more stabilizing agents or dispersing agents. The pharmaceutical carrier may also include one or more pH adjusters or buffers. Suitable buffers include organic salts prepared from organic acids and bases, such as sodium citrate or sodium ascorbate. The pharmaceutical carrier may also include one or more salts, such as sodium chloride or potassium chloride.
Dry powder compositions are typically prepared by blending one or more active agents with a pharmaceutical carrier. Optionally, additional active agents may be incorporated into the mixture. The mixture is then formed into particles suitable for pulmonary administration using techniques  known in the art, such as lyophilization, spray drying, agglomeration, spray coating, extrusion processes, hot melt particle formation, phase separation particle formation (spontaneous emulsion particle formation, solvent evaporation particle formation, and solvent removal particle formation) , coacervation, low temperature casting, grinding, milling (e.g., air-attrition milling (jet milling) , ball milling) , high pressure homogenization, and/or supercritical fluid crystallization.
An appropriate method of particle formation can be selected based on the desired particle size, particle size distribution, and particle morphology. In some cases, the method of particle formation is selected so as to produce a population of particles with the desired particle size, particle size distribution for pulmonary administration. Alternatively, the method of particle formation can produce a population of particles from which a population of particles with the desired particle size, particle size distribution for pulmonary administration is isolated, for example by sieving. It is known in the art that particle morphology affects the depth of penetration of a particle into the lung as well as uptake of the drug particles. As discussed above, drug particles should reach the alveoli to maximize therapeutic efficacy. Accordingly, dry powder compositions is processed into particles having the appropriate mass median aerodynamic diameter (MMAD) , tap density, and surface roughness to achieve delivery of the one or more active agents to the deep lung. Preferred particle morphologies for delivery to the deep lung are known in the art, and are described, for example, in U.S. Patent No. 7,052,678 to Vanbever, et al.
Particles having a mass median aerodynamic diameter (MMAD) of greater than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed. Particles having diameters of about 3 to about 5 microns are small enough to reach the upper-to mid-pulmonary region (conducting airways) , but may be too large to reach the alveoli. Smaller particles, (i.e., about 0.5 to about 3 microns) , are capable of efficiently reaching the alveolar region. Particles having diameters smaller than about 0.5 microns can also be deposited in the alveolar region by sedimentation, although very small particles may be exhaled.
The precise particle size range effective to achieve delivery to the alveolar region will depend on several factors, including the tap density of particles being delivered. Generally speaking, as tap density decreases, the MMAD of particles capable of efficiently reaching the alveolar region of the lungs increases. Therefore, in cases of particles with low tap densities, particles having diameters of about 3 to about 5 microns, about 5 to about 7 microns, or about 7 to about 9.5 microns can be efficiently delivered to the lungs. The preferred aerodyanamic diameter for maximum  deposition within the lungs can be calculated. See, for example, U.S. Patent No. 7,052,678 to Vanbever, et al.
In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 10 microns, more preferably between about 0.5 microns to about 7 microns, most preferably between about 0.5 to about 5 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 0.5 to about 3 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 3 to about 5 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 5 to about 7 microns. In some forms, the dry powder composition is composed of a plurality of particles having a median mass aerodynamic diameter between about 7 to about 9.5 microns.
In some cases, there may be an advantage to delivering particles larger than about 3 microns in diameter. Phagocytosis of particles by alveolar macrophages diminishes precipitously as particle diameter increases beyond about 3 microns. Kawaguchi, H., et al., Biomaterials 7: 61-66 (1986) ; and Rudt, S. and Muller, R.H., J. Contr. Rel, 22: 263-272 (1992) . By administering particles with an aerodynamic volume greater than 3 microns, phagocytic engulfment by alveolar macrophages and clearance from the lungs can be minimized.
In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of less than about 10 microns, more preferably less than about 7 microns, most preferably about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have an aerodynamic diameter of greater than about 0.1 microns.
In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%, of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns and less than about 10 microns, more preferably greater than about 0.5 microns and less than about 7 microns, most preferably greater than about 0.5 microns and less than about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 0.5 microns and less than about 3 microns. In some forms, at least  about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 3 microns and less than about 5 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 5 microns and less than about 7 microns. In some forms, at least about 80%, more preferably at least about 90%, most preferably at least about 95%of the particles in dry powder composition have aerodynamic diameter of greater than about 7 microns and less than about 9.5 microns.
In some forms, the particles have a tap density of less than about 0.4 g/cm 3, more preferably less than about 0.25 g/cm 3, most preferably less than about 0.1 g/cm 3. Features which can contribute to low tap density include irregular surface texture and porous structure.
In some cases, the particles are spherical or ovoid in shape. The particles can have a smooth or rough surface texture. The particles may also be coated with a polymer or other suitable material to control release of one or more active agents in the lungs.
Dry powder compositions can be administered as dry powder using suitable methods known in the art. Alternatively, the dry powder compositions can be suspended in the liquid composition s described below, and administered to the lung using methods known in the art for the delivery of liquid compositions.
b. Liquid Compositions
Liquid compositions contain one or more compounds dissolved or suspended in a liquid pharmaceutical carrier.
Suitable liquid carriers include, but are not limited to distilled water, de-ionized water, pure or ultrapure water, saline, and other physiologically acceptable aqueous solutions containing salts and/or buffers, such as phosphate buffered saline (PBS) , Ringer's solution, and isotonic sodium chloride, or any other aqueous solution acceptable for administration to an animal or human.
Preferably, liquid compositions are isotonic relative to physiological fluids and of approximately the same pH, ranging e.g., from about pH 4.0 to about pH 7.4, more preferably from about pH 6.0 to pH 7.0. The liquid pharmaceutical carrier can include one or more physiologically compatible buffers, such as a phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an aqueous solution for pulmonary administration.
Liquid compositions may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. Liquid  compositions may also include one or more preservatives, such as ethyl or n-propylp-hydroxybenzoate.
In some cases the liquid compositions may contain one or more solvents that are low toxicity organic (i.e., nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol. These solvents can be selected based on their ability to readily aerosolize the composition. Any such solvent included in the liquid composition should not detrimentally react with the one or more active agents present in the liquid composition. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as a freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid composition as desired to increase the volatility and/or alter the aerosolizing behavior of the solution or suspension.
Liquid compositions may also contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, "minor amounts" means no excipients are present that might adversely affect uptake of the one or more active agents in the lungs.
c. Aerosol Compositions
The dry powder and liquid compositions described above can be used to form aerosol compositions for pulmonary administration. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. The term aerosol as used herein refers to any preparation of a fine mist of solid or liquid particles suspended in a gas. In some cases, the gas may be a propellant; however, this is not required. Aerosols may be produced using a number of standard techniques, including as ultrasonication or high pressure treatment.
Preferably, a dry powder or liquid compositions as described above is formulated into aerosol compositions using one or more propellants. Suitable propellants include air, hydrocarbons, such as pentane, isopentane, butane, isobutane, propane and ethane, carbon dioxide, chlorofluorocarbons, fluorocarbons, and combinations thereof. Suitable fluorocarbons include 1-6 hydrogen containing fluorocarbons, such as CHF 2CHF 2, CF 3CH 2F, CH 2F 2CH 3, and CF 3CHFCF 3 as well as fluorinated ethers, such as CF 3-O-CF 3, CF 2H-O-CHF 2, and CF 3-CF 2-O-CF 2-CH 3. Suitable fluorocarbons also include perfluorocarbons, such as 1-4 carbon perfluorocarbons including CF 3CF 3, CF 3CF 2CF 3, and CF 3CF 2CF 2CF 3.
Preferably, the propellants include, but not limited to, one or more hydrofluoroalkanes (HFA) . Suitable HFA propellants, include but are not limited to, 1, 1, 1, 2, 3, 3-heptafluoro-n-propane (HFA 227) , 1, 1, 1, 2-tetrafluoroethane (HFA 134) 1, 1, 1, 2, 253, 3, 3-heptafluoropropane (Propellant 227) , or any mixture of these propellants.
Preferably, the one or more propellants have sufficient vapor pressure to render them effective as propellants. Preferably, the one or more propellants are selected so that the density of the mixture is matched to the density of the particles in the aerosol composition in order to minimize settling or creaming of the particles in the aerosol composition.
The propellant is preferably present in an amount sufficient to propel a plurality of the selected doses of the aerosol composition from an aerosol canister.
d. Devices for Pulmonary Administration
In some cases, a device is used to administer the compositions to the lungs. Suitable devices include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.
Inhalation can occur through the nose and/or the mouth of the patient. Administration can occur by self-administration of the composition while inhaling, or by administration of the composition via a respirator to a patient on a respirator.
Dry Powder Inhalers
The dry powder compositions described above can be administered to the lungs of a patient using a dry powder inhaler (DPI) . DPI devices typically use a mechanism such as a burst of gas to create a cloud of dry powder inside a container, which can then be inhaled by the patient.
In a dry powder inhaler, the dose to be administered is stored in the form of a non-pressurized dry powder and, on actuation of the inhaler, the particles of the powder are inhaled by the subject. In some cases, a compressed gas (i.e., propellant) may be used to dispense the powder, similar to pressurized metered dose inhalers (pMDIs) . In some cases, the DPI may be breath actuated, meaning that an aerosol is created in precise response to inspiration. Typically, dry powder inhalers administer a dose of less than a few tens of milligrams per inhalation to avoid provocation of cough. DPIs function via a variety of mechanical means to administer compositions to the lungs. In some DPIs, a doctor blade or shutter slides across the dry powder composition contained in a reservoir, culling the composition into a flowpath whereby the patient can inhale the powder in a single breath. In other DPIs, the dry powder composition is packaged in a preformed dosage form, such as a blister, tabule, tablet, or gelcap, which is pierced, crushed, or otherwise unsealed to release the dry powder composition into a flowpath for subsequent inhalation. Still others DPIs release the dry powder composition into a chamber or capsule and use mechanical or electrical agitators to keep the dry powder composition suspended in the air until the patient inhales.
Dry powder compositions may be packaged in various forms, such as a loose powder, cake, or pressed shape for insertion in to the reservoir of a DPI. Examples suitable DPIs for the administration of the compositions described above include the 
Figure PCTCN2019090301-appb-000096
inhaler (Astrazeneca, Wilmington, Del. ) , the
Figure PCTCN2019090301-appb-000097
inhaler (Innovata, Ruddington, Nottingham, UK) , the
Figure PCTCN2019090301-appb-000098
inhaler (Glaxo, Greenford, Middlesex, UK) , the 
Figure PCTCN2019090301-appb-000099
(Orion, Expoo, FI) , the
Figure PCTCN2019090301-appb-000100
inhaler (Pfizer, New York, N. Y. ) , the
Figure PCTCN2019090301-appb-000101
 inhaler (Microdose, Monmouth Junction, N.J. ) , and the
Figure PCTCN2019090301-appb-000102
inhaler (Dura, San Diego, Calif. ) .
Pressurized Metered Dose Inhalers
The liquid compositions described above can be administered to the lungs of a patient using a pressurized metered dose inhaler (pMDI) .
Pressurized Metered Dose Inhalers (pMDIs) generally include at least two components: acanister in which the liquid composition is held under pressure in combination with one or more propellants, and a receptacle used to hold and actuate the canister. The canister may contain a single or multiple doses of the composition. The canister may include a valve, typically a metering valve, from which the contents of the canister may be discharged. Aerosolized drug is dispensed from the pMDI by applying a force on the canister to push it into the receptacle, thereby opening the valve and causing the drug particles to be conveyed from the valve through the receptacle outlet. Upon discharge from the canister, the liquid composition is atomized, forming an aerosol.
pMDIs typically employ one or more propellants to pressurize the contents of the canister and to propel the liquid composition out of the receptacle outlet, forming an aerosol. Any suitable propellants, including those discussed above, may be utilized. The propellant may take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas. Chlorofluorocarbons (CFC) were once commonly used as liquid propellants, but have now been banned. They have been replaced by the now widely accepted hydrofluororalkane (HFA) propellants.
pMDIs are available from a number of suppliers, including 3M Corporation, Aventis, Boehringer Ingleheim, Forest Laboratories, Glaxo-Wellcome, Schering Plough and Vectura. In some cases, the patient administers an aerosolized composition by manually discharging the aerosolized composition from the pMDI in coordination with inspiration. In this way, the aerosolized composition is entrained within the inspiratory air flow and conveyed to the lungs.
In other cases, a breath-actuated trigger, such as that included in the
Figure PCTCN2019090301-appb-000103
inhaler (MAP Pharmaceuticals, Mountain View, Calif. ) may be employed that simultaneously discharges a dose of the composition upon sensing inhalation. These devices, which discharge the aerosol  composition when the user begins to inhale, are known as breath-actuated pressurized metered dose inhalers (baMDIs) .
Nebulizers
The liquid compositions described above can also be administered using a nebulizer. Nebulizers are liquid aerosol generators that convert the liquid composition described able, usually aqueous-based compositions, into mists or clouds of small droplets, preferably having diameters less than 5 microns mass median aerodynamic diameter, which can be inhaled into the lower respiratory tract. This process is called atomization. The droplets carry the one or more active agents into the nose, upper airways or deep lungs when the aerosol cloud is inhaled. Any type of nebulizer may be used to administer the composition to a patient, including, but not limited to pneumatic (jet) nebulizers and electromechanical nebulizers.
Pneumatic (jet) nebulizers use a pressurized gas supply as a driving force for atomization of the liquid composition. Compressed gas is delivered through a nozzle or jet to create a low pressure field which entrains a surrounding liquid composition and shears it into a thin film or filaments. The film or filaments are unstable and break up into small droplets that are carried by the compressed gas flow into the inspiratory breath. Baffles inserted into the droplet plume screen out the larger droplets and return them to the bulk liquid reservoir. Examples of pneumatic nebulizers include, but are not limited to, PARI LC
Figure PCTCN2019090301-appb-000104
PARI LC
Figure PCTCN2019090301-appb-000105
Devilbiss
Figure PCTCN2019090301-appb-000106
and Boehringer Ingelheim
Figure PCTCN2019090301-appb-000107
Electromechanical nebulizers use electrically generated mechanical force to atomize liquid compositions. The electromechanical driving force can be applied, for example, by vibrating the liquid composition at ultrasonic frequencies, or by forcing the bulk liquid through small holes in a thin film. The forces generate thin liquid films or filament streams which break up into small droplets to form a slow moving aerosol stream which can be entrained in an inspiratory flow.
In some cases, the electromechanical nebulizer is an ultrasonic nebulizer, in which the liquid compositions is coupled to a vibrator oscillating at frequencies in the ultrasonic range. The coupling is achieved by placing the liquid in direct contact with the vibrator (such as a plate or ring in a holding cup) , or by placing large droplets on a solid vibrating projector (a horn) . The vibrations generate circular standing films which break up into droplets at their edges to atomize the liquid composition. Examples of ultrasonic nebulizers include
Figure PCTCN2019090301-appb-000108
Drive Medical Beetle
Figure PCTCN2019090301-appb-000109
Octive Tech
Figure PCTCN2019090301-appb-000110
and John Bunn
Figure PCTCN2019090301-appb-000111
In some cases, the electromechanical nebulizer is a mesh nebulizer, in which the liquid compositions is driven through a mesh or membrane with small holes ranging from 2 to 8 microns  in diameter, to generate thin filaments which break up into small droplets. In certain designs, the liquid composition is forced through the mesh by applying pressure with a solenoid piston driver (for example, the
Figure PCTCN2019090301-appb-000112
nebulizer) , or by sandwiching the liquid between a piezoelectrically vibrated plate and the mesh, which results in a oscillatory pumping action (for example
Figure PCTCN2019090301-appb-000113
Figure PCTCN2019090301-appb-000114
or
Figure PCTCN2019090301-appb-000115
nebulizer) . In other cases, the mesh vibrates back and forth through a standing column of the liquid to pump it through the holes. Examples of such nebulizers include the AeroNeb
Figure PCTCN2019090301-appb-000116
AeroNeb
Figure PCTCN2019090301-appb-000117
PARI
Figure PCTCN2019090301-appb-000118
Omron
Figure PCTCN2019090301-appb-000119
and Aradigm
Figure PCTCN2019090301-appb-000120
Electrohydrodynamic Aerosol Devices
The liquid compositions described above can also be administered using an electrohydrodynamic (EHD) aerosol device. EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions. Examples of EHD aerosol devices are known in the art. See, for example, U.S. Patent No. 4,765,539 to Noakes et al. and U.S. Patent No. 4,962,885 to Coffee, R.A.
The electrochemical properties of the composition may be important parameters to optimize when delivering the liquid composition to the lung with an EHD aerosol device and such optimization is routinely performed by one of skill in the art.
D. Methods of treatment
Pharmaceutical compositions containing one or more of the compounds described herein can be administered to treat viral infections. In one form the method for treating the viral infection by inhibiting viral nucleoprotein activity with an effective amount of one or more of the compound disclosed herein. In another form a method for inhibiting viral infection including inhibiting viral transcription, replication, protein synthesis, blocking vRNP export, and/or inhibiting virion budding process with an effective amount of one or more of the compound disclosed herein. The method of treating and inhibiting a viral infection where the virus is influenza A virus.
1. Dosages
The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied.
In some forms, one or more of the compounds are in an amount effective to inhibit viral transcription. In some forms, one or more of the compounds are in an amount effective to inhibit viral replication. In some forms, one or more of the compounds are in an amount effective to inhibit viral protein synthesis. In some forms, one or more of the compounds are in an amount effective to suppress the virion budding process. In some forms, one or more of the compounds are in an amount effective to inhibit viral nucleoprotein activity. In some forms, one or more of the compounds are in an amount effective to block vRNP export.
The precise dosage administered to a patient will depend on many factors, including the physical characteristics of the patient (e.g., weight) , the degree of severity of the disease or disorder to be treated, and the presence or absence of other complicating diseases or disorders and can be readily determined by the prescribing physician. In certain forms, the compound (s) is administered at a dosage equivalent to an oral dosage of between about 0.005 mg and about 500 mg per kg of body weight per day, more preferably between about 0.05 mg and about 100 mg per kg of body weight per day, most preferably between about 0.1 mg and about 10 mg per kg of body weight per day.
In some forms, the dosage can be effective to produce a concentration of one or more of the compounds within the range of 0.5μM to 1μM, 0.5μM to 2μM, 0.5μM to 3μM, 0.5μM to 4μM, 0.5μM to 5μM, 0.5μM to 10μM, 0.5μM to 20μM, 0.5μM to 30μM, 0.5μM to 40μM, 0.5μM to 50μM, 0.5μM to 60μM, 0.5μM to 70μM, 0.5μM to 80μM, 0.5μM to 90μM, 0.5μM to 100μM, 0.5μM to 130μM, 0.5μM to 150μM, 0.5μM to 180μM, 0.5μM to 200μM, 0.5μM to 300μM, 0.5μM to 400μM, 0.5μM to 500μM, 0.5μM to 600μM, 0.5μM to 700μM, 0.5μM to 800μM, 0.5μM to 900μM, 0.5μM to 1,000μM, 1μM to 5μM, 1μM to 10μM, 1μM to 20μM, 1μM to 30μM, 1μM to 40μM, 1μM to 50μM, 1μM to 60μM, 1μM to 70μM, 1μM to 80μM, 1μM to 90μM, 1μM to 100μM, 1μM to 130μM, 1μM to 150μM, 1μM to 180μM, 1μM to 200μM, 1μM to 300μM, 1μM to 400μM, 1μM to 500μM, 1μM to 600μM, 1μM to 700μM, 1μM to 800μM, 1μM to 900μM, 1μM to 1,000μM, 5μM to 10μM, 5μM to 20μM, 5μM to 30μM, 5μM to 40μM, 5μM to 50μM, 5μM to 60μM, 5μM to 70μM, 5μM to 80μM, 5μM to 90μM, 5μM to 100μM, 5μM to 130μM, 5μM to 150μM, 5μM to 180μM, 5μM to 200μM, 5μM to 300μM, 5μM to 400μM, 5μM to 500μM, 5μM to 600μM, 5μM to 700μM, 5μM to 800μM, 5μM to 900μM, 5μM to 1,000μM, 10μM to 20μM, 10μM to 30μM, 10μM to 40μM, 10μM to 50μM, 10μM to 60, 10μM to 70μM, 10μM to 100μM, 10μM to 130μM, 10μM to 150μM, 10μM to 180μM, 10μM to 200μM, 10μM to 300μM, 10μM to 400μM, 10μM to 500μM, 10μM to 600, 10μM to 700μM, 10μM to 800μM, 10μM to 900μM, 10μM to 1,000μM,  20μM to 30μM, 20μM to 40μM, 20μM to 50μM, 20μM to 60, 20μM to 70μM, 20μM to 100μM, 20μM to 130μM, 20μM to 150μM, 20μM to 180μM, 20μM to 200μM, 20μM to 300μM, 20μM to 400μM, 20μM to 500μM, 20μM to 600, 20μM to 700μM, 20μM to 800μM, 20μM to 900μM, 20μM to 1,000μM, 30μM to 40μM, 30μM to 50μM, 30μM to 60, 30μM to 70μM, 30μM to 100μM, 30μM to 130μM, 30μM to 150μM, 30μM to 180μM, 30μM to 200μM, 30μM to 300μM, 30μM to 400μM, 30μM to 500μM, 30μM to 600, 30μM to 700μM, 30μM to 800μM, 30μM to 900μM, 30μM to 1,000μM, 40μM to 50μM, 40μM to 60, 40μM to 70μM, 40μM to 100μM, 40μM to 130μM, 40μM to 150μM, 40μM to 180μM, 40μM to 200μM, 40μM to 300μM, 40μM to 400μM, 40μM to 500μM, 40μM to 600, 40μM to 700μM, 40μM to 800μM, 40μM to 900μM, 40μM to 1,000μM, 25μM to 30μM, 25μM to 35μM, 25μM to 40μM, 25μM to 45μM, 25μM to 50μM, 25μM to 60, 25μM to 80μM, 25μM to 100μM, 25μM to 130μM, 25μM to 150μM, 25μM to 180μM, 25μM to 200μM, 25μM to 300μM, 25μM to 400μM, 25μM to 500μM, 25μM to 600, 25μM to 700μM, 25μM to 800μM, 25μM to 900μM, 25μM to 1,000μM, 50μM to 60μM, 50μM to 80μM, 50μM to 100μM, 50μM to 130μM, 50μM to 150μM, 50μM to 180μM, 50μM to 200μM, 50μM to 300μM, 50μM to 400μM, 50μM to 500μM, 50μM to 600, 50μM to 700μM, 50μM to 800μM, 50μM to 900μM, or 50μM to 1,000μM in a body fluid of the subject.
In some forms, the dosage can be effective to produce a concentration of one or more compounds of 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 11μM, 12μM, 13μM, 14μM, 15μM, 16μM, 17μM, 18μM, 19μM, 20μM, 21μM, 22μM, 23μM, 24μM, 25μM, 26μM, 27μM, 28μM, 29μM, 30μM, 31μM, 32μM, 33μM, 34μM, 35μM, 36μM, 37μM, 38μM, 39μM, 40μM, 41μM, 42μM, 43μM, 44μM, 45μM, 46μM, 47μM, 48μM, 49μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 200μM, 300μM, 400μM, 500μM, 600, 700μM, 800μM, 900μM, 1,000μM in a body fluid of the subject.
In some forms, the dosage can be effective to produce a concentration of one or more of the compounds within the range of 1μM to 5μM, 1μM to 10μM, 1μM to 50μM, 5μM to 10μM, 5μM to 50μM, 10μM to 50μM, 20μM to 50μM, 30μM to 50μM, 40μM to 50μM, 10μM to 20μM, 10μM to 30μM, 10μM to 40μM, 20μM to 30μM, 20μM to 40μM, or 30μM to 40μM in a body fluid of the subject.
The efficacy of administration of a particular dose of the compounds or compositions according to the methods described herein can be determined by evaluating the particular aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in  evaluating the status of a subject in need for the treatment of influenza A virus or other diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed) , (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.
Therapeutic Administration
In one aspect, the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
Any of the compounds having the formula I can be used therapeutically in combination with a pharmaceutically acceptable carrier. The compounds described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of compositions of the compounds described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Other compounds will be administered according to standard procedures used by those skilled in the art.
The pharmaceutical compositions described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
The compounds and pharmaceutical compositions described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a compound or pharmaceutical composition described herein can be administered as an ophthalmic solution and/or ointment to the surface of the eye.  Moreover, a compound or pharmaceutical composition can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's , or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Compositions for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
Compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable. Pharmaceutical compositions may be administered, for example, in a single dosage, as a continuous dosage, one or more times daily, or less frequently, such as once a week. The pharmaceutical compositions can be administered once a day or more than once a day, such as twice a day, three times a day, four times a day or more. In certain forms, the compositions are administered orally, once daily or less. The pharmaceutical compositions are administered in an effective amount and for an effective period of time to elicit the desired therapeutic benefit. In certain forms, the pharmaceutical composition is administered for a period of at least one week, two weeks, three weeks, four weeks,  one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer.
The pharmaceutical compositions may also be administered prophylactically, e.g., to patients or subjects who are at risk for infection. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, sex, weight and general condition of the subject, extent of the disease in the subject, route of administration, whether other drugs are included in the regimen, and the like. Thus, it is not possible to specify an exact dosages for every composition. However, an appropriate dosage can be determined by one of ordinary skill in the art using only routine experimentation. For example, effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
1. Co-Administration with Active Agents
In other forms, the compounds disclosed herein can be co-administered with one or more additional therapeutic, prophylactic, or diagnostic agents. Co-administration, as used herein, includes administration within the same dosage form or within different dosage forms. For those forms where the compounds described herein and the one or more additional therapeutic, prophylactic, or diagnostic agents are administered in different dosage forms, the dosage forms can be administered simultaneously (e.g., at the same time or essentially at the same time) or sequentially. “Essentially at the same time” as used herein generally means within ten minutes, preferably within five minutes, more preferably within two minutes, most preferably within in one minute. Dosage forms administered sequentially can be administered within several hours of each other, e.g., with ten hours, nine hours, eight hours, seven hours, six hours, five hours, four hours, three hours, two hours, one hour, 30 minutes, 20 minutes, or 15 minutes.
Examples
Example 1: High throughput screening
Materials and General Methods:
Cell lines, Virus and Chemical Reagents
293T and Madin-Darby canine kidney (MDCK) cell lines were purchased from ATCC and maintained in MEM or DMEM with 10%heat inactivated fetal bovine serum (HI-FBS) . Influenza A virus subtypes A/WSN/33 [H1N1] , A/PR/8/34 [H1N1] were propagated in MDCK cell culture in  either plain MEM supplemented with 0.2%FBS. Other virus strains, including A/Hong Kong/415742/2009 [H1N1] , A/California/NHRC0007/2005 [H3N2] , and clinical isolate [H7N9] were propagated in either MEM or DMEM without FBS. All experiments involving live clinical isolate [H7N9] followed the standard operating procedures of the approved Biosafety Level 3 facility.
Drug libraries and chemicals
Small molecule compounds were purchased from Chembridge. Znamivir was purchased from GlaxoSmithKline.
A Preliminary High throughput screening of the structurally diverse small molecule library was performed to identify an influenza A/WSN/1933 [H1N1] inhibitor. After the initial screening a total of 82 analogues of compound 82 were ordered for further testing. The analogues were tested against an influenza A/WSN/1933 [H1N1] and influenza A/California/NHRC0007/2005 [H3N2] .
Plaque Reduction Assay (PRA) .
The PRA assay was performed in triplicate by seeding confluent MDCK cells in 24-well tissue culture plates (TPP, Switzerland) using MEM (Thermo Fisher Scientific, USA) in 10%FBS, prior to conducting the assay. After 16 to 24 h, cells were infected with either 50 plaque forming unit (P) of influenza virus for 24 well plates with or without the addition of serial diluted compounds. Infected cells were incubate at 37℃ with 5%CO 2 for 1.5 h before removing unbound viral particles by aspiration. After incubation, MEM with 1%FBS and 1μg/mL TPCK-treated trypsin (Thermo Fisher Scientific, USA) containing the corresponding concentration of compounds was mixed with 0.75%low melting agarose (LMA) (Thermo Fisher Scientific, USA) and overlaid onto the infected cells. The plates were incubated at 37℃ with 5%CO 2 for 72 h followed by fixation with 10%formaldehyde (BDH, Poole, England) for 3 h. The fixed plates were submerged into 75%v/v ethanol (Merck, Germany) for 5 min for complete disinfection. Agarose plugs were removed, the monolayers were stained with 0.7%crystal violet (BDH, Poole, England) , and the plaques were counted. The percent inhibition of plaque formation for each compound concentration relative to the control (without compound) was determined, and the median effective concentration, EC 50, representing the concentration of a drug that is required for 50%inhibition in vitro, was calculated by Sigma plot (SPSS, USA) . No FBS was included in the overlying medium for strains: A/Hong Kong/415742/2009 [H1N1] , A/California/NHRC0007/2005 [H3N2] , and the clinical isolate [H7N9] . Plaque reduction assays were conducted at 2 concentrations of analogues, 50μM and 10μM, the protections were normalized with the DMSO group.
Cell Viability Assay
CellTiterGlo kit (Promega) was used to test the cell viability of selected compounds by detection of ATP levels as a function of cell viability according to the manufacturer’s instructions. The assay was performed in triplicate by seeding MDCK cells at 20,000 cells/well in 100μL MEM supplemented with 10%FBS in 96-well cell culture plates one day prior to conducting the assay. After 24 h, cells were washed once with 1×PBS and replenished with MEM before addition of 2X serially diluted compounds mixed in MEM with 1%FBS, starting from 1 mM. The cells were then incubated at 37℃ with 5%CO2 for 24 h. 
Figure PCTCN2019090301-appb-000121
Reagents were added to each well according to manufacturer’s instruction and incubated for 10 min at room temperature. The luminescence signal was monitored by DTX880 multimode plate reader (Beckman coulter) . Cells inoculated to the medium without compounds served as the control. The relative metabolic activity of ATP was calculated by normalizing the mean of raw signals for each concentration of compound to the mean signal for the negative control. This calculation normalizes negative control to 100%ATP activity.
In vivo experiment
All experimental protocols followed the standard operating procedures of approved biosafety level 2 animal facilities and were approved by the Animal Ethics Committee. 5 to 7 week-old BALB/c female mice were kept in biosafety level 2 housing and were provided access to standard pellet feed and water ad libitum. Compound 26 (FA-6005) was dissolved in DMSO to prepare a 250 mM stock solution. The stock solution was kept in the dark at-20℃ to prevent degradation. 30μL of stock solutions containing FA-6005 or DMSO were dissolved in 3 ml 50%v/v methyl cellulose/PBS to make 2.5 mM solutions. Prophylactic treatment of three groups of mice 1 hour prior to infection was carried out as follows: One group (10 mice) was injected intraperitoneally (i.p. ) with 200μl of 10 mg/ml Zanamivir (GlaxoSmithKline) , a second group (10 mice) was injected with 200μl of 2.5 mM methyl cellulose/PBS solution containing compound FA-6005, and the untreated group (10 mice) was injected with 200μl of diluted DMSO in methyl cellulose/PBS solution. The mice were then subjected to intranasal inoculation with 30 LD 50 (6000 PFU) of the influenza A/PR/8/34 H1N1 virus in Zanamivir, FA-6005 or DMSO. The infected mice were then treated with two doses per day of i.p. zanamivir, FA-6005 or DMSO solution for 7 days. The mice were monitored for illness and survival rates for 14 days until death. Five mice from each group were sacrificed 6 th day post-infection to determine viral titre and pathological changes in the lungs by plaque assay (half lung) .
Results
Preliminary High throughput screening of the structurally diverse small molecule library identified Compound 82 to be an influenza A/WSN/1933 [H1N1] inhibitor. A total of 81 analogues to compound 82 were ordered for further testing. The analogues were tested against an influenza A/WSN/1933 [H1N1] , influenza A/California/NHRC0007/2005 [H3N2] and influenza A/Houston/21O2/2009 [H1N1 swine] results are shown in Table 1.
Compound 26 (FA-6005) was finalized for more in depth studies. Compound 26 showed desirable protection against an influenza A/WSN/1933 [H1N1] during the analogue screen and exhibited a low micromolar EC 50against other strains of influenza A, including influenza A/Houston/21O2/2009 [H1N1 swine] , influenza A/Hong Kong/HKU38/2004 [H3N2] , influenza A/Puerto Rico/8/1934 [H1N1] , A/WSN/1933 [H1N1] , and influenza A/Hong Kong/clinical isolate [H7N9] (Figure 1) . Compound 26 was also tested for the CC 50 (Cytotoxic concentration 50%) and estimated to be around 200μm in cell viability assay, giving this compound a good selective index. Since, Compound 26 (F-6005) exhibits broad spectrum antiviral activities against different strains of influenza, we speculate Compound 26 would be advantageous to inhibit possible new outbreaks of influenza.
Compound 26 (FA-6005) in vivo potency was evaluated in murine animal experiments. 5-7 weeks old female BALB/C mice were infected with influenza A/Puerto Rico/8/1934 [H1N1] , divided into three groups and subjected to different treatments twice for 7 days after infection. Survival rate was monitored, groups treated with existing neuraminidase inhibitor drug Zanamivir were 100%rescued, groups with DMSO treatment succumbed to infections starting from day 8 after exposed to the virus, and groups treated with Compound 26 showed 80%survival after the virus challenge. Compound 26 (FA-6005) also demonstrated a delay in time of death (Figure 2A) . Viral titers determination on day 5 post-infection in the mice lungs revealed a significant reduction in viral load in mice treated with Compound 26 (FA-6005) when compared to DMSO group (Figure 2B) . Collectively, the in vivo studies demonstrated that Compound 26 (FA-6005) could offer protection against lethal influenza A virus infection.
Table 1: Shows normalized viral protection of MDCK cells for each analog at 50μM and 10μM concentration against influenza A/WSN/1933 [H1N1] , and influenza A/California/NHRC0007/2005 [H3N2] .
Figure PCTCN2019090301-appb-000122
Figure PCTCN2019090301-appb-000123
Figure PCTCN2019090301-appb-000124
Figure PCTCN2019090301-appb-000125
Example 2: Compound 26 (FA-6005) mechanism of inhibition
Studies of FA-6005 escape mutant
The development of IAV resistant to FA-6005 was generated by serial passages of A/WSN/1933 [H1N1] in MDCK cells in the presence of increasing concentration of FA-6005. The A/WSN/1933 [H1N1] was also passaged in MDCK cells without the addition of FA-6005 as control. Influenza A/WSN/1933 [H1N1] virus was inoculated into MDCK cells at MOI=10 with at two concentrations of compound FA-6005. The lower concentration series were first passage with 5 X the A/WSN/1933 [H1N1] initial EC 50, the higher concentration being 10 X the initial EC 50. 72 hours post-infection, the viruses were harvested and passaged in MDCK at MOI=0.01 with the addition of increasing concentration of compound FA-6005. Escape mutants isolated after 5 passages were further studied. The desired FA-6005 resistant clones were purified by plaque isolation on MDCK cell monolayers in the presence of compound. Viral RNA were purified from FA-6005 escape mutants by viral RNA extraction, complementary DNA (cDNA) of all 8 segments  were obtained by reverse transcriptions using Superscript III reverse transcriptase (Invitrogen) . Genomic sequencing was conducted to identify any mutations. Recombinant influenza virus was generated by pHW2000 eight-plasmid system by reverse genetics. The eight plasmids contain the cDNA of the 8 segments of the influenza A/WSN/1933 [H1N1] genome: pHW2000-PB1, pHW2000-PB2, HW2000-PA, pHW2000-HA, pHW2000-NP, pHW2000-NA, pHW2000-M and pHW2000-NS. Drug resistant mutations were introduced to the parental plasmid using site-directed mutagenesis. The array of plasmids were transfected into co-cultured293T and MDCK cells with TransLT-Oligo Transfection Reagent (Mirus) according to manufacturer's manual. The infectious particles from the supernatants were harvested at 72 hours post-transfection, and the recombinant virus titer was determined by plaque assay.
Time of addition assay (TOA)
MDCK cells were seeded at 5x10 4 cells per well one day prior to the assay. Cells were washed once with PBS then subjected to virus infection on ice at MOI=1 in fresh DMEM containing 0.2%FBS and 1μg/mL TPCK-treated trypsin. The virus adsorption process was allowed for 1 hour on ice (-1 h) . After 1 hour, the cells were washed 3 times with PBS and the fresh DMEM containing 0.2%FBS and 1μg/mL TPCK-treated trypsin was added. The cells are then incubated at 37℃ with 5%CO 2 (time 0) . 20μM of FA-6005 or DMSO were added at indicated time points and removed at the next time point. Samples were collected at 10 h post-infection and subjected to titer determination using plaque assays. Each time point in the TOA was conducted in triplicates in 24-well tissue culture plates.
Luciferase reporter assay for polymerase complex activity
HEK293T cells were seeded in 96-wells plate at 2x10 4 Cells per well one day prior assay. Components of the RNP complex, consist of pHW2000-NP, plus pHW2000-PA, pHW2000-PBI, and pHW2000-PB2, or corresponding mutant plasmids were combined with a luciferase reporter plasmid, pHY-Luci. The plasmids were co-transfected into cultured HEK293T cells using TransLT-Oligo Transfection Reagent (Mirus) according to manufacturer's manual. Plasmid phRL-TK (promega) expressing renilla luciferase, was co-transfected as internal control for data normalization. 2 h post transfection, 2-fold serial-diluted compounds were added into the transfected cells. 24 h post transfection, cell lysates were collected using the Dual-Luciferase Reporter Assay System (Promega) , and luciferase activities were measured using DTX880 multimode detector (Beckman Coulter) .
SDS-PAGE and Western blot
MDCK Cells infected with influenza A/WSN/1933 [H1N1] at MOI=10 in fresh MEM supplemented with 0.2%FBS and 1μg/mL TPCK-treated trypsin with 20μM FA-6005 or DMSO were collected at 2, 4, 6, and 8 h post-infection. Unbounded viruses were removed and cells were washed once with PBS and lysed. The lysate were centrifuged and supernatants were collected, normalized to same amount and subjected to SDS-PAGE. The protein bands were transferred into nitrocellulose membrane, blocked with 10%w/v skim milk in PBST. Membranes were incubated with appropriate dilutions of primary antibodies of α-H1N1 and GADPH in a blocking solution overnight, then washed with PBST three times before incubated with recommended dilution of conjugated secondary antibodies in a blocking solution. The membrane was washed three times with TBST, dried and imaged with Odyssey Imagine system (LI-COR) .
Quantitative RT-PCR assays for different types of influenza RNA
MDCK cells were infected with influenza A/WSN/1933 [H1N1] at MOI=10 in fresh MEM supplemented with 0.2%FBS and 1μg/mL TPCK-treated trypsin with 20μM FA-6005 or DMSO. At 2, 4, 6, and 8 h post-infection, cells were harvested followed by extraction of viral RNAs using the RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. Standard curve of vRNA was generated for quantification of viral RNA. Viral gene segments in the pHW2000 vector were amplified with T7 promoter and T7 terminator, and linearized PCR products were purified by using PureLink PCR Purification Kit (Thermo Fisher Scientific) . RNA standard was synthesized from purified PCR product templates using the RiboMAX Large Scale RNA Production System-T7 kit (Promega Co. ) according to the manufacturer's manual. DNA templates in RNA transcript were digested by DNase I (New England Biolab) at 37℃ for IS min followed by a purification of the RNA transcript by using the RNeasy Mini Kit (QIAGEN) . Concentrations of the RNA transcripts and extracted RNA were determined using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc. ) , molecular copies of the synthetic RNA were then calculated. Reverse transcription was performed in the presence of saturated trehalose using PrimeScript RT-PCR Kit (Clontech) according to manufacturer's manual. Real-time PCR (qPCR) was performed using a FastStart Universal SYBR Green Master (Roche Life Science) and monitored with ABI VIA7 (Thermo Fisher Scientific Inc. ) .
Fluorescence in situ hybridization (FISH) and Immunofluorescence microscopy (IF )
MDCK cells were grown to 70-80%confluence on coverslips. Infections were carried out with by introduction of influenza A/WSN/1933 [H1N1] at MOI=10 in fresh MEM supplemented with 0.2%FBS and 1μg/mL TPCK-treated trypsin. 20μM of FA-6005 or DMSO was added at the  indicated time point. Infections were stopped at indicated time points with 500μl of 4%paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 min. The cells were washed with PBS for three times and permeabilized with 70%ethanol at 4℃ overnight. Custom designed Stellaris (Biosearch Technologies Inc. ) FISH Probes were used for the detection of vRNA and mRNA of segment 5. The slides preparation for simultaneous FISH and IF were carried out according to the manufacturer's instruction. The slides were imaged with Leica DMIL inverted microscope with DC300F digital imaging system (Leica Microsystems) or LSM710 confocal microscopy (Carl Zeiss AG) .
Virus uncoating assay and RNP import assay
Confluent MDCK cells were infected with influenza A WSN/33 virus or a mutant viruses in an infection medium (MEM with 0.2%FBS and lμg/ml TPCK-treated trypsin) at MOI=100 on ice for 1 h. After virus adsorption, the cells were washed with ice cold PBS to remove the unbounded virus particles. Fresh infection medium, with or without compounds, were added to the cells, and allowed to propagate at 37℃ with 5%CO 2 for indicated time. 1 mM cycloheximide was added to the culture to prevent the synthesis of new viral proteins. For the detection of M1 protein, 2 h after the infection, the infected cells were fixed and permeabilized with 0.1%of Triton-X 100 in PBS and further incubated with M1 monoclonal antibody (Abcam) for 1 h. Cells were washed with PBS then incubated with secondary anti-mouse IgG-AF488. Nuclei were stained with ProLong Gold anti-fade reagent with DAPI (Thermo Fisher Scientific) . RNP import was detected at 3 h post infection; the infected cells were stained with NP monoclonal antibodies in PBS and further incubated with anti-mouse IgG-AF488 secondary antibody.
Pulldown assays
Confluent MDCK cells were transfected with PHW2000-FLAG-GFP-NP. After 24 to 48 h, cells were infected with influenza A WSN/33 virus or mutant viruses in an infection medium (MEM with 0.2%FBS and lμg/mL TPCK-treated trypsin) at MOI=10. At 6 hours post-infection, 20μM of FA-6005 or equal volume of DMSO were added into the infected cells. Samples were collected and lysed 8 h post-infection on ice. Cell lysates were pre-cleared with mouse immunoglobulin G (IgG) agarose and the supernatant was bound to mouse
Figure PCTCN2019090301-appb-000126
M2 Magnetic Beads (Sigma-Aldrich Inc. ) on ice for 2 h. The bounded magnetic beads were wash with washing buffer containing 50 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 0.5%NP40, and 10%glycerol. Bounded proteins were eluted by boiling in SDS-PAGE sample buffer. The proteins were subjected to SDS-PAGE and Western blot.
Transmission electron microscopy
MDCK cells were seeded into 10 mm tissue culture dish at 5x10 6 cells per dish one day prior to infection. The cells were washed with PBS once and infected with influenza A/WSN/1933 [H1N1] at MOI=10 in fresh MEM supplemented with 0.2%FBS and lμg/mL TPCK-treated trypsin. At 6 h post-infection, 20μM of compounds or equivalent volume DMSO were added into the infected cells. At 8 hours post-infection, cells were washed with phosphate buffer thrice and incubated at 4℃ in 2.5%glutaraldehyde in 0.1 M phosphate buffer overnight. The monolayer was washed with phosphate buffer, cells were scrapped into phosphate buffer and harvested by centrifugation. The supernatant was replaced with fresh phosphate buffer and processed by Electron Microscope Unit at HKU. Images were acquired by FEI Philips CM100 transmission electron microscope equipped with a Deben AMT digital camera and an EDAX Genesis XM4 EDX system.
Results
Compound 82 and Compound 26 appear to inhibit influenza A virus by a mode of action different from other known antiviral drugs. Raising escape mutants against a drug is a rapid way to decipher its interacting partner, particularly in virus. Similar strategies were employed with gradual increasing of Compound 26 (FA-6005) concentration to generate resistant mutant virus from influenza A/WSN/1933 [H1N1] . The mutants had their genomes sequenced and mutation re-introduced into infectious particles by reverse genetics and tested against Compound 26 (FA-6005) to confirm the mutation sites. The substitution of isoleucine to threonine found on nucleoprotein amino acid41 was identified to be a highly resistant mutant against Compound 26 (FA-6005) in plaque reduction assays (Figure 3) . Molecular docking for NP-Compound interaction was modeled with AutoDock Vina, using the published crystal structures of influenza A/WSN/1933 [H1N1] NP protein and Compound 26 (FA-6005) . The docking model reveals that Compound 26 (FA-6005) could access into a pocket, which is in close proximity to residue 41, and mediate a hydrogen bond interaction with the isoleucine residue (Figure 4) . The threonine substitution might lead to abolishment of the interaction between Compound 26 (FA-6005) and the NP protein, resulting in the resistance of the compound.
NP protein is the most abundantly expressed protein during influenza infection, and serves multiple functions throughout the viral life cycle. Ranging from nuclear import of vRNPs, replication, transcription, to nuclear export of newly synthesized viral genome. Given that NP was likely the molecular target of Compound 26 (FA-6005) , it was speculated that Compound 26 (FA-6005) would exert inhibitory effect on the virus replication and trafficking. To differentiate the effect of the compound on the viral infection cycle, a time of addition experiments was conducted.  Compounds were added at different time points, before and after exposure to virus. A strong inhibitory effect was observed from-1 to 6 h post infection, indicating that Compound 26 (FA-6005) , interferes with the early to late stage of the influenza virus (Figure 5a) . This extends the effect of the compound on viral entry, replication and transcription, NP/vRNP export and release processes. This was in agreement with the replication stage inhibition observed in the modified luciferase reporter based mini genome assay between the wild type virus and the escape mutant virus under different concentration of Compound 26 (FA-6005) (Figure 5b) . To further validate the inhibitory effect of Compound 26 (FA-6005) on replication and/or transcription of influenza A infections. The macromolecule synthesis of viral RNAs and viral proteins were observed by qPCR and western blotting. The production of vRNA was monitor by specific primers, for the wild type and escape mutant virus with or without the addition of Compound 26 (FA-6005) . In the presence of Compound 26 (FA-6005) , wild type virus has significant reduction in the vRNA production (Figure 6) . A similar profile is also observed in the protein expression level, protein production increased over time in the absence of compound but were suppressed in the presence of Compound 26 (FA-6005) . The inhibitory effect of Compound 26 (FA-6005) on overall viral replication process could be correlated in the plaque formation in time of addition assay. The inability of Compound 26 (FA-6005) to inhibit NP 141 T mutant in mini-genome assay, vRNA expression and protein expression indicated that NP is the binding target for Compound 26 (FA-6005) , and possesses a strong correlation with residue 41 for interaction.
Apart from viral transcription and replication processes, NP protein also participates in the RNP translocation between cytoplasm and nucleus. FISH combined immunofluorescence imaging was used to visualize the migration of viral nucleoprotein as well as the vRNA and mRNA of segment 5 (containing NP) under the influence of Compound 26 (FA-6005) . The result indicated that the nuclear export process of vRNPs was inhibited at 8 h post-infection with the addition of Compound 26 (FA-6005) when compared to controls. Similar experiments with the NP 141 T mutants was not affected by Compound 26 (FA-6005) , the nuclear export process of the mutant virus was not inhibited. Further, supporting the role of Compound 26 (FA-6005) in inhibitory function with NP and its interactive site with the 41 isoleucine residue. The addition of Compound 26 (FA-6005) will result in impaired virion budding process (Figure 9) with the generation of defective virions. This defective virion budding process can be correlated with the impede vRNP cytoplasmic trafficking, resulting in the decrease in titre output in TIA assay in the late stage of the viral infection.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises, ” means “including but not limited to, ” and is not intended to exclude, for example, other additives, components, integers or steps.
“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about, ” it will be understood that the particular value forms another, specifically contemplated form that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. It should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. Finally, it should be understood that all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these forms are explicitly disclosed.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by  virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Claims (20)

  1. A compound of Formula I or Formula II or a pharmaceutically acceptable salt thereof or a prodrug thereof:
    Figure PCTCN2019090301-appb-100001
    wherein
    A is a substituted or unsubstituted fused 5 or 6-member heterocyclic ring;
    R 1 is
    Figure PCTCN2019090301-appb-100002
    wherein
    G is independently C=O or SO 2;
    R 2-R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  2. The compound of claim 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein R 2-R 7 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; -COOH, -COO -, -COR 10, -NH 2CO, -CONH 2, -CONHR 9, -CONR 9R 9, -NHCOR 9, -NR 9COR 9, -OCONHR 9; -NHCOOR 9, -OCONR 9R 9; -NR 9COOR 9, -NHCONHR 9; -NR 9CONHR 9, -NHCONR 9R 9, -NR 9CONR 9R 9, -NHCSNHR 8, -CH 2OH; -CHR 9OH, -CR 9R 9OH, -COOR 9, -SH, -NH 2, -NHR 9, -NR 9R 9, -SR 9, -SOR 9, and-SOOR 9;
    wherein each R 8-R 10 of R 2-R 7 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is of Formula I and R 4 is the same as R 1.
  4. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein Formula II is
    Figure PCTCN2019090301-appb-100003
    E is independently O, S, N, NR 15, CR 16R 17, or C=O;
    D and J are independently O, S, N, NR 15, CR 16R 17, or C=O;
    L and M are independently O, S, N, NR 15, CR 16R 17, or C=O;
    R 11 is
    Figure PCTCN2019090301-appb-100004
    wherein G is independently C=O or SO 2;
    each R 12-R 17 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano;  formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  5. The compound of claim 4 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 12-R 17 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl;
    acyl; -COOH; -COO -; -COR 20; -NH 2CO; -CONH 2; -CONHR 19; -CONR 19R 19; -NHCOR 19; -NR 19COR 19; -OCONHR 19; -NHCOOR 19; -OCONR 19R 19; -NR 19COOR 19; -NHCONHR 19; -NR 19CONHR 19; -NHCONR 19R 19; -NR 19CONR 19R 19; -NHCSNHR 18; -CH 2OH; -CHR 19OH; -CR 19R 19OH; -COOR 19; -SH; -NH 2; -NHR 19; -NR 19R 19; -SR 19; -SOR 19; and-SOOR 19,
    wherein each R 18-R 20 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  6. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein when R 4 is-NHCOR 9, then R 9 is
    Figure PCTCN2019090301-appb-100005
    wherein
    U is independently O, S, N, NR 24, CR 25R 26, or C=O;
    Q and T are independently O, S, N, NR 27, CR 28R 29, or C=O;
    V and W are independently O, S, N, NR 27, CR 28R 29, or C=O;
    wherein each R 21-R 29 is independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or  alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  7. The compound of claim 6 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein one or more R 21-R 29 are independently absent or selected from hydrogen; halogen; nitro; substituted or unsubstituted, linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; substituted or unsubstituted aryl or heteroaryl; substituted or unsubstituted alkoxy; hydroxyl; cyano; formyl;
    acyl; -COOH; -COO -; -COR 32; -NH 2CO; -CONH 2; -CONHR 31; -CONR 31R 31; -NHCOR 31; -NR 31COR 31; -OCONHR 31; -NHCOOR 31; -OCONR 31R 31; -NR 31COOR 31; -NHCONHR 31; -NR 31CONHR 31; -NHCONR 31R 31; -NR 31CONR 31R 31; -NHCSNHR 30; -CH 2OH; -CHR 31OH; -CR 31R 31OH; -COOR 31; -SH; -NH 2; -NHR 31; -NR 31R 31; -SR 31; -SOR 31; and-SOOR 31,
    wherein each R 30-R 32 is independently absent or selected from hydrogen; halogen; nitro; linear, branched, hetero, cyclic, or heterocyclic alkyl, alkenyl, or alkynyl; aryl; heteroaryl; alkoxy; hydroxyl; cyano; formyl; acyl; carboxylic acid; carboxylate; carbonyl; primary amide; secondary amide; tertiary amide; secondary carbamate; tertiary carbamate; urea; thiourea; carbinol; ester; thiol; primary amine; secondary amine; tertiary amine; thioether; sulfinyl group; and sulfonyl group.
  8. The compound of claim 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound is a compound selected from the group consisting of:
    Figure PCTCN2019090301-appb-100006
    Figure PCTCN2019090301-appb-100007
    Figure PCTCN2019090301-appb-100008
    Figure PCTCN2019090301-appb-100009
    Figure PCTCN2019090301-appb-100010
    Figure PCTCN2019090301-appb-100011
    Figure PCTCN2019090301-appb-100012
    Figure PCTCN2019090301-appb-100013
    Figure PCTCN2019090301-appb-100014
    Figure PCTCN2019090301-appb-100015
    Figure PCTCN2019090301-appb-100016
    Figure PCTCN2019090301-appb-100017
    Figure PCTCN2019090301-appb-100018
    Figure PCTCN2019090301-appb-100019
    Figure PCTCN2019090301-appb-100020
    Figure PCTCN2019090301-appb-100021
    Figure PCTCN2019090301-appb-100022
    Figure PCTCN2019090301-appb-100023
    Figure PCTCN2019090301-appb-100024
  9. The compound of claim 1 or a pharmaceutically acceptable salt thereof or a prodrug thereof, wherein the compound has the structure:
    Figure PCTCN2019090301-appb-100025
  10. A pharmaceutical composition comprising one or more compounds of any one of claims 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof and one or more pharmaceutically acceptable carriers.
  11. The pharmaceutical composition of claim 10, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof are present in an effective amount to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, or a combination thereof.
  12. The pharmaceutical composition of claim 10 or 11 further comprising one or more pharmaceutically acceptable excipients.
  13. A method of treating a subject in need thereof, the method comprising administering one or more compounds of any one of claims 1-9 or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition of any of claims 10-12 in an amount effective to treat the subject in need thereof.
  14. The method of claim 13, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is in an amount effective to inhibit viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof.
  15. The method of claim 13 or 14, wherein the subject has an infection, which is preferably a viral infection involving a viral nucleoprotein.
  16. The method of claim 15, wherein the viral infection is an influenza A viral infection.
  17. The method of any one of claims 13-16, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition is administered by one or more routes selected from the group consisting of buccal, sublingual, intravenous, subcutaneous, intradermal, transdermal, intraperitoneal, oral, eye drops, parenteral and topical administration.
  18. The method of any one of claims 13-17, wherein the subject has a viral infection.
  19. The method of any one of claims 13-17, wherein the subject is at risk of developing a viral infection.
  20. The method of claim 18 or 19, wherein the one or more compounds or a pharmaceutically acceptable salt thereof or a prodrug thereof or the composition inhibits viral transcription, viral replication, viral protein synthesis, viral RNP export, the virion budding process, viral nucleoprotein activity, or a combination thereof in the subject.
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